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PREPARE AND EXAMINE BLOOD FILMS

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Many practices have in-house haematology analysers. However, these should always be used in

conjunction with a blood film examination since analysers are not always accurate. In particular, the white blood cell differential count is not always reliable nor is the platelet count always correct, especially in cats which have relatively small red blood cells and quite large platelets – most analysers differentiate these cell types by their size. Furthermore, platelet clumping also falsely lowers the platelet count.

Blood film examination will detect these errors and provides a lot of information which is not available from the analyser. For white blood cells: a left shift, toxic neutrophils, reactive lymphocytes and neoplastic cells can all be identified. When assessing anaemic patients, examination of

Elizabeth Villiers, clinical pathologist at Dick White Referrals describes this simple and rewarding in-house diagnostic technique

red blood cell morphology is vital, both to distinguish between regenerative and non-regenerative anaemia and sometimes to identify the cause of the anaemia (e.g. spherocytes in immune-mediated haemolytic anaemia). Finally, by counting platelets seen on the blood film, the analyser count can be verified or corrected.

Making a blood filmPolished glass slides with frosted ends are preferable since pencil can be used for easy labelling. Slides should be handled by their edges/ends since grease from fingers can result in poor smearing. If in doubt the slide can be wiped clean using a tissue before use. Blood sample smears should be prepared soon after taking the blood sample. The blood should be mixed by gently inverting the sample several times.

A spreader slide is required to make ■■

the smear: this is narrower than the smear slide to avoid spreading the cells over the edge of the slide. Spreader slides can be made by breaking off a corner of a normal slide having first scored it with a blade or diamond writer. Spreader slides should be washed in water and dried regularly,

and should be replaced periodically as the edge can become roughened.The blood sample is mixed carefully and ■■

then a sample harvested using a micro haematocrit tube. A drop of blood is placed on to the slide towards the opposite end to the frosted area (Figure 1).The spreader slide is held between ■■

the thumb and second finger, placing the index finger on top of the spreader slide (Figure 2). This helps to apply even pressure on the spreader slide when smearing.The spreader slide is placed at an angle ■■

of about 30° in front of the blood spot and slid backwards until it comes into contact with the blood, which then spreads out along the width of the spreader slide (Figure 3).The spreader slide is advanced forwards ■■

smoothly and quickly. The spreader slide should not be lifted up from the smear slide until after the feathered edge has formed. Ideally the smear should extend approximately 2/3 of the length of the slide, and should a have fairly square feathered edge (Figure 4).The smear is allowed to dry.■■

Figure 1 Figure 2 Figure 3 Figure 4

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Canine red blood cells have a pale area in the centre of the cells (central pallor) which is not obvious in feline red blood cells, which are smaller. If the animal is anaemic, blood film examination helps determine if the anaemia is regenerative.

Features of regenerationPolychromasia – these are young red ■■

blood cells which stain this purple–lilac colour due to the presence of ribosomes in the cytoplasm (Figure 10). Occasional polychromatophils (one every 2–4 1000X field) are seen in films from healthy animals. Several per field would indicate a robust regenerative response.Anisocytosis with large (young) red ■■

blood cells.Howell-Jolly bodies – these are small ■■

but prominent dark inclusions which are remnants of nuclear material (Figure 11).

Figure 8: A large platelet clump seen at the feathered edge of a blood film from a dog

Featherededge

Examination area

Figure 9: Diagrammatic representation of a blood film showing the area of the blood film in which cell examination should be performed

Figure 5: Trouble-shooting poor quality blood smears

Problem Cause Solution

Smear too long (feathered edge has disappeared off the end)

Spreader slide speed too slowLow viscosity blood (i.e. anaemia)

More rapid spreader slide speed

Excess blood applied to slide Apply smaller blood spot

Smear too short/thick

Spreader slide speed too fastHigh viscosity blood (i.e. high PCV)

Slower spreader slide speed

Feathered edge consists of long streaked tails (Figure 6a)

