Mixing Studies- aPTT or PT 1:1 Mix

1. Mixing Studies-aPTT or PT 1:1 Mix

2. Principle • The APTT and/or PT are useful as screening tests for distinguishing between factor deficiencies and coagulation inhibitors. • When patient plasma is mixed with pooled normal plasma whose factor levels are approximately 100%, there should be sufficient factor present (at least 50% normal level) to correct a prolongation caused by a factor deficiency. There is only partial or no correction in the presence of an inhibitor. • Mixing studies can help determine the appropriate next steps to take to diagnose the cause of an abnormal APTT or PT

3. Reagents and Equipment • APTT or PT reagent • CaCl2 (0.02 or 0.025 M)-for aPTT mixing study • Pooled normal plasma

4. Procedure • Collect blood by clean venipuncture technique according to recommended procedures previously described. • Process and store the plasma samples following recommended guidelines. • Prepare the reagents according to the manufacturer's recommendations • Perform the APTT or PT on patient plasma according to the manufacturer's recommendations. • Mix the patient plasma 1: 1 with pooled normal plasma. • Repeat the APTT or PT on the mixture. • Compare the results with the patient's baseline APTT or PT

5. Procedure

6. Notes: • Repeating the mixing study with 4 parts patient sample and 1 part normal pooled plasma may increase the chance of detecting a weak inhibitor. • If a time-dependent inhibitor is suspected, incubated mixing studies should also be performed.

7. PROCEDURE: Incubated Mixing Studies • 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.

8. Incubated Mixing Studies

9. Interpretation • If the APTT or PT is corrected by normal plasma, a factor deficiency is indicated. The addition of normal plasma sup-plies the coagulation factor or factors that are deficient, and thus corrects the APTT or PT. • 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. On incubation, a weak inhibitor progressively inactivates the coagulation factor, thus prolonging the APTT or PT This time-dependent pattern is most typical of a factor VIII inhibitor.

10. Comment • The antibody that inhibits factor VIII is most often a specific IgGantibody. • These antibodies are often present as weak inhibitors but are temperature and time dependent, thus causing only a slightly prolonged APTT on initial testing. • Mixing tests may yield APTT results intermediate between the clotting times of patient and normal control.

11. Comment • On incubation at 37°C, both the undiluted patient plasma and plasma mixtures show prolonged times, but the normal pooled plasma shows little or no change. Because of the nature of the factor VIII inhibitor, the mixture of patient plasma and normal pooled plasma must be incubated for 60 to 120 minutes to allow for the inhibitor's progressive activity.

12. Comment • 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