Leukocytes WBC Morphology Clinical Pathology VTHT 2323 Lori VanValkenburg, RVT.
VTHT 2323 CLINICAL PATHOLOGY KRISTIN CANGA, RVT Thrombocytes and Coagulation.
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Transcript of VTHT 2323 CLINICAL PATHOLOGY KRISTIN CANGA, RVT Thrombocytes and Coagulation.
V T H T 2 3 2 3C L I N I C A L PAT H O L O G Y K R I S T I N C A N G A , RV T
Thrombocytes and Coagulation
Thrombocyte Formation
Production of platelets = ___________________________
Produced in bone marrow by ___________________________________(TPO) = stimuli for
PPSCs to differentiate into thrombocyte precursor.
Platelet parent cell = _________________________________
Thrombocyte Formation
Megakaryocyte undergoes incomplete ___________: (___________________ divides but cytoplasm doesn’t)
Small chunks (~1,000 – 5,000 per megakaryocyte) break off while still in bone marrow, and are sent into circulation as platelets
Some platelets are stored in ______________ for release as needed
Megakaryocytes: Platelet formation
Infoldings develop into plasma membrane that divide marginal _______________ into little compartments.
These compartments break off and enter bloodstream as ______________.
Some platelets are stored in the ___________, while others circulate freely in the blood and live for about ________ days in dogs and just over ___ day in cats.
Megakaryocyte Video
http://www.youtube.com/watch?v=6R-ESPFiKbo&feature=related&ajax=1&nocache=1271011451258
Thrombocytes
Commonly referred to as ________________.Not complete cells (lack a ____________), but
frequently listed as one of the cell types in peripheral blood.
RBCs>PLTs>WBCsHave a greater variety of
_________________than any of the true blood cells. Are responsible for _________________phase of clot
formation AND initiation of ________________phase of clot formation.
Thrombocyte Morphology
Most are _______________than RBCsMost PLTs in circulation are _____________ and
have numerous, small, purple/pink _____________ scattered throughout the cytoplasm.
Occasionally _________ platelets are seen in blood smear Giant platelets are considered more _______________
than smaller platelets
Platelets…
Play a part in both the __________________ and __________________ formation of clots.
Secrete ________________________Form platelet plugsSecrete ____________________Initiate dissolution of blood clots
(“_______________________”)Secrete _______________that attract neutrophils
and monocytes to sites of inflammationSecrete ______________ factors to help maintain
and repair blood vessels
Normal Platelet Values
Canine: 200,000 – 500,000 /µLFeline: 300,000 – 700,000 /µLAll species: ____________ of ____________/µL
Horses = ___________normal concentrationsCattle = ____________normal concentrations
Animals will bleed spontaneously if PLT concentration is ≤ ____________ to ____________ /µL
Function of Platelets
Platelets are essential for ________________.Role of platelets depends on ______________
numbers being present in the blood.There are 3 main functions of platelets:
1) Maintenance of __________________ integrity 2) _________________________ formation 3) ______________________ of plug by contributing to
______________ formation.
Function of Platelets: 1) Vascular Integrity
Platelets attach to _______________________Release endothelial ____________ factor into
endothelial cells. (Prevents leakage of blood in to tissues)
________________ or _______________may occur if there are __________________ numbers of platelets in circulation.
Platelets: 2) Plug Formation
Damaged blood vessel initiates the _________________ process of clot formation.
Platelet _________________The platelets adhere ______________ to the damaged vessel
AND each other.__________ often affects this step in the clotting process
Change shape and form ______________Allows platelets to intertwine with each other to create
platelet __________________.Platelet ______________________
The intertwining of platelets to help stop bleeding and causes the release of _________________ factors.
Initiates the _________________ Function of Platelets
Platelets: 2) Plug Formation, cont’d
Release of platelet factors (PFs) that are necessary for the clotting process to be complete. The aggregation of platelets _______________ the
release of PFsPlatelet _______________ occurs after aggregation
of platelets.This is the beginning of the _______________ phase
of clot formation.
Platelet Activation
Platelets become activated when there is _____________ to the lining of a blood vessel
The platelets are attracted to the damaged area and stick to it. Once the platelet has stuck to the damaged vessel, it
becomes activated.Activated platelets have a ______-like
appearance and form __________“tails” as they try and catch other platelets. Dendritic “tails” are sometimes referred to as
__________________________
Normal Activated Platelets
Platelets that have been slightly activated in the sample or by contact with the glass slide (as is common in feline samples) have a stellate form with dendritic processes ("a" in figure). The inset shows a large platelet with centrally aggregated granules which resemble a nucleus.
