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4DMT

COAGULATION
Hemostasis cellular and biochemical events which function in harmony to keep blood with the veins and arteries. prevent blood loss from injuries. re-establish blood flow.

1. Primary Hemostasis involves platelets and endothelium. activated by small injuries. rapid, short-lived reponse. positive feedback: formation of platelet plug.

A. Platelets Production: Hemocytoblast Megakaryocyte Megakaryoblast Metamegakaryocyte Promegakaryocyte Platelets

Distribution: a. Peripheral blood 70% b. Spleen 30% RR: 150 400 x 10^9 / L Life Span: 8 11 days Size: 2.5 m in diameter Zones: a. Peripheral zone adhesion and aggregation. b. Sol Gel zone shape and contraction. c. Organelle zone metabolic activities. Function: a. Adhesion - platelets roll and cling to non - platelet surfaces. b. Activation - occurs when vWF and collagen binds to glycoprotein Ib receptor on the surface of the platelet. c. Aggregation - platelets adhere to each other. d. Secretion - platelets discharge the contents of their granules. e. Clot Retraction creates more bulk in the clot making it more resistant to stress. f. Cytokine signalling - platelets secrete platelet derived growth factor. B. Endothelial cells - assist in platelet activation. - limits coagulation mechanism. - clot dissolution.

Page |2 2. Secondary Hemostasis involve coagulation factors. activated by large wounds from trauma, surgery, dental procedures. delayed, long-term response. positive feedback: formation of fibrin clot.

A. Coagulation - mechanism whereby after injury to a blood vessel, plasma coagulation factors, tissue factors, and calcium work together on the surface of platelets to form a fibrin clot. B. Coagulation Factors - proteins engaged in formation of a fibrin clot from fibrinogen.
Factor I II III IV V VII VIII IX X XI XII Prekallikrein High Molecular Weight Kininogen XIII Platelet factor 3 Customary name Fibrinogen Prothrombin Tissue factor Ionic Calcium Labile factor Stable Factor Antihemophilic Factor (vWF) Christmas factor Stuart-Prower factor Plasma thromboplastin antecedent (PTA) Hageman factor Fletcher factor, PK Fitzgerald factor, HMWK Fibrin-stabilizing factor (FSF) Phospholipids, phosphatidyl serine PF3 Function Thrombin substrate, polymerizes to form fibrin Serine protease Cofactor Mineral Cofactor Serine protease Cofactor Factor VIII carrier and platelet adhesion Serine protease Serine protease Serine protease Serine protease Serine protease Cofactor Transamidase Assembly molecule

C. Coagulation Cascade - series of biochemical reactions and feedback mechanisms by means of intrinsic and extrinsic pathway, or both, leading to a common pathway forming a fibrin clot. C.1. Intrinsic pathway - utilization of plasma contact factors to initiate coagulation, beginning with the activation of factor XII; all necessary factors required are contained in the circulating blood. - activated partial thromboplastin time (aPTT) test monitors this pathway.

Page |3 - aPTT measures factors XII, XI, X, IX, VIII, V, II and I. C.2. Extrinsic pathway - coagulation pathway that is activated by tissue thromboplastin; necessary components are factor VII and calcium. - prothrombin time (PT) test monitors this pathway. - PT measures factors VII, X, V, II, I. C.3. Common pathway - final stage of the coagulation cascade, beginning with the convergence of the extrinsic and intrinsic pathways (factor X) ending with the formation of fibrin clot.

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3. Specimen Collection and Handling A. Anticoagulants - 3. 8 % or 3.2 % sodium citrate - normal ratio of blood to anticoagulant is 9:1 B. Holding sample - siliconized glassware (glass activates factor XII and platelets will adhere to glass). C. Collection of Blood Sample - venipuncture should be clean and quick with minimal stasis. - if drawing tubes for other tests, draw blue top last, except that if an EDTA tube is needed for a CBC, the purple top tube should be drawn after the blue top coagulation tube. If only drawing blood for coagulation testing, allow some blood to drip into vacutainer before collection. (The first blood drawn in a venipuncture is most likely to be contaminated with small amounts of tissue thromboplastin. This can activate the extrinsic pathway and lead to variable results). - prompt and gentle inversion must be done to bind all calcium immediately. D. Specimen Processing - transportation to the laboratory should be done as quickly because some changes can begin in vitro. - specimens should be centrifuged for 10 minutes to get the cell - free plasma - anticoagulant mixture. - always check for micro-clot formation, if present, specimen is unacceptable for testing. - hemolyzed specimens are also unacceptable for testing because of possible clotting factor activation. 4. Routine Test of Hemostatic Function A. Primary Hemostasis A.1. Bleeding Time - measures the time required for the cessation of bleeding after a standardized capillary puncture to a capillary bed. - the time required will depend on the capillary integrity, number of platelets and the platelet function. - types: a. Duke method: earlobe; RR: 0-6 minutes. b. Ivy method: forearm; RR: 1-6 minutes. c. Template method: forearm; RR: 2-9.5 minutes. - prolonged with aspirin.

