1. General Approach of Haemostasis
Lecture 7:
Mixing Studies

2. 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

3. 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.

4. 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).

5. 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).
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.

6. 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.

7. 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.

9. Values Expected

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. 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.

12. 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

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

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.

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