Platelet Function: 3) Stabilization of Platelet Plug
Often referred to as the “______ Matrix” or “Clotting __________”
Each step must happen in _________ and ________________ in order for the next step to occur. “____________ Reaction”
Converts soluble ______________ to insoluble _________ strands among platelets.
Acts as “scaffolding” to encourage ____________.
Hemostasis
Hemostasis is the process by which blood is prevented from leaking out of _____________ blood vessels.
Depends on three factors: _____________ of blood vessels Presence of adequate ___________________ factors Adequate number of normal circulating ______________.
___________ is a key player!!!1. Manufactures most clotting factors2. Bile = essential for _____________ of vitamin ___
Stages of Coagulation
______________ Hemostasis _________________________ Primary _______________ plug formation
Platelet _______________ Platelet ________________ Does adhesion or aggregation CAUSE platelet
activation?
Stages of Coagulation
_________________ Hemostasis _________________ Cascade
Ultimate goal = __________ for stabilization of platelet plug
Involves three pathways to clotting:__________________ Pathway, __________________ Pathway, and __________________ Pathway
Stages of Coagulation
Tertiary Hemostasis (________________) _________ retraction – occurs after ~___ minutes Platelet Derived ______________ Factor (PDGF) is
secreted during clot retraction. _____________ damage to all tissues involved
Tissue ____________________ Activator is secreted Clot initiates its own ____________________.
Coagulation Simplified
Extrinsic Clotting Mechanism• chemical outside of blood triggers
blood coagulation• triggered by thromboplastin (not found
in blood)• triggered when blood contacts
damaged tissue
Intrinsic Clotting Mechanism• chemical inside blood triggers blood
coagulation• triggered by Hageman factor (found
inside blood)• Triggered when blood contacts foreign
surface
Automated Hemostasis Testing
Samples should be collected very carefully with minimal ________________ damage.
___________ collect sample through indwelling catheters. Can cause ___________ or blow the vein through
manipulation.Anticoagulant of choice = Sodium citrate
Blocks calcium (but not as strongly as EDTA) Blue top tube (a.k.a – turquoise)
Results of some testing may be affected by stress, illness, recent exercise, heat cycle (females)
Clotting Tests
Assess one or more of the phases of ________________ (primary, secondary or tertiary)
Tests involving ______________ hemostasis assess intrinsic, extrinsic and/or common pathways.
All patients should undergo coagulation testing prior to undergoing a __________________ procedure.
Platelet estimation Buccal mucosal bleeding time Activated clotting time (ACT) Prothrombin time (PT) Partial thromboplastin time (PTT) Fibrinogen assay
Platelet Counting Methods
____________ or _______________ (least accurate)Most inaccuracies attributable to
_______________, giant platelets, RBC _____________Always use ___________ sample to minimize
errorManual methods:
1. Platelet estimation during blood film analysis Formula? ALWAYS USE HIGH POWER, _______________________
2. Unopette system & hemocytometer (NOT COMMONLY USED)
Buccal Mucosal Bleeding Time
Tests _____________ hemostasisEvaluates platelet __________ &
_____________(thrombocytopathy, thrombocytopenia)
Evaluates endothelial cell function (__________)
