Measurement of D-dimer: There are several different assays available to measure D-dimer. These include qualitative (positive or negative), semi-quantitative or quantitative methods, such as ELISA (Enzyme Linked Immunosorbent Assay) or LIA (Latex Immunoassay) which use a monoclonal antibody to various epitopes of D-dimer. Quantitative D-dimer measurements obtained by the various assays are not standardized due to the variability in the monoclonal antibody used. D-dimer results must be interpreted based on the assay used. ▲ Reporting units vary between assays, e.g. DDU (D-dimer units) or FEU (Fibrinogen equivalent units). 2. ROUTINE COAGULATION TESTS 16 17 Anti-Xa assay ▲ An Anti-Xa assay can be used to measure the anticoagulant activity of an anticoagulant that inhibits clotting Factor Xa such as heparin, low molecular weight heparin (LMWH), fondaparinux or direct Xa inhibitors (rivaroxaban and apixaban). ▲ Measurement of Anti-Xa activity: A known amount of Factor Xa is added in excess to the plasma sample containing the drug. A complex forms between the drug and factor Xa. A chromogenic substrate is added which hydrolyses the unbound or “residual” factor Xa and the release of colour is measured at a specific wavelength as an optical density (OD). The OD is converted to a drug concentration reported in international units (IU) using a drug-specific calibration curve ▲ The therapeutic level of the anti-Xa is specific for the drug being assessed. Summary ▲ 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) ▲ The PT is most sensitive to the extrinsic and common pathway factors and the APTT to the intrinsic and common pathway factors. ▲ The thrombin time is most sensitive to fibrinogen and presence of inhibitors of thrombin (Factor IIa). ▲ The Anti-Xa assay only assesses the inhibition of factor Xa. 2. Routine Coagulation Tests 17 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 + ! Check that your coagulation laboratory has validated the anti-Xa for the anticoagulant that you wish to assay. A T T E N T I O N ! Reference ranges for common coagulation tests vary between laboratories due to instrument / reagent differences. Patient Plasma Excess Factor Xa Incubate Residual Factor Xa Add Chromogenic Substrate Calibration Curve Release of Colour (measured as optical density) + + Drug Drug Drug Drug Xa Xa 0.3 Anti-Xa (IU/mL) Optical Density (mABS/min) 1 2 0.5 Patient 18 19 3. ANTICOAGULANT DRUGS Yulia Lin & Rita Selby Unfractionated Heparin (UFH) ▲ Unfractionated heparin is a mixture of varying chain lengths of glycosaminoglycans derived from pig intestine. ▲ It is an “indirect” anticoagulant. It exerts its anticoagulant effect by combining with antithrombin (via 5 saccharide units – pentasaccharide) and inhibiting the coagulation factors IIa, Xa, IXa, XIa and XIIa. ▲ It can be administered either intravenously (IV) or subcutaneously (SC). ▲ UFH can be monitored using the APTT or the anti-Xa assay. ▲ Half-life is 60-90 minutes; half-life for SC heparin is longer. ▲ Elective reversal. • Discontinue IV unfractionated heparin 4 hours prior to the planned procedure ▲ Urgent reversal in the setting of significant bleeding. • Antidote: Protamine • Administer 1mg of protamine per 100 units of unfractionated heparin given in the last 2-2.5 hours • Adverse effects of protamine: hypotension, hypersensitivity Low Molecular Weight Heparins (LMWHs) ▲ LMWHs are produced by “fractionating” heparin molecules into smaller chain lengths. ▲ LMWHs are “indirect” anticoagulants. They exert their anticoagulant effect by combining with antithrombin (via 5 saccharide units – pentasaccharide) and inhibiting only the coagulation factors Xa and IIa. ▲ LMWHs are administered subcutaneously (SC). ▲ Several preparations are commercially available – dalteparin, enoxaparin, nadroparin, tinzaparin, etc. They vary in their relative inhibition of factors Xa and IIa also known as the Xa:IIa ratio. ▲ LMWHs