(FII) Thrombin (FIIa) FVIII FVIIIa Fibrinogen Soluble Fibrin Fibrin Clot Formation and Stabilization FXIIIa Insoluble cross-linked !brin Secondary hemostasis, fibrin clot formation and stabilization: 8 9 2. ROUTINE COAGULATION TESTS Elena Brnjac & Rita Selby Evaluating coagulation in the laboratory ▲ In the coagulation laboratory, the coagulation factors are divided into: • Extrinsic pathway factors (Factor VII) • Intrinsic pathway factors (Factors XII, XI, IX, VIII) • Common Pathway factors (Factors X, V, II, Fibrinogen) ▲ Memorizing which factors belong to the extrinsic, intrinsic and common pathways respectively will make evaluating the causes of abnormal coagulation tests easier. Sample collection for coagulation testing ▲ To assess coagulation “in vitro,” the laboratory measures the time taken to form a clot. ▲ Blood is collected into a blue top tube containing sodium citrate anticoagulant (which chelates calcium) to prevent blood clotting in the tube during transport. ▲ Plasma (the liquid component of blood that contains the clotting factors) is then separated from the platelets (phospholipid source) by centrifugation. ▲ Later we will see how adding back phospholipids and calcium is important in standardizing routine coagulation tests. ▲ Some common problems that may result in spurious coagulation test results are: • Blood collected into incorrect type of tube (not a sodium citrate tube) • Incorrect plasma to citrate ratio (e.g. underfilling of tube or patient’s hematocrit > 0.55 L/L) • Heparin contamination of sample (e.g. incorrect order of sample collection or sample collected from central lines) • Clotting in tube from traumatic venipuncture or inadequate mixing • Hemodilution of sample 2. Routine Coagulation Tests 9 Fibrinogen Common V II X VIII IX XI VII XII Pathway Tissue Factor Fibrin Clot Intrinsic Pathway Extrinsic Pathway APTT Common Intrinsic + PT Common Extrinsic + Thrombin Fibrinogen PTT 12 11 9 10 2 1 8 5 7 INR Buy Coat Red Cells Plasma Sodium Citrate Tube (Blue Top) Centrifugation ▲ Here is another picture to help with memorizing the coagulation cascade without the Roman numerals: • The common pathway factors can be memorized by thinking of the denominations of dollars in Canada: factors 10, 5, 2 and 1 • The PT/INR pathway starts with factor 7 and includes the common pathway factors • The APTT pathway starts from the left at factor 12, counts backwards to factor 8 (skipping factor 10) and includes the common pathway factors ! Coagulation testing MUST only be sent in a sodium citrate (blue top) tube. A T T E N T I O N Sodium Citrate Tube (Blue Top) 10 11 2. ROUTINE COAGULATION TESTS Prothrombin Time (PT) ▲ The PT is used to assess deficiencies or inhibitors of the extrinsic pathway factors (Factor VII) and common pathway factors (Factors X, V, II, Fibrinogen). International Normalized Ratio (INR) ▲ The International Normalized Ratio (INR) was developed to standardize the PT to allow for monitoring of oral vitamin K antagonist therapy (e.g. warfarin) across different labs. ▲ The PT time in seconds is used to calculate the INR. ▲ Each lot of PT reagent needs to have an International Sensitivity Index (ISI) determined / assigned, which indicates how sensitive the reagent is to deficiencies in the Vitamin K dependent factors compared to the World Health Organization reference standard. ▲ The INR is the ratio of the patient’s PT value over the geometric mean of the PT (generated from a minimum of 20 normal volunteers) and raised to the power of the ISI of the reagent used to obtain the PT: INR = (PT of patient / geometric mean normal PT)ISI 2. Routine Coagulation Tests 11 Fibrinogen V II X VII Fibrin Clot Common Pathway Extrinsic Pathway ▲ Measurement of PT: PT reagent contains a source of tissue factor (also known as thromboplastin), phospholipids and calcium chloride. Plasma is warmed to 37° C. Pre-warmed PT reagent is added and the time in seconds for clot formation is measured ▲ The PT is dependent on the reagent and instrument used and will vary between laboratories. A normal PT is approximately 9-15 seconds. INR = ISI Patient PT Mean Normal PT Time for clot formation 9 - 15 seconds 0.1 mL Thromboplastin + Ca