Test can be affected by certain ___________________
BMBT Procedure
1. Place _______________ animal in _____________ recumbency.
2. Use a strip of gauze to tie upper lip back and expose mucosal surface. (Also acts as ______________)
3. Using a Surgicutt® or a Simplate® lancet, create a small wound (~1 mm deep)
4. Remove blood with filter paper at 30-second intervals DO NOT TOUCH SKIN
5. Stop timing when there is no more blood.6. Normal = ________ minutes (canine/feline)
Toenail Bleeding Time
An alternative to BMBTClip toenail just past
quick to cause bleedingKeeping animal
undisturbed, monitor for bleeding to cease
Normal = <5 minutes (canine/feline)
Activated Clotting Time (ACT)
Evaluates _________________ hemostasis (all factors except Factor VII)
Requires Vacutainer containing sterile _____________________ earth to activate coagulation pathways 2 mL of blood is collected directly into tube It is important that tube is pre-warmed and kept at
37º C.Test can be affected by significantly ______
platelet numbersNormal = ___ – ___seconds (canine/feline)
Prothrombin Time (PT)
Evaluates adequacy of factors associated with _______________ and ___________ pathways
Routinely done by ___________Factor XIII activity not evaluatedPlatelet substitute added to sample
(thrombocytopenia does not interfere)Normal: Canine = 6.4 - 7.4 seconds;
Feline = 7 - 11.5 seconds
Partial Thromboplastin Time (PTT)
Evaluates adequacy of factors associated with the ___________ and ____________ pathways
Routinely done by machineFactor XIII activity not evaluatedPlatelet substitute addedNormal: Canine = 9-11 seconds;
Feline = 10-15 seconds
Fibrinogen Assay
Can be done by ___________ or ________________ methods
Only evaluates _________________ concentration
Can use ____________ anticoagulated sampleConcentrations may be increased during
__________________ or decreased when consumed during coagulation (_________)
Normal: Canine = 100 – 250 mg/dL Feline = 100 – 350 mg/dL
Other Coagulation Tests
Whole Blood Clotting timeClot Retraction TestOne-Stage Prothrombin Time (OSPT)
Used to confirm warfarin toxicity (rodenticide)Activated Partial Thromboplastin Time
(APTT) PIVKA (proteins induced/invoked by vitamin
K absence)d-Dimer and Fibrin Degradation Products
Coagulopathy
Coagulation defects can be categorized as: Coagulation defects of primary hemostasis Coagulation defects of secondary hemostasis Defects of fibronolysis (tertiary hemostasis)
Coagulation Defects of Primary Hemostasis
Coagulation defects of primary hemostasis _______________________ or ____________________
(Quantity or quality) ________________ bleeding Petechiae, mucosal bleeding, ecchymoses, epistaxis,
melena, prolonged bleeding
Coagulation Defects of Primary Hemostasis
___________________________ Decreased _______ number Can be _________________ or _________________ #1 cause = infectious disease
Ehrlichia, rickettsial diseases, babesiosis, systemic mycoses, toxoplasmosis, mycoplasmosis, Feline retroviruses (FeLV, FIV, FIP), others
Other causes = bone marrow depression; unknown
______________________________ (vWd) Decreased or deficient vWF= decreased PLT _____________ ,
aggregation, and fibrin cross linking Can occur secondary to ______________________ CS: MM hemorrhage, hematuria, GI bleeding, epistaxis Screening test of choice = _____________
Defects of Secondary Hemostasis
Coagulation defects of secondary hemostasis _____________(e.g. pleural, peritoneal, retroperitoneal) __________________formation Delayed bleeding/re-bleeding
Coagulation Defects of Secondary Hemostasis
Congenital clotting factor deficiencies of virtually all known factors have been described. (e.g.: Hemophilia A & B)
_____________ coagulation defects can result from: #1 = ________________ toxicity
Inhibits vitamin K Vitamin K is required to activate factors II, VII, IX, and X One-step prothrombin time = test to confirm
______________ toxicity. Liver disease, infiltrative bowel disease, and biliary
obstruction can also inhibit Vitamin K
Disseminated Intravascular Coagulopathy (DIC)
Not a disease in itself; it is a complex _________________ that results from a pathologic condition.
Involves __________________ activation of platelets, coagulation proteins, and plasmin; evolving into consumption of coagulation proteins, platelets, and inhibitors of fibrinolysis
Some of the many pathologic conditions associated with initiation of DIC include: Trauma and burns Metabolic acidosis/severe shock A large number of infectious diseases _________________________ Systemic infection ________________________ disease _________________________
Sometimes considered an “_______________ ” condition
DIC
Laboratory findings are highly _________________ Classically ACT, PTT, PT, and thrombin time are prolonged; fibrinogen
and platelet counts are decreased _________________________ seen on smear
Diagnosis is based on clinical suspicion and at least 3 abnormal coagulation test results.
Clinical signs depend on the phase in which the patient is experiencing _____________/Subacute phase: may have few to no overt clinical signs ___________ (consumptive) phase: characterized by venipuncture
oozing or modest to severe hemorrhage with inability to form a normal clot
______________ phase: charactized by no clinical signs or oozing of blood
Death is caused by extensive microthrombosis or circulatory failure, leading to single or multiple organ failure
Treatment of DIC
Successful treatment depends on early detection in critically ill animals.
Involves: CORRECTING UNDERLYING _____________________ Support of target organs where microthrombi may cause
hemorrhage Fluid therapy – balanced electrolyte solutions to maintain effective
circulating volume Coagulation factor __________________therapy Administration of _______________ as needed (controversial)
Should be accompanied by administration of _____________ Close monitoring of antithrombin activity
Prognosis is usually _______; depends on underlying causeIf an animal survives an acute DIC event, a ___________
form of DIC can exist
Other Acquired Coagulation Defects of Secondary Hemostasis
_________________ Disease The __________ synthesizes many of the clotting factors
including factors I, II, V, VII, VIII, IX, X, XI, and XII Liver manufacturers __________ which is essential in
absorption of vitamin ___ from diet
Disseminated Intravascular Coagulation (DIC) A complex syndrome with systemically accelerated
coagulation It is clinically difficult to differentiate between hepatic
disease and DIC because PT and PTT are usually prolonged with both.
