Incubate at 37˚C 0.05 mL Plasma ++ 12 13 2. ROUTINE COAGULATION TESTS Activated Partial Thromboplastin Time (APTT) ▲ The APTT is used to assess deficiencies or inhibitors of the intrinsic pathway factors (Factors XII, XI, IX, VIII) and common pathway factors (Factors X, V, II, Fibrinogen). Thrombin Time (TT) ▲ The TT is used to assess deficiencies or dysfunction of fibrinogen or the presence of an inhibitor of thrombin (Factor IIa). The most common cause for TT prolongation is anticoagulant drug therapy (e.g. heparin or direct thrombin inhibitor). Other causes include quantitative or qualitative fibrinogen abnormalities and increased products of clot breakdown (e.g. fibrin degradation products in disseminated intravascular coagulation). 2. Routine Coagulation Tests 13 Time for clot formation 25 - 35 seconds 0.1 mL Contact Activator + PL Incubate at 37˚C 0.1 mL Plasma 0.1 mL CaCI2 Time for clot formation measured 0.1 mL Thrombin Incubate at 37˚C 0.2 mL Diluted Plasma II Fibrinogen Fibrin Clot V II X VIII IX XI XII Intrinsic Pathway Common Pathway Fibrinogen Fibrin Clot ▲ Measurement of APTT: The APTT reagent contains a contact activator (e.g. silica, ellagic acid or kaolin) and phospholipids but does not contain tissue factor or calcium chloride. The intrinsic factors are “activated” when patient plasma is mixed with APTT reagent and incubated at 37° C. Calcium chloride is added and the time in seconds for the plasma to clot is measured ▲ Since the APTT reagent lacks tissue factor it is a “partial thromboplastin” and the test is called an activated partial thromboplastin time. ▲ The APTT is dependent on the reagent and instrument used and will vary between laboratories. A normal APTT is approximately 25-35 seconds. ▲ Measurement of TT: The patient’s plasma is warmed at 37° C and thrombin reagent is added. The time in seconds that it takes for the plasma to clot is measured ▲ The TT is dependent on the reagent and instrument used and will vary between laboratories. 14 15 Fibrinogen ▲ The fibrinogen assay assesses fibrinogen activity. ▲ Hypofibrinogenemia is usually acquired due to loss of fibrinogen (e.g. bleeding), consumption (e.g. hyperfibrinolysis after traumatic injury, disseminated intravascular coagulation) or decreased production (e.g. severe liver disease). Other rare causes include congenital hypofibrinogenemia and dysfibrinogenemia (an abnormal fibrinogen). ▲ Fibrinogen is an acute phase reactant and may be non-specifically elevated with acute or chronic inflammation. ▲ Measurement of fibrinogen based on the Clauss method: The plasma is diluted with a physiological buffer, warmed to 37° C and a high concentration of thrombin is added. The thrombin cleaves fibrinogen to fibrin monomers which polymerize. The time in seconds for the plasma to clot is measured. The time in seconds is inversely proportional to fibrinogen activity which is obtained from a standard calibration curve. The longer the clotting time, the lower the concentration of fibrinogen in the sample D-Dimer ▲ D-dimers are breakdown products generated by the action of plasmin on cross-linked fibrin. A D-dimer contains two cross-linked D fragments. 2. Routine Coagulation Tests 15 2. ROUTINE COAGULATION TESTS Fibrinogen (g/L) Clotting Time (seconds) Patient The Clauss fibrinogen activity is a standardized test as laboratories use a WHO calibrated plasma for the calibration curve. While there may be small differences in the reference ranges between laboratories, the reference range will be approximately 1.5-4 g / L. Fibrin Monomer Fibrin Polymer D D E D-dimer D-dimer E D D E Fibrinogen Plasmin D Cross-linked Fibrin Thrombin ▲ A negative D-dimer can be used to rule out venous thromboembolism (VTE) in selected outpatients (those with low to moderate clinical probability of VTE). Ideally, D-dimer should only be used as part of a validated VTE diagnostic algorithm. ▲ An elevated D-dimer is not specific to thrombosis and may be associated with a host of other non-specific diseases or inflammatory states (e.g. recent surgery or trauma, cancer, acute or chronic infectious or inflammatory diseases, disseminated intravascular coagulation, healthy elderly, normal pregnancy, etc.). ▲