Uneven contact of spreader slide with smear slide

Apply even pressure using index finger on top of spreader slide

Spreader slide has roughened edge Replace spreader slide

Smear has holes or gaps (Figure 6b)

Grease on slideLipaemia

Clean slide before use

Smear thick at feathered edge end of film (Figure 6c)

Blood in front of spreader slide Ensure firm contact between spreader slide and smear slide when pulling spreader slide backwards towards blood spot

StainsRowmanowsky stains include Wrights-Giemsa and rapid dunking kits (Diff-Quik kits). The latter are more than adequate for in-house use (Figure 7). These kits have a three-stage staining procedure, which incorporates a fixative pot (usually 5 dips), an orange–pink dye in the second pot (usually 3 dips), and a blue–purple dye in the third pot (usually 6 dips). The stain is stored in glass jars which should be cleaned regularly to avoid build up of stain precipitate. Periodically they should be scrubbed out using methanol to remove all stain. Stain containing precipitate can be

filtered or discarded and replaced with fresh stain. With time, the red–orange dye can be inadvertently carried over into the blue–purple dye resulting in weak staining. The only solution to this problem is to replace the blue–purple dye.

CoverslipsIt is helpful to add a coverslip to preserve the slide and to improve visual acuity, especially when using the dry 40X lens. Mounting medium glue such as ‘DPX’ can be used to permanently mount the slide. NB if the glue spreads out beyond the edge of coverslip great care should be taken to avoid getting it on the microscope lenses. Preferably, wait for it to set.

Alternatively, immersion oil can be used. A drop of the mounting medium/oil is placed on the slide near the feathered edge and the coverslip is slowly lowered on to the slide.

Film examinationTo ensure all cell lines are examined properly, a set procedure of blood film examination should be routinely followed. Initially, the smear is checked for large platelet clumps (Figure 8) by examining the feathered edge at low power (20X). The smear is then examined at higher power (40X or 100X) in the thin area in from the feathered edge, where the cells are evenly distributed in a monolayer (Figure 9). The red blood cells, white blood cells and platelets are examined in turn.

Examination of red cellsThis should include an assessment of colour, size and shape, and a search for inclusions.

PREPARE AND EXAMINE BLOOD FILMS

Figure 10: Blood film from an anaemic dog showing marked polychromasia (arrowed)

Figure 7: A rapid dunking kit. The stains are stored in clean glass or plastic pots with lids

Figure 6: Slide e is a good quality smear

a b c d e

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Figure 12: Spherocytes are smaller and denser than normal cells and lack central pallor

PREPARE AND EXAMINE BLOOD FILMS

Nucleated red blood cells – do not ■■

confuse these with lymphocytes. Nucleated red blood cells have smaller nuclei and the cytoplasm is a similar colour to polychromatic red blood cells (Figure 11).

Evaluation for a possible cause of anaemiaThe blood film can help identify several causes of anaemia, including immune-mediated haemolytic anaemia, babesiosis, feline infectious anaemia, oxidant injury, microangiopathic haemolytic anaemia and iron deficiency.

Immune-mediated haemolytic anaemiaThis disease usually leads to circulating spherocytes. These appear smaller than normal red blood cells with a darker/denser cytoplasm lacking central pallor (Figure 12). Care should be taken to look in the examination area monolayer and avoid looking at the tail of the smear where cells are flattened and lose their normal central pallor, giving the false impression of spherocytes. Spherocytes are difficult to see in cat blood because normal feline red blood cells have minimal or no central pallor. NB small numbers of spherocytes may be seen with other causes of anaemia.

BabesiosisBabesia canis appear as large pear-shaped organisms, usually in pairs (Figure 13). B. gibsoni organisms are much smaller, circular bodies. These parasites are more often seen in capillary blood (e.g. from an ear prick) and most frequently found along the edges of films. However, these organisms are not always visualized and serology or PCR are more sensitive methods for diagnosis.

