Thrombocytes and Coagulation
VTHT 2323
CLINICAL PATHOLOGY
KRISTIN CANGA, RVT
Reading Assignment
A&P: PG. 230
LAB PRO:
PP. 30-32
(ANTICOAGULANTS)
AND 69-73
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
Megakaryocyte
Megakaryocytes
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=12710
11451258
Thrombocytes
 Commonly referred to as ________________.
 Not complete cells (lack a ____________), but
frequently listed as one of the cell types in peripheral
blood.
 RBCs>PLTs>WBCs
 Have 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 RBCs
 Most 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
Giant Platelet in Peripheral Blood
L
T
Platelets…
 Play a part in both the __________________






and __________________ formation of clots.
Secrete ________________________
Form platelet plugs
Secrete ____________________
Initiate dissolution of blood clots
(“_______________________”)
Secrete _______________that attract neutrophils
and monocytes to sites of inflammation
Secrete ______________ factors to help maintain
and repair blood vessels
Normal Platelet Values
 Canine: 200,000 – 500,000 /µL
 Feline: 300,000 – 700,000 /µL
 All species: ____________ of ____________/µL
 Horses = ___________normal concentrations
 Cattle = ____________normal concentrations
 Animals will bleed spontaneously if PLT concentration
is ≤ ____________ to ____________ /µL
Normal Platelet Morphology
Normal Platelet Morphology
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 PFs
 Platelet _______________ 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
__________________________
Activated Platelets
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 Clumping
Platelet Clumps
Thrombocytosis
Platelet Clumps
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 ____________.
Fun Video Introduction to Coagulation
http://www.youtube.com/watch?v=9QVTHDM90io
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 factors
2.
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
The Coagulation Cascade
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 error
 Manual methods:
1.
Platelet estimation during blood film analysis


2.
Formula?
ALWAYS USE HIGH POWER, _______________________
Unopette system & hemocytometer
(NOT COMMONLY USED)
Buccal Mucosal Bleeding Time
 Tests _____________ hemostasis
 Evaluates platelet __________ &
_____________(thrombocytopathy,
thrombocytopenia)
 Evaluates endothelial cell function
(__________)
 Test can be affected by certain
___________________
BMBT Procedure
1.
2.
3.
4.
5.
6.
Place _______________ animal in
_____________ recumbency.
Use a strip of gauze to tie upper lip back and expose
mucosal surface. (Also acts as ______________)
Using a Surgicutt® or a Simplate® lancet, create a
small wound (~1 mm deep)
Remove blood with filter paper at 30-second
intervals DO NOT TOUCH SKIN
Stop timing when there is no more blood.
Normal = ________ minutes (canine/feline)
Buccal mucosal bleeding time
Toenail Bleeding Time
 An alternative to BMBT
 Clip toenail just past quick to
cause bleeding
 Keeping 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
numbers
 Normal = ___ – ___seconds (canine/feline)
Prothrombin Time (PT)
 Evaluates adequacy of factors associated with




_______________ and ___________ pathways
Routinely done by ___________
Factor XIII activity not evaluated
Platelet 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 machine
Factor XIII activity not evaluated
Platelet substitute added
Normal: Canine = 9-11 seconds;
Feline = 10-15 seconds
Fibrinogen Assay
 Can be done by ___________ or




________________ methods
Only evaluates _________________
concentration
Can use ____________ anticoagulated sample
Concentrations 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 time
 Clot Retraction Test
 One-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
Quick Coagulation Testing
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



Coagulation factor __________________therapy
Administration of _______________ as needed (controversial)


Fluid therapy – balanced electrolyte solutions to maintain effective
circulating volume
Should be accompanied by administration of _____________
Close monitoring of antithrombin activity
 Prognosis is usually _______; depends on underlying cause
 If 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
 Thrombocytopathia
 Most 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 – Halothane
Local - Procain
Antibiotics
Cephalosporins – Cefazolin
Penicillins - 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 top
Commonly used in ________________ medicine
Prevents 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 Anticoagulants
Table 2-2
Name
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