DIC can occur secondary to hepatic disease.
Qualitative Platelet Dysfunction
ThrombocytopathiaMost common cause is inappropriate use of
________________.Can also be caused by:
________________________________ disorders Rare _____________________ problems Certain _______________
Thrombocytopathy: Drugs Causing Platelet Dysfunction
Table 10-3. Drugs Affecting Platelet Function
Anesthetics General – HalothaneLocal - Procain
Antibiotics Cephalosporins – CefazolinPenicillins - Ampicillin
Anticoagulants Heparin
Antihistamines Chlorpheniramine
Cardiovascular drugs Propanolol, Verapamil
Foods and food additives Ethanol, onions
Non-steroidal anti-inflammatory drugs
Aspirin, Phenylbutazone
Oncologic drugs Daunorubicin
Plasma Expanders HetaStarch, Dextrans
Miscellaneous drugs Chlorpromazine
Tertiary Hemostatic Dysfunctions (Defective Fibrinolysis)
The most common dysfunctional state is excessive _____________________. This is an uncommon disease.
Fibrinolysis ______________ can also cause thrombus formation (a condition, not a disease state)
Other Bleeding Disorders
Bleeding disorders may be caused by _____________ or ______________ defects in coagulation proteins, platelets, or vasculature.
Inherited coagulation defects are usually associated with a _________ coagulation protein and often occur at a ____________age.
Acquired coagulation defects often affect ________________ coagulation proteins and can occur at _________ age.
Anticoagulants and Hemostasis
As you already know, anticoagulants _____________ or ____________ the formation of a clot.
Anticoagulants are an important part of blood collection.
Different anticoagulants are utilized depending on the _____________ that are needed.
On the following slides, we are going to talk about each anticoagulant and how it can affect your sample.
Heparin
Heparin is acceptable for most tests requiring ________________. (Green top)
Heparinized tubes should be used for _______________ chemistry analyzers.
Heparin acts on the clotting cascade by preventing the conversion of __________________ to ____________ during the clotting process.
Heparin also may cause _____________of WBCs Heparin interferes with the _______________ of
WBCs and should not be used for blood films.
Ethylenediamine Tetraacetic Acid
Commonly referred to as ___________ (Purple top)Preferred anticoagulant for blood films because it
does not alter cell _______________________.Prevents clotting by binding with _______________ ,
which is necessary for clot formation.Should not be used for ____________ analysis because
it affects the metabolism of certain chemicals in the blood and may ____________________ increase or decrease those chemicals.
______________ EDTA can cause shrinkage of _____ This will invalidate automated chemistry machines.
Oxalates
Available as: _________oxalate, ______________ oxalate, ______________ oxalate, or ______________oxalate.
_____________________ oxalate is most commonly used. (Grey top)
Prevents clotting by binding with __________, which is necessary for clot formation.
Not frequently used as it interferes with potassium tests, alkaline phosphatase tests, and lactate tests.
Citrates
Available as: ______________citrate, or __________________citrate.
Blue topCommonly used in ________________ medicinePrevents clotting by binding with calcium,
which is necessary for clot formation.Interfere with ___________tests and many of
the tests performed by ________________ chemistry analyzers.
Sodium Flouride
Known as __________ preservative but does have anticoagulant properties
Prevents clotting by binding with calcium, which is necessary for clot formation.
May be added to other samples that already have an ______________________.
Also _________top!Interferes with many _______________ tests
performed by automated analyzer.
Commonly Used AnticoagulantsTable 2-2Name
Mode of Action
Advantages
Disadvantages
Uses
Heparin Antithrombin Reversible, nontoxic
Clumps WBCs, expensive, staining interference
Critical RBC measurements
EDTA Chelates calcium
Best preservative
Irreversible, shrinks cells
Hematology
Oxalates (potassium, Na, lithium)
Chelates calcium
Temporary Variable effects
Coagulation
Citrates (Na, lithium
Chelates calcium
Nontoxic, reversible
Interferes with blood chemistry
Transfusions, coagulation
Fluorides (Na)
Chelates calcium
Inhibits cell metabolism
Interferes w/ enzymatic tests
Preserves blood glucose