Figure 13: B. canis organisms are light-staining pear-shaped bodies, usually in pairs

Figure 17: Hypochromic red blood cells with wide central pallor and a thin rim of haemoglobin

Feline infectious anaemia (haemotropic mycoplasma infection)Provided that samples are obtained during an episode of parasitaemia, organisms may be identified using the Rowmanowsky stains. The organisms stain blue–grey to pale purple with Rowmanowsky stains and appear as small cocci singly or in chains. Again PCR is a more sensitive diagnostic technique.

Oxidant injuryIngestion of oxidants such as onions or zinc can lead to the formation of Heinz bodies or eccentrocytes with resultant anaemia. Heinz bodies are seen as non-staining round bodies, usually protruding from the surface of the cell (Figure 14). Eccentrocytes have a pale area on one side of the cell which is devoid of haemoglobin, but with a cell membrane visible around this pale area (Figure 15).

Figure 16: Schistocytes are irregularly shaped red blood cell fragments (black arrow). Acanthocytes are roundish but have several irregular projections (white arrow)

Figure 14: Heinz bodies are round non-staining or light-staining bodies adjacent to the cell membrane or protruding from it

Figure 11: A Howell-Jolly body (black arrow) and a nucleated red blood cell (white arrow) Figure 15: Eccentrocytes have a

clear area on one side of the cell encased in the cell membrane

Microangiopathic haemolytic anaemiaRed blood cell fragmentation, e.g. due to vascular neoplasms such as haemangiosarcoma, results in the formation of schistocytes and acanthocytes (Figure 16). Schistocytes are irregular-shaped, often with elongated fragments and an irregular spiked outline. Acanthocytes also have irregular spiky projections but are roundish in shape.

Iron deficiency anaemiaThis leads to defective haemoglobin synthesis, and so red blood cells are very pale with a wide area of central pallor and a thin rim of haemoglobin (Figure 17).

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PREPARE AND EXAMINE BLOOD FILMS

Examination of leucocytesThe morphological examination and differential count are important to validate (or refute) the analyser differential count, to identify a left shift and/or toxic changes (which are important indicators of inflammation) and to identify atypical, possibly neoplastic cells. It is essential to evaluate a blood film prior to administration of chemotherapy, rather than relying on the neutrophil count produced by the in-house analyser.

A differential white blood cell count is performed by counting leucocytes at both the edges and in the middle of the smear in the examination area because larger cells tend to be pushed to the edges of the smear, and smaller cells tend to be more concentrated in the middle. An example of a battlement meander method of counting is shown in Figure 18. The percentage of each cell type is then multiplied by the total white blood cell count to determine an absolute count for each cell type. Cell counters are useful to speed up this process (Figure 19).

Figure 20: A neutrophil with segmented nucleus and pale blue–grey cytoplasm

Figure 21: A band neutrophil with a U-shaped nucleus with parallel sides

Figure 19: A cell counter used for performing a differential leucocyte count. Each cell type is assigned a button on the counter. A bell is sounded when 100 cells have been counted

Figure 23: A toxic neutrophil with darker blue (basophilic) cytoplasm and small indistinct cytoplasmic granules

Figure 24: A toxic neutrophil with foamy cytoplasm

Figure 25: A small lymphocyte with a round nucleus and scant cytoplasm visible only on one side of the cell

Figure 18: Battlement meander track for performing a white blood cell differential count

Figure 22: A Döhle body (indistinct blue–grey cytoplasmic inclusion) in a neutrophil (arrowed)

NeutrophilsNeutrophils have an elongated, segmented nucleus and light blue–grey cytoplasm without visible granules (Figure 20). Band neutrophils should be counted separately. These have a U-shaped non-lobulated nucleus with parallel sides (Figure 21).

Shallow indentations <50% of the width of the nucleus may be present. Bands indicate acute inflammation.

