F a c u l t y o f V e t e r i n a r y S c i e n c e

VETERINARY CLINICAL PATHOLOGY

VETS 3243

Lecture Notes, Case Reports, Practical Notes

UOS Coordinator Dr Rachael Gray

Professor Paul J Canfield Revised in 2010 by Rachael Gray

© 2010

PREFACE

The discipline of Veterinary Clinical Pathology (Laboratory Medicine) focuses on the laboratory investigation of disease for purposes of diagnosis and prognosis. Whilst Veterinary Clinical Pathology is taught as an independent unit of study, it is important to realise its connection to other disciplines and units of study for purposes of disease investigation. Consequently, this unit of study builds on Veterinary Pathology, Veterinary Microbiology, Veterinary Parasitology; integrates horizontally with Small Animal Medicine and Animal Disease taught within the same semester; and prepares students for both small animal practice and large animal health and production and clinical practice. Because of this, Veterinary Clinical Pathology should be studied in conjunction with these other disciplines for purposes of assessment. These notes are by no means a complete reference for clinical pathology. They are based on lectures and practical classes that are provided to Veterinary Science Students, and are meant to complement learning through formal interaction. They provide information not only on dogs and cats but also on horses and farm animals. Students will find the clinical pathology information on horses and farm animals useful when studying Equine Medicine, Ruminant Health and Production and Pig Health and Production. Moreover, since the notes provide a conceptual framework for the laboratory investigation of disease in all species, they have applicability to other species such as wildlife or laboratory animals. The lecture theory notes are supported by a series of case reports. An index is provided for both of these. There is no index for the practical notes, but a comprehensive table of contents exists. Undoubtedly there will be mistakes in the notes, and I would appreciate the reader's assistance in correcting the contents.

TABLE OF CONTENTS LEARNING OUTCOMES (OBJECTIVES) FOR VETERINARY CLINICAL PATHOLOGY......................................................1

Introduction ........................................................................................................................................................................1 Generic Skills and Graduate Competences ....................................................................................................................1 1. Knowledge skills ......................................................................................................................................................................................1 2.Thinking skills ...........................................................................................................................................................................................1 3.Personal skills...........................................................................................................................................................................................1 4.Personal attributes....................................................................................................................................................................................1 5.Practical skills ...........................................................................................................................................................................................2 What is Veterinary Clinical Pathology? ...........................................................................................................................2 Main Learning Outcomes..................................................................................................................................................2 Further information on main learning outcomes..........................................................................................................................................3

PRINCIPLES OF ASSESSMENT FOR VETERINARY CLINICAL PATHOLOGY ...................................................................5 Assessment Format...........................................................................................................................................................5 Summative assessment ........................................................................................................................................................5 Formative assessment ..........................................................................................................................................................5

Grade Descriptors for Veterinary Clinical Pathology: ....................................................................................................5 Past Examination Question for Veterinary Clinical Pathology, with example of a model answer:............................8 Suggested Approach To Answering Examination Questions................................................................................................8 Model Answer for 2004 Examination Question ...................................................................................................................13

VETERINARY CLINICAL PATHOLOGY (LABORATORY MEDICINE).................................................................................17 Introduction ......................................................................................................................................................................17 Necropsy Technique and Collection of Specimens for Histopathology...................................................................................18 The Nature of Laboratory Tests, Problems for Interpretation and Reference Intervals............................................18 General Remarks on Sampling for Blood Biochemistry ..............................................................................................19 Specimen storage ...............................................................................................................................................................19 Haemolysis..........................................................................................................................................................................19 Gross Lipemia (and its relationship to laboratory detected hyperlipidemia)........................................................................20

Plasma/Serum Enzymology .............................................................................................................................................20 Factors affecting plasma levels of enzymes .......................................................................................................................20 Naming of enzymes ............................................................................................................................................................21 Combining enzymes to detect disease processes ..............................................................................................................23

LABORATORY EVALUATION OF HEPATIC DISEASE........................................................................................................24 Terminology ......................................................................................................................................................................24 Introduction .......................................................................................................................................................................24 Summary of Tests............................................................................................................................................................25 Hepatocellular Damage (Degeneration and/or Necrosis) ...........................................................................................25 Cholestasis.......................................................................................................................................................................26 Bilirubin Metabolism and Analysis ................................................................................................................................27 Tests for Reduced Functional Hepatic Mass ................................................................................................................30 a) Bile acids........................................................................................................................................................................30 b) Blood ammonia determination .......................................................................................................................................30 c) Serum protein estimation ...............................................................................................................................................31

Other Laboratory Tests .........................................................................................................................................................32 A. Bromosulphthalein (BSP, sulfobromophthalein)........................................................................................................32 B. Cholesterol ................................................................................................................................................................32 C. Glucose .....................................................................................................................................................................32

LABORATORY EVALUATION OF URINARY TRACT DISEASE..........................................................................................33 Terminology .....................................................................................................................................................................33 Introduction ......................................................................................................................................................................33 Some of the Laboratory Tests used to Detect Renal Dysfunction..............................................................................34 Assessment of glomerular function .....................................................................................................................................34 Assessment of tubular function ...........................................................................................................................................36

• Miscellaneous Biochemical Alterations that may occur in Various Forms of Renal Disease..............................43

LABORATORY INVESTIGATION OF SOME DIGESTIVE TRACT DISORDERS ................................................................45 Terminology .....................................................................................................................................................................45 Exocrine Pancreatic Disorders.......................................................................................................................................45 Investigation of Colic in the Horse.................................................................................................................................46 Investigation of Problem Diarrheas ...............................................................................................................................47 1. Faecal examination (coprology) ................................................................................................................................47 2. Peritoneal fluid analysis - abdominocentesis (see under fluid analysis) ...................................................................48 3. Absorption tests.........................................................................................................................................................49 4. General haematological and biochemical testing......................................................................................................50 5. Endoscopy, exploratory laparotomy, intestinal washings and biopsy........................................................................50 Additional Notes on Malassimilation .............................................................................................................................51

LABORATORY INVESTIGATION OF ENDOCRINE DISORDERS, INCL. CALCIUM & PHOSPHATE DERANGEMENTS 52 Introduction ......................................................................................................................................................................52 Diabetes mellitus (DM) ....................................................................................................................................................52 Hyperinsulinism...............................................................................................................................................................54 Hyperadrenocorticism.....................................................................................................................................................54 Hypoadrenocorticism (Adrenal Insufficiency - AI) .......................................................................................................55 Hypothyroidism................................................................................................................................................................56 Hyperthyroidism ..............................................................................................................................................................56 Introduction to Calcium and Phosphate Derangements..............................................................................................57 Persistent Hypocalcemia (decreased calcium on two or more occasions) ...............................................................57 Persistent Hypercalcemia (elevated calcium on two or more occasions) .................................................................58 Derangements of magnesium.........................................................................................................................................59 Hypermagnesemia ...........................................................................................................................................................59 Hypomagnesemia ............................................................................................................................................................59

WATER, ELECTROLYTES AND ACID/BASE BALANCE.....................................................................................................60 Introduction ......................................................................................................................................................................60 Hydration Status (Assessment of Total Body Water)...................................................................................................60 Electrolyte Status.............................................................................................................................................................60 Acid/Base Balance............................................................................................................................................................62

HAEMATOLOGY.....................................................................................................................................................................63 Haematological Disturbances and how they reflect Host-Pathogen-Environmental Factor Interactions...............63 Introduction to Haematological Investigation...............................................................................................................66 Some Common Haematological Terms .........................................................................................................................67

DISORDERS/RESPONSES OF THE ERYTHRON.................................................................................................................72 a) Dynamics of erythrocyte production..........................................................................................................................72 b) Anaemia .......................................................................................................................................................................73

i) Laboratory confirmation of anaemia..........................................................................................................................73 ii) Classifying the anaemia on the basis of mechanisms:..............................................................................................73 Some General Comments on Bone Marrow Examination............................................................................................77 Polycythemia....................................................................................................................................................................79

DISORDERS/RESPONSES OF THE LEUKOCYTES ............................................................................................................80 Introduction ......................................................................................................................................................................80 Leukocytosis ....................................................................................................................................................................80 Neutrophilia .....................................................................................................................................................................81 Physiological neutrophilia (leukocytosis).............................................................................................................................81 Corticosteroid induced neutrophilia (leukocytosis)..............................................................................................................82 Neutrophilia due to regenerative anaemia ..........................................................................................................................82 Neutrophilia related to inflammatory demand .....................................................................................................................82 Neutropenia ......................................................................................................................................................................85 Monocytes ........................................................................................................................................................................86 Eosinophils.......................................................................................................................................................................86 Basophils..........................................................................................................................................................................86 Lymphocytes....................................................................................................................................................................87

HAEMATOPOIETIC NEOPLASIA ..........................................................................................................................................88 Introduction ......................................................................................................................................................................88 Lymphoproliferative Disorders.......................................................................................................................................88 a) Lymphosarcoma (malignant lymphoma)........................................................................................................................88 b) Acute lymphoblastic leukemia (ALL)..............................................................................................................................89 c) Chronic lymphocytic leukemia (CLL)..............................................................................................................................89 d) Plasma cell myeloma (multiple myeloma)......................................................................................................................90 Myeloproliferative Disorders ..........................................................................................................................................90

BLEEDING DISORDERS (ABNORMALITIES OF HAEMOSTASIS) .....................................................................................93 Introduction to Haemostatis ...........................................................................................................................................93 Introduction to Bleeding Disorders................................................................................................................................94 Laboratory Evaluation of Bleeding Disorders...............................................................................................................94 a) Vessel wall defect ..........................................................................................................................................................95 b) Thrombocytopenia .........................................................................................................................................................95 c) Platelet function defect...................................................................................................................................................95 d) Clotting factor deficiency................................................................................................................................................96 e) Excessive fibrinolysis .....................................................................................................................................................97 Summary...........................................................................................................................................................................97 Laboratory results for some common bleeding disorders ...................................................................................................98

IMMUNODIAGNOSTICS (for diseases with an immune base – immune-mediated diseases and immunodeficiency)...........................99 Introduction ......................................................................................................................................................................99 Autoimmunity.................................................................................................................................................................100 a) Coombs' test (direct)....................................................................................................................................................100 b) Rheumatoid factor (RF) ...............................................................................................................................................100 c) The antinuclear antibody test (ANA) ............................................................................................................................101 d) Direct and indirect immunofluorescence (IF) of tissue biopsies...................................................................................101 Immunodeficiency .........................................................................................................................................................101

DIAGNOSTIC CYTOLOGY AND BODY FLUID ANALYSIS ................................................................................................102 Diagnostic Cytology (Exfoliative Cytology; Cytopathology) .....................................................................................102

a) Solid tissue cytology ....................................................................................................................................................102 b) Diagnostic cytology as a component of body fluid analysis ..........................................................................................107 Body FLuid Analysis .....................................................................................................................................................107 a) Body cavity effusions ...................................................................................................................................................107 b) Synovial fluid analysis..................................................................................................................................................111 c) Cerebrospinal fluid evaluation......................................................................................................................................113 d) Analysis of airway and pulmonary lesions by respiratory washes (transtracheal aspirates, bronchoalveolar lavages) ...114

CASE REPORTS........................... 119 Introduction ............................ 119

CASE: 1 ......................... 123 CASE: 2 ......................... 125 CASE: 3 ......................... 127 CASE: 4 ......................... 129 CASE: 5 ......................... 132 CASE: 6 ......................... 134 CASE: 7 ......................... 136 CASE: 8 ......................... 138 CASE: 9 ......................... 140 CASE: 10....................... 142 CASE: 11....................... 144 CASE: 12....................... 146 CASE: 13....................... 148 CASE: 14....................... 150 CASE: 15....................... 152 CASE: 16....................... 154 CASE: 17....................... 156 CASE: 18....................... 158 CASE: 19....................... 160 CASE: 20....................... 162 CASE: 21....................... 164 CASE: 22....................... 167 CASE: 23....................... 169 CASE: 24....................... 171 CASE: 25....................... 173 CASE: 26....................... 175 CASE: 27....................... 177 CASE: 28....................... 179 CASE: 29....................... 181 CASE: 30....................... 183 CASE: 31....................... 185 CASE: 32....................... 187 CASE: 33....................... 189 CASE: 34....................... 191 CASE: 35....................... 193 INDEX ............................ 195

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LEARNING OUTCOMES (OBJECTIVES) FOR VETERINARY CLINICAL PATHOLOGY

Introduction

The purpose of this section is to provide students with an idea of the capabilities they are expected to develop during, and the abilities required of them at the end of, VETS 3243 (Veterinary clinical Pathology). In plain English, this section focuses the student on what is important in Clinical Pathology and how this can be applied to the processes of diagnosis and treatment of disease that are essential for practising veterinarians.

Generic Skills and Graduate Competences

Before presenting learning outcomes directly linked to Veterinary Clinical Pathology, it should be mentioned that students are expected to develop certain generic skills during the unit of study. These generic skills are University of Sydney Policy (Academic Board, 1997). These generic skills will come into play during all teaching strategies employed in Veterinary Clinical Pathology and, indeed, are applicable to all units of study in Veterinary Science and in the University. They include:

1. Knowledge skills Graduates should:

a. have a body of knowledge in the field(s) studied; b. be able to apply theory to practice in familiar and unfamiliar situations; c. be able to identify, access, organize and communicate knowledge in both written and oral

English.

2. Thinking skills Graduates should:

a. be able to exercise critical judgment; b. be capable of rigorous and independent thinking; c. be able to account for their decisions; d. be realistic self evaluators; e. adopt a problem solving approach.

3. Personal skills Graduates should have:

a. the capacity for and a commitment to life-long learning; b. the ability to plan and achieve goals in both the personal and the professional sphere; c. the ability to work with others.

4. Personal attributes Graduates should:

a. strive for tolerance and integrity; b. acknowledge their personal responsibility for their own value judgments; and their ethical

behaviour towards others.

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5.Practical skills Graduates should

a. be able to use information technology for professional and personal development; and, where appropriate, be able to:

b. collect, correlate, display, analyse and report observations; c. apply experimentally-obtained results to new situations; d. test hypotheses experimentally; and e. apply technical skills appropriate to their discipline.

These generic skills effectively apply to what is termed ‘Day 1 Competences for Graduating Veterinarians’. Day 1 competences are expected by the accrediting bodies of the Faculty of Veterinary Science (VSAAC; RCVS; AVMA) and include not only generic competences but also practical and knowledge competences. In terms of Veterinary Clinical Pathology, the RCVS has designated the following practical Day 1 competency: C1.6 Collect, preserve and transport samples, perform standard laboratory tests, and interpret the results of those generated in-house, as well as those generated by other laboratories (Commentary: new graduates are expected to have a working knowledge of tests to be undertaken include conditions relating to infectious & contagious diseases; alimentary system; respiratory system; circulatory system; urinary system; nervous system; endocrine system; mucucutaneous system; musculoskeletal system; trauma; poisoning; obstetrics; paediatrics; parturition; reproduction)

What is Veterinary Clinical Pathology?

Veterinary Clinical Pathology is also called Laboratory Medicine. This implies that it is a component of Veterinary Medicine. Veterinary Medicine encompasses the diagnosis and treatment of disease. Veterinary Clinical Pathology assists in the diagnostic process by providing laboratory information/evidence. This information or data is the result of biochemical, Haematological, cytological, pathological, microbiological and parasitological investigations. The sick animal is presented for assessment and the veterinary practitioner decides if the laboratory can provide information that will assist diagnosis. If this information is utilised/interpreted in a logical manner and with common sense, then the disease process (aetiology, host response and effect on host) can be better understood. Laboratory information may also be useful in monitoring an animal’s response to medical or surgical treatment of a disease. Since the course consists of lectures, tutorials and practical classes, there are several broad learning outcomes (or aims) for Veterinary Clinical Pathology. The first and second ones are most important and the written examination will be solely based on these. The third and fourth outcomes will provide the bulk of practical marks:

Main Learning Outcomes

1. At the end of this course of study, students will be expected to have the ability to identify alterations in laboratory data and to critically evaluate their significance for diagnosis and prognosis of disease in animals. 2. At the end of this course of study, students will be expected to have the capacity to think logically, critically and with common sense in relation to the role of Veterinary Clinical Pathology in Veterinary Medicine in providing assistance in diagnosis and prognosis of disease in animals.

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3. At the end of the practical course, with respect to a range of simple laboratory tests commonly utilised in veterinary practice (in-house testing), students will be expected to:

(a) understand the theory of operation for each test (b) have the ability to mechanically perform each test (c) be aware of the limitations and strengths of each test in order to know when to apply

them for the purposes of diagnosis or prognosis of disease in animals. 4. At the end of the practical course, students will be expected to understand the workings of a pathology laboratory and have an awareness of the range of tests available to the veterinary practitioner. Further information on main learning outcomes 1. At the end of this course of study, students will be expected to have the ability to identify

alterations in laboratory data and to critically evaluate their significance for diagnosis and prognosis of disease in animals.

2. At the end of this course of study, students will be expected to have the capacity to think logically, critically and with common sense in relation to the role of Veterinary Clinical Pathology in Veterinary Medicine in providing assistance in diagnosis and prognosis of disease in animals.

To achieve these linked learning outcomes, students will be exposed to animal case reports in lectures and tutorials and provided with a logical, stepwise approach to identifying and interpreting laboratory data alterations. Laboratory data include haematological values; biochemical values; urinalysis; faecal analysis; parasitological, microbiological and pathological results; fluid analysis; and cytological results. Students will not be expected to memorise information but will be expected to be able to retrieve that information efficiently from written sources and to apply it with common sense and understanding. Secondary or expanded learning outcomes of this process are: Students will develop a capacity to understand the limitations of reference intervals, and to utilise reference intervals to identify laboratory data alterations. Reference intervals are established for each laboratory test by collecting samples from clinically healthy animals. • Students will be expected to critically evaluate laboratory data alterations in light of available

clinical information. Clinical information includes history, clinical signs, the results of physical examination, and the results of non-laboratory diagnostic aids (e.g. image analysis)

• Students will be expected to reach a level of diagnosis from the available laboratory data and to be able to recognise the need for further clinical or laboratory testing in order to more specifically define the disease problem.

3. At the end of the practical course, with respect to a range of simple laboratory tests

commonly utilised in veterinary practice (in-house testing), students will be expected to: (a) understand the theory of operation for each test (b) have the ability to mechanically perform each test (c) be aware of the limitations and strengths of each test in order to know when to apply

them for the purposes of diagnosis or prognosis of disease in animals. Secondary or expanded learning outcomes of this process are: • Students will be expected to revise the theory of operation behind simple laboratory tests which

will be provided in the Veterinary Clinical Pathology Handbook and which have been provided in the practical components of earlier units of study (particularly Cell Biology, Animal Structure

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and Function, Principles of Disease and Veterinary Pathology). • Students will be expected to perform simple laboratory procedures relating to biochemistry

(blood urea and glucose analysis), urinalysis, haematology (packed cell volume and total plasma protein determination, and differential leukocyte analysis on blood films), blood coagulation (fibrinogen determination and platelet assessment) and cytology (fine needle cell aspiration and preparation of smears).

• Students will be expected to be able to use a light microscope and to identify different blood cells, elements of urine and basic cell types in cytological smears.

• Students will be expected to understand the usefulness of these simple laboratory procedures in veterinary practice in relation to diagnosis and treatment of disease. Limitations and strengths of each test will be provided in the Veterinary Clinical Pathology Handbook or through written manufacturer’s information.

4. At the end of the practical course, students will be expected to understand the workings of a

pathology laboratory and have an awareness of the range of tests available to the veterinary practitioner.

Secondary or expanded learning outcomes of this process are: • Students will be able to collect appropriate fluid and tissue samples for laboratory evaluation • Students will be able to correctly label samples and fill out laboratory request sheets • Students will be expected to be aware of the range of tests available from a commercial pathology

laboratory. In particular, this refers to an awareness of the types of biochemical and Haematological tests available and how their application varies among animal species.

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PRINCIPLES OF ASSESSMENT FOR VETERINARY CLINICAL PATHOLOGY

Assessment Format Summative assessment Intra-semester assessment consists of: 1. Three case report tutorials will each be marked out of 10% (then an average determined). The three practicals must be attended for students to be eligible for the full 10%. 2. Intra-semester examination of one case report in Week 8 - Monday 13th September

This written assessment contributes 15% of the marks for this unit of study. It will be 45 minutes in duration and open book. Further details of this assessment will be provided at the start of the semester. A feedback session will be provided on this assessment to ensure that this assessment is both summative and formative.

Formal examination period (November 2010): 3. Two hour written examination consisting of two case reports for analysis. This will be an open-book assessment and will contribute 75% of the marks for the unit of study. Formative assessment During lectures, students will be presented with unknown case reports. An approach to evaluation for these case reports will be provided beforehand. This approach is the same as used in tutorials, and is the one expected to be used in the written examinations. Students will be given time to evaluate the case reports before the lecturer analyses the case reports. Students will be given the opportunity to ask questions and to respond to questions during the analysis. Students are encouraged to work through the case reports in the Veterinary Clinical Pathology Handbook and to discuss these with the lecturer. Some practical sessions will be devoted to case report evaluations. This will be done in small groups. Oral presentation will be part of the analysis process.

Grade Descriptors for Veterinary Clinical Pathology

PASS

Denotes satisfactory achievement of main learning outcomes. The student has demonstrated: 1. a basic capacity to recognize and evaluate alterations in laboratory data. To do this requires a

basic capability of retrieving and understanding relevant information from selected information sources; and a basic capability to think logically and critically about the utilization of that relevant information (this will be assessed in a written examination on case reports; and during tutorials and presentations of case reports).

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2. a basic capability to perform and to understand the usefulness of selected simple laboratory tests (this will be assessed by direct observation and questioning of the student in practical classes and tutorials).

3. a basic knowledge of the collection of biological samples for laboratory evaluation. This will involve a simple understanding of the range of biological samples that can be collected, and how they are collected (this will be assessed in a written examination on case reports; and during practical classes, tutorials and presentations of case reports).

CREDIT

This denotes work of predominantly good quality on all specified learning outcomes. The student has demonstrated: 1. a sound capacity to recognize and evaluate alterations in laboratory data. To do this requires an

above basic capability of retrieving and understanding relevant information from selected information sources; and an above basic capability to think logically and critically about the utilization of that relevant information. The emphasis for grading will be more on evaluation rather than recognition of alterations of laboratory data, and a sound understanding of how this provides assistance in general disease diagnosis and prognosis. The student will have a good style (logical, well organized and clear) for written presentations of case reports.

2. a sound capability to perform and to understand the usefulness of selected simple laboratory tests. The emphasis of grading will be more on understanding the limitations and strengths of selected simple laboratory tests.

3. an above basic knowledge of the collection of biological samples for laboratory evaluation. The student will be aware of some of the problems related to collecting various biological samples, and how these affect results of the tests.

DISTINCTION

This denotes work of superior quality on all specified learning outcomes. The student has demonstrated: 1. a superior capacity to evaluate alterations in laboratory data. To do this requires a superior

capability to think logically and critically about the utilization of that relevant information. The student will be expected to fully understand how laboratory information assists general disease diagnosis and prognosis. It is expected that the student will have a strong capability to suggest further laboratory testing and the utilization of other diagnostic aids in order to reach a diagnosis. The student will have a superior style (improved logicality, organization and clarity) for written presentations of case reports.

2. a sound capability to perform and a superior capability to understand the usefulness of selected simple laboratory tests. Students should be able to show evidence of additional reading/information on, and a strong knowledge of the limitations of, the selected simple laboratory tests.

3. a superior knowledge of the collection of biological samples for laboratory evaluation. Students will be aware of many of the important problems related to the collection of biological samples.

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HIGH DISTINCTION

Denotes work of exceptional quality on all specified learning outcomes. The student has demonstrated: 1. an exceptional capacity to evaluate alterations in laboratory data. To do this requires an

exceptional capability to think logically and critically about the utilization of that relevant information. The student will have read widely and be knowledgeable of current debates on the value of utilization of laboratory tests. This will be incorporated in the general interpretation of laboratory data and the discussion of further testing. The student will have an excellent style (exceptional logicality, organization and clarity) for written presentations of case reports.

2. a sound capability to perform and an exceptional capability to understand the usefulness of selected simple laboratory tests. Students should be able to show evidence of considerable reading/information on, and an excellent knowledge of the limitations and strengths of, the selected simple laboratory tests.

3. a superior knowledge of the collection of biological samples for laboratory evaluation. The student will have read widely and be aware of all the important problems of collecting various biological samples.

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Past Examination Question for Veterinary Clinical Pathology, with example of a model answer: Suggested Approach To Answering Examination Questions Think about the information given before looking at the laboratory data. Ask yourself what can be deduced from the history, signalment and clinical findings:

• Is there an indication of time course? • Is age, sex or breed important? • What organs or tissues are involved? • Is there an indication of the pathological process(es) occurring? • Is there an indication of cause?

Keep your deductions in mind when interpreting the laboratory data and thinking about further investigation 1. LIST/HIGHLIGHT Abnormalities (DETECT AND DESCRIBE) This is for your benefit to ensure you don’t forget to discuss any abnormalities. Remember to use the book information on what is regarded as mild, moderate and marked alterations (see the beginning of the Section on Case Reports). 2. GENERAL INTERPRETATION (DEDUCE) Consider the reasons for abnormalities. Do not give complete lists of reasons but only those which could relate to the case. Relate to information (history etc) given to you in the question. You will use this information in reaching conclusions. This can be incorporated in your conclusions if your prefer (i.e. give a conclusion and then add your reasons for reaching it) 3. CONCLUSION(S)/FURTHER INVESTIGATION/IMPLICATIONS FOR MANAGEMENT? (DEDUCE) This is most important and MOST MARKS will come from this section. A. Conclusions These can be in the form of questions or statements (e.g. ‘is the regenerativedisease due to blood loss or blood destruction?’ OR ‘the animal has regenerative anaemia due to either blood destruction of blood loss’) Think about whether there appears to be a main problem. Remember, there may be more than one. The main problem may be presented as organ/tissue dysfunction (e.g. liver disease), a general disturbance/pathological process (e.g. regenerative anaemia; inflammation) or possibly a specific disease (e.g. acute pancreatic necrosis in a dog). Can you explain all the abnormalities in light of the main problem(s)? Don’t forget about what you deduced from the given animal and clinical information when you are reaching your conclusions. B. Further investigation Often you will need to investigate the case further. This should be logical and based on your conclusions reached from analyzing case history and laboratory data. It is important to prioritize investigation i.e. consider what you think is the main conclusion when suggesting further investigation.

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A checklist for further investigation might include: • Do you need to get more history, and if so what? • Do you need to recheck some clinical signs or repeat physical examination? • Do you need to undertake specialist clinical procedures (e.g. ECG, EMG) • Do you need to undertake image analysis? What modality would be most useful? • Do you wish to undertake more laboratory testing?

Sometimes you may consider a response to treatment as part of your further investigation to reach a diagnosis, but this can sometimes be misleading unless you verify your response with further testing. So, you need to state how you would support any response to therapy approach. Sometimes you can sequence your further investigation on the basis of probable findings e.g. ‘I wish to undertake hepatic ultrasound. If I find any masses on ultrasound I would wish to take an FNA or biopsy’. C. Implications of the results for your approach to treatment and prognosis (i.e. management of the case while you are investigating it further) You may not wish to undertake any treatment while investigating the case further. In some situations you may wish to undertake treatment after asking for more tests (e.g. electrolyte analysis for possible fluid therapy). REMEMBER, not all abnormalities may be explicable in light of the case. Be honest, if you don’t know, say so BUT think about how you would find out. Also, put the abnormalities in perspective. Some mild abnormalities may not be very important for the case.

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THE UNIVERSITY OF SYDNEY

FACULTY OF VETERINARY SCIENCE

VETERINARY MEDICINE & CLINICAL PATHOLOGY

VETS 4112 Paper 1 - Clinical Pathology

Semester 1, 2004 Time allowed: 1 hour

INSTRUCTIONS

This is an OPEN BOOK examination

Don’t ignore the HISTORY AND OTHER INFORMATION when writing your case report discussion

Remember to include CONCLUSIONS, FURTHER INVESTIGATION,

if appropriate, and IMPLICATIONS FOR MANAGEMENT for each case report discussion

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102/06 Semester 1 2004 Page 2 of 3

QUESTION 1

Lucy is a 14-year-old, 15.5 kg, female border collie. She has problems sitting and climbing stairs and has wobbly back legs. She is not eating as much as she used to and has lost 3kg in the past 2 years. She has had occasional loose stools all of her life but more frequently in the past 2 years. On examination she has a normal pulse, respiration rate 60/min and temperature 39.1C. She is thin, has a discharge from one eye and a crusty, scaly ear (she has had otitis externa before). Her bladder is large. She has a wide-based stance, reduced patellar reflexes and reduced proprioception in the right hind leg and is sore and stiff on palpation of the hips. As part of the initial work up blood and urine were taken for analysis. Comment on the results, especially in relation to REASONS FOR THE Abnormalities. What CONCLUSION(S) can you draw? What FURTHER INVESTIGATION is required? What IMPLICATIONS are there FOR MANAGEMENT of the animal?

DETECT, DESCRIBE, DEDUCE

TEST SAMPLE REFERENCE INTERVAL AMYLASE IU/L 1704 <1400 ALP IU/L 3301 <110 ALT IU/L 539 <60 CK IU/L 91 <200 Serum protein (biuret) g/L 70 50-70 Albumin (BCG) g/L 38.8 23-43 Globulins g/L 31.3 27-44 Total cholesterol mmol/L 5.24 1.4-7.5 Glucose mmol/L 5.85 3.3-6.4 Urea mmol/L 21.55 3.0-10 Creatinine μmol/L 103 40-120 Calcium mmol/L 2.71 2.1-2.9 Inorganic phosphate mmol/L 1.63 0.8-1.6 Sodium mmol/L 146.6 137-150 Potassium mmol/L 4.2 3.3-4.8 Chloride mmol/L 112.4 105-120 Bicarbonate (TCO2) mmol/L 24.6 18-24 Anion gap mmol/L 13.8 15-25

QUESTION 1 CONTINUED NEXT PAGE

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102/06 Semester 1 2004 Page 3 of 3

QUESTION 1 CONTINUED

TEST SAMPLE REFERENCE INTERVAL Plasma appearance Clear Clear PCV L/L 0.35 .37-.50 Plasma protein g/L (refractometer) 78 55-75 Haemoglobin g/L 128 100-150 Erythrocytes x1012/L 5.3 5-7 MCV fl 66 60-75 MCHC g/L 366 300-350 MCH pg 24.2 20-25 Leukocytes x109/L 12.2 7-12 Neutrophils (seg.) x109/L 9.88 4.1-9.4 Neutrophils (band) x109/L 0.24 0-.24 Lymphocytes x109/L 0.98 .91-3.6 Monocytes x109/L 0.73 .2-.96 Eosinophils x109/L 0.37 .14-1.2 Basophils x109/L 0 0-.36 Platelets x109/L 925 200-600 Reticulocyte % (uncorrected) 0.2 0-1.5 Blood film: Some neutrophils hypersegmented. Some toxic granulation of neutrophils. Occasional macroplatelet

Urinalysis (cystocentesis)

Appearance: slightly cloudy PH: 6.0 Colour: pale yellow Glucose: -ve Specific gravity: 1.015 Ketones: -ve Protein (SSA): trace Blood: trace Centrifuged: not done Bilirubin: + Microscopic findings: moderate bacteria, some cellular debris, 10-20 White blood cells per high power field, 10 Erythrocytes per high power field.

THIS IS THE END OF THE PAPER

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Model Answer for 2004 Examination Question Assessment on signalment and history: Lucy has a history of chronic disease. Musculoskeletal/ neurological, digestive (wt loss, loose stools) and possibly respiratory (increased RR) abnormalities appear to be of main concern. In this old dog, the disease processes could be degenerative, neoplastic or inflammatory reasons; and there is a distinct possibility that there are multiple problems. Abnormalities in test results: There are marked elevations in ALP and ALT, and mild/marginal elevations in urea, AMS, inorganic phosphate and bicarbonate. There is a decreased anionic gap. In haematology, there are mild increases in TPP, MCHC and a mild decrease in PCV. Leukocytes are marginally increased, with very mild/ marginal neutrophilia. The neutrophils are hypersegmented and have toxic granulation. Platelets are increased with occasional megaplatelets viewed on the smear. Urinalysis reveals a specific gravity close to isosthenuria, with a trace of protein and 1+ bilirubin. WBC are present in significant numbers (pyuria) and high in proportion to RBC numbers (hematuria). There are “moderate” numbers of bacteria. Reasons for changes: The dog has significant cholestasis and hepatocellular damage. This combination might suggest hepatic disease. Another possibility is that the ALP elevation is unrelated to cholestasis and due to corticosteroid induction (e.g. hyperadrenocorticism). However, clinical signs are not highly supportive of HyperA (we do not have any information about the animal has been medicated with corticosteroids). The elevation of blood urea in combination with hyperphosphataemia and a specific gravity close to isosthenuria (in a dog with possible dehydration – elevated TPP) might suggest a degree of renal disease. The increased AMS could be related to renal disease, but may be related to pancreatic/intestinal disease (chronic diarrhoea). The elevated bicarbonate is difficult to assess. Respiratory, intestinal, renal and hepatic disease could all be contributing to electrolyte and acid/base levels. The dog has a marginal non-regenerative anaemia which could be simply related to chronic disease. Leukocyte changes are minimal, but do not rule out ongoing mild inflammatory disease. The toxic neutrophils could be due to inflammation or metabolic disturbances. The hypersegmented neutrophils are often due to corticosteroid effects. The high platelets (with some megaplatelets) might be related to inflammatory disease. Intestinal disease could be contributing? The urinalysis shows a 1+ bilirubin, which at this sp gr could be significant and reflect the cholestasis. The trace of blood is related to mild hematuria. The pyuria is significant and probably related to the bacturia (a fresh cystocentesis sample should be free of bacteria) and suggests urinary tract infection. Considering the large bladder and likely neurological defects, the dog is likely to have cystitis. The ratio of WBC to RBC numbers is roughly 2:1 and suggests that the hematuria is related to inflammation rather than hemorrhage. Conclusions and further testing: The dog certainly has strong indications of multisystemic disease. Many could be due to old age (e.g. degenerative joint/spinal disease). The likely conclusions from the clinical pathology results are hepatic disease, renal disease and cystitis. Pancreatic/gut disease has not been assessed effectively. The possibility of respiratory disease has not assessed. Suggested further testing may include image analysis of the thoracic (lungs) and abdominal (pancreas, liver, gut, bladder, adrenals) cavities. A bile acid analysis could be done to further assess liver. A history of corticosteroid medication might negate any interest in the possibility of HyperA. Image

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analysis of the spine and joints would be useful. The urine sample should be cultured and assessed for antibiotic sensitivity.

Implications for management and prognosis?: Many of the changes are related to chronic disease, but the urinary tract infection should be treated as soon as possible. Further management really hinges on what is detected on image analysis. Whatever eventuates, it is likely that the prognosis should be guarded. (Postscript: Ultrasound of the abdomen revealed an irregularly-shaped liver with variable echogenicity. An ultrasound-guided fine needle cell aspirate revealed cells that could have been neoplastic (diagnosis of possible hepatoma/ hepatocarcinoma). The neoplastic cells were obtained from several sites suggesting widespread involvement of the liver. Ultrasonography of the kidneys revealed bilateral dilation of the renal pelvices and hyperechoic renal cortices. The dog was given a poor prognosis and euthanasia was recommended. Unfortunately, the owners returned to the referring veterinarian for euthanasia of the dog and a necropsy was not performed.)

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GENERAL REFERENCES

Some of these references were utilised in the preparation of both the lecture and practical notes. They can be used as an adjunct to the notes. Some references cover all species. The preferred references are in bold.

Archer RK. Jeffcott LB. Comparative Clinical Haematology. Blackwell Scientific Publications, Oxford, UK. 1st Edn, 1977. ISBN 0-632-00289-1.

Baker R. Lumsden JH. Eds. Color Atlas of Cytology of the Dog and Cat. Mosby Inc., St Louis, USA. 1st Edn, 2000. ISBN 0-8151-0402-2.

Cowell RL. Tyler RD. Meinkoth JH, DeNicola DB. Eds. Diagnostic Cytology and Haematology of the Dog and Cat. Mosby Elsevier, St Louis, USA. 3rd Edn, 2008. ISBN 978-0-323-03422-7.

Cowell RL. Tyler RD. Eds. Diagnostic Cytology and Haematology of the Horse. Mosby Inc., St Louis, USA. 2nd Edn, 2002. ISBN 0-323-01317-1.

Day MJ. Clinical Immunology of the Dog and Cat. Manson publishing Ltd London UK; 2nd Edn, 2008. ISBN:978-1-84076-098-9.

Davidson M. Else R. Lumsden J. Eds. Manual of Small Animal Clinical Pathology. British Small Animal Veterinary Association, Cheltenham, UK. 1998. ISBN 0-905214-41-2.

Eade SC. Bounous DI. Ed. Pratt PW. Laboratory Profiles of Equine Diseases. Mosby Inc., St Louis, USA. 1st Edn, 1997. ISBN 0-8151-1731-0.

Feldman BF. Zinkl JG. Jain NC. Eds. Schalm’s Veterinary Haematology. Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, USA. 5th Edn, 2000. ISBN 0-683-30692-8.

Harvey J. Atlas of Veterinary Haematology. WB Saunders Co., Philadelphia, USA. 1st Edn, 2001. ISBN 0-7216-6334-6.

Hawkey CM. Dennett TB. Color Atlas of Comparative Veterinary Haematology. Iowa State University Press, Ames, Iowa, USA. 1st Edn, 1989. ISBN 0-8183-0449-3.

Jain NC. Schalm’s Veterinary Haematology. Lea & Febiger, Philadelphia, Pennsylvania, USA. 4th Edn, 1986. ISBN 0-8121-0942-2.

Kaneko JJ. Harvey JW. Bruss ML. Clinical Biochemistry of Domestic Animals. Eds. Academic Press Inc., San Diego, California, USA, 5th Edn, 1997. ISBN 0-12-396305-2.

Latimer KS. Mahaffey EH. Prasse KW. Eds. Duncan & Prasse’s Veterinary Laboratory Medicine – Clinical Pathology. Iowa State University Press, Blackwell Publishing Co., Ames, Iowa, USA. 4th Edn, 2003. ISBN 0-8183-2070-7.

Meyers DJ. Coles EH. Rich LJ. Veterinary Laboratory Medicine. WB Saunders Co, Philadelphia, Pennsylvania, USA. 1st Edn, 1992. ISBN 0-7216-2654-8.

Raskin RE. Meyer DJ. Eds. Atlas of Canine and Feline Cytology. WB Saunders Co., Philadelphia, USA. 1st Edn, 2001. ISBN 0-7216-6335-4.

Reagan WG, Rovira ARI. DeNicola DB. Veterinary Haematology – Atlas of common domestic species. Wiley Blackwell, Ames, Iowa. 2nd Edn, 2008. ISBN 978-0-8138-2809-1.

Rebar AH. MacWillliams PS. Feldman BF. Metzger Jr FL. Pollock RVH. Roche J. Ed. Cann CC. A Guide to Haematology in Dogs and Cats. Teton NewMedia, Jackson Wyoming. USA. 1st Edn, 2002. ISBN 1-893441-48-2.

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Sodikoff CH. Laboratory Profiles of Small Animal Diseases. Mosby Inc., St Louis, USA. 3rd Edn, 2001. ISBN 0-323-00956-5.

Thrall MA. Baker DC. DeNicola D. Fettman MJ. Lassen ED. Rebar A. Weiser G. Ed Troy DB. Veterinary Haematology and Clinical Chemistry. Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, USA. 1st Edn, 2004. ISBN 0-683-30415-1.

Willard MD. Tvedten H. Turnwald GH. Small Animal Clinical Diagnosis by Laboratory Methods. WB Saunders Co, Philadelphia, Pennsylvania, USA. 3rd Edn, 1999. ISBN 0-7216-7160-8.

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VETERINARY CLINICAL PATHOLOGY (LABORATORY MEDICINE)

Introduction

Clinical pathology covers the use of laboratory aids (histopathology, microbiology, parasitology, immunology, biochemistry, cytology and haematology) to assess the health, and/or detect a disease state, of an animal. Clinical pathology is often referred to as laboratory medicine because it is a component of the investigative process that leads to the diagnosis and treatment of medical conditions. Laboratory aids may be used to:

a. assess breeding or racing performance b. assess the disease status of an animal that is to be transported c. aid a clinical diagnosis d. aid prognosis and treatment

Laboratory aids are selected on the basis of clinical information (signalment, history, clinical signs and physical examination) and sometimes on the results of other diagnostic tests (e.g. image analysis). The type of test selected will depend also on whether you are dealing with the diagnosis of individual animal or group animal disease. Individual animal disease (mainly small animals and horses) relies on biochemistry and haematology primarily to detect organ or tissue dysfunction (although haematology can detect host response to tissue damage). Cytology, histopathology and microbiology assist in this process but in addition investigate aetiopathogenesis (the time course and development of disease; it includes causes and pathological processes [degeneration and necrosis; inflammation and repair; vascular disturbance; disorder of growth; and pigmentations/deposits]). Group animal disease (mainly production animals) relies less on biochemistry and haematology as necropsies on one or more dead animals usually provide significant information on aetiopathogenesis. Microbiology and toxicology are important for aetiological diagnosis in group animal disease and samples are commonly taken at necropsy. The use of biochemical and Haematological tests, especially for the diagnosis of individual animal disease, is not always necessary. If they are required, they are selected either to provide information that cannot be determined on clinical signs (i.e. none or few clinical signs available or the clinical signs are non-specific) or to investigate clinical possibilities. Clinical possibilities may involve either a specific disease entity or apparently unrelated clinical signs. In general, a combination of biochemical and Haematological tests is used to investigate clinical disease. Profiles (a broad range of biochemical tests and full Haematological investigation) are commonly used when investigating non-specific clinical signs or specific organ diseases. Whatever the reason for the use of laboratory tests, their interpretation must be done in light of history, signalment, clinical signs, physical examination and the results of other diagnostic tests. This is especially so for Haematological and biochemical tests which are rarely specific for particular diseases.

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Necropsy Technique and Collection of Specimens for Histopathology

Basically there are two common necropsy techniques: midline and flank. It is best to learn one basic technique and then adapt that to suit the animal and the suspected disease. Variations in technique usually occur due to size of the animal and the differing gastrointestinal tract (GIT). Samples for histopathology are usually placed in the fixative 10% neutral buffered formalin (10 parts formalin to 1 part tissue) in a proportion of about 1 volume tissue to 9 volumes fixative. The section of tissue will depend on whether it is biopsy or necropsy material. Samples are not supposed to be thicker than 1 cm but thicker samples may be taken if several slices (for penetration of the fixative) are made in the collected tissue or if the sample is to be further selected shortly after. Additional information on necropsy technique and collection of tissue for histopathology is provided in the handbooks for Principles of Disease and Veterinary Pathology (VETS 2013 and 3011).

The Nature of Laboratory Tests, Problems for Interpretation and Reference Intervals

Most haematological and biochemical tests are non-specific and are commonly used in combination to acquire meaningful information. Other laboratory aids are more specific but their interpretation still depends on the clinical information. Biochemical tests need to be varied depending on what species is being investigated. Species variation in interpretation of tests also exists. But perhaps the greatest difficulty in interpretation is due to the limitations of establishing a so called normal or reference interval (range) for a quantitative test within an animal population. The reference interval is usually determined from a clinically normal population of mature animals regardless of sex, breed and activity. It is expressed as the mean + 2 S.D., which essentially covers 95% of the population tested (this assumes that the distribution of results is Gaussian). In theory, individual laboratories should establish their own reference interval for a particular constituent but for several reasons they may not be willing to do so. Reference intervals quoted in texts can be used with reservation for haematology and end point biochemistries but should be used cautiously for kinetic assessment of enzymes. It is best for the laboratory to assess enzyme levels in 5-10 normal animals and use those results as a guide if proper statistical analysis cannot be done. There is always some overlap between results for a normal population and those from a diseased population for a particular biochemical or haematological constituent. The degree of overlap varies for different constituents but useful tests tend to have low levels of false positives and false negatives (i.e. a limited degree of overlap) (see figure following).

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FIGURE:  Values for a test for a healthy and a sick group of animals. The grey triangle denotes the small overlap for the two groups and suggests that this test might be useful to delineate healthy and sick animals. In this case, the distribution of the healthy group of animals could be used to establish the reference intervals (mean + 2 standard deviations)  Because of these limitations, the reference interval should be used as a guide to abnormality rather than as an absolute indication. Values just outside (or inside) the reference interval need to be treated with care. This is when other available information is most important for interpretations. What can be said is that the more distant a particular value falls from the reference interval, the more likely it reflects a definite abnormality.

General Remarks on Sampling for Blood Biochemistry

What samples are required for particular tests will depend on the commercial laboratory being used. In general, both serum and plasma can be utilised with some exceptions (see practical notes on sampling for blood biochemistry): Serum – is the fluid component derived from clotted blood. Plasma – is the fluid component derived from whole blood containing an anticoagulant (i.e. still contains clotting proteins such as fibrinogen). Heparin (mostly used for biochemistry), EDTA (primarily used for haematology), citrate and oxalate are commonly used anticoagulants. Specimen storage How long a biochemical constituent remains stable in a sample varies with: a. temperature of storage e.g. many constituents are more stable if chilled (4 degrees Centigrade) or

frozen ( -20 degrees Centigrade) b. what biochemical constituent it is e.g. some enzymes are particularly unstable once stored c. species e.g. some enzymes vary in stability between species The rule of thumb is to store at 4 degrees Centigrade unless the commercial laboratory instructs otherwise. Haemolysis Can interfere with the estimation of many biochemical constituents by: a. changing the concentration of the biochemical constituent (depends on the relative concentrations

of the constituent within the plasma and within the erythrocyte) and b. interfering with the performance of the biochemical test (commonly due to free haemoglobin) To avoid haemolysis, carefully handle blood and separate the plasma/serum promptly from the erythrocytes if there is to be any prolonged delay in biochemical analysis e.g. overnight.

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Gross Lipemia (and its relationship to laboratory detected hyperlipidemia) Hyperlipidemia refers to a measured increase in plasma/serum lipid levels (i.e. the levels are above reference intervals for the laboratory). These can be triglycerides, cholesterol or phospholipids (usually, only the first two are measured). If the hyperlipidemia is caused by significant increases in triglyceride rich lipoproteins (e.g. chylomicra [chylomicrons] and very low density lipoproteins [VLDL]) then there can be a milky appearance to the serum or plasma (this may be a diffuse white cloudiness or just a plug of white material at the top of the serum or plasma – the latter is related to chylomicra and can be accentuated by refrigeration at 4 degrees Centigrade). Historically, this gross milky appearance has been referred to as lipaemia (hyperlipaemia less commonly used – in the horse the term ‘hyperlipaemia syndrome’ is even more specific and refers to a disease of mainly obese ponies that commonly occurs during pregnancy and lactation), even though the term is a perhaps a little misleading. Gross lipaemia, apart from occurring in certain disease conditions e.g. diabetes mellitus), is a common finding in monogastric animals fed prior to collection of blood samples (postprandial lipaemia due to the presence of chylomicra). Gross lipaemia can affect the measurement of biochemical constituents by elevating optical density and thus interfering with colorimetric techniques of analysis. In addition, gross lipaemia can enhance the fragility of erythrocytes leading to haemolysis. To avoid excessive gross lipemia in monogastrics take blood samples after fasting (8 hours minimum).

Plasma/Serum Enzymology

Enzymes detected in the serum/plasma can be divided into: a. plasma specific. They are rarely measured (exceptions include glutathione peroxidase to detect

selenium/Vitamin E deficiency in ruminants and cholinesterase reductions to detect organo-phosphate poisoning in a variety of species)

b. plasma non-specific. They have no known function in the plasma and are present in the plasma at levels much lower than those that exist in tissues/cells. They can be divided into:

i) enzymes associated with cellular metabolism. These can be divided further into organ specific and organ non-specific. Organ specific enzymes have relatively high levels in certain organs and therefore on release usually reflect damage to those organs e.g. ALT in hepatocellular damage in the dog or cat. Organ non-specific enzymes are usually in comparable levels in a number of tissues/organs e.g. AST in liver and skeletal muscle. Organ non-specific enzymes can be made more organ-specific by separating into isoenzymes (different forms of the same enzyme that perform the same function e.g. CK isoenzymes in striated muscle, gut and brain)

ii) secretory enzymes. These may elevate in blood if there is damage to the cells producing the enzyme or there is blockage to the secretory route e.g. AMS from the pancreas

Factors affecting plasma levels of enzymes These are incompletely understood. Consequently the effect of disease on certain plasma enzyme levels is not always predictable. a. Those affecting enzyme release i) alterations in cell membrane permeability or internal cell organisation. Those enzymes

of low molecular weight (e.g. CK) and high solubility (cytoplasmic in situation e.g. LD, CK) are usually released more readily than others during changes in membrane alteration.

ii) cell death. Usually marked increases in plasma enzyme levels occur, irrespective of molecular weight or enzyme situation

iii) altered enzyme synthesis (induction e.g. ALP)

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b. Those affecting enzyme removal i) circulatory impairment ii) obstruction of normal excretory route iii) increased catabolism of enzymes c. Those affecting measurement i) technique of the enzyme assay- varying techniques (especially kinetic reactions) can

have marked effects on reference intervals ii) collection and storage of the sample. Enzymes are proteins which can be easily

denatured by rough handling or by repetitive freezing and thawing iii) time of collection. Each enzyme will rise and fall at different rates after a disease

process affects a particular organ Naming of enzymes The overall reaction catalysed by the enzyme is the basis of the name. All enzymes have a designated numerical code (4 numbers), an official name, a trivial name and an abbreviation. The latter 2 are used most widely in veterinary circles. The following is an example: trivial name: Lactic dehydrogenase, abbreviation: LDH (LD), numerical code: 1.1.1.27, official name: L lactate: NAD+ oxido-reductase

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TABLE:  Some of the more common enzymes used in domestic animals   

ENZYME USAGE IN DOMESTIC ANIMALS   Dog/Cat  Horse  Sheep/Cattle  Pig 

A. GENERAL DETECTION OF CELL DAMAGE 

AST (mainly muscle/Liver), LD (present in most cells) for all species 

B1. HEPATIC DISEASE ‐ HEPATOCYTE DAMAGE 

ALT (ARG ‐ can be used for all species but rarely done) 

ID or GD (ARG)  GD or OCT (ARG)  OCT (ARG) 

B2. HEPATIC DISEASE ‐ CHOLESTASIS 

ALP, GGT  GGT,  ALP  (less commonly used) 

GGT  GGT 

C. CARDIAC/ SKELETAL MUSCLE CELL DAMAGE 

CK, AST, LD (especially isoenzyme analysis) for all species. NB. it has been reported that certain skeletal muscle diseases in some dogs and cats may cause elevations of ALT 

D. PANCREATIC ACINAR CELL DAMAGE 

AMS, LPS (other laboratory aids more useful) 

AMS  not done  not done 

E. GUT DAMAGE  AMS?, ALP? other laboratory aids are of greater use for all species 

F. CNS DAMAGE  CK (in CSF) for all species, other laboratory aids of greater use 

G.REPRODUCTIVE TRACT DAMAGE 

other laboratory aids of greater use 

H. JOINT DISEASE  Other laboratory aids of greater use (AST, ICD, LD can be measured in the synovial fluid, especially in the horse) 

I. RENAL DAMAGE  other laboratory aids of greater use (GGT has been measured in urine in dogs and cats to detect renal cell damage) 

J. ENDOCRINE DISORDERS 

laboratory confirmation usually requires hormonal assays ‐ enzymes may detect secondary organ dysfunction (e.g. diabetes mellitus may cause elevation in liver enzymes) NBs. hyperadrenocorticism in the dog can induce a specific isoenzyme of ALP; hypothyroidism in the dog can be characterised by increases in CK 

 ALP ‐ Alkaline Phosphatase, ALT ‐ Alanine Transaminase, AMS ‐ Amylase, ARG ‐ Arginase, AST ‐ Aspartate Transaminase, CK ‐ Creatine Kinase, GD ‐ Glutamate Dehydrogenase, GGT ‐ Gamma Glutamyl Transferase, ID ‐ Iditol Dehydrogenase (old name SDH‐Sorbitol Dehydrogenase), ICD ‐ Isocitrate Dehydrogenase, LD ‐ Lactate Dehydrogenase, LPS ‐ Lipase, OCT ‐ Ornithine Carbamoyl Transferase 

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Combining enzymes to detect disease processes Example: In the horse CK has been used in combination with AST to detect active damage in muscle. CK return to normal in about 2-4 days whereas AST may be elevated for up to 1 week.

Time

Level of enzyme

Single episode of muscle damage

CKAST

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LABORATORY EVALUATION OF HEPATIC DISEASE

Terminology

Jaundice (icterus) yellowing of the skin and other tissues due to deposition of bilirubin Cholestasis bile without motion Hyperbilirubinemia increased circulating levels of bilirubin in the blood. Retention form

refers to increases in unconjugated, regurgitation form refers to increases in conjugated and combined refers to increases in both

Hepatic encephalopathy signs of central nervous disease related to liver disease. In the dog and

cat it is invariably related to congenital or acquired vascular shunting Acute phase reactants refers to altered (mostly increased) levels of specific proteins (mostly

globulins) in the blood. They can include clotting proteins (e.g .fibrinogen), haptoglobin and C-reactive protein. They occur in response to a variety of acute/active degenerative/inflammatory conditions

Post prandial after feeding

Introduction

In individual animal disease initial laboratory evaluation of hepatic disease depends on biochemical analysis. Biochemical tests, (liver "function" tests) can detect 3 basic processes that can occur in liver disease: hepatocellular damage, cholestasis and reduced functional mass. Haematological analysis is usually of limited use in the diagnosis of liver disease but a cytological aspirate and/or biopsy can more specifically define the hepatic problem (i.e. investigate the aetiology as well as the pathogenesis). In group animal hepatic disease biochemical tests still have a role to play but histopathological, microbiological and toxicological examination of hepatic tissue taken at necropsy are of greater importance (many liver diseases in group animals, especially in farm animals, are caused by infectious agents and environmental poisons [including plants], which may be detected by gross and microscopic examination of the liver, and by microbiological and toxicological analysis). However, the significance of detected liver lesions must be determined in light of history, signalment, clinical signs and physical examination because not all may produce clinical disease (due to the large reserve capacity of the liver). Biochemical tests employed to detect liver disease are selected on the basis of what disease processes might be occurring. For example, significant hepatocellular damage is more likely to occur in acute or sub-acute disease rather than in chronic; reduced functional hepatic mass is more likely to be of importance in chronic disease; cholestasis can occur at any stage of liver disease. All the tests have limitations and consequently, they are commonly used in combination. Liver dysfunction may occur as a primary disease or secondary to another organ/tissue dysfunction

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(e.g. endocrine abnormalities leading to secondary liver damage). In many cases biochemical tests cannot differentiate primary and secondary hepatic disease. Liver "function" tests can be affected by extrahepatic factors e.g. blood flow to the liver can influence the results for bile acid testing. By serial sampling, liver "function" tests can trace the course of a hepatic disease (e.g. the use of enzymes with different T1/2’s to determine the persistence of hepatocellular damage). For convenience, liver "function" tests will be presented according to their main usage, i.e. detecting hepatocellular damage, cholestasis or reduced hepatic cell function. However, many tests will detect, and be affected by, more than one of these basic processes. Also, it is worthwhile remembering that a liver disease will often involve more than one of these basic processes.

Summary of Tests

PRINCIPAL USAGE  BIOCHEMICAL TEST 

ACTIVE LIVER CELL DAMAGE  AST, ALT, LD, ID, ARG, OCT, GD, ICD (depending on species) 

CHOLESTASIS  ALP, GGT (both induced enzymes) 

CHOLESTASIS, CELL DAMAGE  Bilirubin analysis (total; unconjugated; conjugated) 

REDUCED FUNCTIONAL  HEPATIC MASS 

Bile acids*, Bromsulphopthalein (BSP) test*, blood ammonia/urea, serum proteins 

 *The BSP  test and bile acid analysis will detect all  three basic processes but are more  commonly used to detect reduced functional hepatic mass as other tests are cheaper and simpler to do for the detection  of  hepatocellular  damage  and  cholestasis.  The  BSP  test  is  no  longer  used  now,  being largely replaced by bile acid analysis. 

Hepatocellular Damage (Degeneration and/or Necrosis)

For levels of serum enzymes to be elevated in liver disease, an active damaging process to a significant number of hepatocytes is required. This is more likely to occur in acute/sub-acute disease rather than in chronic disease. Elevated levels of enzymes may be seen with reversible degenerative changes as well as in cell necrosis. Consequently, the levels may not be an accurate assessment of prognosis. a) ALT - Reasonably specific in the dog and cat for hepatocellular damage although it may be

elevated in certain muscle diseases in certain breeds of dogs and some cats (therefore, combine with CK if in doubt). It is present in the cytoplasm; therefore, even changes in cell permeability can increase levels. Consequently, elevated ALT does not necessarily mean cell death. In the dog, corticosteroids and some anticonvulsants may induce ALT. It is rarely used in farm animals (horses, cattle and sheep) due to limited elevations in hepatic disease, and the fact that elevations may also occur in muscle disease. ALT has been used in combination with arginase, which is liver-specific for many species, to detect progressive damage (arginase, returns to normal more quickly than ALT- in the dog, T1/2‘s of ARG and ALT are less than 12 hours and 40 to 60 hours respectively).

b) ID - Iditol dehydrogenase (old name SDH-sorbitol dehydrogenase) - Reasonably liver specific in

most farm animals but returns to normal rapidly after a non-progressive bout of hepatic damage.

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It is commonly used only for the horse. It can be used in the dog.

c) OCT - Liver specific in most species. It is rarely used except in swine.

d) GD - Reasonably liver specific in cattle and sheep. It can be used in the horse and dog.

e) ARG - Liver specific in most species but not widely used.

f) LD, AST, ICD – They are not liver specific enzymes but can be used to detect hepatocellular damage if other organ diseases can be ruled out (especially muscle). They are commonly used if more specific enzymes are not available.

NOTE: Enzymes are often used in combination to detect hepatocellular damage as expected increased serum levels in hepatocellular damage do not always occur for a particular enzyme.

Cholestasis

The term cholestasis literally means "bile without motion". It refers to reduced excretion of bile which may occur due to hepatocellular damage (often interferes with the energy production required to excrete bilirubin into the bile canaliculi) or due to physical obstruction of intrahepatic or extrahepatic bile canals. The enzymes ALP and GGT are the most sensitive indicators of cholestasis and elevations are supposed to precede the onset of hyperbilirubinemia or significant bilirubinuria. In fact, cholestasis may never become severe enough to cause hyperbilirubinemia even though enzyme levels may become high. On the other hand, most cases of hyperbilirubinemia due to cholestasis are accompanied by high enzyme levels. Serum levels of ALP and GGT are high in neonatal puppies and kittens due to high levels in ingested colostrum. Consequently, measurement is of little use until about two weeks of age. ALP isoenzymes do exist for bone and gut. The gut isoenzyme has a very short half life and gut disease, therefore, causes little in the way of blood increases. Bone growth and diseases can cause 2-3X increases in blood ALP. ALP is the enzyme of choice in the dog and cat. In the dog, mild to marked increases can occur in cholestasis (e.g. 200 up to, and exceeding, 1000 IU/L). In the dog moderate to high levels of ALP may be present in the serum in cases of hyperadrenocorticism due to induction of a specific hepatic isoenzyme (may be in excess of 2000 IU/L). Exogenous corticosteroids in the dog also induce the specific isoenzyme but this normally takes a few days. The cat appears not to have the specific isoenzyme for corticosteroids. Moreover, in the cat the magnitude of elevation of ALP in cholestasis is much less than in the dog. This is due to the lower levels in hepatocytes and biliary epithelium and the shorter plasma T1/2 in the cat (6 hours compared to 72 hours in the dog). Consequently, any elevation in ALP in the cat, irrespective of the magnitude, is considered significant and warrants further investigation. In the dog, ALP can be induced by anticonvulsants, volatile anaesthetics and barbituates. ALP is rarely used in the horse but elevations can occur in cholestasis. GGT is more commonly used in the horse to detect cholestasis. ALP is not reliable in sheep and cattle and GGT is the enzyme of choice for cholestasis in farm animals. GGT can be used in the dog (questionable usage in the cat) to detect cholestasis and is not apparently affected by barbiturates or anticonvulsants. However, it is induced by corticosteroids in the dog. GGT in renal epithelium is only released in the urine on damage; therefore, increased urine levels may indicate tubular damage.

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Bilirubin Metabolism and Analysis

This is a general discussion of bilirubin metabolism and will mention both hepatic and extrahepatic diseases that can cause derangements. When analysing bilirubin metabolism, serum bilirubin, urine bilirubin, urine urobilinogen and faecal bile pigments can be measured. Most emphasis is placed on the first two. Serum bilirubin levels can be visually assessed by the icterus index (II) technique (by visually assessing the colour of plasma or serum). Dogs and cats have an II less than 5. Horses have a value less than 15 but this can be up to 25 if the animal is on green feed or has a high PCV (0.40-0.55 L/L). The ox usually has less than 15 but can have levels up to 25 presumably due to serum carotenoid levels. The sheep and pig have an II of less than 5. The II technique is rarely used these days, but it is still worthwhile assessing the yellowishness of plasma or serum. Accurate assessment of serum bilirubin requires the Van den Bergh's test. This measures total bilirubin, but can be modified to measure the proportions of unconjugated to conjugated bilirubin (total bilirubin on its own is of limited use in determining the reason for hyperbilirubinemia, but there is some debate over the usefulness of measuring conjugated to unconjugated proportions). Bilirubin metabolism in the body in the dog (view diagram) can be applied to other domestic species, but with some minor variations (NB. birds form biliverdin instead of bilirubin from the breakdown of heme, but biliverdinemia due to hepatic disease is apparently rare in birds). Urine bilirubin levels provide an indication of blood levels of conjugated bilirubin. Only conjugated bilirubin is passed by the kidney. However, the male dog's kidney apparently has good ability to break down haemoglobin and to conjugate bilirubin. In addition, the dog has a low renal threshold to conjugated bilirubin. Consequently, bilirubinuria in the dog needs to be interpreted with care e.g. a one plus to two plus may be of little significance in a male dog or in concentrated urine over specific gravity of 1.030. Urine urobilinogen as a test has fallen out of favour. Increased urobilinogen can be seen in haemolysis and certain types of liver disease. Decreased or no urobilinogen can occur with disturbances of the enterohepatic circulation or as a chance finding in a normal animal. Faecal bile pigments are rarely measured. Increases may occur in haemolytic crises, decreases may occur in biliary obstructions. Traditionally, icterus (jaundice [yellowing of the tissues due to the presence of significant hyperbilirubinemia]) has been divided into 3 types: a. Haemolytic - is non-cholestatic and essentially involves an excess production due to haemolysis

of erythrocytes. b. Hepatocellular - is partly due to non-cholestatic mechanisms (reduced cell conjugation) and

partly due to intrahepatic cholestasis (reduced cell excretion due to lack of energy and blocked bile canaliculi primarily due to cell swelling).

c. Obstructive - principally refers to post-hepatic obstruction of bile flow or obstruction to the larger bile ducts. The jaundice is completely due to cholestasis, at least in the earlier stages.

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FIGURE:  Bilirubin Metabolism in the Dog 

Other haemoproteins

15%

Erythrocytes 85%

Free indirect reacting, unconjugated, water insoluble bilirubin in plasma (bound to protein for transport)

Direct acting, conjugated, water soluble bilirubin in the hepatocytes

Most excreted in the bile (minor levels in the urine

In the intestine bacteria reduce conjugated bilirubin to compounds of similar structure called urobilinogens

10-15% 85-90%

Further conversion and excreted in the faeces (faecal bile pigments)

Urobilinogens and conjugated bilirubin in urine

General circulation

Portal blood system

20% 80% recycled

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This classification system is useful but has limitations when being applied to specific domestic species. Consequently, another classification system has been developed that attempts to cover all possibilities: a. Retention hyperbilirubinemia

This is characterised by unconjugated bilirubin as the predominant form in the blood. Bilirubinuria may or may not occur. Haemolytic crises are the main cause but a variety of extrahepatic and hepatic diseases can interfere with hepatic uptake and conjugation (in horse, cattle, sheep and swine anorexia associated with many diseases can give rise to retention hyperbilirubinemia. Jaundice may not develop. This cause of retention hyperbilirubinemia occurs irregularly and mildly in the dog and cat).

b. Regurgitation (cholestatic) hyperbilirubinemia This is characterised by conjugated bilirubin as the predominant form. Bilirubinuria is usually present. Both intrahepatic and extraheptic obstruction to bile flow produce regurgitation hyperbilirubinemia.

c. Combined hyperbilirubinemia This develops due to both retention and regurgitation methods; therefore, both conjugated and unconjugated bilirubin levels in the blood are increased. Bilirubinuria is usually present. This type of hyperbilirubinemia can occur in both acute and chronic hepatic diseases. In the dog and cat most hyperbilirubinemias are combined, with the conjugated bilirubin being anything between 20% and 60%. Pure retention can occur in haemolytic crises (although elevated conjugated bilirubin generally occurs in the later stages). In post-hepatic obstruction the hyperbilirubinemia can be mainly regurgitation although some unconjugated bilirubin is nearly always present. In the cat, bilirubinuria always means increased conjugated bilirubin in the blood stream. In the horse hyperbilirubinemia is commonly retention in type due to a variety of extrahepatic diseases inducing anorexia and causing significant increases in unconjugated bilirubin (rarely do you get jaundice with this cause of hyperbilirubinemia). Combined hyperbilirubinemia occurs in both hepatic disease and post-hepatic obstruction, but with the unconjugated bilirubin always predominating (possibly still due to the impact of anorexia). However, there is a greater percentage of conjugated bilirubin in primary cholestatic disease in the horse. Bilirubinuria always signifies increased conjugated bilirubin in the blood. In farm animals hyperbilirubinemia is commonly retention in type due to haemolytic crises and due to a variety of extrahepatic diseases inducing anorexia (especially in cattle). In ruminants, bilirubin elevations are inconsistent in hepatic disease and post hepatic obstruction and hence used sparingly; but can be interpreted similarly as in dogs and cats. It is best to combine bilirubin analysis with other tests for laboratory diagnosis of hepatic disease in ruminants. Naturally, this classification system requires measurement of total, conjugated and unconjugated bilirubin levels. When interpreting the proportions of conjugated to unconjugated it is also important to look at their total amounts (i.e. the magnitude of the rise).

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Tests for Reduced Functional Hepatic Mass

a) Bile acids Bile acids are synthesized by hepatocytes from cholesterol and then conjugated prior to excretion in the bile. They are stored and concentrated in the gall bladder during the interdigestive period. Upon feeding, cholecystokinin release occurs following gastric emptying. This stimulates gall bladder contraction and expulsion of bile into the duodenum. In the small intestine bile acids are necessary for fat assimilation. Most secreted bile acids are actively reabsorbed in the ileum (90% efficient) and returned directly to the liver via the portal system (i.e. an enterohepatic circulation similar to that for bilirubin exists which allows for about 90% of the bile acids to be extracted by the liver). The liver has a tremendous reserve capacity to produce bile acids and failure of bile acid synthesis is rare. Any process that impairs the hepatocellular, biliary or portal components of the enterohepatic circulation of bile acids results in increased serum levels. Consequently, serum levels of bile acids can be used to check for a wide variety of liver related diseases. Increased plasma levels can occur in hepatocellular damage, cholestasis, reduced functional hepatic mass and in portosystemic shunting, but the levels usually cannot be used to distinguish the type of disease. Decreased serum bile acids may occur in the final stages of end stage liver disease (only when greater than 90% of functional mass has gone).

Serum bile acids can be measured prior to feeding (fasting levels) or 2 hours after feeding a high protein or fatty meal (post-prandial). Post-prandial levels can be significantly elevated compared to fasting levels in dogs with vascular shunting and cirrhosis (reduced functional mass). Consequently, serum bile acid analysis can be used instead of other tests currently used to detect these abnormalities (e.g. BSP or other dyes and NH3 tests). NB. bile acid analysis is unreliable in Maltese dogs in determining shunting or liver disease as healthy Maltese can have levels far in excess of reference intervals for other dogs.

b) Blood ammonia determination NH3, produced by bacterial action in the gut, is converted by the liver to urea.

Elevated blood NH3 may be due to: i) terminal hepatic insufficiency (i.e. significantly reduced functional hepatic mass) ii) circulatory bypass of the liver (i.e. intrahepatic or extrahepatic shunting - a

common use for the test) iii) urea cycle enzyme deficiency iv) excessive vomiting v) shock

In practice only (i) and (ii) are important in causing significant elevations of NH3. Urea is commonly low. In the dog, fasting blood NH3 levels (8-12 hrs after feeding) are normally elevated in significant shunting of blood around the liver (congenital or acquired due to hepatic disease). If chronic liver disease is unassociated with significant acquired shunting then fasting blood NH3 levels may be normal. The ammonia tolerance test (ATT-oral or rectal) can be performed and may produce marked increases in NH3 due to reduced functional hepatic mass in terminal hepatic insufficiency (5 x fasting blood NH3). In significant shunting, where there usually is a relatively high fasting level, 2-2.5 x fasting blood NH3 levels may occur with the ATT (these figures are for oral ammonia tolerance test). Ammonia and urea investigations should be done in tandem (as there is often an inverse relationship) and any suggestion of shunting should be supported by vascular studies.

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Congenital shunts have been recorded in the dog, cat, horse and cow. They can produce what is termed hepatic encephalopathy. In ruminants and sometimes the horse, however, signs of hepatic encephalopathy can also occur in a variety of liver diseases (e.g. hepatic lipidoses, necrosis, chronic progressive hepatitis) not usually associated with significant vascular shunting. In these diseases it may be necessary to combine blood NH3 determination with other tests (e.g. in the horse liver biopsy is extremely useful) to determine the etiopathogenesis. NB. In dogs, because of the difficulty of performing the ATT and technical problems with NH3 analysis, bile acid analysis (fasting and post-prandial) may be used, with care, to detect congenital or acquired vascular shunting.

c) Serum protein estimation

Serum protein estimation (total and types of protein) can be useful in detecting a chronic hepatopathy. It is rarely of use in early hepatopathies (but there may be increased levels of globulins in acute hepatic disease. These increased proteins are usually acute phase reactants and include clotting proteins, haptoglobin, transferrin and many others). Mild hypoalbuminemia has been recorded in acute inflammatory disease of the liver but this is usually insignificant and probably related to the fact that albumin is a ‘negative’ acute phase reactant (nb. T1/2 of albumin in the serum is about 8 days).

In a chronic hepatopathy with marked reduction in functional hepatic mass a significant hypoalbuminemia may occur. The beta and gamma globulins may be increased but this depends on the disease process. Total serum protein may be decreased. Fibrinogen levels may be lowered in advanced hepatic insufficiency.

Clotting factors (other than fibrinogen) may have reduced production in both acute and chronic liver disease (they have short plasma half lives). Rarely are they sufficiently suppressed to produce spontaneous hemorrhage (but they could contribute to a bleeding disorder with another disease process - see Bleeding Disorders). Decreased levels have been detected by modified clotting factor tests (OSPT, APTT). Serum protein analysis is commonly incorporated in general biochemical profiles, as abnormalities can occur in a variety of extrahepatic diseases. Usually, this involves direct measurement of total serum protein and serum albumin, with a derived value for total globulins. If abnormalities are detected through these simple biochemical methods, then serum protein electrophoresis can be performed to better delineate changes to albumin and the various classes of globulins (e.g. to detect reasons for hyperglobulinemia other than due to dehydration). Some specific proteins, apart from albumin, can be measured in the laboratory; and most of these are acute phase reactants. Blood levels of these proteins are often altered in acute degenerative/inflammatory conditions, although some can be increased in other disease situations. Most of these proteins increase but some, such as pre-albumin, albumin and transferrin, may decrease (called major negative acute-phase proteins) in response to acute degenerative/inflammatory conditions (transferrin may increase in acute liver disease). Major positive acute-phase proteins (i.e. those that increase in the blood) vary amongst species, but most are detected as alpha and beta globulins through serum protein electrophoresis. Fibrinogen (and some other non-enzymic clotting proteins) is useful in most domestic species, but especially in ruminants and horses. C-reactive protein is a major responder in dogs and pigs, serum amyloid A is useful in dogs, horses, cattle and birds, Haptoglobin is a major responder in farm animals, whilst TNF-alpha is supposedly useful in cats. There are many other positive acute phase proteins, and it is likely that their usage will increase in delineating disease processes (e.g. Porcine major acute protein; alpha 2-Macroglobulin in cattle).

