General Approach of Haemostasis Lecture 7: Mixing Studies

Mixing studies: Mixing studies are tests performed on blood plasma used to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant, or specific factor inhibitors, such as antibodies directed against factor VIII. 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 coagulation screening tests are relatively insensitive to protein concentration

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.

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. Nonspecific inhibitors like the lupus anticoagulant usually are not time dependent; the immediate mixture will show prolongation. 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).

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

Procedure Prepare a 1:2 dilution of patient plasma using pooled plasma as the diluents, by mixing equal volumes of each of the plasmas. (make sufficient quantities to run the test in duplicate) Label two test tubes for each test plasma to be re-tested (Mixture, NPP) Add 0.1 ml of patient plasma to 0.1 ml of NPP in one of the two labeled tube Carefully mix the plasmas using the pipette, aspirating and expelling the solution several times (avoid making bubbles). Transfer 0.1 mL of the diluted patient plasma to the second labeled test tube. Measure the APTT or PT for the mixed and incubated tube, and the control tube.

In cases where time and temperature dependent inhibitors are suspected, repeat testing should also be performed on incubated mixes: patient plasma – pooled plasma mix incubated for 1 to 2 hours at 37° C prior to testing. Mix patient plasma with pooled normal plasma in equal volumes in a plastic test tube. In two separate tubes, pipet a volume of patient plasma and a volume of pooled normal plasma. Incubate all 3 tubes for 1 to 2 hours at 37°C. Combine the incubated patient plasma tube and the incubated pooled normal plasma and use as the control tube. Measure the APTT or PT for the mixed and incubated tube, and the control tube.

Values Expected

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).

Interpretation 1:1 Mixing Study Results Not incubated Correction Table A Differentiation of Factor Deficiency and Inhibitors By Mixing Studies 1:1 Mixing Study Results Not incubated Incubated Factor deficiency Correction Immediate acting inhibitor No correction Time/temperature dependent inhibitor Correction (Falsely) Table adapted from McKenzie, S.,, Clinical l Laboratory Hematology, 2004, p. 790.

Possible Interpretations Coagulation Screen Results: PT prolonged PT mixing study results: PT corrects Most likely interpretation: Factor VII deficiency Probable cause(s): Early response to warfarin, early vitamin K deficiency Rare cause: Congenital factor VII deficit PTT prolonged PTT mixing study results: PTT corrects Factor deficit Factor VIII or IX (male) deficiency, or von Willebrand Disease (female) Possible cause Factor inhibitor PTT markedly prolonged (>200 seconds) Severe Contact Factor deficit Factor Prekallikrein, HMWK, XI, or XII PT and PTT prolonged PT & PTT mixing study results: PT and PTT correct Acquired, multiple factor deficiency DIC, Liver Disease, Vitamin K deficiency Possible cause: Warfarin therapy PTT slightly – moderately prolonged No correction Immediately reacting antibody inhibitor Lupus anticoagulant

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. *Antihemophilic factor (AHF ) : A commercially prepared source of factor VIII.

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.

Thank you http://site.iugaza.edu.ps/ialaswad/ http://site.iugaza.edu.ps/wael Next Lecture: Coagulation-instruments