Toxic changes in neutrophils and bands are seen in severe inflammation, especially associated with bacterial infection. Toxic changes are listed below:

Döhle bodies – indistinct blue–grey ■■

cytoplasmic inclusions (Figure 22)Increased cytoplasmic basophilia (but ■■

not as dark as monocytes) (Figure 23)Foamy vacuolated cytoplasm (Figure 24)■■

Cytoplasmic granules (Figure 23)■■

Giant neutrophils■■

Nuclear swelling■■

Doughnut-shaped nuclei.■■

LymphocytesLymphocytes have a round nucleus with condensed, smudged chromatin and a narrow rim of basophilic cytoplasm. Lymphocytes vary in size but most are small, only slightly larger than canine red blood cells (Figure 25). They have sparse cytoplasm, which is not visible all the way round the nucleus. Medium-sized lymphocytes may approach the size of neutrophils and have more abundant cytoplasm, often completely encircling the nucleus (Figure 26). Reactive lymphocytes are larger still with a nucleus approximately 1.5 times the diameter of a canine red blood cell and abundant deeply basophilic cytoplasm, often with a darker tinge at the periphery. (Figure 27).

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Figure 28: A monocyte with a broad U-shaped nuceus and basophilic cytoplasm with clear vacuoles

Figure 29: A canine eosinophil with many round pink cytoplasmic granules. (These granules are rod-shaped in cats)

Figure 30: A canine basophil with dark purplish cytoplasm

Figure 31: A feline basophil with more abundant granules, which are grey–lavender in colour

Figure 32: Large atypical blast cells in a peripheral blood sample from a dog with acute leukaemia. The cells have large nuclei containing stippled chromatin and nucleoli. Cytoplasmic granules are seen in some cells

Figure 33: Platelets are small circular cells without a nucleus and with pink grainy cytoplasm

MonocytesMonocytes are larger than neutrophils and have abundant sky blue cytoplasm, often containing clear discrete vacuoles and sometimes fine pink dust-like granules. The shape of the nucleus is very variable and can be round, kidney bean-shaped, lobulated, U-shaped (Figure 28) or S-shaped.

PREPARE AND EXAMINE BLOOD FILMS

Figure 26: A medium-sized lymphocyte with a slightly larger nucleus and more abundant cytoplasm

Figure 27: A markedly reactive lymphocyte with an enlarged nucleus and more abundant cytoplasm, which is deeply basophilic

EosinophilsEosinophils are slightly larger than neutrophils, and are characterised by numerous prominent pink cytoplasmic granules (Figure 29).

BasophilsBasophils are rare in blood smears from normal animals. They are a similar size to eosinophils and have an elongated ‘ribbon-like’ segmented nucleus and variable numbers of cytoplasmic granules. In dogs these granules are sparse and dark purple (Figure 30), in cats they are abundant and pale lilac, sometimes with a few dark purple granules (Figure 31).

Examination of plateletsThese are small round structures with no nucleus. They are 1/4–1/2 the diameter of red blood cells with pink cytoplasm and fine granules. Platelet numbers can be estimated by counting the number of platelets seen in a 1000X field (i.e. 10X eye piece and 100X objective), having first determined that no platelet clumps are present. 5 fields are counted and a mean value is calculated. The normal count is 10–30 platelets per 1000X field (Figure 33). Each platelet per 1000X field equates to approximately 15 x 109/l. Thus if 10 platelets are seen per 1000X field, the platelet count is approximately 10 x 15 = 150 x 109/l. Animals with severe thrombocytopenia (< 30 x 109/l) have only 0–3 platelets per field. Large ‘shift’ platelets suggest active thrombopoiesis. ■

Atypical cellsAtypical cells suggest leukaemia or ‘overspill’ of lymphoma into the blood, especially when such cells are present in large numbers. Features which would suggest leukaemia include large cells with large round or convoluted nuclei, containing coarse or stippled chromatin and sometimes nucleoli (Figure 32). Suspicious cases should be reviewed by an experienced cytopathologist.

AcknowledgementsThe Figures in this article have been reproduced from the BSAVA Manual of Canine and Feline Clinical Pathology, 2nd edition.