Chapter 059. Bleeding and Thrombosis (Part 9)

Mixing StudiesMixing studies are used to evaluate a prolonged aPTT or, less commonly PT, to distinguish between a factor deficiency and an inhibitor. In this assay, normal plasma and patient plasma are mixed in a 50:50 ratio, and the aPTT or PT is determined immediately and after incubation at 37oC for varying times, typically 30, 60, and/or 120 min. With isolated factor deficiencies, the aPTT will correct with mixing and stay corrected with incubation. With aPTT prolongation due to a lupus anticoagulant, the mixing and incubation will show no correction. In acquired neutralizing factor antibodies, such as an acquired...
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Chapter 059. Bleeding and Thrombosis (Part 9) Chapter 059. Bleeding and Thrombosis (Part 9) Mixing Studies Mixing studies are used to evaluate a prolonged aPTT or, less commonlyPT, to distinguish between a factor deficiency and an inhibitor. In this assay,normal plasma and patient plasma are mixed in a 50:50 ratio, and the aPTT or PTis determined immediately and after incubation at 37oC for varying times,typically 30, 60, and/or 120 min. With isolated factor deficiencies, the aPTT willcorrect with mixing and stay corrected with incubation. With aPTT prolongationdue to a lupus anticoagulant, the mixing and incubation will show no correction.In acquired neutralizing factor antibodies, such as an acquired factor VIIIinhibitor, the initial assay may or may not correct immediately after mixing butwill prolong or remain prolonged with incubation at 37oC. Failure to correct withmixing can also be due to the presence of other inhibitors or interfering substancessuch as heparin, fibrin split products, and paraproteins. Specific Factor Assays Decisions to proceed with specific clotting factor assays will be influencedby the clinical situation and the results of coagulation screening tests. Precisediagnosis and effective management of inherited and acquired coagulationdeficiencies necessitate quantitation of the relevant factors. When bleeding issevere, specific assays are often urgently required to guide appropriate therapy.Individual factor assays are usually performed as modifications of the mixingstudy, where the patients plasma is mixed with plasma deficient in the factorbeing studied. This will correct all factor deficiencies to >50%, thus makingprolongation of clot formation due to a factor deficiency dependent on the factormissing from the added plasma. Testing for Antiphospholipid Antibodies Antibodies to phospholipids (cardiolipin) or phospholipid-binding proteins(β2-microglobulin and others) are detected by ELISA. When these antibodiesinterfere with phospholipid-dependent coagulation tests, they are termed lupusanticoagulants. The aPTT has variable sensitivity to lupus anticoagulants,depending in part on the aPTT reagents used. An assay utilizing a sensitive reagenthas been termed an LA-PTT. The dilute Russell Viper Venom test (dRVVT) andthe tissue thromboplastin time (TTI) are modifications of standard tests with thephospholipid reagent decreased, thus increasing the sensitivity to antibodies thatinterfere with the phospholipid component. The tests, however, are not specific forlupus anticoagulants, as factor deficiencies or other inhibitors also result inprolongation. Documentation of a lupus anticoagulant requires not onlyprolongation of a phospholipid-dependent coagulation test but also lack ofcorrection when mixed with normal plasma and correction with the addition ofactivated platelet membranes or certain phospholipids, e.g., hexagonal phase. Other Coagulation Tests The thrombin time and the reptilase time measure fibrinogen conversion tofibrin and are prolonged when the fibrinogen level is low (usually Laboratory assays to detect thrombophilic states include moleculardiagnostic, immunologic and functional assays. These assays vary in theirsensitivity and specificity for the condition being tested. Furthermore, acutethrombosis, acute illnesses, inflammatory conditions, pregnancy, and medicationsaffect levels of many coagulation factors and their inhibitors. Antithrombin isdecreased by heparin and in the setting of acute thrombosis. Protein C and S levelsmay be increased in the setting of acute thrombosis and are decreased by warfarin.Antiphospholipid antibodies are frequently transiently positive in acute illness. Asthrombophilia evaluations are usually performed to assess the need to extendanticoagulation, testing should be performed in a steady state, remote from theacute event. In most instances warfarin anticoagulation can be stopped after theinitial 3–6 months of treatment, and testing is performed at least 3 weeks later.Furthermore, sensitive markers of coagulation activation, notably the D-dimerassay and the thrombin generation test, hold promise as predictors, when elevated,of recurrent thrombosis when measured at least 1 month from discontinuation ofwarfarin, although further study is needed to better support this application. Measures of Platelet Function The bleeding time has been used to assess bleeding risk; however, it has notbeen found to predict bleeding risk with surgery, and it is not recommended foruse for this indication. The PFA-100 and similar instruments that measure platelet-dependent coagulation under flow conditions are generally more sensitive andspecific for platelet disorders and vWD than ...