Hemostasis Testing

Question 1

Hemostasis
+
Primary v Secondary

Answer 1

arrest of bleeding or interruption of blood flow

abnormalities cause hemorrhage or prothrombus

Primary = endothelial response + formation of platelet plug
Secondary = coagulastion cascade + clot resolution

Question 2

Major players in hemostasis

Answer 2

blood vessels
platelets
coagulation factors
fibronilytic factors

Question 3

Healthy Endothelium

Answer 3

Normally in antithrombotic state

1) antioplatelet factors:
- blo subendothelial extracellular matrix
- produce prostacyclin + NO

2) anticoagulant factors
- heparin like molecules
- thrombomodulin
- tissue factor pathway inhibitor

3) fibrinolytic factors
- tissue-type plasminogen activator

Question 4

Injured Endothelium

Answer 4

local neurologic response after injury 🡒 arteriolar constriction
- attempt to ↓ defect size

prothrombotic properties during trauma + inflammation

subendothelial EC matrix now exposed to platelets 🡒 adhesion thru vWF
- vWF synthesized + stored in endothelial cells

procoagulant tissue factor produced in response to inflammation

release of plasminogen activator inhibitor
- inhibits fibrinolysis
- favors thrombosis

Question 5

Megakaryopoiesis + Regulation

Answer 5

• almost all mature forms in marrow = < 1% nucleated cells
• multinucleate cell with lobulated nucleus = forms by endomitosis
• maturation time 2-10 days

Thrombopoietin
- from liver + kidneys + marrow stromal cells
- produced at constant rate in health
- 🡑 # of progenitor cells
- 🡑 megakaryocyte size + ploidy
- 🡑 releases of platelets

🡑 thrombopoeitin production stimulated by:
- 🡓 in megakaryocyte + platelet mass
- IL-6, GM-CSF, stem cell factor

Question 6

Platelets

Answer 6

• anucleate cytoplasmic fragments
• released directly into blood
• 30-40% in spleen
• life span = 5-9 days
• removal 🡒 phagocytosis by macrophages

Question 7

Avian + Reptiles + Fish + Amphibians
Thrombocytes

Answer 7

platelets known as thrombocytes instead

• nucleated
• high nuclear to cytoplasmic ratio
• may be difficult to distinguish from lymphocytes
• from distinct mononuclear cell line

Question 8

Platelet Phases of Hemostasis

Answer 8

1) Adhesion 🡒 mediated primarily by vWF
- bridge between platelet + exposed collagen
- release from endothelial cells + platelets

2) Shape Change
- from discound to spherical
- formation of pseudopodia

3) Aggregation
- activated platelet receoptor binds fibrinogen + vWF 🡒 forms bridge between platelets
- Thromboxane A2 amplifies 🡒 produced during platelet activation

4) Granule Release
- activates platelet phospholipases 🡒 TXA2 production
- release Ca2+ 🡒 componenet of coag cascade
- release of factors V + XI + vWF
- translocation of phosphotidylserine 🡒 binds Ca2+ = focused coagulation

Question 9

Platelet Count

Answer 9

measured in EDTA anticoagulated blood

clumping may falsely 🡓
- very common in cats + cattle

can be estimated on blood smear
- avg # in monolayer (100X objective) x 15-20K
- can look for large platelets

< 20,000/uL concern for spontaneous bleeding

May get other parameters:
- mean platelet volume (MPV)
- platelet distribution width (PDW)
- thrombocrit

Question 10

Thrombocytopenia Mechanisms

Answer 10

1) 🡓 production
- drugs = chemo, chloramphenicol, sulfa drugs, etc.
- toxins = aflatoxin, bracken fern
- infectious = FeLV, parvo, ricketssial diseases etc.
- proliferation of neoplastic cells in bone marrow

2) 🡑 destruction
- immune mediated = IMHA or ricketssial disease
- increased activation = DIC, infection

3) Sequestration
- splenic congestion/neoplasia

4) 🡑 consumption/utilization
- hemorrhage/trauma rare
- DIC

Question 11

Macrothrombocytopenia of Cavalier King Charles Spaniels

Answer 11

release of platelets from megakaryocytes abnormal

counts 30,000 - 100,000/uL (200K-700K/uL

have large platelets

plateletcrit often normal

doesn’t cause bleeding

Question 12

Thrombocytosis Mechanisms

Answer 12

1) splenic contraction

2) reactive thrombocytosis
- inflammation
- neoplasia
- iron deficiency

3) megakaryocytic leukemia / myeloproliferative disorders

Question 13

Thrombopathy

Answer 13

functional platelet disorder

Question 14

Buccal Mucosal Bleeding Time

Answer 14

evaluates primary hemostasis

requires special lancet tool

do not use if thrombocytopenic

helpful to confirm thrombopathy 🡒 may be prolonged in von Willebrand disease

in dogs 🡒 usually < 4 min

Question 15

Von Willebrand Disease (vWD)

