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General Approach of Haemostasis MIXING STUDIES

General Approach of Haemostasis MIXING STUDIES. Khoa Xet Nghiem Huyet Hoc. Mixing studies:. Mixing studies are tests performed on blood plasma used to distinguish factor deficiencies from factor inhibitors

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General Approach of Haemostasis MIXING STUDIES

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  1. General Approach of Haemostasis MIXING STUDIES Khoa Xet Nghiem Huyet Hoc

  2. Mixing studies: • Mixing studies are tests performed on blood plasma used to distinguish factor deficiencies from factor inhibitors • Ex: lupus anticoagulant, or specific factor inhibitors, such as antibodies directed against factor VIII.

  3. Mixing studies: • Mixing studies take advantage of the fact that factor levels that are 50 percent of normal should give a normal Prothrombin time (PT) or Partial Thromboplastins time • Mixing studies can help determine the appropriate next steps to take to diagnose the cause of an abnormal APTT or PT

  4. Test method • The patient plasma is mixed 1:1 with Normal pooled plasma • That contains 100% of the normal factor level results in a level ≥ 50% in the mixture (say the patient has an activity of 0%; the average of 100% + 0% = 50%). • Therefore, correction with mixing indicates factor deficiency; failure to correct indicates an inhibitor.

  5. Test method • Some inhibitors are time dependent. • The clotting test performed immediately after the specimens are mixed may show correction because the antibody has not had time to inactivate the added factor (false positive). • A test performed after the mixture is incubated for 2 hours at 37°C will show prolongation.

  6. Test method • Many specific factor inhibitors are time dependent, and the inhibitor will not be detected unless the test is repeated after incubation (factor VIII inhibitors are notorious for this). • Nonspecific inhibitors like the lupus anticoagulant usually are not time dependent; the immediate mixture will show prolongation.

  7. Reagents and Equipment • Pooled Plasma - platelet-poor plasma from 20 or more healthy, male and female adult donors. • DO NOT use a single-sourced normal plasma. • Pooled plasma must be used to ensure approximately 100% of all factors are present. • Do Not Use Lyophilized Normal Control. • Other reagents required to perform the screen test(s) (i.e., PT or PTT).

  8. Reagents and Equipment • Quality Control The pooled plasma must be evaluated for the test to be performed and results must fall within the reference range or testing is repeated with a fresh aliquot of the pooled plasma.

  9. ValuesExpected

  10. Interpretation • The first step when evaluating unexpected prolonged PT or PTT results is to rule out preanalytical interference, e.g., presence of contaminating heparin. • If the APTT or PT is corrected by normal plasma, a factor deficiency is indicated. • If the APTT or PT is not corrected by the addition of nor-mal plasma immediately, a strong inhibitor is indicated. • A weak or time-dependent inhibitor is indicated by a prolonged APTT or PT following incubation at 37°C for 1 to 2 hours ( factor VIII inhibitor).

  11. Table A Differentiation of Factor Deficiency and Inhibitors By Mixing Studies Table adapted from McKenzie, S.,, Clinical l Laboratory Hematology, 2004, p. 790.

  12. PossibleInterpretations

  13. Comment • The antibody that inhibits factor VIII is most often a specific IgG antibody (temperature and time dependent) , which will cause only a slightly prolonged APTT on initial testing. • If a factor VIII inhibitor is present, it is important to determine the initial level of factor activity because the development of an inhibitor complicates the management of a patient with hemophilia A when therapy involves AHF* concentrates. These should be monitored periodically. • Repeating the mixing study with 4 parts patient sample and 1 part normal pooled plasma may increase the chance of detecting a weak inhibitor.

  14. Notes: • Be careful when thawing the pooled plasma because prolonged incubation at 37°C will selectively decrease Factor V, prolonging the results and making interpretation of the 1:1 mix test results difficult. • The pooled normal plasma is stable for ~2 hours at room temperature. Initial test results for the pooled normal plasma must be within the reference range or the mix should be repeated with a fresh aliquot of pooled normal plasma.

  15. Next Lecture: Coagulation-instruments Thankyou http://site.iugaza.edu.ps/ialaswad/ http://site.iugaza.edu.ps/wael

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