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Other Laboratory Tests A. Bromosulphthalein (BSP, sulfobromophthalein)

The BSP test is rarely used as it has been replaced by bile acid analysis. It is primarily used as a research tool today. The BSP test measures hepatocyte functional integrity and hepatic blood flow. BSP is transported in the serum bound to albumin, is conjugated by the hepatocytes and excreted in the bile (75%- 25% is cleared by skeletal muscle and kidney). This is analogous to bilirubin hepatic metabolism. Consequently, in jaundice, the BSP test is limited by competition between BSP and bilirubin for excretory pathways. Bile acid determination has replaced the BSP test to a great extent. The BSP test involves some expense and difficulty. Consequently, simple tests, if available, are often used to define liver problems - cholestasis, ALP and bilirubin; active hepatocellular damage, ALT and other specific enzymes. But the BSP test may be used to confirm these conditions, and in the case of chronic hepatopathies with low levels of active damage and cholestasis, used to detect the conditions. In the horse (and cattle ) a BSP clearance rather than retention method is used. One gram of BSP is given intravenously and between 5-12 minutes later 2 heparinized samples are taken 4-6 minutes apart. The T1/2 is determined from the samples and should be between 2 and 3.7 minutes for the normal horse. The interpretation of the BSP test in the horse is similar to that in the dog and cat. Indocyanine Green is another substance that can be injected to assess liver problems. Unlike BSP, ICG is solely cleared by the liver, but the disadvantage of ICG is that it is more expensive than BSP.

B. Cholesterol

Cholesterol is primarily produced by hepatocytes, intestinal epithelium, adrenal cortex and gonads. Cholesterol is utilised for the synthesis of bile acids. Hypercholesterolemia can occur in cholestatic conditions but may also occur in the nephrotic syndrome, acute pancreatitis, congenital hyperlipidemias and certain endocrinopathies (DM, hyperadrenocorticism, hypothyroidism). Increases may also occur post prandially. Increased blood levels of cholesterol alone will not cause gross turbidity of the serum or plasma. Decreased serum cholesterol can occur in hepatic shunting and in gut problems (Exocrine Pancreatic Insufficieancy, malabsorption, Protein Losing Gastroenteropathy).

C. Glucose In chronic liver disease there may be fasting hypoglycemia and prolonged post-prandial hyperglycemia.

*SEE CASE REPORTS 1-6

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LABORATORY EVALUATION OF URINARY TRACT DISEASE

Terminology

Azotemia the presence of excessive amount of nitrogenous compounds in the blood Uraemia a clinical syndrome occurring in kidney failure characterized by a variety of

systemic and neurological signs. Numerous biochemical abnormalities exist of which azotemia is one

Polyuria the passage of an excessive quantity of urine Oliguria a reduction in the quantity of urine produced Anuria suppression or arrest of urinary output Isosthenuria continued inability of the kidney to produce either concentrated or dilute urine Hyposthenuria the constant secretion of urine of low specific gravity (below 1.008 and often

as low as 1.001) -uria a combining form denoting either condition of urine or the presence of a

substance in urine e.g. hematuria, crystalluria, bacteriuria Cylindruria the presence of casts in the urine Pyuria the presence of increased leukocytes (’pus’) in the urine

Introduction

The basis for understanding renal disease and the tests used to detect that disease depends on knowledge of normal structure and function. The primary function of the kidney is to maintain the volume and composition of the extracellular fluid. It does this by: a) regulating water levels b) elimination of metabolic waste products and certain foreign toxic substances c) regulating electrolyte and acid/base balance. Urine is the end result of these processes and is dependent on glomerular filtration, tubular function and interstitial integrity. The glomerular filtrate is an ultrafiltrate of plasma and has a specific gravity of 1.008-1.012. The rate of formation of the glomerular filtrate within a nephron (i.e. the glomerular filtration rate - GFR) is dependent on 2 factors: a) effective filtration pressure in the glomerulus which is equal to the blood pressure within the

glomerular capillary minus the osmotic pressure of plasma protein and the back pressure within Bowman's capsule

b) renal blood flow.

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Total glomerular filtrate produced is dependent on the number of functioning glomeruli (nephrons). In the proximal tubules, 85% of the water and solids are reabsorbed. From that point onwards, the tubular fluid is either diluted or concentrated. Water resorption occurs primarily in the distal and collecting tubules and is dependent on the presence of ADH and a hypertonic interstitium (i.e. concentrating power of the kidney is dependent on the integrity of the tubules and interstitium, and the presence of ADH). The tubules are also important in acid/base balance and sodium retention. When evaluating urinary tract disease, examination of the urine is essential. Urinalysis will provide information on renal disease which may or may not be producing renal failure. Urinary tract disease lower than the kidney can in many circumstances be adequately assessed by urinalysis. Urinalysis is commonly combined with a variety of plasma chemical tests to detect renal disease and failure (e.g. Blood urea and creatinine). Acute and chronic renal failure will give rise to uraemia (the clinical condition related to renal failure). Azotemia, oliguria and isosthenuria characterise acute renal failure. The polyuric phase of chronic renal failure is commonly accompanied by loss of tubular concentrating ability (e.g. continued excretion of urine at less than a specific gravity of 1.030 for the dog, less than 1.035 for the cat [value not agreed on and some veterinary urologists would prefer less than 1.040 for the cat – this may depend on the refractometer used and whether it has a separate scale for the cat], less than 1.025 for the horse and probably for the cow [value not agreed on, some suggest 1.030 for the horse whilst others suggest 1.020 for the horse and cow) but may or may not be accompanied by azotemia (e.g. in the dog isosthenuria usually has to be reached before significant azotemia is present. This is not always the case in the cat where azotemia can accompany values of 1.020 or less). In the oliguric/anuric phase of chronic renal failure azotemia and isosthenuria are present. The animal is invariably dehydrated. If the chronic renal disease primarily involves the glomeruli and has limited effects on the tubules it will be characterized by azotemia with variable loss of tubular concentrating ability. Commonly there is a significant proteinuria in glomerular disease (often 3-4 + on the urine dipstick). Renal disease progressing to renal failure (i.e. chronic renal disease/failure) is most common in the dog and cat, less common in the horse and rare in farm animals. In farm animals renal disease/failure is often acute and due to infections or poisons. Because a group of farm animals is normally affected, necropsy is often possible and this reduces the need for detailed biochemical or haematological investigations. However, the approach to diagnosis of renal disease/failure in farm animals is similar to that employed for companion animals when only individual animals are affected and they are valuable (e.g. suspected pyelonephritis in a stud or good milking cow).

Some of the Laboratory Tests used to Detect Renal Dysfunction

Assessment of glomerular function Generally involves measurement of the glomerular filtration rate or related tests.

a) Clearance tests Renal clearance of a substance measures the amount of substance cleared from the plasma in a certain time. Urea, creatinine and mannitol are substances that can be measured. However, the clearance value is highly specific for glomerular function only if tubular reabsorption or excretion is minimal e.g. creatinine in the dog is minimally reabsorbed by the tubules; hence the endogenous creatinine clearance test is useful. Clearance tests are commonly used when renal disease is suspected but the animal does not appear to be in renal failure; that is, clinical signs may be vague or intermittent.

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Urinalysis is also useful to investigate renal disease that has not yet progressed to renal failure.

NB: Sulfanilate clearance test: Sodium sulfanilate is eliminated from the blood primarily by glomerular filtration. The test is suggested to be of value in the dog for detecting decreases in renal function before there is development of azotemia or concentration abnormalities.

b) Blood concentration levels These are less accurate than clearance tests in estimating glomerular function, but are easier to perform. i) Blood urea (sometimes called blood urea nitrogen). Urea is the end product of

catabolism of protein and is formed in the liver. It is filtered by the glomeruli, but a certain percentage is reabsorbed by the proximal tubules. Consequently, blood urea levels are affected by hepatic function, protein metabolism, glomerular function and tubular function.

ii) Blood creatinine. Creatinine is formed in the metabolism of muscle. Its blood level is

relatively constant, although it can be affected minimally by diet and exercise, and it is excreted by the glomeruli with minimal reabsorption by the tubules (nb. some may be secreted by tubules but this is usually limited). Consequently, it has been suggested that it is a better indicator of glomerular function than urea. It has been suggested that creatinine is more useful than urea in acute renal disease but it is probably wise to utilise both analytes when investigating suspected renal disease of any duration. As most renal failures in dogs and cats are chronic in form, both analytes are likely to be increased. A common method used for the measurement of creatinine (the kinetic Jaffé reaction) is affected by substances in the serum (non-creatinine chromagens) which can cause false increases or decreases. Examples of non-creatinine chromagens include acetoacetic acid (ketone body), glucose, cephalosporin antibiotics and bilirubin (nb. hyperbilirubinemia may give false lows).

Nb. Elevations of urea and/or creatinine warrant a diagnosis of azotemia but don't necessarily mean that the animal is uraemic (i.e. in renal failure). However, all uraemic animals will be azotemic.

Azotemia can be divided into prerenal, renal and post-renal in origin. Prerenal causes of azotemia include: i) increased protein intake or catabolism (mainly elevates urea) ii) reduced glomerular filtration rate e.g. shock, haemorrhage, congestive heart failure and

dehydration (affects both urea and creatinine).

Pre-renal azotemia is common in a variety of diseases. In small animals, the magnitude of urea elevation in prerenal azotemia is usually limited (rarely greater than 40 mmol/L) when compared to renal and post-renal causes. However, the problems are that any level of azotemia can be present in renal disease and that there may be concomitant pre-renal and renal azotemia. Values for urea can reach or pass 100 mmol/L in renal disease and post renal obstruction in the dog and cat.

Renal azotemia occurs due to glomerular or tubular damage reducing glomerular filtration rate or total glomerular filtrate. Post-renal azotemia occurs due to urinary obstruction/retention interfering with glomerular filtration and tubular function via backpressure.

In farm animals the same principles apply to azotemia. However, elevation of urea in renal

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failure may not be as marked and consequently, there may be more overlap with pre-renal causes of azotemia. In the horse, blood urea levels in renal failure commonly vary between 18-80 mmol/L. In pre-renal causes of azotemia (especially GIT disturbances) urea levels can reach or pass 50 mmol/L. Consequently, in GIT disturbances in horses the level of pre-renal azotemia is often used for prognostic purposes. In cattle, prerenal causes of elevated urea are important and the levels overlap markedly with those for renal azotemia (10-100 mmol/L of urea in renal failure; 10-60 mmol/L of urea in other disturbances). In ruminants, the rumen has a limited capacity to utilize urea and this may lead to a 'tempering' effect on early elevation of urea with renal failure (the reason why creatinine is more useful in early or mild renal failure). It is unlikely to have an effect when renal failure is advanced.

c) Urinalysis Urinalysis may be helpful in determining glomerular dysfunction, e.g. haemorrhage or significant protein loss from glomeruli. However, urinalysis is more useful in detecting tubular dysfunction.

Assessment of tubular function

a) Dye excretion tests - used rarely

b) Blood urea level

c) Tests based on water elimination and reabsorption (urine concentration tests) e.g. water deprivation tests - generally used to detect concentrating ability in two situations:

1. in animals with polyuria, low specific gravity (<1.030 dog, <1.035 cat [some researchers suggest <1.040], <1.025 horse and cow [some researchers suggest <1.020 for the cow and horse]) and no azotemia

2. in animals with non-azotemic isosthenuria. ADH is released in response to the induced dehydration (usually 3% or more clinical dehydration) and its effect on urine concentration is assessed by determining urine specific gravity or plasma to urine osmolality. Suckling neonates have poor concentrating ability and the above figures do not apply. If the animal is azotemic and/or dehydrated then urine concentration tests are not warranted and could be dangerous. In dehydrated animals the finding of a specific gravity around the isosthenuric range usually suggests renal azotemia. If the urine is concentrated (i.e. above the values mentioned for water deprivation tests) then the animal probably has prerenal azotemia.

d) Aberrations of acid/base balance Renal tubular acidosis (RTA) syndrome can produce metabolic acidosis due to either defective bicarbonate reabsorption from the proximal tubules (Type I) or defective H+ excretion from the distal tubules (Type II). There is also a Type IV RTA, which involves defective secretion of potassium and hydrogen ions from the collecting ducts due to aldosterone deficiency or resistance.

e) Urinalysis Apart from its obvious uses in detecting renal disease and lower urinary and genital tract disease, it is useful also in analysing many extra-renal problems e.g. diabetes mellitus. Therefore, urinalysis will be presented in a form that covers its uses for all possible abnormalities.

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Urinalysis involves the following procedures and should be done completely in the first instance as often the results are interrelated.

i) observation of physical properties ii) estimation of solute concentration iii) chemical analysis iv) sediment examination v) optional examinations based on the results of the first 4 procedures

The way urine is collected will alter the results of urinalysis. Ideally, a 24 hour sample is necessary for accurate quantitative analysis, but in practice a single sample is analysed. Once the sample has been collected it should be analyzed quickly (within the first 2-3 hours - store at 4 degrees C if analysis cannot be done straight away).

A) Observation of Physical Properties 1) Volume - volume estimation ideally should be done on urine collected over a 24 hour

period. This will allow the detection of increased volume (polyuria) or decreased volume (oliguria). Volume changes can be transient or persistent (pathological)*. Normally an indication of volume is gained from the measurement of specific gravity (or osmolality). The generalization is that low specific gravity means high volume (exception: acute renal failure, oliguric phase of chronic renal failure).

*Transient polyuria (e.g. diuretic therapy, increased fluid intake, parental administration of fluids and administration of ACTH and/or corticosteroids) or pathological (e.g. pyometra, hyperadrenocorticism, diabetes insipidus, diabetes mellitus, polyuric phase of renal failure). Transient oliguria (e.g. decreased water intake, high environmental temperature, hyperventilation in the dog) or pathological (e.g. shock, fever, dehydration, urinary tract obstruction, oliguric phase of renal failure).

2) Colour - The normal colour of urine is dependent on the concentration of urochromes

(amorphous pigments and metabolic waste products). Normal urine for most of the domestic species is varying shades of yellow or light amber (Note: horse's urine may turn brown on standing).

3) Turbidity - Normal urine is clear to mildly turbid. Turbidity, if present, indicates

crystals, cells, mucus and other formed elements in high concentrations. Turbidity may develop on standing of urine due to the precipitation of crystals or bacterial overgrowth.

4) Odour - The normal odour of urine is derived from volatile organic acids. Abnormal

odours include ammonia and ketone bodies.

If abnormal gross characteristics are detected then the rest of urinalysis should detect the reasons for the changes. If the gross characteristics are normal it does not mean that the urine is normal and the other components of urinalysis should still be performed.

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B) Solute Concentration This gives an indication of the ability of the kidney tubules to dilute or concentrate excretory products (i.e. a true function test). In addition, an estimation of solute concentration is essential for the correct interpretation of levels of chemicals and cells. Solute concentration can be determined by measuring the specific gravity of urine. Specific gravity is dependent on the types of solutes, their numbers, molecular size and weights e.g. salts can affect specific gravity significantly because of their low molecular weight and large numbers. Specific gravity is measured today with a refractometer. If the urine is excessively turbid, it should be centrifuged prior to determining specific gravity (if not the specific gravity may be falsely elevated by 002-004 units). Solute concentration (as determined from specific gravity) is inversely related to urine volume in health and in many disease processes. Isosthenuria can be defined as continued excretion of urine at the specific gravity of the glomerular filtrate i.e. 1.008 - 1.012. A specific gravity reading of 1.008 - 1.012 from a single urine sample may indicate: 1) a chance finding in a normal animal (e.g. the dog kidney is able to dilute urine to 1.001, and concentrate urine to 1.060 [1.080 for the cat]) 2) renal failure. Unlike (1), repeated urine samples will have a specific gravity of 1.008 to 1.012 or close to it. Proteinuria or glucosuria may elevate the specific gravity reading (usually by 001-005 units depending on the levels). In renal failure with proteinuria this may take the reading out of the isosthenuric range. Hyposthenuria can be defined as the continued excretion of urine of low specific gravity i.e. less than 1.008. A low specific gravity may be found occasionally in a normal animal but if it is constant it points to such conditions as diabetes insipidus, psychogenic polydipsia and nephrogenic diabetes insipidus. At times the specific gravity may be in the isosthenuric range for these conditions. Beware, healthy suckling animals may have continued excretion or urine below 1.008! Rather than measure specific gravity for an estimation of solute concentration, the osmolality of urine can be determined. This appears to give a better estimation of the solute concentration as it is dependent only on the number of molecules rather than their molecular size or weight. Osmolality is expressed in milliosmoles and is measured by an osmometer (the measurement commonly involves the degree of freezing point depression which is directly proportional to solute concentration). Urine osmolality assessment is normally done in conjunction with plasma/serum osmolality to determine the concentrating ability of the kidney (the ratio of urine to plasma osmolality on single blood and urine samples will provide an accurate assessment of renal concentrating ability e.g. a plasma reading of 297 and a urine reading of the same indicates no concentration by the kidney). Osmometers, however, are expensive and specific gravity will continue to be used for estimation of solute concentration in veterinary practice.

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C) Chemical analysis of urine Reagents strips and tablets are commonly used to detect chemicals in urine. They are screening tests and consequently have limitations. If more accurate measurement of a urinary chemical constituent is required, a 24 hour sample of urine should be collected. NB: Chemical constituents of urine are determined according to concentration. Increases in concentration may occur due to increases in total amount of the chemical constituent excreted (true elevation) or be due to decreased urinary volume which is indicated by an increased specific gravity reading (relative or false elevation).

1) Protein - Urine from a normal animal has minimal levels of protein (mainly albumin).

Reagent strips are used as a screening test for protein as they are sensitive to albumin but they are less sensitive to globulins (which will include Bence Jones protein). Highly alkaline urine and urine with high haemoglobin or myoglobin may give false positives on the reagent strip. Consequently, if a positive is recorded on the reagent strip, it is best to check it by another method e.g. sulfosalicylic acid (SSA) precipitation (nb. all herbivores commonly have alkaline urine). SSA is less sensitive to albumin but detects all protein.

The magnitude of proteinuria can be assessed relatively accurately by determining the ratio of urine protein to urine creatinine (UP/UC or UPC). Protein is usually assessed by a microprotein method. A UPC ratio less than 0.5 is usually considered normal, a ratio between 0.5-1.0 is minimal or of questionable significance, whilst values equal or greater than 1.00 indicate significant proteinuria. Proteinurias related to glomerular disease usually give the greatest UPC ratios.

Proteinuria can be transient/physiological (e.g. exercise, oestrus) or pathological (e.g. cardiac disease, glomerular/tubular damage, lower urinary or genital tract inflammation). Physiological causes of proteinuria usually produce minimal elevations (rarely greater than 1+).

NOTE: Haemorrhage: the protein strip may show a 1 to 3+ reading if the blood strip is showing a maximal reading.

2) Glucose - Glucose is passed by the glomerulus and usually completely reabsorbed by

the tubules unless the renal threshold is exceeded (cats around 15-16 mmol/L, dogs and horses around 10-11 mmol/L, cattle around 5-6 mmol/L).

The 2 main methods of glucose estimation are reagent strips utilizing glucose oxidase and the Clinitest® reagent tablets utilising the copper reducing power of glucose and other sugars. The reagent strips can give false negatives in the presence of high ketone or ascorbic acid levels. The Clinitest® may give false positives in the presence of antibiotics, salycylates and other sugars.

Glucosuria can be transient (e.g. stress, heavy meal of carbohydrate – related to transient hyperglycemia) or pathological (most related to persistent hyperglycemia). Transient glucosuria is particularly common in ruminants (as mentioned cows have a low renal threshold of about 5-6 mmol/L) and can occur not only in physiological changes but also in diseases that cause transient hyperglycemia (e.g. neurological diseases, bovine milk fever, ovine enterotoxemia). Transient glucosuria in the cat can occur due to excitement or stress. By the time it is identified the hyperglycemia may have disappeared.

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In dogs, cats and horses glucosuria is usually related to diseases causing persistent hyperglycemia, and, therefore, is persistent itself. In the dog and cat diabetes mellitus is the common cause but in horses it can occur in both hyperadrenocorticism and diabetes mellitus.

Glucosuria unrelated to hyperglycemia is uncommon but in the dog, cat and horse has been recorded related to specific tubular diseases.

3) Ketones - Blood ketones increase whenever there is an increase in lipolysis or an

interference with lipogenesis of the TCA cycle (i.e. defective carbohydrate metabolism). Since the renal threshold is low for ketones, ketonuria is often detected before a significant ketonemia develops.

Ketonuria in dogs and cats in commonly related to diabetes mellitus. It may be seen also in puppies and kittens that have been starved (rarely in adults). In ruminants ketonuria is non-specific and can occur in starvation, high fat diet, bovine ketosis, ovine pregnancy toxemia and in a variety of diseases that induce anorexia. Ketonuria is uncommon in the horse.

Ketones in the urine can be detected by reagent strips (specific for acetoacetate) or by Acetest® tablets (reacts with acetone and acetoacetate).

4) Bilirubin - Detected by the Ictotest® or by the bilirubin strip which are mainly reactive

with conjugated bilirubin and rather insensitive to unconjugated bilirubin. The significance of bilirubinuria has been discussed in the laboratory evaluation of liver disease. In all common domestic species except for the dog, bilirubinuria means conjugated hyperbilirubinemia. In the dog, especially the male, bilirubinuria is not highly specific for conjugated hyperbilirubinemia because of the low renal threshold and a renal capacity to conjugate and even breakdown haemoglobin to bilirubin. However, the presence of marked amounts of bilirubin in the urine of a dog usually means hyperbilirubinemia.

5) pH - In the dog and cat the pH is normally 5.5-7.5. In the normal horse the pH is close

to 8. Ruminants have a pH 7.4-8.4. Most young animals suckling, irrespective of species, will have acid urine.

Urine pH must be measured promptly and is the result of renal regulation of blood pH. It should not be used alone to evaluate acid/base status as it is heavily influenced by diet, therapy and urinary tract disease.

Aciduria occurs with a carnivorous diet, starvation, fever, metabolic acidosis and renal tubular acidosis. Occasionally, aciduria occurs with metabolic alkalosis because of the kidney concentrating more on correcting related electrolyte imbalances (hypochloremia and hypokalemia) and dehydration. This is called ‘paradoxical aciduria’, as it is unexpected, and can be seen in severe vomiting, gastric or abomasal reflux (the last two are so called ‘internal vomiting’ in herbivores) where both HCl and other electrolytes (such as potassium) can be lost to the body. Alkaline urine may be due to a herbivorous diet, cystitis (depends on the type of bacteria present - urea splitting bacteria will commonly produce alkaline urine), urine retention, metabolic alkalosis or extended standing of urine before testing (bacterial action).

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6) Blood - The reagent strip test is based on the peroxidase activity of haemoglobin or myoglobin. The test is more sensitive to free haemoglobin than to haemoglobin within the erythrocyte although the two are supposed to be able to be distinguished on the strip (don’t rely on the strip for this - examine the sediment for erythrocytes).

A positive test means haematuria, haemoglobinuria or myoglobinuria. Haematuria can be distinguished on sediment examination while the other 2 can be distinguished on ammonium sulphate precipitation and plasma colour (plasma needs to be saturated with haemoglobin i.e. obviously red, before haemoglobin appears in urine. This is not the case with myoglobin as it is a smaller molecule and more easily passed by the kidney).

Haematuria indicates urogenital tract disease (especially if the sample has been voided), haemoglobinuria indicates intravascular haemolysis or lysed erythocytes (may occur at low specific gravities), and myoglobinuria indicates muscle necrosis.

D) Sediment examination What structures are present in the sediment will depend not only on disease but also on how the sample of urine is collected (i.e. voided, catheterisation or cystocentesis). Structures present in urine can be divided into 3 groups: Group 1 consists of those formed elements that are present in health in low numbers. Increased numbers of these structures may have little significance unless indicated by other information e.g. most epithelial cells, many types of crystals, lipid, sperm, mucus, fungi, bacteria. Group II, like group I, consists of formed elements present in health in low numbers. However, increased numbers commonly have significance e.g. casts, erythrocytes, leukocytes. Group III consists of formed elements of sediment that are not normally present in urine e.g. neoplastic epithelial cells, unusual crystals, unusual casts. Their presence in any numbers are significant. Below is information on elements that may be found in urine. Usually urine is concentrated by centrifugation prior to examination to ensure recognition of structures. In this laboratory 10mls of urine is centrifuged and the sediment is resuspended in about 0.5mls of supernatant. Other laboratories may centrifuge 5mls of urine and resuspend sediment in 0.5mls of supernatant: this will alter normal levels of cells presented below. Irrespective of the amount it is important to maintain a standard technique to ensure direct comparability of results.

1) Epithelial Cells

*Renal epithelium. In the dog and cat, their numbers are not a reliable indication of renal disease unless they are in clusters and associated with casts.

*Caudate epithelium. Primarily derived from the renal pelvis, ureter and prostatic urethra. Increased numbers may indicate disease of the areas mentioned if other indicators of disease are also present e.g. inflammatory cells.

*Transitional epithelium. Principally from the bladder. Their significance is questionable.

*Squamous epithelium. Derived from the lower urogenital tract and of little significance. They usually indicate contamination.

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*Abnormal cells e.g. carcinoma cells. These are of obvious significance.

2) Erythrocytes Indicates haematuria if greater than 5 per high powered (x 40 obj) field in moderately concentrated urine.

3) Leukocytes

Increased numbers (i.e. greater than 3-5 per high powered [x 40 obj] field) in moderately concentrated urine indicates inflammation in the urogenital tract. In cows, it has been suggested that greater than 8 per high powered [x 40 obj] field is more appropriate for a voided sample. Beware, leukocytes tend to disintegrate in alkaline or hypotonic urines that are left to stand (even if stored at 4oC). Consequently, in some bacterial cystitides with highly alkaline urine there could be few leukocytes present. Additionally, some bacteria may cause direct lysis of leukocytes and lead to false low numbers. Remember, a voided sample of urine may have leukocytes from the genital tract as well as the urinary tract.

4) Casts

Casts are precipitates of protein formed in the distal convoluted tubules and the collecting tubules. They conform to the shape of the tubules and may contain any material present in the tubular lumen at the time of formation. A small number of casts, especially hyaline casts, may be found in normal urine (up to one per low powered [10x objective] field).

The significance of cylindruria depends on the numbers and their types. However, in moderately concentrated urine greater that 1 cast per low powered (x 10 0bj) field is suggestive of active renal damage, decreased urine flow or renal perfusion, or a response to renal damage. Persistent cylindruria is more significant than transient cylindruria in identifying renal damage.

*Hyaline casts. Increased numbers have been noted in mild renal tubular damage, fever conditions, and as a consequence of surgery or exercise.

*Granular casts. When hyaline casts have refractile particles embedded in them, they are called fine or course granular casts. The granules were thought to be derived from disintegrating tubular epithelium and, therefore, indicate tubular degeneration and necrosis, but recently the granules were shown to be composed of fractions of various serum proteins. Therefore, granular casts have the same significance as hyaline casts. These are the most common type in dogs.

*Waxy casts. They are thought to be derived from granular casts and indicate advanced renal disease. The presence of any waxy casts is significant.

*Fatty casts. Granular casts with a high degree of fat. These are the common type in the cat.

*Renal epithelial casts. Well preserved renal epithelium within the cast may indicate acute tubular damage. Any casts are deemed significant.

Casts may also contain erythrocytes and leukocytes (any number are significant and suggest cell origin in the tubules), and may be stained with bilirubin.

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5) Lipid droplets Increased amounts may occur in obesity, high fat diet, hypothyroidism and diabetes mellitus. Lipid may be seen in normal dog and cat urine (very common in the cat). The lipid is derived from the renal tubular epithelium.

6) Spermatozoa

7) Fungi

The yeasts and hyphae are nearly always contaminants. Significance will depend on the presence or absence of inflammation. Fungal cystitis can occur, mainly with Aspergillus spp, and should be accompanied by increased leukocytes.

8) Bacteria

The significance of bacteria in the urine will depend on determining an association with inflammation in the urogenital tract. Occasionally, leukocytes will not be obvious due to the destruction by bacterial toxins, low specific gravity or highly alkaline urine. Consequently, inappropriate bacteruria without pyuria should be investigated further. Cystocentesis should provide a sterile urine sample, whereas a voided urine sample will be invariably contaminated with bacteria from the lower urethra or genitalia

9) Crystals

Crystals are frequently found in urine and their precipitation is dependent on their concentration and the pH of the urine. They are usually of little significance but leucine/tyrosine/ammonium biurate (the last may occasionally occur in healthy dogs, especially Dalmatians) crystals may be indicative of chronic liver disease and/or porto-systemic shunting, oxalates (mainly calcium oxalate in the monohydrate form) of ethylene glycol toxicity, sulphonamides of nephrosis, and bilirubin or haemoglobin crystals of acute haemolytic states or haemorrhage.

Miscellaneous Biochemical Alterations that may occur in Various Forms of Renal Disease

1. Non-regenerative anaemia. Commonly occurs in chronic renal failure due to a combination of

factors including decreased erythropoietin action, toxic depression of bone marrow and enhanced red cell fragility. The degree of anaemia is extremely variable and can vary from mild to life threatening.

2. Metabolic acidosis. This is due to the retention of acids (titration acidosis) and is dealt with in

the section on acid-base inbalances. Metabolic acidosis is common in renal failure in dogs and cat and a little more variable in horses. In cattle, there is likely to be normal acid-base balance (as measured by total CO2 or Bicarbonate) or metabolic alkalosis because of rumen atony and HCl sequestration (‘internal vomiting’). Similar mechanisms may operate for some cases of renal failure in dog, cat (excess external vomiting) and horse (excess ‘internal vomiting’), with normal acid-base balance the consequence. In those cases, there is still retention of so called uraemic acids, which can only be detected by determination of an increased anion gap (see later section on acid-base imbalances).

3. Hypercholesterolemia. May be seen in certain renal diseases and conditions (e.g. the nephrotic

syndrome), probably due to derangements of lipoprotein metabolism and or the presence of acute phase reactants (mainly lipoproteins).

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4. Hyperamylasemia/hyperlipasemia. May be seen with high azotemia in uraemia in dogs. The

reasons for elevation are not completely clear. 5. Hyperphosphatemia. This is common in chronic renal failure in dogs and cats. Persistent

hyperphosphatemia can lead to renal secondary hyperparathyroidism and variable effects on serum calcium levels (they may be decreased, normal or rarely increased). In horses, hyperphosphatemia is not common in renal failure except in young horses (usually less than 1 yo) where there might also be hypocalcemia; in fact, hypophosphatemia (or normal levels) is more common in chronic renal failure in the adult horse. In contrast to dogs and cats, hypercalcemia is not uncommonly seen in oliguric or anuric chronic renal failure in the adult horse (the adult horse's kidney may be more important in calcium excretion rather than retention in relation to calcium rich diets). In acute renal failure in the horse the blood calcium levels are commonly low to normal.

In cattle, serum calcium levels are extremely variable in renal disease, but hypocalcemia is probably more common. Hyperphosphatemia is common in acute renal failure, but is rarely dramatic.

6. Hyperkalemia. This is more commonly seen in acute renal failure and may be related to

metabolic acidosis as well as renal shutdown. It is not as common in chronic renal failure, but can occur in the oliguric and anuric stages. In cats, hypokalemia can actually accompany chronic renal failure (possibly due to chronic metabolic acidosis, renal loss and decreased intake) and lead to a specific myopathy (mainly muscle weakness) if the potassium is extremely low. This syndrome has not been noted in other domestic species. Hypokalemia may also be seen in the recovery phase of acute renal failure due to kaliuresis. Cattle may get hypokalemia in renal failure due to decreased intake, but hyperkalemia is more common in acute renal failure.

7. Hyponatremia and hypochloridemia may occur with renal failure, particularly in the horse

and cow. Hypermagnesemia may occur in renal failure in monogastric animals and occasionally in ruminants.

*SEE CASE REPORTS 7-13

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LABORATORY INVESTIGATION OF SOME DIGESTIVE TRACT DISORDERS

Terminology

Malassimilation Collective term for maldigestion and malabsorption Maldigestion abnormality resulting in the failure of normal digestive processes Malabsorption abnormality resulting in the failure of normal absorptive processes Diarrhea increased frequency of defecation and volume/fluid consistency of the

faeces Melena the discharge of faeces coloured black by altered blood. Usually indicates

small intestinal bleeding Steatorrhea increased amounts of fat in the faeces Creatorrhea increased amounts of nitrogen (protein) in the faeces. In dogs and cats it is

usually detected by increased amounts of undigested muscle in the faeces Amylorrhea increased amounts of starch in the faeces Lipemia milky appearance of serum or plasma caused by increased levels of

triglyceride-carrying lipoproteins (e.g. chylomicrons) Hyperlipidemia is an increase in serum or plasma lipids (triglycerides, cholesterol or

phospholipids)

Exocrine Pancreatic Disorders

These can be divided into acute pancreatic necrosis (necrotizing pancreatitis) and pancreatic insufficiency. Pancreatic necrosis commonly presents as an acute abdominal problem while pancreatic insufficiency is a major cause of maldigestion. Pancreatic insufficiency will be discussed under problem diarrheas and malassimilation. Pancreatic Necrosis In the dog, acute pancreatic necrosis presents as a distinct entity and its cause is still poorly understood (although diet is an important factor). It has been recorded to a lesser extent in the cat and rarely for the horse. Most cases in the dog involve extensive necrosis and consequently, produce severe clinical signs. Lower grade pancreatic necrosis/inflammation can occur with limited clinical signs. The syndrome has also been called acute necrotizing pancreatitis due to the intense inflammation that relates to pancreatic and surrounding fat necrosis (this should not be confused with pancreatitis in most species which can occur due to a variety of known causes such as bacteria or parasites).

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To confirm acute bouts of pancreatic necrosis in the dog and other species requires:

a) Elevation of serum enzymes. In the dog and cat elevations of amylase and lipase often occur in pancreatic necrosis. Lipase appears more reliable. The elevations for both enzymes are not consistent and there are other causes of elevations. Consequently, the 2 enzymes are often used in tandem. In the dog they can rise within 1-3 hours of damage. The extent of the elevation depends on the degree of damage to the pancreas (low up to 3-4x for AMS; low up to 2-3x for LPS). A canine pancreas-specific lipase immunoassay may become the test of choice in the future.

In the cat the enzymes are less reliable to confirm pancreatic necrosis/inflammation (this may be partly due to the fact that most cases are probably low grade). In the horse AMS elevations have been recorded in the few reports on acute pancreatic necrosis but, as in other species, need to be supported by other laboratory tests.

The trypsin-like immunoreactivity (TLI) test (primarily used to detect pancreatic insufficiency) has been suggested as useful when investigating acute pancreatic necrosis in the dog and cat, but appears less useful than the canine pancreas-specific lipase immunoassay. Increased levels for TLI (e.g. >35 ug/L in the dog) may occur with acute pancreatic necrosis. Like all other tests, it is best used in combination.

b) Other supportive laboratory findings for acute pancreatic necrosis (primarily reported in the dog but inconsistent): i) Hamatology. Stress reaction with a left shift; hamoconcentration ii) hyperlipidemia (fasting) iii) fluid imbalance (due to vomiting and loss of HCl) iv) pre-renal azotemia v) non-septic exudate in peritoneal cavity vi) transient, mild hyperglycemia vii) transient hypocalcemia

NOTE: Hepatocellular necrosis and cholestasis may occur secondarily to the acute pancreatic necrosis and these need to be investigated appropriately.

Investigation of Colic in the Horse

Colic (abdominal pain) in the horse is potentially lethal, has numerous causes and is a common condition primarily because of anatomical factors related to the arrangement of the gastrointestinal tract. Consequently, laboratory aids to diagnosis are extremely important to differentiate cause as well as to consider prognosis. Hamatocrit and total plasma protein determinations are important to support assessment of hydration status; blood gas analysis and electrolyte determination are important to detect metabolic acidosis or alkalosis (rapid deterioration in acid-base balance is a poor prognostic sign) or electrolyte disturbances, urea and creatinine determinations to primarily detect the degree of pre-renal azotemia; and serum enzyme determination (e.g. ALP may rise in complicated abdominal disease presumably due to release from intestinal cells; AST and CK will increase due to muscle damage related to rolling). Abdominal paracentesis is perhaps one of the more useful laboratory tests in investigating colic. In the horse, assessment of peritoneal fluid can be done even when an effusion is not present. It is a useful diagnostic tool for the investigation of abdominal disease, and reflects changes occurring on mesothelial surfaces and, to a lesser extent, in abdominal organs. The approach to interpretation of abdominal fluids is the same as for dogs and cats and will be discussed under the section of Body Fluid Analysis

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Investigation of Problem Diarrheas

Problem diarrheas refer to persistent diarrheas that are refractory to simple treatment or correction. In the dog and cat primary small intestinal disease gives rise to voluminous, fluid faeces and is often divided into those diseases that produce significant steatorrhea and weight loss (malassimilation) and those diseases that are relatively non-steatorrheic. Primary large intestinal disease in small animals does not produce a profuse watery diarrhea and does not significantly interfere with assimilation. However, the faeces may be soft or slightly fluid and there is a marked increase in frequency of defecation. In the horse profuse, watery diarrhea is produced only when the large intestine is involved. Primary small intestinal disease may give rise to malassimilation but diarrhea is limited to a soft consistency of faeces ('dung pat' faeces). The reasons for these differences between the horse and small animals are related to differing functions of the large intestine. In the horse the large intestine is responsible for breakdown of plant material to absorbable substances; water is absorbed primarily along with the volatile acids derived from carbohydrate breakdown. Problem diarrheas in all species should be investigated in the laboratory by the following methods:

1. Faecal examination - used also in acute diarrheas

2. Peritoneal fluid analysis - normally limited to the horse (sometimes cattle) and applied in all types of diarrhea

3. Absorption tests - used for the detection of malabsorption

4. Haematological and serum biochemical tests - these provide supportive evidence for diagnosis e.g. low serum protein in a suspected protein-losing gastroenteropathy (PLG).

5. Exploratory laporotomy, intestinal washings and biopsy (mainly dog, cat and horse).

1. Faecal examination (coprology) This is the starting point for the investigation of any gastrointestinal disturbance:

i) Gross characteristics Involves examination for odour, colour, consistency, volume and presence of unusual material. In the dog and cat steatorrhea will commonly give a rancid odour (may be masked by blood and other faecal components). If blood is suspected of being present but is not obvious, then the Hematest® can be employed.

ii) Microscopic examination a. Parasites (worms, protozoa) - these should not be overlooked as a cause of diarrhea,

especially in farm animals.

b. Faecal smears - stained for morphology. These may be useful in detecting large intestinal inflammatory or neoplastic disease (cells) and for detecting certain protozoal organisms.

c. Faecal Occult blood – blood in the faeces is not always obvious grossly, so if intestinal

bleeding is suspected then it is useful to test with the Hematest® or something similar. d. Examination for undigested/unabsorbed food particles - examine for the presence of

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steatorrhea (undigested [unsplit] and unabsorbed [split] fat), creatorrhea (undigested muscle) and amylorrhea (undigested starch). They are screening tests for malassimilation and are best interpreted when compared to control smears of faeces from normal animals on the same diet. They are useful in the dog and cat for preliminary investigation of malassimilation. They are not of use in the horse with malassimilation (LI microbial activity degrades all the undigested/unabsorbed food particles).

iii) Measurement of Trypsin (protease) in faeces and Trypsin-like Immunoreactivity (TLI) in serum for exocrine pancreatic insufficiency (EPI) In faeces, trypsin can be estimated by gelatin digestion methods or determined quantitatively by a dye binding technique (azoalbumin method). Trypsin activity varies between, and within, animals. Also, diet can have an effect on the trypsin level. A fresh stool is required for analysis as trypsin activity falls rapidly. It is best to check trypsin activity in faeces on 3-4 consecutive days, and take into account results from microscopic examination for undigested/unabsorbed food particles, before deciding on whether an abnormality exists (increased starch, undigested muscle and undigested [unsplit] fat suggests maldigestion commonly due to EPI).

A more accurate assessment of pancreatic function can be determined by feeding the dog or cat n-benzoyl-l-tyrosol-p-aminobenzoic acid (BT-PABA). Chymotrypsin attacks the tripeptide to produce PABA which is absorbed and measured in the blood stream (or urine). This test apparently is not affected by malabsorption but is more difficult to perform than trypsin.

Another test for EPI in the dog and cat, and currently the test of choice, involves a radioimmunoassay for trypsin-like immunoreactivity (TLI) in serum. The test requires a single fasting serum sample and detects trypsinogen plus other related substances which normally leak into blood from the pancreas. The test is species specific, so there are distinct dog and cat assays for suspected EPI. Dogs with EPI, even if already supplemented with pancreatic enzyme extract, are expected to have serum TLI levels of less than 2.5 µg/L (normal range 5-35 µg/L). This must be a fasting sample as feeding may increase the level of TLI in dogs with EPI into the low normal range. Increased levels of TLI are supposedly seen in dehydrated or dogs in renal failure due to reduced glomerular filtration, and in dogs with pancreatic inflammation (i.e. acute pancreatic necrosis - see that section). Once again, the test should be combined with other findings and tests for a definite diagnosis of EPI.

EPI is most common in the dog but cases have been recorded in the cat. It is the major cause of maldigestion in small animals. In the horse EPI is rare and most cases of malassimilation are due to malabsorption involving the small intestine.

iv) Faecal culture Usually a low return diagnostic procedure due to the presence of normal flora. More commonly done in horses and farm animals with prolific diarrhea (e.g. for Salmonella spp or Mycobacterium paratuberculosis in ruminants).

2. Peritoneal fluid analysis - abdominocentesis (see under fluid analysis)

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3. Absorption tests Absorption tests are often difficult and tedious to perform but do provide a more specific assessment of digestion and absorption of specific components of the diet.

i) Tests for carbohydrate digestion and absorption In the dog and cat they were used to investigate malabsorption usually after simpler tests have been performed (e.g. smears for unabsorbed [split] fat), but today endoscopic biopsies are more likely undertaken. In the horse they are employed after faecal examination as initial tests to investigate malabsorption.

a. Oral glucose tolerance test (OGTT) - for the dog after a 16 - 24 hour fast, 2g/Kg body weight of glucose is administered orally as a 20% solution. Blood samples are collected at 0, 30, 60, 90, 120 and 180 minutes and assayed for blood glucose. For the horse, glucose is administered via nasogastric tube at 1g/kg as a 20% solution. A normal response is accepted as an increase in blood glucose levels of more than 100% of the baseline levels after 1-2 hours This procedure measures the monosaccharide absorptive capability of the small intestine and can be used effectively as long as glucose metabolism has not been disturbed by other diseases.

b. Xylose absorption test (XAT) - a similar test. Measures the monosaccharide absorptive capability of the duodenum and cranial jejunum. The XAT is often preferred to the OGTT in the horse as the latter test can be affected by endocrine controls and disturbances of glucose metabolism. Moreover, xylose is not normally found in equine blood. In the horse, food is withheld for 12 hours and 0.5g/kg of d-xylose as a 10% solution is administered via nasogastric tube. For a normal horse, d-xylose should be detected in the blood at a level of 1.0 mmol/L or greater after 1-2 hours.

Other carbohydrate absorption tests may test both digestion and absorption but are not commonly used e.g. lactose tolerance test, starch tolerance test, sucrose tolerance test.

ii) Tests for fat digestion and absorption (for dog and cat not horse) a. Post-prandial lipemia - the animal is fasted and then fed 4 ml of corn oil/Kg body weight

mixed with a small amount of dog food. Blood samples are taken at 2, 4 and 6 hours and the plasma/serum appearance compared with that of a sample taken prior to feeding. Plasma/serum should be clear in the fasting state and develop an obvious turbidity 2 - 6 hours after feeding (the test is normally done by collecting blood in EDTA and utilizing microhematocrit tubes for centrifugation).

Nb: The determination of triglyceride levels in serum is better than a visual assessment of turbidity.

This test measures the functional integrity of the entire fat digestion and absorption process.

Nb: By adding a pancreatic enzyme supplement to the corn oil prior to administration, information can be gained on whether there is a lipase deficiency (and, therefore, the presence of EPI).

Assessment for post-prandial lipemia may be done before performing such tests as faecal smears for indigested/unabsorbed food particles. However, the results are not always conclusive and other tests are usually needed to support the diagnosis of malassimilation.

b. Fat balance determination - fat levels are determined in the feed and faeces over a certain

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period of time e.g. 3 days. In the normal dog, 95-98% of ingested fat is absorbed. With malabsorption 80-90% is absorbed. With maldigestion 40-80% is absorbed.

Fat balance studies are rarely done for obvious reasons.

4. General haematological and biochemical testing These are generally of limited use in investigating problem diarrheas but may detect changes that could be related to GIT diseases (e.g. eosinophilia associated with eosinophilic enteritis; anaemia associated with prolonged GIT bleeding).

In protein losing gastroenteropathy (PLG), biochemical testing is necessary to confirm hypoproteinemia (all globulins and albumin usually depressed) and to determine if the gut is the source of loss. As it is difficult to determine protein loss from the gut other causes of hypoproteinemia are eliminated first (e.g. renal disease, hepatic failure and malnutrition). Since many of the causes of PLG can, under the right circumstances, produce malabsorption, appropriate testing will establish a diagnosis by implication (for example, it is highly unlikely in an animal with malabsorption that a hypoproteinemic state is produced by disease other than in the GIT).

PLG can develop due to chronic GIT conditions. Such conditions as chronic gastric ulceration, chronic enteritis and colitis, eosinophilic gastroenteritis, intestinal neoplasms (e.g. cat lymphosarcoma), and lymphatic obstruction (e.g. lymphangiectasia) may give rise to PLG. Many of these diseases may give rise to a chronic uncomplicated diarrhea or malabsorption. Consequently, preliminary investigations must have a common basis (e.g. faecal examination). PLG is common in farm animals in relation to enteritis and parasitism, and can develop quickly in the horse with persistent diarrhea and inflammatory bowel disease.

Serum folate and vitamin B12 (cobalamin) levels have been measured in cases of malassimilation in the dog. Folate is absorbed in the proximal small intestine while vitamin B12 is absorbed primarily in the ileum. Prolonged anorexia, small intestinal disease and EPI may produce reduced serum levels of folate and vitamin B12. Bacterial overgrowth, which may accompany small intestinal disease (or EPI), is supposed to reduce serum vitamin B12 (by binding and interfering with absorption) but increase serum folate (involves bacterial synthesis). This is not consistent, but can be used to investigate the possibility of bacterial overgrowth.

5. Endoscopy, exploratory laparotomy, intestinal washings and biopsy In some cases of chronic SI diarrhea with or without malabsorption, an endoscopic procedure or exploratory laparotomy is undertaken as other procedures have been unable to provide an exact diagnosis. A similar situation commonly occurs with PLG. Intestinal washings may be useful if a parasitic cause is suspected (e.g. giardiasis in dogs and cats). In cases of malabsorption it may be useful to remove tissue for histopathological examination from stomach, liver, duodenum, jejunum and ileum. In large intestinal disease the collection of biopsy material is a simpler procedure due to coloscopy/protoscopy. This procedure may be undertaken early on in the investigation of a diarrhea but should not precede simpler tests such as faecal examination. Large intestinal biopsies should include the full depth of the mucosa and preferably some submucosa.

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Additional Notes on Malassimilation

Malassimilation, for simplicity, is commonly divided into maldigestion and malabsorption. To understand the significance of tests used in investigating maldigestion/malabsorption, knowledge of the normal mechanisms of digestion and absorption is necessary. It is essential to remember that there are complex interactions between organs (stomach, liver, pancreas, gut, lymphatics) in the process of digestion and absorption. Consequently, disease processes often affect both digestion and absorption in varying proportions (with obvious effects on the results of laboratory tests). Some generalisations can be made on disease processes that lead to maldigestion/malabsorption: a) diseases that affect the production of bile salts or the lymphatics have a principal effect on fat

digestion and absorption. b) diseases that affect the pancreas or gut usually have an effect on fat, protein and carbohydrate

metabolism. However, in most clinical cases in dogs and cats, the manifestations of carbohydrate and protein maldigestion/malabsorption are grossly overshadowed by those of fat maldigestion/malabsorption.

c) in dogs and cats diagnosis is based on the detection of significant steatorrhea. EPI is the

common cause of maldigestion in the dog but congenital or acquired deficiencies of the small intestinal brush border enzymes have been recorded (e.g. lactase). In cats, malabsorption due to small intestinal disease is more common than maldigestion due to EPI.

d) in the horse most cases of malassimilation are due to malabsorption of SI origin. Steatorrhea is

not a feature and diagnosis is primarily based on absorption tests. e) malassimilation is not well recognised in ruminants and pigs but chronic diarrheas with or

without PLG are common and should be approached in a similar manner (usually investigations are undertaken when a group of animals are affected - e.g. certain parasites can cause PLG in cattle, sheep and pigs).

*SEE CASES 19,28,29

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LABORATORY INVESTIGATION OF ENDOCRINE DISORDERS, INCLUDING CALCIUM AND PHOSPHATE DERANGEMENTS

Introduction

Information on laboratory investigation of endocrine disorders is available primarily for the dog and the cat, and to a lesser extent for the horse. In farm animals, endocrine disorders are of consequence only if they affect a significant proportion of the herd or flock. Most are dietary related affecting endocrine gland operation, but some could be heritable e.g. calcium homeostasis and the parathyroid gland; hypomagnesemia; intake of goitrogenic substances or inherited goiter. Many of the tests outlined in these notes, especially the hormonal assays, can be expected to change and develop in the future.

Diabetes mellitus (DM)

Diabetes mellitus (hypoinsulinism) is important in both the dog and cat. Diabetes mellitus has been recorded in the horse. This is commonly defined as a disorder of carbohydrate metabolism due to disturbance of the normal insulin mechanism. There may be an absolute or relative lack of insulin. In people DM is divided into insulin-dependent (IDDM - Type I), non-insulin dependent (NIDDM – Type II) and secondary DM. IDDM usually develops in children and requires insulin treatment. NIDDM occurs in older people who are predisposed to abnormal interactions of insulin with peripheral tissue receptors. Delayed insulin secretion and impaired insulin action occur and obesity may trigger overt DM. These patients are normally managed by diet and oral hypoglycemic agents. Secondary DM develops because of predisposing disease such as pancreatitis, hyperadrenocorticism, acromegaly, prolonged administration of certain drugs or hormones (e.g. progestogens or glucocorticoids). These patients are treated for the underlying condition but still may require insulin treatment on a long term basis. Many of the causes of DM in the dog and cat are poorly understood. Some are likely to have a genetic basis, but it is well accepted that immune-mediated destruction of islets may occur for both dogs and cats (although the latter has been better documented for dogs). In addition, obesity, infection, pancreatitis and drugs (e.g. glucocorticoids in dogs; megestrol in cats) may predispose. As in people, DM can be loosely divided into IDDM (absolute lack of insulin) and NIDDM (relative lack of insulin; usually due to insulin resistance at tissues or a combination of this and reduced insulin production) based on the animal’s need for insulin (it should be emphasized that this classification is not strictly followed by all veterinary endocrinologists). IDDM, because it is characterised by beta cell destruction/Loss (immune-mediated destruction [most frequent cause of DM in the dog]; islet amyloid deposition leading to beta cell loss or destruction [common in the cat]), requires insulin treatment. It may be sudden in onset or take some time to develop clinically. NIDDM, as mentioned, is characterised by insulin resistance and, sometimes, ‘dysfunctional’ beta cells. Consequently, insulin levels may be increased, normal or sometimes decreased. The distinction between IDDM and NIDDM is not always clear-cut as some affected animals (especially cats) may appear to fluctuate between the two during the course of clinical disease. In the dog, IDDM accounts for most cases and tends to occur in middle-aged or older dogs, and ketoacidosis can be a complication. A form similar to NIDDM does occur in the dog but is uncommon and tends to occur in juveniles. In the cat, most cases occur in middle-aged or older cats and more than half appear to be commonly responsive to insulin (i.e. most are IDDM). However, unlike the dog, NIDDM is more common and may account for 30-50% of cases of DM. These cats tend to be obese and resistant to the development of

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ketosis. Insulin therapy may not be required in these cats and management involves weight reduction, dietary control and oral hypoglycemics. DM in cats (rarely in dogs) may fluctuate from being clinical to sub-clinical and is sometimes referred to as Transient Clinical Diabetes Mellitus. Basically, insulin is an anabolic and is especially important for glucose uptake and/or metabolism in specific organs (liver, skeletal muscle, fat). The actions of insulin in the body are antagonised by a variety of hormones and other substances ('diabetogenic factors'). They are responsible for diabetes mellitus due to a relative lack of insulin (although some reduced insulin production by the pancreas may be involved as well). Hormones that oppose the effects of insulin include glucagon, glucocorticoids, catecholamines, growth hormone, progesterone and thyroxine. In addition, such conditions as azotemia and obesity can be diabetogenic. In the dog and cat the hallmark of DM is persistent fasting hyperglycemia and glucosuria (glucosuria in the dog is uncommon in other conditions due to the high renal threshold [approximately 10-11 mmol/L]; cats may have transient glucosuria in stress but this is not consistent as the renal threshold can be up to 15-16 mmol/L). In these cases it may not be necessary to perform a glucose tolerance test to confirm DM. However, there are earlier/mild forms of DM that do not present with a severe hyperglycemia and glucosuria; and an intravenous glucose tolerance test can be performed to diagnose DM (a flat curve or low glucose disappearance co-efficient suggests glucose intolerance). In all the dogs with DM, the insulin response to the IV glucose tolerance test will determine whether there is an absolute or relative lack of insulin (i.e. if diabetogenic factors are involved). In the horse, although the disease is less common, DM can result from absolute or relative lack of insulin as well as being a secondary form. The most common reason appears to be Secondary DM due to Pituitary adenoma, commonly of the pars intermedia (i.e. pituitary dependent hyperadrenocorticism called Equine Cushing’s Disease; pituitary adenoma of the pars intermmedia may also give rise to Diabetes Insipidus). NIDDM has been recorded in relation to obese ponies. In most cases of IDDM some increase in ketone production will occur but normally this is controlled by peripheral utilisation of ketones. If this peripheral utilisation of ketones is impaired or exceeded then ketoacidosis may develop. Ketoacidosis appears to be a rare complication of NIDDM in dogs and cats. The test for ketones in urine is not sensitive for the main type of ketone produced early on in DM (hydroxybutyrate). Consequently, early ketonuria may be missed nb. in adult dogs (and probably cats) DM is about the only condition that will give rise to ketonuria; in suckling puppies and kittens ketonuria is sometimes seen with starvation. In the horse, ketonuria related to diabetes mellitus has been recorded, but its commonness in relation to IDDM, NIDDM and Secondary DM has not been distinguished (most likely due to Secondary DM). Other laboratory abnormalities that may occur in DM include hyperlipidemia (nearly all cases and due to increases in tryglycerides, cholesterol and free fatty acids – gross lipemia, therefore, is commonly evident), increased liver enzymes (due to fatty change), increased PCV and total plasma protein (dehydration) and electrolyte loss (due to osmotic diuresis), and proteinuria (due to lower urinary tract infection and/or glomerular disease). Other laboratory abnormalities may occur in Secondary DM depending on the other disease (e.g. hyperadrenocorticism will give rise to its own set of laboratory abnormalities) Blood glucose levels can be monitored in treated DM by measuring glycosylated haemoglobin (HbA1) or fructosamine in the blood. HbA1 concentration reflects blood glucose concentrations over the previous 2-3 months while fructosamine levels are supposed to give an indication of blood glucose levels over shorter periods (normally 2-3 weeks).

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Hyperinsulinism

This is caused by a productive islet cell tumour of the B cells. It is only of significance in the dog. An insulin-secreting neoplasm may be diagnosed by demonstrating a severe hypoglycemia at the time of appropriate clinical signs. However, in most cases confirmation requires the demonstration of an inappropriately elevated plasma insulin concentration at the time when plasma glucose is within or below the reference interval (use of the amended insulin to glucose ratio after fasting the dog).

Hyperadrenocorticism

This refers to the clinical syndrome produced by prolonged and excessive blood levels of glucocorticoids. It can be produced iatrogenically (long term corticosteroid administration), but commonly it is due either to adrenal tumours (usually unilateral) or to adrenal hyperplasia secondary to excessive pituitary stimulation (PDH). Hyperadrenocorticism is most common in the dog, uncommon in the horse and rare in the cat. In the dog and horse most cases of hyperadrenocorticism are PDH. PDH can result from hyperplasia or neoplasia of ACTH secreting cells in the anterior pituitary (and occasionally pars intermedia, especially common type in the horse in which it can lead also to decreased ADH secretion and Diabetes Insipidus, as well as predisposing to Secondary DM ). In the cat both PDH and adrenal tumours have been noted but they have not been well studied because of the rareness of hyperadrenocorticism. If hyperadrenocorticism is suspected clinically then laboratory confirmation requires:

• Routine laboratory tests (results are inconsistent and species dependent) - elevated ALP isoenzyme (dog) and other liver enzymes (e.g. GGT in the horse), elevated blood glucose (glucosuria in the horse, cat), white cell changes in the blood (a ‘stress response’), hypercholesterolemia (in small animals).

• Hormonal evaluation - this aims at confirming hyperadrenocorticism and differentiating PDH and adrenal tumours. Confirmation and then differentiation should always include correlation with clinical signs and other tests, as all hormonal tests have limitations.

To confirm hyperadrenocorticism:

1) Basal plasma cortisol level - in the dog there is some overlap of levels between normal and hyperadrenocortical dogs. Consequently, other tests may need to be used to confirm (ACTH response test and low dose dexamethasone test). It is also important to remember that basal plasma cortisol immunoassays may cross react with prednisolone and other exogenously-provided steroids (except dexamethasone) to give a false elevation. Horses with PDH have inconsistent increases in baseline cortisol.

2) ACTH response (stimulation) test - plasma cortisol levels are determined pre- and post-ACTH (synthetic ACTH is utilised). In dogs and cats, the post-ACTH sample is taken at one hour in the dog and cat, and 2 and 4 hours in the horse. In normal dogs there is a 2-3x increase in cortisol after ACTH whilst many PDH dogs will show an exaggerated response (4x baseline levels) Nb. some endocrinologists prefer to deal with absolute values for cortisol rather than percentage increases, for example accept a baseline level of cortisol as 28-110 mmol/L and a post ACTH cortisol level for PDH cases as greater than 55 mmol/L for dogs. In the small number of PDH and for the large number of adrenal tumours (less than 50%) that have a normal response to exogenous ACTH and which have normal baseline cortisol levels, the low dose

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dexamethasone test can be used in the dog. Iatrogenic hyperadrenocorticism will have little or no response to exogenous ACTH administration, so it is the test of choice to confirm the condition.

3) Low dose dexamethasone suppression test - plasma cortisol levels are determined pre- and post-dexamethasone (usually 8 hours in the dog and cat). In normal dogs there is an extended drop in cortisol. In most cases of PDH and nearly all cases of adrenal tumours there will be no or little suppression of cortisol. One disadvantage of the test in dogs for confirmation is that many non-adrenal diseases may have no or little suppression of cortisol.

Nb. The urine cortisol/creatinine ratio has been advocated as a screening test for

hyperadrenocorticism (i.e. confirmation or rejection). Increased ratios will not only occur in hyperadrenocorticism but also in stressed dogs due to non-adrenal disease.

To differentiate PDH from adrenal tumours:

4) High dose dexamethasone suppression test - plasma cortisol is measured pre- and post-high dose dexamethasone (commonly 8 hours after). The difficulty with this test is to find the level of dexamethasone that suppresses PDH cases but has little effect on adrenal tumours. A small percentage of PDH dogs will not show adequate suppression (less than 50% of baseline cortisol) and may be misdiagnosed as adrenal tumours (but there is always image analysis.

In the horse there is some contention as to whether it effectively identifies PDH cases. It is not commonly used though, because adrenal tumours are rare and there is little need to differentiate them from PDH.

5) Circulating endogenous ACTH - provides accurate differentiation of PDH and adrenal tumours but the test is more difficult to perform. In PDH in dogs, plasma ACTH is usually normal to increased; in adrenal tumours in dogs, plasma ACTH is usually decreased.

Hypoadrenocorticism (Adrenal Insufficiency - AI)

This occurs mainly in dogs due to deficient adrenal production of glucocorticoids and/or mineralocorticoids. Primary AI involves adrenal cortex destruction. Secondary AI is due to pituitary dysfunction. In the horse, there is a lack of substantive evidence for true AI, but it has been suggested as a cause of poor performance in racehorses and endurance horses. Laboratory findings in AI are extremely variable and depend on the degree of glucocorticoid and/or mineralocorticoid disturbance.

i) Routine laboratory findings - Aldosterone lack leading to increased potassium and decreased sodium (a ratio of less than 23:1 for sodium to potassium is suggestive of AI in the dog, less than 18:1 is close to conclusive), white cell changes due to lack of glucocorticoids (e.g. lymphocytosis in some cases) and hypoglycemia, anaemia, hypercalcemia, pre-renal azotemia and acidosis.

ii) Hormonal assays - these will detect reduced glucocorticoids e.g. resting plasma cortisol, ACTH response test. Dogs with naturally occurring AI (either pituitary or adrenal based) will have little or no response to ACTH administration.

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Measurement of plasma ACTH may be useful to differentiate primary nad secondary AI e.g. dogs with pituitary dependent (secondary) hypoadrenocorticism generally have decreased ACTH in the blood while those with the primary adrenal dependent form generally have increased levels.

Hypothyroidism

This is a commonly diagnosed endocrine disorder in the dog and is usually associated with immune-mediated lymphocytic thyroiditis (tests for detecting autoantibodies against thyroglobulin, T3 and T4 are available) or idiopathic thyroid atrophy. Secondary hypothyroidism (due to a pituitary lesion) in the dog is uncommon. In the cat it usually occurs secondarily to treatment for hyperthyroidism but a familial form has been noted in Abyssinians. Hypothyroidism occurs rarely in the adult horse (and this has been poorly documented), but neonatal hypothyroidism has been documented in foals born to mares that have ingested excessive iodine during pregnancy. Laboratory diagnosis (mainly applies to the dog) requires: • Routine laboratory tests – non-regenerative anaemia, hypercholesterolemia, and elevated CK (all

these changes are inconsistent e.g. about half of the cases have anaemia, about 80% hypercholesterolemia and about 10% increased CK).

• Hormonal assessment - most assays for thyroid function involve the measurement of T4 (T3 can be

measured but has no distinct advantage over T4). The measurement of serum free T4 has been suggested as being superior to the measurement of serum T4; however, there are still limitations.

1) Baseline T4 (and/or T3) -a major problem in the dog is that 20% of euthyroid dogs have low levels of T4 (due to physiological variation, hypoproteinemia, drug therapy - e.g. corticosteroids, hyperadrenocorticism, diabetes, chronic renal failure).

2) TSH (thyrotropin) stimulation test - levels of T4 are determined pre- and post-TSH. Failure of response suggests hypothyroidism (most normal dogs show 3-4x increases in baseline levels, but there are still some euthyroid dogs with other illnesses that fail to respond to TSH). This test not only can be used to confirm most cases of hypothyroidism but also it may help differentiate primary and secondary hypothyroidism (secondary hypothyroidism -which is uncommon and usually due to pituitary lesions causing reduced TSH secretion - will respond to repeated doses of TSH but rarely to one).

Hyperthyroidism

This occurs most commonly in the cat and is usually due to functional adenomas. In the dog , only a small group of thyroid tumours are functional, and not all of these will give rise to clinical hyperthyroidism. It can be confirmed in the laboratory by:

• Routine laboratory tests (variable) – erythrocytosis (about 50% of cats), increased ALT and ALP (about 60% of cats), hyperphosphatemia.

• Hormonal evaluation - elevated resting T4 (in most cases but because of day to day fluctuation more than one sample may be needed), reduced increase in T4 in response to TSH (stimulation test).

*SEE CASES 31-34

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Introduction to Calcium and Phosphate Derangements The controls over serum calcium levels are complex. Changes in blood levels cause rapid alteration in the rate of calcium mobilisation from bone (via parathormone and thyrocalcitonin). Consequently, transient hypo- or hypercalcemias only become persistent if the control mechanisms are exceeded in capacity. In addition to bone mobilisation, blood calcium levels are controlled by gut absorption and renal excretion/retention. Vitamin D, phosphorous levels, thyrocalcitonin (from thyroid follicular cells – C cells) and parathormone (from parathyroid gland) interact at all the key tissues to maintain blood levels. In contrast, blood levels of inorganic phosphorus are inexactly and primarily controlled by renal excretion/retention. Increased serum phosphorus will cause a rapid increase in renal excretion (therefore, persistent hyperphosphatemia will not occur until these control mechanisms are exceeded). Decreased serum phosphorus levels, however, are more difficult to correct as the kidney takes time to convert to phosphorus retention. Fortunately, persistent hypophosphatemia is an uncommon occurrence in disease (exceptions include hyperinsulinism, milk fever, in some forms of neoplasia and renal failure in the adult horse). Calcium can be measured by assessing total serum levels (unbound plus that bound to protein [mainly albumin]) or ionised levels. Ideally, ionised levels should be measured as these provide a direct indication of the calcium available for neuromuscular activity and for other body functions. If total serum calcium is measured, altered serum protein levels will have an impact. Many laboratories correct total serum calcium for the level of serum albumin (e.g. corrected calcium = measured calcium + [average albumin - measured albumin, the resultant value is then divided by 40]; where average albumin is 33 g/L for dog, 29 for cat and 30 for horse).

Persistent Hypocalcemia (decreased calcium on two or more occasions) This can occur in: a) hypoalbuminemia (decreases the protein bound component - use correction formula provided) b) renal secondary hyperparathyroidism (decreased calcium is related to phosphorus retention but

at times the calcium may be normal or increased – mainly for the dog and cat) c) eclampsia in the bitch, mare or ewe d) parturient paresis (milk fever) in cattle e) hypomagnesemic tetany in cattle (75% of cases have hypocalcemia) f) acute pancreatic necrosis in the dog (50% of cases) g) nutritional hyperparathyroidism ( now less common; related to diets low in calcium and/or high

in phosphorous) h) miscellaneous e.g. ethylene glycol toxicity, small intestinal enteropathies.

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Persistent Hypercalcemia (elevated calcium on two or more occasions) This can occur due to: a) pseudohyperparathyroidism (related to certain types of neoplasia and operates through multiple

mechanisms, which vary with the type of neoplasm). Pseudohyperparathyroidism is seen more commonly in the dog and less so in the cat and horse. In lymphosarcoma (most common cause) and plasma cell myeloma (multiple myeloma) in the dog, cat and horse, hypercalcemia may result from osteolysis through release of a tumour factor (e.g. osteoclast-activating factor in multiple myeloma in the dog) and stimulation of release of parathyroid hormone because of paraprotein binding to ionised calcium. In the dog, anal sac adenocarcinoma is the second commonest neoplasm to cause hypercalcemia, in this case through release of PTH-related peptide. Hypercalcemia has also been reported in other carcinomas, and in some thymomas.

b) primary hyperparathyroidism (rare) Hypophosphatemia commonly accompanies (a) and (b) as long as the hypercalcemia has not caused significant renal damage (then the phosphate begins to rise) c) hypoadrenocorticism (in dog - small percentage of cases) d) renal failure - hypercalcemia is more common in the adult horse than in small animals (see

section on renal disease) e) osteolytic metastatic tumours of bones f) miscellaneous e.g. ingestion of certain plants. In (c) to (f) hyperphosphatemia commonly accompanies the hypercalcemia; one exception is in renal disease in the horse where hypophosphatemia is the rule. *SEE CASE 35

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Derangements of magnesium

Blood magnesium levels are primarily dependent on dietary intake, tissue requirements and renal excretion. They may play a role in regulating PTH release. Blood magnesium abnormalities are of greatest concern for ruminants, and are usually related to feed intake. In dogs, magnesium depletion may be related to insulin treatment for diabetic ketoacidosis.

Hypermagnesemia

Persistent hypermagnesemia is uncommon because the kidneys can readily excrete excess magnesium in the blood. Hypermagnesemia can occur due to:

a) Renal failure in ruminants and horses b) Iatrogenic (related to treatment with magnesium-rich substances [e.g. magnesium rich

laxatives or antacids] to patients already in renal failure.

Hypomagnesemia

Persistent hypomagnesemia is of greatest concern in calves, adult cattle and sheep. Hypomagnesemia may complicate the situation of hypocalcemia in cattle because of its role in regulating PTH release. Hypomagnesemia can occur due to:

a) Rapid change in diet in cattle, which may be related to alterations in rumenal magnesium transport

b) Calves fed milk only diets low in magnesium (hypomagnesemic tetany – ‘milk tetany’) c) Feed deprivation in lactating ewes with twins (hypomagnesemic tetany) d) Adult cattle (mostly old cows) grazing cereal crops or lush grass-dominant pasture

(hypomagnesemic tetany – ‘grass tetany’). Grass tetany has a multifactorial aetiology and is incompletely understood.

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WATER, ELECTROLYTES AND ACID/BASE BALANCE

Introduction The laboratory can assess hydration status, electrolyte levels and acid/base disturbances for ill patients. This is more useful for management of the illness rather than diagnosis. However, some electrolyte and acid/base analysis do have implications for the diagnosis of some diseases (these will be discussed in the following sections).

Hydration Status (Assessment of Total Body Water)

Dehydration commonly causes hypovolemia which will artificially raise most laboratory biochemical and hamatological values. However, it is PCV, TPP and urine specific gravity that respond significantly to hypovolemia; and, consequently, they are useful laboratory tests to assess hydration. Additionally, urea and creatinine are commonly increased by dehydration due to reduce renal perfusion (i.e. decreased glomerular filtration) and is the major reason for pre-renal azotemia. The measurement of osmolality of blood may provide an indication of changes to hydration status, but only when there is pure water loss. Sodium and chloride are the main contributors to osmolality and if their levels are disturbed in disease the osmolality may not be a good indicator of hydration status. Therefore, electrolyte analysis should always accompany the measurement of osmolality. Osmolality can be measured directly with an osmometer or indirectly by using a formula that utiliss levels of electrolytes and other solutes (e.g. mOsmol/Kg = 2[Na+ + K+]; mOsmol/Kg = 1.8[Na+ + K+] + glucose + urea [if urea or glucose is elevated]). Osmolality of blood is roughly 270-330, with some variation between species (e.g. the cat is normally 308-336). Hypo-osmolality is nearly always due to hyponatremia while hyperosmolality is commonly associated with hypernatremia. Hypo-osmolality, if developing quickly, can cause brain oedema and intravascular haemolysis due to rapid movement of water from the extracellular fluid (ECF) to the intracellular fluid (ICF). A similar situation may occur if hyperosmolality is corrected rapidly. Rarely do other molecules significantly change osmolality (an exception: glucose increases in DM may significantly alter osmolality). Urea is not an effective osmole in the body (i.e. does not add to tonicity) as it freely moves between ECF and ICF (therefore little effect on water movement) but does influence laboratory measurement of osmolality (hence its use in some of the formulae used to derive osmolality).