Answer 15

vWF in entholial cells + platelets
- responsible for platelet adherance
- binds Factor VIII 🡒 🡑 half-life

small + medium + large multimers
- large most active

common in dogs, rare in other species

clinical signs when conc. < 20-35%

3 types

Question 16

3 types of vWD

Answer 16

Type I = 🡓 in multimer sizes
- plasma conc. < 50%
- multimers present structurally + functionally normal
- majority of canine cases (doberman pinschers)

Type II = disproportionate loss of large multimers
- Rare

Type III = severe decreases in all multimers
- conc. often < 0.1% of normal

Question 17

vWD testing

Answer 17

need plasma sample
- citrate or EDTA sample
- free of clots + hemolysis
- recommended to seperate plasma, place in plastic tube + freeze

measured by species specific Ab to vWF
multimeric analsysis possible
most dogs that bleed have values < 35%
genetic testing available

Question 18

Intrinsic Disorders of Platelet Function

Answer 18

Chediak-Higashi syndrome
- platelets lack dense granules 🡒 lack platelet/coagulation activators

Cyclic Hematopoiesis of Grey Collies
- platelet reactivity to some activators defective
- dense granules absent

Question 19

Acquired Disorders of Platelet Function

Answer 19

Cyclooxygenase inhibiting drugs
- inhibits TXA2 production
- aspirin = irreversible (up to 5 days)
- non-selective NSAIDS = reversible (usually only hours)

Uremia
- inhibits fibrinogen binding
- alters interaction with vWF
- 🡑 NO + prostacyclin activity

Hyperglobulinemia
- interferes with platelet adhesion

Question 20

Clinical Signs of Primary Hemostatic Disorder

Answer 20

• petechia
• ecchymoses
• bruising
• spontaneous hemorrhage when platelets < 20K/uL
• vWD 🡒 mucosal hemorrhage, prolonged bleeding from wounds

Question 21

Clinical Signs of Secondary Hemostatic Disorder

Answer 21

large accumulations of blood:
- hematoma
- bleeding into cavities
- melena
- rebleeding from wounds
- hematuria

Question 22

Coagulation

Answer 22

activation of pathways
- intrinsic with collagen exposure
- extrinsic with endothelial cell disruption

activation of FX
formation of thrombin
formation of fibrin

Question 23

Intrinsic Pathway

Coagulation

Answer 23

stimulated by exposure of collagen (subendothelial ECM)
- negatively charged surface

Factor XII (FXII) initiates 🡒 activated by contact with neg. charges

cascade of enzymatic reaction 🡒 results in activation FX

Question 24

Extrinsic Pathway

Coagulation

Answer 24

stimulated by tissue factor:
- from damaged tissue
- from activated cells (monocytes/macrophages)

TF + activated FVII activate FX
- can also activate FIX

Factor VII 🡒 shortest half-life of factors

Question 25

Common Pathway | Coagulation

Answer 25

starts with activation of FX converts prothrombin to thrombin w/ activated FV - thrombin feeds back to activate FV + FVIII + FXI thrombin cleaves fibrinogen to fibrin thrombin activates FXIII 🡒 cross-links fibrin

Question 26

Inhibitors of Coagulation

Answer 26

**Antithrombin III (ATIII)** - produced by hepatocytes - binds + inactivates enzymes 🡒 thrombin (FIIa), FIXa, Fxa, FXIa, FXIIa - activity enhanced by heparin Protein C - produced by hepatocytes - activated by thrombin/thrombomodulin complex - inactivates FVa + FVIIIa 🡒 help from protein S Tissue factor pathway inhibitor - made by endothelial cells, monocytesm, macrophages, hepatocytes - inhibits TF-FVIIa in presense of Ca2+

Question 27

Fibrinolysis

Answer 27

Plasmin degrades fibrin - releases FDPs - D-dimers specific for fibrinolysis of cross-linked fibrin plasminogen binds fibrin - tissue plasminogen activator converts plasminogen to plasmin 🡒 from endothelial cells - digestion of fibrin opens new sites for plasminogen binding - also degrades 🡒FVa, FVIIIa, vWF