Page |5 A.2. Clot Retraction - evaluates platelet function, fibrinogen, red cell volume and fibrinolytic activity. - whole blood is allowed to clot and observed for retraction. - platelets of adequate number and viability are required for clot retraction to occur. - normal value: evidence of retraction within 2 hours (varies with methodology). B. Secondary Hemostasis B.1. Coagulation time/ Clotting Time - measures the period required for the free flowing blood to clot or solidify after it has been removed to the body. - types: a. Drop or Slide method: capillary blood; NV: 2-4 minutes b. Lee and White method (tube method): venous blood: NV: 7-15 minutes B.2. Prothrombin Time (PT) - test for extrinsic and common pathway. - measures factors I, II, V, VII and X. - monitors oral anticoagulants (warfarin, coumarin, dicoumarol). - reagent: tissue thromboplastin and CaCl2. - sensitive to vitamin K factors. - International normalized ratio (INR)

INR = (patient result)ISI

(mean of the reference range) *ISI = International Sensitivity Index from manufacturer

- reference range: < 14 seconds a. Therapeutic goal: INR 2.0 3.5 B.3. Activated Partial Thromboplastin Time - test for intrinsic and common pathway. - measures all factors except factor VII and XII. - monitors heparin therapy. - reagents: activator (kaolin, celite or ellagic acid), platelet phospholipid (PF3) and CaCl2. - reference range: 20 40 seconds

Page |6 OTHER TESTS: Laboratory Tests for Primary Hemostasis 1. Capillary Resistance / Fragility/ Tourniquet /Rumpel Leedes or Hess Test 2. 3. 4. 5. Platelet Adhesiveness Test Platelet Aggregation Test Platelet Count Platelet Morphology and MPV Laboratory Tests for Secondary Hemostasis 1. 2. 3. 4. Plasma Recalcification Time Activated Clotting Time Stypven Time Thrombin Time / Thrombin Clotting Time 5. Reptilase Time 6. Substitution Test (Mixing Studies) 7. Prothrombin Consumption/Serum Prothrombin Test 8. Thromboplastin Generation Test 9. Specific Factor Assay 10. Assay of vWR:Ag and vWR:Reo Rockett/Laurel 11. Duckerts or Clot Solubility Test 12. Tests for Circulating Inhibitors of Coagulation Laboratory Tests for Fibrinolysis 1. Determination of Fibrinolytic Products 2. Lysis Time 3. Proteins involved in Fibrinolysis

5. Bleeding Disorders

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Disorder of Primary Hemostasis

Platelet Disorders Vascular Disorders

Qualitative

Quantitative

Disorders of Platelet Secretion Disorders of Platelet Adhesion Disorders of Platelet Aggregation Glanzmanns thrombasthenia Acquired von Willebrand Disease

Thrombocytopenia

Thrombocytosis primary

Hereditary Hereditary Hemorrhagic Telangiectasia/ Rendu-WeberOsler, HemangiomaThrombocytopenia

Disorders related to distribution or dilution /big spleen

reactive

Acquired

Bernard Soullier/ Giant Platelet Syndrome

Increased platelet destruction/ utilization Impaired/ decreased platelet production BM failure Thromboxane Pathway Disorders TTP, drug induced, non immune mechanisms, DIC Kasabach-Merit, Ehler-Danlos, Marfan, Osteogenesis imperfect, Pseudoxanthoma elasticum

von Willebrand Disease Storage Pool Diseases Primary granule deficiency Hemmeler anomaly

Congenital hypoplasia, MHA, WAS, BS, Fanconi, TAR

Anaphylactoid purpura, Henoch Schonlein, Senile purpura, Scurvy, Purpura simplex, Infectious purpura, Drug induced purpuras associated with paraprotenemiea s, Amyloidosis, Idiopathic purpuras

Electron dense/ delta granules deficiency

Neonatal hypoplasia - drugs, infections Acquired Acquired hypoplasia ionizing radiation, drugs -

Hereditary

Pudlak, Wiskott Aldrich, Chediak Higashi, TAR

aspirin like defects

Alpha granules deficiency

due to inhibitors of prostaglandin pathway (chronic aspirin intake or inhibitors of thromboxane or cyclooxegenase pathway

Gray Platelet Syndrome, Quebec Platelet Disorder

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References:
McPherson, R. & Pincus, M. (2006). Henrys Clinical Diagnosis and Management by Laboratory Methods. US: Saunders Elsevier Linne, J. & Ringsrud, K. (1999). Clinical Laboratory Science The Basics and Routine Techniques Harmening DM: Clinical Hematology and Fundamentals of Hemostasis, ed 3. Philadelphia, FA Davis Co, 1997. Turgeon ML: Clinical Hematology: Theory and Procedures, ed 3. Philadelphia, Lippincott-Raven Publishers, 1998.

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