Electrolyte Status

Commonly Na+, K+ and Cl- are measured. Serum/plasma Na+ is a good reflection of total body Na+. Hyponatremia, normonatremia and hypernatremia may occur with dehydration, normal hydration and overhydration (any combination is possible). The common situations are: a) hyponatremia and decreased extracellular (ECF) water (hypotonic dehydration-hypo-osmol

[except for DM where glucose increases may override the sodium loss] as electrolytes are lost in excess of water) e.g. DM (osmotic diuresis), diarrhoeas in horses, well developed hypoadrenocorticism [hypoaldosteronism], renal disease in many cattle.

b) Hyponatremia and normal ECF water (normal hydration) e.g. salt deficiency in cattle; excess saliva loss in horses; sustained exercise in the dog or horse; early hypoaldosteronism

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c) normonatremia and decreased ECF water (isotonic dehydration - normally isosmol as electrolytes are lost at the same rate as water) e.g. vomiting, most diarrhoeas in small animals, exudation, hemorrhage, gut fluid sequestration.

d) normonatremia with increased ECF water (edema - water and electrolytes are retained) e.g. congestive heart failure, liver disease in the dog.

e) normonatremia and normal hydration - health A word of warning, the examples listed above are inconsistent in their effects on sodium levels. Other combinations are less common and occur in particular conditions e.g. hypernatremia and normal hydration in salt poisoning in pigs. Changes in serum/plasma chloride commonly parallel changes in Na+ but with loss of gastric HCL hypochloridemia and normonatremia (or hypernatremia) may occur. Likewise, hyperchloridemia may occur with normonatremia due to bicarbonate loss in metabolic acidosis. Serum/plasma potassium levels are not always a good indicator of total body K+ as much of the K+ is intracellular. However, the circulating levels are important as they indicate the levels available for normal functions (e.g. neuromuscular activity). Serum K+ levels can be altered by internal shifts (extracellular to intracellular and vice versa) and/or by external factors (intake, loss). Commonly, if both internal and external factors are influencing blood levels of K+, the external factors are more important in determining the overall changes. Hyperkalemia due to internal shifts include: a) metabolic acidosis b) hyperosmolality c) tissue degeneration/necrosis d) insulin deficiency e) hamolysis (variable depending on species and breeds within species e.g. erythrocytes of horses

and pigs are usually high in K+; in cattle and sheep moderate to low; in most breeds of dogs and cats low), the same effect can occur through high leukocyte or thrombocyte counts (through leakage)

f) diarrhoea (mainly calves) Hyperkalemia due to external factors include: a) anuria (usually in acute renal failure or post-renal obstruction – not as common in dogs or

ruminants for the latter condition) b) hypoadrenocorticism (hypoaldosteronism) c) various drugs e.g. high doses of crystalline penicillin d) polyuric renal disease in the horse e) marked pleural or abdominal effusions in dogs and cats Hypokalemia rarely occurs due to internal shifts but can occur due to external factors such as: a) increased GIT losses (diarrhoea [especially horses] and persistent vomiting; in cattle abomasal

sequestration may act the same as persistent vomiting) b) increased urinary loss (kaliuresis) e.g. polyuric renal disease, especially in cat and cow but not

usually in the horse. c) decreased oral intake (e.g. anorexia due to disease; mainly in herbivores as they commonly have

excess K+ in their diet and their kidneys are used to excrete. With a drop in intake of K+, the kidneys take a few days to adapt to K+ conservation).

d) various drugs e.g. insulin, diuretics, amphotericin B e) profuse sweating in the horse f) prolonged exercise in the dog or horse

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Acid/Base Balance The following is probably an oversimplification of a complex situation, but does provide a starting point to understanding the usefulness of acid/base investigation in the laboratory. Acid/base balance in the body is maintained by blood buffer systems (e.g. bicarbonate buffer), respiratory function (CO2 removal) and renal function (acid secretion, bicarbonate retention). In limited disturbances the blood buffers will keep the pH within the acceptable range (roughly 7.3 - 7.5). In more severe disturbances respiratory adaptation will occur quickly while renal adjustment will occur over a few days. Commonly acid/base disturbances are classified as metabolic, respiratory and mixed. Evaluation of respiratory disturbances requires blood gas analysis. The evaluation of metabolic disturbances commonly requires the determination of blood pH (alterations are called acidemia and alkalemia) and/or bicarbonate (alterations are called metabolic acidosis and metabolic alkalosis). In addition, by determining Na+, K+ and Cl- the anion gap (=[Na+ + K+] – [Cl- + HC03-]) can be calculated and used to further elucidate forms of metabolic acidosis and mixed metabolic disturbances.

The anion gap (AG) is based on the concept of electrical neutrality in the ECF i.e. anions = cations. In health most cations are Na+ and K+ whereas most anions are Cl- and HCO3. Unmeasured cations (e.g. Ca+, Mg+) are always exceeded by unmeasured anions (e.g. protein, organic acids, inorganic acids) i.e. Na+ + K+ > HCO3- + Cl- to maintain electrical neutrality. The difference is called the anion gap and indirectly measures the level of unmeasured anions. In health, the AG in dogs and cats is 15 to 25mmol/L and in other domestic animals about 10 to 20 mmol/L, but these values will vary from laboratory to laboratory.

        FIGURE:  Anion  Gap  –  based  on  concept  of  electrical neutrality  in  the  blood  stream  and  that  unmeasured anions commonly exceed unmeasured cations  In metabolic acidosis ([MAc] - lowered bicarbonate, commonly less than 20 mmol/L for most species, but probably a little lower for dogs and cats) the AG may be normal or increased. In MAc due to secretion (loss of HCO3 e.g. diarrhoea, excess loss of saliva in cattle, some cases of renal tubular acidosis) the AG will invariably be normal due to compensatory increases in Cl-. In MAc due to titration (acid excess and titration of bicarbonate e.g. uraemic acids in renal failure; lactic acidosis in shock, hypoxia, grain overload in ruminants, neonatal diarrhoea in calves, some cases of grain induced laminitis in horses ; diabetic ketoacidosis and ketosis in ruminants) the AG increases due to an increase in unmeasured anions (derived from acids). Bicarbonate decreases, but there is little need for chloride increases as the unmeasured anions have increased. In metabolic alkalosis ([MAk] - raised bicarbonate, commonly above 25 mmol/L for most species, but usually not significant until closer to 30 mmol/L) the AG is normal to slightly increased. Most cases of MAk are due to vomiting (or abomasal reflux) and involve decreases in Cl- and compensatory increases in bicarbonate. In mixed MAc (mainly titration) and MAk a low serum Cl- and high AG commonly accompanies the close to normal HCO3 (e.g. vomiting and renal failure).

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HAEMATOLOGY

Haematological Disturbances and how they reflect Host-Pathogen-Environmental Factor Interactions

Haematology, the study of blood, has been traditionally approached from its usage in the diagnosis and treatment of disease. The reason why it can be approached in such a manner, is that the haematopoietic system has fundamental systemic roles through the export of its cellular components. The haematopoietic cells, primarily produced by bone marrow and a lesser extent by spleen and liver, interact with all body tissues through the circulatory system; hence, the circulatory system is a portal for viewing the haematopoietic system. Haematopoietic cells can be primarily divided into erythroid (erythrocytes) and myeloid (leukocytes) series. Megakaryocytes, which produce platelets, are another hematopoietic cell line. The erythrocytes and platelets can perform their major roles, namely gaseous exchanges for tissue cells and haemostasis, respectively, without leaving the circulatory system; but leukocytes have their primary roles after moving into tissues. Their primary roles are very much related to host defences, with leukocytes contributing to both innate and adaptive immunity through inflammation and repair. These roles can be better understood by taking a host-pathogen-environment interaction (HPEI) view of disease (Figure 1). Disease refers to an effect on the host because of detectable (clinical or subclinical) dysfunction or altered structure. There are four basic pathological processes that arise from or are present in disease: degeneration and necrosis, vascular disturbances, disorder of growth and pigmentations and deposits (Table 1). Pathogens are causes of disease. Although, pathogens have become to mean living agents of disease, particularly micro-organisms, by definition physical, chemical, heritable and immune-based causes can also be referred to as pathogens (Table 2). For pathogens to cause disease, there is a need for them to overcome host defences, which are commonly divided into innate and adaptive immunity. Inflammation and repair form part of that immunity after local and systemic damage due to an agent of disease and is regarded as the fifth major pathological process (Table 1). The external environment influences both the capacity of the pathogen to cause disease and the capacity of the host defences to resist disease. Hence, a changing environment can have both positive and negative impacts on the host. Environmental factors may act as direct ‘pathogens’ (e.g. fire).

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FIGURE 1:  A conceptual framework for the interaction of host, pathogen and environmental factors to produce disease (effect on host)  Table 1. Five basic pathological processes arise from HPEI: EFFECT ON HOST

1. Degeneration and necrosis 2. Disorder of growth

a. Developmental abnormality b. Hyperplasia/hypertrophy, atrophy, metaplasia, dysplasia c. Neoplasia

3. Circulatory (vascular) disturbances 4. Pigmentation/deposit

HOST DEFENCE

5. Innate and adaptive Immunity (includes adaptive behaviour, surface defence mechanisms and response to local and systemic damage [Inflammation and repair])

Nbs

• The key pathological process, sometimes time course, and the tissue affected are the bases for the morphological diagnosis (e.g. chronic dermatitis)

• there may be more than one pathological process occurring in a disease; however, one will predominate or be the reason for the presence of the others (e.g. degeneration and necrosis and inflammation may accompany neoplasia – neoplasia is still most important and is the basis for the morphological diagnosis)

Agent of disease (pathogen)

Effect on host(DISEASE)

Host defence/response

Environment

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Table 2. There are five main groups of causes of disease (i.e. aetiological agents; ‘pathogens’)

1. Living agents of disease a. Multicellular (e.g. metazoan such as nematodes; predators?) b. Micro-organisms (e.g. bacteria, protozoa, fungi, viruses, algae)

2. Physical agents (e.g. heat, motor vehicle accidents) 3. Chemical agents

a. poisons/toxins b. metabolic disturbances c. nutritional deficiencies and excesses

4. Heritable diseases (genetic based) 5. Immune-based disease (i.e. host immunity is the major cause of the effect on the host through

an excessive response e.g. immune mediated allergy, autoimmunity; lack e.g. immunodeficiency and immunosuppression). Immune-based diseases are often ‘triggered’ by one of the other four groups of aetiological agents

Nbs

• These form the basis for the name for the aetiological diagnosis. Once again, if more than one agent of disease is involved then the key aetiological agent may be determined as the initiating agent or perhaps the agent that has the most impact on the development of disease (as long as they can be identified). For example, immune-mediated glomerulonephritis is named because most of the damage to the host is immune-based. However, the trigger may be living agents (e.g. viruses) or chemicals (e.g. certain drugs). An animal with an intussusception (physical blockage of the bowel) actually dies from invasion of living agents and through necrosis of the intestine releasing chemicals. Some diseases may be named after two agents of disease if both have made a significant contribution (e.g. a dog hit by a car, which has led to lacerations of the intestine and the development of septicaemia and death would have an aetiological diagnosis of trauma with secondary bacterial complications).

• Some agents of disease operate in more than one category (e.g. bacteria may produce toxins; nutritional deficiencies may cause immunodeficiency; genetic disease may express itself through metabolic disturbance or autoimmunity). This is part of the pathogenesis of the disease and raises the concept of agents operating in tandem to cause disease and perhaps death

• Environmental factors may operate directly as pathogens e.g. motor vehicle accidents (physical damage)

• Iatrogenic disease (disease induced through inappropriate treatment of disease) is commonly through chemical agents

• Idiopathic disease (unknown cause) will eventually be placed in one or more of the five known categories

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Why then is a fundamental understanding of HPEI useful for the analysis of circulating blood? Alterations in erythrocytes can be a reflection of disease agents affecting a wide ranges of tissues that lead to impairment of circulation, oxygenation and provision of chemicals (e.g. hormones, iron, protein) vital for effective erythrocyte production (effect on host due to H-P interactions); or due to disease agents that directly act on erythrocyte production or viability (H-P interaction). This is very much a host-pathogen interaction leading to abnormalities of erythrocyte production. Platelets (thrombocytes in non-mammalian species) are principally involved in vascular disturbances (primary haemostasis), but they also provide chemicals that can contribute to the innate immune (inflammatory) response (H-P interactions and effect on host). Leukocytes are fundamentally associated with both innate and adaptive immunity to pathogens and the subsequent tissue damage (Host response - the fifth basic pathological process; a H-P interaction for disease –). Neutrophils are a key cell for phagocytosis of microbes (mainly bacteria) and minor cell damage, monocytes are capable of becoming macrophages that have important roles in macromolecular phagocytosis (particulate cell debris, microbes and foreign material) and antigen-presenting to activate adaptive immunity. Lymphocytes are quintessential for adaptive immunity. B lymphocytes and resultant plasma cells that produce antibodies are responsible for the humoral arm of adaptive immunity, whilst T lymphocytes are responsible for cell-mediated adaptive immunity (host response to pathogens). Null Killer (NK) lymphocytes appear to play a role in innate immunity but can also be recruited in adaptive immunity. Bone marrow is regarded as a primary lymphoid organ as B lymphocyte differentiation originates and continues there into adulthood. Plasma cells are also present in adult bone marrow. Immature T lymphocytes are also produced in bone marrow during early development, but are then transported to the thymus for maturation and export to secondary lymphoid organs such as lymph nodes. Consequently, bone marrow has the potential, even in adulthood to produce both B and T lymphocytes. Eosinophils have a specific role to play in adaptive immunity on mucosal surfaces and to certain groups of pathogens. Usually, prolonged antigenic stimulation (e.g. such as through parasitic, fungal or protozoal) is required before blood levels of eosinophils are consistently elevated. Mast cells can produce chemicals that attract eosinophils in tissues. Basophilia commonly accompanies eosinophilia, probably because both relate to prolonged antigenic stimulation leading to the formation of Ab-Ag complexes. Haematological analysis of circulating blood, and sometimes bone marrow, should then be undertaken in the context of alterations providing vital information on the cause of disease, effects on the host, the host response and environmental influences (Figure 1). It is only through this awareness, and an understanding of how these factors interact, can haematological analysis be utilised to effectively assist diagnosis and treatment of disease. Although the following information is organised along traditional lines, there will be a continual reminder of how haematological disturbances shed light on HPEI, and how this understanding can be utilised in the diagnostic (interpretive) process by the veterinary clinician for the purposes of effective treatment.

Introduction to Haematological Investigation

Blood is studied either (1) to detect disease processes that are primarily affecting the hematopoietic system or (2) to detect disease processes that the hematopoietic system is indirectly affected by or reacting to (e.g. neutrophil response related to inflammation). For (2), it is only by combining the results of haematological analysis with other findings that they become useful e.g. the finding of a non-regenerative anaemia in a dehydrated dog with azotemia and isosthenuria supports a diagnosis of chronic renal failure.

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When analysing the hematopoietic system it is important to remember that there are four components: bone marrow (hematopoiesis); spleen and liver (phagocytic activity and hematopoiesis); peripheral circulation (main site of erythrocyte and platelet function); and tissues (main site of leukocyte action). Results of analysis of the peripheral blood component will be a reflection of what is occurring in all four components. In other words, peripheral blood analysis will provide information on the dynamic equilibrium between production, phagocytic activity and tissue utilization. However, at times the other components will need to be examined to provide a comprehensive evaluation of the problem (e.g. bone marrow analysis).

Some Common Haematological Terms

Acanthocytes: Irregularly spiculated red cells with projections of varying length and position. Seen in derangements of lipid metabolism (e.g. certain liver diseases) and splenic disorders. They are reported to be common in haemangiosarcoma in the dog. Anaemia: 1) A reduction below the reference interval for the total circulating red cell mass. 2) A decrease in the haemoglobin value, the packed cell volume, or the erythrocyte count below

normal limits. Anisocytosis: Variation in the size of cells, usually of the erythrocytes. Aplastic: Cessation of blood cell formation. Azurophil: Applied to granules seen typically in the cytoplasm of cells of the lymphocytic and monocytic series and the progranulocyte (promyelocyte) of the granulocytic series. The term refers to an affinity for a particular dye and not to the colour of the granules (but the granules are commonly deep red). Basophilia (basophilic): Term that, when applied to a cell or cells of the erythrocytic series, indicates that the cell shows no trace of the characteristic haemoglobin colour and that the cytoplasm shows a strong affinity for basophilic dyes. Basophilic stippling (punctate basophilia): Erythrocyte that shows blue, basophilic granules and lines scattered throughout (on Romanowsky staining). The material is usually aggregates of ribosomes. Basophilic stippling can occur in lead poisoning and in intense regenerative anaemias (in the latter, the stippling often appears more distinct). Blood dyscrasia: An abnormal condition of the blood or the blood-forming organs. Buffy coat: The layer of leukocytes, platelets, and nucleated erythroid cells, if any, that collect immediately above the erythrocytes in sedimented or centrifuged whole blood.

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Burr cells: See Crenation. Codocytes: See Target cells Crenation: Shrinkage of cells in a hypertonic solution with the formation of irregular margins and a number of regular spikes. Often seen when cells have been in prolonged contact with EDTA. Also seen in smears affected by dirty glassware, slow drying, extreme temperatures, and poor smearing technique. Crenation should be distinguished from echinocytes (burr cells) that occur in kidney failure (they usually have blunt projections). Dacryocytes ("tear drop" cells): Have an elongated point and commonly occur in myelophthisic anaemias and in myelofibrosis. Disintegrated, smudge or basket cell: Any cell of any series in which the cytoplasmic outline has been disrupted or the nuclear chromatin is no longer surrounded by a membrane. Usually caused by the smear technique but increased numbers may indicate aged or rapidly proliferating cells. Döhle bodies: Small (1-2 microns) round or oval, gray-blue bodies in the cytoplasm of neutrophilic leukocytes, thought to be due to incomplete utilisation of RNA during maturation of the cytoplasm. They may be due to toxicity or due to an administered drug affecting metabolism. Nb. domestic cats may contain small Döhle bodies in some of their neutrophils in health Echinocytes: See Crenation. Elliptocyte: Erythrocytes that are elliptical (rod shape) in wet or dry films. Also called ovalocytes. These may be seen in a myriad of unexplained anaemias. A hereditary form can occur in dogs. Elliptocytes are normal in Camelidae. Granulocyte: A leukocyte that contains specific cytoplasmic granules (neutrophils, eosinophils, basophils) regardless of the stage of differentiation. Heinz bodies: An intraerythrocytic mass of denatured globin, irregular in shape and appearing as refractile granules when out of focus (in unstained smears). The latter property is responsible for their being called erythrocyte refractile (ER) bodies. In stained smears, intraerythrocytic Heinz bodies present as clear areas near or bulging from the surface. In ruptured erythrocytes Heinz bodies often remain attached to the membrane and appear bluish and round. They are due to certain oxidant drugs and chemicals that cause haemolytic anaemias. Nb: E.R. bodies may be seen in 10% of feline erythrocytes in health (need to stain with 0.5% brilliant cresyl blue in normal saline or another supravital stain to visualise them effectively) and may increase in size and number in various diseases affecting cats (i.e. relatively non-specific). They are most significant when associated with haemolysis.

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Howell-Jolly bodies: Small, round, densely staining bodies in the erythrocytes that are considered to be nuclear remnants and are usually eccentric in location. More frequently observed in regenerating anaemias and in splenic disorders (the spleen is responsible for removing them). Common in normal horse and cat blood. Hypochromasia or hypochromatic: Terms which, when applied to the microscopic appearance of a cell or cells of the erythrocyte series, indicate a significant decrease in density of the characteristic haemoglobin colour. This may be due to either thinness of the cell, decreased concentration of haemoglobin, or both. Normochromasia or normochromatic means normal haemoglobin colour. Hypochromic: An adjective describing a blood picture in which the erythrocytes have a saturation index (MCHC) and usually a colour index (MCH) below the reference interval (MCHC is the more important index). Normochromic refers to MCHC and MCH within the reference interval. Leptocyte: A thin erythrocyte of decreased volume in relationship to its diameter, often characterised by abnormality of shape. Observed in chronic debilitating diseases and anaemias. They are common in diseases that alter lipid metabolism (e.g. endocrinopathies, liver disease). In dogs, they commonly occur in lead poisoning. Target cells and folded cells are types of leptocytes. Target cells are also called codocytes. Leukemia: Neoplastic disease commonly arising in hematopoietic tissue (but not always) in which the type cells commonly appear in the peripheral blood. Leukemoid: Resembling leukemia by having a marked leukocytosis and/or having many immature leukocytes in the peripheral blood. Lupus erythematosus cell: A neutrophil that is distended by an intracytoplasmic, homogeneous, red-purple body and has the lobes of its nucleus compressed at the periphery of the mass. It is an uncommon occurrence but may occur in Systemic Lupus Erythematosus and sometimes other immune-mediated diseases. Macrocyte: An erythrocyte having a diameter exceeding that of the reference interval. Microcyte has a diameter below the reference interval. Macrocytic: An adjective describing a blood picture in which the erythrocytes have a volume index (MCV) above the reference interval. Microcytic refers to a MCV below the reference interval; while normocytic refers to one within the reference interval. Macroplatelet: Abnormally large platelets that may occur in abnormal production or increased turnover (i.e. a marrow response). Also called megaplatelets and giant platelets. Nb. Thrombocyte equals platelet. All non-mammalian species have thrombocytes.

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Myelofibrosis: Displacement of bone marrow by fibrous tissue. Myelophthisis: Displacement or crowding out of bone marrow by abnormal cells or tissues. Myelofibrosis is a form of myelophthisis. Platelet (thrombocyte): A fragment of a megakaryocyte (mammal) involved in haemostasis. It is non-nucleated in the mammal. Poikilocytosis: Refers to an abnormality in shape of circulating erythrocytes. Commonly observed in chronic blood loss, iron deficiency and diseases that increase erythrocyte fragility. In many cats, marked poikilocytosis may occur in response to non-specific severe illness (especially liver diseases). Poikilocyte: An erythrocyte with an abnormal shape, not to be confused with distortion that results from faulty technique. Poikilocytes are seen in many anaemias. This term encompasses all misshapen erythrocytic forms, e.g. elliptocytes, dacryocytes. Polychromasia, polychromatic, polychromatophilic: Erythrocytes that show a faint bluish tint due to an admixture of the characteristic colours of haemoglobin and basophilic erythrocytic cytoplasm. Polycythemia: A state of the blood characterized by increased numbers of erythrocytes (therefore, commonly increased PCV and Hb levels). - penia: Combining form denoting deficiency of (i.e. reduced numbers). - philia: Combining form denoting affinity for (i.e. increased numbers). Reticulocyte: Any non-nucleated cell of the erythrocytic series containing RNA, which when supravitally stained with new methylene blue or brilliant cresyl blue will have discernable granules or a diffuse network of fibrils. Rouleaux Formation: The arrangement of erythrocytes in a column, with their flat surfaces facing, in which they appear as figures resembling stacks of coins. It is most common in the horse and cat. Schistocytes (Schizocytes): Are red cell fragments often seen in vascular disorders (e.g. D.I.C., haemangiosarcoma, vasculitis) due to collision with fibrin strands. Keratocytes (horn cells) are distorted cells that have 2 or more large points and probably form from ‘blister erythrocytes’; they too can occur in vascular disorders. Sedimentation rate: The rate at which the erythrocytes will fall in their own plasma in a given length of time.

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Siderocyte: An erythrocyte in which blue granules, 1-20 or more in number, can be demonstrated to contain iron (haemosiderin) by the Prussian Blue reaction. Spherocyte: A spheroid erythrocyte of decreased diameter in relationship to its volume and having the microscopic appearance of a hyperchromatic microcyte, commonly seen in autoimmune haemolytic anaemias in abundance. They are supposed to form through phagocytic removal of part of their membrane. Occasional spherocytes can be seen in a variety of diseases associated with erythrocyte damage or enhanced activity of phagocytes. Spherocytes are more obvious in canine rather than feline smears because of the size of erythrocytes. Stomatocyte: Erythrocyte with a shape deformity in which there is a linear rather than a central area of pallor. May be seen in certain liver diseases and hereditary stomatocytosis of Alaskan Malamutes. Supravital staining: Staining of cells with a stain of low toxicity that will not cause death to the living cells, so that vital and functional process may be studied in the live cells (e.g. supravital stains show RNA in reticulocytes). Target cells (codocyte): An erythrocyte with a central rounded area of pigmentation surrounded by a clear zone, with a dense ring of cytoplasm about the periphery of the erythrocyte. They are also called ‘bullseye’ cells. Nb. They are a form of leptocyte. Folded cells are other forms. Thrombocyte: See platelet. Toxic neutrophil: A neutrophil characterised by toxic granules, basophilia of the cytoplasm, or vacuoles. Doehle bodies can be loosely called a form of toxic change. Toxic neutrophils commonly suggest metabolic disturbances or infective processes.

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DISORDERS / RESPONSES OF THE ERYTHRON

 Erythron:  In  haematology,  a  concept  of  the  peripheral  blood  erythrocytes  and  the erythropoietic bone marrow as one functional unit. 

a) Dynamics of erythrocyte production Erythrocyte production occurs primarily in the bone marrow (BM) but other organs/tissues (especially the spleen and liver) are capable of production in times of stress in certain species (this is called extramedullary erythropoiesis). The release of erythrocytes from the BM is orderly: mainly normal erythrocytes are released (most abnormal cells are phagocytosed) while immature erythroid cells enter the peripheral circulation in limited numbers (mainly reticulocytes, but some nucleated erythroid cells). Those abnormal cells that do enter the circulation are quickly removed by phagocytes in the spleen and liver (mainly spleen). These phagocytes are also important in removing aging erythrocytes and for ‘pitting’ the nuclei from released nucleated erythroid cells. Extramedullary hematopoiesis or increased phagocytosis of erythrocytes, if excessive, can cause enlargement of liver and spleen (hepatomegaly and splenomegaly), but splenomegaly is usually more obvious.

In times of increased demand for erythrocytes the BM becomes 'leaky' in certain species and releases more immature erythroid cells into the circulation (reticulocytes, nucleated red cells). The release is usually orderly with a greater proportion of later immature cells. If extramedullary erythopoiesis is significantly contributing to erythroid numbers then the proportions of immature cell types may not be as orderly. Distinct disorderly release of immature erythroid cells occurs in many cases of erythroid neoplasia and in dysplasias of the BM (e.g. lead poisoning in the dog). In splenic disorders, it may appear that there has been disorderly release of immature erythroid cells as nucleated red cells may not be ‘pitted’ (i.e. the nucleus removed by phagocytes) at the usual rate. In addition, there may be increased numbers of Howell-Jolly bodies (nuclear remnants).

Erythropoiesis in the BM is under hormonal control mainly through erythropoietin. Other hormones can have an effect on erythropoiesis, usually by altering erythropoietin levels (e.g. thyroxine can enhance levels by altering cell requirements for oxygen; oestrogens can inhibit levels). Androgens may have a direct enhancing effect on erythropoiesis.

The naming of erythroid cells is variable between countries. In all terminologies the mature red cell is called an erythrocyte while the immature non-nucleated red cell is called a reticulocyte. Nucleated erythroid cells vary in their terminology (e.g. normoblast, erythroblast etc). In bone marrow, erythroid cells can be divided into those that are capable of division and those that are simply capable of maturation. When the haemoglobin content reaches a critical level in the cytoplasm of a nucleated red cell division no longer occurs and the cell simply matures. One stem cell tends to produce about 16 mature cells in about 3-5 days (this is a generalisation and may not apply to all species). In times of demand this time period can be reduced in the BM and cells will be released earlier into the peripheral blood. Increased production requires increased stem cell input, which usually takes a few days.

Erythrocytes have different life spans in the circulation depending on the species (e.g. dogs 110-120 days, cats 70, horses 145, ox and sheep 150-160). This must be taken into consideration when assessing how long it takes for a significant anaemia to develop due to interference with bone marrow production. The erythrocytes age and are removed primarily by splenic macrophages, although some do rupture in the circulation. Antibody coating of aging erythrocytes may be important for macrophage destruction even in health.

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b) Anaemia When assessing an anaemia, variations in breed (for the species under investigation), sex and age must be taken into consideration, as all these will alter the accepted reference range for a species. For example, the packed cell volume (as determined by the microhematocrit method) for a normal kitten can be as low as 0.25 but in an adult cat it would rarely be below 0.30. In fact the young of most domestic species (especially pre-weaning) will have lower reference values for red cell measurements. In foals even the mean corpuscular volume will be lower than in an adult and has led to misdiagnoses of microcytic anaemia. In some species males have higher reference values than females, while training can enhance values in horses and greyhounds.

Anaemia can be simply defined as a reduction in the circulating red cell mass as measured by reduced packed cell volume, haemoglobin concentration and red cell number below the reference interval. This will lead to decreased oxygenation of cells in tissues and a reduced capacity to remove carbon dioxide and transport it to the lungs for expiration. Depending on the level of anaemia, there may be clinical or sub-clinical disease related to cardio-respiratory compensation, and compromised tissue perfusion (effect on host). Anaemia may be the primary presenting complaint (agent of disease directly targets the erythron) or secondary to another disease process (i.e. an effect on the host leads to the development of anaemia e.g. chronic renal disease leading to anaemia through several mechanisms). To understand the etiopathogenesis of anaemia, knowledge of the controls and dynamic equilibrium of erythrocyte production and destruction is required. Some of this information has been presented before under ‘(a) Dynamics of Erythrocyte Production’. The following is a suggested approach to investigation of the problem of anaemia (suspected on clinical signs). The goal is to characterise anaemia so that a cause or causes (agent of disease acting directly [primary] or indirectly through another effect on host [secondary]) can be detected to allow effective treatment and control:

i) Laboratory confirmation of anaemia Packed cell volume (microhematocrit reading) in conjunction with the total plasma protein (to determine hydration status) measurement can confirm a diagnosis of anaemia. Haemoglobin estimation and the total erythrocyte count may support the laboratory confirmation of anaemia, but they are more useful in classifying the anaemia by determination of the red cell indices.

ii) Classifying the anaemia on the basis of mechanisms: 1) determine whether the anaemia is regenerativeor non-regenerative 2) regenerative anaemias are due to either blood loss or haemolysis 3) non-regenerative anaemias are due to either reduced or defective erythropoiesis 4) further classification according to etiological agent (often difficult)

Nb: This is an over simplified classification as some agents of disease, either directly or indirectly through other effects on host, cause anaemia through a variety of mechanisms. For example: chronic renal failure in the dog produces anaemia via some haemolysis, some blood loss (e.g. in GIT) and some bone marrow depression; Equine Infectious Anaemia (EIA) produces anaemia primarily by haemolysis but other factors operate that can give rise to bone marrow depression (secondary). In both these cases the net effect can still be classified as either regenerative or non-regenerative on the basis of simple criteria, but variability occurs e.g. in chronic renal failure bone marrow depression, due to lack of erythropoietin, predominates leading to non-regenerative anaemia; in EIA some cases are regenerative but many are non-regenerative.

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1) Establishment of a diagnosis of regenerative anaemia A) In the dog and cat the hallmark of regeneration is an increased reticulocyte count (red cells that supravitally stain for RNA). This can be called an adaptive response by the host (i.e. part of the host response to anaemia; repair through regeneration). If the reticulocyte response is significant this will be accompanied by polychromasia, macrocytosis and, possibly, nucleated red cells in the peripheral blood film. After simple blood loss or haemolysis there is a lapse of 2-3 days before the reticulocyte response occurs. In the horse, even though the process of regeneration is similar to that in the dog and cat, reticulocytes do not occur in peripheral blood in health or in anaemias (except occasionally in intense regeneration in adult horses and even then there are low numbers; young foals may show some circulating reticulocytes in regeneration). Consequently, a diagnosis of regenerative anaemia has to rely on serial packed cell volumes (PCV) or bone marrow examination (where the regeneration is primarily occurring). In adult ruminants, reticulocytes do not occur in health but will be present in peripheral blood if intense haemorrhage or haemolysis has occurred. Consequently, any circulating reticulocytes suggest regeneration. However, the level of reticulocytosis does not always give an indication of the intensity of the regeneration, and in mild blood loss or haemolysis, reticulocytes may be absent. In ruminants with regenerative anaemia basophilic stippling is common. This can occur in dogs and cats but is uncommon. Another form of basophilic stippling occurs in lead poisoning, commonly in the dog - this is rarely related to an anaemia. Pigs respond with reticulocytosis in regenerative anaemia. In the adult dog 0-1.5% aggregate reticulocytes occur in health. This can be increased to over 10% in intense regeneration. This figure is an expression of the percentage of red cells in the circulation which are reticulocytes (500-1000 red cells are counted on a peripheral blood smear stained with a supravital stain such as brilliant cresyl blue) i.e. it is a relative figure. It should be corrected for the level of anaemia (and, if possible, maturation times [these are probably not really necessary and besides, only possible for the dog]). By obtaining a corrected reticulocyte count, an anaemia can be called a truly regenerative one. Another way of correcting is by multiplying the reticulocyte percentage by the erythrocyte count to get an absolute reticulocyte count. This is then compared to the reference intervals. Correction for the level of anaemia: Corrected reticulocyte count (%) = observed reticulocyte count x (Patient PCV/average PCV [0.45 for dog; 0.37 for cat]) A corrected reticulocyte count of greater than 1.5% for the dog (and 1% for the cat) is indicative of regeneration. Less than these values suggests an inadequate response by the bone marrow (i.e. the bone marrow is not responding adequately to restore the packed cell volume to its original level). Correction for the level of anaemia and maturation time (reticulocyte production index [RPI] in the dog: RPI = observed reticulocyte count x (Patient PCV/Average PCV) x (1/maturation) Maturation times for the dog - 1.5 days at a PCV of 0.35, 2 days at a PCV of 0.25, and 2.5 days at a PCV of 0.15. These are extrapolated from figures for humans. No values are apparently available for cats.

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A RPI greater than 2 is definitely regenerative, a RPI of 1-2 is an inadequate (sub-optimal) response by bone marrow (i.e. the packed cell volume will not be restored to its original level), while a RPI less than 1 is definitely non-regenerative. Some of the anaemias classified as regenerative on correction for the level of anaemia alone, may turn out to be sub-optimal by this correction method. Note: RPI has only been used in the dog, and then sparingly. It is usually acceptable just to correct the reticulocyte count for the level of anaemia. In the adult cat 0-1% aggregate reticulocytes occur in health. In addition punctate reticulocytes can occur in up to 10% of cells. Usually only aggregate reticulocytes are counted but large numbers of punctate reticulocytes may indicate regeneration even in the absence of aggregate reticulocytes. The reticulocyte count should be corrected for the level of anaemia (average PCV is taken as 0.37 L/L for an adult). Absolute reticulocyte counts: This is becoming the correction of choice for dogs and cats. The upper level of the reference range for dogs is 0.075 (grey zone to 0.105) x 1012/L and for cats 0.060 (grey zone to 0.100) x 1012/L. If a dog has 4% reticulocytes and an erythrocyte count of 3.0 x 1012/L then the absolute reticulocyte value is 0.120 x 1012/L. This suggests regeneration.

In the dog and cat, associated with reticulocytosis, there are related changes to red cell morphology and indices.

B) Alterations in red cell morphology on the peripheral blood smear: When a Romanowsky stain (e.g. Giemsa, Diff Quik) is used on a peripheral blood smear, reticulocytes appear as large, bluish cells (macrocytes, polychromatophilic). Consequently, a blood smear in a regenerative anaemia exhibits anisocytosis and polychromasia. Nucleated red cells may also be presented in peripheral blood smears in a regenerative anaemia. However, if they occur unaccompanied by reticulocytes, a non-regenerative anaemia with abnormal marrow or a splenic disorder should be suspected. C) Alterations in the red cell indices. On the basis of mean cell volume (MCV), anaemias can be designated microcytic, normocytic or macrocytic. On the basis of mean cell haemoglobin concentration (MCHC) (and, to lesser extent, mean cell haemoglobin [MCH]) anaemias can be designated as hypochromic or normochromic. Regenerative anaemias, depending on the numbers of reticulocytes, are often macrocytic and hypochromic (depressed MCHC, normal or increased MCH). The changes in the red cell indices last as long as there is a regenerative response; for this reason, a regenerative anaemia is often referred to as a transitory or pseudomacrocytic anaemia. The majority of non-regenerative anaemias are normochromic and normocytic. However, a non-regenerative anaemia associated with myeloproliferative disorders in the cat may be macrocytic, while iron deficiency anaemia is usually microcytic and hypochromic (depressed MCHC and MCH).

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2) Classification of regenerative anaemias A) Blood loss anaemia (haemorrhagic anaemia)

Primary blood loss anaemia may be caused by physical agents (e.g. trauma), living agents (e.g. blood-sucking agents such as fleas), chemicals and heritable disease (the last two mainly affecting blood clotting thereby leading to increased susceptibility to bleeding). It may also occur secondary to another disease (pathological) process such as degeneration and necrosis of tissues or neoplasia affecting the integrity of vessels or clotting mechanisms. The site of haemorrhage should be found before classifying the anaemia as blood loss. This may be difficult in internal hemorrhage, especially if continuous and low grade.

Blood loss anaemia can be further classified depending on whether it is external or internal hemorrhage and acute or chronic. These factors affect the capacity for regeneration and, therefore, laboratory findings in blood loss anaemia e.g. external hemorrhage: loss of iron and plasma protein, if chronic may develop into an iron deficiency non-regenerative anaemia; internal hemorrhage: the components are mainly reabsorbed (about 1/3 of the erythrocytes are reabsorbed and 2/3 phagocytosed) and reutilised.

In an acute episode of external blood loss, the PCV is normal initially due to equal loss of components, but splenic contraction may transitorily elevate PCV. After 2-3 hours and to about 48-72 hours, the blood volume is restored by the addition of interstitial fluid which dilutes the erythron leading to lowered PCV and erythrocyte count i.e. laboratory signs of anaemia. Increased erythroid production is usually evident after 2-3 days and should reach a maximum 7 days after the onset of haemorrhage i.e. reticulocytosis. The peripheral blood picture usually returns to normal 1-2 weeks following a single acute haemorrhagic episode. If the reticulocytosis persists longer than 2-3 weeks, continual bleeding should be suspected.

In chronic external blood loss the anaemia develops slowly and hypovolemia is not seen. The PCV can reach quite low levels before clinical signs of anaemia become obvious because of adaptive mechanisms by the animal. The animal can develop hypoproteinemia and low body iron, which can cause the regenerative response to develop into a non-regenerative one.

B) Accelerated erythrocyte destruction can occur as intravascular haemolysis or as extravascular haemolysis (mainly due to splenic macrophage activity). In a haemolytic anaemia, destruction usually occurs in both sites but one predominates.

In haemolytic anaemia there is usually a marked regenerative response (greater than for blood loss) as iron and other components from the red cells can be reutilised quickly (availability of iron decides the magnitude of the regenerative response). If destruction is of a significant magnitude, haemoglobinemia or hyperbilirubinemia may develop.

Diseases causing principally intravascular haemolysis usually occur as per-acute or acute episodes wth haemoglobinemia a predominant feature. Hyperbilirubinemia and icterus will occur if the haemolysis is of sufficent duration and excessive. Neutrophilia, sometimes with left shift (see Disorders/responses of Leukocytes) is common in haemolytic anaemias in the dog, but the mechanisms for the neutrophilia are poorly understood.

Diseases principally causing extravascular haemolysis (i.e. phagocytosis by the mononuclear phagocytic system – especially in the spleen) are usually more chronic with an insidious onset. Haemoglobin is not present in the blood or urine and hyperbilirubinemia occurs only in a minority of cases. Depending on the cause, some of these diseases can ultimately produce a non-regenerative anaemia due primarily to bone marrow depression (the mechanisms are not fully understood). In other cases the regenerative response may

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adequately compensate for the destruction, thereby producing a normal PCV (compensated haemolytic anaemia). The blood smear and bone marrow should show the regenerative response.

Causes (agents of disease) of haemolysis include living agents (e.g. haemobartonellosis), chemicals (e.g. acute copper poisoning), physical (e.g. microangiopathic haemolytic anaemia – fibrin strand damage to circulating erythrocytes), genetic (e.g. pyruvate kinase deficiency) and immune-mediated (e.g. auto-immune haemolytic anaemia). Whether these agents cause primarily intravascular or primarily extravascular haemolysis depends on the degree of damage to the erythrocyte. Many agents can cause both intravascular and extravascular haemolysis, but one usually predominates. Some agents can predominantly cause direct haemolysis (e.g. acute, high dose copper poisoning in sheep) whilst others may predominantly cause sub-lethal damage to the erythrocyte membrane (e.g. some blood parasites such as Anaplasma spp) which predisposes to splenic macrophage scavenging and destruction.

3) Classification of non-regenerative anaemias Non-regenerative anaemias due to reduced erythropoiesis are usually slow in onset and have a chronic clinical course (the rate of reduction commonly relates to the life span of the circulating erythrocytes). The agent of disease may be causing some blood loss or destruction, but depression of bone marrow erythropoiesis is more important and produces the overall non-regenerative anaemia. In other cases the non-regenerative anaemia is due to defective erythropoiesis rather than reduced erythropoiesis. These are less common and, depending on the cause, can have a variable time course. Whether reduced or defective erythropoiesis, BM examination is warranted when investigating non-regenerative anaemias particularly of undetermined origin. A classification for, and some causes of, non-regenerative anaemia are provided under the General Comments on Bone Marrow Examination.

Some General Comments on Bone Marrow Examination

Bone marrow is the primary site for haematopoetic production. Consequently, bone marrow examination allows assessment of production of all cell lines.

− When peripheral blood examination reveals the presence of persistent unexplained leukopenia, non-regenerative anaemia, thrombocytopenia, or abnormal cell types, bone marrow examination should be considered.

− Bone marrow examination can often confirm a leukemia and help differentiate it from

a leukemoid reaction in the dog.

− In the horse, bone marrow examination is useful also in establishing a diagnosis of regenerative anaemia (although serial PCV’s are more commonly done).

A number of deficiencies/Limitations exist for bone marrow examination. They include the difficulty of obtaining representative samples of bone marrow, the problem of peripheral blood 'contaminating' bone marrow, the lack of a method for quantitative evaluation of cellular elements in bone marrow and inter-individual variability in differentiating various bone marrow cell types. Bone marrow smears are usually in the form of 'squash' preparations and are stained with a

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Romanowsky stain (e.g. Giemsa, commercial ‘Diff Quik’). Bone marrow particles are identified under a lower power objective (4-10x) and megakaryocyte numbers assessed (commonly 5-10 in a particle per 10x objective is considered normal for the dog). Individual nucleated cell types are differentiated under the oil objective (100x). A minimum of 500 cells are differentiated and a myeloid (granulocytes) to erythroid ratio (M:E) obtained. This term expresses the proportion of total granulocytes to nucleated erythroid cells. For healthy animals, the following M:E ratios apply: Dog M:E of 0.75-2.53:1 (average 1.25:1) Cat M:E of 0.6 -3.9:1 (average 1.6:1) Horse M:E of 1.1 -10.2:1 (average 2.43:1) Specific notes on bone marrow examination and M:E ratios: a) Authors very greatly in the values presented for M:E ratios. The above figures are a guide only.

They are derived from Schalm’s Veterinary Haematology (4th Edition) and supported by our laboratory’s findings.

b) Within the myeloid and erythroid cell lines it is useful to differentiate the maturity of cells and to divide them into proliferating and non-proliferating pools. In health, approximately 80% of granulocytes should be in the non-proliferating pool (mainly metamyelocytes onwards - no division, just maturation), and approximately 90% of erythroid cells should be in the non-proliferating pool (mainly intermediate to late normoblasts [nucleated erythroid cells]).

c) The M:E ratio should not be interpreted without knowledge of peripheral blood cell counts. The M:E in regenerative anaemia is usually reduced (i.e. marked increase in erythroid series, normal to slight increase in myeloid series) with an increase in the proportion of erythroid cells in the proliferating pool (greater than 10%).

d) The M:E in non-regenerative anaemias is variable. Non-regenerative anaemias can be classified as follows:

i) Reduced (hypoproliferative) erythropoiesis In domestic animals, causes of non-regenerative anaemia commonly tend to be in this category.

Normocytic normochromic anaemia; normal neutrophil and platelet production; increased M:E (hypocellular erythroid) A common type 1) Anaemia of erythropoietin lack - chronic renal disease and certain

endocrinopathies. 2) Anaemia of chronic disease (inflammatory or neoplastic) - iron is unavailable

for erythropoiesis due to sequestration into macrophages (availability of iron determines the intensity of regeneration). Other factors may operate depending on the cause. The anaemia is mild to moderate and may become hypochromic, microcytic in the later stages. A common cause of mild anaemia, especially in farm animals. Note: a mild to moderate anaemia can occur in acute inflammatory disease (e.g. within 4-5 days after abscess induction in a cat) and appears to be primarily due to accelerated erythrocyte destruction and utilisation of iron by the bacteria. The accelerated destruction is possibly due to antibody coating of erythrocytes and destruction by macrophages. This mechanism may also contribute to anaemia in chronic disease.

3) FeLV-associated non-regenerative anaemia (FIV may also produce something similar).

4) Immune mediated red cell aplasia (intramedullary destruction of erythroid cells).

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Normocytic, normochromic anaemia; decreased neutrophil and platelet production; M:E difficult to determine due to too few cells: 1) Aplastic anaemia - certain chemicals and a range of therapeutic drugs (effects

on neutrophil and platelet production seen earlier as these cells have shorter life spans).

2) Myelophthisic anaemia - replacement of BM with abnormal cells and/or fibrous tissue (myelofibrosis).

3) FeLV induced anaemia. 4) Feline infectious panleukopenia virus induced anaemia (inconsistent late

finding in the disease).

Normocytic, normochromic anaemia; increased production of neutrophils; increased M:E 1) Granulocytic leukemia. 2) Feline infectious panleukopenia virus in recovery.

ii) Defective (hypercellular) erythropoiesis

The M:E is usually reduced because erythroid precursors are numerous in the bone marrow. Abnormal size, haemoglobinisation and/or asynchronous nuclear-cytoplasmic maturation may characterize the erythroid precursors.

Microcytic, hypochromic anaemia; Variable M:E due to variable effects on myelopoiesis 1) Iron, pyridoxine and copper deficiences.

Macrocytic, normochromic anaemia; M:E usually reduced 1) Erythremic myelosis or erythroleukemia.

NB: lead poisoning in dogs gives rise to defective erythropoiesis but rarely gives rise to anaemia and is usually diagnosed without the examination of bone marrow. *SEE CASES 21-24

Polycythemia

Polycythemia refers to an increased red cell mass i.e. the PCV, total erythrocyte count and haemoglobin concentration are increased. Polycythemia can be divided into: a) Spurious or relative polycythemia

An increased PCV but the red cell mass is normal e.g. haemoconcentration through dehydration or shock; splenic contraction in a young horse or cat.

b) Absolute polycythemia An increased PCV due to an increased red cell mass (i.e. increased erythropoiesis). Plasma volume and protein are normal. Absolute polycythemia may be regarded as primary (myeloproliferative disorder of unknown etiology) or secondary (due to increased erythropoietin production). Secondary polycythemia may be appropriate (e.g. in chronic hypoxia) or inappropriate (e.g. erythropoietin producing tumour of the kidney).

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DISORDERS / RESPONSES OF THE LEUKOCYTES

Introduction

Leukocytes have fundamental roles to play in the innate and adaptive immune responses to agents of disease (host response), but like all cells can be affected by degenerative processes and disorders of growth (primarily neoplasia). The numbers and the appearance of leukocytes can be easily examined by taking a sample of peripheral blood, which can be supported by bone marrow examination. Peripheral blood levels of leukocytes are a reflection of bone marrow supply (occasionally extramedullary leukopoiesis in the spleen and liver), tissue demand and their distribution within the circulation (marginal or circulating pool). This particularly applies to neutrophils (Figure 1). Only the circulating pool of leukocytes is measured in peripheral blood.

FIGURE 1:   Leukocytes (neutrophils, eosinophils and monocytes) can move backwards and forwards between the circulating pool (CP) and the marginal pool (MP ‐ capillary beds). This is mainly important for neutrophils, where the pools are referred to as marginal and circulating neutrophil pools (MNP and CNP, respectively). The  storage  pool  of  bone  marrow  is  an  important  supply  of  leukocytes  to  the  CP.  Extramedullary hematopoiesis can contribute to the CP when necessary. Most neutrophils are lost in the tissues. 

Leukocytosis

Note: the evaluation of peripheral blood levels of leukocytes is determined on absolute values rather than relative values (i.e. the percentages). The main exception to this is the ratio of the relative values of segmented to band neutrophils in determining whether a left shift exists. Another exception is perhaps the neutrophil to lymphocyte ratio used in farm animals. Leukocytosis per se is not of significance; what is important is the reason for leukocytosis: is it due to

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neutrophilia (most common cause in dogs, cats, horses and pigs), lymphocytosis (contributes more so in ruminants), eosinophilia, basophilia or monocytosis? The detection of leukopenia must be treated in the same manner; that is, the cell types involved must be determined. In the dog, cat and horse leukopenia is usually due to neutropenia, although lymphocytopenia may contribute. In ruminants, lymphocytopenia can be the reason for leukopenia, either alone or in conjunction with a neutropenia.

Neutrophilia

Neutrophils are produced in the bone marrow from a precursor cell that is capable, under the right stimuli of also producing monocytes. Neutrophil production is regulated by granulopoietin, which is produced by a variety of cells primarily in response to depletion of the bone marrow storage pool (there are about 4-5 days supply of mature neutrophils stored in the bone marrow). Granulopoietin stimulates neutrophil production and replenishes the storage pool. Some bacterial products stimulate granulopoietin production. Neutrophil release from the BM storage pool can be promoted by a plasma factor (called either leukocytosis inducing factor or neutrophil releasing factor). The factor's production is stimulated by bacterial products and neutropenias. The factor causes a rapid neutrophilia and a depletion of the bone marrow storage pool (this in turn causing granulopoietin release). If the process continues (e.g. continued tissued demand), then immature neutrophils appear in the circulation. Neutrophils survive about 10 hrs in the circulation where they are distributed between a measurable circulating neutrophil pool (CNP) and a non-measurable marginal neutrophil pool (MNP - slow moving cells normally adjacent to small vessel walls, especially in capillary beds). Most neutrophils are lost in tissues or secretions/excretions (see Figure 1). Neutrophilia may occur in response to physiological reasons, release of corticosteroids, tissue (inflammatory) demand and regenerative anaemias. Changes to the numbers of other cell types may help to differentiate the types of neutrophilia. In most situations neutrophilia due to inflammatory demand is of importance (indicating a host response to either a disease agent or to damaged tissue); other causes of neutrophilia merely interfere with its detection. Basically, the mechanisms for neutrophilia are: a) mobilisation of the cells in the MNP b) increased rate of release from the storage department (due to leukocytosis inducing factor). (a) and (b) are the reasons for rapid neutrophilia (few minutes to 2-3 days) c) increased neutrophil production (controlled, in part, by granulopoietin). Increased stem cell input

will take 3-5 days before changes appear in the CNP. Increased effective granulopoiesis (additional divisions) takes 3 days before changes appear in the CNP.

Physiological neutrophilia (leukocytosis) Physiological leukocytosis (principally due to neutrophilia) may occur in such situations as parturition, lactation or after eating (e.g. 1 hr post-prandial in the dog), but the significant increases occur due to release of epinephrine in response to exercise, fear or excitement. Physiological leukocytosis is thought to be a pro-active protective host response, but does not always prove useful. Physiological leukocytosis due to excitement supposedly only occurs in healthy animals and is

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common in the young cat and horse (levels of leukocytes can reach 20-25 x 109/L), in which both neutrophilia and lymphocytosis occur. The neutrophilia occurs because of shift of cells from the MNP to the CNP. In the cow eosinopenia appears to occur in physiological leukocytosis. Corticosteroid induced neutrophilia (leukocytosis) This response commonly occurs in sick animals with severe illness (this is usually seen in acute illness, but may occur during intense episodes of recurrent or chronic disease, or in terminal disease). It is often produced by pain or temperature extremes (e.g. fever) and is more common in the dog than in the cat and horse. In the horse excessive (over-exertion) muscular activity can produce typical changes, whilst in cattle a variety of conditions (e.g. ketosis, milk fever, dystocia, GIT disturbances) have been recorded as producing corticosteroid induced changes. In pigs and cattle strenuous exercise and difficult parturition may cause corticosteroid induced changes. Corticosteroid induced neutrophilia has as its basis protection for the host, but often this goes awry because of the alterations to other leukocytes. The neutrophilia is accompanied by lymphocytopenia (lymphopenia), eosinopenia and, in some species (e.g. dog and inconsistently in the cat), by monocytosis. In the horse and cow monocytopenia is apparently more common in acute stress. This is often followed by a later monocytosis. A left shift for the neutrophilia does not occur unless there is another disease process causing inflammatory demand. Lymphocytopenia is rarely dramatic in horses, whilst eosinopenia is not easy to detect in cattle. The neutrophilia occurs due to a shift from the MNP to CNP, decreased migration from peripheral blood and release from the BM storage pool. Lymphocytopenia (may just be low normal) occurs due to a redistribution of lymphocytes in the short term (held in the lymphoid tissue) and a selective lymphocytolysis in the long term. Eosinopenia is probably due to a shift from the CP to the MP and an inhibition of bone marrow release (eosinopoiesis in BM still continues and a rebound eosinophilia may occur if the animal recovers). Monocytosis is probably due to a shift from the MP to the CP. In the dog total leukocytes can reach 40 x 109/L or greater, but, commonly, levels are between 15-25 x 109/L. In the cat values of 30 x 109/L or greater have been recorded, while in the horse levels are usually around 20 x 109/L. Cattle may show up to 18 x 109/L total leukocytes. These are guidelines only and assessment must be supported by appropriate changes in levels of individual cell types. Nb. Hyperadrenocorticism in dogs will produce similar changes in white cells. However, in long term hyperadrenocorticism the magnitude of the neutrophilia will be diminished. Neutrophilia due to regenerative anaemia Neutrophilia commonly accompanies both haemorrhagic and haemolytic anaemia if there is moderate to intense regeneration. The response is more pronounced in haemolytic anaemia, in which neutrophilia with left shift may occur (this has been primarily detected in the dog). The mechanism for the neutrophilia is not well understood Neutrophilia related to inflammatory demand This usually occurs due to purulent inflammation due to infection, but can occur due to tissue damage (i.e. degeneration and necrosis) related to another disease process e.g. neoplastic necrosis; pancreatic necrosis. The degree of neutrophilia and left shift present in the peripheral blood depends on: • the degree of tissue demand for neutrophils • the reserves of neutrophils available in the MNP and BM storage pool • the ability of BM to increase production.

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In some types of inflammation (e.g. cystitis, catarrhal enteritides) where there is limited tissue demand, neutrophilia may not be necessary as the existing circulating levels of neutrophils can cope. As tissue demand for neutrophils increases variable levels of neutrophilia develop due to a shift from the MNP and release from the BM storage pool. The level of neutrophilia depends on the balance between BM production and storage, and the loss of neutrophils from peripheral blood into the tissues (see Figure 1). For example, in some purulent diseases an initial neutropenia may occur due to demand exceeding supply (i.e. more neutrophils are moving into tissues than are moving into the CNP). In good prognostic cases this is quickly followed by neutrophilia due to enhanced production and release in the CNP. A left shift (the release of an increased proportion of immature neutrophils into the peripheral circulation) will occur in tissue demand if the BM storage pool becomes depleted for any length of time and granulopoietin levels increase. The left shift will disappear as soon as increased production restores storage pool numbers.

If a left shift doesn't accompany a neutrophilia it doesn't mean that tissue demand is not occurring (the BM storage pool may not have been depleted or it may have recovered), but it is possible also that the neutrophilia is due to another reason such as corticosteroid release (this is more likely in acute, intense illness i.e. clinical signs will provide invaluable information for differentiation).

In acute onset, intense inflammation there is likely to be a corticosteroid-induced component to the inflammatory demand neutrophilia (e.g. acute pancreatic necrosis in the dog). This will only be determined by detecting lymphocytopenia and eosinopenia.

What is classified as a left shift? There is a normal proportion of immature to mature neutrophils in the peripheral blood. As the proportion of immature cells increases (due to demand causing depletion of the bone marrow storage pool) then the likelihood of a left shift developing exists. When the proportion drops less than 1:16-18 for the dog, 1:10-12 for the cat and 1:12-16 for the horse (there is great variation in the literature for these values and they should be used with caution) then it is definite that a left shift has developed. Proportions just above these, however, should not be taken to mean that a left shift is not present or developing. Serial samples are useful in deciding whether a left shift is developing if the ratio is around or just above its upper limit. Ruminants rarely have immature neutrophils circulating in health, therefore, their presence indicates a left shift without the need for ratios. Nb. In the dog, and possibly in the cat, it is accepted by many haematologists that greater than 1 x 109/L bands is definitely a left shift irrespective of the ratio. In the horse, some are happy to accept greater than 0.3 x 109/L bands as indicating a left shift. Consequently, it is not clear cut. Perhaps it is best to use the ratios when values for bands are below those designated acceptable levels for left shifts? Common sense is the answer. It is important to remember, also, that the lack of a left shift does not rule out inflammatory demand!

What constitutes a favourable prognosis (in relation to neutrophil numbers and types in the peripheral blood)? Neutropenia in the early stages of overwhelming demand cannot always be regarded as a poor prognostic sign. However, the persistence of neutropenia, especially without the development of a left shift, is a poor prognostic sign, as it indicates bone marrow failure and a compromise of an important part of innate immunity. Morphological changes can occur in neutrophils related to inflammatory demand. These are loosely called 'toxic changes' and most probably interfere with neutrophil function. Toxic changes include:

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a) Döhle (Doehle) bodies (bluish angular cytoplasmic inclusions). These are related to disturbances of neutrophil maturation and occur in many cases of increased neutrophil production. Although they can be taken as an indication of mild toxemic change, they may occur unrelated to toxemia e.g. certain drugs can cause them.

b) Cytoplasmic basophilia and vacuolation. This represents a more severe manifestation of toxemia

and often occurs in severe infections and intense inflammation (e.g. pancreatic necrosis in the dog).

c) Cytoplasmic granulation (purplish). Prominent purplish granulation of neutrophils in the horse

can indicate toxemia. Persistent toxic changes to neutrophils, especially after the initiation of treatment, in conjunction with low CNP neutrophil levels, may suggest a poor prognosis.

What levels of neutrophilia can accompany tissue demand? In dogs any level is possible but most total WBC counts are within 10-50 x 109/L (corticosteroid induced WBC counts are usually less than 25 x 109/L but can occur higher). In certain purulent infections (e.g. pyometra) the count may be between 50-100 x 109L and would commonly be accompanied by an extreme left shift (i.e. myelocytes, metamyelocytes start to circulate in the peripheral blood). This is called a 'leukemoid response' as it may resemble chronic neutrophilic (granulocytic) leukemia. In cats, total WBC levels in inflammatory demand rarely exceed 35 x 109/L (corticosteroid induced total WBC levels are usually less than 30 x 109/L). The frequency of left shift parallels that in the dog. In horses total WBC levels in inflammatory demand are similar to those that occur in the cat (corticosteroid induced total WBC levels are usually less than 20 x 109/L). In adult horses left shifts in inflammatory demand are uncommon and rarely spectacular. Most cases of inflammatory demand are reflected as simple neutrophilia. In foals, left shifts are more common especially in purulent infections. In the dog, cat and horse, as mentioned, a neutropenia may develop in early intense inflammatory demand, and may take a few days to become a neutrophilia. Gram-negative bacteria causing overwhelming sepsis and endotoxemia commonly produce early neutropenia (horses are particularly sensitive to small amounts of endotoxin). A rebound neutrophilia is likely later. In adult cattle, early neutropenia is the rule in inflammatory demand (as reserve levels of neutrophils in the MNP and bone marrow storage pool are low). Therefore, in addition to endotoxemia related to Gram-negative bacteria, a wide range of acute purulent inflammations commonly induce early neutropenia in cattle. This is commonly accompanied by a left shift and lymphocytopenia (thereby causing a leukopenia). Toxic changes in neutrophils are commonly observed but, as mentioned, are only a poor prognostic sign if they persist. Although the adult ox appears to have low reserves of neutrophils to combat purulent infections (hence the early neutropenia), the BM responds quickly (hence the moderate left shift) and by 2-3 days neutrophil levels may be starting to recover. The left shift normally persists as long as the inflammatory demand persists, while total WBC levels (due to neutrophilia and a return to normal lymphocyte levels) can reach up to 30 x 109/L or greater in long standing cases (commonly levels of total WBCs in inflammatory demand will be in a range up to 20 x 109/L; but corticosteroid induced leukocytosis is rarely prominent due to the lymphocytopenia). Young calves may have inflammatory demand leukocyte changes similar to dogs and cats. This is presumably due to differing reserve levels of neutrophils.

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In fibrinous, non-purulent inflammation cattle may show little or no neutrophil response and high plasma fibrinogen levels may be the only laboratory sign of inflammatory disease. Consequently, fibrinogen levels (in association with total protein estimation to exclude a false elevation due to dehydration) have been used in many animal species to indicate inflammatory or degenerative disease. Fibrinogen is an 'acute phase reactant' protein (and the major clotting protein) and rises rapidly in a variety of diseases. It appears to be the main protein that alters erythrocyte sedimentation rate. It has been well used in ruminants (and less so in horses). In cattle, total serum or plasma protein to fibrinogen ratios of 10 to 1 or less indicates a significant increase in fibrinogen and suggests disease (i.e. it is a non-specific indicator). Nb: In dogs and cats, fibrinogen levels may be elevated in inflammatory/degenerative disease; but other acute phase reactant proteins (e.g. haptoglobin, C-reactive protein) are more commonly used to detect inflammatory or degenerative disease. In pigs, neutrophilia due to inflammatory demand is of moderate intensity when compared to the dog and cat but may be missed due to the wide reference range. Left shifts are common in purulent infections and may be accompanied by total WBC levels of up to 100 x 109/L, but most are between 20-35 x 109/L. Pigs, like cattle, may develop little neutrophil response in non-purulent inflammation and fibrinogen may be of use to indicate inflammation.

Neutropenia

Neutropenia may occur through a variety of mechanisms. These include reduced survival, reduced production, increased ineffective granulopoiesis, unknown mechanisms, and sequestration. Reduced survival (or increased utilisation) neutropenia occurs when destruction of mature neutrophils or excess tissue utilisation exceed bone marrow supply e.g. immune-mediated neutropenia, overwhelming sepsis, bovine acute purulent diseases. Recovery to a neutrophilia usually takes a few days. Reduced production neutropenia may be transient or persistent and is usually due to an agent of disease directly affecting cell production. Transient causes include certain acute infectious diseases such as feline infectious panleukopenia, canine adenovirus. As with reduced survival, rebound neutrophilia can occur. Persistent causes of neutropenia (usually indicating bone marrow failure) have a poor prognosis and may be due to myelophthisis, drug toxicity or irradiation. Increased ineffective granulopiesis produces neutropenia by failure of release of cells from the bone marrow, intramedullary destruction, and arrest of granulocyte maturation (e.g. myelophthisis, feline leukemia virus). It has a poor prognosis. Some neutropenias develop through unknown mechanisms in a variety of infectious (mainly viral) diseases in the acute stages (e.g. canine distemper, equine infectious anaemia, sporadic bovine encephalomyelitis). Commonly, they are transient. Sequestration neutropenia (pseudoneutropenia) is due to a sudden and transient margination of neutrophils. It lasts for a few hours and is followed by rebound neutrophilia e.g. common in endotoxemia and anaphylactic shock. It should be obvious from the preceding discussion that some infections may cause neutropenia by a variety of mechanisms and that many are transient.

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Monocytes

Monocytes are formed in the bone marrow, are transported in the circulation and are capable of being transformed into macrophages to complement fixed tissue macrophages when there is increased demand for macro-molecular phagocytosis (innate immunity). They have additional roles to play in adaptive immune responses (antigen-presenting cells; effector cells when activated by cytokine release from T lymphocytes). Monocytes, like the neutrophils, may be unevenly distributed in the circulation (i.e. there are marginal and circulatory pools: MP and CP). Monocytosis may occur in intense stress due to corticosteroid release (primarily in the dog; it is variable in the cat), in disorders characterised by tissue demand for phagocytosis of macromolecular molecules, in conditions in which cellular immunity is potentiated, and in neutropenic states related to granulocytic hypoplasia (probably a compensatory mechanism). In the horse and cow monocytopenia is apparently more common in acute stress. This is often followed by a later monocytosis. Monocytopenia can occur in endotoxemia. Monocytopenia is not of consequence in itself and may be difficult to detect in the individual animal as reference ranges for most species have low limits.

Eosinophils

Bone marrow storage of eosinophils is minimal and cells are usually in the circulation for a short time. Eosinophils are attracted by and inhibit chemical mediators released from mast cells (principally histamine). They are also parasiticidal. Eosinophils have weak bactericidal properties. Eosinophilia can occur in response to histamine release or antigenic stimulation via sensitized T lymphocytes (may be involved in stimulation of eosinopoiesis). Consequently, eosinophilia can occur in hypersensitivities especially involving skin, lung, GIT and the reproductive tract. Prolonged parasitic contact can induce eosinophilia. Localised eosinophilic lesions do not necessarily produce blood eosinophilia. Some eosinophilias are quite marked and inexplicable (hypereosinophilic syndrome). Eosinophilia has been seen during oestrus in a number of species, especially the bitch. Eosinophilia may also accompany some neoplastic conditions (e.g. mast cell tumours, some lymphosarcomas) Eosinopenia is commonly associated with corticosteroids but it can be caused by adrenaline release, and it can occur in acute infections by additional mechanisms independent of corticosteroids. With corticosteroid release, blood histamine levels are reduced and mast cell regranulation is inhibited. This causes eosinophils to probably move to the marginal pool and possibly to remain in the bone marrow. Prolonged release of corticosteroids reduces bone marrow eosinophil production.

Basophils

Basophils contain many mediators of inflammation and release these in response to immune complexes on their surface (i.e. in adaptive immunity) and to various chemicals and physical agents. The reasons for basophila are poorly understood but it is accepted that basophilia usually accompanies eosinophilia as in some parasitic and allergic diseases (both adaptive immune responses). Basophilia may occur without eosinophilia (e.g. in horses), but is rare. Basopenia is not of significance.

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Lymphocytes

Lymphopoiesis mainly occurs in the lymphoid tissue but some production continues in the bone marrow (especially for B lymphocytes). Lymphocytes when stimulated transform into lymphoblasts which proceed to prolymphocytes and then to T effector cells, B lymphocytes or memory lymphocytes. Activated B lymphocytes are the source for plasma cells. Lymphocytes are the most important cells for adaptive immunity. In circulating blood the majority of lymphocytes are of the T cell variety (there is some variation between species). Plasma cells, lymphoblasts and prolymphocytes are rarely seen in peripheral blood. However, transformed lymphocytes (`immunocytes' or ‘activated hyperbasophilic lymphocytes’) of both the B and T cell lines are seen in peripheral blood and their numbers can increase in periods of antigenic stimulation. Blood lymphocyte numbers are most often influenced by effects on cell recirculation. Mild lymphocytosis may occur in physiological leukocytosis, chronic infections and hypoadrenocorticism. It does not have to accompany lymphoid hyperplasia due to antigenic stimulation, but is common in prolonged situations. Lymphopenia (lymphocytopenia) can be due to corticosteroid-induced redistribution of recirculating lymphocytes, but can be related also to acute systemic infection (usually viraemias). Recirculating lymphocytes are entrapped in lymph nodes by the presence of antigenic material. Other causes of lymphopenia are less common and include T cell deficiencies and loss of lymphocyte rich lymph. Lymphoid leukemia may show circulating small lymphocytes but more often lymphoblasts and prolymphocytes are the predominant cell types (see notes under hematopoietic neoplasia.) *SEE CASES 14, 16-20, 25,26

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HAEMATOPOIETIC NEOPLASIA

Introduction

Haematopoietic neoplasia can be divided into lymphoproliferative and myeloproliferative forms. Lymphoproliferative disorders involve either proliferation of lymphocytes or plasma cells. Commonly, this leads to infiltration and enlargement of organs or tissues. Tissues affected vary but if BM is involved then a leukemic manifestation is likely (circulating neoplastic cells). Myeloproliferative disorders usually present as leukemias as the BM is always involved. Lymphoproliferative disorders are more common than myeloproliferative disorders in most species. In farm animals, myeloproliferative disorders are rare.

Lymphoproliferative Disorders

a) Lymphosarcoma (malignant lymphoma) This is a malignant solid neoplasm of lymphocytes or precursor cells (i.e. affecting lymphoid organs or infiltrating other tissues of organs). Benign neoplasms of lymphocytes have not been recognised in animals, so many are willing to drop the ‘malignant’ from ‘malignant lymphoma’ and just use ‘lymphoma’ to designate the condition. Lymphosarcoma may become leukemic in the later stages (figures range from 25-60% for the dog and up to 25% for the cat). Lymphoid leukemia can occur on its own (primary acute and chronic lymphoid leukemia – see below), but usually, at least in the cat and dog, occurs in combination with lymphosarcoma. Anaemia is not uncommon in dogs with advanced lymphosarcoma and may be related to immune-mediated destruction and/or chronic disease. Hypercalcemia and a gammopathy may also be detected in dogs with lymphosarcoma. Solid masses can occur in variable sites in lymphosarcoma and this forms the basis of an anatomical classification (e.g. nodal; abdominal; cranial mediastinal; miscellaneous; primary leukemia without solid masses). Nodal in association with involvement of internal organs is common in the dog (called multicentric). The cranial mediastinal form is more commonly seen in the cat, but the most common form in the cat appears to be alimentary. The horse has a low incidence of lymphosarcoma, which is predominantly multicentric with high involvement of abdominal (alimentary tract mainly) tissues. If external lymph nodes are involved then there is usually marked enlargement as opposed to mild enlargement which may occur in myeloproliferative and primary lymphoid leukemias. Lymphosarcoma, particularly affecting lymph nodes, can be classified on architectural pattern of the neoplastic cells (diffuse or nodular). Lymphosarcoma can also be classified according to the predominant cell type (e.g. lymphoblast, prolymphocyte, lymphocyte), and in relation to the main immunophenotype (e.g. B or T cell). Immunophenotyping in people is important for prognosis but, at this stage, immunophenotyping, on its own, appears to have limited prognostic value for the dog and cat. This may alter with increasing sophistication of testing and with increased treatment. There are several classification systems for lymphosarcoma in animals. These include the National Cancer Institue Working Formulation (based on architectural pattern and cellular morphology), the Updated Kiel System (based on architectural pattern, cellular morphology and immunophenotyping) and the Revised European-American Classification of Lymphoid Neoplasms (REAL – based on cellular morphology, immunophenotyping and sometimes cytogenetics). The first two, have been better utilised so far (especially for canine tomours) and classify lymphosarcomas into low, intermediate and high grade forms.

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In the cat some cases of lymphosarcoma appear to be associated with Feline Leukemia Virus (FeLV) or Feline Immunodeficiency Virus (FIV), but the numbers vary from country to country. FIV could well have its impact through altering the immune response rather than directly causing transformation of lymphocytes. If viruses are involved in the transformation, anaemia commonly accompanies the disease (may be either regenerativeor non-regenerative). It has been reported from overseas that FeLV negative lymphosarcomas are more likely to be abdominal (alimentary) in form and not be associated with anaemia. Heritable factors may exist for some forms of feline lymphosarcoma Treatment of lymphosarcoma in dogs and cats has developed dramatically and several successful protocols of chemotherapeutic agents are available. In horses, lymphosarcoma has variable cell morphology and the multicentric form is commonly B cell in origin (although they may be associated with many mature T lymphocytes). Subcutaneous and alimentary forms are also usually composed of B lymphocytes, whilst the mediastinal form is composed of neoplastic T lymphocytes. Lymphocytosis and leukemia are uncommon, but a monoclonal gammopathy has been recorded in the multicentric form. Anaemia (due to either immune-mediated destruction or chronic disease) is often the most common laboratory finding and may be the presenting complaint. In cattle lymphosarcoma presents as two main forms: sporadic and enzootic. Sporadic lymphosarcoma tends to affect animals less than four years of age. A sporadic multicentric form occurs in calves, a sporadic mediastinal form in yearlings and a sporadic cutaneous form in young cattle. Enzootic bovine lymphosarcoma (EBL) tends to affect older animals and, commonly, several animals in one herd are affected. EBL is commonly multicentric in form, involves B lymphocytes, and is transmissible as a retrovirus has been incriminated (Bovine Leukemia Virus). Persistent lymphocytosis can precede EBL for several years, but a true leukemic manifestation (abnormal lymphoid cells usually in high numbers) occurs in less than 30% of affected cattle.

b) Acute lymphoblastic leukemia (ALL) ALL can occur as a primary leukemia or as a manifestation of late stage lymphosarcoma. It is not always easy to distinguish these two conditions in terminal disease, but the lack of solid tissue masses should suggest the leukemia is a primary manifestation. Immunophenotyping has shown many are B cells, but T and NK (natural killer) cell types have also been recorded for dogs and cats. ALL is commonly rapid in onset, causes myelophthisis and usually presents with low to medium increases in circulating lymphoblasts. c) Chronic lymphocytic leukemia (CLL) CLL tends to occur in older animals and most occur in dogs. The time course is months to years and enlargement of lymph nodes may occur in the later stages so as to make it difficult then to decide if this is a primary leukemia or a form of small cell lymphosarcoma with a leukemic manifestation. Most CLLs are T cells, but B cell forms with monoclonal gammopathy have been recorded. Circulating levels of neoplastic lymphocytes can vary from 10-100+ x 109/L Large granular lymphocyte (LGL) leukemia is a distinct form, whose presentation is similar to CLL. LGL lymphosarcoma also occurs. LGLs are cells with disctinct, often large, azurophilic granules in the cytoplasm. In humans they are thought to be related to either activated cytotoxic T lymphocytes (T cell LGL) or natural killer (NK) cells (NK cell LGL). In dogs with LGL neoplasia they appear mostly to be T cell LGL, whilst in cats both T cell LGL and NK cell LGL have been implicated. In horses, both LGL leukemia and lymphosarcoma (mainly affecting gut) have been reported, with T cell LGL being implicated.

