VTHT 2323

1
Thrombocytes and Coagulation

• VTHT 2323
• CLINICAL PATHOLOGY
• KRISTIN CANGA, RVT

2
Reading Assignment

• AP PG. 230
• LAB PRO
• PP. 30-32 (ANTICOAGULANTS)
• AND 69-73

3
Thrombocyte Formation

• Production of platelets ________________________
  ___
• Produced in bone marrow by __________
• _________________________(TPO) stimuli for
  PPSCs to differentiate into thrombocyte
  precursor.
• Platelet parent cell ___________________________
  ______

4
Thrombocyte Formation

• Megakaryocyte undergoes incomplete ___________
• (___________________ divides but cytoplasm
  doesnt)
• 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

5
Megakaryocytes
6
Megakaryocyte
7
Megakaryocytes
8
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.

9
Megakaryocyte Video

• http//www.youtube.com/watch?v6R-ESPFiKbofeature
  relatedajax1nocache1271011451258

10
Thrombocytes

• Commonly referred to as ________________.
• Not complete cells (lack a ____________), but
  frequently listed as one of the cell types in
  peripheral blood.
• RBCsgtPLTsgtWBCs
• 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.

11
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

12
Giant Platelet in Peripheral Blood
L
T
13
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

14
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

15
Normal Platelet Morphology
16
Normal Platelet Morphology
17
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.

18
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.

19
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

20
Platelets 2) Plug Formation, contd

• 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.

21
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
  __________________________

22
Activated Platelets
23
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.
24
Platelet Clumping
Platelet Clumps
Platelet Clumps
Thrombocytosis
25
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 ____________.

26
Fun Video Introduction to Coagulation

• http//www.youtube.com/watch?v9QVTHDM90io

27
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!!!
• Manufactures most clotting factors
• Bile essential for _____________ of vitamin ___

28
Stages of Coagulation

• ______________ Hemostasis
• _________________________
• Primary _______________ plug formation
• Platelet _______________
• Platelet ________________
• Does adhesion or aggregation CAUSE platelet
  activation?

29
Stages of Coagulation

• _________________ Hemostasis
• _________________ Cascade
• Ultimate goal __________ for stabilization of
  platelet plug
• Involves three pathways to clotting
• __________________ Pathway,
• __________________ Pathway, and
• __________________ Pathway

30
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 ____________________.

31
(No Transcript)
32
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

33
The Coagulation Cascade
34
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)

35
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

36
Platelet Counting Methods

• ____________ or _______________ (least accurate)
• Most inaccuracies attributable to
• _______________, giant platelets, RBC
  _____________
• Always use ___________ sample to minimize error
• Manual methods
• Platelet estimation during blood film analysis
• Formula?
• ALWAYS USE HIGH POWER, _______________________
• Unopette system hemocytometer
• (NOT COMMONLY USED)

37
Buccal Mucosal Bleeding Time

• Tests _____________ hemostasis
• Evaluates platelet __________
  _____________(thrombocytopathy, thrombocytopenia)
• Evaluates endothelial cell function
• (__________)
• Test can be affected by certain
  ___________________

38
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)

39
Buccal mucosal bleeding time
40
Toenail Bleeding Time

• An alternative to BMBT
• Clip toenail just past quick to cause bleeding
• Keeping animal undisturbed, monitor for bleeding
  to cease
• Normal lt5 minutes (canine/feline)

41
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)

42
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

43
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

44
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

45
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

46
Quick Coagulation Testing
47
Coagulopathy

• Coagulation defects can be categorized as
• Coagulation defects of primary hemostasis
• Coagulation defects of secondary hemostasis
• Defects of fibronolysis (tertiary hemostasis)

48
Coagulation Defects of Primary Hemostasis

• Coagulation defects of primary hemostasis
• _______________________ or ____________________
  (Quantity or quality)
• ________________ bleeding
• Petechiae, mucosal bleeding, ecchymoses,
  epistaxis, melena, prolonged bleeding

49
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 _____________

50
Defects of Secondary Hemostasis

• Coagulation defects of secondary hemostasis
• _____________(e.g. pleural, peritoneal,
  retroperitoneal)
• __________________formation
• Delayed bleeding/re-bleeding

51
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

52
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

53
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

54
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 cause
• If an animal survives an acute DIC event, a
  ___________ form of DIC can exist

55
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.

56
Qualitative Platelet Dysfunction

• Thrombocytopathia
• Most common cause is inappropriate use of
  ________________.
• Can also be caused by
• ________________________________ disorders
• Rare _____________________ problems
• Certain _______________

57
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

58
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)

59
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.

60
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.

61
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.

62
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.

63
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.

64
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.

65
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.

66
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