Mixing studies involve mixing a patient's plasma sample with normal pooled plasma in a 1:1 ratio. This distinguishes between factor deficiencies and inhibitors. If the abnormal coagulation test corrects to normal with mixing, it indicates a factor deficiency. If there is no correction, it suggests an inhibitor. Some inhibitors are time-dependent, so tests are repeated after incubation. Mixing studies help determine the cause of abnormal coagulation test results and guide further diagnosis and treatment.

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

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.  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).
Test
method

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.
1. 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.
2. Incubate all 3 tubes for 1 to 2 hours at 37°C.
3. Combine the incubated patient plasma tube and the
incubated pooled normal plasma and use as the control tube.
4. 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 Incubated
Factor deficiency Correction Correction
Immediate acting inhibitor No correction No correction
Time/temperature
dependent inhibitor
Correction
(Falsely)
No correction
Table A Differentiation of Factor Deficiency and Inhibitors By Mixing Studies
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
Coagulation Screen Results: PTT prolonged
PTT mixing study results: PTT corrects
Most likely interpretation: Factor deficit
Probable cause(s): Factor VIII or IX (male) deficiency, or von Willebrand Disease (female)
Possible cause Factor inhibitor
Coagulation Screen Results: PTT markedly prolonged (>200 seconds)
PTT mixing study results: PTT corrects
Most likely interpretation: Severe Contact Factor deficit
Probable cause(s): Factor Prekallikrein, HMWK, XI, or XII
Coagulation Screen Results: PT and PTT prolonged
PT & PTT mixing study results: PT and PTT correct
Most likely interpretation: Acquired, multiple factor deficiency
Probable cause(s): DIC, Liver Disease, Vitamin K deficiency
Possible cause: Warfarin therapy
Coagulation Screen Results: PTT slightly – moderately prolonged
PTT mixing study results: No correction
Most likely interpretation: Immediately reacting antibody inhibitor
Probable cause(s): 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.

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
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