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d) Plasma cell myeloma (multiple myeloma) This is a malignant neoplasm of older animals derived usually from a single clone of plasma cells and their precursors. Solid masses usually develop in bones or visceral tissues, and the neoplastic cells often produce an immunoglobulin or immunoglobulin sub unit in excess. Most cases have been recorded in the dog but any animal species may be affected. Dogs can respond successfully to chemotherapy. NB: plasmacytoma - refers to a benign neoplastic mass of plasma cells. It is rare in most domestic animals, but skin plasmacytomas are common for the dog. Laboratory findings in plasma cell myeloma may include: a) non-regenerative anaemia (30% of cases in dogs due to myelophthisis) b) circulating plasma cells (20% of cases in dogs) c) hyperglobulinemia (majority of cases in the dog) Hyperglobulinemia is the most consistent finding. It most commonly presents as a narrow-based peak either in the beta or gamma zone of the electrophoretogram (hence the name monoclonal gammopathy – occasional biclonal forms have been reported). The abnormal globulin is often referred to as a paraprotein or M-component. If the paraprotein is a light chain of an immunoglobulin it may be passed in the urine (Bence-Jones protein). If the globulin produced is IgM (macroglobulinemia) or polymerized IgA, then the hyperviscosity syndrome can occur (can lead to a bleeding diathesis). Derangements of haemostasis may also occur because of thrombocytopenia (related to myelophthisis) and paraprotein binding to platelets (thrombocytopathy). Hypercalcemia in the dog may result from osteolysis through release of a tumour factor and stimulation of release of parathyroid hormone because of paraprotein binding to ionised calcium. NB: Occasionally lymphosarcoma may produce a hyperglobulinemia which may be a polyclonal, biclonal or monoclonal gammopathy. Hypercalcemia through pseudohyperparathyroidism (see section under derangements of calcium metabolism for mechanisms) is probably more common in lymphosarcoma than in multiple myeloma.

Myeloproliferative Disorders

Medically, myeloproliferative disorders are far less common than lymphoproliferative disorders, and include a variety of non- and pre-neoplastic conditions as well as neoplastic conditions. In veterinary medicine little is known about myeloproliferative disorders other than those that are obviously neoplastic. However, a pre-neoplastic condition called ‘Myelodysplastic syndrome’ (MDS) is recognised most commonly for the cat (can be FeLV positive), and infrequently in the dog and horse. Myelodysplastic syndrome is characterised by less than 30% blasts in BM, dyserythropoiesis (abnormal maturation and morphology) and cytopenia affecting more than oen cell line. Invariably MDS progresses to AML. Myeloproliferative disorders are all characterised by BM hyperplasia and a variable degree of myelophthisis. Consequently, there is variable disturbance of all BM cell types. The neoplastic cells will be circulating and commonly in high numbers. However, at times they may be low in number or even absent (so called ‘aleukemic’ or ‘subleukemic’ forms – occurs because the neoplastic cells, for some reason, are released from BM spasmodically or not at all). Variable infiltration of systemic organs (spleen, liver, lymph nodes) will occur. Acute and chronic forms are recognised (this also applies to primary lymphoid leukemia). Solid tumours (chloromas) are rare. Myeloproliferative disorders are more common in the cat in which some are associated with FeLV.

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However, even in cats they are becoming less common. They are occasionally seen in the dog, but are rare in other domestic species. Most appear to respond poorly to chemotherapeutic agents, although chronic leukemias have a better prognosis (that also includes CLL). 1. Acute myeloid leukemia (AML) is recognised primarily in cats and dogs and develops rapidly and is invariably fatal because of myelophthisis and related effects. The system of classification is based on the human French-American-British Group (FAB) classification, but there are some unclassifiable and hybrid forms in animals. AML is diagnosed when >30% of the nucleated cells in bone marrow are blasts and the total non-erythroid population of bone marrow cells is >50%. a) Acute undifferentiated leukemia (AUL). This term is used when it is difficult to classify the

cells. Included in this category are the previously described cases in cats called reticuloendotheliosis.

b) Myeloblastic leukemia (This was originally called acute granulocytic leukemia.). This form is divided into two subtypes: myeloblastic leukemia without differentiation (AML-M1 – 90% of non-nucleated cells in BM are blasts, with less than 10% differentiating into neutrophilic or eosinophilic promyelocytes); and myeloblastic leukemia with differentiation (AML-M2 – 30-90% of non-nucleated cells are blasts, with more than 10% differentiating into promyelocytes and later forms of granulocytes [neutrophils, eosinophils and, rarely, basophils). Eosinophilic differentiaton is rare in the cat and non-existent in the dog. Basophilic differentiation is extremely rare for all species.

In the cat some cases are FeLV positive (some are possibly related to FIV but more research is required to confirm this - this virus may allow a variety of Haematopoietic malignancies to develop through immunosuppression or produce them directly). In the cat, acute granulocytic leukemia may be the terminal stage of a disease that has progressed from another form such as erythroleukemia. In both the dog and cat, acute granulocytic leukemia is commonly accompanied by non-regenerative anaemia and thrombocytopenia with bizarre platelets.

c) Promyelocytic leukemia (AML-M3). This form is extremely rare and has only been reported in

the pig. d) Myelomonocytic leukemia (AML-M4). This is the most common form of AML in dogs, cats

(some are FeLV positive) and horses. Both neutrophilic and monocytic cell lines are affected (both come from the bipotential precursor blast) and myeloblasts and monocytoblasts comprise more than 30% of the nucleated BM cells. More than 20% of the nucleated cells of BM are differentiating neutrophilic and monocytic cells.

e) Monocytic leukemia (AML-M5). This can be further typed as M5a (more than 80% of nucleated cells in BM are monoblasts or promonocytes) and M5b (30-80% nucleated cells in bone marrow are monblasts and promonocytes, with prominent monocyte differentiation also). In the dog this form may progress to M4 (and possibly M1-3 for the granulocytic line)

f) Erythroleukemia (AML-M6). This is characterised by a mixture of erythroid precursors and myeloblasts/monoblasts in BM (usually greater than 30% of all nucleated cells). If erythroid precursors exceed 30% of the nucleated cells in bone marrow, then AML-M6Er is used instead of M6. Originally, M6Er was designated erythremic myelosis and in cats was often FeLV positive. In cats, M6 can sometimes progress to M1 or M2.

g) Megakaryoblastic leukemia (AML-M7). This has been recorded in both the dog and cat (FeLV positive in the cat) and more than 30% of all nucleated cells in BM are megakaryoblasts. Abnormal forms circulate. Myelofibrosis is common.

Nb. Another form of megakaryocytic neoplasia involving mainly the platelets is called Essential

Thrombocythemia and is really a chronic myeloproliferative disorder.

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2. Chronic Myeloproliferative disorders In contrast to CLL, these disorders are rare. They are characterised by blasts being less than 30% of all nucleated cells in the BM. MDS is sometimes included in this group.The group includes polycythemia vera (primary erythrocytosis or essential erythrocytosis – described in cats, dogs, cattle and horses), chronic granulocytic (myelogenous) leukemia (described in cats, dogs and horses), chronic eosinophilic leukemia (described in cats only, but there is the problem of differentiating this from the hypereosinophic syndrome in cats and dogs), chronic basophilic leukemia (has been described in dog and cat, but rare), essential thrombocythemia (rare, but seen in dogs and cats), chronic myelomonocytic leukemia, and chronic monocytic leukemia. 3. Mast cell leukemia. In the dog this can be a primary haematopoietic neoplasm, distinct from mastocytomas. Circulating mast cells apparently occur with advanced forms of mastocytoma (sometimes called mastocytosis), but this is distinct from primary mast cell leukemia. In the cat, splenic mast cell tumours have a systemic mastocytosis (sometimes called a leukemic manifestation). *SEE CASES 27 & 35

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BLEEDING DISORDERS (ABNORMALITIES OF HAEMOSTASIS)

Introduction to Haemostatis

Disorders of haemostasis can lead to either bleeding (haemorrhage) or thromboemobolism (‘hypercoagulation’). This section will focus mostly on bleeding disorders. To understand the causes and investigation of bleeding disorders it is important to have a general knowledge of haemostatis. The following is a simplistic view of the complex topic of haemostatis, but should suffice for the investigation of bleeding disorders. Haemostatis depends upon a complete interaction among vascular endothelium, platelets and clotting (coagulation) factors. In normal haemostatis vascular injury leads to platelet adherence (temporary haemostatic plug), which in turn promotes clotting. The result is a fibrin clot that stabilises the platelet plug. At the same time of activation of clotting there is activation of fibrinolysis that aims to remodel and remove formed clots to maintain vascular channels. Endothelial cells normally form a non-thrombogenic surface, but when damaged they release substances that activate clotting. Platelets adhere to exposed sub-endothelial collagen upon endothelial damage. Upon adherence and aggregation, platelets cause further platelet activation and release non-enzymic clotting factors associated with their membranes. Platelet phospholipid in the membrane accelerates the intrinsic pathway of clotting while other substances are released from the platelets that contribute to the formation of a stable clot. Platelets also play an essential role in inflammation, through the release of vasoactive substances and the production of cytokines, and tissue repair. Platelets appear to provide a factor that `nourishes' endothelial cells. In prolonged or extreme thrombocytopenias endothelial disturbances can lead to petechiation. Platelet production is under control of a hormone named thrombopoietin which enhances platelet production upon reduced platelet mass. Coagulation and fibrinolytic systems are intricately involved as well as having complex interactions with platelets and endothelial cells. Coagulation basically involves sequential factor activation, which yields thrombin, an enzyme that enhances conversion of fibrinogen to fibrin. Clotting activation can occur via an intrinsic or extrinsic pathway. Both activate a common pathway which gives rise to fibrin. The intrinsic pathway can be activated in vivo by endothelial cell damage, collagen exposure, pyrophosphate from platelets and endotoxin. The extrinsic pathway is activated in vivo by the release of tissue thromboplastin (Factor III), which is a lipoprotein present in most cells. The extrinsic system accelerates the whole process of blood clotting and is probably the initiator of coagulation in most disease. In many disease situations, it is likely that both the intrinsic and extrinsic pathways are activated. Most clotting factors are produced by the liver and can be divided into enzymic and non-enzymic types. Enzymic factors are usually circulating in inactive forms (FVII is the exception) and activated forms are inhibited by natural inhibitors. This is necessary as enzymic factors are not consumed by the clotting reaction. Enzymic factors II, VII, IX and X are dependent on Vitamin K for production by the liver. The level of the enzyme determines clotting ability. Therefore, partial deficiencies lead to partial loss of clotting ability. Non-enzymic factors (Calcium, Factor V, VIII and fibrinogen) primarily become associated with platelet membranes and are localised during haemostatis by platelet aggregation. With clotting these factors are consumed. Partial deficiencies of most of these factors do

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not interfere with clotting as only small amounts are required. Therefore, almost complete absence is required for clotting defects (although clotting factor tests may be altered if fibrinogen is less than 0.5 g/L). Factors V, VIII and fibrinogen are all 'acute phase reactants' and rapid increases can occur in a variety of inflammatory or neoplastic diseases. Factor VIII is a complex factor which is composed of 2 main components: Factor VIII:C (procoagulant activity - most important in the intrinsic pathway of coagulation) and Factor VIII:vW (von Willebrand's Factor, also called Factor VIII:related antigen). Factor VIII:vW is actually produced by endothelial cells. It is absorbed by circulating platelets (like most non-enzymic factors) and is neccessary for their adherence to collagen. Circulating naturally occurring anticoagulant proteins downregulate the coagulation cascades by inhibiting the procoagulant proteins of the intrinsic, extrinsic and common pathways i.e. there is a balance between anticoagulant and procoagulant proteins. This ensures some control of the degree of haemostatis occurring during vascular damage. The major anticoagulant protein is Antithrombin III, a protein produced in the liver and which requires heparin to operate at endothelial surfaces in order to bind to and inactivate thrombin. Antithrombin III (AT III), because of its low molecular weight, can be lost in forms of renal disease (e.g. nephrotic syndrome), protein losing enteropathy and severely ischemic bowel disorders in horses. Inherited or acquired (e.g. DIC) deficiencies of Antithrombin III can predispose to increased risk of thrombosis (hypercoagulability). Consequently, there are veterinary tests for AT III. Other important naturally occurring anticoagulants include Protein C, Protein S and alpha 2 macroglobulin. Protein C is a vitamin K-dependent protein and can also decrease in acquired hypercoagulable disorders.

Introduction to Bleeding Disorders

A bleeding disorder is characterised by uncontrollable/inexplicable hemorrhage because of altered haemostatis. This excludes hemorrhage through trauma that is controlled by surgical intervention because, apart from the damage to vessels, there is normal haemostatis. Bleeding disorders are characterised by the occurrence of petechiation, ecchymoses and haematomas. Petechiation often occurs on mucous membranes and, therefore, can give rise to epistaxis, haematuria and melena. What type of haemorrhage occurs will depend on what component of the haemostatic process has been disturbed. Vessel wall defects, platelet problems and vW factor deficiency will commonly give rise to petechiation and ecchymoses (all involved in the initial platelet plug – primary haemostatis). General clotting factor deficiencies will commonly give rise to internal tissue haematomas or massive bleeding into cavities (involved in producing the fibrin plug that stabilises the temporary platelet plug – secondary haemostatis). Laboratory investigation of bleeding disorders needs to take into consideration the clinical signs, especially the type of haemorrhage. The following provides basic information on laboratory tests.

Laboratory Evaluation of Bleeding Disorders

Bleeding disorders can involve one or more of the following: a) Vessel Wall Defect (Uncommon) b) Thrombocytopenia c) Platelet Function Defect d) Clotting Factor Deficiency e) Excessive Fibrinolysis

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a) Vessel wall defect Laboratory evaluation aims at determining platelet numbers and function and coagulation factors (and possibly fibrinolysis). Vessel wall defects are more difficult to investigate, and because of their rarity, at least in small animals, are usually considered after other causes have been eliminated. Clinical signs are important to decide on what tests to perform. As mentioned, if petechiation, small ecchymoses or bleeding from mucous membranes predominate then thrombocytopenia or platelet function defects should be considered (von Willebrand's disease due to an inherited absolute deficiency of Factor VIII:vW will also cause similar clinical signs. Rarely does it interfere with the intrinsic clotting pathway unless it is completely absent). Vascular defects will also cause petechiation. If massive internal bleeding or external haemorrhage is occurring unrelated to injury then clotting factor defects should be considered. In some diseases more than one component of haemostatis may be affected and this will give rise to a variety of clinical signs (e.g. disseminated intravascular coagulation [DIC]).

b) Thrombocytopenia

Thrombocytopenia is a common cause of a bleeding disorder. Platelet numbers are usually assessed by direct manual counting, although they can be indirectly assessed on peripheral blood films (e.g. in the dog less than 3-4 platelets per oil objective field is considered a significant thrombocytopenia). From manual counts, dogs have platelet numbers greater than 200 x 109/L (cat > 300 x 109/L, horses > 100 x 109/L), but values less than these do not cause spontaneous (as opposed to bleeding after trauma) haemorrhages unless either the value reaches 25-50 x 109/L or there are associated platelet function defects. If values are above these levels (say 50 to 100 x 109/L) and spontaneous bleeding is occurring then other defects may be working in conjunction with the thrombocytopenia. At levels below 100 x 109/L (and especially 75 x 109/L) bleeding with light trauma is possible without other defects in haemostatis being present (Nb. healthy greyhounds can have levels between 100-200 while cavalier King Charles spaniels may occasionally have levels below 100 x 109/L without clinical disease). Macro (mega, giant) platelets have more activity than normal sized platelets (overall activity depends on platelet mass rather than platelet numbers); therefore, lower numbers of these types of platelets can occur without predisposing to spontaneous bleeding.

Causes of thrombocytopenia include collection artefact (i.e. platelet aggregation in the tube), disorders of production (e.g. oestrogen and other drug [e.g. phenylbutazone] toxicity, any cause of myelophthisis), distribution (splenic disorders may cause sequestration but usually not dramatic drops in numbers), utilisation (e.g. DIC) and destruction (e.g. immune mediated; many drugs cause shortened life spans for platelets). Macroplatelets (also called shift platelets) are prominent in peripheral blood and megakaryocytes increase in bone marrow when there is either excessive utilisation or destruction of platelets. When platelet numbers drop below 10 x 109/L the Activated Clotting Time test will usually be affected as it relies on adequate platelet phospholipid to be present. The Activated Partial Thromboplastin Time and the One Stage Prothrombin Time tests will not be interfered with by absolute thrombocytopenia as an exogenous source of platelet phospholipid (or its equivalent) is added (but they will be affected in vivo).

Primary immune-mediated thrombocytopenia may occur alone or in combination with other immune-mediated diseases. The anti-PF3 test is an indirect assay for anti-platelet antibodies. Platelet numbers and function are affected (the antibodies coat the platelets thereby allowing destruction of some and interfering with function of others).

c) Platelet function defect

Thrombocytopathies may be congenital (rare in domestic animals) or acquired (drug induced e.g. non-steroidal anti-inflammatory agents, or secondary to other diseases e.g. chronic liver or

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kidney disease, myeloproliferative disorders, SLE). As a consequence, platelet dysfunction is not uncommon. However, the level of dysfunction may not be severe enough to cause clinical signs of spontaneous haemorrhage. Platelet function assessment is difficult but tests include:

i) Bleeding time (BT This is determined from a standard incision made in mucous membrane (buccal mucosal bleeding time) or hairless skin. BT is prolonged in thrombocytopenia, platelet function defects, some cases of von Willebrand's disease, severe fibrinolysis and vessel wall defects. BT is a measurement of small vessel haemostatis which does not require clotting factors in the first instance.

Nb. deficiencies of clotting factors will not prolong BT but because there is no fibrin stabilisation of the platelet plug re-bleeding may occur a few hours later.

ii) Clot retraction A contractile protein, thrombasthenin, is produced by platelets and is responsible for strengthening the haemostatic plug by pulling the fibrin strands taut. This leads to clot retraction in vivo and in vitro. Failure of clot retraction (i.e. serum separation) in vitro (the basis of the clot retraction test) can be due to platelet function defects, thrombocytopenia and lack of fibrinogen. This test is rarely done.

iii) Others These are sophisticated laboratory tests that either detect defects of platelet adhesion or aggregation. Usually they are not asked for unless the other causes of a bleeding disorder have been excluded.

d) Clotting factor deficiency

These can be initially assessed by the Activated Clotting Time (ACT). The test measures the time required for fibrin clot formation in non-anticoagulated blood in vitro. It is prolonged in deficiencies or inhibition of intrinsic or common clotting factors. The test requires a standardised technique and has low sensitivity (the factor deficiency must be less than 5% of the normal level to prolong ACT). Greater sensitivity is provided by the citrated plasma clotting tests. They include: • Activated Partial Thromboplastin Time (APTT) • One Stage Prothrombin Time (OSPT) • Thrombin Clotting Time (TCT)

Other citrated plasma clotting tests are used once a problem has been detected e.g. specific factor analysis (add specific factors and re-perform APTT or OSPT). The results of all tests should be compared to the results for control animals.

Activated Partial Thromboplastin Time (APTT) APTT measures the time required for fibrin clot in recalcified (calcium is bound by citrate and is essential for clotting), fresh, citrated plasma after addition of a contact activator and phospholipid in vitro. The APTT yields the same, but more precise, information as clotting time (CT) i.e. it measures the intrinsic and common pathways of clotting factors (which includes fibrinogen). Note the APTT

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is prolonged only when the deficient factor is less than 30% of the normal level i.e. haemophiliac carriers having 40-60% of the normal level of factor VIII are not detected by APTT. Fibrinogen of less than 0.5 g/L may also prolong APTT. Fibrinogen, factors VIII and V may increase in inflammatory disease (acute phase reactants) leading to a shortened APTT.

One Stage Prothrombin Time (OSPT) OSPT measures the time required for Fibrin clot formation in recalcified, fresh, citrated plasma after addition of tissue thromboplastin in vitro (tissue thromboplastin activates extrinsic system of clotting factors in vivo). A prolonged OSPT occurs in Factor VII deficiency (extrinsic clotting factor) or in common system factor deficiencies (factors need to be less than 30% of normal level). Fibrinogen of less than 0.5 g/L may also prolong OSPT.

Thrombin Clotting Time (TCT) TCT measures the time required for fibrin clot formation in recalcified, fresh, citrated plasma after addition of thrombin (activated factor II that converts fibrinogen to fibrin). The TCT measures the common pathway. By adding excess thrombin, the TCT can be modified to measure fibrinogen. It is one of the more sensitive ways of detecting decreases in fibrinogen concentration. A crude method of detecting changes in fibrinogen concentration (especially increases) is by measuring the difference between total serum and plasma protein or by direct measurement after heat precipitation of fibrinogen in a microhematocrit tube (see practical notes). e) Excessive fibrinolysis

Fibrinolysis is activated at the same time as coagulation by plasminogen activators released from injured endothelium. Plasmin acts locally on the clot to degrade fibrin and factors V and VIII. Fibrin (fibrinogen) degradation products (FDP) are normally removed by macrophages but if there is excessive fibrinolysis, excess fibrin (Fibrinogen) degradation products can inhibit clot formation (affect platelet plugging and fibrinogen polymerisation).

FDP can be measured and the test is mainly used to detect increased levels in DIC (increases also occur in severe internal bleeding and in thrombotic disease such as acute pulmonary thrombosis). D-dimer is a specific by-product of plasma degradation of cross-linked fibrin (i.e. one type of FDP) and can be measured in dogs to detect DIC or pulmonary thrombosis.

Summary

All the tests for bleeding disorders have inadequacies but if they are used in combination they can help determine the likely cause. Vascular defects and platelet function defects are most difficult to assess, but fortunately they are rare as primary causes of bleeding disorders. At the University of Sydney a platelet count, fibrinogen estimation, OSPT and APTT are used to initially assess a bleeding disorder. This helps differentiate common disorders such as pure thrombocytopenia, DIC, Vitamin K deficiency or antagonism [coumarin poisoning], and hereditary factor deficiency. Hepatic insufficiency may contribute to a bleeding disorder but usually the other effects of hepatic insufficiency are more important. On the basis of the results of these tests more specific testing can be undertaken. History is always very useful in differentiating causes of bleeding disorders. The following table shows the likely laboratory results for some of the more common bleeding disorders.

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Laboratory results for some common bleeding disorders   Platelet 

count APPT  OSPT  Fibrinogen  FDP 

Thrombocytopenia  Decrease  Normal  Normal  Normal  Normal Platelet  function defect Thrombocytopathy) 

Normal  Normal  Normal  Normal  Normal 

Vit  K  antagonism  or deficiency 

Normal  Extended  Extended  Normal  Normal 

Heritable factor deficiency 

Normal  Variable (often  normal for FVIII:vW) 

Variable (often normal  for FVIII:vW) 

Normal unless primary deficiency 

Normal 

Acute DIC  Decrease  Extended  Extended  Decrease  Increase Hepatic insufficiency  Normal  Extended  Extended  Decrease 

(variable) Normal 

Hereditary coagulation disorders in domestic animals are well documented, especially in the dog. They are not common in the cat and, in all species; they are probably not as important as other causes of bleeding disorders in the population as a whole. In the dog, haemophilia A (F VIII:C), haemophilia B (F IX) and von Willebrand's disease (FVIII:vW) are the more common hereditary factor deficiencies. Breed dispositions do occur and this may be useful in diagnosis. Disseminated intravascular coagulation (DIC) can occur in a variety of conditions characterised by extensive tissue or vessel damage (trauma, surgery, neoplasia, infections). The damage leads to widespread activation of clotting mechanisms as well as fibrinolysis (consumptive coagulopathy), which eventually becomes self-perpetuating. Initial activation may be due to the release of factors, such as tissue thromboplastin (or a thromboplastin like substance) from neoplastic cells or inflammatory altered moncytes, directly into the blood stream. The initial effect is widespread microvascular thrombosis (also aided by decreased levels of anticoagulants such as Antithrombin III and Protein C; and impaired fibrinolysis) which can give rise to tissue hypoxia and dysfunction, as well as incitement of systemic inflammation through the release of inflammatory mediators. Eventually, the consumptive coagulopathy may give rise to widespread bleeding because of the lack of platelets, clotting factors and increased fibrinolysis. In dogs, bleeding is commonly seen in clinical DIC, but is less common in cats and horses. In those species, clinical DIC often presents with non-specific signs related to tissue hypoxia due to the microvascular (small and medium vessels) thrombosis. DIC also varies in degree and, consequently, can give variable laboratory test results. In the severe cases there is thrombocytopenia, prolonged APTT and OSPT, decreased fibrinogen and increased FDP and D-dimer. Schistocytes (and occasional keratocytes and spherocytes) are common on peripheral blood films due to fibrin strand damage in small vessels. Sub-clinical DIC is probably more common than clinical DIC. It may only be detected by FDP/D-dimer analysis or by the examination of peripheral blood films for schistocytes.

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IMMUNODIAGNOSTICS (for Diseases with an Immune Base

– Immune-Mediated Diseases & Immunodeficiency)

Introduction

In immune mediated (related) diseases most of the damage done to the tissues is due to the action of antibodies, complement and effector cells (i.e. the host’s immune response). The agent (antigen) triggers the immune response but often does limited direct damage itself. Hypersensitivities have been loosely referred to as immune mediated disease but most immune mediated disease relates to so called ‘autoimmunity’. Hypersensitivities are also called allergies and they describe a variety of reactions that produce inadvertent significant tissue damage (i.e. the immune response to the foreign antigen causes inflammation which secondarily damages tissue to the extent that clinical signs are produced - any immune response to a foreign antigen will cause some secondary tissue damage but it is usually limited). Autoimmunity causes direct tissue damage because of a reaction (usually involving antibodies) to attached antigens regarded as ‘foreign’ by the host’s immune system. In the past few years researchers have come to realise that cases of ’autoimmunity’ may not only be due to the production of true autoantibodies (primary) against normal tissue antigens (self-antigens), but also due to the production of antibodies directed against an antigen (foreign) which is bound to tissues or cells (secondary). The mechanisms of immune reactions in autoimmunity are the same as occur in hypersensitivity reactions and in normal immune responses which aim to remove foreign antigens with minimal tissue damage. Types I, II and III mechanisms are antibody mediated while Type IV is mediated by T lymphocytes. However, for these mechanisms to operate effectively, there often needs to be co-operation between humoral factors and T lymphocytes. Type I involves the synthesis and binding to mast cells and basophils of IgE (reaginic or homocytotropic antibody) in response to exposure to allergens. Degranulation of the bound cells leads to the clinical signs (e.g. bronchoconstriction in allergic bronchitis). This is a common type of hypersensitivity in skin, gut, gastrointestinal tract and urogenital tract (anywhere Mast cells are in abundance). Systemic anaphylaxis, canine and feline respiratory and gastrointestinal allergies, bovine allergic rhinitis and equine urticaria are all examples of Type I hypersensitivities. Type I can also operate in conjunction with other mechanisms in certain hypersensitivities (e.g. canine flea allergy has elements of both Type I and Type IV reactions). Kits for measuring IgE are available. Type II reactions are mediated by complement fixing antibodies (usually IgG and IgM and called cytotoxic antibodies) directed to cell surface antigens. Autoimmune haemolytic anaemia and neonatal isoerythrolysis are examples of Type II reactions. Drug and parasitic antigens attached to cell surfaces (e.g. Haemobartonellosis and erythrocytes) will initiate a Type II reaction. Pemphigus in dogs may involve a Type II reaction. Glomerulonephritis due to autoantibodies against glomerular basement membrane is another example of a Type II reaction. Type III reactions are mediated by immune complexes formed from antigen and antibody (often IgG). Sometimes the antigen is a pathogenic agent, other times it is a self-antigen. The immune complexes, when large enough, lodge in glomeruli and small blood vessels. Complement is activated and local neutrophilic inflammation occurs. This causes tissue damage and dysfunction (vasculitis in various

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organs or generalised and glomerulonephritis). Examples of Type III mediated disease include glomerulonephritis in a wide range of domestic species, dermatitis due to immune complex-mediated vasculitis and systemic lupus erythematosus (SLE). Type IV reactions are mediated by sensitised T lymphocytes. These in turn release a number of cytokines that directly or indirectly (often via macrophages) lead to death of tissue cells. Type IV may be involved in canine flea allergy and in canine lymphocytic thyroiditis.

Autoimmunity

It is not always possible to specifically define an autoimmune disease but available tests should allow the detection of immune mediated disease (e.g. a polyarthritis can be assessed as being immune mediated but it is not always possible to determine whether this is rheumatoid arthritis, systemic lupus erythematosus or some other form). To diagnose autoimmune disease, history and clinical examination are essential. Laboratory investigation can be divided into two parts: the first part relies on the use of routine laboratory tests to establish which organs or tissues are involved and that the disease is inflammatory (and, therefore, likely to be immune mediated): a) Haematology (general organ/tissue assessment) b) Biochemistry (general organ/tissue assessment) c) Urinalysis (specifically for glomerulonephritis), joint fluid analysis etc Once these tests are done, it may be worthwhile to look specifically at immunoglobulin. d) examination of humoral immune function:

i) serum protein electrophoresis ( globulins) ii) immunoelectrophoresis of immunoglobulins iii) single radial immunodiffusion (immunoglobulin quantities) iv) examination of the complement cascade v) examination of cell mediated immunity

Once these tests have suggested that the disease is immune mediated then the second part of laboratory investigation can be undertaken. This involves the use of specialised immunodiagnostic tests to more specifically define the problem. a) Coombs' test (direct)

This test aims at detecting antibodies or complement on the surface of circulating erythrocytes or red cell precursors present in the bone marrow. The test is used to diagnose autoimmune haemolytic anaemia (AIHA) which can occur as a primary condition or as a component of a multisystem autoimmune disease. It may also occur secondary to a wide range of non-specific diseases e.g. neoplasia such as lymphosarcoma. Titres of antibodies in AIHA are highest for the primary form of the disease. Autoimmune thrombocytopenia may accompany AIHA (called Evans Syndrome), but it can also be a distinct entity in itself. AIHA occurs in the dog, cat, horse and cow but is more common in the dog. Haemolysis can occur intravascularly or extravascularly and the formation of spherocytes is common. Before performing the Coombs' test evidence for a haemolytic anaemia should be obtained.

b) Rheumatoid factor (RF)

This is an IgM (or IgG) autoantibody directed against the Fc of IgG that has been altered. The resulting complex fixes complement whose activation leads to cartilaginous damage (rheumatoid

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arthritis RA). RF detection aids in the diagnosis of rheumatoid arthritis, although RF may be present in many autoimmune disorders and RF negative rheumatoid arthritis has been documented. RA diagnosis depends on characteristic radiological changes, synovial fluid changes as well as on the presence of RF. In the dog 40-75% of cases of RA have RF.

c) The antinuclear antibody test (ANA)

This test has a non-specific usage in the diagnosis of autoimmune disease but is essential for the confirmation of systemic lupus erythematosus (SLE). The basis of the test is the detection of antibody directed against various nuclear proteins. This test requires specific antisera. In the dog ANA occurs in a low percentage of the normal population but at a low titre. Autoimmune diseases other than SLE may have low titres of ANA (hence its non-specific usage). The ANA test has replaced the LE cell test (40% of SLE cases will be negative for LE cells). SLE has only been positively diagnosed in the dog and laboratory rodents, although probable cases have been reported in the cat.

d) Direct and indirect immunofluorescence (IF) of tissue biopsies

This is useful to detect various autoantibodies to various cell types, e.g. autoimmune skin disease such as pemphigus vulgaris; glomerulonephritis. Direct IF is supposedly better than indirect IF. Instead of immunofluorescence to detect the autoantibodies, tissue sections can be labelled by an immunoperoxidase method: autoantibodies present in tissues will then stain brown when viewed with a conventional light microscope.

Immunodeficiency

Immunodeficiency may be heritable or acquired and involve defects of one or more arms of the immune system (commonly cell mediated and humoral defects). The dog and horse are most commonly affected by immunodeficiency. Immunodeficiency should be suspected in recurrent cases of increased susceptibility to low grade pathogens or to unusual pathogens, and poor response to therapy. Laboratory assessment of humoral function (Ig quantitation, lymph node biopsy, quantitation of circulating B cells) cell mediated immune function (total blood lymphocytes, lymph node biopsy, blastogenesis), complement levels and phagocytic function (e.g. as in cyclic haematopoiesis in the Collie dog) should be undertaken if appropriate. *SEE CASE 21

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DIAGNOSTIC CYTOLOGY AND BODY FLUID ANALYSIS

Diagnostic Cytology (Exfoliative Cytology; Cytopathology)

Diagnostic cytology is the examination of individual cell details for the purpose of diagnosis and prognosis. Although more information can usually be gained from the examination of tissues (histopathology) rather than individual cells, cytology does have distinct advantages over histopathology: a) Cytological samples are cheaper and easier to obtain b) Cytological samples are simpler to process. Consequently, the result from a cytological

preparation is quickly obtained. c) Agents of disease are sometimes`easier to detect because of better resolution at 100 x

magnification Cytological preparations may be stained with blood stains, e.g. Giemsa, Diff Quik®. Commonly, a diagnosis on the basis of cytological data is limited and broad. Histopathological examination usually needs to be done to support the cytological findings (by biopsy or at necropsy), and is the only way that tissue architecture (including margins of a lesion) can be examined. a) Solid tissue cytology

i) Tissue imprints/scrapings are useful for examining accessible soft tissues (in the living animal, from biopsy material, from necropsy material). The surface of the tissue is blotted free of debris and blood and a slide touched onto the exposed surface. The imprint allows examination of individual cell details, cell relationships (if the imprint is performed carefully) and examination for the presence of etiological agents. In firm tissues or in sites inaccessible to imprints (e.g. conjunctiva) tissue scrapings can be done. The tissue is cleaned as for an imprint and then material collected on the `scraper' is then gently spread over a slide (‘buttering’) or transferred to a slide and spread as for a peripheral blood film.

ii) Fine needle cell aspirates are principally of use in the living animal (examination of internal and external solid/fluid masses). The cellular material is aspirated with a fine gauge needle (usually 21-23) and syringe (2 ml or 5 ml commonly) and then placed on a slide and gently smeared (as a squash preparation or as a for a peripheral blood film).

Solid tissue cytology is approached the same way as histopathology: detect, describe and deduce (the three ‘Ds’ for diagnosis). Since cytological smears are often examined under the 100x objective, the focus for description is cellular detail, although both acellular material and living and non-living agents of disease may be detected. The interpretation is based firstly on the five basic pathological processes (degeneration and necrosis; inflammation and repair; vascular disturbance; disorder of growth; and pigmentation/deposit). In most cases, the lesions are proliferative (hyperplastic or neoplastic – i.e. a disorder of growth), inflammatory or degenerative (the last including pigments and deposits) (see Table 1). Further differentiation will depend on cytological and acellular features. Neoplastic lesions can often be differentiated into benign or malignant epithelial, mesenchymal (connective tissue) and round cell types. Solid tissue cytology is particularly useful for detecting and identifying round cell neoplasms (e.g. mastocytoma, lymphosarcoma, benign cutaneous histiocytoma). Inflammatory lesions may be differentiated into acute and chronic (including granulomatous) processes. Cytology is also useful for detecting agents of disease. These may be physical (e.g. foreign bodies), living (e.g. acid fast bacteria, toxoplasmal organisms, chlamydial organisms) or chemical (e.g. calcification of tissue).

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An example of the use of solid tissue cytology would be to investigate lymphadenomegaly. A fine needle cell aspirate of an enlarged lymph node may be able to distinguish among benign lymphoid hyperplasia, lymphadenitis, lymphosarcoma and metastatic neoplasia.

1. Primary degenerative lesions These include those lesions that are cystic in nature (e.g. epidermal cyst, renal pseudocyst or cyst), those that are due to deposits of minerals or pigments (e.g. calcium in calcinosis circumscripta, haemosiderin in haemorrhages, silicon or asbestos in lung macrophages), and those that are due to damage (e.g. physical trauma or toxic substances).

2. Inflammatory lesions Inflammatory lesions are characterised primarily by the presence of inflammatory cells or by the presence of infectious agents (e.g. bacteria, viruses, fungi). Cytology is extremely useful in detecting infectious agents and may at times provide positive identification without further investigation; for example the detection of Distemper virus or chlamydial inclusions in conjunctival smears. However, in most instances, further investigation (e.g. culture, molecular studies) is required to positively identify the infectious agent.

Lesions caused by infectious agents often feature large numbers of inflammatory cells. Occasionally, as in some cases of cryptococcosis or clostridial hepatitis, few inflammatory cells are present. These lesions are still classified as inflammatory. Not all inflammatory lesions are caused by infectious agents. Other causes include foreign material (e.g. sterile penetration wound; injected irritant) or endogenous irritants (e.g. ruptured epidermal cyst and release of keratin; bile or urine escape into the abdomen).

Inflammatory lesions, apart from being characterised by the presence of their cause, can be further classified by the types of inflammatory cells present. This can provide clues to the cause of the problem.

Neutrophils indicate acute inflammation in response to damage or to infective agents such as bacteria. Neutrophils can also form a component of ongoing ('chronic') inflammation if the chemotactic factor (such as bacteria) is persistent in the lesion (active chronic inflammation). The term purulent exudate refers to a neutrophil predominance and is often further classified as septic (bacteria are present, especially within neutrophils or non-septic (other causes such as fungi, sterile irritants or immune-mediated disease).

Based mainly on nuclear morphology, neutrophils can be described as lytic or nonlytic (degenerate and non-degenerate). The nucleus of lytic neutrophils appears faded and can have ragged or distorted edges. Lytic neutrophils develop under the influence of toxins and are often interpreted as indicating the presence of bacteria. Lytic neutrophils, however, can also be seen in some viral infections (feline infectious peritonitis) or as a result of exposure to chemical toxins as in bile peritonitis or uroperitoneum. Not all septic exudates feature lytic neutrophils. The term “degenerate” is often used interchangeably with “lytic”.

Eosinophils commonly accompany lesions which have a strong immune component and which usually take some time to develop (e.g. response to some parasites, allergies). They may also be present in lesions involving mast cells (e.g. mastocytoma in dogs; inflammatory lesions involving the skin, respiratory tract, urogenital tract or gastrointestinal tract) or other neoplastic cells (paraneoplastic phenomenon due to the production of chemicals that stimulate eosinopoiesis and/or attract eosinophils). Some predominantly eosinophilic inflammatory lesions are of unknown cause (e.g. the eosinophilic granuloma complex in cats, idiopathic pulmonary infiltrates with eosinophils in dogs).

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Macrophages (also called histiocytes) are important for the phagocytosis of macromolecular material. They usually form a component of chronic inflammatory lesions and are enlisted by a demand for phagocytosis and by lymphocyte derived cytokines. Macrophages can also be present very early on in an inflammatory reaction if the need for phagocytosis is great e.g. in the face of much tissue damage or foreign debris. This is especially so in certain organs or tissues such as the lower airways.

The predominance of macrophages, especially large, aggregated or multinucleate macrophages, in an inflammatory lesion may suggest a granulomatous response. This is a special form of chronic inflammation defined by a particular histopathological arrangement of macrophages that may be caused by certain agents of disease such as foreign debris, certain fungi, protozoa, multicellular parasites and bacteria (e.g. staphylococcal, mycobacterial, nocardial organisms). Many of these agents of disease can also cause a pyogranulomatous response in which both neutrophils and macrophages are found in significant numbers.

Lymphocytes and plasma cells are present in lesions due to adaptive immune stimulation and commonly accompany prolonged inflammation. However, in some skin lesions, such as allergies, they can accumulate quickly. While their presence suggests an immune component to the inflammation, it does not automatically mean that the immune response is the primary cause of the problem (i.e. autoimmunity or hypersensitivity).

Fibroblasts are normally present in chronic inflammation (therefore, also in granulomatous inflammation) and in healing wound. Be careful, reactive fibroblasts can be quite pleomorphic and can overlap in appearance with neoplastic spindle cells.

3. Proliferative (hyperplastic/neoplastic) lesions Because hyperplastic and neoplastic lesions tend to produce lumps and bumps that are relatively easy to sample, this is the most common category to be diagnosed by cytology.

As mentioned previously, inflammation can accompany many tumours and can be the more prominent feature. Neoplasms can cause damage to surrounding tissue and many tumours have a high cell turnover. Both these processes produce factors that are chemotactic for inflammatory cells. Examples include the intense inflammation that commonly accompanies a squamous cell carcinoma (the irritant nature of free keratin also contributes to the inflammation); the nonseptic exudates that accompany neoplastic cells exfoliating into the pleural, pericardial and peritoneal cavities; septic inflammation due to clostridial infection of necrotic liver tumours and septic peritonitis secondary to a perforating neoplasm of the gastrointestinal tract. If tumour necrosis is extensive, the neoplastic cells can be difficult to find or identify. Experienced cytopathologists realise this and often warn the practitioner with the phrase 'suspect underlying neoplasia as a cause'.

Much has been written about diagnosing neoplasia by cytology, especially in relation to certain tumours such as mast cell neoplasia. The suggested approach involves a logical, stepwise approach using general principles. It allows identification of the neoplastic process and often leads to diagnosis of the specific neoplasm. Questions to be asked, in order of importance, are:

1. Is the lesion primarily proliferative (disorder of growth) and, if so, is it hyperplastic,

benign or malignant?

Proliferative lesions can be hyperplastic or neoplastic, benign or malignant. Differentiation can be extremely difficult and is not always straightforward based on nuclear and cytoplasmic features.

It is probably not critical or possible to distinguish between hyperplastic and benign neoplastic processes on cytology. Detection of malignancy, however, is highly desirable but, again, not always possible. The hallmarks of malignancy are anaplasia, invasion, and metastasis. The presence of

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invasion into surrounding tissue can only be assessed on gross and histological examination. Metastases can be detected by imaging and cytology e.g. aspiration of lymph nodes, organ masses. Anaplasia, which refers to the fact that the neoplastic cells are poorly differentiated and, consequently, highly variable in appearance, can be assessed cytologically. Anaplasia is a common feature of malignancy, but does not have to be present. Some malignant neoplasms can have relatively 'benign' cellular features (i.e. the cells look like well differentiated origin cells and show only slight nuclear and cytoplasmic variation). A common example would be some thyroid carcinomas in the dog. Even the cells from their metastases can look relatively well differentiated and be in a glandular pattern!

Despite this limitation, many tumours can be designated as malignant on cytological features. Although it is difficult to make rules for cells that break rules to become established and spread, it can generally be said that the more bizarre the cells appear the more likely are they to be malignant (exceptions – plasmacytomas, fibroblasts). Cytological features that are most likely to indicate malignancy include: numerous and abnormal mitoses, cell and nuclear pleomorphism, cytoplasmic basophilia, enlarged or bizarre nucleoli, and multinucleation (especially with variably sized nuclei).

If you suspect neoplasia from the history but your cytological sample is not supportive, don’t disregard your suspicion. Re-aspirate if the answer is not clear cut. Neoplastic cells may be difficult to detect, especially if there has been blood dilution, tissue necrosis or masking by inflammatory cells as mentioned previously.

2. Are the predominant cells round, spindle, or epithelial?

These are the three basic cell groups responsible for proliferative lesions and are classified according to cell shape and embryonic origin.

Round cells include all haematopoietic cells (including lympho-histiocytic cells) and mast cells. As suggested, the cells are round in shape. They usually show little tendency to aggregate in smears (unless the smear is thick) and are of embryonic mesoderm origin.

Spindle cells suggest a mesenchymal (embryonic mesoderm) origin (i.e. all connective tissues, muscle, vessels etc). Not all spindle cells are tapering, some can appear rounded or angular. While spindle cells typically occur as individual cells in smears, some mesenchymal neoplasms exfoliate well to produce epithelial-like aggregates of cells e.g. haemangiopericytomas.

Epithelial tumours are derived from ectoderm or endoderm. Cells vary from square to columnar, and at least some should be found in groups in smears.

Some specific tumours derived from neuroectoderm (melanocytes and Schwann cells) can present in either spindle or epithelial forms. Malignant melanocytes may even present as round cells.

3. Can the cell types be subcategorised?

Subcategorisation depends on the detection of specific arrangements of cells or the production of a cell product. For example, if the epithelial cells are in a duct or acinus arrangement or if they are in a solid clump but have an intracellular product, then they are identified as glandular. Intracytoplasmic melanin will suggest a melanotic tumour while intracytoplasmic, purple granules could suggest mast cell neoplasia.

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TABLE  1:  Approach  to  interpretation  of  cytological  smears  (based  on  predominant pathological process) 

 Predominant pathological process 

 Main feature for characterisation 

 Usefulness of cell type 

 Basis for further characterisation 

 Primary degenerative changes 

 cysts (e.g. renal cyst) 

  

  

  

 damage (e.g. burn, trauma) 

  

  

  

 deposits and pigments (e.g. calcinosis circumscripta) 

  

  

 Primary inflammatory lesions (i.e. not secondary to primary degenerative lesions or neoplastic conditions) 

 On predominant cell type 

 neutrophils (purulent inflammation ‐ septic due to bacteria; non‐septic due to other causes) 

  

  

  

 eosinophils (strong immune component in the inflammation) 

  

  

  

 macrophages (histiocytes) ‐ epithelioid cells and multinucleates may indicate granulomatous inflammation 

  

  

  

 lymphocytes and plasma cells indicate chronic immune stimulation 

  

 glandular or ductular in arrangement 

 Hyperplastic and neoplastic lesions   

 On main cell type   

 Epithelial     

squamous differentiation   

  

 Spindle 

 pattern and acellular deposits may allow differentiation 

  

  

 Round 

 mastocytoma, lymphosarcoma, haematopoietic tumours, histiocytic tumours 

  

  

 variable shape (some mesodermal and neuroectodermal tumours) 

 melanomas should have cytoplasmic pigment; osteosarcomas should be producing osteoid 

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b) Diagnostic cytology as a component of body fluid analysis Cytological preparations for fluids can be obtained by direct smearing of the fluid or by centrifuging the fluid and smearing the sediment. Fluids of an inflammatory nature should be placed in anticoagulant (e.g. EDTA) as clotting interferes with smear preparation. Interpretation of the cell smears will depend on the site of origin of the fluid and will be discussed under body fluid analysis. Nb. Cells deteriorate rapidly in body fluids. If a delay is envisaged before the sample reaches the laboratory then smears should be made directly from the fluid prior to despatch.

Body FLuid Analysis

a) Body cavity effusions This is mainly referring to abdominal and thoracic fluids but there is applicability to pericardial and other cavities)

An effusion refers to a pouring out of any fluid into a body cavity or tissue, i.e. excess fluid. By assessing gross characteristics and determining protein levels (by refractometer or by the chemical Biuret method), total nucleated cell count, and differential cell count (on a smear). Abdominal and thoracic cavity fluid is commonly assessed when there is an effusion. In cattle and horses, abdominal cavity fluid may be assessed as part of an abdominal cavity disease investigation even when increased fluid (an effusion) is not present. Effusions can be divided into:

i) Pure transudates These may occur in hypoproteinemic states due to lowered plasma osmotic pressure and in some forms of chronic liver disease in the dog. They are really normal body fluid in excess (therefore low protein and low cells). These are non-inflammatory and uncommon.

ii) Modified transudates These are still non-inflammatory in origin. They are modified by the addition of protein or cells (but limited compared to values for exudate), e.g. ascites due to chronic passive congestion, neoplasia, chylothorax. These are very common.

Nb. A process may start off as a pure transudate and, with time, progress to a modified transudate and even to a non-septic exudate (once serum protein components, neoplastic cells etc start to accumulate they may create chemotaxis for phagocytes, thereby causing secondary inflammation). Consequently, it may be difficult at times to separate some modified transudates from early non-septic exudates due to variable alterations of protein and cells. Chronic heart failure in the dog tends to cause thoracic and abdominal effusions that quickly pass from pure transudates to modified transudates (erythrocytes are common in the fluid). Occasionally they may progress to non-septic exudates. Chronic cardiac disease in the cat can cause thoracic effusions, which are commonly classified as chylous (i.e. chylothorax).

iii) Chylous effusions These most commonly occur in the thorax (but can be present in the abdomen) and are assumed to be due to leakage from major or minor lymphatics. Consequently, chylous effusions are characterised by many small lymphocytes and high triglycerides (higher values than in plasma).

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A variety of conditions can give rise to chylothorax (e.g. neoplastic interference to lymphatic flow, trauma to lymphatics, heartworm, idiopathic), but the most common cause in the cat is chronic heart disease. In contrast, chylous effusion in the thorax of the dog is less common than in the cat and is rarely associated with chronic heart disease. Instead, it is most commonly due to thoracic duct compression (rarely rupture), but may occasional occur in other diseases affecting lymphatics. Most chylous effusions are milky white but exceptions do occur. In addition, some milky white fluids may be pseudochylous. These are extremely rare. They are turbid fluids that can be caused by exfoliating neoplasms or tissue inflammation. With cell breakdown large amounts of cholesterol build up in the fluid. However, the milky colour of the fluid is not due to fat but due to cell debris and chemical complexes). To differentiate chylous from pseudochylous effusions, it is best to support gross and cytological findings with simultaneous analyses of triglycerides and cholesterol in both effusion and plasma (or just effusion and determine the ratio). Chylous effusions will have high triglycerides and low cholesterol effusion levels when compared to plasma levels; and vice versa for pseudochylous effusions.

iv) Exudates (high protein and high total nucleated cell numbers)

1) non-septic exudate This type of effusion either develops from a modified transudate or is due to direct inflammation caused by irritants that usually produce few toxic changes in neutrophils, e.g. sterile foreign bodies, ruptured urinary bladder (this more commonly causes a modified transudate), bile peritonitis, Feline Infectious Peritonitis (FIP- it has been suggested that this condition gives rise to a modified transudate rather than a non-septic exudate as cell counts are often limited. However, since both cells and protein are increased, and it is known to be an inflammatory process, it is more appropriate to call it a non-septic exudate).

2) septic exudate This type of effusion is the product of inflammation caused by a wide variety of microbes that are toxic to neutrophils (principally causing karyolysis due to interference with water balance). Values for protein and total nucleated cells are high compared to the other categories.

v) Haemorrhagic effusion (recent or long standing) Recent haemorrhage will be characterised by clear supernatant on centrifugation and platelet clumps on examination of the smear. With time, the supernatant will become red-brown or yellow due to degradation of erythrocytes. A smear will show erythrophagocytosis and/or haemosiderin in macrophages.

It is important to remember that the basis for the classification of body fluid effusions depends upon protein estimation, total nucleated cell count and differential cell count on a smear. Values are provided for abdominal fluid analysis (probably can be applied to pleural and pericardial fluid analysis with care): Refer to the following table. Values provided by texts for protein estimations by refractometer and total nucleated cell numbers for the different categories may vary from these. Consequently, the following values should be used as guides and not be regarded as absolute.

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TABLE: Characteristics of body fluid effusions for the dog, cat and horse (mostly applies to peritoneal fluid)

  

Pure transudate*  Modified transudate  Chylous effusion 

Non‐septic exudate 

Septic exudate  Haemorrhagic effusion 

Gross characteristics 

clear/colourless (S); clear/pale yellow (H) 

variable but often cloudy  often turbid white or pink 

Cloudy  marked cloudiness  cloudy red or brown 

Total protein g/L (refractometer) 

<25  25‐50 (S); 25‐30 (H)  >25 (inaccurate due to presence of the fat) 

30‐70  30‐70  variable but usually high 

Total nucleated cell count (x106/L) 

500‐1500 (S); 1500‐10,000 (commonly <7,500) (H) 

300‐5000 (S); 5000‐12,000 (H) 

variable but usually 500‐5000 

5000‐100,000 (S); >12,000 (H) 

5000‐100,000 (S); >12,000 (H) 

variable but usually high 

Differential cell count (smear) 

monocytes, lymphocytes and non‐lytic (non‐degenerate) neutrophils 

as for pure transudate but increased non‐lytic (non‐degenerate) neutrophils and some erythrocytes and reactive mesothelial cells 

Small lymphocytes usually predominate 

mainly non‐lytic (non‐degenerate) neutrophils, some erythrocytes and mesothelial cells 

mainly lytic (degenerate) neutrophils, microbes, some macrophages and erythrocytes 

mixed peripheral blood cells plus macrophages and mesothelial cells 

*also applies to values for normal abdominal fluid 

H = horse; S = dog and cat; if not stated applies to both 

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NOTES ON BODY FLUID EFFUSIONS i) Horse abdominocentesis: The procedure can be done even though significant effusion may not be present (e.g. in evaluation of GIT disturbances, colic). In horses, lactate levels in peritoneal fluid may be analysed in conjunction with plasma levels to assess abdominal problems. In healthy horses lactate levels in peritoneal fluid are usually lower than in plasma. With abdominal crisis lactate levels in peritoneal fluid exceed those in plasma (especially when there is gut hypoperfusion [e.g. strangulation] or sepsis), and the wider the gap, the poorer the prognosis (lactate levels are not used for diagnosing abdominal catastrophes, just for prognosis and response to therapy). Nb. General notes on the use of blood lactate in the horse: Blood lactate (an end product of anaerobic glycolysis) can be measured in the horse as part of a general health assessment. Physiological hyperlactaemia can occur with strenuous exercise, but blood levels are rapidly removed by the liver and kidney. Pathological hyperlactemia occurs when oxygen delivery to tissues is inadequate to meet their demands (tissue hypoxemia due to hypovolemia leading to tissue hypoperfusion e.g. acute blood loss and severe anaemia; plasma volume depletion; septic/endotoxic shock; heart failure), and lactate production exceeds blood buffering and liver/renal clearance (e.g. the commonest reasons are severe liver disease, malignancy, sepsis and endotoxemia, and excess catecholamine release). Blood lactate elevations are, therefore, non-specific and may be elevated in a wide range of diseases. However, levels can be useful in prognosis and response to therapy, especially for estimating inadequacy of tissue perfusion. At the University Veterinary Teaching Hospital Camden, normal lactate concentrations (for L-lactate, which is the mammalian cell form) measured in whole blood in adult horses are <2.0 mmol/L. Normal levels in newborn neonatal foals <36 hours of age are slightly higher at <2.5 mmol/L. Pathological elevations in blood lactate between 2.0-5.0 mmol/L (termed hyperlactatemia) are regarded as mild, moderate between 5.0-7.0 mmol/L (termed lactic acidosis), and severe when >7.0 mmol/L (strenuous exercise is an exception, when under physiological conditions blood lactate levels reach greater than 20.0mmol/L). ii) Pathological haemorrhage needs to be differentiated from iatrogenic hemorrhage. If blood vessel penetration has occurred at the time of abdominocentesis then the blood is often streaky. Splenic penetration will produce a high PCV and many lymphocytes. Erythrocytes can accompany modified transudates and exudates (via diapedesis), but will never reach the levels experienced in haemorrhage due to rhexis. iii) In cattle abdominocentesis has been used to diagnose peritonitis. Total nucleated cells and protein levels, however, are not as useful as the differential. In healthy animals protein levels, as determined by the refractometer, are up to 30 g/L while total nucleated cells are up to 10,000 x 106/L (10 x 109/L). Eosinophils are commonly high in normal peritoneal fluid (>30%) while neutrophils are usually less than 50% (nb. healthy cows, less than two weeks post-partum may have a nucleated cell count greater than 10,000 x 106/L, while either neutrophils or eosinophils may predominate in the differential). In peritonitis, protein levels vary from 20 g/L upwards (top of around 60) while total nucleated cells vary from 1,000 x 106/L upwards (top can be over 200,000). However, more importantly, the proportion of neutrophils increases while the proportion of eosinophils drops. The presence of appropriate bacteria and high fibrinogen levels support the diagnosis of peritonitis. iv) Neoplastic effusions have an extremely variable appearance. Diagnosis is dependent on examination of the cytological smear and identification of neoplastic cells. Malignant neoplasms which commonly exfoliate into body cavities include mesotheliomas, adenocarcinomas and lymphosarcoma. If neoplastic cells have not exfoliated, then the effusion can present as a modified transudate, chylous effusion, an exudate or as a haemorrhagic effusion.

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b) Synovial fluid analysis Introduction Except for septic arthritis, synovial fluid analysis rarely provides a specific diagnosis. What it does do is to provide information that allows placement of the joint problem into a general category (e.g. degenerative, inflammatory). When this is combined with other physical, radiological and laboratory findings a specific diagnosis may be obtained. Synovial fluid is a dialysate of plasma to which mucus is added by synovial cells as the plasma diffuses through the synovial membrane into the joint cavity.

Laboratory evaluation of synovial fluid i) Normal gross characteristics

1) volume: 0.01 - 1 ml for the dog (less in the cat, more in horse)

2) colour: colourless (may be light yellow in the horse) 3) transparency: clear 4) viscosity: noticeably viscous (due to hyaluronic acid content) 5) mucin content: determined by the mucin clot test: One ml of synovial fluid is mixed with 5 ml of 2.5% acetic acid and left for at least 1/2 hour. Normal synovial fluid, which is high in mucin, produces a tight ropy clot in a clear solution (good mucin clot). A fair mucin clot is a soft mass in a slightly turbid solution. A poor mucin clot is a small friable mass in a turbid solution. A very poor mucin clot is a few flecks in a turbid solution.

ii) Normal cytological characteristics A total nucleated cell count is determined from synovial fluid collected in EDTA. A differential cell count is determined from a smear of synovial fluid (preferably made directly from withdrawn synovial fluid). The dog has a total nucleated cell count of less than 3000x106/L (this depends on the joint sampled) while a horse usually has less than 500x106/L. In health, 90% of the nucleated cells are lymphocytes and monocytes.

iii) Other characteristics (investigated when indicated by disease) e.g. Protein levels (mainly of use in the horse- although not completely accurate, the

refractometer can be used to estimate total protein – in a healthy horse the value is usually 10-15 g/L), glucose levels (in health, are often equal or slightly higher than in the serum), certain enzymes (e.g. AST, ALP).

When collecting synovial fluid, place some in an EDTA tube and some in a plain tube (sterile if you suspect septic inflammation). Make a smear immediately from withdrawn fluid. Often in the dog and cat not enough fluid is obtained to do all tests. Priority should be given to the smear (for differential cell count) and a total nucleated cell count, unless the history indicates otherwise

Changes in synovial fluid characteristics with disease: Refer to table on following page. Basically, synovial fluid changes due to disease are categorised as acute degenerative/traumatic, low grade/Long term degenerative or primary inflammatory (non-septic or septic). Note: lytic (degenerate) neutrophils, which are common in septic inflammation anywhere, are sometimes not obvious in septic synovial fluid due to the apparent protective nature of chemicals present. Neoplasms involving joints and causing synovial fluid changes are uncommon.

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TABLE: Synovial fluid analysis showing changes in disease

 Acute degenerative/ traumatic conditions 

Low grade/Long term degenerative conditions 

Primary inflammatory  (non‐septic) 

Primary inflammatory  (septic) 

Volume  Increased  normal to increased  increased  increased 

Colour  normal to discoloured (red) normal to discoloured (red‐

brown) discoloured  discoloured 

Turbidity  slight to marked  normal to slight  slight to marked  marked 

Fibrin clot  ‐ve to +ve  ‐ve  +ve  +ve 

Viscosity  Variable  normal to slight  decreased  decreased 

Mucin  fair to poor  normal to fair  fair to very poor  poor to very poor 

Total nucleated cells  slight to moderate  normal to slight increase  moderate to marked increase 

Differential cell count  erythrocytes, neutrophils macrophages (cartilage fragments may be seen) 

mainly neutrophils (non‐lytic (non‐degenerate) for non‐septic, usually lytic (degenerate) for septic) plus erythrocytes (via diapedesis), macrophages and 

lymphocytes (microbes for septic) 

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c) Cerebrospinal fluid evaluation

Introduction Cerebrospinal fluid (CSF) is produced partly by diffusion from plasma and partly by active secretion from the choroid plexi and ependymal linings. In the dog and cat, CSF is collected usually from the subarachnoid space at the atlanto-occipital articulation. In the horse and dog the subarachnoid space at the lumbosacral articulation can be used.

Laboratory evaluation of CSF

i) Physical Examination 1) Colour

In the healthy dog, cat and horse CSF is colourless. Xanthochromia (yellowing of the CSF) may occur in a jaundiced animal or in an animal that has had previous haemorrhage in the CSF. Red CSF may indicate recent haemorrhage (pink to orange supernatant) or haemorrhage at the time of collection (clear supernatant). A grey-green CSF may indicate suppuration.

2) Turbidity

Normal CSF is clear. A turbid CSF suggests excess cells (turbidity is only observed when the cell count is in excess of 500x106/L) or perhaps protein or bacteria.

3) Coagulation

Normal CSF doesn't clot. Clotting may be due to inflammation or haemorrhage.

ii) Total nucleated cell count CSF is collected in an EDTA tube (in case of risk of clotting) and in a plain serum tube (for protein estimation). If infection is suspected some CSF should be collected in a sterile tube. The total nucleated cell count (usually done on the EDTA tube) has to be performed quickly as there is rapid degeneration of cells (usually within a few hours). Pleocytosis (an increase in the number of nucleated cells in CSF-in health: the healthy dog, cat and horse have counts usually 5 x 106/L or below; 5-8 is a grey zone for increases) may be due to trauma, primary inflammatory conditions, toxic conditions or neoplasia. Pleocytosis requires the performance of a differential cell count (if total cell count is greater than 500x106/L then a smear should be made directly from the CSF, if the total count is less than 500x106/L then ideally a smear should be made of the sediment of CSF after unit centrifugation).

iii) Differential cell counts In practice, smears should be made at the time of collection, irrespective of total nucleated cell count. This is because cells in CSF deteriorate rapidly. In a normal sample most cells are small lymphocytes (around 60-70%) and monocytes/macrophages (20-30%). In pyogenic infections the cells are mostly lytic (degenerate) neutrophils. In haemorrhage the cells are mostly non-lytic (non-degenerate) neutrophils. In viral, uremic, some chronic bacterial and toxic conditions, lymphocytes and monocytes usually predominate. Neoplastic cells may be seen in meningeal tumours.

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iv) Protein estimation Normal CSF has a low level of protein (less than 0.3 g/L for dog, 0.2 g/L for cat and 0.67 g/L for horse). Most of the protein is albumin. Increases in CSF protein may occur in a variety of inflammatory conditions (primary or secondary) and principally involve increases in globulins. To detect increased levels of protein, either total protein (ponceau S method) or globulin (Pandy test) levels can be determined (urine protein reagent strips will provide an indication of increased protein).

General comments on disease affecting CSF The greatest alterations to CSF usually occur with meningeal disease. Nervous parenchymal disease may cause minimal changes to the CSF. CSF analysis is an aid to diagnosis and requires to be analysed in conjunction with history, clinical signs and other diagnostic procedures performed. Commonly, CSF changes are categorised as degenerative (e.g. disc disease), inflammatory (sub-categories include non-suppurative, suppurative and granulomatous depending on proportions of cells present) and neoplastic (primary or metastatic). Sometimes, infectious agents may be detected on the cytological smear (e.g. cryptococcal and toxoplasmal agents).

*SEE CASES 14-16, 25, 26, 30 d) Analysis of airway and pulmonary lesions by respiratory washes (transtracheal aspirates and bronchoalveolar lavages) Respiratory washes for airway and pulmonary disease can be performed in any animal species, but is commonly done in the dog, cat and horse

Sampling techniques Collection of cells from the tracheal and bronchoaveolar surfaces can be done in a number of ways. Bronchoaveolar lavage (BAL), via a bronchoscope or catheter, provides the best cellular samples of the lower respiratory tract (alveolar spaces and smaller airways). Occasionally, oropharyngeal contamination may occur by this method (usually related to contamination from the endotracheal tube). Transtracheal aspiration (TTA) will usually provide sterile samples for microbiological investigation but cell retrieval, especially from the alveolar spaces, may be limited.

Oropharyngeal contamination of respiratory washes This will be recognised by the presence of squamous epithelium (often with associated bacteria). Specific oropharyngeal bacteria, such as Simonsiella sp, may be present. This will jeopodise the investigation of a possible infectious process affecting the lower respiratory tract. Moreover, any neutrophils present may be related to oropharyngeal inflammation rather than lower respiratory inflammation.

Types of lining cells and other structures present in respiratory washings Cellular features will vary depending on whether the sample has been taken by TTA or by BAL. BAL: will contain a greater proportion of alveolar macrophages and lower airway epithelial cells, and will not usually have oropharyngeal contamination.

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Ciliated columnar epithelium The majority of tracheal and bronchial cells will be ciliated columnar cells. They have small round to oval nuclei situated at the end of the cell not displaying the plate of cilia. Cilia are often lost in the washing process and will be found free: they can be mistaken for filamentous bacteria.

Mucosecretory (goblet) cells These are large oval to bulging, elongated cells containing numerous large deep-pink granules in their cytoplasm.

Bronchoalveolar cells These refer to the small, round to square cells, often present in clusters. Their nuclei are usually round and they have moderate amounts of blue-grey cytoplasm. Some of these cells are basal cells while others may be lining cells from the lower airways. Some may be pneumocytes just becoming macrophages.

Alveolar macrophages Alveolar macrophages are usually large with obvious cytoplasmic vacuoles or ingested material (e.g. blue granules of haemosiderin, black-brown granules of carbon). There are always some alveolar macrophages present in washes and, rather than indicate disease, may simply indicate that the sample is truly representative of alveolar spaces. Of course, increased numbers, their appearance and the presence of specific inclusions may indicate disease.

Mucus This is commonly present in washes and will be pink to light blue. Excessive and prolonged secretion of mucus will occur in a variety of chronic respiratory conditions (inflammatory or neoplastic). In these circumstances the mucus often stains a darker blue. In addition there may be dark blue to purple spirals of inspissated mucus called Curschmann's spirals.

Inflammatory cells Inflammatory cells (neutrophils, eosinophils, mast cells and lymphocytes) may be present in normal respiratory washes but they are usually in low numbers (e.g. less than 5% for neutrophils, only occasional mast cells). An exception is eosinophils in cats where levels of up to 15% can be normal.

Commonly diagnosed airway and pulmonary conditions on the basis of respiratory washes Many of these are simply divided into pathological processes, the main ones being inflammatory, vascular or neoplastic.

Acute (active) inflammation In the dog, cat and horse this will be characterised by high levels of neutrophils and lesser numbers of alveolar macrophages. In some cases this will be related to bacterial infection (with appropriate intracellular bacteria – septic inflammation). At other times there will no bacteria visible, nor will any be cultured. This type of sterile (non-septic) inflammation may accompany neoplastic conditions but can also be idiopathic (even though some appear to respond to antibiotics in dogs and cats!).

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Active inflammation can be present in chronic conditions of the respiratory tract. Septic inflammation is often a sequellae of viral pneumonia.

Allergic or parasitic (hypersensitivity) inflammation This will commonly be characterised by significant numbers of eosinophils. Mast cells and neutrophils will be present in variable numbers. Often, many of the eosinophils and mast cells will degranulate, thereby producing a granular background. Parasites, such as lungworm or microfilaria, may be visible in the washes. Chronic Obstructive Airway Disease in horses is regarded as a hypersensitivity. However, the presence of increased numbers of eosinophils is variable, and may just be a feature of early disease. Most features are consistent with those seen for chronic respiratory disease

Mycotic (fungal) inflammation Eosinophils may also be a prominent component of mycotic (fungal) inflammation due to similar hypersensitivity mechanisms which occur in allergies or parasitic infections. However, the inflammatory profile is more variable depending on the agent and the duration of the disease. Both purulent and pyogranulomatous responses may occur. Yeasts may be present in Cryptococcus neoformans infection, while fungal hyphae may be present in Aspergillus spp infection. Multinucleated macrophages may be present but are not specific for this type of infection. They can also occur in foreign body reactions and be present non-specifically in any form of chronic lung disease.

Pulmonary haemorrhage This is not usually a specific feature and can occur in most conditions affecting the lungs. However, it can be a prominent feature in trauma, heart failure, infarction (e.g. heartworm), bleeding disorders, certain types of neoplasia and certain poisons (e.g. paraquat toxicity). Erythrophagocytosis by alveolar macrophages will be visible in acute haemorrhage, while haemosiderin-laden macrophages are more of an indication of past or chronic haemorrhage. Exercise-Induced Pulmonary Haemorrhage (EIPH) occurs in horses as a consequence of strenuous exercise. TTA, and especially BAL will support the diagnosis. In the early stages erythrophagocytosis will be prominent, while haemosiderin-laden macrophages will develop and remain for weeks after the event.

Chronic respiratory disease The features of chronic respiratory disease are variable and depend on the cause (i.e. these are non-specific findings that may be related to a wide variety of causes). Mucin and increased numbers of macrophages are common features but are not highly specific. Goblet cell and general epithelial hyperplasia are common if there is airway involvement. There may be squamous metaplasia with chronic irritation to lining cells. Primary and secondary neoplasia Neoplasia of the lungs cannot always be diagnosed on respiratory washes. There is more chance of diagnosing primary epithelial lung tumours (commonly adenocarcinomas) than metastatic tumours. Fine needle aspiration or biopsy of the pulmonary tissue is usually required to diagnose neoplasia. Consequently, most of the information on pulmonary neoplasia has been presented in the next part. Primary and metastatic neoplasia are rare in the horse.

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CASE REPORTS  

  Introduction 

The  following  case  reports  are  primarily  derived  from  clinical  cases  presented  to  the  University Veterinary  Teaching Hospitals.  The  clinical  pathology  for  the  case  reports was  performed  by  the Veterinary Pathology Diagnostic Services  laboratory  in  the Faculty of Veterinary Science. They are meant to complement the preceding lecture notes. 

 

The case reports are organised in two parts: 

1.  Information on  the animal, history,  clinical  signs and physical examination  findings, and clinical pathology results 

2. Case report analysis Case  reports  form  the  basis  for  the written  form  of  assessment  in  Veterinary  Clinical  Pathology. These  cases  are  designed  to  provide  information  about  specific  conditions  as  well  as  allowing students to  test themselves on case report analysis. The analysis part  for each case  is placed on a separate page.  It  is suggested  that  the  readers attempt  their own case  report analysis  first before reading the provided analysis. Note that the style of analysis has been modified for some from the form expected in the examination. The suggested examination/assessment style for interpretation is given below  in summarised form. Please also see the examination question and model answer under ASSESSMENT  as  there  is more  detailed  discussion  of  approach  to  answering  case  report examination questions. 