Question 28

Coagulation + Fibrinolysis testing

Answer 28

**Sodium citrate blood tube 🡒 light blue tube** - citrate chelates calcium - **blood to anticoagulant ratio 9:1 ** - clean stick 🡒 tissue damage / hemolysis activate clotting - immediate gentle mixing - aPTT, PT, TT, fibrinogen, FDPs, D-dimers Tests: aPPT - activated partial thromboplastin time PT - prothrombin time TT - thrombin time ACT - activated clotting time Fibrinogen FDPs - fibrin(ogen) degradation products D-dimers specific factor test

Question 29

Activated Partial Thromboplastin Time (aPTT)

Answer 29

**intrinsic + common pathway** time in seconds til clot formation: - kaolin - platelet phospholipid - calcium **🡑 time with factor deficiency in either pathway** Prolonged: - factor activity < 30% - affected hemophilliacs - hereditary FXI deficiency - hereditary FXII deficiency 🡒 no clinical bleeding tendencies, birds + reptiles naturally lack FXII Shortened: - inflammation 🡒 🡑 in fibrinogen + FVIII ## Footnote tip : brad aP**I**TT

Question 30

Prothrombin Time (PT)

Answer 30

**extrinsic + common pathway** time in seconds til clot formation: - tissue factor - calcium **🡑 time with factor deficiency in either pathway** ## Footnote tip : P**E**T

Question 31

Prolongation of BOTH aPTT + PT

Answer 31

- deficiency in common pathway - acquired vit. K deficiency/antagonism - disseminated intravascular coagulation (DIC) - liver failure - 🡓 fibrinogen (< 50mg/dL) - heparin treatment

Question 32

Thrombin Time (TT)

Answer 32

**measure of functional fibrinogen** - add thrombin + Ca2+ Prolonged: - hypofibrinogenemia - dysfibrinogenemia - 🡑 FDPs can measure fibrinogen conc. directly - heat precipitation not accurate for 🡓 ## Footnote *not actually measuring thrombin*

Question 33

Activated clotting time (ACT)

Answer 33

**intrinsic + common pathway** measures time for fresh whole blood to clot - exposure to contact activator 🡒 glass tube, diatomaceous earth prolonged: **- severe factor activity < 10%** - severe thrombocytopenia - therapy 🡒 heparin, aspirin therapy - inadequate warming of tube

Question 34

Fibrin(ogen) Degradation Productions (FDPs)

Answer 34

degradation of fibrinogen + fibrin by plasmin 🡑 with DIC

Question 35

D-dimers

Answer 35

degradation of cross-linked fibrin 🡑 with DIC 🡒 more specific than FDPs

Question 36

Coagulopathies

Answer 36

hereditary disorders - hemophilia A + B - other factors less common acquired disorders: - vit. K deficiency or antagonism - liver disease - DIC

Question 37

Hemophilia A

Answer 37

**deficiency FVIII** - **most common inherited coagulopathy** - commonly affects GSDs clinical signs depend of factor activity - < 30% prolong aPTT - carriers 40-60% activity - spontaneous bleeding possible 2-5%, likely < 2% - bleeding with minor trauma likely 2-5%, for sure < 2%

Question 38

Hemophilia B

Answer 38

**deficiency FIX** - spontaneous internal hemorrhage more common 🡒 very low activity - carriers 40-60% activity - **same lab abnormalities as hemophillia A**

Question 39

Vitamin K deficiency/antagonism

Answer 39

**Vit K dependent factors/proteins** -** II (thrombin), VII, IX, X** - Protein C **Associated with:** - 🡓 absorption 🡒 severe GI dz, liver dz, exocrean pancreatic insuffiency - rodenticide toxicity clinical signs variable

Question 40

Liver Disease | Coagulation

Answer 40

**most coagulation factors made by liver** **severe disease** - PT usually elevated first - similar test pattern to vit K deficiency

Question 41

Disseminated Intravascular Coagulation

Answer 41

**primary + secondary hemostatic disorder** - 🡑 utilization of platelets + clotting factors 🡒 excessive clotting - 🡑 fibrinolysis 🡒 excess bleeding **clinical signs of both:** - petechiae - hematoma formation - hematuria **DIC always secondary to another condition** - severe tissue necrosis, sepsis, heatstroke, parvovirus, disseminated neoplasia, pancreatitis, more Need at least 3 of 4 for diagnosis: - 🡓 platelet count - 🡑 aPTT - 🡑 PT - 🡑 FDPs/D-dimers Other abnormalities: - prolonged TT - hypofibrinogenemia - shistocytes - 🡓 ATIII

Question 42

Coagulation

Answer 42