Case report analysis should be organized into: 

(a)  detection and highlighting of abnormalities 

(b) general  interpretation  of  the  abnormalities,  keeping  in  mind  any  information  (history, clinical  signs,  physical  examination)  provided  for  the  animal.  In  other  words,  general interpretation must be appropriate for the case in hand 

(c) integration of all the data to reach conclusion(s) or a specific diagnosis. In many situations it  is  not  possible  to  reach  a  specific  diagnosis  until  further  investigation  is  undertaken. Sometimes  only  several  general  conclusions  can  be  drawn  from  the  data,  and  further investigation  is  required  to  follow  several  lines.  Think medicine when  considering  further investigation  i.e.  don’t  restrict  investigation  to  laboratory  testing,  include  image  analysis, exploratory  laparotomy  etc.  A  consideration  of  the  implications  of  the  conclusions  for management of the case should be included in an answer 

This style emphasises that (a) and (b) provide the basis for (c), and it is (c) that is the most important; i.e. conclusions,  further  investigation and  implications  for management are  the key outcomes of laboratory investigation. Remember to assess the clinical information provided for the animal, as this is likely to give certain clues to the disease problem. 

Please note that for the following case reports often only the results of relevant biochemical tests are provided. The results of haematology are usually provided in full. 

Interpretation of clinical pathology results is assisted by the following information on assessment of the levels of biochemical and Haematological increases in disease. The table can be referred to while attempting  interpretation. The table provides an  indication of what  increases or decreases  in some 

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of the more commonly used analytes are needed to call a change mild, moderate or marked. They are  subjective  (i.e.  based  on  the  author’s  experience)  and  based  on  values  detected  by  the Veterinary Pathology Diagnostic Services  laboratory at The University of Sydney. The equine values were determined  from  the University Veterinary Teaching hospital Camden. They do not apply  to values from other diagnostic laboratories. The values can be used for the Examination in Veterinary Clinical Pathology. 

 

Remember:  

1. The degree of change  in the value often  indicates the severity of the disease process, but there are exceptions (e.g. moderate to marked increases in CK can occur in exercise as well as disease).  

2. Some of the analytes are non‐specific and changes may be caused by several disease processes in several  organs  or  tissues  (e.g.  amylase  can  be  increased  in  GIT,  renal  and  exocrine  pancreatic disease). 

3. These do not apply to certain analytes for some breeds of dogs. This is because the breeds have either different reference intervals or different responses in disease e.g. bile acids in Maltese terriers are of  little use  in disease as  reference  intervals are extremely  large; greyhounds and some other sight hounds have much lower reference levels for total leukocytes and neutrophils (can be as low as 3‐4 x 109/L  for  total  leukocytes), and  sometimes  increases  in disease may be muted compared  to other breeds.  

 Analyte  Mild change  Moderate change  Marked change CK IU/L  Increases 

201‐800;  400‐1,000 (H) 

 801‐2000;  1001‐2000 (H) 

 >2000* 

Amylase IU/L  Increases 1401‐1800 (D&C) 

 1801‐2800 (D&C) 

 >2800 (D&C) 

Lipase IU/L  Increases 301‐500 (D) 101‐300 (C) 

 501‐800 (D) 301‐500 (C) 

 >800 (D) >500 (C) 

ALP IU/L  Increases 111‐500 (D);  51‐100 (C);  260‐400 (H) 

 501‐1000 (D);  101‐200 (C);  401‐600 (H) 

 >1000 (D);  >200 (C);  >600 (H) 

ALT IU/L  Increases 61‐200 (D&C) 

201‐500 (D&C)  >500 (D&C) 

GGT IU/L  Increases 8.3‐20 (D); 36‐50 (H) 

21‐30 (D);  51‐70 (H) 

>31 (D);  >71 (H) 

GLDH IU/L  Increases 4.8‐20 (H) 

21‐50 (H)  >50 (H) 

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Increases 71‐80 (D);  74‐80 (C);  77‐80 (H) 

 81‐90  

 >90 

Serum Protein g/L 

Decreases 45‐49 (D);  45‐53 (C);  53‐60 (H) 

 35‐44;  45‐52 (H) 

 <34;  <45 (H) 

Bile Acids (fasting) μmol/L 

Increases 11‐25 (D);  6‐15 (C) 

 26‐50 (D);  16‐50 (C) 

 >50 

Total Bilirubin μmol/L  Increases 8.2‐20 (D);  3.6‐20 (C);  51‐75 (H) 

 21‐100;  76‐100 (H) 

 >100 

Glucose mmol/L  Increases 6.5‐8.0 (D);  6.7‐15 (C);  6.4‐8.0 (H) 

 8.1‐15 (D&H);  15.1‐20 (C) 

 >15 (D&H);  >20 (C) 

Creatinine μmol/L  Increases 121‐180 (D);  181‐220 (C);  150‐200 (H) 

 181‐500 (D);  221‐500 (C);  201‐500 (H) 

 >500 

Urea mmol/L  Increases 10.1‐25 (D);  10.8‐25 (C);  8.3‐25 (H) 

 25.1‐40 

 >40 

Increases 4.9‐5.2 (D);  4.7‐5.2 (C);  5.1‐5.5 (H) 

 5.3‐7.0;  5.6‐7.0 (H) 

 >7.0 (death at 10‐12 for D&C) 

Potassium mmol/L 

Decreases 3.0‐4.8 (D);  3.5‐4.6 (C);  2.5‐2.7 (H) 

 2.5‐2.9 (D);  2.5‐3.4 (C);  2.2‐2.4 (H) 

 <2.5;  <2.2 (H) 

PCV L/L  Decreases 0.30‐.36 (D);  0.20‐.29 (C);  0.25‐.31 (H) 

 0.20‐.29 (D);  0.15‐.19 (C);  0.20‐.24 

 <0.20 (D&H);  <0.15 (C) 

Total Plasma Protein g/L  

Increases 76‐85 (D);  79‐90 (C);  85‐90 (H) 

 86‐100 (D);  91‐100 (C&H) 

 >100 

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Increases 12.1‐20 (D);  14.1‐20 (C);  13.1‐18.0 (H) 

 20.1‐50 (D);  20.1‐40 (C);  18.1‐30.0 (H) 

 >50 (D);  >40 (C);  >30 (H) 

Leukocytes x109/L 

Decreases 5‐7 (D);  6‐8 (C);  4‐6 (H) 

 3.0‐4.9 (D);  3.0‐5.9 (C);  3.0‐3.9 (H) 

 <3.0 

Increases 9.4‐18 (D);  10.1‐18 (C);  7.0‐14.0 (H) 

 18.1‐35 (D);  18.1‐30 (C);  14.1‐25 (H) 

 >35 (D);  >30 (C);  >25 (H) 

Neutrophils x109/L 

Decreases 3.0‐4.0 (D);  3.0‐3.7 (C);  2.0‐2.5 (H) 

 2.0‐2.9 (D&C);  1.5‐1.9 (H) 

 <2.0 (D&C);  <1.5 (H) 

Platelets x109/L  Decreases 101‐200 (D);  101‐300 (C);  60‐79 (H) 

 50‐100 (D&C);  40‐59 (H) 

 <50 (D&C);  <40 (H) 

 *The values apply to dog, cat and horse unless otherwise stated D = dog; C = cat; H = horse  

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CASE  1  ANIMAL: 3 year old male Beagle.  PRESENTING COMPLAINTS:  Severe weight loss, inappetence and occasional vomiting for 4 months. The dog had had a history of recurrent bouts of vomiting and acute abdominal pain before the four months. Radiographs during one episode suggested hepatomegaly.  LABORATORY RESULTS  BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Amylase IU/L  ND*  <1400 Lipase IU/L  ND  <60 ALP IU/L  165  <110 ALT IU/L  48  <60 AST IU/L  ND  <40 CK IU/L  ND  <100 GGT IU/L  ND  0.6‐8.2 Serum protein (biuret) g/L  ND  50‐70 Albumin (BCG) g/L  ND  23‐43 Globulins g/L  ND  7‐16 Serum protein (refract.) g/L  41  50‐70 Albumin (EPG) g/L  17.3  23‐39 α globulins (EPG) g/L  4.7  7‐16 β globulins (EPG) g/L  10.7  9‐16 γ globulins (EPG) g/L  8.3  4‐12 Bile acids (fasting) μmol/L  45  <10 Total bilirubin μmol/L  32  1.2‐5.1 Unconjugated bilirubin μmol/L  0  1.2‐5.1 Conjugated bilirubin μmol/L  32  1.2‐5.1 

 URINALYSIS (voided sample) Appearance  Cloudy  PH  6 Colour  Yellow  Glucose  ‐ve Specific gravity  1.026  Ketones  ‐ve Protein  Trace  Blood  4+     Bilirubin  4+ Microscopic findings: erythrocytes 50‐60 per HPF, leukocytes 2‐3 per HPF, many struvite crystals 

 *ND – not done

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INTERPRETATION OF LABORATORY FINDINGS 

The information provided suggests a chronic disease that could involve the digestive tract, especially the liver.  (a)  Detected  laboratory  abnormalities:  mild  elevation  of  ALP,  mild  to  moderate  conjugated hyperbilirubinemia, hypoproteinemia due  to decreases  in albumin and alpha globulins,   moderate elevation of bile acids, haematuria, hyperbilirubinuria.  (b) & (c) general interpretation, conclusions, further investigation and implications for management: The  animal  has  evidence  of  cholestasis  (mild  elevation  of  ALP,  mild  to  moderate  conjugated hyperbilirubinemia  and  hyperbilirubinuria)  but  no  hepatocellular  damage  (normal  ALT).  This  in combination  with  a  moderate  elevation  of  bile  acids  (will  increase  in  a  wide  variety  of  liver conditions)  suggests  that  the  hypoproteinemia  may  be  due  to  liver  disease  (conclusion).  Hypoalbuminemia in liver disease (decreased production) usually means chronic disease and hepatic failure  (conclusion). This was  supported by  the history. Further  investigation  could have been an ultrasound guided fine needle aspiration of the liver    In end stage  liver disease there  is often a decrease  in all protein production. Gamma globulins are not normally decreased (production is by plasma cells) and in many cases is increased due to antigen stimulation. The beta globulins  in this case were not decreased, which  is a  little unusual, but could be due to the fact that some  immunoglobulins were detected  in this zone on electrophoresis. The cause of the haematuria was not determined and is probably not important. However, it could have been investigated further through image analysis and collection of urine by cystocentesis.  Implications for management: The owners were given a poor prognosis for treatment and elected for euthanasia.  FINAL DIAGNOSIS: Hepatic failure (cirrhosis). Confirmed at necropsy 

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CASE: 2  

ANIMAL: 7 year old female neutered Persian cat.  PRESENTING  COMPLAINTS:    Jaundice  and  depression  for  about  3  weeks.  Previous  history  was unhelpful.  There  had  been  occasional  vomiting  and  the  cat  had  probably  lost  weight.  The  cat appeared to be dehydrated on presentation and was very thin.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Amylase IU/L  ND  <1400 ALP IU/L  213  <50 ALT IU/L  567  <60 Total bilirubin μmol/L  263  2.5‐3.5 Unconjugated bilirubin μmol/L  57  <3.5 Conjugated bilirubin μmol/L  206  <3.5 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Yellow  Clear PCV L/L  0.28  0.30‐0.45 Plasma protein g/L  86  59‐78 Haemoglobin g/L  98  80‐140 Erythrocytes x1012/L  6.7  6‐10 MCV fl  42  40‐45 MCHC g/L  350  310‐360 Leukocytes x109/L  7.8  8‐14 Neutrophils (seg.) x109/L  6.2  3.8‐10.1 Neutrophils (band) x109/L  0  0‐0.4 Lymphocytes x109/L  1.1  1.6‐7.0 Monocytes x109/L  0.2  0.1‐0.6 Eosinophils x109/L  0.3  0.2‐1.4 Basophils x109/L  0  0‐0.2 Blood film: moderate poikilocytosis and anisocytosis of erythrocytes Platelets x109/L  ND  300‐700 Reticulocyte % (uncorrected)  0.3  0‐1 

 URINALYSIS (cystocentesis) Appearance  Cloudy  PH  6 Colour  Yellow  Glucose  ‐ve Specific gravity  1.050  Ketones  ‐ve Protein  Trace  Blood  ‐ve     Bilirubin  4+ Microscopic findings: many transitional cells and much lipid 

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INTERPRETATION OF LABORATORY FINDINGS 

The  information provided suggests ongoing disease. The  jaundice could be pre‐hepatic, hepatic or post‐hepatic.  (a) Detected laboratory abnormalities: Marked hyperbilirubinemia (approximately 80% conjugated), moderate elevations of ALP and ALT, mild non‐regenerative, normocytic, normochromic anaemia, hyperproteinemia, mild lymphocytopenia, hyperbilirubinuria.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The cat has evidence of hepatocellular damage and cholestasis. The level of ALP is significant for the cat but would probably mean  little  in a dog. The hyperbilirubinemia  is of a  combined  form  (both retention  and  regurgitation  forms  are  elevated)  but  significant  regurgitation  (cholestasis)  exists. Since  the  hepatocellular  damage  is moderate,  the  cholestasis was  interpreted  as  being  primarily intra‐hepatic.  The hyperbilirubinuria is marked and, unlike the dog, is a direct reflection of the levels of conjugated bilirubin  in  the  blood  stream.  The  anaemia  is  non‐specific  and  possibly  related  to  the  chronic disease. Poikilocytosis can be common in a variety of severe diseases in certain breeds of cat and is relatively  non‐specific.  However,  it  is  reported  more  commonly  in  liver  disease.  The hyperproteinemia  was  possibly  due  to  dehydration  (mild  dehydration  was  noted)  but hyperglobulinemia could not be ruled out.  The  initial conclusion was simply hepatopathy. Further  investigation would require  image analysis and cytological/histological samples from affected tissue. The owners elected for euthanasia.  POSTSCRIPT AND DIAGNOSIS: Necropsy  revealed  a pancreatic  carcinoma which had  caused bile duct  obstruction. On  reflection,  the  hepatocellular  damage  probably  occurred  secondarily  to  the marked  and  prolonged  cholestasis  but  part  could  have  been  due  to  the  non‐specific metabolic derangements that cancer occasionally causes. 

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CASE  3  ANIMAL: 7 year old male Siamese cat.  PRESENTING  COMPLAINTS:  Depression,  vomiting  of  greater  than  5  days  duration.  This  cat  was presented vastly overweight. There was no previous history of problems.  LABORATORY RESULTS  BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Amylase IU/L  ND  <1400 ALP IU/L  230  <50 ALT IU/L  987  <60 Total bilirubin μmol/L  30  2.5‐3.5 Unconjugated bilirubin μmol/L  15  <3.5 Conjugated bilirubin μmol/L  15  <3.5 Urea mmol/L  4  7.2‐10.7 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Yellow  Clear PCV L/L  0.26  0.30‐0.45 Plasma protein g/L  64  59‐78 Haemoglobin g/L  87  80‐140 Erythrocytes x1012/L  6.0  6‐10 MCV fl  43  40‐45 MCHC g/L  334  310‐360 Leukocytes x109/L  26  8‐14 Neutrophils (seg.) x109/L  24  3.8‐10.1 Neutrophils (band) x109/L  0.3  0‐0.4 Lymphocytes x109/L  1.3  1.6‐7.0 Monocytes x109/L  0.4  0.1‐0.6 Eosinophils x109/L  0.13  0.2‐1.4 Basophils x109/L  0  0‐0.2 Blood film: normal Platelets x109/L  ND  300‐700 Reticulocyte % (uncorrected)  ND  0‐1 

 

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INTERPRETATION OF LABORATORY FINDINGS 

The information provided points to a myriad of diseases that might cause vomiting in an overweight cat (e.g. diabetes, pancreatitis, hepatitis, GIT disease).  (a) Detected laboratory abnormalities: Moderate elevation of ALP, moderate to marked elevation of ALT,  mild  combined  hyperbilirubinemia,  mild  normocytic,  normochromic  anaemia,  leukocytosis primarily due to a neutrophilia without left shift, lymphocytopenia and eosinopenia.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The  cat  had  significant  hepatocellular  damage  with  some  cholestasis.  The  combined hyperbilirubinemia (albeit low) supported the interpretation of active hepatopathy (conclusion). The anaemia was assumed to be non‐regenerative (no polychromasia on the blood film), but could not be explained in terms of the apparent short clinical course. The leukocyte changes were considered to be due to stress (corticosteroid‐induced). Further investigation could have involved image analysis and ultrasound‐guided fine needle cell aspiration. Bile acid analysis may have been useful.  Liver  disease  was  confirmed  by  image  analysis,  which  showed  diffuse  hepatomegaly.  However, further investigation was not undertaken.  DIAGNOSIS AND POSTSCRIPT: The animal’s condition worsened and it died despite fluid therapy. At necropsy the animal had severe hepatic steatosis and necrosis.  The condition of hepatic steatosis is well documented in over fat cats but the pathogenesis is poorly understood. Commonly it presents as  a  chronic  condition  leading  to  clinical  signs  of  hepatic  failure.  However,  occasionally  severe necrosis develops leading to dramatic clinical signs. On reflection, the mild anaemia could have been related to the liver disease. 

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CASE  4 ANIMAL: 9 year old male Schnauzer dog. 

PRESENTING  COMPLAINTS:  Long  history  of  spasmodic  inappetence,  polydipsia  and  polyuria.  Last four days collapsed and vomiting. Presented jaundiced and dehydrated. 

LABORATORY RESULTS  

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL ALP IU/L  479  <110 ALT IU/L  190  <60 Total bilirubin μmol/L  170  1.2‐5.1 Unconjugated bilirubin μmol/L  45  1.2‐5.1 Conjugated bilirubin μmol/L  125  1.2‐5.1 Total cholesterol mmol/L  2.3  1.4‐7.5 Glucose mmol/L  4.7  3.3‐6.4 Urea mmol/L  30.7  3‐10 Sodium mmol/L  144  137‐150 Potassium mmol/L  3.8  3.3‐4.8 

 

HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Yellow‐pink  Clear PCV L/L  0.34  0.37‐0.50 Plasma protein g/L  62  55‐75 Haemoglobin g/L  127  100‐150 Erythrocytes x1012/L  5.3  5‐7 MCV fl  64  60‐75 MCHC g/L  373  300‐360 Leukocytes x109/L  89  7‐12 Neutrophils (seg.) x109/L  70.2  4.1‐9.4 Neutrophils (band) x109/L  0.4  0‐0.24 Lymphocytes x109/L  3.3  0.9‐3.6 Monocytes x109/L  14.7  0.2‐1.0 Eosinophils x109/L  0.4  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: normal Reticulocyte % (uncorrected)  1  0‐1.5 Eosinophils (direct count) x109/L    0.1‐1.0 Fibrinogen g/L    2‐4 

 

URINALYSIS (catheterised) Appearance  Cloudy  PH  5 Colour  Yellow  Glucose  ‐ve Specific gravity  1.015  Ketones  ‐ve Protein  ‐ve  Blood  2+     Bilirubin  4+ Microscopic findings: 2‐3 erythrocytes per HPF, 2‐3 leukocytes per HPF, 2‐3 granular casts per LPF 

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INTERPRETATION OF LABORATORY FINDINGS 

The information provided suggests chronic disease that may have reached a crisis or end point. The polyuria  and  polydipsia might  suggest  renal,  endocrine  or  liver  disease.  The  developed  jaundice could be related to pre‐hepatic, hepatic or post‐hepatic disease. 

(a) Detected laboratory abnormalities:  Mild non‐regenerative anaemia, marked leukocytosis due to neutrophilia  (without  left  shift)  and  monocytosis,  mild  elevation  in  ALP, mild  elevation  in  ALT, marked  hyperbilirubinemia  (approximately  70%  conjugated),  moderate  azotemia  (only  urea),  a specific  gravity  close  to  the  isosthenuric  range,  possible  haemoglobinuria  and  maximum bilirubinuria. 

(b) & (c) General interpretation, conclusions, further investigation and implications for management: The animal has marked  cholestasis but minimal hepatocellular damage. Bilirubin analysis  shows a combined  type  but  with  regurgitation  predominating.  The  4+  bilirubinuria  reflects  the hyperbilirubinemia but, unlike most other species, is not purely reflective of the levels of conjugated bilirubin (dogs apparently have some capacity to degrade haemoglobin to bilirubin, and to conjugate bilirubin). These results suggest a primary cholestatic disease, which could be biliary (extrahepatic or intrahepatic) in site (conclusion). 

The anaemia is probably related to chronic disease. The finding of an elevated MCHC suggests either laboratory error (technical error or interfering substances) or haemolysis prior to or after collection. The  leukocyte changes may  reflect an  intense continuing  inflammatory demand. The  lack of a  left shift might be due to bone marrow production adequately adapting  to the  increased demand  (i.e. the bone marrow storage pool has been replenished over time). The marked monocytosis could be due  to  demand  for  macromolecular  phagocytosis.  Although  there  is  no  lymphocytopenia  or eosinopenia, corticosteroid release may be contributing to the neutrophilia (the clinical signs suggest a period of intense illness). 

Azotemia in combination with clinical dehydration and a sp. gr. close to isosthenuria suggests renal failure (conclusion). This appears  in addition to the hepatic disease. The 2+ blood on the strip and yet lack of significant erythrocytes in the sediment, suggests either free haemoglobin or myoglobin. The  differentiation  usually  requires  specific  chemical  testing  but  in  this  case  history  appears  to exclude the possibility of muscle damage. Assuming that the blood is haemoglobin, this may be due to haemoglobinemia (usually the plasma has to be saturated before the haemoglobin is filtered) or due to breakdown of erythrocytes in the urine related to delays in testing or extremes of pH or sp. gr. The latter is more likely. However, considering the PCV, it is probably not of great significance. 

Further  investigation  might  have  been  aimed  at  investigating  the  probable  liver/renal  disease. Diagnostic  imaging  through  radiographs or ultrasonographs would have been useful. Further  tests would have been depended on the findings of image analysis. 

The implications for management suggested a poor prognosis.  

 

DIAGNOSIS AND POSTSCRIPT: The owner elected euthanasia and at necropsy the dog had extensive cholangiohepatitis and a large hepatic abscess.  In addition, there was evidence of nephrosis. With these  liver changes  it  is unusual not  to get a greater elevation of ALT. However, other  laboratory changes are consistent with these findings. The anaemia probably developed due to a combination of  factors  including  increased  turnover  of  erythrocytes  and  unavailability  of  iron  stored  in macrophages. 

The laboratory finding of renal failure (lack of concentrating ability) was supported by the detection 

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of  nephrosis.  This  probably  developed  secondarily  to  the  liver  diseases  (nb.  in  older  dogs,  it  is common to have some chronic renal disease which is not a problem unless other disease develops. Then the renal disease may be pushed towards renal failure. This may have been suspected  in the present case but was not so). 

No definite conclusions can be drawn on the suspected haemoglobinemia and haemoglobinuria. It is possible  that some haemolysis was occurring related  to  toxic or metabolic disturbances caused by the hepatic abscess (clostridial organisms were isolated from the abscess). 

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CASE  5  ANIMAL: 8 year old Sydney silkie dog.  PRESENTING COMPLAINTS: Long periods of vomiting. Now jaundiced and depressed.  LABORATORY RESULTS  

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Amylase IU/L  3340  <1400 Lipase IU/L  ND  <60 ALP IU/L  942  <110 ALT IU/L  64  <60 Total bilirubin μmol/L  154  1.2‐5.1 Unconjugated bilirubin μmol/L  0  1.2‐5.1 Urea mmol/L  103  3‐10 Creatinine μmol/L  860  40‐120 Sodium mmol/L  147  137‐150 Potassium mmol/L  3.8  3.3‐4.8 Chloride mmol/L  71  105‐120 Bicarbonate (TCO2) mmol/L  ND  17‐24 

 

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INTERPRETATION OF LABORATORY FINDINGS 

The information has highlighted vomiting and jaundice in a middle‐aged dog. This could be related to liver disease, pancreatic disease or GIT disease.  (a)  Detected  laboratory  abnormalities: Moderate  elevation  of  ALP,  unconvincing  change  in  ALT, marked hyperamylasemia, marked hyperbilirubinemia, marked azotemia  (both urea and creatinine elevated) and hypochloridemia.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The  dog  has  significant  cholestasis  which  is  reflected  in  the  ALP  and  bilirubin  levels.  The hyperbilirubinemia  is totally regurgitation  in origin, which could point to  large biliary vessel or post hepatic obstruction.  The marked  azotemia  could be due  to both pre‐renal  and  renal  factors, but there  is not  enough  information  to  identify  all  the  factors. Vomiting,  if  causing  significant  loss of fluid, will elevate both urea and creatinine due to decreased renal blood flow. The hypochloridemia probably has developed because of the vomiting.  The  hyperamylasemia  could  be  due  to  pancreatic  necrosis  or  possibly  due  to  small  intestinal obstruction  (amylase  is  known  to  elevate  in  cases  of  intestinal  obstruction which  cause marked vomiting).  Because of the limited tests performed, it is not possible to be definite about interpretation (i.e. no strong conclusions. Post hepatic obstruction, possibly due to swelling caused by pancreatic necrosis, was considered and could have been further investigated by image analysis.   DIAGNOSIS AND POSTSCRIPT: At necropsy,  surprisingly, an acquired pyloric  stenosis  (obstructive jaundice) was detected. Extension of  the disease process  into  the proximal part of  the duodenum possibly interfered with bile outflow. The pancreas appeared normal but this does not rule out some interference with pancreatic duct outflow. The kidneys had mild interstitial nephritis. 

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CASE  6  ANIMAL: 8 month old Sydney silkie dog.  PRESENTING COMPLAINTS: Poor growth and vague neurological signs related to lack of awareness.  LABORATORY RESULTS  BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL ALP IU/L  229  <110 ALT IU/L  50  <60 Bile acids (fasting) μmol/L  65  <10 Bile acids (post‐prandial) μmol/L  350  <25 NH3 (fasting) μmol/L  234  0‐50 NH3 (post NH4Cl) μmol/L  591  50‐90 Urea mmol/L  2.7  3‐10 

 URINALYSIS (voided) Appearance  Cloudy  PH  6 Colour  Yellow  Glucose  ‐ve Specific gravity  1.012  Ketones  ‐ve Protein  ‐ve  Blood  ‐ve     Bilirubin  Trace Microscopic findings: many transitional cells 

 

135

INTERPRETATION OF LABORATORY FINDINGS 

The  information  suggests possibly  a  congenital problem  that  is producing neurological  signs. This could mean primary neurological disease or secondary neurological signs due to systemic disease.  (a) Detected laboratory abnormalities: Mild elevation of ALP, marked elevations of ammonia and bile acids. Urine specific gravity within the isosthenuric range and a trace bilirubin.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The finding of an elevated fasting level of ammonia in combination with a low level of urea suggests a  lack of  conversion by  the  liver. Post NH4Cl  levels  supports  this  lack of  conversion  and  suggests shunting (conclusion). The bile acid values also support this possibility. The lack of change in ALT and the minimal change in ALP are not uncommon in porto‐systemic shunting. Further investigation will normally involve diagnostic imaging and vascular studies.  The urine  is unremarkable but  commonly, ammonium biurate  crystals may be present  in urine  in cases of shunting and  in certain other hepatopathies. The trace of bilirubin has no significance and the sp. gr. reading may be a chance finding.  DIAGNOSIS  AND  POSTSCRIPT:  Hepatic  encephalopathy  due  to  porto‐systemic  shunting.  A congenital porto‐systemic shunt was confirmed through image analysis and treated surgically. 

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CASE  7  ANIMAL: 8 year old male Bull terrier dog.  PRESENTING COMPLAINTS: Prolonged history of polydipsia and polyuria, now not eating or drinking. It appeared dehydrated on presentation.  LABORATORY RESULTS  BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Urea mmol/L  27  3‐10 Creatinine μmol/L  600  40‐120 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.27  0.37‐0.50 Reticulocyte % (uncorrected)  0.5  0‐1.5 

 URINALYSIS (voided) Appearance  Clear  PH  6.5 Colour  Yellow  Glucose  ‐ve Specific gravity  1.013  Ketones  ‐ve Protein  Trace  Blood  ‐ve     Bilirubin  ‐ve Microscopic findings: occasional transitional cell, 1 leukocyte per HPF 

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INTERPRETATION OF LABORATORY FINDINGS 

The  information suggests a prolonged disease producing polyuria and polydipsia. Renal, hepatic or endocrine conditions should be considered.  (a) Detected laboratory abnormalities: Marked azotemia, moderate non‐regenerative anaemia, close to isosthenuric sp. gr.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: A close  to  isosthenuric  reading  for  sp.gr.  in combination with azotemia and probable dehydration suggests  renal  failure  (conclusion). A non‐regenerative anaemia  is commonly  found  in association with chronic renal failure and occurs due to a variety of mechanisms (lack of erythropoietin – most important,  enhanced  haemolysis  and  direct  bone  marrow  depression).  The  history  supports  a diagnosis  of  chronic  renal  failure.  Further  investigation might  include  diagnostic  imaging  of  the kidneys and electrolyte analysis (could have implications for management).  DIAGNOSIS: Chronic renal failure. 

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CASE  8  ANIMAL: Aged Holstein cow.  PRESENTING COMPLAINTS:   Acute onset of depression,  listlessness, anorexia, unwilling  to drink  and  haematuria.  The  cow  appeared  mildly  dehydrated  on  examination  and  was reluctant to move.  LABORATORY RESULTS  BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Serum protein (biuret) g/L  80  68‐75 Glucose mmol/L  7.4  2.5‐4.2 Urea mmol/L  24  7‐11 Creatinine μmol/L  400  88‐117 Sodium mmol/L  110  132‐152 Potassium mmol/L  2.3  3.9‐5.8 Chloride mmol/L  48  97‐111 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.29  0.24‐0.46 Plasma protein g/L  97  70‐80 Leukocytes x109/L  9.7  4‐12 Neutrophils (seg.) x109/L  7.8  0.6‐4.0 Neutrophils (band) x109/L  0.3  0‐0.1 Lymphocytes x109/L  1.0  2.5‐7.5 Monocytes x109/L  0.5  0‐0.9 Eosinophils x109/L  0.1  0‐2.4 Basophils x109/L  0  0‐0.2 Blood film: normal 

 URINALYSIS (voided) Appearance  Cloudy  PH  7 Colour  Amber  Glucose  2+ Specific gravity  1.016  Ketones  ‐ve Protein  3+  Blood  4+     Bilirubin  ‐ve Microscopic findings: greater than 50 erythrocytes per HPF, 3‐4 granular casts per HPF 

 

139

INTERPRETATION OF LABORATORY FINDINGS 

The information provided suggests acute severe disease. The haematuria could be caused by urinary or genital disease.  (a) Detected  laboratory abnormalities: Hyperproteinemia, mild hyperglycemia, moderate azotemia, hyponatremia, hypokalemia, hypochloridemia, leukocytosis due to neutrophilia (probable left shift), lymphocytopenia, low normal eosinophils, haematuria, proteinuria and glucosuria.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The cow has azotemia with a sp. gr. close to isosthenuria (it is probably truly isosthenuric as protein and glucose falsely elevate the specific gravity reading). Considering the cow was not drinking and mildly  dehydrated,  this  suggests  renal  failure  (conclusion).  The  presence  of  casts  suggests  that perhaps the haematuria is related to renal disease rather than to lower tract abnormalities. All these findings, with the history, point to acute renal failure (conclusion).  The proteinuria may be related to renal disease. However, when the diagnostic strip for blood shows a maximum 4+ often the protein strip is positive to variable degrees. The glucosuria is related to the hyperglycemia  as  the  renal  threshold  for  bovine  animals  is  low  (about  5.5  ‐  6  mmols/L). Hyperglycemia  in  cattle  is  common  and  transient,  and  commonly  due  to  corticosteroid  or catecholamine  release.  In  this  case,  corticosteroid  release  is  supported by  the  leukocyte  changes (neutrophilia and lymphocytopenia, but not a possible left shift) (conclusion).  All electrolytes are  low. Hyponatremia and hypochloridemia are common  in renal disease  in cattle. The  hypokalemia  is most  likely  due  to  decreased  intake  and  increased  loss  (kaliuresis  –  but  the history  did  not  provide  evidence  for  polyuria).  The  hypochloridemia  is  most  likely  due  to gastrointestinal  stasis  and  abomasal  loss  of  HCl  (a  parallel  can  be  drawn  with  vomiting  in monogastrics), which can occur in a wide variety of conditions in ruminants.  Total plasma protein and total serum protein are distinctly different. The difference (17 g/L) can be assumed  to  be  primarily  fibrinogen  (although  two  different methods were  used  to measure  the proteins and this may be partly the reason for the difference). Fibrinogen is an 'acute phase reactant' protein which  rises  non‐specifically  in  a wide  range  of  degenerative  and  inflammatory  diseases. Increases  are  larger  and more  consistent  in  farm  animals  than  in  dogs  and  cats.  In  this  cow  the increase can be assumed to be related to acute renal disease. Nb. in dehydrated animals all proteins are falsely elevated ‐ this includes fibrinogen. To correct for this and to determine if a true increase in fibrinogen has occurred, the ratio of total plasma protein to fibrinogen should be determined. A ratio of less than 10 to 1 (e.g. 9 to 1) definitely indicates increased fibrinogen. This is the case in hand where the ratio is roughly 5.6 to 1.   The cow died and at necropsy acute renal nephrosis was diagnosed. The cause was assumed to be an ingested toxin.  DIAGNOSIS: Acute renal failure. 

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CASE  9  ANIMAL: 1 year old, female DSH cat.  PRESENTING COMPLAINTS: Weight loss and generalised oedema over three weeks.  LABORATORY RESULTS  BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Serum protein (refract.) g/L  50  54‐73 Albumin (EPG) g/L  8.1  24‐30 α globulins (EPG) g/L  21.4  9‐21 β globulins (EPG) g/L  10.1  8‐15 γ globulins (EPG) g/L  10.4  9‐23 Total cholesterol mmol/L  6.0  1.9‐3.9 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.24  0.30‐0.45 Plasma protein g/L  58  59‐78 Haemoglobin g/L  85  80‐140 Erythrocytes x1012/L  5.0  6‐10 MCV fl  48  40‐45 MCHC g/L  354  310‐360 Leukocytes x109/L  14.2  8‐14 Neutrophils (seg.) x109/L  7.9  3.8‐10.1 Neutrophils (band) x109/L  0  0‐0.4 Lymphocytes x109/L  5.4  1.6‐7.0 Monocytes x109/L  0.3  0.1‐0.6 Eosinophils x109/L  0.4  0.2‐1.4 Basophils x109/L  0.2  0‐0.2 Blood film: normal 

 URINALYSIS (voided) Appearance  Cloudy  pH  7.0 Colour  Light yellow  Glucose  ‐ve Specific gravity  1.016  Ketones  ‐ve Protein  3+  Blood  ‐ve     Bilirubin  ‐ve Microscopic findings: 1‐2 leukocytes per HPF, 1 fatty cast per HPF, much lipid 

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The information suggests sub‐acute to chronic illness in a young cat. The generalised oedema could be due to hypoproteinemia (reduced intake, decreased production by liver, increased losses through GIT or kidney) or increased hydrostatic pressure (e.g. heart failure).  (a) Detected  laboratory abnormalities: mild hypoproteinemia, marked hypoalbuminemia, marginal increase  in  alpha  globulins,  hypercholesterolemia,  anaemia,  possible  hyperfibrinogenemia (difference  between  total  plasma  protein  and  total  serum  protein  –  both  measured  by  the refractometer  in this case),  increased MCV, mild  leukocytosis, mild basophilia?, poor concentrating ability and proteinuria.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: A  significant  decrease  in  the  albumin  suggests  that  this  could  be  the  primary  cause  of  the generalised oedema  (although other  factors are probably contributing  to  the oedema).  It appears that  the main  reason  for  low  albumin  is  proteinuria.  This  would  point  to  nephrotic  syndrome. Glomerular disease  in  the  cat  is more  likely  to  give  rise  to  the nephrotic  syndrome  (conclusion). Azotemia might be expected with this but unfortunately urea or creatinine were not analysed. The low  urine  sp.  gr.  (close  to  isosthenuric  range),  if  persistent,  could  indicate  tubular  dysfunction (conclusion).  The anaemia could be due to renal disease (conclusion), but unfortunately it has not been classified. The high MCV does not make sense and is possibly due to laboratory error (FeLV may be a possibility for macrocytic anaemia). The  leukocytosis  is not of significance as  the  individual  leukocyte groups have not risen (the marginal basophilia is not of relevance).  Derangements  of  lipid  metabolism  often  occur  in  nephrotic  syndrome  in  man.  Apparently, diminished plasma oncotic pressure stimulates hepatic lipoprotein synthesis, although urinary loss of plasma protein  factors  regulating  lipoprotein  synthesis or  catabolism may also play a  role. This  is reflected  in hypercholesterolemia and  lipoprotein peaks  in the protein electrophoretograph.  In the dog and cat hypercholesterolemia occurs  inconsistently  in  the nephrotic  syndrome but other  lipid changes have not been investigated.  In the present case hypercholesterolemia  is present  in the cat.  In man,  lipiduria  is also common  in the nephrotic syndrome but since the cat normally has lipid in the urine this is not useful (nb. fatty casts are the common type found  in cats). The mild  increase  in alpha globulins may be due to the presence  on  acute  phase  reactant  proteins  that  rise  in  a  wide  variety  of  inflammatory  and degenerative  conditions.  However,  lipoprotein may  also  be  contributing  (one would  have  to  do individual protein analysis to be sure). Hyperfibrinogenemia supports the presence of degenerative or  strong  inflammatory  disease  (increases  in  fibrinogen  in  the  cat  and  dog  related  to  these conditions are not as common as  in  farm animals and horses). However,  the  leukogram  seems  to rule out significant inflammation or tissue damage (i.e. we are present with inconsistent findings).  Further  investigation  could  include  diagnostic  imaging,  fine  needle  aspiration  and  biopsy. Implications  for management could depend on  the  findings of biopsy; however,  the oedema and low protein will need to be managed.  DIAGNOSIS: Nephrotic syndrome. 

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CASE  10  ANIMAL: 12 month old, female Labrador dog.  PRESENTING  COMPLAINTS: Depression,  vague  abdominal  pain  and  frequent micturition  of  about two weeks duration. The dog had possible cystitis on two occasions over the last six months  LABORATORY RESULTS  BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Urea mmol/L  17.1  3‐10 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.42  0.37‐0.50 Plasma protein g/L  80  55‐75 Haemoglobin g/L  136  100‐150 Erythrocytes x1012/L  7.1  5‐7 MCV fl  59  60‐75 MCHC g/L  323  300‐360 Leukocytes x109/L  29.5  7‐12 Neutrophils (seg.) x109/L  23.3  4.1‐9.4 Neutrophils (band) x109/L  2.8  0‐0.24 Lymphocytes x109/L  1.2  0.9‐3.6 Monocytes x109/L  2.2  0.2‐1.0 Eosinophils x109/L  0  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: normal 

 URINALYSIS (voided) Appearance  Cloudy  pH  6 Colour  Yellow  Glucose  ‐ve Specific gravity  1.022  Ketones  ‐ve Protein  Trace  Blood  ‐ve     Bilirubin  ‐ve Microscopic findings: abundant bacteria and 60 leukocytes per HPF 

143

INTERPRETATION OF LABORATORY FINDINGS 

The information suggests lower urinary tract disease, but the depression and pain is a little unusual.  (a)  Detected  laboratory  abnormalities:  mild  azotemia,  mild  hyperproteinemia,  erythrocytes  just above  reference  interval,  MCV  just  below  reference  interval,  leukocytosis  primarily  due  to neutrophilia with left shift (ratio of less than 1 to 10 ‐ definite left shift in the dog if the ratio is less than  1  to  16‐18  or  the  total  band  count  is  greater  than  1  x  109/L),  monocytosis,  absolute eosinopenia, pyuria and bacteruria.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: From  the urinalysis  it can be deduced  that  inflammation  is occurring somewhere  in  the urogenital tract  (conclusion).  The  trace  protein  may  be  significant  at  this  relatively  low  sp.  gr.,  but  the leukocytes are the key. Presumably the  inflammation  is due to a bacterial  infection. Azotemia and neutrophilia with a  left  shift are more  likely  to occur  in either  renal or uterine disease as  cystitis rarely  causes  these  changes  (conclusion).  The  fact  that  TPP  is  elevated  could  suggest  some haemoconcentration  but  since  there  is  no  evidence  of  clinical  dehydration,  an  increase  in  a particular protein must also be considered  (e.g. gamma globulins or  fibrinogen).  If  the animal was dehydrated  then  the  fact  that  there  is  azotemia  and  a  less  than  optimal  concentration  of  urine would more likely point to the kidney as being the site of disease (conclusion). The young age of the dog would go against the possibility of endometritis/pyometron.  The monocytosis, absolute eosinopenia and low normal lymphocytes could suggest that some of the neutrophilia  is  due  to  stress  (i.e.  inflammatory  demand  is  on  top  of  this).  The  high  erythrocytes (relative to PCV and Hb) could be a laboratory error. Consequently, the MCV is low.  Further  investigation could  involve diagnostic  imaging of  the urinary  tract. Any detection of  renal involvement  has  implications  for management.  Obviously,  the  urine  should  be  cultured  and  an appropriate antibiotic regime determined as this has implications for management.    DIAGNOSIS AND POSTSCRIPT: The dog died and at necropsy a bilateral pyelonephritis was found. However,  further histological  investigation  showed  that  the  inflammation probably  started  in  the bladder (i.e. cystitis was also present). 

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CASE  11  ANIMAL: 5 year old female crossbred dog.  PRESENTING COMPLAINTS: Red urine of one week duration.  LABORATORY RESULTS  URINALYSIS (voided) Appearance  Cloudy  pH  5.5 Colour  Amber  Glucose  ‐ve Specific gravity  1.056  Ketones  ‐ve Protein  2+  Blood  4+     Bilirubin  ‐ve Microscopic findings: greater than 200 erythrocytes per HPF, 5‐15 leukocytes per HPF 

 

145

INTERPRETATION OF LABORATORY FINDINGS 

Red  urine  in  a  middle‐aged  female  dog  provides  little  information,  but  haematuria  or haemoglobinuria should be considered.  (a) Detected laboratory abnormalities: proteinuria and positive blood strip due to haematuria.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The  dog  has  haematuria, which was  the  cause  of  the  positive  blood  strip  (the  strip will  detect erythrocytes, free haemoglobin and myoglobin). The proteinuria may be due to leakage from vessels (this  level  of  red  cells  would  suggest  some  rhexis  of  vessels  ‐lower  levels  can  occur  just  with diapedesis e.g. 30 per HPF) or due to interference from the blood strip ‐ it has been shown that any reaction can occur on the protein strip if the reaction on the blood strip is maximal i.e. 4+.  The level of leukocytes, although above the reference interval (roughly 2‐5 leukocytes per HPF) are probably related to the bleeding (i.e. the conclusion is simply bleeding). By determining the ratio of leukocytes  to  erythrocytes,  one  can  get  a  reasonable  idea whether  the  increased  leukocytes  are related to frank haemorrhage or due to true inflammatory demand. In the dog, circulating blood has a leukocyte to erythrocyte ratio of one to 200‐400 or greater.  In most fluids e.g. peritoneal fluid, a ratio of less than one to 200 is suspicious of true increases in leukocytes but to be certain the ratio should be below one  to 50.  In urine, normal  levels of  leukocytes should be subtracted before  the ratio is determined. In the present case subtraction leaves between 5 and 10 leukocytes.  The ratio cannot be accurately determined as the erythrocytes are given as being greater than 200 per HPF. However, it is highly unlikely that the ratio will be low enough to be significant.  The cause of the haematuria could be further investigated through diagnostic imaging and biopsy of the  bladder.  It may  be  worthwhile  to  collect  a  urine  sample  by  cystocentesis.  Detection  of  an underlying cause will have implications for management.   DIAGNOSIS AND POSTSCRIPT: The cause of the haematuria was not determined for this dog and it cleared up without treatment or further investigation, and without a significant anaemia occurring. It was presumed that the bleeding had occurred in the bladder and was idiopathic. 

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CASE  12  ANIMAL: 8 year old female neutered Labrador dog  PRESENTING COMPLAINTS: Red urine of ten days duration  LABORATORY RESULTS  URINALYSIS (voided) Appearance  Cloudy  PH  6 Colour  Red  Glucose  ‐ve Specific gravity  1.039  Ketones  ‐ve Protein  3+  Blood  4+     Bilirubin  ‐ve Microscopic findings (uncentrifuged): greater than 200 erythrocytes per HPF, greater than 100 leukocytes per HPF 

147

INTERPRETATION OF LABORATORY FINDINGS 

Red urine  in  an  aged  female dog provides  little  information, but haematuria or haemoglobinuria should be considered.  (a) Detected laboratory abnormalities: Haematuria, pyuria.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: Although  the chemical analysis of  the urine  shows  similar  results  to case 12,  the  sediment  shows distinct differences. There were so many cells that it was not necessary to concentrate the urine 1 in 20. However, this does not interfere with the determination of the leukocyte to erythrocyte ratio. In this  case  it  could  be  as  low  as  1  to  2.  Thus  true  inflammatory  demand  (conclusion)  is  present somewhere  in the urinary tract (the dog has been spayed). The fact that the  levels of erythrocytes are  so  high  suggests  that  some  of  the  bleeding  is  occurring  through  rhexis  rather  than  through simple inflammatory diapedesis. This, for example, could be related to urinary calculi in the bladder or to neoplasia causing secondary inflammation and haemorrhage. Occasionally severe infection and inflammation will cause rhexis leading to high levels of erythrocytes (all these are conclusions).  Further  investigation  could  involve  diagnostic  imaging  and  biopsy  of  the  bladder.  Culture  of  the urine  (perhaps  on  a  cystocentesis  sample)  should  be  considered.  Determining  the  cause  of  the inflammation and bleeding will have obvious implications for management.  DIAGNOSIS AND POSTSCRIPT:   Radiographs of this dog found bladder calculi (analysed as struvite) and the dog was treated for cystitis and the calculi (i.e. haematuria due to cystitis related to urinary calculi). 

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CASE  13  ANIMAL: 4 month old female Thoroughbred foal  PRESENTING COMPLAINTS: Depression, swollen joints, pyrexia for over one week  LABORATORY RESULTS 

HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Variable PCV L/L  0.23  0.32‐0.52 Plasma protein g/L  64  58‐84 Haemoglobin g/L  78  110‐190 Erythrocytes x1012/L  6.7  8‐12.5 MCV fl  34  41‐49 MCHC g/L  339  300‐360 Leukocytes x109/L  39  6.0‐13 Neutrophils (seg.) x109/L  31  2.5‐7 Neutrophils (band) x109/L  2  0‐0.2 Lymphocytes x109/L  4.5  1.6‐5.4 Monocytes x109/L  1.3  0‐0.7 Eosinophils x109/L  0.2  0.2‐1 Basophils x109/L  0  0‐0.4 Blood film: vacuolation of neutrophils, some Howell‐Jolly bodies present Fibrinogen g/L  10  1‐4 

Other tests: 

STIFLE FLUID ANALYSIS 

Appearance:  slightly cloudy, yellow green (usually clear and light yellow) Protein (g/L):  45 (usually less than 20) Total nucleated cells (x106/L): 

19,100 (usually less than 3,000) 

Erythrocytes (x106/L):  32,000(usually none) Differential:  97% neutrophils with intracellular bacteria and 3% 

macrophages (usually most cells are lymphocytes or monocytes with neutrophils being less than 10%) 

 E. coli recovered from joint fluid 

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INTERPRETATION OF LABORATORY FINDINGS 

In  a  young  foal with  pyrexia  and multiple  joint  effusions,  an  infectious  arthritis would  be  a  top consideration.  (a) Detected  laboratory abnormalities: Anaemia, decreased MCV,  leukocytosis due  to neutrophilia and  mild  monocytosis,  absolute  eosinopenia,  toxic  neutrophils  (vacuolation  of  cytoplasm), hyperfibrinogenemia, and septic exudate in the stifle joint.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The foal has anaemia, which according to reference values is microcytic. However, foals between 1‐9 months often have low MCVs (mean is actually around 34.5 for thoroughbreds foals of this age) i.e. the MCV  is actually normal for this age foal. Always remember that the young of most species will have different  reference  intervals  for many analytes when compared  to adult values  (it  requires a quick  check of  the  texts!). The anaemia  cannot be  further  categorised without  serial  sampling or bone marrow examination (unwarranted at this stage).  The leukocyte changes are probably partly due to stress but there appears to be more occurring. The neutrophilia  is quite high  for  stress alone  (levels  for  total  leukocytes  rarely exceed 20.0x 109/L  in cases of stress in adult horses) and this in conjunction with a definite left shift (see below) and toxic changes  to neutrophils  suggest an  inflammatory demand component  (conclusion). This  is possibly supported by the increased fibrinogen.  The  joint  fluid  shows  increased  cells  (which  are  predominantly  neutrophils  with  intracellular bacteria) and increased protein (as measured by the refractometer ‐ a crude method for determining protein  in  joint  fluid).  This  indicates  a  septic  exudate  (conclusion)  (nb.  septic  exudates  in most cavities are characterised by  lytic (degenerate) neutrophils. However, this  is rarely the case  in  joint fluid due to the apparent protective nature of synovial secretions).  On reflection, the anaemia was probably secondary to the inflammation (enhanced haemolysis and utilisation of  iron by bacteria). Although  the  ratio of bands  to  segmented neutrophils  is 1  to 15.5 (grey zone for a  left shift  in horse), some veterinarians would be happy to definitely call this a  left shift as there are more than 0.3 x 109/L bands.  Further  investigation  requires culture and determination of an appropriate antibiotic  regime. The finding of a septic exudate has obvious implications for management. One may decide on antibiotic treatment based on past experience while waiting for the laboratory to provide information on the micro‐organisms involved.  DIAGNOSIS AND POSTSCRIPT: Generalised septic arthritis due to Escherichia coli. 

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CASE  14  ANIMAL: 10 year old female Weimeraner  PRESENTING COMPLAINTS: Depression, swollen joints (both stifles, elbows, carpi and tarsi), pyrexia of greater than one week’s duration  LABORATORY RESULTS 

 STIFLE FLUID ANALYSIS 

Appearance:  slightly cloudy, colourless and clear Protein (g/L):  30 (usually less than 25) Total nucleated cells (x106/L): 

3,200 (usually less than 2,900) 

Erythrocytes (x106/L):  1,000 (usually none) Differential:  77% neutrophils, 20% monocytes and 3% macrophages 

(usually most cells are lymphocytes or monocytes with neutrophils being less than 10%) 

 No bacteria recovered from joint fluid 

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INTERPRETATION OF LABORATORY FINDINGS 

The information provided suggests polyarthropathy in an aged dog. This could be inflammatory and both infectious and non‐infectious (e.g. immune‐mediated) causes need to be considered.  (a) Detected laboratory abnormalities: Non‐septic exudate in the stifle joint.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The dog had multiple  joints  affected.  Inflammatory  joint disease  is  commonly divided  into  septic (bacterial)  and  non‐septic.  Non‐septic  forms  can  include  infectious  agents  such  as mycoplasmal organisms,  fungal  agents  (Cryptococcus neoformans)  and protozoal organisms but  these  are  rare. Most non‐septic forms are non‐infectious. These are classified as immune‐mediated (‘autoimmune’) or non‐immune‐mediated  and  generally  involve  a number of  joints. Non‐immune‐mediated  types are  uncommon  and may  be  due  to  crystal  deposition  (e.g.  certain  forms  of  calcium)  or, more commonly,  due  to  haemoarthrosis  due  to  a  bleeding  disorder.  Immune‐mediated  types  include erosive  forms  (e.g.s  rheumatoid  arthritis  in  the  dog,  polyarthritis  in  the  greyhound),  proliferative types  (e.g. chronic progressive polyarthritis  in the cat) and non‐erosive  forms  (e.g.s systemic  lupus erythematosus,  polyarthritis/polymyositis  syndrome,  and  idiopathic  polyarthritis).  The  idiopathic types of immune‐mediated arthritis are probably related to immune complex deposition, which may occur  due  to  infections  or  neoplasia  elsewhere  in  the  body.  There  appears  to  be  a  particular association between various gastro‐intestinal diseases and this type of arthritis.  In this case the non‐septic polyarthritis was considered to be immune‐mediated because of the lack of  bacteria,  but  no  extra‐joint  disease was  detected  (conclusion).  There would  be  little  need  for further  investigation,  but  one  could  do  haematology  (to  detect  inflammatory  demand),  a rheumatoid factor analysis and an anti‐nuclear antibody test.  DIAGNOSIS AND POSTSCRIPT: The dog was considered to have immune‐mediated polyarthritis. The dog responded to anti‐inflammatory medication. 

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CASE  15  ANIMAL: 7 year old male Japanese Chin dog  PRESENTING  COMPLAINTS:  Respiratory  distress,  abdominal  effusion  slowly  developing  over  a month. The heart had a distinct valve murmur.  LABORATORY RESULTS 

HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.30  0.37‐0.50 Plasma protein g/L  49  55‐75 Haemoglobin g/L  104  100‐150 Erythrocytes x1012/L  4.7  5‐7 MCV fl  64  60‐75 MCHC g/L  347  300‐360 Leukocytes x109/L  21.6  7‐12 Neutrophils (seg.) x109/L  19.5  4.1‐9.4 Neutrophils (band) x109/L  0.1  0‐0.24 Lymphocytes x109/L  0.8  0.9‐3.6 Monocytes x109/L  1.1  0.2‐1.0 Eosinophils x109/L  0  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: slight polychromasia, 1 NRBC per 100 leukocytes 

Other tests: 

PERITONEAL FLUID ANALYSIS 

Total protein:  19 g/L (normal < 25 g/L) Nucleated cells:  5000 x 106/L (normal < 500 x 106/L) Erythrocytes:  40,000 x 106/L (none normally) Smear:  70% non‐lytic (non‐degenerate) neutrophils, 30% 

mononuclear cells (normally mainly mononuclears) 

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INTERPRETATION OF LABORATORY FINDINGS 

The provided information could indicate a cardiac related abdominal effusion.  (a) Detected  laboratory  abnormalities: Mild  anaemia, mild  hypoproteinemia,  leukocytosis  due  to neutrophilia  and  mild  monocytosis,  lymphocytopenia,  absolute  eosinopenia  and  a  modified transudative fluid.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The identification of the peritoneal fluid as a modified transudate could support a diagnosis of heart failure in the dog (conclusion). Often the fluid starts off as a pure transudate but quickly becomes a modified transudate, and may progress to a non‐septic exudate. The erythrocytes are present due to diapedesis related to increased hydrostatic pressure over a prolonged period of time.  The anaemia has not been classified but in chronic heart failure is commonly non‐regenerative. The slight polychromasia and presence of a normoblast  suggest attempts at  regeneration  in  this case. The  mild  hypoproteinemia  may  possibly  be  due  to  chronic  disease  and  interference  with  liver function.  However,  it  is  possible  that  the  levels  of  individual  classes  of  proteins  are  still within reference  intervals  i.e.  the  hypoproteinemia  is  not  of  significance.  The  leukocyte  changes  are consistent with stress i.e. corticosteroid release. This is common in the dog related to intense illness. Possibly this dog has reached a crisis in the disease process which has led to enhanced corticosteroid release (conclusion).  Further investigation requires diagnostic imaging and electrocardiography.  DIAGNOSIS AND POSTSCRIPT: Cardiac  imaging detected valvular  lesions (endocardiosis suspected). The  diagnosis  was  heart  failure  due  to  advanced  endocardiosis.  The  animal  was  given  a  poor prognosis and the owner’s elected euthanasia. 

154

CASE  16  ANIMAL: 4 year old male Siamese cat.  PRESENTING  COMPLAINTS:  Depression,  pyrexia  and  abdominal  effusion.  The  cat  had  suddenly returned home after a three day absence.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Urea mmol/L  9.3  7.2‐10.7 Creatinine μmol/L  137  98‐180 Sodium mmol/L  131  147‐156 Potassium mmol/L  4.5  4‐4.6 Chloride mmol/L  103  115‐130 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.39  0.30‐0.45 Plasma protein g/L  73  59‐78 Haemoglobin g/L  131  80‐140 Erythrocytes x1012/L  8.6  6‐10 MCV fl  45  40‐45 MCHC g/L  333  310‐360 Leukocytes x109/L  25.1  8‐14 Neutrophils (seg.) x109/L  21.4  3.8‐10.1 Neutrophils (band) x109/L  1.4  0‐0.4 Lymphocytes x109/L  0.9  1.6‐7.0 Monocytes x109/L  1  0.1‐0.6 Eosinophils x109/L  0.2  0.2‐1.4 Basophils x109/L  0  0‐0.2 Blood film: toxic granulation of the neutrophils 

Other tests: 

PERITONEAL FLUID ANALYSIS 

Total protein:  57 g/L (normal < 25 g/L) Nucleated cells:  64,100 x 106/L (normal < 500 x 106/L) Erythrocytes:  20,000 x 106/L (none normally) Smear:  90% lytic (degenerate) neutrophils (with intracellular bacteria), 

10% macrophages (normally mainly mononuclears)  Culture revealed mixed anaerobic bacteria 

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INTERPRETATION OF LABORATORY FINDINGS 

The  information of pyrexia and abdominal effusion  in a  four year‐old cat could  suggest  infectious disease (although pyrexia is not exclusive to inflammatory disease).  (a) Detected  laboratory  abnormalities: Moderate  leukocytosis due  to neutrophilia  (with  left  shift) and  monocytosis,  lymphocytopenia,  toxic  granulation  of  neutrophils,  hyponatremia, hypochloridemia and septic exudate within the peritoneal cavity.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The peritoneal fluid analysis indicates increased protein and nucleated cells. Since most of the cells are  lytic  (degenerate) neutrophils and contain bacteria, a diagnosis of septic exudate can be made (conclusion). This was most  likely the result of a cat bite or a penetration wound (conclusion). The erythrocytes are probably there due to diapedesis.  One would expect the cat to be stressed i.e. increased corticosteroid release, and this is reflected in the  lymphocytopenia  and  probably  part  of  the  neutrophilia.  The  low  normal  eosinophils  and increased monocytes may also be a  reflection of  this  (although monocytosis  is not as common  in stress as in the dog). A 1 to 15 ratio of band to segmented neutrophils may indicate a left shift (one can't be certain until the ratio drops below 1 to 10‐12 but for this cat 1 to 15 may be adequate, but as mentioned some veterinarians would automatically accept this as a left shift as there are over 1 x 109  /L  bands.  The  monocytosis  may  also  be  due  to  inflammatory  demand  for  macromolecular phagocytosis as  stress  induced monocytosis  is not as  common  in  the  cat as  in  the dog. The  toxic changes in the neutrophils are a reflection of the bacterial infection.  The hyponatremia is possibly due to the extensive effusion and redistribution of sodium. The drop in chloride often goes hand in hand with a drop in sodium. The lack of azotemia in this case is unusual but  is  possibly  a  good  prognosis  (the  development  of  shock with  subsequent  reduction  of  renal blood flow is a common reason for decent increases in urea and creatinine in pre‐renal diseases).  Further  investigation would  require  culture  and  determination  of  appropriate  antibiotic  therapy. One  may  place  the  animal  on  a  broad  spectrum  antibiotic  (and  probably  drain  and  flush  the abdomen) while waiting for laboratory analysis (implications for management).  DIAGNOSIS: Bacterial peritonitis (mixed micro‐organisms). 

156

CASE  17 ANIMAL: 6 year old Thoroughbred mare.  PRESENTING  COMPLAINTS:  Depression  and  pyrexia  after  foaling.  Possible  increase  in  abdominal fluid.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Urea mmol/L  43  3.7‐8.2 Creatinine μmol/L  656  87‐149 Sodium mmol/L  134  132‐150 Potassium mmol/L  5.5  2.8‐5.0 Chloride mmol/L  92  99‐110 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Variable PCV L/L  0.54  0.32‐0.52 Plasma protein g/L  75  58‐84 Haemoglobin g/L  197  110‐190 Erythrocytes x1012/L  12.3  6‐12 MCV fl  44  41‐49 MCHC g/L  364  300‐390 Leukocytes x109/L  8.1  6‐13 Neutrophils (seg.) x109/L  3.7  2.5‐7 Neutrophils (band) x109/L  0  0‐0.2 Lymphocytes x109/L  3.5  1.6‐5.4 Monocytes x109/L  0.9  0‐0.7 Eosinophils x109/L  0  0.2‐1 Basophils x109/L  0  0‐0.4 Blood film: moderate granulation of neutrophils 

 

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INTERPRETATION OF LABORATORY FINDINGS 

The limited information could suggest a genital problem post‐foaling.  (a) Detected  laboratory abnormalities: Polycythemia, absolute eosinopenia, possible  toxic changes to neutrophils, moderate azotemia, hyperkalemia and mild hypochloridemia.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The polycythemia  is most  likely due  to haemoconcentration  (despite  the TPP still being within  the reference  interval) related to fluid  imbalances.  In this case we have eosinopenia and possible toxic granulation of neutrophils. The latter could be due to toxaemia or infection (conclusion).  The  moderate  azotemia  is  related  to  the  catabolic  state  of  the  animal  and  the  expected derangements in renal blood flow. In horses, it is accepted that the higher the values, the poorer the prognosis  (i.e.  an  indicator  of  the  development  of  shock).  The  hyperkalemia  could  be  related  to possible acid‐base imbalance (metabolic acidosis may cause a shift of potassium from cells into the plasma  in exchange  for excess H+) and widespread  cell damage  (due  to  the  infection and  related tissue damage). The mild drop in chloride may be related to the low normal level of Na+ (i.e. this Na+ level  may  be  low  for  this  particular  horse).  Both  could  be  due  to  the  peritoneal  effusion  and redistribution.   Further investigation will involve genital examination and peritoneal effusion analysis (if a ruptured uterus  is  suspected).  It  will  also  involve  bacterial  culture  and  appropriate  antibiotic  therapy  if infection is identified (implications for management).   DIAGNOSIS AND POSTSCRIPT: Ruptured uterus and  subsequent peritonitis diagnosed on  further investigation. The horse died during early treatment.  The  lack  of  leukocyte  changes  is  unusual  in  this  case.  A  stress  response  (neutrophilia, lymphocytopenia and eosinopenia), a response to endotoxemia (neutropenia and lymphocytopenia ‐ the horse is particularly sensitive to small amounts of endotoxin produced by certain Gram‐negative rods)  or  a  response  to  infection  (early  ‐  neutropenia  due  to  over  whelming  demand, lymphocytopenia  due  to  entrapment  of  lymphocytes  within  lymph  nodes,  eosinopenia  due  to mechanisms unrelated to corticosteroid release; later ‐ neutrophilia with left shift, although left shift development  is not as common  in the adult horse as  it  is  in dogs and cats or ruminants) would be expected. 

158

CASE  18  ANIMAL: 2 year old male Labrador dog.  PRESENTING COMPLAINTS: Vomiting and abdominal pain, for about 4 days duration.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Amylase IU/L  7500  <1400 Lipase IU/L  850  <60 ALP IU/L  1202  <11 ALT IU/L  170  <60 Urea mmol/L  9  3‐10 Sodium mmol/L  149  137‐150 Potassium mmol/L  3.8  3.3‐4.8 Chloride mmol/L  103  105‐120 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.57  0.37‐0.50 Plasma protein g/L  83  55‐75 Haemoglobin g/L  201  100‐150 Erythrocytes x1012/L  8.9  5‐7 MCV fl  64  60‐75 MCHC g/L  352  300‐360 Leukocytes x109/L  15.8  7‐12 Neutrophils (seg.) x109/L  12.8  4.1‐9.4 Neutrophils (band) x109/L  1.7  0‐0.24 Lymphocytes x109/L  0.5  0.9‐3.6 Monocytes x109/L  0.8  0.2‐1.0 Eosinophils x109/L  0  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: Doehle bodies in neutrophils 

 

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INTERPRETATION OF LABORATORY FINDINGS 

Acute vomiting and abdominal pain might suggest acute hepatopathy, acute pancreatic necrosis, GIT inflammation or obstruction, peritonitis/splenitis, or less likely acute nephropathy.  (a) Detected  laboratory abnormalities: Marked hyperamylasemia, marked hyperlipasemia, marked elevation of ALP, mild elevation of ALT, mild hypochloridemia, polycythemia, mild hyperproteinemia, mild leukocytosis due to neutrophilia (with left shift ‐ ratio less than 1 to 10 and total value greater than 1 x 109/L), lymphocytopenia and absolute eosinopenia.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The marked hyperamylasemia and hyperlipasemia are suggestive of pancreatic necrosis or perhaps high  intestinal obstruction (conclusion). The presence of the stress  leukogram  in combination with true  inflammatory  demand  (left  shift)  would  support  the  possibility  of  pancreatic  necrosis.  The marked  cholestasis  and mild  hepatocellular  damage  could  be  explained  by  bile  duct  obstruction through pancreatic swelling and mild toxemia.  The polycythemia appears  to be  spurious due  to haemoconcentration  (probably  through vomiting and shock). The hypochloridemia is probably due to vomiting. These changes are common in, but not specific for pancreatic necrosis. The lack of a pre‐renal azotemia is unusual in pancreatic necrosis.  In  summary, most  of  the  laboratory  changes  can  be  explained  by  the  animal  having  pancreatic necrosis  (conclusion).  Further  investigation  could  involve  diagnostic  imaging  (especially ultrasonography).  DIAGNOSIS  AND  POSTSCRIPT:  Acute  pancreatic  necrosis.  It  responded  well  to  fluids  and symptomatic therapy. 

160

CASE  19  ANIMAL: 2 year old female greyhound.  PRESENTING COMPLAINTS: Pain and reluctance to move after race.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL AST IU/L  213  <40 CK IU/L  5207  <100 Serum protein (refract.) g/L  78  50‐70 Albumin (EPG) g/L  43.1  23‐39 α globulins (EPG) g/L  7  7‐16 β globulins (EPG) g/L  11.4  9‐16 γ globulins (EPG) g/L  16.5  4‐12 Urea mmol/L  65  3‐10 Sodium mmol/L  116  137‐150 Potassium mmol/L  4.8  3.3‐4.8 Chloride mmol/L  77  105‐120 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.67  0.37‐0.50 Plasma protein g/L  77  55‐75 Haemoglobin g/L  274  100‐150 Erythrocytes x1012/L  11.7  5‐7 MCV fl  66  60‐75 MCHC g/L  356  300‐360 Leukocytes x109/L  18.7  7‐12 Neutrophils (seg.) x109/L  14.6  4.1‐9.4 Neutrophils (band) x109/L  0  0‐0.24 Lymphocytes x109/L  0.75  0.9‐3.6 Monocytes x109/L  3.3  0.2‐1.0 Eosinophils x109/L  0.1  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: hypersegmentation of the neutrophils 

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INTERPRETATION OF LABORATORY FINDINGS 

The information is too limited, but could suggest trauma through racing.  (a)  Detected  laboratory  abnormalities:  Mild  elevation  of  AST,  marked  elevation  of  CK,  mild hyperproteinemia due  to elevations  in both albumin and globulins, marked azotemia  (urea only), hyponatremia,  hypochloridemia,  elevated  PCV,  Hb  and  erythrocytes  (polycythemia),  leukocytosis (moderate to marked  for a greyhound as they commonly have  lower reference  intervals) due to a neutrophilia and monocytosis, lymphocytopenia and eosinopenia.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The significantly elevated CK after a  race suggests muscle damage. Of course CK will elevate after any muscular activity, and sometimes quite high, but  in  this case  the elevation  is accompanied by other  changes which  point  to  true  rhabdomyolysis  (pain  and  reluctance  to move  after  the  race) (conclusion). The AST is only mildly elevated but often this takes longer to rise than CK after damage (the advantage of using enzymes in tandem).  The  dog  appears  to  be  haemoconcentrated  (elevated  red  cell  parameters  and  TPP),  which  is supported  by  the  protein  electrophoresis  (albumin  could  only  be  increased  due  to haemoconcentration, i.e. dehydration). There is a stress leukogram. The level of neutrophilia is quite significant for a greyhound as they tend to have lower circulating levels in health compared to other dog breeds, and their response to disease is more limited. Hypersegmentation of neutrophils occurs due  to  aging  in  the  circulation  as  corticosteroids  released  in  stress  have  inhibited  the  escape  of neutrophils into the tissues.  The dog is significantly azotemic which could be primarily due to pre‐renal factors (tissue breakdown and  derangements  of  renal  blood  flow)  but  renal  disease  cannot  be  excluded without  urinalysis (conclusion). The  low  sodium  is difficult  to  explain but may be  related  to  renal disease. Chloride changes often  follow sodium changes. The upper normal potassium could be  related  to metabolic acidosis through tissue breakdown or renal disease, or directly due to release from damaged cells.  All  these  changes  support  a  diagnosis  of  paralytic  rhabdomyolysis.  Further  investigation  through urinalysis would have been useful  in determining  the presence of  free myoglobin  (positive on  the blood  strip)  and  the  extent  of  possible  kidney  damage which  has  been  recorded with  extensive rhabdomyolysis (so called myoglobinuric nephrosis).  DIAGNOSIS: Paralytic rhabdomyolysis. 

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CASE  20  ANIMAL: 8 year old female neutered crossbred cat.  PRESENTING  COMPLAINTS:  Motor  car  accident  (broken  pelvis)  one  week  before  presentation. Check‐up.  LABORATORY RESULTS 

HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.26  0.30‐0.45 Plasma protein g/L  76  59‐78 Haemoglobin g/L  93  80‐140 Erythrocytes x1012/L  5.8  6‐10 MCV fl  45  40‐45 MCHC g/L  358  310‐360 Leukocytes x109/L  25.1  8‐14 Neutrophils (seg.) x109/L  20.4  3.8‐10.1 Neutrophils (band) x109/L  0  0‐0.4 Lymphocytes x109/L  3.3  1.6‐7.0 Monocytes x109/L  0.5  0.1‐0.6 Eosinophils x109/L  0.9  0.2‐1.4 Basophils x109/L  0  0‐0.2 Blood film: moderate anisocytosis and polychromasia, 3 NRBC per 100 leukocytes Reticulocyte % (uncorrected)  9.2  0‐1 

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INTERPRETATION OF LABORATORY FINDINGS 

 (a) Detected  laboratory abnormalities: Mild anaemia,  leukocytosis due  to neutrophilia without  left shift, reticulocytosis.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: This  is a regenerative anaemia. The blood changes are characteristic of blood  loss about one week previously (maximum response usually occurs at 7‐9 days) (conclusion). The presence of  immature cells  within  the  circulation  (reticulocytes)  produces  the  anisocytosis  and  polychromasia.  The circulating NRBCs  (normoblasts) are accompanying  the regenerative response, and are  in  the right proportion  for  an  orderly  response.  They  are  more  common  in  the  regenerative  response  to haemolytic  anaemia.  The  high  normal  MCV  may  be  a  reflection  of  the  level  of  circulating reticulocytes  (larger  cells).  The  reticulocyte  count,  9.2%,  because  of  its magnitude  doesn't  really need to be corrected to ensure that the response  is truly regenerative. For the exercise, correction for the level of anaemia will give a value of 6%. The absolute level of reticulocytes is 0.530 x 1012/L (RR up to 0.100 x 1012/L) The neutrophil response is likely to be related to the intense regenerative anaemia but some could be due to true tissue demand (despite the absence of a left shift) related to the  pelvic  injuries.  The  total  plasma  protein  is  now  normal  due  to  increased  production  and replacement.   Further investigation of the anaemia, at this stage, is probably not warranted.  DIAGNOSIS: Blood loss anaemia. 

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CASE  21  ANIMAL: 3 year old male Cocker Spaniel.  PRESENTING COMPLAINTS: Depression and weakness started 5 days earlier. Now jaundiced.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL ALP IU/L  409  <110 ALT IU/L  238  <60 Total bilirubin μmol/L  203  1.2‐5.1 Unconjugated bilirubin μmol/L  19  1.2‐5.1 Conjugated bilirubin μmol/L  184  1.2‐5.1 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Yellow  Clear PCV L/L  0.21  0.37‐0.50 Plasma protein g/L  77  55‐75 Haemoglobin g/L  80  100‐150 Erythrocytes x1012/L  2.5  5‐7 MCV fl  84  60‐75 MCHC g/L  380  300‐360 Leukocytes x109/L (corrected for NRBC)  25.1  7‐12 Neutrophils (seg.) x109/L  17.5  4.1‐9.4 Neutrophils (band) x109/L  2.6  0‐0.24 Lymphocytes x109/L  0.7  0.9‐3.6 Monocytes x109/L  4.0  0.2‐1.0 Eosinophils x109/L  0.3  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: marked anisocytosis and polychromasia, 30 nucleated erythroid cells (NRBC) per 100 leukocytes, many spherocytes Reticulocyte % (uncorrected)  10.2  0‐1.5 

 Other tests:  Coomb's test was positive (end point 1/32 i.e. highest dilution of serum 

showing agglutination). 

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INTERPRETATION OF LABORATORY FINDINGS 

The  limited  information  suggests  a  relatively  intense,  recent disease.  The  jaundice points  to pre‐hepatic, hepatic or post‐hepatic disease. 

(a) Detected  laboratory abnormalities: Moderate regenerative anaemia, neutrophilia with  left shift, lymphocytopenia, monocytosis,  spherocytes,  positive  Coomb's  test, mildly  elevated ALP  and ALT, hyperbilirubinemia (predominantly conjugated). 

(b) & (c) General interpretation, conclusions, further investigation and implications for management: The anaemia  is  regenerative on  the basis of a high  reticulocyte  count. Correction  for  the  level of anaemia gives a value of 4.8% (.21/.45 [as average PCV] x 10.2), correction for both level of anaemia and erythroid maturation time (taken as 2 days for this PCV) gives a value of 2.4 (>2  is regarded as truly regenerative, a value of 1‐2 indicates less than optimum regeneration while a value less than 1 is  considered  non‐regenerative.    The  absolute  reticulocyte  count  is  0.255  x  1012/L  (RR  less  than 0.105  x  1012/L).  The  polychromasia  and  anisocytosis  are  a  reflection  of  the  high  numbers  of circulating reticulocytes (polychromatophilic macrocytes in a smear stained by Giemsa or 'Diff Quik'). These  large  cells,  because  of  their  significant  numbers,  have  raised  the  MCV  above  reference interval, and this will be maintained until reticulocytosis subsides. Normally  in  intense regenerative anaemia the MCHC will be depressed due to the fact that the reticulocytes are immature and have too little Hb for their size (the so called pseudomacrocytic, pseudohypochromic response in intense regenerative  anaemia).  However,  in  this  case  the  MCHC  is  actually  increased  above  reference interval. A high MCHC  is not possible as erythrocytes cannot be oversaturated with Hb. Therefore, the value is due to laboratory error or due to the presence of free Hb (as in haemolytic states – most likely in this case). 

The  large  number  of  circulating  nucleated  erythroid  cells  (NRBC)  is  appropriate  in  regenerative anaemia when reticulocytosis is marked. This is more likely to occur in haemolytic rather than blood loss  regenerative  anaemias  as  iron  is  more  easily  re‐utilised  (conclusion).  The  presence  of spherocytes  (dense  cells  without  a  central  pallor  which  occur  because  of  the  pinching  off  of damaged  or  antibody‐coated  surface membrane  by macrophages)  and  the  positive  Coomb's  test suggest that the haemolytic anaemia is immune mediated (conclusion). In this case, because of the high titre in the Coomb's test and the lack of other disease processes that may give rise to antibody coating of erythrocytes (e.g. erythrocyte parasites, drugs etc) it is likely that the problem is primary immune‐mediated (auto‐immune) (conclusion and final diagnosis). 

The total leukocyte count has been corrected for a falsely high count created by nucleated erythroid cells being included in counting by the automatic cell counter. Normally, the total leukocyte count is not  corrected  unless  the  circulating  nucleated  erythroid  cells  reach  5  per  100  leukocytes  (total leukocyte  count  x 100/100 + no. of  circulating nucleated erythroid  cells per 100  leukocytes). The neutrophilia, lymphocytopenia and monocytosis (eosinophils are within reference interval but at the lower end) could be interpreted as stress induced (the animal was still intensely ill). However, there is  a  left  shift  to  the  neutrophilia,  which  suggests  true  inflammatory  demand  for  part  of  the neutrophilia. This  is probably  in response to the haemolytic anaemia. Intense haemolytic anaemias commonly cause left shifts, especially in the dog, but the exact mechanisms are poorly understood. Non‐specific  stimulation of  the macrophage system may be  involved as may be  released products from cell breakdown. Part of the monocytosis in this case may be directly related to the haemolytic anaemia. 

ALP elevation  suggests mild  cholestasis, ALT elevation  suggests mild hepatocellular damage  (fatty change  for example). Both of  these could be related  to hepatic hypoxia created by acute onset of anaemia. The markedly high total serum bilirubin is to be expected in haemolytic anaemias that are 

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predominantly  intravascular. Free Hb produced by  intravascular erythrocytic destruction  is quickly converted to bilirubin. In the early stages most of the bilirubin is unconjugated but as time goes on an  increase  in  conjugated may occur.  In  this  case  the  conjugated  levels are marked. This may be partly due to some cholestasis caused by hepatocyte swelling but is most likely to be due to simple regurgitation related to enhanced production. 

The support diagnosis in this case was straight forward. The prognosis for the auto‐immune anaemia appears  good  as  regeneration  is  substantial. However,  the  process  of  destruction  appears  to  be continuing  (the high MCHC probably means  free HB  through present erythrocyte destruction) and treatment needs to be continued. 

Further  investigation  is  really  related  to  following  the  course  of  the  disease  and  response  to therapy. This would be done through routine haematology. 

DIAGNOSIS: Auto‐immune haemolytic anaemia. 

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CASE  22  ANIMAL: 5 year old female pony.  PRESENTING COMPLAINTS: Weakness, rapid respiration, dark urine and jaundice. Noticed after a 3‐day period when the horse was left in a paddock.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Total bilirubin μmol/L  160  0‐50 Unconjugated bilirubin μmol/L  154  0‐6.8 Conjugated bilirubin μmol/L  6  3.4‐50 Urea mmol/L  16  3.7‐8.2 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Red‐yellow  Variable PCV L/L  0.10  0.32‐0.52 Plasma protein g/L  82  58‐78 Haemoglobin g/L  47  130‐190 Erythrocytes x1012/L  2.1  6.5‐12.5 MCV fl  47  41‐49 MCHC g/L  470  300‐390 Leukocytes x109/L  17.1  6.0‐13 Neutrophils (seg.) x109/L  14.6  2.5‐7 Neutrophils (band) x109/L  0.2  0‐0.2 Lymphocytes x109/L  1.4  1.6‐5.4 Monocytes x109/L  0.9  0‐0.7 Eosinophils x109/L  0  0.2‐1 Basophils x109/L  0  0‐0.4 Blood film: many ghost erythrocytes and Heinz bodies 

 URINALYSIS (voided) Appearance  Cloudy  PH  6.0 Colour  Red  Glucose  ‐ve Specific gravity  1.021  Ketones  ‐ve Protein  4+  Blood  4+     Bilirubin  ‐ve Microscopic findings: 0‐2 erythrocytes per HPF 

 

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INTERPRETATION OF LABORATORY FINDINGS 

The provided information suggests an acute onset disease that has given rise to jaundice, weakness and  red  urine  (Hb, Mb  or  haematuria?).  Considerations would  be  pre‐hepatic,  hepatic  and  post‐hepatic  causes  of  jaundice.  If  the  red  urine  is  directly  related  to  jaundice  then  pre‐hepatic haemolysis might be a reason.  (a)  Detected  laboratory  abnormalities:  Marked  anaemia,  hyperproteinemia,  elevated  MCHC, leukocytosis  due  to  neutrophilia,  lymphocytopenia,  eosinopenia,  ghost  cells  and  Heinz  bodies, marked serum hyperbilirubinemia (primarily due to  increased unconjugated), mild azotemia (urea), possible haemoglobinuria.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The results (the  low PCV, high MCHC [through the presence of free HB], haemoglobinuria and high unconjugated  bilirubin)  suggest  that  the  pony  has  had  a  recent  intravascular  haemolytic  crisis (conclusion). Some of the increase in unconjugated bilirubin is probably due to anorexia. Ghost cells (i.e.  ruptured  erythrocytes)  and  Heinz  bodies  (denatured  Hb)  suggest  haemolysis  through  Hb denaturation mechanisms. Drugs and plants could be causes (conclusion).  The hyperproteinemia  is possibly partly spurious, perhaps related to  free Hb elevating  the reading on  the  refractometer.  However,  some  haemoconcentration  can't  be  ruled  out.  The  leukocyte changes can be explained by corticosteroid release (monocytosis rarely occurs  in horses). The mild azotemia  is  probably  related  to  protein  catabolism  and  reduced  renal  blood  flow  through  any haemoconcentration. The positive urinary blood strip is assumed to be due to free Hb as there is no indication  of muscle  damage  and  intact  erythrocytes  are  few  in  the  sediment.  The  4+  protein  is probably related to the 4+ blood. No bilirubin is being passed as it is almost all unconjugated in the blood (only conjugated is passed in the urine of most domestic species ‐ the dog is an exception).  Further investigation would involve obtaining more history from the owner about the environment and  the  past  history  for  the  horse.  Did  it  have  access  to  poisonous  plants?  Had  it  access  to chemicals?   DIAGNOSIS  AND  POSTSCRIPT:  Heinz  body  haemolytic  anaemia.  On  further  questioning  of  the owner,  it was  found  that  the  horse  had  been  given  phenothiazine  (anthelmintic)  prior  to  being released in the paddock. If given a high enough dose, it could have caused the haemolysis. The horse died a day later.  

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CASE  23  ANIMAL: 4 year old male crossbred dog.  PRESENTING COMPLAINTS: Weight loss, inappetence and depression for a period of three weeks.  LABORATORY RESULTS 

HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.46  0.37‐0.50 Plasma protein g/L  87  55‐75 Haemoglobin g/L  150  100‐150 Erythrocytes x1012/L  6.7  5‐7 MCV fl  69  60‐75 MCHC g/L  326  300‐360 Leukocytes x109/L (corrected for NRBC)  13.4  7‐12 Neutrophils (seg.) x109/L  8.7  4.1‐9.4 Neutrophils (band) x109/L  0  0‐0.24 Lymphocytes x109/L  1.9  0.9‐3.6 Monocytes x109/L  2.5  0.2‐1.0 Eosinophils x109/L  0.3  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: 8 nucleated erythroid cells (NRBC) per 100 leukocytes, moderate numbers of target cells, basophilic stippling of erythrocytes Reticulocyte % (uncorrected)  2  0‐1.5 

 Other tests  Urinary delta aminolevulinic acid (ALA) analysis: 190 (reference interval <38) at a urinary specific gravity of 1.030 (i.e. moderately concentrated urine)  

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INTERPRETATION OF LABORATORY FINDINGS 

The  provided  information  provides  little  in  the way  of  clues  for  specific  diseases  or  even what organs/tissues may be involved.  (a) Detected laboratory abnormalities: Mild leukocytosis due to monocytosis, basophilic stippling of erythrocytes, moderate target cells (leptocytes), circulating nucleated erythroid cells, mildly elevated reticulocyte count (in the absence of anaemia), elevated delta ALA.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The  moderately  elevated  urinary  delta  ALA  is  suggestive  of  lead  poisoning  (conclusion  and diagnosis). Delta ALA elevates in urine when there is interference with porphyrin metabolism (as in heme  synthesis  for erythrocytes). Lead  is capable of doing  this as  it  interferes with  several of  the enzymatic  steps. Blood  lead  levels would have given an  indication of  recent exposure  to  lead but would not have indicated past exposure (lead is quickly stored in tissues and blood levels will drop). Delta ALA is an indication of the effect of increased body lead, whether obtained now or in the past.  Basophilic  stippling of erythrocytes,  the presence of  target  cells and  the occurrence of  circulating nucleated erythroid cells in the absence of anaemia support the diagnosis of lead poisoning. These are the viewed effects of altered erythropoiesis which  lead produces through several mechanisms. These  changes  are  not  always  present  in  lead  poisoning  and  moreover,  may  occur  in  other derangements of erythropoiesis.  Basophilic stippling can occur in intense regenerative anaemias, especially in ruminants and pigs. In dogs and cats  it rarely occurs and  its appearance  is slightly different to that  in  lead poisoning. The presence of circulating nucleated erythroid cells with a low reticulocyte count and/or in the absence of the appropriate  level of anaemia  indicates  inappropriate (defective or disorderly) erythropoiesis and can occur in bone marrow or splenic disorders (e.g. myelofibrosis, leukemia, hemangiosarcoma). Target  cells  are  a  form  of  leptocyte  that  have  a  large  surface  area  for  their  volume.  They may develop  through  membrane  lipid  derangements  such  as  in  certain  types  of  hepatic  disease  or endocrinopathies (e.g. hypothyroidism).  The mild monocytosis in this case is difficult to explain. It does not seem to be of importance in the diagnosis.  Further investigation might involve further questioning of the owners about possible access to lead. If ingestion of solid lead (e.g. fishing sinker) is considered, then it might be useful to radiograph the abdomen. A blood  lead analysis might be considered. This will be  increased during acute  ingestion and absorption, but may not be much altered  if most of the  lead has been deposited  in bone and other  tissues  (past  exposure).  The  diagnosis  of  lead  poisoning  has  direct  implications  for management. While determining the source of lead, the animal could be treated with lead chelating agents.  DIAGNOSIS AND POSTSCRIPT: Lead poisoning. On further questioning, the owner admitted that he had been renovating an old home and there may have been the possibility of access to  lead‐based paint. The dog responded well to treatment. 

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CASE  24  ANIMAL: One year old male Burmese cat.  PRESENTING COMPLAINTS: Respiratory distress, coughing and elevated temperature for 3 weeks.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Serum protein (refract.) g/L  78  54‐73 Albumin (EPG) g/L  30.9  24‐30 α globulins (EPG) g/L  17.7  9‐21 β globulins (EPG) g/L  9.8  8‐15 γ globulins (EPG) g/L  19.6  9‐23 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.29  0.30‐0.45 Plasma protein g/L  84  59‐78 Haemoglobin g/L  95  80‐140 Erythrocytes x1012/L  6.7  6‐10 MCV fl  44  40‐45 MCHC g/L  328  310‐360 Leukocytes x109/L  29.3  8‐14 Neutrophils (seg.) x109/L  23.6  3.8‐10.1 Neutrophils (band) x109/L  3.1  0‐0.4 Lymphocytes x109/L  2.3  1.6‐7.0 Monocytes x109/L  0.1  0.1‐0.6 Eosinophils x109/L  0.2  0.2‐1.4 Basophils x109/L  0  0‐0.2 Blood film: toxic granulation and Doehle bodies in neutrophils 

Other tests: 

Trans‐tracheal aspirate: Cytology revealed numerous alveolar macrophages and clusters of lytic (degenerate) neutrophils. A pure growth of Staphylococcus intermedius was obtained from the fluid.  The animal was positive for Feline Leukemia Virus (antibody test on serum). 

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INTERPRETATION OF LABORATORY FINDINGS 

Respiratory distress and coughing could suggest a primary respiratory disease affecting airways and pulmonary alveoli; but underlying cardiac disease should also be considered. The pyrexia may occur with any severe  inflammatory or degenerative disease, but  is most commonly related to  infectious diseases. 

 (a) Detected  laboratory  abnormalities:  Septic  exudate  from  respiratory  tract, borderline  anaemia (on  PCV  alone),  mild  hyperproteinemia,  leukocytosis  due  to  neutrophilia  with  left  shift,  toxic granulation and Doehle bodies in neutrophils. 

 (b)  &  (c)  General  interpretation,  conclusions,  further  investigation  and  implications  for management:  The  trans‐tracheal  aspirate  findings  support  a  diagnosis  of  pneumonia  (conclusion and diagnosis). Alveolar macrophages  suggest  that  the  inflammation has  reached  the  level of  the pulmonary alveoli and  is not  just a simple tracheitis or bronchitis. The  isolation of S.  intermedius  is unusual as this part of the skin's normal flora. It is obviously acting as an opportunistic pathogen but how it got into the respiratory tract is difficult to answer. The animal is probably haemoconcentrated (elevated  total  protein), which  could mean  that  the  cat  probably  has  a  true mild  anaemia.  The anaemia could go along with inflammatory disease (bacterial utilisation of iron or, in the long term, iron sequestration by macrophages), but since the cat is FeLV positive, the possibility of direct bone marrow depression by  the  virus  cannot be excluded. The neutrophilia with  left  shift  is  consistent with  an  inflammatory  process  of  3  weeks  duration.  The  neutrophil  response  is  not  entirely satisfactory as the bacteria are obviously having an effect on neutrophil function (toxic granulation and  Doehle  bodies),  but  at  least  the  bone  marrow  is  responding  well.  If  the  changes  in  the neutrophils persist with treatment this may be a poor prognostic sign but they are to be expected in severe  bacterial  disease  in  the  early  stages.  The  Doehle  bodies  are  an  indication  of  altered neutrophil maturation and suggest toxemia or the effects of certain drugs (i.e. they are not specific for  toxemia).  It  should be  remembered  that  in  the cat  small  'Doehle' bodies may occur  in a  small number of neutrophils in health. Therefore only large Doehle bodies in a significant number of cells (say 30% plus) are considered indication of altered neutrophil function. 

Both  the plasma and serum proteins would have been measured by  refractometer. Consequently, the values can be directly compared and the difference (indirect measurement) is usually fibrinogen. In  this  case,  it  is  elevated  (6  g/L  –  normally  2‐4  g/L).  Fibrinogen  is  an  acute  phase  reactant  and elevations may be  seen  in  inflammatory or,  less  so, acute degenerative disease. This occurs more consistently in ruminants and horses than in dogs and cats. Other acute phase reactant proteins are being  developed  as  tests  for  inflammatory  disease  in  the  dog  and  cat  (see  notes  under  liver diseases).  If the serum protein  is measured by the chemical method  (Biuret)  then  it  is not directly comparable to plasma protein measured by refractometer and fibrinogen must be determined by a different method (see practical notes on Haematology). 

The cat's pneumonia may well be a result of immune‐ compromise. The finding of an opportunistic pathogen  and  the  fact  that  the  cat  is  FeLV  positive  could  well  indicate  this  fact  (conclusion). Implications for management are as for any bacterial pneumonia. Further investigation is probably not required once appropriate antibiotic therapy is initiated, but repeat haematology may be useful to ensure  that  the  inflammatory demand  and  toxic  changes  are diminishing  (i.e.  good prognostic signs). Presumably, diagnostic imaging had been performed at the same time as the pulmonary wash was undertaken. 

DIAGNOSIS AND POSTSCRIPT: Bacterial pneumonia. The owner was warned of the possibility that the cat might be predisposed to unusual  infections  in the future. The cat recovered from antibiotic treatment and supportive therapy. 

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CASE  25  ANIMAL: One year old female Burmese cat.  PRESENTING COMPLAINTS: Fever, depression, peritoneal and pleural effusions.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Serum protein (refract.) g/L  77  54‐73 Albumin (EPG) g/L  32.2  24‐30 α globulins (EPG) g/L  10.3  9‐21 β globulins (EPG) g/L  8.9  8‐15 γ globulins (EPG) g/L  25.6  9‐23 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.16  0.30‐0.45 Plasma protein g/L  85  59‐78 Haemoglobin g/L  53  80‐140 Erythrocytes x1012/L  3.4  6‐10 MCV fl  51  40‐45 MCHC g/L  331  310‐360 Leukocytes x109/L  25.5  8‐14 Neutrophils (seg.) x109/L  20.4  3.8‐10.1 Neutrophils (band) x109/L  1.3  0‐0.4 Lymphocytes x109/L  2.5  1.6‐7.0 Monocytes x109/L  0.7  0.1‐0.6 Eosinophils x109/L  0.5  0.2‐1.4 Basophils x109/L  0  0‐0.2 Blood film: moderate polychromasia, one NRBC per 100 leukocytes 

Other tests: 

PERITONEAL FLUID ANALYSIS 

Total protein:  60 g/L (normal < 25 g/L) Nucleated cells:  3000 x 106/L (normal < 500 x 106/L) Erythrocytes:  600 x 106/L (none normally) Smear:  78% non‐lytic (non‐degenerate) neutrophils, 22% mononuclear 

cells (including reactive mesothelial cells) (normal: mainly mononuclears) 

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INTERPRETATION OF LABORATORY FINDINGS 

Body cavity effusions and pyrexia  in a young cat might suggest  infectious disease. Feline  Infectious Peritonitis  is  one  such  cause  (i.e.  ‘pattern  recognition’),  and  will  need  to  be  considered  in investigation.  (a) Detected laboratory abnormalities: Moderate anaemia, mild hyperproteinemia, leukocytosis due to  neutrophilia with  left  shift,  hyperglobulinemia,  hyperfibrinogenemia,  peritoneal  fluid  is  a  non‐septic exudate.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The  laboratory changes suggest multiorgan  inflammatory disease that could be consistent with FIP (conclusion and specific diagnosis).  The  cat  has  an  anaemia  which  has  not  been  investigated  further.  Changes  on  the  smear (polychromasia, nucleated erythroid cell could possibly indicate regeneration but this would need to be  confirmed with  a  reticulocyte  count.  In  FIP  the  anaemia  can  be  quite  variable.  Inflammatory disease tends to give a mild to moderate non‐regenerative anaemia. The neutrophilia  is suggestive of  inflammatory demand. A  left shift  is present, considering that total numbers of bands exceed 1x 109/L.  The cat has hyperfibrinogenemia  (8 g/L  ‐ difference between total serum protein and total plasma protein,  both  measured  by  the  refractometer)  and  hyperglobulinemia  which  are  suggestive  of inflammatory disease with a strong immune response. The globulin increase was broad based on the EPG  (i.e.  increase  in a variety of gamma globulins). Fibrinogen  increases  can occur  in a variety of degenerative and inflammatory conditions but are more consistent in farm animals. Fibrinogen is an 'acute  phase  reactant' which  is  produced  and  stored  by  the  liver.  Therefore,  rapid  increases  can occur. In FIP, fibrinous inflammation is a feature, possibly due to the vasculitis.  The peritoneal fluid changes are consistent with FIP. An extremely high protein (much of this would be gamma globulins)  in combination with a subdued increase in nucleated cells is highly suggestive of FIP in the cat. On the basis of increased protein and nucleated cells I have called this an exudate (non‐septic). However, some pathologists might prefer  to call  it a modified  transudate because of the limited elevations that can occur in the nucleated cells in some cases. This difference in opinion is not important. It is important to realise that the changes indicate an inflammatory process of non‐bacterial origin. The reactive mesothelial cells are more likely to be seen in modified transudates and non‐septic exudates.  Further investigation requires confirmation for the coronavirus that causes FIP. There are serological tests, but many of  these have poor specificity  (overlap with  the  intestinal coronavirus of cats  that causes little disease). Coronavirus antigen detection can now be done on cytological and histological preparations.  Management of FIP is difficult and most affected cats are euthanised. If the cat is from a cattery or multi‐cat household, then it is important to develop a plan of management for the owners.  DIAGNOSIS AND POSTSCRIPT: Feline  Infectious Peritonitis. Because  this case occurred before  the development  of  the  coronavirus  antigen  detection  test,  confirmation  of  the  disease  was  not obtained. Necropsy confirmed typical FIP lesions of vasculitis and pyogranulomatous inflammation. 

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CASE  26 ANIMAL: 6 year old male Chihuahua.  PRESENTING  COMPLAINTS:  Generalised  lymphadenomegaly,  hepatomegaly,  splenomegaly,  pale mucous membranes.  LABORATORY RESULTS 

HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.13  0.37‐0.50 Plasma protein g/L  52  55‐75 Haemoglobin g/L  45  100‐150 Erythrocytes x1012/L  1.7  5‐7 MCV fl  76  60‐75 MCHC g/L  346  300‐360 Leukocytes x109/L (corrected for NRBC)  203  7‐12 Neutrophils (seg.) x109/L  7.9  4.1‐9.4 Neutrophils (band) x109/L  0  0‐0.24 Lymphocytes x109/L  187  0.9‐3.6 Monocytes x109/L  3.1  0.2‐1.0 Eosinophils x109/L  0  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film:  many lymphoid cells were blasts, neutrophils were hypersegmented, poikilocytosis and mild polychromasia of erythrocytes Reticulocyte % (uncorrected)  2  0‐1.5 

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The pale mucous membranes could suggest anaemia or shock. The generalised lymphadenomegaly, hepatomegaly  and  splenomegaly  could  occur  with  widespread  infiltrative  disease  of  either inflammatory or neoplastic nature.  (a)  Detected  laboratory  abnormalities:  Marked  non‐regenerative  anaemia,  hypoproteinemia, leukocytosis due to lymphocytosis, circulating lymphoblasts, hypersegmented neutrophils.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The diagnosis of  lymphoid  leukemia (circulating neoplastic  lymphoid cells)  is obvious from the high level of  lymphocytes and  the  fact  that significant numbers of  lymphoblasts are circulating  (usually there are none)  (conclusion and  specific diagnosis). The non‐regenerative anaemia  (corrected  for the level of anaemia the reticulocyte value is less than 0.6%; corrected for the level of anaemia and maturation  time,  i.e.  the  Reticulocyte  Production  Index,  the  value  is  less  than  one;  absolute reticulocyte count  is 0.035 x 1012/L)  is probably due  to a combination of  factors but bone marrow replacement by the production of neoplastic lymphoid cells (myelophthisis) is likely to be important. Hypersegmented neutrophils are indicative of retention within the circulation that can occur due to the release of corticosteroids  in stress. The hypoproteinemia  is possibly due to neoplastic cachexia and possibly hepatic dysfunction (the liver and spleen are often infiltrated in cases of leukemia ‐ of any cell type).  Lymphoid neoplasia  in the dog often presents as solid masses  in organs and enlarged  lymph nodes (hence  the  name  lymphosarcoma).  A  lymph  node  cell  aspirate  or  biopsy  should  confirm  the diagnosis (further investigation) but often blood examination is undertaken to assist prognosis and treatment. The leukemia could be further investigated through bone marrow examination.  An animal with lymphosarcoma and that is leukemic and with a marked non‐regenerative anaemia is less  likely to respond well to chemotherapy because of the bone marrow effects and the advanced stage;  however,  whether  treatment  is  undertaken  will  depend  on  the  owners  (implications  for management). Lymphoid  leukemia can develop  in an animal with  lymphosarcoma or  it can be an entity in itself as either acute lymphoblastic or chronic lymphocytic leukemia.  DIAGNOSIS  AND  POSTSCRIPT:  Lymphosarcoma  and  lymphoid  leukemia.  The  owner  elected  for euthanasia. 

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CASE  27  ANIMAL: 2 year old male Springer spaniel.  PRESENTING COMPLAINTS: Diarrhoea, inappetence and weight loss of 6 weeks duration.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Serum protein (refract.) g/L  17  50‐70 Albumin (EPG) g/L  8.8  23‐39 α globulins (EPG) g/L  2.9  7‐16 β globulins (EPG) g/L  4.7  9‐16 γ globulins (EPG) g/L  0.6  4‐12 Calcium mmol/L  1.6  2.1‐2.9 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  clear  Clear PCV L/L  0.49  0.37‐0.50 Plasma protein g/L  23  55‐75 Haemoglobin g/L  162  100‐150 Erythrocytes x1012/L  7.4  5‐7 MCV fl  66  60‐75 MCHC g/L  331  300‐360 Leukocytes x109/L  14.8  7‐12 Neutrophils (seg.) x109/L  11.2  4.1‐9.4 Neutrophils (band) x109/L  0  0‐0.24 Lymphocytes x109/L  3.1  0.9‐3.6 Monocytes x109/L  0  0.2‐1.0 Eosinophils x109/L  0.5  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: normal 

Other tests: 

Serum trypsin/trypsinogen like Immunoreactivity (TLI) μg/L: 12 (reference interval 5‐35)  FAECAL ANALYSIS 

Trypsin (really all faecal proteases): 5 Azoalbumin units (reference interval >7) Blood: present Smears for undigested/ unabsorbed food particles: no increase in starch, undigested muscle or undigested fat (unsplit, primarily triglycerides); large increase in unabsorbed fat (split, primarily free fatty acids) 

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Chronic  diarrhea  with  significant  weight  loss  could  suggest malassimilation  associated  with  the intestinal disease.  (a)  Detected  laboratory  abnormalities:  Marked  hypoproteinemia,  elevated  Hb  and  erythrocyte count,  mild  leukocytosis  due  to  neutrophilia,  monocytopenia,  all  protein  fractions  decreased, hypocalcemia, decreased trypsin level, faecal blood, increased unabsorbed fat.  The dog has a significant hypoproteinemia  (all classes), which  in  this case  is most  likely due  to an enteropathy (faecal tests and the clinical signs suggest this) (conclusion). End stage liver disease can cause a hypoproteinemia affecting most protein groups, although gamma globulins are not usually affected  and may  actually  be  increased.  Severe  starvation may  cause  a  hypoproteinemia.  Kidney disease  may  cause  hypoproteinemia  but  this  commonly  involves  only  albumin  (globulins  are generally too large to be lost by the glomeruli or tubules). In fact some globulins may increase in the blood.  The hypocalcemia in this case is possibly due to loss of protein bound calcium through damaged gut (most kits detect total calcium which includes protein bound and free ionised forms).  Increased unabsorbed fat in the faeces suggests an enteropathy as most cases of malabsorption are due to enteric disease (conclusion). The slightly decreased trypsin activity has no significance in this case  as  the  smears  for  food particles  do not  suggest maldigestion.  This  is  also  supported by  the normal TLI  (less  than 2.5  μg/L  is diagnostic  for maldigestion due  to EPI). The TLI has  replaced  the faecal trypsin test for maldigestion due to EPI. The positive faecal blood may  indicate mild  leakage associated with a gut disease. However, the test  is not terribly reliable as  it can give false positives due  to  the presence of other  substances having peroxidase  activity.  The mild neutrophilia  in  the absence of other white cell changes may suggest  limited  inflammatory demand, perhaps related to gut  disease.  The monocytopenia  has  no  significance  in  any  circumstances.  The  elevated  Hb  and erythrocyte  count  can  be  best  explained  by  haemoconcentration.  Commonly,  non‐regenerative anaemia  occurs  in  chronic  bowel  diseases  but  perhaps  this  one  has  not  been  occurring  for  long enough.  In  summary,  the  persistent  diarrhoea  and  evidence  of  malabsorption  suggest  that  the hypoproteinemia is due to gut disease (main conclusion).   Further  investigation  could  involve  diagnostic  imaging,  intestinal  absorption  tests,  endoscopic examination and gut biopsies The hypoproteinemia could be investigated by injecting protein bound to radioactive chromium, and checking for losses in the faeces.    DIAGNOSIS  AND  POSTSCRIPT:  Malabsorption  and  protein  losing  enteropathy  due  to  severe inflammatory bowel disease (IBD).  IBD is a poorly understood, probably multifactorial condition of dogs and cats and, depending on intensity, can cause a variety of clinical signs relating to dysfunction of the small intestine. 

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CASE  28  ANIMAL: One year old female neutered German shepherd.  PRESENTING COMPLAINTS:  Weight loss, normal to increased appetite, soft voluminous faeces. Gradually developing over 3 months.  LABORATORY RESULTS 

BIOCHEMISTRY 

Trypsin/trypsinogen like Immunoreactivity (TLI) μg/L: 1.5 (reference interval 5‐35)  FAECAL ANALYSIS 

Trypsin (really all faecal proteases): 2.6 Azoalbumin units (reference interval >7) Blood: absent Smears for undigested/ unabsorbed food particles: no increase in starch or unabsorbed fat (split, primarily free fatty acids), Large increases in undigested muscle and undigested fat (unsplit, primarily triglycerides).  

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INTERPRETATION OF LABORATORY FINDINGS 

Significant weight  loss and voluminous faeces, especially  in an animal that has normal to  increased appetite, could suggest malassimilation. The fact that  it  is a young dog might suggest congenital as well as acquired disease.  (a) Detected  laboratory  abnormalities:  Low  faecal proteolytic  (trypsin)  activity,  low  TLI,  increased undigested muscle and fat.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The  results  are  highly  suggestive  of  maldigestion  (conclusion).  Lowered  TLI,  faecal  trypsin  and increased unsplit  fat  in  the  faeces are  the most  important  results and suggest Exocrine Pancreatic insufficiency (EPI) as a cause of the maldigestion (conclusion and diagnosis). Results for undigested starch and muscle are extremely variable depending in the diet. If possible it is best to analyse faeces from a healthy dog placed on the same diet. In cases of suspected maldigestion where contradictory results  between  trypsin  levels  and  smears  for  food  particles  occur  (i.e.  low  trypsin  and  normal smears)  repeat  testing must  be  employed.  This  is  because  low  trypsin  values  (less  than  7)  will occasionally be detected in healthy dogs. The TLI has replaced the need for faecal trypsin analysis.  This dog had classic stools for EPI, i.e. rancid, soft but semi‐formed and voluminous. In cases of EPI (the most common cause of maldigestion) the faeces are rarely fluid. Fluid diarrhoea is more likely to occur with an enteropathy, with or without malabsorption.  Further investigation might involve ultrasonography of the pancreas.   The  laboratory  results  have  direct  implications  for management  as  pancreatic  extract  and  diet control are warranted  DIAGNOSIS AND POSTSCRIPT: Exocrine pancreatic insufficiency (EPI). Because of its young age and the  fact  that  it was  a German  shepherd,  pancreatic  hypoplasia was  considered  best  fit  (pattern recognition). The animal responded to therapy for EPI. 

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CASE  29  ANIMAL: 6 year old male cross Labrador.  PRESENTING COMPLAINTS: Depression, fitting, ataxia  LABORATORY RESULTS  CEREBROSPINAL FLUID ANALYSIS 

Gross characteristics clear and colourless (normal) Protein  0.54 g/L (reference interval < .4 g/L) Cells  erythrocytes 9 x 106/L; nucleated cells 47 x 106/L (reference interval 

for nucleated < 8 x 106/L, no erythrocytes normally seen) Differential  65% lymphocytes, 35% monocytes and macrophages (normal ‐ 

usually a mixture of mononuclear cell types) 

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INTERPRETATION OF LABORATORY FINDINGS 

The ataxia and fitting could suggest primary neurological disease, but metabolic disorders or poisons should probably be considered also.  (a) Detected laboratory abnormalities: Elevated protein and cells within cerebrospinal fluid.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The changes in CSF are suggestive of chronic (non‐suppurative) inflammation (conclusion). The mild increase  in protein  is  significant  as  the  concentrations of  constituents  are  strictly  controlled. This level of protein can only be detected by microanalytic methods (e.g. ponceau red S), but the protein strip on the urinary dipstick may give a rough guide to  levels (can detect values above  .1 g/L). The increase in protein is probably due to inflammatory leakage but in some cases of distemper, protein increases  can occur without  increases  in  cells  (so  called albumino‐cytologic dissociation  ‐  this  can also  occur  in  some  tumours  and  in  vascular  lesions  i.e.  protein  leakage  apparently  unrelated  to inflammation). The level of increased nucleated cells is quite significant and is not greatly influenced by the fact that erythrocytes are present (i.e.  iatrogenic bleeding does not usually elevate  levels of nucleated  cells until erythrocyte values exceed 30, and not  significantly until values exceed 500 x 106/L ‐ in these cases increases in nucleated cells are due to increased neutrophils). The differential suggests chronicity (immune response cells and macrophages) but it is important to remember that these cells are normally present in low numbers in CSF in health.  CSF  changes do not occur  in all  cases of neurological disease. Diseases affecting  the meninges or adjacent layers are more likely to cause changes. Interpretation is basically as for other fluids.  Further investigation would be to determine specific causes of non‐suppurative inflammation. One might consider culture of CSF  for protozoal or  fungal organisms  (although not detected  in  routine examination and usually causing a pyogranulomatous cell response). Serological tests are available for toxoplasmal, neosporal and cryptococcal disease.   DIAGNOSIS  AND  POSTSCRIPT:  Necropsy  revealed  severe  meningoencephalomyelitis  with  viral inclusions. This was suggestive of Canine Distemper. 

183

CASE  30  ANIMAL: 9 year old female neutered miniature collie dog.  PRESENTING COMPLAINTS: Polydipsia, polyuria and weight  loss  for  some months. Now  collapsed with vomiting and diarrhoea.  LABORATORY RESULTS 

Blood sample markedly lipemic.  BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL ALP IU/L  135  <110 ALT IU/L  95  <60 Triglycerides mmol/L  5.3  0.5‐2.0 Total cholesterol mmol/L  13.65  1.4‐7.5 Glucose mmol/L  42.7  3.3‐6.4 Urea mmol/L  27.6  3‐10 

 URINALYSIS (voided) Appearance  Cloudy  pH  5.0 Colour  Yellow  Glucose  2% Specific gravity  1.051  Ketones  2+ Protein  Trace  Blood  ‐ve     Bilirubin  Trace Microscopic findings: hyaline casts 50‐60 per LPF, fine granular casts 10‐15 per LPF, much lipid, abundant transitional epithelium. 

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INTERPRETATION OF LABORATORY FINDINGS 

The polyuria and polydipsia could occur with renal, hepatic or endocrine diseases. The fact that it is now  vomiting  and  severely  unwell  could  suggest  a  terminal  event  or  a  totally  unrelated  disease process to the one that caused polyuria/polydipsia.  If the first possibility  is considered (this makes more sense – Occam’s Razor: ‘that the fewest possible assumptions are to be made  in explaining a thing’), then chronic renal failure and diabetes mellitus with developed ketoacidosis are most likely.  (a) Detected  laboratory abnormalities: Mildly elevated ALP and ALT, marked hypertriglyceridemia, moderate hypercholesterolemia, marked hyperglycemia, moderate azotemia, ketonuria, glucosuria and cylindruria.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: Laboratory diagnosis of diabetes mellitus requires the identification of persistent hyperglycemia and glucosuria (2 or more samples). However, in this case the marked elevation of blood glucose is highly suggestive  of  diabetes  (conclusion  and  diagnosis).  The  glucosuria  is  to  be  expected  as  the  renal threshold (about 11mmols/L for the dog) has been exceeded. The hyperlipidemia commonly occurs and is due to derangements of carbohydrate‐lipid metabolism and the inhibition of lipoprotein lipase activity. Elevations  in  triglyceride are  commonly marked whilst elevations  in  cholesterol are more variable. ALT and ALP minimal elevations are probably due to fatty change in the liver or just due to the severity of illness.  The  presence  of  ketones  in  the  urine  and  the  low  pH  are  highly  suggestive  that  this  dog  is ketoacidotic (in an adult dog or cat ketonuria rarely accompanies other diseases) (conclusion). This is probably  contributing  to  the  azotemia  as  would  be  the  vomiting  and  inappetence.  Although cylindruria  (increased  casts)  may  suggest  tubular  damage,  the  high  specific  gravity  rules  out  a concentrating  problem  for  the  kidney  (i.e.  adequate  function  of  the  tubules,  interstitium  and adequate release of ADH). Perhaps the cylindruria  indicates a period of decreased renal blood flow due to haemoconcentration followed by the first voiding of urine? Abundant transitional epithelium may support this. The trace protein and bilirubin are of doubtful significance in this case and at this concentration  level.  Lipiduria  is  a  reflection  of  changes  in  tubular  cells  and  can  occur  in  healthy animals. In this case it would be expected.  Most cases of advanced diabetes mellitus are relatively easy to diagnose but earlier cases are more difficult. Blood glucose levels may not be markedly elevated, and elevations may be inconsistent. In those cases, an intravenous glucose tolerance test (further testing) may be necessary to support the diagnosis  of  diabetes  mellitus  (a  flat  curve  or  low  glucose  disappearance  co‐efficient  suggests glucose intolerance). In all dogs with diabetes mellitus, the insulin response to the tolerance test will help decide whether there  is an absolute or relative  lack of  insulin (i.e.  insulin dependent diabetes mellitus [IDDM] or non‐insulin dependent diabetes mellitus [NIDDM]).  Implications for management are for controlling the ketoacidosis and then stabilising the DM  DIAGNOSIS  AND  POSTSCRIPT:  Diabetes  mellitus  with  ketoacidosis.  The  dog  was  treated successfully and recovered. The animal required  long‐term  insulin as most cases of DM  in the dog are IDDM. Most causes of IDDM are obscure. 

185

CASE  31  ANIMAL: 8 year old female poodle.  PRESENTING COMPLAINTS: Prolonged history of lethargy, dry, shortened coat, obese, non‐ cycling.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL CK IU/L  135  <200 Total cholesterol mmol/L  16.8  1.4‐7.5 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Lipemic  Clear PCV L/L  0.28  0.37‐0.50 Plasma protein g/L  Lipemia 

interfered 55‐75 

Haemoglobin g/L  Lipemia interfered 

100‐150 

Erythrocytes x1012/L  4.5  5‐7 MCV fl  62  60‐75 MCHC g/L  ND  300‐360 Leukocytes x109/L  26.6  7‐12 Neutrophils (seg.) x109/L  22.3  4.1‐9.4 Neutrophils (band) x109/L  0.3  0‐0.24 Lymphocytes x109/L  2.9  0.9‐3.6 Monocytes x109/L  1.1  0.2‐1.0 Eosinophils x109/L  0  0.14‐1.2 Basophils x109/L  0  0‐0.4 Reticulocyte % (uncorrected)  2.1  0‐1.5 Blood film: marked numbers of target cells 

Other tests: 

Baseline T4: 8nmol/L (reference interval 20‐40 nmol/L)   

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INTERPRETATION OF LABORATORY FINDINGS 

The  clinical  signs  suggest  a  chronic disease  that might be affecting more  than body  system  (skin, genital  tract) Obesity  is  a  little  unusual  in  chronic  disease  related  to  neoplasia,  degeneration  or inflammation (inappetence and direct effects of the pathological process often  lead to cachexia). A metabolic/endocrine disturbance might be considered (e.g. hypothyroidism).  (a) Detected  laboratory abnormalities:   Mild elevation of CK, moderate hypercholesterolemia, non‐regenerative  mild  to  moderate  anaemia,  leukocytosis  due  primarily  to  neutrophilia,  mild monocytosis and absolute eosinopenia, target cells, low baseline T4.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The  history  is  suggestive  of  hypothyroidism  and  changes  in  non‐specific  tests  support  this (conclusion  and  diagnosis).  The  mild  elevation  in  CK  has  been  recorded  in  a  proportion  of hypothyroid  dogs  and  is  apparently  due  to  a  myopathy.  Hypercholesterolemia  can  occur  in hypothyroidism  due  to  decreased  utilisation  and  reduced  plasma  clearance  through  decreased lipoprotein  lipase  activity.  The  visible  lipemia  present  in  this  case  is  probably  due  to  increased triglyceride  within  circulating  lipoproteins  as  increased  cholesterol  rarely  gives  visible  turbidity. Lipemia  and  the  composition  of  the  lipids  are  extremely  variable  in  hypothyroidism.  The  non‐regenerative anaemia (corrected for the  level of anaemia, the value  is 1.3, and the RPI  is  less than one)  is  likely due to  interference with erythropoietin production or activity. The white cell changes are  likely due  to  stress. This  is uncommon  in  low grade chronic disease unless a clinical crisis has occurred. Target cells commonly occur when there is derangement of plasma membrane structure. Diseases  that cause derangements of  lipid metabolism may cause  target cell production  (e.g.  liver diseases, endocrinopathies).  All  these changes are consistent with hypothyroidism, but  the problem  is  that  their occurrence  in hypothyroidism  is  inconsistent.  Therefore,  the  performance  of  specific  hormonal  assays  is  often necessary.  Baseline  T4  is  an  appropriate  test  in  the  first  instance  (T3 measurement  has  no  real advantage over T4). However, a problem  is  that 20% of euthyroid dogs  can  show  low  levels of T4 (physiological variation, hypoproteinemia, certain drugs,  in some cases of hyperadrenocorticism). If this  is the case then a TSH stimulation test can be performed (further  investigation). A rise of  less than 3 x the baseline level about 4 hours after administration of TSH is suggestive of hypothyroidism. In the case discussed, the history, changes in non‐specific tests and the low baseline value for T4 are enough to be certain of the presence of hypothyroidism.   DIAGNOSIS  AND  POSTSCRIPT:  Hypothyroidism.  The  dog  was  successfully  treated  for hypothyroidism. 

187

CASE  32  ANIMAL: 7 year old female neutered corgi.  PRESENTING COMPLAINTS: Anorexia and weight loss, occasional vomiting, possibly dehydrated.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL Urea mmol/L  31  3‐10 Sodium mmol/L  122  137‐150 Potassium mmol/L  7.1  3.3‐4.8 Chloride mmol/L  91  105‐120 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.35  0.37‐0.50 Plasma protein g/L  68  55‐75 Haemoglobin g/L  119  100‐150 Erythrocytes x1012/L  5.9  5‐7 MCV fl  59  60‐75 MCHC g/L  340  300‐360 Leukocytes x109/L  12.6  7‐12 Neutrophils (seg.) x109/L  6.3  4.1‐9.4 Neutrophils (band) x109/L  0  0‐0.24 Lymphocytes x109/L  4.9  0.9‐3.6 Monocytes x109/L  0.6  0.2‐1.0 Eosinophils x109/L  0.8  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: normal 

188

INTERPRETATION OF LABORATORY FINDINGS 

The clinical signs are vague and of little help in pinpointing specific organ/tissue disease  (a) Detected laboratory abnormalities: Mild anaemia, borderline leukocytosis due to lymphocytosis, moderate azotemia, hyponatremia, hyperkalemia, hypochloridemia.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: Many  of  the  results  could  be  consistent  with  hypoadrenocorticism  (conclusion  and  diagnosis). Vague  clinical  signs  often  accompany  hypoadrenocorticism.  The  changes  are  related  to  variable decreases  in aldosterone and/or cortisol. Consequently, the  laboratory findings are variable. In this case the hyponatremia and hyperkalemia are highly suggestive of a lack of aldosterone (conclusion). The  ratio of  approximately 17  to 1  is well below  the 23  to 1  that  some workers  accept  as being unequivocal evidence  for hypoaldosteronism  (although some  think 23‐18  to 1  is a grey zone). The changes are due  to urinary  loss of sodium  (the chloride  loss  follows  this) and urinary  retention of potassium. Some gastrointestinal disturbances may affect the Na:K ratio  in a similar manner which may create difficulty in diagnosis as some hypoadrenocortical dogs may present with diarrhoea.  The azotemia  is consistent with hypoaldosteronism  (80% of cases) and  is due  to pre‐renal  factors such  as  protein  catabolism,  hypovolemia mainly  due  to  electrolyte  disturbances  and  subsequent reduced  renal  perfusion.  Some  animals may  have  impaired  tubular  concentrating  ability  with  a specific gravity  less than 1.025. This often means that primary renal failure  is one of the diagnoses under  consideration. We are unable  to  comment on  this  case and  further  investigation  for  renal disease may be warranted.  The mild anaemia has not been categorised but  is normocytic, normochromic and possibly worse than it seems if the animal is haemoconcentrated. In hypoadrenocorticism, a mild non‐regenerative anaemia  can  occur  due  to  cortisol  lack  (conclusion),  which  is  an  essential  hormone  for erythropoiesis. The lymphocytosis in a sick animal, which possibly should be stressed, is unusual and a  further  indication  that  there  may  be  a  lack  of  cortisol  release.  In  some  cases  of  adrenal insufficiency eosinophilia may also occur.  All  these  laboratory changes are  inconsistent  in adrenal  insufficiency  (e.g. up  to 26% of dogs with confirmed adrenal  insufficiency can have normal electrolyte  levels). Therefore,  it  is often necessary to measure baseline cortisol  levels and  levels after exogenous ACTH administration to confirm the diagnosis  of  adrenal  insufficiency  (further  investigation).  Of  course,  if  it  is  a  primary  lack  of aldosterone  then  the  values may  be  normal  but most  cases  of  primary  adrenal  insufficiency  do involve abnormalities of both cortisol and aldosterone.  DIAGNOSIS  AND  POSTSCRIPT:  Hypoadrenocorticism  (adrenal  insufficiency)  was  confirmed  by measuring baseline cortisol levels (low). The animal was treated with corticoid replacers. 

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CASE  33  ANIMAL: 11 year old female Scottish terrier.  PRESENTING COMPLAINTS: Polyuria, polydipsia, pot bellied and bilaterally symmetrical alopecia.  LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL ALP IU/L  4905  <110 ALT IU/L  63  <60 Total cholesterol mmol/L  10.2  1.4‐7.5 Glucose mmol/L  4.9  3.3‐6.4 Sodium mmol/L  153  137‐150 Potassium mmol/L  3.9  3.3‐4.8 

 HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.46  0.37‐0.50 Plasma protein g/L  80  55‐75 Haemoglobin g/L  168  100‐150 Erythrocytes x1012/L  7.2  5‐7 MCV fl  64  60‐75 MCHC g/L  365  300‐360 Leukocytes x109/L  15.6  7‐12 Neutrophils (seg.) x109/L  13.9  4.1‐9.4 Neutrophils (band) x109/L  0.5  0‐0.24 Lymphocytes x109/L  0.3  0.9‐3.6 Monocytes x109/L  0.9  0.2‐1.0 Eosinophils x109/L  0  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: normal Eosinophils (direct count) x109/L  .006  0.1‐1.0 

 URINALYSIS (voided) Appearance  Clear  PH  6.5 Colour  Light yellow  Glucose  ‐ve Specific gravity  1.014  Ketones  ‐ve Protein  ‐ve  Blood  ‐ve     Bilirubin  Trace Microscopic findings: little seen 

 

190

INTERPRETATION OF LABORATORY FINDINGS 

The dog probably has multiorgan disease that has given rise to polyuria/polydipsia, symmetrical skin changes and possible abdominal enlargement. A number of diseases could contribute to  individual clinical  signs,  but  the  clinical  signs  in  combination make  it  highly  likely  that  this  breed  of  dog (predisposed)  has  hyperadrenocorticism.  This  pattern  recognition  is  something  that  increases  in importance for veterinarians as they become more experienced. However, for the  inexperienced  it may be more prudent  to consider  likely organ/tissue dysfunction  from  the clinical signs and other information for further investigation rather than rely on pattern recognition. If pattern recognition is utilised,  then  it  is  important  for  the  proposed  diagnosis  to  be  fully  supported  and  confirmed  by further investigation. 

 (a)  Detected  laboratory  abnormalities:  Marked  increase  in  ALP,  moderate  hypercholesterolemia,  mild hypernatremia,  mild  hyperproteinemia,  mild  leukocytosis  due  to  neutrophilia  without  left  shift, lymphocytopenia, eosinopenia (on differential and direct count), a specific gravity reading close to isosthenuria. 

 (b)  &  (c)  General  interpretation,  conclusions,  further  investigation  and  implications  for management:  All  these  laboratory  changes  could  be  consistent  with  hyperadrenocorticism (conclusion  and  diagnosis).  Elevations  of  ALP  occur  due  to  activation  of  a  specific  ALP  liver isoenzyme are  common  in hyperadrenocorticism.  In  this  case  the value  is marked;  in other  cases values can be absent or minimal. Occasionally, mild elevations of ALT occur which could be partly related  to  corticosteroid  induction  and  partly  due  to  altered  hepatocyte  metabolism.  The hypercholesterolemia reflects altered carbohydrate‐lipid metabolism (rarely  is there  lipemia due to increased  triglyceride unless  there  is concomitant diabetes mellitus). The mild hypernatremia and hyperproteinemia are possibly related to haemoconcentration due to polyuria. The specific gravity reading may also be a  reflection of persistent polyuria and  resultant medullary washout.  In  some cases  renal  disease  cannot  be  ruled  out  without  a  gradual  water  deprivation  test  (gradual  to counteract medullary washout) (further investigation). Low specific gravity readings are not always the  case  in  hyperadrenocorticism,  despite  the  polyuria.  The  normal  glucose  level  is  occasionally encountered  in  hyperadrenocorticism  but more  commonly  the  level  is mildly  increased.  In  small animals  with  hyperadrenocorticism,  the  value  of  glucose  is  not  expected  to  surpass  the  renal threshold (which is relatively high for dogs and cats). If marked hyperglycemia and glucosuria occurs then one  should consider  the possibility of concomitant diabetes mellitus  (this has been primarily recorded in the USA; it appears not to be as common here). The mild neutrophilia, lymphocytopenia and eosinopenia are all consistent with the effects of corticosteroids (stress response). However, in prolonged  release  of  corticosteroids,  such  as  in  hyperadrenocorticism,  often  the  magnitude  of neutrophilia drops. The most useful and consistent leukocyte abnormality seems to be eosinopenia. This can be assessed on the smear but  it  is more appropriate to do direct eosinophil counts. Often serial eosinophil counts are employed to monitor the effect of treatment on corticosteroid release.  

Because all  these  routine  tests are  inconsistent and  relatively non‐specific, confirmation of clinical diagnosis usually requires direct evaluation of the pituitary‐adrenal axis using plasma cortisol levels (further  investigation). Often single measurements of basal cortisol  levels are not good enough as there  can  be  considerable  overlap  between  normal  and  hyperadrenocortical  dogs.  This  is  due  to episodic  release  of  cortisol  and  the  effects  of  stress  on  sick  dogs with  normal  adrenal  function. Consequently,  dynamic  testing  of  the  pituitary‐adrenal  axis  is  necessary  to  confirm  (e.g.  ACTH response Test and/or low‐dose dexamethasone suppression tests) and to help differentiate adrenal tumours  from  pituitary  dependent  hyperadrenocorticism  (PDH)  (high‐dose  dexamethasone suppression test). It should be noted that  low resting (basal) T4 can occur  in hyperadrenocorticism, but a TSH response test will usually show a normal thyroid gland. 

DIAGNOSIS AND POSTSCRIPT: Hyperadrenocorticism. Dynamic testing suggested PDH. 

191

CASE  34 ANIMAL: 7 year old male crossbred dog.  

PRESENTING  COMPLAINTS:  Prolonged  history  of  polyuria,  polydipsia.  The  dog  had  pale mucous membranes, lethargy and inappetence. Splenomegaly and hepatomegaly were detected.  

LABORATORY RESULTS 

BIOCHEMISTRY  SAMPLE  REFERENCE INTERVAL ALP IU/L  80  <110 ALT IU/L  55  <60 Serum protein (biuret) g/L  58  50‐70 Albumin (BCG) g/L  30  23‐43 Globulins g/L  28  27‐44 Glucose mmol/L  5.0  3.3‐6.4 Urea mmol/L  34  3‐10 Calcium mmol/L  3.1  2.1‐2.9 Inorganic phosphate mmol/L  4.2  0.8‐1.6 

 

HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.20  0.37‐0.50 Plasma protein g/L  60  55‐75 Haemoglobin g/L  68  100‐150 Erythrocytes x1012/L  3.1  5‐7 MCV fl  64  60‐75 MCHC g/L  340  300‐360 Leukocytes x109/L  5.8  7‐12 Neutrophils (seg.) x109/L  2.0  4.1‐9.4 Neutrophils (band) x109/L  0.8  0‐0.24 Lymphocytes x109/L  2.7  0.9‐3.6 Monocytes x109/L  0.3  0.2‐1.0 Eosinophils x109/L  0  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: 1 NRBC per 100 leukocytes, most of the lymphoid cells are blasts Platelets x109/L  390  200‐900 Reticulocyte % (uncorrected)  0.2  0‐1.5 

 

URINALYSIS (voided) Appearance  Clear  pH  6.0 Colour  Light yellow  Glucose  ‐ve Specific gravity  1.019  Ketones  ‐ve Protein  ‐ve  Blood  ‐ve     Bilirubin  Trace Microscopic findings: little seen 

192

INTERPRETATION OF LABORATORY FINDINGS The  polyuria  and  polydipsia  could  be  due  to  renal/hepatic/endocrine  disease.  The  pale mucous membranes  could  suggest  anaemia.  The  spleno/hepatomegaly  could  be  related  to  infiltrative disease. At this stage, there does not seem to be a specific pattern emerging to explain all the clinical signs in this chronically ill dog.  (a)  Detected  laboratory  abnormalities:  Moderate  non‐regenerative  anaemia,  mild  leukopenia, moderate neutropenia with  left  shift,  eosinopenia,  circulating  lymphoblasts  (leukemia), moderate azotemia (urea), hyperphosphatemia and hypercalcemia.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: Because  the  animal  was  leukemic  (circulating  lymphoid  blasts),  it  was  assumed  that  the  non‐regenerative  anaemia  and  neutropenia  was  primarily  due  to  myelophthisis  (conclusion  and diagnosis).  This  was  confirmed  by  bone  marrow  aspirates  that  showed  numerous  neoplastic lymphoid cells and few immature myeloid or erythroid cells (further investigation). The appearance of  the  immature  erythroid  and myeloid  cells  in  the  circulation  was  inappropriate  but  probably occurred due to disruption of the bone marrow architecture. The hepatomegaly and splenomegaly were  assumed  to  be  due  to  neoplastic  lymphoid  infiltration.  The  clinical  signs,  non‐regenerative anaemia,  leukopenia  and  leukemic  manifestation  suggested  that  the  prognosis  was  poor (implication for management).  In addition, the presence of hypercalcemia and suspected renal disease (clinical signs, azotemia and poorly  concentrating  urine) were  further  poor  prognostic  signs  (conclusion  and  implications  for management). Hypercalcemia can occur in cases of lymphoid neoplasia, and occasionally in multiple myeloma.  It  is  thought  to  be  primarily  due  to  the  production  of  substances  that  enhance  bone resorption  (parathormone‐like  protein,  prostaglandins,  Vitamin  D‐like  steroid  and  osteoclast activating factor ‐ hence the name pseudohyperparathyroidsm). Other tumours can produce similar substances in the dog such as apocrine gland adenocarcinoma of the anal sacs. Commonly, inorganic phosphate is not elevated in cases of pseudohyperparathyroidism unless there is some interference in  renal outflow. This appears  to be  the case  in  this dog and  is probably due  to  the deposition of calcium in the kidney interstitium and tubules. Some tumours cause hypercalcemia by metastasising to the bones and causing osteolysis. This may be by direct action or by activating osteoclasts. It is still referred  to  as  pseudohyperparathyroidism.  In  these  situations  the  inorganic  phosphate may  be elevated.  Nb.  Hypercalcemia may be  transient or persistent. Only persistent hypercalcemia  is  important; 

therefore, ideally two or more elevated levels should be detected.  DIAGNOSIS  AND  POSTSCRIPT:  The  dog  was  euthanised  rather  than  given  chemotherapy  The diagnosis  of  pseudohyperparathyroidism  and  hypercalcemic  nephropathy  associated  with lymphosarcoma  and  lymphoid  leukemia  was  confirmed  at  necropsy  and  subsequent histopathology. Abdominal lymph nodes were also affected. 

193

CASE  35  ANIMAL: 7 year old male Boxer.  PRESENTING COMPLAINTS:  Lethargy,  lack of exercise  tolerance and  inappetence  in excess of one month.  On  presentation,  the  dog  had  pale  mucous  membranes,  possible  splenomegaly  and hepatomegaly.  LABORATORY RESULTS 

HAEMATOLOGY  SAMPLE  REFERENCE INTERVAL Plasma appearance  Clear  Clear PCV L/L  0.19  0.37‐0.50 Plasma protein g/L  50  55‐75 Haemoglobin g/L  72  100‐150 Erythrocytes x1012/L  2.9  5‐7 MCV fl  65  60‐75 MCHC g/L  379  300‐360 Leukocytes x109/L (corrected)  58.5  7‐12 Neutrophils (seg.) x109/L  43.9  4.1‐9.4 Neutrophils (band) x109/L  5.4  0‐0.24 Lymphocytes x109/L  4.1  0.9‐3.6 Monocytes x109/L  4.8  0.2‐1.0 Eosinophils x109/L  0.3  0.14‐1.2 Basophils x109/L  0  0‐0.4 Blood film: moderate polychromasia, moderate acanthocytes and schistocytes (schizocytes), 10 NRBC per 100 leukocytes Platelets x109/L  100  200‐900 Reticulocyte % (uncorrected)  5  0‐1.5 

194

INTERPRETATION OF LABORATORY FINDINGS 

A chronic  illness with hepato/splenomegaly could suggest  infiltrative disease (neoplastic or chronic inflammatory). The pale mucous membranes and lack of exercise tolerance suggest anaemia.  (a)  Detected  laboratory  abnormalities:  Regenerative  anaemia;  mild  hypoproteinemia;  increased MCHC; marked  leukocytosis  (the  leukocyte  levels  are  always  corrected  for  circulating  nucleated erythroid cells [normoblasts]  if the numbers exceed 5 per 100  leukocytes) due to neutrophilia with left  shift,  lymphocytosis  and  monocytosis;  circulating  normoblasts  (NRBC),  acanthocytes  and erythrocyte fragments (schistocytes); mild to moderate thrombocytopenia.  (b) & (c) General interpretation, conclusions, further investigation and implications for management: The  regenerative  anaemia  (corrected  reticulocyte  count  for  the  level  of  anaemia was  2.1%) was assumed to be due to blood destruction rather than blood loss due to the presence of the circulating nucleated erythroid cells and the strong neutrophilia with left shift (conclusion). The lymphocytosis and monocytosis were  considered  the  result of  antigenic  stimulation  and erythrocyte destruction occurring over a period of time. The presence of moderate numbers of schistocytes usually indicates a  microangiopathic  haemolytic  process  (fibrin  strands  laid  down  in  small  vessels  damage erythrocytes  in transit). This can occur  in vasculitis, disseminated  intravascular coagulation (due to numerous microthrombi formation  in small vessels), some splenic disorders and abnormal vascular proliferation  (e.g.  vascular  tumours)  (conclusion).  Acanthocytes  also  occur  in  splenic  and microangiopathic diseases. They are often common in haemangiosarcoma (possible diagnosis). The high MCHC was  considered  not  to  be  due  to  laboratory  error,  but  due  to  the  presence  of  free haemoglobin (although this was not detected grossly in plasma) related to low grade haemolysis.  The  low  plasma  protein  did  not  fit  in  with  blood  destruction  (more  with  blood  loss),  but  was considered to be due to chronic disease, inappetence and probable liver disease.  Further  investigation  could  involve  diagnostic  imaging  of  the  spleen  and  liver.  Fine  needle  cell aspiration or biopsy might be considered.   DIAGNOSIS  AND  POSTSCRIPT:  Haemangiosarcoma  of  the  spleen.  The  animal  was  investigated further  by  radiography  and  ultrasound.  Multiple  masses  were  detected  in  liver  and  spleen.  A tentative diagnosis of neoplasia was given to the owners and the dog was euthanased. Necropsy and histopathology  revealed  metastatic  haemangiosarcoma.  Haemangiosarcoma  is  one  of  the  few diseases  that  commonly  gives  high  levels  of  circulating  nucleated  erythroid  cells. Other  diseases include  lead  poisoning,  erythroid  neoplasia,  other  bone  marrow  disorders,  occasional  splenic disorders.  In  contrast  to  common  causes  of  haemolytic  anaemia, where  the  levels  of  nucleated erythroid  cells  are  always  proportional  to  the  levels  of  reticulocytes,  these  diseases  may  give disproportional levels. In haemangiosarcoma, for example, the anaemia may be non‐regenerative or regenerative,  but  circulating  nucleated  erythroid  cells  are  common.  This  is  probably  due  to  the variable  effects  of  this  disease  (it may  cause  some  internal  bleeding  and/or  it may  cause  bone marrow depression). 

195

INDEX  

Abdominocentesis‐bovine, 110 Abdominocentesis‐equine, 110 Acid/base balance, 62 Acid/base disturbances 

metabolic disturbances, 62 Activated Clotting Time (ACT), 96 Activated Partial Thromboplastin Time (APTT), 96 acute myeloid leukemia, 91 Acute phase reactants, 94 Adrenal insufficiency 

hormonal assays, 55 Routine Laboratory findings, 55 

Ammonia (blood), 30 ammonia tolerance test, 30 fasting, 30 

Amylorrhea, 45 Anemia, 73 

non‐regenerative, 73 regenerative, 73 

Anemia of chronic disease, 78 Anion gap (AG), 62 Antinuclear antibody test (ANA), 101 Anuria, 33 Aplastic anemia, 79 APTT, 96 Autoimmune hemolytic anemia (AIHA), 100 Autoimmunity, 100 Azotemia, 33 

post‐renal, 35 prerenal, 35 renal, 35 

Basophila, 86 Basophilia, 81 Basophilic stippling, 74 Basophils, 86 Bile acids, 30 

fasting, 30 post‐prandial, 30 

Bilirubin, 28 Fecal bile pigments, 27 Urine bilirubin, 27 Urine urobilinogen, 27 

bilirubin metabolism, 27 Bilirubinuria, 40 Bleeding disorder 

clotting factor deficiency, 96 excess fibrinolysis, 97 platelet function defect, 95 Thrombocytopenia, 95 vessel wall defect, 95 

Bleeding Disorders, 94 

Bleeding time (BT), 96 Body FLuid Analysis, 107 Bone marrow examination, 77 

M:E, 78 Bone marrow storage pool, 81 BSP, 32 

Horse and cattle, 32 Calcium 

hypercalcemia, 58 hypocalcemia, 57 

Calcium and Phosphate Derangements, 57 Casts, 42 cholestasis, 26 chronic myeloproliferative disorders, 92 Chylous, 107 Chylous effusions, 108 Circulating neutrophil pool (CNP), 81 Clot retraction, 96 Clotting factors, 93 Colic 

Abdominal paracentesis (horse), 46 Corticosteroid induced neutrophilia (leukocytosis), 

82 Creatinine, 35 CSF ‐ protein, 114 Cylindruria, 33, 42 Diagnostic Cytology (exfoliative cytology, 102 D‐dimer, 97 Defective (hypercellular) erythropoiesis, 79 Diabetes mellitus, 52 Diabetes mellitus (DM) 

intravenous glucose tolerance test, 53 ketones, 53 persistent hyperglycemia and glucosuria, 53 

Diagnostic cytology Solid tissue cytology, 102 

Diarrhea, 45, 47 dog and cat, 47 horse, 47 

Disseminated intravascular coagulation (DIC), 98 Döhle bodies, 84 Electrolyte status 

chloride, 61 Na+, 60 potassium, 61 

Enzootic bovine lymphosarcoma (EBL), 89 Enzyme 

ALP, 26 ALT, 25 ARG, 26 AST, 26 

196

GD, 26 GGT, 26 ICD, 26 ID, 25 LD, 26 OCT, 26 

Enzymes, 20 Eosinopenia, 86 Eosinophilia, 81, 86 Eosinophils, 86 Erythron, 72 Exocrine pancreatic insufficiency (EPI), 48 extramedullary hematopoiesis, 72 exudates 

non‐septic exudate, 108 septic exudate, 108 

Exudates, 108 Eythrocyte production, 72 Fecal Examination, 47 Fibrin (fibrinogen) degradation products  (FDP), 97 Fibrinogen, 85 Fibrinolysis, 97 Fine needle cell aspirates, 102 Folate, 50 Glucosuria, 39 Granulopoietin, 81 hematological terms 

glossary, 67 Hematopoietic neoplasia, 88 Hematuria, 41, 42 Hemoglobinemia, 76 Hemoglobinuria, 41 Hemostasis 

normal mechanisms, 93 Hepatic encephalopathy, 31 Hereditary coagulation disorders, 98 Hydration status 

osmolality, 60 hyperadrenocorticism 

urine cortisol/creatinine ratio, 55 Hyperadrenocorticism, 54, 82 

Hormonal evaluation, 54 PDH and adrenal tumour differentiation, 55 Routine laboratory tests, 54 

Hyperbilirubinemia, 76 cat, 29 Combined, 29 dog, 29 farm animals, 29 horse, 29 Regurgitation (cholestatic), 29 Retention, 29 

Hypercalcaemia case report, 192 

Hypercalcemia, 90 Hyperkalemia, 61 hyperlipidemia, 20 Hypermagnesemia, 59 Hyperthyroidism, 56 Hypoadrenocorticism (adrenal insufficiency ‐ AI), 

55 Hypokalemia, 61 Hypomagnesemia, 59 Hyposthenuria, 33, 38 Hypothyroidism, 56 Icterus, 27 Icterus (Jaundice) 

Hemolytic, 27 Immune mediated (related) diseases, 99 Immunodeficiency, 101 Indocyanine Green, 32 Insulinoma (hyperinsulinism), 54 Isosthenuria, 33, 38 Ketonuria, 40 lactate, blood, 110 Left shift, 83 Leukemoid response, 84 Leukocytosis, 80 Leukopenia, 81 lipemia, 20 Liver disease 

Cholesterol, 32 Clotting factors, 31 Glucose, 32 

Lymphatic leukemia, 88 Lymphocytes, 87 Lymphocytosis, 81, 87 Lymphopenia (lymphocytopenia), 87 Lymphoproliferative disorder 

Acute lymphoblastic leukemia, 89 chronic lymphocytic leukemia, 89 lymphosarcoma, 88 

Lymphoproliferative Disorders, 88 Malabsorption, 45, 49 

Fat balance determination (dog and cat), 49 Oral glucose tolerance test (OGTT), 49 Post‐prandial lipemia (dog and cat), 49 Xylose absorption test (XAT), 49 

Malassimilation, 45 Maldigestion, 45 Marginal neutrophil pool (MNP), 81 Mast cell leukemia, 92 Metabolic acidosis, 62 Metabolic alkalosis, 62 Monocytes, 86 Monocytosis, 81, 86 Mucin clot test, 111 Myelofibrosis, 79 

197

Myelophthisic anemia, 79 Myeloproliferative disorders, 90 Myoglobinuria, 41 Neoplastic effusions, 110 Nephrotic syndrome 

Hypercholesterolemia, 43 Neutropenia, 85 

inflammatory demand, 83 Neutrophilia, 81 Neutrophilia due to regenerative anemia, 82 Neutrophilia related to inflammatory demand, 82 Neutrophils 

toxic changes, 83 Non‐regenerative anemia, 75, 78 

defective erythropoiesis, 77 reduced erythropoiesis, 77 

Nucleated red cells, 75 Oliguria, 33, 37 One Stage Prothrombin Time (OSPT), 97 Osmolality, 38 OSPT, 97 Pancreatic necrosis, 45 

amylase, 46 canine pancreatic‐specific lipase immunoassay, 

46 lipase, 46 

Pancreatic necrosis (acute pancreatitis) dog, 46 

phosphorus hyperphosphatemia, 57 

Physiological Neutrophilia (leukocytosis), 81 Plasma cell myeloma (multiple myeloma), 90 Pleocytosis, 113 Polycythemia, 79 Polyuria, 33, 37 Protein losing gastroenteropathy (PLG), 50 Proteinuria, 39 

Urinary Protein to Creatinine Ratio, 39 Pseudochylous effusion, 108 Pseudomacrocytic anemia, 75 Red cell indices, 75 Reduced (hypoproliferative) erythropoiesis, 78 Reference Interval, 18 Regenerative anemia, 74 

Blood loss, 76 hemolytic, 76 reticulocyte count, 74 

Renal clearance tests, 34 creatinine, 34 Sodium sulfanilate, 35 

Renal failure acute, 34 chronic, 34 

farm animals, 34 Hyperamylasemia, 44 hypercalcemia, 44 Hyperkalemia, 44 Hyperphosphatemia, 44 hypokalemia (cats), 44 hyponatremia and hypochloridemia, 44 hypophosphatemia, 44 Metabolic acidosis, 43 Non‐regenerative anemia, 43 

Renal tubular acidosis, 36 Respiratory washes, 114 Reticulocyte count 

correction, 74 Reticulocytes 

aggregate, 74 Reticulocytosis 

anisocytosis and polychromasia, 75 Rheumatoid arthritis RA, 101 Rheumatoid factor (RF), 100 Serum protein, 31 Serum proteins 

acute phase reactants, 31 Fibrinogen, 31 globulins, 31 hypoalbuminaemia, 31 

SLE LE cell test, 101 

Steatorrhea, 45 Synovial fluid, 111 

Normal cytological characteristics, 111 Normal gross characteristics, 111 Other characteristics, 111 

Synovial FLuid Analysis, 111 Synovial fluid changes due to disease, 111 Systemic lupus erythematosus (SLE), 101 TCT, 97 Thrombin Clotting Time (TCT), 97 Thrombocytopathies, 95 Thrombocytopenia, 95 Tissue imprints, 102 Trypsin‐like immunoreactivity (TLI), 48 Urea, 35 Uremia, 33 Urinalysis, 34, 36 

Physical Properties, 37 Sediment examination, 41 Solute Concentration (specific gravity), 38 

Urine pH, 40 Aciduria, 40 

Vitamin B12, 50 Von Willebrand's disease, 95 Xanthochromia, 113 

 

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F a c u l t y o f V e t e r i n a r y S c i e n c e

VETERINARY CLINICAL PATHOLOGY

VETS 3243

Practical Notes and Information on Techniques

Developed by: Professor Paul J Canfield &

David LP Griffin (Professional Officer)

© 1988, revised 2009

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PREFACE

These notes have been designed for veterinary

undergraduates but will have application for

laboratory medicine in veterinary practice. Only

simple laboratory techniques are described, with

some theory given where appropriate. The practical

notes complement the previously provided lecture

notes but can also stand on their own. Although

most of the laboratory techniques are standard and

will change little with time, others will need to be

upgraded. This revision does occur regularly, but

sometimes certain laboratory techniques are

inadvertently overlooked. Consequently, the

reader’s assistance is required in detecting errors or

flaws in the presented techniques.

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T A B L E O F C O N T E N T S

SAFETY IN THE LABORATORY .............................................................................................205 SUMMARY OF PRACTICAL CLASSES ....................................................................................207 ACTIVITIES 1, 3 & 5 – CASE REPORT ANALYSIS TUTORIALS ...................................................208 ACTIVITY 2 PRACTICAL CLASS - HAEMATOLOGY ..................................................................220 ACTIVITY 4 PRACTICAL CLASS – A. BIOCHEMISTRY ..............................................................224 ACTIVITY 4 PRACTICAL CLASS – B. URINALYSIS ...................................................................225 ACTIVITY 6 PRACTICAL CLASS - CYTOLOGY.........................................................................227 REQUEST SHEETS AND COLLECTION OF SAMPLES FOR CLINICAL PATHOLOGY .....................229

1 INTRODUCTION ..........................................................................................................................229 2 REQUEST SHEETS .....................................................................................................................229 3 COLLECTION OF SAMPLES .......................................................................................................229 4 WHAT TO DO IF TAKING SPECIMENS FOR LABORATORY EXAMINATION AFTER HOURS..236 5 INFORMATION ON MEASUREMENT UNITS AND ENZYMES....................................................238 6 CONVERSION OF OLD ENZYME UNITS TO INTERNATIONAL UNITS (IU) .............................239 7 TEMPERATURE CONVERSION FACTORS FOR ENZYMES TO 37°C ......................................239 8 CONVERSION FACTORS............................................................................................................240 9 REFERENCE RANGES FOR BIOCHEMISTRY.........................................................................242 10 LOCATION OF CLINICAL PATHOLOGY CLASSROOM AND VPDS LABORATORIES IN MCMASTER B14 ..243

HAEMATOLOGY PRACTICAL NOTES.....................................................................................245

1 INTRODUCTION ..........................................................................................................................245 2 SOME ANTICOAGULANTS .........................................................................................................245

EDTA ...........................................................................................................................................245 Heparin ........................................................................................................................................245 Sodium Citrate .............................................................................................................................245

3 ERYTHROCYTE ANALYSIS ........................................................................................................246

a) The Microhaematocrit (PCV) ..................................................................................................246 b) Haemoglobin Estimation Using the Cyanmethaemoglobin Method ........................................246 c) Erythrocyte Count - (Unopette) ...............................................................................................247 d) Erythrocyte Indices .................................................................................................................248 e) Reticulocyte Count..................................................................................................................249 f) Coombs Antiglobulin Test for Autoimmune Haemolytic Anaemia (AIHA) ..................................250 a) Leukocyte Count (Unopette method) ......................................................................................251 b) The Peripheral Blood Smear (Film) ........................................................................................251 c) Total Eosinophil Count (Direct Method) ..................................................................................256

4 LEUKOCYTE ANALYSIS.............................................................................................................251

a) Leukocyte Count (Unopette method) ......................................................................................251 b) The Peripheral Blood Smear (Film) ........................................................................................251

5 LABORATORY EVALUATION OF HAEMOSTASIS .....................................................................257

a) Bleeding Time.........................................................................................................................257

204

b) Clotting Time...........................................................................................................................258 c) Clot Retraction ........................................................................................................................259 d) Fibrinogen Estimation .............................................................................................................260 e) Prothrombin Time (ProT, PT or OSPT)...................................................................................261 f) Partial Thromboplastin Time (PTT)..........................................................................................262 g) Platelet (Thrombocyte) Count.................................................................................................263 h) Fibrin (fibrinogen) degradation products – including D-dimer .................................................265

6 MISCELLANEOUS PROCEDURES .............................................................................................266

a) Bone Marrow Examination......................................................................................................266 b) Cross-matching Blood for Transfusion....................................................................................267 c) Blood typing for cats and dogs................................................................................................268 d) The Blood/Erythrocyte Sedimentation Rate (BSR:ESR).........................................................269 e) The Lupus Erythematosus (LE) Cell Test (For Systemic LE) .................................................270

7 REFERENCE RANGES FOR HAEMATOLOGY...........................................................................271 8 THE HAEMOCYTOMETER..........................................................................................................272

U R I N A L Y S I S ................................................................................................................277

1 INTRODUCTION ..........................................................................................................................277 2 OBSERVATION OF PHYSICAL PROPERTIES ...........................................................................277 3 ESTIMATION OF SOLUTE CONCENTRATION...........................................................................277 4 CHEMICAL ANALYSIS.................................................................................................................277 5 SEDIMENT EXAMINATION..........................................................................................................279

C Y T O L O G Y ...................................................................................................................281 1 SOLID TISSUE CYTOLOGY (SURFACE LESIONS, BIOPSIES, AUTOPSY MATERIAL) ...........................281

a) Tissue impression (imprint). ................................................................................................281 b) Tissue scraping. ..................................................................................................................281 c) Fine needle aspirate............................................................................................................281

2 FLUID CYTOLOGY (CSF, synovial, body cavity effusions, tracheal aspirate, fluid masses)........283 3 VAGINAL CYTOLOGY .................................................................................................................283 4 SEMEN ANALYSIS ......................................................................................................................284

F A E C A L A N A L Y S I S ...................................................................................................285 1 INTRODUCTION ..........................................................................................................................285 2 GROSS EXAMINATION ...............................................................................................................285 3 MICROSCOPIC EXAMINATION ..................................................................................................285 4 HEMATEST - FOR OCCULT BLOOD...........................................................................................287 5 EXAMINATION FOR TRYPSIN (PROTEASE) .............................................................................287 6 FAECAL STAIN ............................................................................................................................288

U R I N A R Y C A L C U L I ...................................................................................................289 1 INTRODUCTION ..........................................................................................................................289 2 PRACTICAL EXAMINATION OF CALCULI ..................................................................................291

a) Physical examination ..........................................................................................................291 b) Chemical Analysis (Based on the Merck Urinary Calculus Analysis) ................................292

205

SAFETY IN THE LABORATORY Safety is important in the laboratory. It is designed to protect you, other members of your class, and staff. It forms part of your education in being an effective practitioner. If you injure yourself in practice through poor safety procedures then you lose time and often income! If learnt now, it should carry over into your professional life.

Personal safety is your own responsibility The regulations in this laboratory are:

1 Laboratory coats to be worn at all times; gloves to be worn when handling biological samples

2 No smoking, drinking or eating in laboratory and/or corridor

3 Adequate protective footwear i.e. non slip, and fully enclosed footwear.

4 No pipetting by mouth

5 Washing of hands after practical classes

6 No improper use of safety or fire fighting equipment

7 No horseplay or reckless behaviour in the laboratory

Never run in the laboratory or along corridors.

8 Care in handling materials a) All material should be treated as infective b) Needles to be sheathed at all times c) Other sharp objects to be disposed of after use d) Broken glassware disposed of e) Used slides disposed of

9 All material should be labelled correctly

10 Do not leave bags in corridors where people can trip over them

11 Care with bunsen burners and heating plates

12 Care with water near electrical equipment

13 Care with animal handling

14 Report all accidents to the member of staff

Some of these sound trivial, but they all play a role in protecting yourself from serious injury and disease.

Should the fire alarms sound, the building is to be evacuated immediately. The member of staff present will show you how to leave. Do not panic!

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Personal safety is your own responsibility.

Check that any glassware that you are to use is not broken or cracked.

Long hair should be tied or held back.

By this time, you should all have had tetanus and hepatitis injections and necessary boosters.

Also, you should have had a Mantoux test and, if negative, a B.C.G.

General Laboratory Safety Rules • Never adopt a casual attitude in the laboratory and always be conscious of the potential

hazards.

• Always wear eye protection when in the laboratory area.

• Always exercise care when opening and closing doors and entering or leaving the laboratory.

• Always use a fume cupboard, fume cabinet or glove box when working with highly toxic, volatile or odoriferous substances.

• Do not work in isolation in a laboratory, ensure that at least a second person is with in call.

• Do not store food or drink in a refrigerator which is used to store laboratory materials.

• Regard all substances as hazardous unless there is definite information to the contrary.

• Never undertake any work unless the potential hazards of the operation are known as precisely as possible, and the appropriate safety precautions are adopted.

• Take additional care when carrying any potentially hazardous substances.

• Maintain the minimum required quantities of hazardous substances in the laboratory work area.

• Wash skin areas which come in contact with chemicals, irrespective of concentration.

• Keep all fire escape routes completely clear at all times.

• Ensure that all safety equipment remains accessible to the laboratory personal at all times.

• Clean up spills immediately.

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SUMMARY OF PRACTICAL CLASSES SEMESTER 6

Practical Classes/Tutorials One two hour session per week: Tuesdays 11am-1pm, Weeks 1-12 inclusive

Half the students per session, divided into three groups for three separate activities

(Groups A-F)

Group allocations will be posted on the Clinical Pathology Sydney eLearning site on Monday of Week 1.

  Activity 1  Activity 2  Activity 3  Activity 4  Activity 5  Activity 6 

Week 1  Group A  B  C       

Week 2  D  E  F       

Week 3  B  C  A       

Week 4  E  F  D       

Week 5  C  A  B       

Week 6  F  D  E       

Week 7        Group D  E  F 

Week 8        A  B  C 

Week 9        E  F  D 

Week 10        B  C  A 

Week 11        F  D  E 

Week 12        C  A  B  Topic and site for Activity

Activity 1: Tutorial (case report analysis) - Seminar room 223, Level 2 Gunn Building B19

Activity 2: Haematology Practical Class – CP Lab Room 141, McMaster Building B14

Activity 3: Tutorial (case report analysis) – Seminar room 353, Level 3 Gunn Building B19

Activity 4: Biochemistry/Urinalysis Practical - CP Lab Room 141, McMaster Building B14

Activity 5: Tutorial (case report analysis) – Seminar room 223, Level 2 Gunn Building B19

Activity 6: Cytology Practical/Tutorial – Level 4 Lab A, Gunn Building B19

Note: The  three  case  report  tutorials will each be marked out of 10% (then an average determined). The three practicals must be attended for students to be eligible for the full 10% (see summative assessment). 

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ACTIVITIES 1, 3 & 5 – CASE REPORT ANALYSIS TUTORIALS

INTRODUCTION TO INTERPRETATION OF LABORATORY RESULTS The interpretation of laboratory results is an integral part of the diagnostic process utilised in clinical medicine. Diagnosis of disease, in its simplest form, relies on the three ‘D’s’: Detect, Describe, Deduce. The first two ‘D’s’ are objective and rely on an understanding of normal structure and function (therefore, able to detect abnormalities) and a development of descriptive powers. The last ‘D’, namely deduce, is partly subjective as it is influenced by one’s past knowledge and experiences, but relies heavily on an integrative, sequential and common sense approach to interpreting abnormalities. Evidence-based interpretation (support for all conclusions, ‘hunches’ and suspicions) can diminish the subjective impact on the deductive process. In a sense, the diagnostic process is akin to a ‘detective story’ with ‘clues’ being utilised effectively to reach evidence-based ‘convictions’. Every practicing veterinarian has a bit of the ‘vetective’ in them and searches for clues that can be integrated to reach a diagnosis.

Laboratory tests are just one group of diagnostic aids available to the practicing veterinarian. Their usefulness is enhanced by careful choice based on clinical detection and deduction and by careful interpretation based on all other available information of the case. The three ‘D’s’ equally apply to the use and interpretation of laboratory tests. It is a logical process based on the detection and description of laboratory abnormalities, and then an interpretative (deductive) process drawing on all other information available to you about the case. The interpretive process is commonly one starting from the general then working to the specific (i.e. think of general disease processes before considering specific diseases). Remember also, laboratory results should not be interpreted in isolation of other case information.

Once laboratory results are interpreted, then there is often a need for further investigation to support the conclusions. This may lead to complex further investigations, such as MRI to support abnormalities detected on CSF analysis, or simple further investigations, such as returning to the owner and asking them whether the animal had exposure to chemicals that may have contributed to the liver disease detected by elevations in liver enzymes. The plan for further investigation must be primarily based on the main conclusions.

Apart from assisting in drawing conclusions and a plan for further investigation, laboratory results often have implications for management of the case while undertaking further investigation, for example detected electrolyte disturbances for fluid therapy.

This session introduces the concepts behind the use of laboratory tests in the diagnostic process and will provide a technique to detect, describe and deduce. It will also emphasise the use of laboratory test interpretation in planning for further investigation and disease management while doing so.

SUGGESTED TECHNIQUE FOR INTERPRETATION OF LABORATORY RESULTS AND LINKAGE WITH THE OVERALL DIAGNOSTIC PROCESS Think about the information given before looking at the laboratory data. Ask yourself what can be deduced from the history, signalment and clinical findings:

• Is there an indication of time course? • Is age, sex or breed important? • What organs or tissues are involved? • Is there an indication of the pathological process(es) occurring? • Is there an indication of cause?

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Keep your deductions in mind when interpreting the laboratory data and thinking about further investigation

1. LIST/HIGHLIGHT ABNORMALITIES (DETECT AND DESCRIBE)

This is for your benefit to ensure you don’t forget to discuss any abnormalities.

Remember to use the information on what is regarded as mild, moderate and marked alterations (see the beginning of the Section on Case Reports).

2. GENERAL INTERPRETATION (DEDUCE)

Consider the reasons for abnormalities. Do not give complete lists of reasons but only those which could relate to the case. Relate to information (history etc) given to you in the question. You will use this information in reaching conclusions. This can be incorporated in your conclusions if your prefer (i.e. give a conclusion and then add your reasons for reaching it)

3. CONCLUSION(S)/FURTHER INVESTIGATION/IMPLICATIONS FOR MANAGEMENT? (DEDUCE)

This is most important and MOST MARKS will come from this section.

A. Conclusions

These can be in the form of questions or statements (e.g. ‘is the regenerative disease due to blood loss or blood destruction?’ OR ‘the animal has regenerative anaemia due to either blood destruction of blood loss’)

Think about whether there appears to be a main problem. Remember, there may be more than one. The main problem may be presented as organ/tissue dysfunction (e.g. liver disease), a general disturbance/pathological process (e.g. regenerative anaemia; inflammation) or possibly a specific disease (e.g. acute pancreatic necrosis in a dog). Can you explain all the abnormalities in light of the main problem(s)? Don’t forget about what you deduced from the given animal and clinical information when you are reaching your conclusions.

B. Further investigation

Often you will need to investigate the case further. This should be logical and based on your conclusions reached from analysing case history and laboratory data. It is important to prioritise investigation i.e. consider what you think is the main conclusion when suggesting further investigation.

A checklist for further investigation might include: • Do you need to get more history, and if so what? • Do you need to recheck some clinical signs or repeat physical examination? • Do you need to undertake specialist clinical procedures (e.g. ECG, EMG)? • Do you need to undertake image analysis? What modality would be most useful? • Do you wish to undertake more laboratory testing?

Sometimes you may consider a response to treatment as part of your further investigation to reach a diagnosis, but this can sometimes be misleading unless you verify your response with further testing. So, you need to state how you would support any response to therapy approach.

210

Sometimes you can sequence your further investigation on the basis of probable findings e.g. ‘I wish to undertake hepatic ultrasound. If I find any masses on ultrasound, I would wish to take an FNA or biopsy’.

C. Implications of the results for your approach to treatment and prognosis (i.e. management of the case while you are investigating it further).

You may not wish to undertake any treatment while investigating the case further. In some situations you may wish to undertake treatment after asking for more tests (e.g. electrolyte analysis for possible fluid therapy).

REMEMBER, not all abnormalities may be explicable in light of the case. Be honest,  if you don’t know,  say  so BUT  think about how you would find  out.  Also,  put  the  abnormalities  in  perspective.  Some  mild abnormalities may not be very important for the case. 

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CASE 1:  3‐year‐old male neutered DSH cat called “Mory”  

BACKGROUND 

Mory had been obtained by his owners as a kitten. There were 2 other cats in the household both of whom were healthy. Mory had been neutered at 6 months of age and had been vaccinated regularly against FIE, FCV and FHV. He had free access outside and was known to be a hunter. He was fed tinned cat food.  HISTORY OF ILLNESS 

Three months before referral the owners had noticed swelling of his  lower  limbs, face and ventral abdomen. This had resolved following treatment with frusemide (a diuretic). Some weight loss had been noticed since then and he was in poor condition. The swelling had now recurred and he was referred at this stage.  CLINICAL AND PHYSICAL EXAMINATION FINDINGS 

He was  bright  but  in  poor  condition,  thin with  a  dull  coat.  Rectal  temperature was  38.6 degrees C and heart rate 160 bpm. Pitting subcutaneous oedema was present affecting the distal limbs, face and ventral abdomen. His abdomen appeared to be slightly distended and there was a suspicion of a fluid thrill.  LABORATORY RESULTS 

Haematology TEST  RESULT  UNITS  REFERENCE INTERVAL Hb  48  g/L  80‐140 PCV  0.23  L/L  0.30‐0.45 RBC  7.3  X1012/L  5.5‐10 Platelets  310  X109/L  300‐700 WBC  27.8  X109/L  8‐14 Neutrophils  24.7  X109/L  3.76‐10.8 Lymphocytes  1.7  X109/L  1.6‐7 Monocytes  1.1  X109/L  0‐08‐0.56 Eosinophils  0.3  X109/L  0.16‐1.4  

Serum Biochemistry TEST  RESULT  UNITS  REFERENCE INTERVAL Total proteins  60.9  g/L  54‐73 Albumin  16.0  g/L  19‐38 Globulins  44.9  g/L  26‐51 Urea  13.7  Mmol/L  7.2‐10.7 Creatinine  207  μmol/L  90‐180 ALT  4  IU/L  <60 ALP  21  IU/L  <50 Sodium  150  mmol/L  147‐156 Potassium  4.1  mmol/L  4.0‐4.6 Calcium  1.8  mmol/L  1.75‐2.6 Cholesterol  9.7  mmol/L  1.9‐3.9  

212

Urinalysis (cystocentesis) Appearance: slightly cloudy Colour: dark yellow Urine specific gravity: 1.050 Protein: 3‐4+ pH: 7.0 Other chemistries on dipstick negative  Urinary sediment: occasional transitional epithelial cells and low number of triple phosphate crystals  Urine creatinine: 6.4 mmol/L (=72.4 mg/dl) Urine protein (microprotein assay): 8.84 g/L (884 mg/dl) UP/UC ratio is over 12 (8.84/6.4 X by conversion factor of 8.839 – if working in SI units)  

213

CASE 2:  5‐year‐old male Old English Sheepdog called Oscar”  BACKGROUND 

Oscar  had  been  obtained  as  a  puppy.  The  owners  had  no  other  pets.  He was  regularly vaccinated. His diet consisted largely of canned food supplemented with table scraps.  HISTORY OF ILLNESS 

Oscar’s owners noticed  that he had become  less  interested  in exercise over  the previous two weeks and become very  reluctant  to walk more  than a  short distance. He was quite lethargic.  CLINICAL AND PHYSICAL EXAMINATION FINDINGS 

Oscar appeared reasonably bright but his mucus membranes were pale. Rectal temperature was 38.2 degrees C and heart rate 135 bpm. His pulse was strong and CRT under 2 seconds. A systolic murmur was audible loudest over the left apex. He appeared to resent abdominal palpation although pain could not be localised.  LABORATORY RESULTS 

Haematology TEST  RESULT  UNITS  REFERENCE INTERVAL Hb  74  g/L  100‐150 PCV  0.21  L/L  0.37‐0.55 RBC  2.6  x1012/L  5‐7 MCV  80  FL  60‐75 MCHC  352  g/L  300‐350 Platelets  220  x109/L  200‐600 WBC  24.6  x109/L  7‐12 Neutrophils  19.3  x109/L  4.06‐9.36 Band Neutrophils  0.6  x109/L  0‐0.24 Lymphocytes  3.4  x109/L  0.91‐3.6 Monocytes  0.7  x109/L  0.21‐0.96 Eosinophils  0.3  x109/L  0.14‐1.2 Nucleated red blood cells 

0.4  x109/L  0‐0.1 

Blood film: moderate polychromasia and marked anisocytosis. There are nucleated erythroid cells and some basophilic stippling of polychromatophilic cells. Some erythrocytes lack central pallor, but this may be partly artefactual.  

Serum Biochemistry TEST  RESULT  UNITS  REFERENCE INTERVAL Total proteins  78.8  g/L  50‐70 Albumin  33.6  g/L  23‐43 Globulins  45.2  g/L  27‐44 Urea  8.3  mmol/L  1‐10 Creatinine  161  μmol/L  40‐120 ALT  89  IU/L  <60 ALP  143  IU/L  <110 

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GGT  5  IU/L  0.6‐8.2 Total bilirubin  5  μmol/L  1.2‐8.1 Sodium  146  mmol/L  137‐150 Potassium  3.8  mmol/L  3.3‐4.8  

Urinalysis (voided) Appearance: clear Colour: dark yellow Urine specific gravity: 1.045 Proteins:Trace (0.13 g/L) pH: 6.5 Bilirubin: + Blood: ‐ve Glucose:‐ve Ketones: ‐ve  Urinary sediment: 1‐2 leukocytes per HPF, occasional squamous and transitional cells. 

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CASE 3:  9‐year‐old male neutered Labrador called “Poacher”  BACKGROUND 

Poacher had been obtained as a puppy. The owners had no other pets. He was  regularly vaccinated. His diet consisted largely of canned food supplemented with table scraps.  HISTORY 

Three  days  before  presentation  to  our  clinic,  Poacher  became  anorectic  and  started vomiting  2‐3  times  every  day.  The  vomit  had  a  liquid  consistency,  a  yellow  colour  and contained no food material.  CLINICAL FINDINGS 

Poacher was markedly jaundiced but bright. Rectal temp was 38.0 degrees C and heart rate 120 bpm.  LABORATORY RESULTS 

Haematology TEST  RESULT  UNITS  REFERENCE 

INTERVAL Hb  161  g/L  100‐150 PCV  0.45  L/L  0.37‐0.55 RBC  6.87  x1012/L  5‐7 MCV  65.1  FL  60‐75 MCHC  334  g/L  300‐360 Platelets  341  x109/L  200‐600 WBC  10.8  x109/L  7‐12 Neutrophils  5.62  x109/L  4.06‐9.36 Band Neutrophils  0.11  x109/L  0‐0.24 Lymphocytes  2.27  x109/L  0.91‐3.6 Monocytes  1.08  x109/L  0.21‐0.96 Eosinophils  1.73  x109/L  0.14‐1.2 Basophils  0  x109/L  0‐0.36 Blood film: unremarkable 

Serum Biochemistry TEST  RESULT  UNITS  REFERENCE 

INTERVAL Total proteins  75.9  g/L  50‐70 Albumin  38.2  g/L  23‐43 Globulins  37.7  g/L  27‐44 Urea  3.2  mmol/L  1‐10 Creatinine  91  μmol/L  40‐120 ALT  535  IU/L  <60 ALP  2065  IU/L  <110 GGT  85  IU/L  0.6‐8.2 Cholesterol  11.5  mmol/L  1.4‐7.5 Total bilirubin  49  μmol/L  1.2‐8.1 Amylase  2756  IU/L  <1400 Lipase  400  IU/L  <60 

216

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CASE 4:  Adult (13‐15 yrs) gelded male thoroughbred horse called ‘Trojan’  BACKGROUND 

Trojan had been owned for two years. Before that time he had had various owners. He was bought for recreational riding on the weekends, but in the past six to nine months had been reluctant to trot or gallop.  HISTORY 

Trojan  had  been  increasingly  losing weight  for more  than  3 months  and  the  owner  had noticed a decrease in food intake and an increase in water intake. Trojan was kept in a large paddock with one other horse that was in good health.  CLINICAL FINDINGS 

On examination, Trojan was in poor condition with a loss of muscle mass and a dry coat. The horse  could  have  possibly  been  dehydrated  with  slightly  sunken  eyes.  Vital  signs  (HR. Temperature, RR) were within reference intervals.  LABORATORY RESULTS 

Serum Biochemistry TEST  RESULT  REFERENCE INTERVAL ALP IU/L  96  <210 GGT IU/L  18  5.6‐22 Serum protein (refract.) g/L  63  53‐76 Albumin (EPG) g/L  27.1  28‐36 α globulins (EPG) g/L  11.3  8‐13 β globulins (EPG) g/L  15.1  8‐15 γ globulins (EPG) g/L  9.6  7‐14 Glucose mmol/L  5.6  3.3‐6.1 Urea mmol/L  98  3.6‐6.5 Creatinine μmol/L  1398  110‐170 Calcium mmol/L  3.7  2.8‐3.4 Inorganic phosphate mmol/L  2.1  1‐2.3 Sodium mmol/L  118  131‐147 Potassium mmol/L  4.8  2.1‐4.8 Chloride mmol/L  86  99‐108 

  

Haematology TEST  RESULT  REFERENCE INTERVAL Plasma appearance  Clear  Variable PCV L/L  0.38  0.32‐0.53 Plasma protein g/L  66  55‐78 Hemoglobin g/L  135  130‐160 Erythrocytes x1012/L  6.4  8‐11 MCV fl  59  41‐49 MCHC g/L  355  300‐360 Leukocytes x109/L  11.9  6.5‐12 

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Neutrophils (seg.) x109/L  9.6  2.5‐7 Neutrophils (band) x109/L  0  0‐0.2 Lymphocytes x109/L  2.1  1.6‐5.4 Monocytes x109/L  0.2  0‐0.7 Eosinophils x109/L  0  0.2‐1 Basophils x109/L  0  0‐0.4 Blood film: normal 

  

Urinalysis (voided sample) Appearance  Cloudy  pH  7.3 Colour  Brown  Glucose  ‐ve Specific gravity  1.014  Ketones  ‐ve Protein  1+  Blood  ‐ve     Bilirubin  ‐ve Microscopic findings: much mucin and calcium carbonate crystals 

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TUTORIAL STRATEGY for approach to the technique for interpretation of laboratory results and linkage with the overall diagnostic process: 1. Work individually to look at clinical information and identify laboratory

abnormalities (about 5-10 minutes) 2. Pair up and discuss possible reasons for laboratory abnormalities (about 10-15

minutes) 3. Form two groups and see if you can reach some (A) conclusions, (B) what further

investigation may be necessary and, (C) any implications for management of the case (about 15-20 minutes). One person should keep a record of this.

4. The two groups come together and a scribe from each writes the main

conclusions (in order of importance), any further investigation and any implications for case management onto the whiteboard. Everyone will read and reflect on the other group’s statements before discussing any differences. Hopefully, a consensus will be reached and a plan agreed on by the end of this session (about 20-30 minutes).

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ACTIVITY 2 PRACTICAL CLASS – HAEMATOLOGY 1. Demonstration of PCV, TPP and peripheral blood smear preparation. Demonstration of

automated haematology analyser.

2. Perform microhaematocrit on sample. (Check buffy coat for platelets and leukocytes.)

3. Determine total plasma protein (TPP).

4. Make blood smear and stain with "Diff-Quik".

5. Perform differential leukocyte count on your smear and also note number of normoblasts per 100 leukocytes as well as the morphology of the erythrocytes, leukocytes and platelets.

6. Calculate absolute values from differential and provided leukocyte count.

7. Calculate erythrocyte indices from provided erythrocyte count and haemoglobin, and own PCV.

8. Clean microscope and work area.

9. Check your results against the laboratory's results.

N.B. Total cell counts – you will be supplied with values of leukocytes, erythocytes and platelets for the practical class Staining Smears Diff-Quik Fix over 30 secs in methanol, then 5 one second dips in each of the two following stains, rinse in tap water. Do not rinse between stains. Giemsa Fix for 2 mins. with stain (1 vol). Add 1 vol buffer, mix then allow to stain for 7 mins. Rinse with tap water.

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HAEMATOLOGICAL RESULT SHEET FOR ACTIVITY 2 PRACTICAL CLASS    Animal..............................    Age..............Sex................    I.D. ...................................   Results: 

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MICRO HAEMATOCRIT SCALE  

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HAEMATOLOGICAL REFERENCE INTERVALS 

Veterinary Pathology Diagnostic Services Faculty of Veterinary Science 

University of Sydney   

    DOG  CAT  HORSE (adult) 

PCV   L/L  0.37‐0.55  0.30‐0.45   0.32‐0.52 

Plasma protein 

g/L  55‐75  59‐78  58‐84 

Haemoglobin  g/L  100‐150  80‐140  110‐160 

Erythrocytes  x1012/L  5‐7  6‐10  8‐11 

MCV  fl  60‐75  40‐45  41‐49 

MCHC  g/L  300‐350  310‐350  300‐360 

MCH  pg  20‐25  13‐17  13‐16 

Fibrinogen  g/L  2‐4  1‐3  2‐4 

Leukocytes  x109/L  7‐12  8‐14  6.0‐13.0 

Neutrophils  Seg. cells/109/L  4.06‐9.36  3.76‐10.08  2.47‐6.96 

  band cells/109/L  0‐0.24  0‐0.42  0‐0.24 

Lymphocytes  cells/109/L  0.91‐3.6  1.6‐7.0  1.6‐5.4 

Monocytes  cells/109/L  0.21‐0.96  0.08‐0.56  0.0‐0.72 

Eosinophils  cells/109/L  0.14‐1.2  0.16‐1.4  0.16‐0.96 

Basophils  cells/109/L  0‐0.36  0‐0.14  0‐0.36 

Platelets  x109/L  200‐900  300‐700  100‐300 

Reticulocytes  %  0‐1.5  0‐1.0  0  *There may be marked variations in normal values due to age, sex, breed and use.  These are crude approximations only.  They are not necessarily applicable to results from other laboratories. 

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ACTIVITY 4 PRACTICAL CLASS

A. BIOCHEMISTRY

1) Perform glucose estimation using the dextrostix reagent strips.

2) Perform urea estimation using the azostix reagent strips.

3) Perform glucose estimation using the glucometer or equivalent.

4) Demonstration of biochemical analysis using equipment available in practice such as Vetscan and Vetest.

BIOCHEMICAL ANALYSIS RESULT SHEET FOR ACTIVITY 4

Animal............................ Age................................. Sex.................................. I.D. ................................ Results:

GLUCOMETER..........................................

DEXTROSTIX.............................................

AZOSTIX.....................................................

Comments: ....................................................................................................……………………………….. ...........................................................................................………………………………......... ...............................................................................................………………………………....... ...................................................................………………………………..................................

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ACTIVITY 4 PRACTICAL CLASS

B. URINALYSIS See theory and practical notes on urinalysis.

1. Perform biochemical analysis on urine using: Multistix 10SG for

1) Glucose 2) Ketones 3) Blood 4) PH 5) Protein 6) Bilirubin

Use refractometer for specific gravity (don’t rely on the Multistix). Use SSA to confirm protein on the Multistix

2. Examine centrifuged sediment

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URINALYSIS RESULT SHEET FOR ACTIVITY 4 PRACTICAL CLASS 

  Animal...........................    Age..............................     Sex..............................    I.D. ............................     Results:  

  URINE    Catheterised    Voided    Cystocentesis 

  ROUTINE     ANALYSIS   Biochemical   Appear        pH       Colour        Glucose       Sp. Gr        Ketones       Protein        Blood               Bilirubin       MICROSCOPIC    EXAMINATION    CENT    UNCENT 

                                                                                                       

Comments:  ....................................................................................................................... ....................................................................................................................... ....................................................................................................................... .......................................................................................................................

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ACTIVITY 6 PRACTICAL CLASS – CYTOLOGY See cytology theory and practical notes.

A demonstration will be provided of techniques to collect, prepare and stain cytological samples. 1) Perform imprints, scrapings and fine needle aspiration on tissue supplied

2) Make smears

3) Stain by Diff Quik

4) View slides

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CYTOLOGICAL RESULT SHEET FOR ACTIVITY 6 PRACTICAL CLASS 

Comments:  ....................................................................................................................... ....................................................................................................................... ....................................................................................................................... .......................................................................................................................

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REQUEST SHEETS AND COLLECTION OF SAMPLES

FOR CLINICAL PATHOLOGY

1 INTRODUCTION The way samples are collected, and in what, will vary depending on the laboratory and what tests they employ. Therefore, familiarisation with individual laboratory requirements is an essential prerequisite to the collection of samples. Private laboratories will provide you with this information and do not expect you to remember it but do expect you to review it when taking samples. The list below is a summary of what request sheets and samples are required for tests performed by the Veterinary Pathology Diagnostic Services (VPDS) in The Faculty of Veterinary Science. Although many samples are acceptable, preferences are indicated. VPDS provides a service to external practices but also for the University Veterinary Teaching Hospital at Sydney (UVTHS). UVTHS employs the AIS (Animal Intelligence System) on-line package for tracking and retrieving animal cases.

2 REQUEST SHEETS All request sheets are to be clearly labelled with the UVTHS’s AIS animal number (no name) and the owner’s name (no number) and the registry number. The UVTHS label covers all this signalment. At present there is a single request sheet for most of the tests except for endocrine tests. Case information should be given in the space provided on the request sheets when interpretation is required. It is important that the clinician's initials are on the request sheet as well as your own. This is for two main reasons, firstly, the sample may be inappropriate and will need to be recollected e.g. unfortunate clotting of a sample for haematology; secondly, the results may need to be communicated immediately if one or more values are outside the reference range and the animal requires immediate attention. Reference intervals (‘normal values or range’) are expressed with the results in the AIS report. These apply to this laboratory only, although they can be used as a rough guide for figures received from other laboratories. Reference intervals vary between laboratories due to the type of test performed, the conditions of the test and the way the test is performed.

3 COLLECTION OF SAMPLES A result is only as good as the material that is presented to the laboratory. Use the correct type of container for the analyte to be examined. Do not go by the colour of the top but by the worded label of the container. Make sure that the container is mixed quickly and gently after collection. Specimens should reach the laboratory as quickly as possible and may not be processed the same day unless received by 12.30 pm. The specimen(s) with the request form(s) should be left on the bench in the VPDS Clinical Pathology laboratory in the McMaster Building.

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All containers, tubes and swabs (two from each site, one for gram stain, one for culture) must be labelled with the animal’s AIS number and the owner's name (i.e. 51165; Mr MacGregor: not ‘Peter’, ‘Flopsy’, ‘Mopsy’ or ‘Cottontail’). During out-of-hours, many specimens need to be processed without delay. This processing should be done by the intensive care/weekend duty students. In particular, blood smears should be made immediately and heparin blood samples should be centrifuged within two hours and plasma removed into a plain blood tube. These samples are stored in the pharmacy or near the blood-gas machine in the refrigerator. The sample(s) must then be taken to Clinical Pathology the following morning. For details regarding processing required for samples collected after-hours (see Section 4). Samples and Safety Often syringes with needles attached are sent off to the Clinical Pathology Laboratory for processing. While it is highly desirable for the needle to be removed to avoid needle stick injuries, some samples, as in the case of Fine Needle Aspirates, may contain vital diagnostic material still in the needle. For this purpose special plastic containers have been provided for the transport of these potential dangerous items. Please use these containers for the transport of these items. These containers can also be used for the transport of slides as well to prevent the slides falling onto the ground or getting wet. Please note that this procedure, of leaving needles on syringes, is not acceptable in sending samples to outside pathology laboratories and in some case they may refuse to accept the sample. To help with diagnosis the laboratory offers some profiles. Basic small animal profile (Profile A) (70016 – Biochem Profile A) Amylase, Alkaline Phosphatase (ALP), Alanine Aminotransferase (ALT, GPT),

Creatine Kinase (CK, CPK), Protein, Albumin, Globulins, Cholesterol, Glucose, Total Bilirubin, Creatinine, Urea, Calcium, Phosphate, Sodium, Potassium, Chloride, Bicarbonate.

Alimentary small animal (Profile B) (70017 – Biochem Profile B) Amylase, Lipase, ALP, ALT, Urea. Pre-anaesthetic profile (Profile F) (70021 – Biochem Profile F) ALP, ALT, Albumin, Glucose, Creatinine, Urea. Kidney/Renal Profile (70136 –Profile Renal/Kidney) two different ones Albumin (Kidney only), Creatinine, Urea, Calcium, Phosphate, Potassium (Renal

only). Liver Profile (70144 –Profile Liver) Alkaline Phosphatase (ALP), Alanine Aminotransferase (ALT, GPT), Protein,

Albumin, Globulins, Cholesterol, Total Bilirubin, Urea, VSP-D Comprehensive Diagnostic (70118 – Biochem Profile VSD)

Amylase, ALP, ALT, Protein-Total, Albumin, Globulins, Bilirubin-Total, Glucose, Creat., Urea, Ca, Phosphorus, Sodium, Potassium.

VSP-P Prep profile II (70119 – Biochem Profile VSP)

ALP, ALT, Protein-total, Glucose, Creat, Urea.

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Individual information on each type of analyte is continued below: Blood Biochemistry  

Analyte  Serum  In Heparin (plasma) 

In Fluoride oxalate (plasma) 

Amylase  X(P)  X   ALP  X  X(P)   ALT  X  X(P)   AST  X  X(P)   CPK  X  X(P)   GGTP  X(P)  X   Lipase  X  X   Bilirubin  X(P)  X   Total Protein  X(P)  X   SPE  X     Cholesterol  X(P)  X   Glucose  X  X  X(P) Urea  X  X   Creatinine  X  X   Bile Acids  X     Calcium  X  X   Inorganic Phosphate  X  X   Sodium  X  X   Potassium    X  X Chloride    X  X Magnesium  X  X   Triglycerides  X(P)  X   T4  X(P)  X   Phenobarbital  X  X   Iron  X  X   UIBC  X     Fructosamine  X  X   ∃ Hydroxybutyrate  X  X   LDH  X  X   Uric Acid  X  X   Ammonia   Heparin ‐ Collect on Ice 

(Contact laboratory prior to sample collection) 

    X ‐‐ Acceptable sample      P ‐‐ Preferred by our laboratory  For glucose estimation, samples in fluoride oxalate are preferred (fluoride is an enzyme poison and prevents glycolysis). The samples have to be presented to the laboratory promptly so that the plasma can be removed quickly from the cells. For ammonia estimation, the blood should be collected in anticoagulant (contact laboratory - one type of reagent kit requires lithium heparin, while the other requires EDTA) and placed in a beaker of ice, and transported to the laboratory promptly so the plasma can be removed quickly from the cells. It is preferred that the anticoagulant container is chilled prior to collection.

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Urinalysis Urine containers supplied to the UVTHS should be used. It is best if the sample is collected fresh at the UVTHS, than brought in by the owner. If not collected in the UVTHS please mark on the sheet. ALA (delta-aminolevulinic acid) -approx. 5 ml urine General examination -approx. 10 ml urine (smaller samples accepted) General exam comprises: specific gravity, dipstick chemistry and sediment microscopy Urinary Protein/Creatinine Ratio - approx 2ml urine

Faecal analysis Stools should be placed in the provided urine containers. For special requests in parasitology, please contact that section. General exam -appox. 5 g fresh faeces Occult blood -approx. 1 g fresh faeces Protase only -approx. 1 g fresh faeces General exam comprises: microscopy for undigested fat, starch and muscle.

Haematology For WBC and RBC Counts, PCV, Hb and TPP estimation, blood collected in EDTA (ethylenediamine tetra-acetic acid. Synonyms include versenate, versene, sequestrene) is preferred. It is important that the correct amount of blood be placed in the tube i.e. the tube is filled to the calibrated mark. Too much blood and the anticoagulant will not be effective and a clot will form, too little blood and the EDTA will affect the smear morphology. The tube is gently mixed and checked for any clotting. Peripheral blood smears can be made from blood collected in EDTA; however, for the best preservation of morphology, smears should be made directly from freshly collected blood. The anticoagulant Heparin is not acceptable for making blood smears as it interferes with the staining and morphology of leukocytes.

For microfilarial examination, blood collected in Heparin is preferred as it does not interfere with the enzymatic procedure that is employed to differentiate Dipetalonema and Dirofilaria microfilarae by the acid phosphatase test. (refer to your parasitology notes)

For coagulation studies (e.g .fibrinogen, prothrombin time and partial thromboplastin time), the anticoagulant 3.8% sodium citrate is used (contact the laboratory for this anticoagulant). D Dimer (for fibrinogen degradation products) requires plasma or whole blood that has been treated with an anticoagulant.

Blood Grouping requires an EDTA sample.

The requirements for other tests can be obtained by contacting the VPDS clinical pathology laboratory.

Bacteriological samples All containers and swabs (two from each site - one for Gram stain, one for culture) must be labelled with unit number and owner’s name. All specimens must be collected in an appropriate manner (see relevant sections in 2nd and 3rd year Principles of Disease and Veterinary Microbiology handbooks) and handled so that specimen viability is maintained and so that samples do not spill or discharge into the environment.

If in doubt please contact the bacteriological section of the laboratory. 

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a. Urine Samples, CSF and Other Liquids

Place in sterile, screw-capped containers which are kept upright and do not leak. Transport to the laboratory as quickly as possible. If a delay of greater than 2 hours occurs, store at 50C.

b. Pus, Synovial Fluid

These are best transported in a syringe from which all the air has been expelled. Remove the needle from the syringe and cap effectively to prevent leakage. If bottles are used, tape lids to prevent leakage.

c. Solid Material

Place in a small screw-capped bottle, with a swab moistened with sterile saline, if transport is required. Seal edge of Petri dish if transport is directly from surgery. If some distance from the laboratory, place tissue into the transport medium of a swab container (NB. Some commercial transport swabs have a plastic swab case filled with transport medium).

d. BS (Blind Screen) Urine Culturing

This is a crude test to determine the possibility of bacteria being present in urine.

This does not provide for the identification and antibiotic sensitivity testing if an infection is present. Nor is it a substitution for the normal sediment examination of urine, nor does it include a gram stain.

Sample - The sample must be a cystocentesis sample.

The minimum amount is 1 ml.

e. LCAT

This is for assessment of cryptococcal antigen. This requires a minimum of 1ml of serum after clotting.

Virology FeLV and FIV - Serum 1ml but Plasma (EDTA or Lithium Heparin) can be used

FIP Testing Definitive diagnosis of FIP currently relies on histopathology and immunohistochemistry or its adaptation into immunofluoresece. Immunohistochemistry uses a monoclonal antibody against Feline Coronavirus to identify macrophages infected with the mutant form of this virus (known as FIP virus) within tissues. VPDS offers different options for the analysis of effusions (immunofluoresence) or tissues (immunohistochemistry)

With effusions, it should be submitted as quickly as possible to reduce false negatives.

Cytology Cytology should be submitted to the laboratory within three hours of preparation or, once air-dried on a glass slide, dip in absolute ethanol or methanol for 15-60 seconds. CSF * - EDTA and sterile plain tubes. FNA - Smear, and/or syringe with needle still on and covered. Fluids - EDTA and sterile plain tubes. Smears - At least two smears of each site.

*To be taken to the laboratory as soon as possible

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Other Special Examinations Cross match - Recipient - EDTA and plain tubes.

- Donor(s) - EDTA, plain and lithium heparin tubes.

Blood Grouping - EDTA sample

Bone marrow * - Bone marrow on watch glass in EDTA solution.

Calculi - Calculi in sterile container.

Semen * - Semen in calibrated glass container.

Immunology - contact laboratory or refer to UVTHS handbook.

* To be taken to the laboratory immediately after collection.

Histopathology Biopsy

These should be submitted with the request form to the Clinical Pathology laboratory and given to the laboratory staff. During out-of-hours the specimen and request form are placed in the Clinical Pathology “In Tray” in the Treatment Room.

Place the sample in 10% buffered formalin (in the ratio of 1 volume of sample to 10 volumes of formalin). Using a pencil, write the owner’s name and the UVTHS animal’s identity number clearly and neatly on a piece of white cardboard and place the cardboard inside the container.

FROZEN SECTIONS - If frozen sections are required, notify the histopathology laboratory so that the cryostat can be chilled down to operating temperature. This will allow the tissue to be processed as soon as it is brought to the histopathology laboratory. The request form is taken to the Clinical Pathology laboratory for processing. Generally, needle aspirates and crush preparations taken for intra-operative cytology are more useful than frozen sections at the UVTHS.

Post Mortem

Please remember that no post mortem examination can be carried out unless the owner’s permission has first been obtained. All bodies must be accompanied by request forms which are completed in full as described above. The necessity for a full report of the relevant history and clinical signs is stressed.

Before any animal is destroyed, the students should consult the pathologist to determine the time at which euthanasia would be preferred and whether special procedures are desired (e.g. ante mortem perfusion of the eye). The body is taken to the cold room of the post mortem room and the request form is immediately taken to Clinical Pathology. Identity tags are available in the cold room and should be filled in and attached to the animal. If no labels are available use a white cardboard label with a UVTHS printed label attached to it and attach it to the cadaver. Place on the correct rack in the PM Cold Room – These racks are all labelled for each purpose.

During out-of-hours, the body, appropriately labelled, is placed in the post mortem cold room and the request form is placed in the Clinical Pathology “In Tray” in the Treatment Room.

When the cadaver is finished with, it is placed in the correct bin for disposal. 

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External Analytes VPDS will provide a service for the following analytes to be tested at other laboratories on the provision of the following requirements.

Blood lead - 5ml blood in EDTA (do not centrifuge)

Digoxin, phenobarbitone - 5ml in lithium heparin

Von Willebrand or clotting factors - 4.5ml blood in 0.5ml 3.8% sodium citrate and separated with plastic pipettes into plastic tubes

Cholinesterase - 5ml blood in EDTA or Lithium Heparin Do not separate

Trypsin Like Immunoreactivity (TLI) - 10ml blood with no anticoagulant

Folate, Vitamin B12,Bromide, FIP - 10ml blood with no anticoagulant

Samples should be  labelled with client's name, AIS animal number and date and accompanied by a pathology  request  form with  the client's name and clinic unit number, clinician's name, date, name of test requested. 

Endocrine These have a yellow Endocrine Request form in UVTHS.

Samples should be labelled with client’s name, the animal’s AIS number and date and accompanied with a yellow endocrine request form with the client’s name and AIS number, clinician’s name, date and name of test requested.

Please contact the VPDS lab in the McMaster Building to organise PTH and Catecholamines. All anticoagulant blood collected for hormone analysis should be separated and plasma frozen within 1 hour of collection (15 mins for an accurate PTH value).

Please take separated plasma/serum samples, unseparated clotted blood tubes and/or urine samples to the VPDS lab in the McMaster Building. If there are any problems please ring Dot Lewis on 13718 or 17456.

ACTH response test -5ml in lithium heparin or 5ml clotted blood (2x Cortisol at 0 and 1h)

Cortisol -5ml in lithium heparin or 5ml clotted blood

Dexamethasone suppression test -5ml in lithium heparin or 10ml clotted blood Canine - (3x Cortisol at 0,4 and 8h,) Equine – (2 or 3 samples 0, (15) and 19h)

UCCR (at 0h) - 5ml Urine

Conversion Analyte  Old Unit  New Unit  Conversion Factor 

Old ‐‐> New Cortisol  μg/dl  nmol/L  27.59 

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4 WHAT TO DO IF TAKING SPECIMENS FOR LABORATORY EXAMINATION AFTER HOURS

(This has more relevance for the practice situation than to the UVTHS) Ensure that the specimens are labelled prior to storage. Initial processing of specimens is essential to diminish the risk of erroneous results. SAMPLES FOR BIOCHEMISTRY

Separate the serum*/plasma from the red cells and store at 4ºC (if overnight delay) or at -18ºC (longer delays) in the refrigerator.

* allow the sample to clot properly before separation ROUTINE FULL BLOOD COUNTS - Make a peripheral blood film (we prefer 2), label it, and leave at room temperature. - Perform PCV and TPP determination. - Leave rest of sample in the refrigerator (4ºC). For other specialist haematology tests contact the laboratory for advice. MICROFILARIA CHECK - Store heparin sample at 4ºC. URINE FOR URINALYSIS - The results of urinalysis can alter for delays of more than 4 hours, even if the urine is stored

at 4ºC. Out of hours samples should be examined by the collector. Any difficulty with interpretation can be followed up on subsequent samples.

URINARY ALA - Acidify the urine (a few drops of glacial acetic acid) and store at 4ºC. i.e.. 100μL/10mls

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FAECES

Fresh stools should be:

1. frozen (-18ºC) for TRYPSIN analysis

2. chilled (4ºC) for stains for undigested/unabsorbed food particles and for routine parasitology.

CELL ASPIRATES - Smears should be made, dried, fixed in alcohol, preferably methanol, and left at room

temperature. FLUID ANALYSIS - A smear for cytology should be made, dried, fixed in alcohol, preferably methanol, and left

at room temperature. Store the sample at 4ºC.

N.B. CELLS DETERIORATE RAPIDLY IN BODY FLUIDS (CAVITIES, SYNOVIAL, CEREBROSPINAL) AND PROLONGED STORAGE (e.g. OVERNIGHT) MAY CAUSE FALSE LOW TOTAL CELL COUNTS. IN ADDITION BACTERIA WILL PROLIFERATE AND ALTER THE RESULTS.

HISTOPATHOLOGY - Place the sample in 10% buffered formalin. (In the ratio of 1 volume of sample to 10

volumes of formalin) Label with pencil on cardboard inside the container. POST MORTEMS - Place the labelled sample at 4ºC in the cold room. BACTERIOLOGY

SWABS AND FLUIDS

Make a smear, dry and leave at room temperature. Store the moist swab (if not moist add sterile saline) or fluid at 4ºC.

N.B. BACTERIAL FLORA CAN ALTER SIGNIFICANTLY WITH PROLONGED DELAYS

The above suggestions will diminish but not  remove the possibility of erroneous results. 

Where possible, avoid taking samples out of hours. 

Ensure that the specimens are labelled prior to storage.

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5 INFORMATION ON MEASUREMENT UNITS AND ENZYMES In this laboratory most test results are expressed in S.I. units ("Systeme Internationale") or derivatives of such, which is a system of metric units. The litre (L) is the unit of volume used in clinical pathology but is outside the SI units. Enzymes are expressed in IU (international units) per litre. One international unit of enzyme (IU) is that amount which catalyses the transformation of one micromole (μmol) of substrate per minute under defined conditions. Most chemical substance concentrations are expressed in moles or fractions of moles per litre. Although not necessary when using VPDS, the old units of measurement and their conversion factors to the new units will be given (Many Veterinary Clinical Pathology texts are using the old units - particularly North American texts). See page 16 for conversion factors. Enzyme Terminology ALP Alkaline phosphatase AMS alpha Amylase ALT Alanine transaminase. Synonym is (S)GPT [(Serum) Glutamic-pyruvic

transaminase] AST Asparate (transaminase) (aminotransferase) Synonym is (S)GOT [(Serum)

Glutamic-oxalo acetic transaminase] CPK Creatine phosphokinase. Synonym is CK (Creatine kinase) GGT Gamma glutamyl transpeptidase HBDH 2 Hydroxybutyrate dehydrogenase (Not done in this laboratory) LDH (LD) Lactate or Lactic dehydrogenase (Not normally done in this

laboratory) LPS Lipase SDH Sorbitol dehydrogenase (Synonym ID - Iditol dehydrogenase) (Not done in

this laboratory) (NB. Before the standard international units (IU) were accepted, numerous units of measurement were employed. They cannot be easily converted to the new units.)

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6 CONVERSION OF OLD ENZYME UNITS TO INTERNATIONAL UNITS (IU)

These are for enzymes measured at 37ºC and expressed as IU/L.

Enzyme  Old Unit  Conversion Factor Amylase  Somogyi/Caraway  1.85   Huggins‐Russell  5.7   Street‐Close  5.7   Roche Dye unit  1.59   u‐Katal  60      A.L.P.  Bessey‐Lowry‐Block  16.7   Bodansky  5.4   King‐Armstrong  

(U.S.A.‐ Phenolphthalein phosphate) 3.55 

  (Kind‐King  7.10   u‐Katal  60      A.L.T./ A.S.T.  Babson‐Transac  1.0   Karmen  0.483   Reitman‐Frankel  0.483   Sigma‐Frankel  0.483   Wroblewski‐LaDue  0.483   u‐Katal  60      C.P.K./ G.G.T.P.   u‐Katal  60      L.D.H.  Wacker  0.483   Wroblewski  0.483   u‐Katal  60      Lipase  Roe‐Byler  16.7   Cherry‐Crandall  278      S.D.H.  Sigma‐Frankrl  0.0167 

7 TEMPERATURE CONVERSION FACTORS FOR ENZYMES TO 37°C Enzyme  Result at 25°C   Result at 30°C Amylase  Unknown  1.8 A.L.P.  1.63  DEA buffer 1.29, AMP buffer 

1.35 A.L.T.  2.00  1.40 A.S.T.  2.10  1.48 C.P.K.  2.35  1.50 G.G.T.P.  1.70  1.35 L.D.H.     (Lactate )  2.50  1.64 (<2mm pyr)  2.15  1.55 (>2mm pyr)  2.74  1.69 

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8 CONVERSION FACTORS

Biochemistry Analyte  Old Unit  New Unit  Conversion Factor  

Old ‐‐> New Albumin  g/dl  g/L  10 Bilirubin (total, free‐conjugated) 

mg/100ml (dl)  μmol/L  17.103 

Protein (total, EPG) 

g/dl  g/L  10 

Cholesterol  mg/dL  mmol/L  0.0259 Glucose  mg/dL  mmol/L  0.0555 

mg/dL  mmol/L  0.1665 Urea Blood Urea Nitrogen (BUN) in mg/dL 

mmol/L (Urea)  0.3561 

Creatinine  mg/dL  μmol/L  88.402 Triglycerides  mg/dl  mmol/L  0.0114 

mg/dL  mmol/L  0.2495 Calcium mEq/L  mmol/L  0.5 

Inorganic Phosphate 

mg/dL  mmol/L  0.323 

mEq/L  mmol/L  1 Sodium mg/dl    0.435 mEq/L  mmol/L  1 Potassium mg/dl    0.256 mEq/L  mmol/L  1 Chloride mg/dl    0.282 

Bicarbonate  mEq/L  mmol/L  1 Ammonia  μg/dL  μmol/L  0.588 Bromide  mg/ml  mmol/L  12.5 Magnesium  mg/dl  mmol/L  0.411 Uric Acid  mg/dl  mmol/L  0.0595 Xylose  mg/dl  mmol/L  0.067 Cortisol  μg/dl  nmol/L  27.6 Digoxin  ng/ml  nmol/L  1.28 Phenobarbital  μg/ml  μmol/L  4.3 T4  μg/dl  nmol/L  12.9 

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Haematology Parameter  Old Unit  New Unit  Conversion 

Factor  Old ‐‐> New 

PCV (packed cell volume)  %  L/L  0.01 TPP (Total plasma protein)  g/dL  g/L  10 

g/dL  g/L  10 Hb (Haemoglobin) g/dL  mmol/L  0.621 

Erythocyte Count  106/μL(mm3) 

1012/L  1 

MCV (mean corpuscular or cell volume) 

μm3(μ3)  fl (femtolitre) 

1 

MCHC (mean cell haemoglobin concentration) 

g/dL  g/L  10 

MCH (mean cell haemoglobin)  μ2g (picogram) 

pg  1 

BSR (ESR) (blood or erythocyte sedimentation rate) 

mm1/2mmlhr 

The same no conversion 

 

Leukocyte count  103/μL  109/L  1  NB. milli (m) = 10-3 micro (μ) = 10-6 nano (n) = 10-9

pico (p) = 10-12 femto (f) = 10-15 Reference: SI Units in Clinical Pathology Printed by the Health Commission of New South Wales. These are based upon World Health Organization (1977) The SI for the Health Professions. WHO, Geneva.

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9 REFERENCE RANGES FOR BIOCHEMISTRY Veterinary Pathology Diagnostic Services 

The Faculty of Veterinary Science The University of Sydney 

Unless stated, these values have been determined in the VPDS laboratory 

ANALYTE/ ENZYME  UNIT  DOG  CAT  HORSE AMYLASE   IU/L  <1,400  <1,400   ALP  IU/L  <110  <50  <260 ALT  IU/L  <60  <60  <50 AST  IU/L  <40  <40  <400 CK  IU/L  <200  <200  <400 GGT  IU/L  0.6 ‐ 8.2   1‐10  <36 LDH  IU/L  <105  <105  <250 Lipase  IU/L  <60     Bilirubin total  μmol/L  1.2‐5.1  2.5‐3.5  0‐50  

(non‐ fasting)  ‐ conjugated  μmol/L  1.2‐5.1  0‐5.0  0‐6.8  ‐ unconjugated  μmol/L  1.2‐5.1  2.5‐8.0  3.4‐50 Protein,total   g/L  50‐70  54‐73  60‐76 Albumin BCG  g/L  23‐43  19‐38  29‐38 Cholesterol  mmol/L  1.4‐7.5  1.9‐3.9  1.7‐3.1 Glucose  mmol/L  3.3‐6.4  3.6‐6.6  4.4‐6.3 Urea  mmol/L  3.6‐10.0  7.2‐10.7  3.7‐8.2 Creatinine  μmol/L  40‐120  90‐180  87‐149 Ammonia (fast)   μmol/L  0‐50  0‐50  0‐60 Bile Acids (fast)  μmol/L  0‐10  0‐5  0‐20 Bile Acids (pfeed)  μmol/L  <25  <15            ELECTROLYTES         Calcium  mmol/L  2.1‐2.9  1.75‐2.6  2.8‐3.4 Magnesium  mmol/L  0.73‐0.98  0.82‐1.23  0.73‐1.23 Inorg Phosp.  mmol/L  0.8‐1.6  1.3‐2.3  0.80‐1.77 Sodium  mmol/L  137‐150  147‐156  132‐150 Potassium  mmol/L  3.3‐4.8  4.0‐4.5  2.8‐4.8 Chloride  mmol/L  105‐120  115‐130  99‐108 Bicarbonate  mmol/L  18‐24  17‐21  20‐28 SPE         Protein Alb  g/L  23‐39  24‐30  28‐36          α  g/L  7‐16  9‐21   8‐13          ß  g/L  9‐16  8‐15  8‐15          γ   g/L  4‐12  9‐23   7‐14          SPECIAL*         BSP  T2 or % ret  <5% (2 hr)    2.0‐3.7 m Delta‐ALA  μmol/L  <38     Faecal Tryp.  Azo.Alb.U  >7.0     

*most are now obsolete 

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10 LOCATION of Clinical Pathology Classroom and VPDS Laboratories in the McMaster Building B14

244

245

HAEMATOLOGY PRACTICAL NOTES

1 INTRODUCTION These notes, which are not intended to be intensive or extensive, present some simple manual haematological techniques that can be used to evaluate common haematological disturbances. For convenience, the techniques are categorised according to their usefulness in erythrocyte analysis, leukocyte analysis, coagulation analysis and in miscellaneous problem analysis. Some techniques, however, have general applicability to haematological analysis.

2 SOME ANTICOAGULANTS Ideally, dry anticoagulants of known proportions should be used to minimize dilution, artefacts and alteration of staining properties. No one anticoagulant is entirely satisfactory.

• EDTA - disodium or dipotassium salt of ethylenediamine tetra-acetic acid (synonyms: sequestrene, versenate or versene) EDTA acts by chelating calcium ions required in the clotting mechanism. At a concentration of 1 mg per ml of blood, it is the anti-coagulant of choice for routine blood counts and smears. NB. excess EDTA, i.e. 2 mg per ml of blood, will cause shrinkage of the red blood cells and a significant decrease in PCV. So be careful to fill the tube to the graduated level only. With EDTA, thrombocyte (platelet) counts and cell counts may be performed up to several hours after the collection of blood. Refrigerated EDTA blood samples are suitable for cell counts, PCV and haemoglobin estimations up to 24 hours post-collection.

• Heparin Heparin prevents coagulation by interfering with the conversion of prothrombin to thrombin and with the action of thrombin on fibrinogen. Heparin is naturally occurring and found abundantly in the liver. It does not alter erythrocyte cell size, even in excess; but it does interfere with the staining of leucocytes (the cells appear hazy). This is due to its strong affinity for basic dyes. Heparin is used at a concentration of 10-50 international units per ml of blood.

• Sodium Citrate Sodium citrate is used for some coagulation studies at the rate of one part of 3.8% aqueous solution to 9 parts of blood. Citrate removes the calcium which is essential for clotting.

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3 ERYTHROCYTE ANALYSIS

a) The Microhaematocrit (PCV) This makes use of high speed centrifugation, using capillary tubes, which ensures complete packing of erythrocytes in a minimum time. The speed of microhaematocrit centrifuges range from 8000 to 16,000 r.p.m. Method: After filling capillary tubes to 66-75% capacity by capillary attraction, the opposite end is sealed either by a small flame or by plasticine (preferred). The sealed tube is centrifuged for the requisite time - 2 minutes at 16,000 r.p.m. or 5 minutes at 8,000 r.p.m. - to give three distinct layers: (i) the mass of erythrocytes at the bottom called the packed cell volume (PCV) (ii) a mixed layer immediately above the erythrocytes, composed of leukocytes,

platelets and other nucleated cells, called the buffy coat. The top white layer of the buffy coat is composed primarily of platelets. The leukocyte layer is immediately below the platelet layer and is usually an opaque dark red. This layer is difficult to visualise unless there are increased numbers of leukocytes.

(iii) the top layer composed of plasma. The microhaematocrit value is determined by

use of a reader supplied with the microhaematocrit centrifuge and is expressed as a fraction

microhaematocrit value (PCV) = red cell volume (litres)

total volume of blood (litres)

Notes: The microhaematocrit reading is a good indication of the presence of anaemia or polycythaemia as long as the total plasma protein (TPP) concentration is determined - usually by refractometer. For instance, dehydration (TPP above normal units), by reducing plasma volume, may mask an anaemia or produce a spurious or relative polycythaemia. The colour of the plasma layer provides useful information. For instance, it may depict the presence of jaundice (icterus), lipaemia or haemolysis.

b) Haemoglobin Estimation Using the Cyanmethaemoglobin Method The reagent used is called Drabkins reagent. Reagent: Place 500 mls of distilled water in the 1 litre volumetric flask. Add Sodium bicarbonate 1.00 gm

Potassium cyanide 0.05 gm Potassium ferricyanide 0.02 gm

Dissolve the contents, and make up to the 1 litre mark with distilled water. Principle: Haemoglobin is oxidised to methaemoglobin by ferricyanide, and the methaemoglobin is converted into the stable cyanmethaemoglobin by the addition of KCN. The absorbance of cyanmethaemoglobin is measured at 540 nm, where cyanmethaemoglobin exhibits a broad absorbance peak. Because of this broad peak it is acceptable to use a colorimeter (such as the unimeter) using a yellow-green filter (e.g. Ilford 625).

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Method: 0.02 ml (20μl) of blood is added to 5 ml of reagent and mixed by inversion. The solution is then stood at room temperature for 10 minutes, then the absorbance is read at 540 nm against a reagent blank. This absorbance is then converted to grams of haemoglobin per litre from a standard curve. The curve is constructed using a commercially available standard. The unimeter method is the same as this except different amounts of reagent and sample are used.

c) Erythrocyte Count - (Unopette) This method uses a pre-measured diluent reservoir of 1.99 ml (normal saline with sodium azide as a growth inhibitor) and a disposable 10 μl micropipette. A haemocytometer is also required (see appendix 1). Note: Azide is dangerous The protective shield for the pipette is used to puncture the diaphragm of the reservoir. NB. Discard shield after this step. i) The pipette is filled with well mixed whole blood and the excess wiped off. ii) The reservoir is squeezed and the pipette placed onto the reservoir and locked in. iii) The index finger is placed over the top of the pipette and the pipette rinsed with

diluent by releasing then squeezing the reservoir several times. iv) The finger is removed and the apparatus is mixed by inversion. v) The pipette is turned around and used as a dropper to fill the haemocytometer. NB. A few drops of diluent are discarded before filling the chamber. Five of the 25 intermediate squares in the central large square are counted (4 corners and centre). Both ruled areas are counted using 40 x objective and the mean is used in the calculation.

count Calculation: 100 x 1012/Litre

(see haemocytometer notes for more detail) Notes: Haemoglobin estimation and the erythrocyte count are usually not essential for establishing the presence of anaemia or polycythaemia but can provide supportive evidence. Also, they allow the determination of red cell indices which may be useful in categorising an anaemia.

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d) Erythrocyte Indices These determine the relationships between the erythrocyte count, haemoglobin concentration, cell volume and cell thickness.

1. Mean corpuscular volume (MCV)

This is the average volume of a single cell expressed in femtolitres (fl- a femtolitre is 10-15 of a litre). It is obtained from the following formula:

MCV (fl) = PCV (L/L) x 1000 (expressed as a whole number) Erythrocyte count (x1012/L)

2. Mean corpuscular haemoglobin (MCH)

This measures the mean or average haemoglobin content of a cell in picograms (pg - a picogram is 10-12 of a gram). In some older and American texts it is refered to as the color index.

MCH (pg) = Haemoglobin (g/L) (expressed to one decimal place) Erythrocyte count (x 1012/L)

3. Mean corpuscular haemoglobin concentration (MCHC)

This measures the ratio of the weight of the haemoglobin to the volume of the erythrocyte, expressed in grams per litre. It takes into account the variation in size of erythrocytes, which is often present in anaemias. In some older and American texts it is referred to as the saturation index.

MCHC (g/L) = Haemoglobin (g/L) (expressed as a whole number) PCV (L/L)

Notes: On the basis of MCV, anaemias can be designated macrocytic, normocytic or microcytic. On the basis of MCHC, and to a lesser extent MCH, anaemias can be designated hypochromic or normochromic. Regenerative anaemias (see explanation under reticulocyte count) are often macrocytic (due to the large numbers of reticulocytes) and, although the MCH is normal or increased, hypochromic due to lowered MCHC. However, these changes in red cell indices, if they do occur, are transient and return to normal on restoration of the red cell mass. For this reason, regenerative anaemias are often referred to as transitory or pseudomacrocytic anaemias. True macrocytic anaemias may be found in non-regenerative anaemias associated with myeloproliferative disorders in the cat (due to elevated levels of nucleated red blood cells). Iron deficiency anaemia, a non-regenerative anaemia, is usually microcytic and hypochromic. However, the majority of non-regenerative anaemias are normocytic and normchromic.

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e) Reticulocyte Count Reticulocyte stain: Brilliant cresyl blue 1.0 gm

Sodium citrate 0.4 gm Normal saline 100.0 mls

Dissolve the stain in saline, add sodium citrate, mix and filter. The stain should always be filtered before use. Method: Equal quantities (i.e. 2 drops) of well mixed blood (in EDTA) and stain are mixed in a test tube and allowed to stain for approximately 20 minutes. If the animal is severely anaemic (e.g. PCV 0.10) then 3 quantities of blood to 1 quantity of stain is used. A blood film is prepared in the usual manner and can be counterstained with Giemsa/Diff Quik although it is not advisable as the reticulocyte stain may be removed. Select an area of the smear where there is a monolayer of cells and count the number of reticulocytes. The mature erythrocytes are usually yellow-green while the reticulocytes contain dots or strings of blue staining material (residual cytoplasmic RNA). The reticulocyte count is performed, under an oil immersion lens, by differentiating at least 1000 red blood cells (sometimes it may be possible to count only 500) and expressing the result as a percentage of the erythocytes. If the reticulocyte count is to be expressed as a percentage, ideally it should be corrected for the reduced PCV present in anaemia (fewer erythrocytes are present to dilute reticulocytes released from the bone marrow). Corrected reticulocyte % = observed reticulocyte % x patient's PCV

normal PCV For this calculation the "average" PCV for a) Dogs is 0.45, b) Cats is 0.37. (In medical science, the reticulocyte percentage is corrected also for variation in maturation times of erythroid cells. However, maturation times have not been adequately determined in most animals (except dog - see lecture notes). The reticulocyte % can also be expressed as an absolute value: Absolute reticulocyte count(x 1012/L) = observed reticulocyte % X erythrocyte cell count. This is becoming the correction of choice for dogs and cats. The upper level of the reference interval for dogs is 0.075 (grey zone to 0.105) x 1012/L and for cats 0.06 (grey zone to 0.100) x 1012/L. If a dog has 4% reticulocytes and an erythrocyte count of 3.0 x 1012/L then the absolute reticulocyte value is 0.120 x 1012/L. This suggests regeneration. Notes: In all domestic species, except the horse, the reticulocyte count is an indication of whether the anaemia is regenerative (i.e. an adequate bone marrow response to anaemia). In regenerative anaemia the marrow responds, from 3 days after the onset of anaemia, by elevating the numbers of circulating immature erythrocytes (primarily reticulocytes but some may be nucleated red blood cells).

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The dog has small numbers of circulating reticulocytes in health (0-1.5%). The cat has a reticulocyte percentage of 1.4 - 10.8% in health. The marked variation in reticulocyte numbers in the cat is partly due to the fact that two main types of reticulocytes exist. The aggregate type (strands or clumps of reticulum) is similar to other species and high values indicate current erythropoietic activity. The punctate type (dotted reticulum) may occur in large numbers in health (9-10%) and are increased in regenerative anaemias. They remain increased at least two weeks after the aggregate count has returned to normal; therefore, they can indicate a bone marrow response as much as four weeks previously. (In our laboratory we count only the aggregate type in cats which are 0-1.0% in health). The adult horse in health and in anaemia rarely has reticulocytes in the peripheral circulation (foals may develop reticulocytosis in response to anaemia); therefore, the response of bone marrow has to be assessed from changes in serial microhaematocrits and from, if required, bone marrow aspirate examination.

f) Coombs Antiglobulin Test for Autoimmune Haemolytic Anaemia (AIHA) The basis of the Coombs test is the detection of antibodies or complement directed against the patient's own erythrocytes by the addition of species specific antiglobulin (or anticomplement) and the development of agglutination. The Coombs test forms the basis for all antibody detection and cross-matching tests.

1. Direct Coombs Test.

This detects antibodies attached to the erythrocyte membrane and is the method of choice for AIHA. A clotted sample is obtained from the patient and a control animal. Equal volumes of 0.1 ml of cell suspension and antiglobulin are combined in test tubes, set up in and incubated at 37°C (and at 22°C and 4°C if cold agglutinins suspected) for 15 minutes. After incubation, the tubes are centrifuged at 1500 r.p.m. for 15 seconds and then gently shaken to check for agglutination. NB. it is best to use serial dilution of antiglobulin to distinguish between false and true positives. The test requires appropriate negative controls i.e. cells minus anti-globulin must be included for comparison.

2. Indirect Coombs Test (rarely used for AIHA)

This determines whether autoantibodies are in the patient's serum and have an affinity for a determinant on the red cells of normal dogs. Washed saline cell suspension are prepared from normal dogs. The cells are sensitised by combining equal volumes of cells suspension with the patient's serum and incubating for 30 minutes at 37ºC. The cells are then washed several times to remove unbound protein. The species specific anti-globulin is then added to the washed sensitised cells and incubated again for 15 minutes at the various temperatures. The samples are then centrifuged and checked for agglutination. This indirect Coombs test may be used in cross-matching (modified) and detecting neonatal isoerythrolysis.

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4 LEUKOCYTE ANALYSIS

a) Leukocyte Count (Unopette method) The Unopette method employing a 20 μl capillary tube, haemocytometer counting chamber and Unopette reservoir containing 1.98 ml of diluent mixture (glacial acetic acid 3%, concentration made up in distilled water). The protective shield for the pipette is used to puncture the diaphragm of the reservoir. NB. Discard shield after this step. i) The pipette is filled with well mixed whole blood and the excess wiped off. ii) The reservoir is squeezed and the pipette placed onto the reservoir and locked in. iii) The index finger is placed over the top of the pipette and the pipette rinsed with

diluent by releasing then squeezing the reservoir several times. iv) Let the apparatus stand for 10 minutes, then mix thoroughly, convert the Unopette

to the dropper assembly and fill the haemocytometer counting the chamber (ideally, both chambers should be filled and counted, and the average cell number per chamber used.)

Using 100 x magnification (i.e. 10 x objective) count the leukocytes in all nine large squares of the haemocytometer counting chamber (see appendix). Calculation:

Count + 10% x 109/Litre 10

(see haemocytometer appendix for more detail)

Notes: Total white blood cell count indicates the presence of leukocytosis or leukopenia. In the dog, cat and horse, leukopenia/leukocytosis are usually due to changes in the number of neutrophils. The total white blood cell count is essential for determining the absolute values of leukocyte types seen on the peripheral blood smear.

b) The Peripheral Blood Smear (Film)

1. Preparation of a peripheral blood smear.

Using chemically clean slides, free from dust, films may be made in the following manner: A small drop of blood (ideally the smear should be made directly from withdrawn blood rather than blood placed in anticoagulant) is placed on the slide 1 cm from the end. The spreading slide is placed at an angle of 30-45 degrees to the slide and then moved back to make contact with the drop. The drop should then spread out quickly along the line of contact of the spreader with the slide. The moment this occurs, the film should be spread by a smooth forward movement of the spreader. Films are then rapidly air dried. It is essential that the spreader should have an absolutely smooth edge and should be narrower in breadth than the slide on which the film is to be made. If the edge is rough, films with ragged tails, containing many leukocytes, result.

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The angle of the spreader determines the thickness of the film, i.e. the greater the angle, the thicker the film. The ideal thickness is such that there is some overlap of red cells throughout much of the smear's length with separation and lack of distortion towards the tail of the film.

2. Staining of a peripheral blood smear.

Flood the slide with blood Giemsa and leave to fix for 2 minutes. Dilute and mix the Giemsa with an equal quantity of water (pH7). Leave to stain for 7 minutes. Rinse the slide in tap water and allow to dry.

Blood Giemsa: Giemsa 4 gm Methanol (A.R.) 1000 ml Glycerine 15 ml

Blood Giemsa is placed in an incubator at 37ºC for approximately 1 week before use. The solution is mixed daily while incubating.

3. Staining the smear for Haemoplasma (originally called Haemobartonella) spp.

The blood smear is fixed for 4 minutes in methanol. It is then stained in a coplin jar in 6% stock Giemsa in tap water overnight. The coplin jar is flooded with water to remove the Giemsa. The slide is then dried, rinsed in methanol, rinsed in tap water, redried and examined.

Stock Giemsa: Giemsa 7.6 gm Glycerine 250 ml Methanol (AR) 750 ml

The solution is treated as for blood Giemsa Notes: 1. A commercial stain ("Diff Quik" – Lab-Aids Pty Ltd) can be used for 2 and 3.

2. Mycoplasma (Haemobartonella) canis and Mycoplasma spp. in cats are usually found on the erythrocytes and may take one of several forms including chains of cocci, rods, bows and rings, or it may appear as a single coccus.

4. Examination of the Peripheral Blood Smear

Under low power note: (i) if the leukocytes are well distributed.

A bad film will show uneven distribution, with cells collecting at the margins or in the tail of the smear;

(ii) if there is a rouleaux formation; (iii) if there is red cell agglutination; (iv) if there are leukocyte clumps; (v) if there are platelet or fibrin clots; (vi) if there are any extracellular parasites present.

Under the oil objective (100x) note: (i) Leukocyte Morphology

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Note the presence of any primitive or immature leukocytes e.g. myelocytes, metamyelocytes. Note the presence of any atypical or abnormal leukocytes, e.g. atypical mononuclears, plasma cells. Note if the neutrophils show any toxic granulation, vacuolation and any inclusions. For the appearance of cell types please consult posters (supplied) and text books. Once the slide has been scanned under high power, a differential leukocyte count can be performed, with the oil lens, using the modified battlement technique (takes in account that larger cells tend to collect at the extreme margins of the film while lymphocytes are usually more centrally placed). Commence counting at one edge of the slide, traverse across the slide to the other margin, move 4-5 horizontal edge fields then reverse the traverse (towards the tail).

In the differential leukocyte count, an attempt is made to determine the percentage distribution of the various leukocyte types in peripheral blood. The procedure is subject to considerable error, since an extremely small portion of the total number of leukocytes is observed. This error can be decreased by counting a large number of cells: at least 100 cells for every 10 x 109/L cells in the total white blood cell count. From the results each leukocyte type is expressed as a percentage of the total count. However, the important value for each leukocyte type is the absolute number per litre of blood. Absolute value for a leukocyte type = total leukocyte count x percentage of leukocyte type. The absolute values determine the following: the presence of neutrophilia, neutropenia, eosinophilia, eosinopenia, lymphocytosis, lymphopenia and monocytosis. However, the presence of a left shift can be determined from the percentage values for the segmented and immature neutrophils e.g. the dog has a normal ratio of > 16-18 segmented neutrophils to 1 immature form; for a left shift to be definitely present, the ratio would have to be below that normal ratio. For instance, 60% segmented neutrophils and 5% band neutrophils. The ratio is 12:1, thus a left shift is presumed to be present. For dogs and cats, it is accepted that bands greater than 1.0 x 109/L indicate a left shift. The ratio of bands to segmented is only used for values less than 1.0 x 109/L. If there are any nucleated red cells present in the smear, they are usually recorded either as numbers per 100 WBCs counted or as an absolute value derived from the initial leukocyte count. If there are over 5 nucleated red cells per 100 WBC’s present, the total leukocyte count should be corrected before determining the absolute values for leukocyte types. Corrected total = initial total leukocyte count x 100 leukocyte count [ 100 + nucleated RBCs (per 100 WBCs)]

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Leukocyte description This table is modified from one in Outline of Veterinary Clinical Pathology by Maxine M. Benjamin, 2nd Edition, The Iowa State University Press. 1964 

MORPHOLOGY OF BLOOD CELLS USING ROMANONSKY TYPE STAINS 

  NUCLEUS  CYTOPLASM CELL  Size 

(microns) Shape  Staining 

Quality Chromatin  Nucleoli  Relative 

Amount Colour  Granules 

1. GRANULOCYTES                 a. Myeloblast  15‐20  Round or oval  Light 

purple Finely reticulated 

2 ‐ 5 Pale Blue 

Small  Deep blue  ‐‐ 

b. Progranulocyte  14‐20  Round or oval  Purple  Slightly coarse 

 1‐3  Small  Blue  Few azurophilic 

c. Myelocyte  10‐18  Round or oval  Purple  Fairly coarse  ‐‐   Moderate  Bluish pink  Start differentiating d. Metamyelocyte      (Juvenile) 

10‐18  Indented oval, resembling kidney or bean 

Deep purple 

Coarse  ‐‐  Large  Pink  Neutrophilic, eosinophilic, basophilic 

e. Band cell (Stab)  10‐15  Curved with parallel sides 

Purplish blue 

Coarse  ‐‐  Large  Pink  Neutrophilic, eosinophilic, basophilic 

f. Segmented                 ‐ Neutrophil  10‐15  Ragged or 

divided into lobes 

Purplish blue 

Coarse  ‐‐  Large  Faint pink  Violet or absent in animal blood 

‐ Eosinophil  10‐15  2‐3 lobes  Purplish blue 

Coarse  ‐‐  Large  Bluish pink  Bright red, Horse ‐ very large, Dog ‐ varying sizes, Cat ‐ rod‐shaped 

‐ Basophil  10‐15  Outline covered with granules 

Pale blue  Indistinct  ‐‐  Moderate  Bluish pink  Bluish black, coarse in horse and cattle, moderate in dog and cat 

                 2. LYMPHOCYTES                 a. Lymphoblast  10‐18  Round or oval  Lt. 

reddish purple 

Fine  1‐2 Pale blue 

Small  Deep blue  ‐‐ 

b. Prolymphocyte  10‐18   Oval or sl. indented 

Deep reddish blue 

Fine to sl Coarse. 

Occasional  Large  Dark to med. blue 

Few azurophilic 

c. Lymphocyte  6‐18  Round, oval or sl. indented 

Deep purplish blue 

Large coarse clumps 

‐‐  Small to moderate 

Sky blue  Few large azurophilic 

                 3. MONOCYTES                 a. Monoblast  14‐18  Round or oval  Deep 

purple Fine retic.  1‐2  Moderate  Deep blue  ‐‐ 

b. Promonocyte  14‐18  Irregular  Purple  Fine retic.  0‐1  Moderate  Gray blue  Few fine lilac c. Monocyte  12‐18  Round, 

indented, band or lobed 

Pale purple 

Fine strands  ‐‐  Large  Gray or gray blue 

Fine azurophilic 

                 4. ERYTHROCYTES                 a. Rubriblast  14‐19  Round  Purple  Fine 

stippled Usually 1‐2  Small  Deep blue 

to gray blue ‐‐ 

b. Prorubricyte  10‐15  Round  Purple  Coarse  Usually 1  Moderate  Blue  ‐‐                  c. Rubricyte   8‐12  Round  Deep 

purple Definite   ‐‐  Moderate  Blue to pink  ‐‐ 

d. Metarubricyte    (Late Normoblast) 

 7‐10  Pyknotic, fragmented, or partially extruded 

Dense purple 

Solid  ‐‐  Moderate  Pink  ‐‐ 

e. Erythrocyte   4‐8          Total  Pink  ‐‐                  5. THROMBOCYTES          (Platelets) 

 2‐4          Total  Pale blue  Azurophilic 

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(ii) Erythrocyte Morphology

Note any variation in size (anisocytosis) and the presence of microcytes, macrocytes. Note any variation in shape (poikilocytosis) and the presence of target cells, burr cells (acanthocytes) and spherocytes. Note any variation in colour (polychromasia) and the presence of hypochromatic or polychromatic (polychromatophilic) cells. Note the presence of erythrocyte inclusions e.g. Howell Jolly bodies, punctate basophilia (basophilic stippling) and parasites.

NB. E.R. bodies (Heinz bodies) can be stained with 0.5% brilliant cresyl blue in normal saline.

Reticulocytes, in the Giemsa stained peripheral blood smear, will appear blue and will be larger than the mature erythrocytes. Accordingly, a blood smear from a regenerative anaemia will show anisocytosis and polychromasia. The presence of nucleated red cells in the blood smear may also occur in regenerative anaemias. However, if in an anaemia, nucleated red cells appear in the blood unaccompanied by anisocytosis and polychromasia, a non-regenerative anaemia associated with abnormal marrow should be suspected.

 Erythrocyte Morphology ‐ number of cells per oil field of 200 ‐ 250 erythrocytes 

ABNORMALITY  SLIGHT  MODERATE  MARKED 

Anisocytosis       

 Dog  7‐10  11‐20  >21 

 Cat  5‐8  9‐20  >20 

 Horse   1‐3  4‐10   >10 

Polychromasia       

 Dog  1‐2  3‐10  >11 

 Cat  1‐2   3‐15  >15 

 Horse  rarely observed 

   

Poikilocytosis       

 All species  8‐25  26‐150  >150 

Hypochromasia       

 All species  1‐2  3‐10  >10 

Codocytes        

 Dogs only  1‐2  3‐6  >6 

Spherocytes       

 All species*  1‐2  3‐10  >10 

Echinocytes       

 All species   1‐2  3‐10  >10 

Acanthocytes, schizocytes, keratocytes, elliptocytes, dacryocytes, depranocytes, stomatocytes All species  1‐2  3‐5  >5 

* spherocytes are not easily identified in species with small erythrocytes (e.g. cats, ruminants) 

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(iii) Platelets

Note their size and numbers.

See coagulation section g (ii) for indirect count methods.

c) Total Eosinophil Count (Direct Method) Sample: Collect blood in EDTA. Total count should be performed within a few hours. Stain:

2% Eosin Y (aqueous) 5 ml Acetone (A.R.) 5 ml Distilled water 90 ml

Prepare on day of use. This fluid is said to be stable for 2-3 weeks but it is best to prepare fresh. Method: Dilute well mixed blood 1 in 10 with stain and mix for no more than 30 seconds. Fill haemocytometer, allow to settle, then count all 9 squares on both sides. Calculation:

Total no. of eosinophils counted = Count x 109/Litre 180

NB. To check count, a smear should be made and stained by Giemsa and examined. The absolute eosinophil count determined by this indirect method should approximate or be slightly greater than the direct count. If there is doubt the direct method should be repeated. The total eosinophil count is of importance in evaluating adrenocortical activity. For instance, eosinopenia is often present hyperadrenocorticism. There is a Unopette available for direct counting of eosinophils.

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5 LABORATORY EVALUATION OF HAEMOSTASIS

Haemostasis is dependent on vascular integrity, platelet function and numbers, and clotting factors. Consequently, laboratory evaluation of haemostasis involves the determination of these components.

Although there are many tests available, these notes concentrate on the simpler and more commonly used coagulation tests. It is advisable to provide samples from control (normal) animals along with the test sample.

a) Bleeding Time The interval between the occurrence of injury to the blood vessel and the cessation of bleeding is called the bleeding time.

The test may be performed on the skin of the ear, the nose, the pad of the foot, or the inside of the lip.

Method:

A thin coat of petroleum jelly is spread over the area, and a moderately deep puncture is made with a number II Bard Parker blade so that the blood flows freely. Do not use pressure.

Drops of blood are absorbed onto a filter paper at 30 second intervals, and the time at which bleeding stops is noted.

Normal bleeding time for most animals is 1-5 minutes.

Notes:

Bleeding time is an estimation of vascular integrity and platelet function and numbers, and is not dependent on clotting factors. Therefore, prolonged bleeding times occur in thrombocytopenia, platelet function defects and vessel wall defects. Von Willebrand’s factor deficiency (allows platelet adhesion) will also cause an extended bleeding time. A normal bleeding time occurs in health and in deficiencies of clotting factors.

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b) Clotting Time This test measures the time required for fibrin clot formation in non-anticoagulated blood in vitro. Method: A 5 ml syringe is rinsed with normal saline solution immediately before use. 3 ml of blood is withdrawn from a vein. The blood is divided into 1 ml lots and is transferred to 3 clean glass test tubes (8 mm bore). The tubes are maintained in a water bath at 25-37ºC. After 3 minutes, the tubes are tilted at 30 second intervals, starting with tube 1; when this clots, i.e. when the tube can be inverted without blood flowing out, the time is recorded. Clotting times are noted for tubes 2 and 3. The average time for coagulation in all 3 tubes is then determined. Normal values:

3-13 minutes for the dog 8 minutes for the cat 4-15 minutes for the horse

Notes: Clotting (coagulation) time is not dependent on vascular integrity or platelet function and numbers (as long as glass tubes are used). Clotting in vitro is primarily due to activation of the intrinsic pathway of clotting factors by direct contact with glass. However, in siliconized or plastic tubes, platelet factors are important in activating the intrinsic pathway of clotting factors). Also, clotting time is not affected by the extrinsic system of clotting factors as this system is activated only by the release of tissue thromboplastin from injured vascular endothelium. Prolonged clotting times occur in *deficiencies or inhibition of intrinsic or common pathways of clotting factors. Normal clotting times occur in health, thrombocytopenia, platelet function defects and vessel wall defects. *the deficiency must be less than 5% of the normal level before clotting time is extended.

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c) Clot Retraction Clot retraction in vitro is primarily a function of platelets, although the fibrinogen content of the blood does have an influence. Method: 5 ml of blood is placed into a clean, dry glass test tube (with no added anticoagulant) and incubated at 37°C. A normal clot will retract markedly within 2-4 hours and by the end of 24 hours will be a compact mass. Notes: The above is purely a screening test and more quantitative methods are available. Clot retraction is impaired primarily by platelet function and number defects. It may be impaired in afibrinogenaemia and anaemia. When fibrinolysis is very active, the fibrin clot may be dissolved as quickly as it is formed, and clot retraction will obviously be impaired.

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d) Fibrinogen Estimation Method: A 2 microhaematocrit tubes are filled with blood containing EDTA and centrifuged for 5 minutes as would routinely be done for determining packed cell volume. The plasma protein level is determined from a tube by using the refractometer. The second tube is placed in a water bath at 56°C for 3 minutes. This will precipitate fibrinogen, which is removed from the plasma by further centrifugation for 5 minutes. The total plasma protein of the sample in the heated tube is determined with the refractometer. Fibrinogen concentration is calculated by subtracting the protein value of the plasma in the heated tube from that of the unheated tube. Method B (Millar Method - suggested method)) A microhaematocrit tube is filled with blood containing EDTA and centrifuged for 5 minutes as would routinely be done for determining packed cell volume. The tube is placed in a water bath at 56°C for 3 minutes. Making sure that all the fluid is immersed. This will precipitate fibrinogen, which is packed on top of the buffy coat by further centrifugation for 5 minutes. The microhaematocrit tube is then attached to a slide so that measurements can be made of the different interfaces in the tube using the microscope's scale and its vernier. If the microscope does not have a scale, an ocular micrometer eyepiece can be used. For this calculation the interface between the buffy coat and the precipitated fibrinogen is called A. The interface between the precipitated fibrinogen and the serum is called B. The base of the meniscus of the plasma (now serum) is called C. The length of the column of the precipitated fibrinogen (AB) is measured in relation to the original length of the column of plasma (AC). Fibrinogen concentration (g/L) is calculated from the formula:

( )( ) 100×

−−

ACAB

Normal fibrinogen (Factor I) levels:

2-4 g/L for the dog 1-3 g/L for the cat 2-4 g/L for the horse

Notes: (These methods are limited in their ability to detect reduced levels. Other more sensitive methods are available to detect reduced fibrinogen.) Fibrinogen (Factor I) levels are increased in a wide variety of inflammatory and tissue destructive conditions. They can be decreased in certain chronic liver diseases, D.I.C. and genetic disorders. Fibrinogen levels can have an effect on the results of certain coagulation tests.

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e) Prothrombin Time (ProT, PT or OSPT) Measures the time required for fibrin clot formation in recalcified, fresh, citrated plasma after addition of tissue thromboplastin (which should be absent if the sample was taken correctly) in vitro i.e. it measures the extrinsic (tissue) and common pathways' clotting factors. Sometimes referred to as one stage prothrombin time (OSPT) Methods: Add 4.5 ml whole blood (control and patient) to 0.5 ml of 3.8% (0.13M) trisodium citrate. Centrifuge and remove the plasma. Place the plasma and simplastin in a bath for 5 minutes set at 37ºC (plasma can be stored at 4ºC for a maximum of 4 hours before being used in the test). Label 3 control and 3 patient serological tubes. Add 0.2 ml of Simplastin to each tube. Add 0.1 ml of plasma (control and patient) to each tube and time the clot formation. Determine the average clotting time for the control and patient and determine any significant difference. Reference range values:

7-12 seconds in the dog 7-10 seconds in the cat 10-15 seconds in the horse

Notes: A prolonged PT occurs in deficiencies or inhibitions of extrinsic and common system clotting factors, and in heparin inhibition (if heparinized blood had been given by transfusion). A normal PT occurs in deficiencies or inhibitions of intrinsic system clotting factors and in health. With automation doing most of the coagulation work in larger laboratories and point of care instruments becoming more common in smaller laboratories and practices, there was some form of standardisation required in the measurement of the coagulation times. Some instruments measure when the clot first starts to form while others wait till the clot is fully formed. To overcome this and other abnormal determinations from instruments and reagents, International Normalised Ratios (INR) were determined. With the PT test, this is based upon International Sensitivity Indexes (ISI) (supplied by the reagent manufacturers) and Mean Normal Prothrombin Time (MNPT) (supplied by the instrument maker). You should be aware that some laboratories report the result as INR. The calculation for the INR is found in the reagent insert with a table for ISI values.

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f) Partial Thromboplastin Time (PTT) Measures the time required for fibrin clot formation in recalcified, fresh, citrated plasma after addition of a contact activator in vitro (which activates the intrinsic [intravascular] pathways of clotting factors). The PTT yields the same, but more precise, information as clotting time, i.e. it measures the intrinsic and common pathways of clotting factors. Method: 4.5 ml of whole blood (from a control animal as well as the patient) is placed into tubes containing 0.5 ml of 3.8% trisodium citrate and mixed. The sample is then centrifuged and the plasma removed. Label 3 control and 3 patient serological tubes. Pipette 0.1 ml of Platelin Plus Activator into the tubes and place in a 37ºC water bath for 2 minutes. Add 0.1 ml of the appropriate plasma to the tubes and incubate 5 minutes. Transfer 0.1 ml of 0.025M calcium chloride to each tube and simultaneously time for clot formation. Determine the average clotting time for control and patient, and any significant difference. Reference range values:

12-25 seconds for the dog 12-25 seconds for the cat 37-54 seconds for the horse

Note: The PTT is prolonged only when the deficient factor is less than 30% of the normal level. The PTT is very low in birds and reptiles.

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g) Platelet (Thrombocyte) Count I. DIRECT METHOD Platelet counting fluid: Ammonium oxalate 1.0 gm Brilliant Cresyl Blue 0.1 gm Distilled water 100.0 mls i.e. 20 drops of 0.5% brilliant cresyl blue in 1% ammomium oxalate. (Filter prior to using). Method: A normal red cell dilution of 1 in 200 of well mixed whole blood in the fluid is made. It is mixed for approximately 5 minutes before being used to fill the haemocytometer counting chamber. The haemocytometer counting chamber is then placed in a moist chamber and left for about 15 minutes to allow the platelets to settle. Using the 40x objective, platelets in the entire centre 1.0mm2 area of both counting chambers of the haemocytometer are counted; this gives the number in 0.2ul. Counting is best carried out using a phase contrast microscope if possible. Make sure the objective is lined up the correct condenser. Calculation: Number of platelets = total platelets counted X 200 X 5 X 106

(x 109) per litre i.e. count x 109 /Litre Also, platelets may be counted using a special Unopette chamber. The leukocyte dilution is prepared, the leukocytes counted, and then the platelets counted and calculated. The direct method of counting platelets is plagued with difficulties. Platelets are small and are often confused with other particles such as dust and debris. Also, they may disintegrate or agglutinate, making counting difficult. Hence, direct platelet counts are often poorly reproducible. Reference range values (using the direct method of counting):

200-900 x 109/L for the dog 300-700 x 109/L for the cat 100-300 x 109/L for the horse

Notes: A moistened chamber may be produced from a petri dish with two sticks placed in the base (to support the haemocytometer) with a piece of moistened blotting paper. If using phase contrast the haemocytometer should have a flat base.

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II. INDIRECT METHODS To cross check or estimate platelet numbers, the indirect methods can be employed. These methods have clinical application only when more sophisticated methods are not available. A. From the stained blood film, under oil immersion, the number of platelets per 100 leukocytes can be estimated. Calculation: platelets(x 109/L) = number of platelets per 100 leukocytes x total leukocyte count B. When using the Olympus microscope with the 100X oil immersion lens, an estimate of the number of platelets, in the peripheral blood smear, can be made by taking a average of 5 scattered fields and referring to the table below. This mainly works for the dog, and when there is not a severe anaemia present (upsets the relativity). This is an estimate only because the count does vary on the type of microscope, objective size and quality, and the ocular size and quality.

Number  Estimate X 109/L 

5  103 

10  200 

15  298 

20  396 

25  494 

30  592 

35  690 

40  780  Notes:

The platelet count assesses the presence of thrombocytopenia e.g. less than 200 x 109 per litre for the dog. However, spontaneous haemorrhages (thrombocytopenia purpura) will not usually occur until the count is much lower than the minimum e.g. below 50-70 x 109 per litre for the dog, or there is a concomitant platelet defect.

Thrombocytopenia may occur due to marrow damage, splenic disorders (stores a third of the platelets at any one time), accelerated use e.g. D.I.C. (disseminated intravascular coagulation), and immune-mediated destruction e.g. idiopathic thrombocytopenia purpura. Thrombocytopenia due to marrow damage is accompanied by decreased numbers of megakaryocytes, thrombocytopenia due to other causes usually has normal to increased numbers of megakaryocytes.

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h) Fibrin (fibrinogen) degradation products – including D-dimer present in cross-linked fibrin degradation products During blood coagulation, fibrinogen is converted to fibrin by the action of thrombin. The resulting fibrin monomers polymerize to form a soluble network of non cross-linked fibrin. This fibrin gel is then converted to cross-linked fibrin by thrombin activated Factor XIII to form an insoluble clot. Once the insoluble clot is formed, fibrinolysis is activated. This requires the activation of plasmin, which is the major clot-lyzing enzyme. Plasmin facilitates the conversion of both fibrinogen and fibrin to degradation products. Those degradation products from cross-linked fibrin contain D-dimer. Consequently, measurement of D-dimer levels is very useful to determine the extent of fibrinolytic activity. Fibrinolytic activity is likely to be enhanced in any state of hypercoagulability (Disseminated Intravascular Coagulation [DIC]; thrombolytic disease such as in pulmonary thromboembolism). Consequently levels of D-dimers are enhanced (even in cases of sub-clinical DIC). A number of tests have been developed to measure fibrin (fibrinogen) degradation products, but the more-specific D-dimer tests appear to be the present ones of choice. A simple specific canine D-dimer test kit (Agen Canine D-dimer Test), based on rapid immunochromatography utilising specific murine monoclonal antibodies against D-dimer, will detect elevated levels (negative or positive result). A specific cat one is likely to be developed if demand becomes high.

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6 MISCELLANEOUS PROCEDURES a) Bone Marrow Examination Bone marrow samples can be collected from the iliac crest, femur, rib and sternum. The site will depend on the species and size of the animal. (i) Making the smear Methods of aspiration are available in most texts. As bone marrow rapidly clots, a sample is usually withdrawn in a syringe washed out with EDTA (make an aqueous solution of 2 ml from a vial with dried EDTA normally used for blood collection). Only a small amount of bone marrow should be aspirated (stop when it enters the main barrel of the syringe) and transferred to a watchglass. The bone marrow particles (white flecks) are carefully aspirated with a pasteur pipette so as to leave behind as much of the peripheral blood as possible. They are transferred to a cleaned glass slide, gently pushed into a mound and then squashed between two slides ("T" or "L" method). The pressure of squashing will vary between individual but it is usually mild to moderate. A correctly made bone marrow smear should be elliptical and have any peripheral blood present around the periphery of the squashed bone marrow particles. The slide is rapidly air dried, fixed in methanol and stained routinely with Giemsa but may be stained with Prussian blue (for iron) and peroxidase (for granulocytes). (ii) Examination of the Stained Smear The degree of cellularity can be assessed under low power. Under oil immersion, a differential cell count can be performed. The most reliable results are obtained in the tail area of the smear, where 500-1000 cells are counted. From the differential cell count: the myeloid to erythroid (M:E) ratio (expresses the proportion of total granulocytes to nucleated erythroid cells), the proportions of myeloid and erythroid cells in the proliferating pools (promyelocytes and myelocytes for myeloid, usually less than 20%; pronormoblasts and early normoblasts for erythroid, usually less than 10%), and the proportion of megakaryocytes and other cell types can be determined. M:E ratios: 1.2:1 for the dog

1.5:1 for the cat 1.1-10.20:1 for the horse

Notes: Bone marrow examination is useful for:

1. Investigating non-regenerative anaemias in most species. In the horse it can be useful for investigating all anaemias.

2. Establishing or confirming the presence of a myeloproliferative disorder. 3. Investigating a platelet disorder. 4. Investigating a myelophthisic disorder.

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b) Cross-matching Blood for Transfusion

Saline Cross-Match (dog) This is the simplest method available. Although it is not as sensitive as the antiglobulin test, reagents are readily available whereas for the antiglobulin method they are hard to obtain and very expensive.

a) Samples The sample of choice is clotted blood. From this sample both serum and cells may be obtained. After the sample has clotted it should be centrifuged to separate both serum and the cells. The serum is stored at 4ºC. The clotted blood should not be refrigerated until the serum and cells have been removed. The serum is only stable for up to 24 hours due to loss of complement. It is best to use fresh serum, but plasma must not be used as anticoagulants are anti-complement. Red cells are teased from the clot and used within 24 hours. If there is any difficulty in obtaining cells from the clot then cells from the EDTA sample may be used. Cells obtained in such a manner should be washed three times in buffered normal saline before use. If there is no alternative, cells collected in ACD (Acid Citrate Dextrose) may be used and these are stable for several days at 4ºC.

b) Technique The clotted blood sample is centrifuged and the serum removed. The clotted samples need to be completely clotted - if the request is urgent, then the clotting process may be speeded up by placing the samples in the water bath at 37°C. After the serum is removed a little normal saline is added to the clot and the clot broken up with a glass rod to free the cells. Some of the freed red cells are placed into a test tube and centrifuged at 200G for one minute. Take care that there is no clots in the cells. The saline is replaced and the process repeated twice more. The saline on the last wash is removed and a 3% suspension of red cells is made in LISS (1 drop of cells and 32 drops of LISS ). If LISS is not available saline may be used. Both donor and recipient samples are treated in the same manner.

Duplicates of each of the following tubes are prepared.

1) Saline Control - 1 drop Recipient Cells + 2 drops saline (LISS)

2) Auto-Control - 1 drop Recipient Cells + 2 drops Recipient Serum

3) Donor Test - 1 drop Donor Cells + 2 drops Recipient Serum

4) Donor Control - 1 drop Recipient Cells + 2 drops Donor Serum

Tubes 3 and 4 are repeated for each additional donor.

One set of tubes is placed in a 37°C water bath and the other left at room temperature for 30 minutes. The room temperature should be in the range of 20-24°C , preferably closer to 24°C as below 20°C the test is affected by cold agglutinins. It is best to run at both temperatures as the literature is in conflict as to the ideal temperature and until this is determined it is best to cover all possibilities.

At the end of 30 minutes the tubes are centrifuged for 1 minute at 750 G (slow speed) and then examined for haemolysis and/or macro-agglutination. Negative tubes should then be examined for micro-agglutination. If any doubt exists with the result then the blood is declared incompatible.

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Notes: Care must be taken not to confuse rouleaux for agglutination.

The donor dogs are commonly checked for microfilaria prior to use.

All animals having a second transfusion must be cross-matched. Breeding bitches that have already had litters should be cross-matched using the antiglobulin test as well as the saline cross-match.

The horse is cross-matched using the antiglobulin test or the haemolysin test.

The cat is commonly blood typed for transfusions since the saline cross-match often produces inadequate results.

c) Blood typing for cats and dogs Blood typing for dogs and cats is now available for the veterinary practitioner and has, in many cases, replaced the need for cross matching. It is recommended that all cats undergoing a transfusion be typed as there may be high levels of circulating isoantibodies to incompatible blood groups. Dogs undergoing a transfusion for the first time usually do not have significant levels of isoantibodies to incompatible blood groups. These usually develop after the initial transfusion. Consequently, typing of dog erythrocytes is optional for first transfusions but essential for second and subsequent transfusions. RapidVet-H (Feline) (Kansas State University and dms/agrolabo products ag, Neuhausen am Rheinfali, Switzerland): is intended for use to classify cats as blood group Type A, Type B or Type AB. This is one blood group system in the cat, with the majority of cats possessing the A antigen. One third of the cats possessing A antigen have naturally occurring, low-titre, anti-B antibody. All Type B cats have naturally occurring, highly titred anti-A antibody. Type AB cats are rare and do not have or develop anti-A or anti-B antibodies. Cats with B erythrocytes will develop a marked anaphylactic and haemolytic response to Type A blood because of their natural, high titre anti-A antibodies. Cats with A erythrocytes and natural, low titre anti-B antibodies will exhibit only a mild reaction when transfused with Type B blood for the first time (but significant haemolysis can occur over a period of time). Cats with AB erythrocytes do not usually develop transfusion reactions. The kit assay is based on the agglutination reaction that occurs when an erythrocyte which contains either a Type A, Type B or a Type AB antigen on its surface membrane interacts with lyophilized specific antisera present on the test card. Whole blood collected in EDTA and test diluent are added to the Auto-agglutination Saline Screen well, the Patient Type A well and the Patient Type B well and mixed. The test card is rocked until agglutination occurs. The Patient Type A well agglutinates if the cat is Type A, the Patient Type B well agglutinates if the cat is Type B. Both wells agglutinate if the cat is AB. There are limitations to the test which are discussed in the information sheet. RapidVet-H (Canine DEA 1.1) (Kansas State University and dms/agrolabo products ag, Neuhausen am Rheinfali, Switzerland): is intended for use to classify dogs as DEA 1.1. Eight specific antigens have been identified on the surface of canine erythrocytes. The internationally accepted canine blood group system, the DEA= (Dog Erythrocyte Antigen), is based on these antigens. The antigens are DEA 1.1-1.8. DEA 1.1 and 1.2 are the most significant. Both are highly antigenic but DEA 1.1 is the primary lytic factor in canine transfusions. Dog erythrocytes with DEA 1.1 have the greatest potential to stimulate formation of isoantibodies. Thus most reactions resulting from the transfusion of incompatible cells occur when DEA 1.1 positive blood is given to DEA 1.1 negative

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recipients. DEA 1.2 and 1.7 may produce minor transfusion reactions. Greyhounds are particularly low in DEA 1.1, 1.2 and 1.7 antigens and, therefore, are useful donors. About 40% of dogs are DEA 1.1 positive. A DEA 1.1 positive dog can receive both DEA 1.1 positive and negative blood. A dog that is DEA 1.1 negative should not receive DEA 1.1 positive blood. The kit assay is based on the agglutination reaction that occurs when an erythrocyte which contains a DEA 1.1 antigen on its surface membrane interacts with a murine monoclonal antibody proven specific to DEA 1.1 which is lyophilized on the test card. Whole blood collected in EDTA and test diluent are added to the Auto-agglutination Saline Screen well. Positive Control fluid is added to the DEA 1.1 Positive Control well. Whole blood collected in EDTA is added to the Patient Test well and mixed. The test card is rocked until agglutination occurs. The Positive Control should agglutinate. If the Patient Test agglutinates then the animal is DEA 1.1 positive. There are limitations to the test which are discussed in the information sheet. d) The Blood/Erythrocyte Sedimentation Rate (BSR:ESR) When blood containing an anti-coagulant is allowed to stand in a tube in a vertical position, the red cells will gradually settle, leaving the plasma as a clear supernatant. The rate of settlement over a specified time is known as the sedimentation rate. Method: The Wintrobe method entails the use of a tube, in which 10 cm are graduated from the base in mm. The bore is 2.5 mm to 3 mm and the volume approximately 0.7 ml. The tube is filled to the 10 cm mark with blood and allowed to stand vertically for one hour, for the dog and cat, at which time a reading is taken of the sedimentation of the cells (for the horse, readings are taken at 10, 20, and 30 minutes). The test should be set up within 2 hours of collection of the sample (in EDTA). As variations in temperature affect the sedimentation rate, care should be taken to ensure that the test is performed under standard conditions (usually 22-27°C). The rate of sedimentation is obtained by reading from the top of the blood plasma to the top of the layer of sedimented erythrocytes in mm. Ideally, the result should be corrected for PCV as the ESR varies inversely with PCV. Significant ESR difference = observed ESR - anticipated ESR at that PCV

(read from a table) Notes: The usefulness of the ESR has been questioned. The ESR is a non-specific reaction which does not aid differential diagnosis. Even a normal ESR does not exclude the possibility that a disease process exists. Today, it is only utilised by a few equine veterinarians. Elevated ESR may be found in infections, skin alterations, tissue injury or destruction and pregnancy. A depressed ESR may occur when there are significant numbers of abnormal or immature red cells, and when there is a low total plasma protein e.g. chronic liver disease, poor nutrition.

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e) The Lupus Erythematosus (LE) Cell Test (For Systemic LE) The LE cell test is based upon the principle that a substance (LE factor) in the gamma globulin fraction of plasma of affected patients reacts with the nuclei of injured leukocytes. A nucleoprotein transformed by the presence of the LE factor acquires chemotactic properties and attracts phagocytes, which are usually segmented neutrophils. Consequently, the phagocytes ingest the nuclei. The final result, that is the LE cell, is a neutrophil (less commonly a monocyte or eosinophil) that has ingested a mass of nuclear material. The ingested nuclear material is the size and shape of a lymphocyte nucleus and is homogenous. The ingesting leukocyte has a peripherally displaced nucleus. Care must be taken to differentiate LE cells from Tart cells (monocytes containing foreign nuclei that retain some characteristic nuclear structure). Forty per cent of SLE cases will be negative for LE cells. Consequently, the Anti-nuclear Antibody (ANA) test is replacing the LE cell test. Method 1: One ml of the patient's blood is added to a test-tube containing 4 glass beads. This is then shaken vigorously to prevent clotting and rotated to 33 r.p.m. at room temperature for 30 minutes. (This provides the necessary trauma to leukocytes as LE factor appears to be incapable of acting upon healthy living leukocytes). The tube is then placed at 37°C for 10-15 minutes after which the contents are transferred to a haematocrit tube. Buffy coat films are made after centrifugation. The films are air dried and stained by Romanowsky method (Giemsa). Method 2: Freshly drawn blood is permitted to clot and remain at room temperature for two hours. The serum and clot are pressed through a fine wire mesh screen into a petri dish. A wintrobe haematocrit tube is filled with mashed clot and centrifuged at 2000 G for 10 minutes. The supernatant is discarded and films made from the buffy coats then stained as for Method 1. NB. For both methods, slides should be examined for at least 15 minutes before a negative report is given.

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7 REFERENCE RANGES FOR HAEMATOLOGY Veterinary Pathology Diagnostic Services 

The Faculty of Veterinary Science The University of Sydney 

    DOG  CAT  HORSE (adult) 

PCV   L/L  0.37‐0.55  0.30‐0.45   0.32‐0.52 

Plasma protein 

g/L  55‐75  59‐78  58‐84 

Haemoglobin  g/L  100‐150  80‐140  110‐160 

Erythrocytes  x1012/L  5‐7  6‐10  8‐11 

MCV  fl  60‐75  40‐45  41‐49 

MCHC  g/L  300‐350  310‐350  300‐360 

MCH  pg  20‐25  13‐17  13‐16 

Fibrinogen  g/L  2‐4  1‐3  2‐4 

Leukocytes  x109/L  7‐12  8‐14  6.0‐13.0 

Neutrophils  Seg. cells/109/L  4.06‐9.36  3.76‐10.08  2.47‐6.96 

  band cells/109/L  0‐0.24  0‐0.42  0‐0.24 

Lymphocytes  cells/109/L  0.91‐3.6  1.6‐7.0  1.6‐5.4 

Monocytes  cells/109/L  0.21‐0.96  0.08‐0.56  0.0‐0.72 

Eosinophils  cells/109/L  0.14‐1.2  0.16‐1.4  0.16‐0.96 

Basophils  cells/109/L  0‐0.36  0‐0.14  0‐0.36 

Platelets  x109/L  200‐900  300‐700  100‐300 

Reticulocytes  %  0‐1.5  0‐1.0  0 

*There may be marked variations in normal values due to age, sex, breed and use.  These are crude approximations only. They are not necessarily applicable to results from other laboratories. 

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8 THE HAEMOCYTOMETER The improved Neubauer haemocytometer is used here (see Figure 1). A special optical plane coverslip must be used which is thicker than an ordinary coverslip. The depth between the coverslip and the ruled area is 0.1 mm. The ruled area is 9 mm2 and is divided into nine large squares. Each of the four large corner squares is divided into 25 smaller squares. The central large square is divided into 400 tiny squares arranged in 25 groups of 16 by triple boundary lines. There are two ruled areas per haemocytometer separated by a moat. When filling a haemocytometer the clean coverslip is placed on the clean haemocytometer and the diluted sample run in each side. The chamber must be filled quickly (i.e. reduce error in distribution) and must fill the raised area completely without the sample flowing into the moat. When counting, those cells touching or lying on the upper or left boundaries of the squares are included while those on the right and lower boundaries disregarded (or vice versa). For cell counts it is the usual routine for both chambers to be counted and the mean count used for the calculation. The total volume of the ruled area is 0.9 μl (mm3) with each large square 0.1 μl. From these values and the dilution factor it is a simple matter to calculate the cells per litre of blood. For Leukocytes (when using Unopettes) this is: count x 10 x D.F. x 106/Litre

9 10 corrects to one μl. 9 D.F. (Dilution factor) = 100 106 brings the total volume to one litre

A simple calculation although not totally correct is:

count + 10% x 109/Litre 10

For Erythrocytes the volume counted is 5/25 of 0.1μl (i.e. 1/ 50μl). The calculation is thus:

count x 50 x D.F. x 106/Litre if D.F. = 200 then

count x 50 x 200 x 106 Put simply

count x 1012/Litre 100

The same approach may be used for all other cells counts using the haemocytometer.

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FIGURE 1 

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275

MICRO HAEMATOCRIT SCALE

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277

URINALYSIS

1 INTRODUCTION Urinalysis should always be performed when a urinary tract disorder is suspected. Also, it is useful in investigating and confirming many extra renal problems. Urinalysis should be performed completely as the results are often interrelated, e.g. the chemical detection of blood and the microscopic examination of sediment for the presence of erythrocytes. The laboratory procedure for urinalysis in VPDS is a standard one and involves:

2 OBSERVATION OF PHYSICAL PROPERTIES For this, 10 ml of urine (well mixed as for all analyses) is placed in a centrifuge tube and examined for clarity (appearance), colour, odour and foam. Before the urine is sent to the laboratory, a note is made of the volume collected so that the results can be interpreted in relationship to that volume.

3 ESTIMATION OF SOLUTE CONCENTRATION Generally this involves the determination of specific gravity by using the refractometer. If the urine is markedly turbid it should be cleared by centrifugation as cells, crystals and large macromolecules refract a disproportionate amount of light when compared to their true effect on specific gravity. Remember protein and glucose (see later and lecture notes) will also elevate urine specific gravity up to about 4 units. An estimation of solute concentration, by determining specific gravity, is essential before interpreting the results of chemical and microscopic examination.

4 CHEMICAL ANALYSIS Generally this involves reagent strips and tablet methods, which are semi-quantitative and as such, are really only useful as screening tests. Instructions for these tests are apt to change from time to time. Therefore, the instructions that accompany the test should be read carefully. Multistix 10 SG: The reagent strips containing multiple squares are dipped into the urine and read at specific times, glucose 30 seconds later, bilirubin 30 seconds later ketones 40 seconds later, blood 60 seconds later pH & Protein 60 seconds later (The squares for specific gravity, urobilinogen, nitrite and leukocytes are of little use) NB. Care is to taken on removing the strips from the urine sample so that excess does not interfere with the different reagent squares.

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The pH square may be confirmed by pH paper as an alkaline buffer in the protein test strip may flow over onto the pH strip and interfere with the result. If the test for protein records a trace or above, the salicylsulphic acid test is performed (add 1 volume of supernatant of centrifuged urine to 2 volumes of salicylsulphonic acid in a tube. Assess the cloudiness and precipitate). The Ames Multistix test depends upon pH for reaction and sometimes an alkaline pH will give false positives due to quaternary ammonium salts.

Result Appearance Approximate value negative no change <0.05 gms/L

Trace mild cloudiness 0.20 + moderate cloudiness 0.50

++ cloudiness and fine precipitate 2.00 +++ cloudiness and moderate precipitate 5.00

++++ cloudiness and heavy precipitate 10.00 gms/L

If there is any doubt to the actual value or a more accurate value is required, then the urinary protein can be determined by analytical (Spectrophotometerical) methods. If the test for glucose records a trace or above, or if it is negative and the test for ketone bodies is positive, the “Clinitest” can be performed (to a round bottom tube, 5 drops of urine and 10 drops of water are added; a “clinitest” tablet is added; after the effervescence has stopped, the tube is shaken and colour compared to that on a chart). “Clinitest” is less sensitive for glucose, sensitive to other reducing substances, and is not affected by ketones. It may give false positives with large amounts of ascorbic acid which may also depress the colour development in the Multistix The ketone reagent square can be double checked by the “Acetest”. Place a tablet on clean white paper after resealing the bottle. Place one drop of urine on the tablet. After 30 seconds compare colour of tablet to colour chart provided. False positives can be caused by L.dopa, BSP and large quantities of phenylketones. Blood - large amounts of ascorbic acid may inhibit the reaction and cause false negatives. Myoglobin causes false positives and some contaminating oxidizing agents can give false positives. Results are expressed as per the label on the Multistix bottle, except for protein which is explained above and glucose and ketone if clinitest and acetest are used. Ictotest: A test for bilirubin. (sometimes it is difficult to read the bilirubin reagent square). Ten drops of urine are dropped onto an absorbent test mat. A reagent tablet is placed on the mat, one drop of water is placed on the tablet, then after 5 seconds another drop of water is added so as to overflow onto the absorbent mat. After 60 seconds the colour of the mat around the tablet is noted. Results are expressed as; negative - no colour change, or orange is a negative. positive - Any formation of a purple colour is a positive for bilirubin.

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5 SEDIMENT EXAMINATION 10 ml of urine is centrifuged for 5 minutes at 400-500G. The supernatant is removed by inverting the tube. The sediment is mixed with the remaining supernatant (about 0.5 ml) and examined (if the urine is already turbid, concentrated, centrifugation may not be necessary to examine the sediment). One drop of sediment is placed on one end of a slide and two drops on the other end. To the one drop of sediment add one drop of either toluidine blue, nile blue or new methylene blue, mix and overlay with a cover slip. Add a coverslip to the other end of the slide. The unstained preparation should be examined first, preferably under low power with reduced light (drop the condenser and close the iris diaphragm. Objects will appear refractile). Casts are examined under low power (10 x objective), > 1 in a moderately concentrated urine is usually considered abnormal. Cells are examined and counted (average of 4-5 scattered H.P. fields) under dry high power (40 x objective). Erythrocytes >5 per high power field is suggestive of haemorrhage, leukocytes >3 per high power field is suggestive of active inflammation (moderately concentrated urine – may be up to 5 if voided urine). Other elements, such as crystals, fat droplets, etc., should be noted. The stained preparation should be used to further identify structures seen in the unstained preparation.

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281

CYTOLOGY

1 SOLID TISSUE CYTOLOGY (surface lesions, biopsies, autopsy material)

a) Tissue impression (imprint) Useful only for soft tissues that are exposed (biopsies, ulcerated skin lesions, and necropsy material). The surface must be cleaned of all debris and as much blood as possible. The tissue can be touched onto the cleaned slide or vice versa. Two or three impressions can be placed on the same side. b) Tissue scraping Useful for exposed, hard as well as soft tissues (e.g. connective tissue tumour) and poorly accessible areas (e.g. conjunctiva). Once again, the surface must be cleaned. Scrapings can be done with a variety of instruments to suit the tissue (e.g. spatula, scalpel blade). The surface is gently scraped a few times and the material transferred to a cleaned slide. The material can be squashed between 2 slides or smeared (as for a peripheral blood smear) using a slide or the scraping instrument. c) Fine needle aspirate Useful for non-ulcerated surface masses or internal organs/masses in the living animal. The size of the needle used will vary depending on the site and the species but commonly a 21-23 gauge is used. Aspiration must be done carefully so as not to cause excess haemorrhage. Once blood appears in the syringe stop aspirating. It is important to aspirate from several sites in the mass so as to acquire a representative sample. The material, if ample, can be dropped on a slide after removing the needle from the syringe and smeared as for a peripheral blood smear or squashed between 2 slides. Occasionally, little material is obtained via aspiration and most is in the hub of the needle. To obtain a smear from this situation either: (a) disengage the needle and fill the syringe with air. Reconnect the needle and blow the contents onto the end of a slide. A smear is then made as for a peripheral blood smear or as for a squash preparation. (b) Disengage the needle and aspirate some saline in the syringe. The saline can be used to remove the contents from the needle which can then be smeared. The disadvantage of this method is that the contents are diluted. *A CYTOLOGICAL SMEAR SHOULD BE A MONOLAYER AND HAVE MINIMAL DAMAGE TO THE CELLS IF IT IS TO BE DIAGNOSTIC. 

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CYTOPATHOLOGY – suggested approach to diagnosis when examining a solid tissue cytological preparation 1. Detect 2. Describe 3. Deduce – general to the specific  Detect, Describe (select an area that is a monolayer with minimal damage; avoid thick areas) • Low power (4x, 10x and 20x objectives) more for scanning to detect the right area; little

description o Areas of cellularity – are they different in appearance, if so, note down? o Acellular areas? o Unusual colours? o Blood contamination?

• High Power (100x oil objective; 40x objective can only be used with coverslip) for most description o Appearance of constituent cells (nuclear and cytoplasmic detail) o Appearance of any inflammatory or abnormal cells (beware of artefactual changes

to damaged cells) o Appearance of any acellular material o Can you detect agents of disease? o Is there foreign material present?

Deduce (on the basis of description and clinical information) • What basic pathological processes are occurring?

o Degeneration and necrosis o Inflammation o Disorder of growth (e.g. neoplasia) o Vascular disturbance (usually impossible to determine and included under

degeneration) o Pigments/deposits

The first three are most important. Often there is more than one basic pathological process occurring so: • Which pathological process is most important (basis for first part of morphological

diagnosis)? If it is degeneration/necrosis then further questions are: • Is it a primary lesion (cyst, haemorrhage)? • Could it be related to any inflammation present? • Could it be secondary to a disorder of growth?

If it is inflammatory then further questions are:

• Is it acute (active), chronic or both (cell types determine this)? If chronic, is it granulomatous?

• Can you detect a cause? o Physical (e.g. foreign material) o Pathogen o Chemical (unlikely to detect cytologically, but history may suggest) o Immune-mediated? o Genetic (history might suggest predisposition)

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If it is a disorder of growth then further questions are: • What cell type is it? Round, epithelial or spindle? • Is it hyperplasia/benign neoplasia? • Is it malignant (on cellular detail and clinical information)? • Cause is debatable, but familial predisposition (genetic), chemical, physical and

pathogens may be considered on all information

2 FLUID CYTOLOGY (CSF, synovial, body cavity effusions, tracheal aspirate, fluid masses) The material is usually aspirated if the fluid has a high content of cells, smears can be made directly from the fluid; if the cells are low to moderate in content, the fluid* should be first centrifuged (400-500G) and the plug of cells resuspended in a small amount of fluid. If cell counts are to be performed, the latter method is done after those procedures. For both, it is best to smear as for a peripheral blood smear after some of the fluid has been taken up in a microhaematocrit tube. * THE FLUID SHOULD BE PLACED IN EDTA TO PREVENT POSSIBLE CLOTTING  NB. Cells in fluid deteriorate rapidly; therefore smears have to be made quickly. *ALL CYTOLOGICAL PREPARATIONS HAVE TO BE FIXED AND STAINED.  IF THE SOLUTIONS ARE TO BE METHANOL AND GIEMSA THEN THE SLIDES SHOULD BE RAPIDLY AIR DRIED FIRST (LIKEWISE FOR "DIFF QUIK"). 

3 VAGINAL CYTOLOGY

Samples are obtained from the anterior vagina either using a cotton swab stick or blunt spatula. If a cotton swab stick has been used it is gently rolled over the surface of a cleaned slide and the smear fixed and stained either by the Giemsa method or with Shorr stain. For the dog, the following characteristics are noticed during the different stages of the oestrous cycle.

1. Anoestrus - Non-keratinized round epithelial cells (parabasal and some intermediate). Minimal debris. Neutrophils may be present but less than in dioestrus. Occasional erythrocytes.

2. Pro-oestrus - keratinized epithelia begin to appear (superficial cells with or without nuclei). Neutrophils decrease. Erythrocytes usually present. Debris and bacteria present.

3. Oestrus - nearly all epithelial cells are superficial, keratinized. Debris minimal. Neutrophils are absent until the final one or two days. Erythrocytes are absent after the first couple of days.

4. Dioestrus (metoestrus) - abundance of neutrophils. Reappearance of round non-keratinized cells (parabasal and intermediate cells).

For the cat, epithelial cell types are similar to those seen in the dog. In the follicular stage (pro-oestrus and oestrus) there is a gradual increase in the proportion of anucleated superficial cells (keratinized) and a drop in intermediate and parabasal cells. Erythrocytes may be seen. In the luteal phase (dioestrus), anucleated superficial cells disappear and intermediate and parabasal cells increase. In anoestrus intermediate and parabasal cells predominate. Neutrophils may be seen.

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4 SEMEN ANALYSIS

Fertility examination of the male involves semen examination. The results of semen examination vary with the method of collection. Basically, semen examination aims to evaluate the concentration, morphology and viability of the spermatozoa. Seminal fluid as such is of secondary importance. Despite species differences, laboratory methods of semen analysis are standardised and include the following:

a. Gross examination - volume, colour, density, foreign material.

i. Volume. Graduated collecting vessels should be used.

ii. Colour, density. Semen varies from watery white to milky to creamy depending on the concentration of spermatozoa (e.g. creamy is equivalent to greater than 2 million per l). Certain bulls deliver semen of yellowish-green colour.

iii. Foreign material. Semen should contain only spermatozoa and occasional leukocytes.

b. Microscopic examination.

i. Wave motion. This refers to mass activity of the sperm and is a reflection of both the concentration and motility of sperm. Dog sperm has no or limited wave motion.

ii. Motility. The percentage of sperm motile is assessed on a warmed stage of a microscope.

iii. Live-dead differentiation. This is done by using selective stains such as eosin-nigrosin.

iv. Spermatozoal morphology. Generally in semen with adequate concentration and motility, the majority of spermatozoa will have normal morphology. Certain abnormalities will appear in a proportion of spermatozoa even in normal semen. Consequently, it is important to recognise not only the type of abnormalities but also the number of spermatozoa affected.

Primary abnormalities reflect disturbances of spermatozoal production and include head abnormalities, mid-piece abnormalities and coiled tails.

Secondary abnormalities reflect disturbances during storage of the spermatozoa in the epididymis or during the process of ejaculation. These include loose normal heads (i.e. tail-less heads), detachment of Galea Capitus, proximal droplets and bent tails.

c. Spermatozoa concentration. This is done by direct cell counting in a chamber.

TABLE:  Minimum standards for semen 

  Volume (mls) 

Concentration (μl) 

Motility  Primary  Secondary abnormalities 

Bull  2  50,000  >50%  <15%  <15% Stallion  50‐100  10,000  >50%  <15%  <6% Boar  100  10,000  >50%  <15%  NA* Ram  1  100,000  >50%  <10%  NA Dog  5  10,000  >50%  <15%  NA 

*Not Available 

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FAECA L ANA LYSIS

1 INTRODUCTION

Faeces, although unpleasant to examine, can provide important information about digestive tract disorders. To obtain the maximum amount of information from faeces, examination of a fresh stool is essential, especially as certain chemical characteristics may change with aging. Rectal samples may be necessary for certain parasitological tests. All tests should be interpreted in light of the diet of the animal. The technique of faecal examination employed in the Veterinary Clinical Pathology Laboratory at The University of Sydney involves the following:

2 GROSS EXAMINATION

Note the amount, colour and odour of faeces. Examine for worms, blood and unusual foreign material.

3 MICROSCOPIC EXAMINATION

a. Examination for Parasites (Metazoan parasites and Protozoa). (See Parasitology course).

b. A faecal smear can be stained with Giemsa /Diff Quik and examined for the

presence of cells, microorganisms, etc. For instance, the presence of many neutrophils may indicate active inflammation of the colon or rectum.

c. Chemical examination (The values presented are applicable to the dog and probably

to the cat). Add a small amount of faeces, about as big as a match-head, to each of 4 slides and emulsify with a drop of saline (not necessary if the stool is liquid).

(i) for starch: Add a few drops of Lugol's iodine to the emulsified faeces on one slide and mix. Examine under low power (10X objective) for blue-black starch granules, with the condensor fully open. Greater than 3 granules per field (count a few scattered fields and determine the average) is considered abnormal but normal dogs and cats can show higher numbers at times.

(ii) for protein (muscle fibres):

Add a few drops of 1% alcoholic eosin to the emulsified faeces on one slide and mix. Examine first under low power (10X objective) with the condensor slightly dropped and closed to find the fibres and then use high power (40X objective) for the presence of muscle with visible cross striations.

NB. Lugol's iodine will stain undigested muscle fibres.

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(iii) for fat: Fat in the faeces may be in the form of neutral fats, fatty acids and soaps (the last two are called split fats). To determine the component proportions, add a few drops of Sudan IIIA (alcoholic stain) to the emulsified faeces on one slide, and a few drops of Sudan IIIB (acetic acid stain) to the emulsified faeces on the other slide, and mix.

Sudan IIIA will stain the neutral fats, which are in the form of droplets and plaques, an orange-red. Fatty acids, which are in the form of droplets, plaques and needles, and soaps, which are in the form of plaques, needles and crescents, are colourless with Sudan IIIA. Normal faeces have less than 10 droplets, smaller than 10 μm, per high powered field (40 x objective). NB. This test is inconsistent.

On heating the Sudan IIIB faecal mixture (warm the slide over a bunsen burner until it is just too hot to touch), much of the fat will melt and stain as deep orange globules, i.e. both the neutral fat and free fatty acids will stain. The soaps are melted on heating because of the presence of the acetic acid, but do not stain.

When the slide cools the orange droplets of fatty acids transform to needles and spicules. Colourless droplets of soaps also transform into needles and spicules. Neutral fat droplets remain unchanged.

In normal faeces less than 10 droplets, smaller than 20 μm, are usually visible per high powered field. 0.5% Nile Blue is another stain that can be used for faecal fat. It stains droplets and plaques of neutral fat red and plaques of fatty acids blue-violet (all the rest are colourless).

Although the results are not always easy to interpret, generally (at least for the dog) fat maldigestion leads to a greater increase in neutral fats rather than split fats, while fat malabsorption increases split fats more than neutral fats. Ideally, the results should be supported by fat absorption tests.

Results expressed as:

Starch Protein Fat-unsplit Fat-split Normal <3 r/wd <10 droplets

of <10μm <10 droplets of<20μm

+ ++ +++ ++++

r = rounded wd = well digested

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4 HEMATEST - FOR OCCULT BLOOD

Will react with myoglobin, haemoglobin or dietary substances having peroxidase-like activity. This is not a very good test and may be later replaced.

5 EXAMINATION FOR TRYPSIN (PROTEASE)

Diseases of the exocrine portion of the pancreas may cause a reduction in the output of trypsin into the faeces. Little or no trypsin in the faeces mean severe exocrine pancreatic deficiency. However, normal animals may have low trypsin on single sampling; multiple samples are, therefore, sometimes necessary to confirm a case of exocrine pancreatic insufficiency. A simple screening test that can be employed for the detection of trypsin involves the use of x-ray film (a gelatin digestion test). One gram of well-mixed faeces is emulsified with 9 ml of 1% sodium carbonate in a centrifuge tube to give a 1/10 dilution. From the 1/10 dilution, after it has settled, 1/20, 1/100, 1/200, 1/1000, 1/2000 dilutions are prepared. A square of x-ray film is divided into smaller squares and 1 drop of sodium carbonate (blank) and 1 drop of each dilution of faeces are placed in separate squares. An alternative method is the placement of strips of x-ray film directly into tubes containing the different dilutions. The x-ray film is incubated at 37ºC for 30-60 minutes (or room temperature for 2-4 hrs). The x-ray film is allowed to harden (place in a refrigerator for 5 minutes) and washed with a gentle stream of water. The titre of trypsin is designated as the highest dilution showing complete digestion of the gelatin (i.e. when the blue celluloid backing becomes totally visible). NB. instead of a square of x-ray film, individual strips can be substituted and immersed in the tubes containing the dilutions of faeces - 'tube gelatin digestion test'. Interpretation: The titre of trypsin should be interpreted in light of the findings of the microscopic examination for undigested food particles. A titre of 1/100 or greater with minimal undigested food particles suggests that the animal does not have exocrine pancreatic insufficiency. A titre of less than 1/100 with abnormal numbers of undigested food particles (starch/muscle/neutral fats) is suggestive of exocrine pancreatic insufficiency. A titre of less than 1/100 but with minimal undigested food particles is inconclusive, and the faeces should be retested at least three times before making a decision. NB. A normal dog can have a trypsin titre from 1/20 to 1/2000 (may be lower). More sophisticated tests for the detection of faecal trypsin are available but restricted to laboratories. In addition, for the dog and cat, tests for the presence of trypsin-like immunoreactivity in the serum are available in laboratories, and which do not have the limitations of trypsin.

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6 FAECAL STAIN

Lugols Iodine Iodine crystals 1.0 gm Potassium iodide 2.0 gm Distilled water 100 mls Grind solids together then dissolve in water. Eosin Eosin yellow (alcohol & water sol.) 0.5 gm 70 % ethanol 100 mls Dissolve stain, then filter. Sudan lllA (alcoholic) Sudan 111 about 0.5 gm 95 % ethanol 45 mls Heat in a water bath (not over flame) to 30-35ºC to dissolve stain then filter. Sudan lllB (acetic acid sol.) Sudan 111 0.2 gm 95 % ethanol 10 mls Acetic acid(glacial) 90 mls Mix then filter

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URINARY CALCULI

1 INTRODUCTION

Calculi are solid concretions formed within ducts and hollow organs. Their physical presence may cause obstruction to the flow of secretions or excretions. There are several types of calculi; urinary, gall, intestinal, salivary and pancreatic. All/most are comprised of substances that are normally excreted. There are 3 main groups: 1) Simple - single constituent. 2) Mixed - two or more constituents - most common type. 3) Foreign body - rare usually start the formation i.e. nucleus such as fibrin. Urinary calculi (uroliths) may occur in the renal pelvis, ureter, bladder and urethra. They usually consist of an organic matrix and minerals, and if there is more than one main constituent they are referred to as mixed calculi. The type of constituent in a calculus is dependent upon 2 main factors: i) The species: urinary calculi in the dog and cat are commonly composed of

MgNH4PO4.6H2O (MAP; struvite; "triple phosphate"). The horse has urinary calculi commonly composed of calcium carbonate with magnesium phosphate as a minor constituent.

ii) The pH of the urine: Certain types of calculi are more likely to occur in acid or alkaline

urine, principally due to the effect on solubility of their constituents. However, some calculi occur in both acid and alkaline urine (e.g. struvite calculi).

            Urinary calculi more likely to be found in: 

Species  Acid Urine  Alkaline Urine 

Dog  Oxalate  Calcium Carbonate & Phosphate 

  Cystine  Struvite (MAP) 

  Uric Acid/Urates  Ammonium Urate 

Cat  Cystine  Calcium Carbonate & Phosphate 

  Oxalate  Struvite (MAP) 

  Uric Acid  Other Phosphates 

Horse  Silica  Magnesium Phosphate 

  (Silicon Dioxide)  Calcium Carbonate & Phosphate 

    Struvite (MAP) 

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Other factors that are involved with urinary calculus formation include: i) Increased urinary output of one or more of the insoluble constituents For example, in the dog phosphate stones may be associated with elevated excretion of calcium. Cystinuria or increased urinary levels of urates (especially in the Dalmation where there is an inherent breed failure to resorb urates present in the glomerular filtrate) may lead to calculus formation. However, concentrations of minerals in excess of their solubility don't always mean stone formation. Although all the reasons for this are not clear, it may be due in part to the colloidal properties of urinary proteins and polysaccharides stabilising large concentrations of minerals in solution and/or due to the presence of substances that form soluble complexes with the insoluble constituents of calculi, e.g. citrate stabilising calcium ions, thereby interfering with the formation of calcium phosphate/carbonate calculi. Hyperparathyroidism may cause enhanced deposition of calcium. ii) Infection Bacterial degradation, principally of urea, leads to elevation of urinary pH. iii) Urinary stasis Urinary stasis allows bacterial growth and the retention of substances that could act as nuclei for stone formation. Both ii) and iii) operate when there is interference to urine outflow. iv) Reduced water intake Means concentrated urine in a healthy animal i.e. a supersaturated state is reached. This is common with calcium oxalate but a nucleus is still required. v) Nutrition Ruminant urolithiasis is thought to be largely nutritional in origin. Vitamin A deficiency has been implicated in urate calculi in Dalmations. Not all the factors involved with urinary calculus formation are known. And it is quite obvious that those factors which are recognised, are not fully understood. Most probably, urinary calculus formation is dependent on a combination of factors rather than any single one.

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2 PRACTICAL EXAMINATION OF CALCULI

The constituents of urinary calculi can be determined using simple methods. The results may be useful in treating the condition, e.g. acidifying the urine for struvite (MAP) calculi.

a) Physical examination i) Shape and form of urinary calculi

With distilled water, wash the calculus free of debris. Dry and examine.

Many bladder calculi have characteristic physical appearances depending on their constituents but there is overlap, therefore, other types of analyses may be necessary at times, e.g. heat, chemical analysis. Chemical analysis is always necessary for mixed calculi.

Phosphate Calculi

May be:

(i) a large single calculus with a coral like surface e.g. struvite, calcium phosphate or a mixture of phosphates.

(ii) numerous, variably sized, tetrahedral, smooth stones e.g. struvite. NB. Struvite (MAP) calculi in the cat are usually in the form of "sand",

microcalculi.

(iii) small, spherical stones which may be impossible to distinguish from cystine stones by naked eye examination.

Phosphate calculi are usually white or cream and have a chalk-like appearance when broken i.e. the softest when broken. Phosphate calculi commonly occur in alkaline pH when there is infection of the urinary tract.

Cystine Calculi

These are usually small, spherical, cream to yellow, smooth stones and waxy in appearance. Occasionally they may be brownish yellow to light green.

Oxalate Calculi

These are usually small. The dihydrate form of calcium oxalate calculi may have large shelf-like projections from its surface. Because of this rough surface, the calculi may be blood stained. Other forms of oxalate usually produce smooth calculi.

Oxalate stones are variable in colour, may be brittle and have obvious crystals when crushed. Calcium oxalate is the hardest stone and therefore is not so easily crushed. It is usually the chief constituent of the small stones from the renal pelvis.

Urate Calculi

These are usually in the form of grey to yellow, highly polished spherical stones which when broken consist of thin concentric crystal layers.

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Carbonate Calculi

Carbonate calculi are dull white to red-brown, chalky and usually hard. They often occur as large, spherical to ovoid calculi, having a coral-like surface.

ii) Heat

The calculus is split and checked to see if it is homogeneous. If different layers are apparent all should be analysed.

Some of the stone is ground using a mortar and pestle. The ground stone is then burnt in a flame. Organic elements will tend to burn and disappear. Inorganic elements will remain. NB. nearly all calculi have some organic components.

If there is some smoke and a "burnt protein" (sulphurous) smell, the calculus is probably composed of cystine. If there is a tongue-like flame and a small residue, it could be oxalate. If there is an ammonia smell and a small residue, it is probably composed or urates. MAP (struvite) calculi burn very little and usually leave a grey fused mass.

The organic residues may be used for chemical analysis but as certain inorganic compounds may change form during heating, it is probably best to use freshly ground stone.

b) Chemical Analysis (Based on the Merck Urinary Calculus Analysis)

i) Grind sample of calculus very finely and mix well.

ii) Transfer a full white spatula to the red boat; add 5 drops of Reagent 1 (Conc. Sulphuric Acid) and stir to achieve complete dissolution.

iii) Foaming indicates carbonate.

iv) Transfer sample solution in the boat into the plastic graduated beaker, which is filled to one third with distilled water. Make up to 50 mls with distilled water, and stir several times with the boat.

v) To carry out individual analyses, fill the reaction vessels with sample solution up to the 5ml calibration mark (except for magnesium which requires only 1ml of sample solution).

vi) Calcium (5ml).

Add 2 drops of Reagent 2 (27% Sodium Hydroxide) while shaking. Then add 1 white spatula full of Reagent 3 (Calcon carboxylic acid) and add Reagent 4 (Titriplex III solution) drop by drop until the colour of the solution changes from red to blue.

Count the drops required: the number of drops required, multiplied by 5, gives the percentage of calcium content.

vii) Oxalate (5ml).

Add while shaking, 2 drops of Reagent 5 (Borate Buffer solution), 3 drops of Reagent 6 (Ferric Chloride solution), and 3 drops of Reagent 7 (Sulphosalicylic acid).

After 2 minutes, carry out colour comparison on the colour chart.

viii) Ammonium (5ml).

Add, while shaking, 3 drops of Reagent 8, (Potassium mercury iodide), and 3 drops of Reagent 2 (27% Sodium Hydroxide). Carry out colour comparison on the colour chart.

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ix) Phosphate (5ml).

Add, while shaking, 5 drops of Reagent 9 (Ammonium molybdate) and 5 drops of Reagent 10 (Reduction solution).

After 5 minutes carry out colour comparison on the colour chart.

x) Magnesium (1ml).

Pipette 1 ml of sample solution into a reaction vessel and make up to the 5ml calibration mark with distilled water. Add, while shaking, 10 drops of Reagent 11 (Buffer solution) and 10 drops of Reagent 12 (Dye solution).

After 1 minute, carry out colour comparison on the colour chart.

xi) Uric Acid (5ml).

Add 3 drops of Reagent 13 (Molybdate phosphoric acid), shake and allow to stand for 2 minutes; then add 2 drops of Reagent 5 (Borate buffer solution) and shake.

Within 10 seconds, carry out colour comparison.

xii) Cystine (5ml).

Add, while shaking, 10 drops of Reagent 14 (Ammonia solution) and 1 red dosing-spoonful of Reagent 15 (Reducing agent). After 1 minute, add 1 black dosing-spoonful of Reagent 16 (Sodium nitroprusside) and shake.

After 30 seconds, carry out colour comparison on the colour chart.

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Veterinary Historical Notes

Arthur William Turner OBE DVSc DSc 1900-1989

Arthur Turner was born, educated and worked for most of his career in scientific research in Melbourne. He graduated BVSc (Hons) in 1923 at the University of Melbourne where he was appointed to the teaching staff of the Veterinary school. During that year he qualified for the higher degree of MVSc for research built on the work of Dr HE Albiston on infectious necrotic hepatitis of sheep (black disease).

In 1924, he became Walter and Eliza Hall Fellow in Veterinary Science. He was awarded a Rockefeller Foundation Fellowship in 1926 and studied at the Pasteur Institute in Paris. There he received training and inspiration such as Dr JA Gilruth had received at that Institute at a similar age. In Paris, he continued his studies on black disease and ultimately developed an effective vaccine for the disease, which was responsible for heavy losses of sheep in eastern Victoria and southern New South Wales. In 1928, before returning to Australia to accept an invitation to join the staff of the newly-formed Animal Health Division of the Council for Scientific Research and Industry, he spent a period at the Institute of Animal Pathology at the University of Cambridge.

On his return, Gilruth, who was Chief of the division of Animal Health, appointed Turner as Officer in Charge of the new laboratory at Oonoonba, Queensland. Here he was introduced to an environment where contagious bovine pleuropneumonia (CBPP) was enzootic and where nutritional and protozoan diseases of cattle required investigation. He established the foundation for a lifetime interest in CBPP and his studies on the causal organism, serological diagnosis, vaccination and the general pathology of the disease were invaluable in this country and overseas. In 1930, Turner was awarded the degree of DVSc for his outstanding research on black disease by the University of Melbourne, where he was subsequently awarded the degree of DSc.

Returning to Melbourne in 1935, where Dr LB Bull had succeeded Gilruth as Chief of the Division of Animal Health of CSIRO was headed by Dr LB Bull at Parkville, Turner was appointed Officer in Charge of the Parkville laboratory. Always irked by the interruption of his research by administrative matters, Turner transferred the position to Dr TS Gregory and, until his retirement in 1960, spent his time on his first love, research. He earned a unique place in the estimation of veterinarians throughout Commonwealth for his achievements.

He was a Foundation Fellow of the Australian Academy of Science and a Foundation Life Fellow of the Australian College of Veterinary Scientists. He was awarded the Gilruth Prize and the Syme Prize, and was a Fellow of the AVA.

RI Taylor March 2002 References Albiston HE. Obituary. Aust Vet Assoc News, 6 June 1990, p N223. Citation for the Gilruth Prize Aust Vet J 1958;34:262-264. Schedvin CB Shaping science and industry, a history of Australia's Council for CSIR 1926-1949. Allen & Unwin, Sydney. 1989.pp 137-139, 141-142.

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