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
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 37C 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.
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.
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.
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 37C.
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.
Expected Values
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 37C for 1 to 2 hours ( factor VIII inhibitor).
Interpretation

Table A Differentiation of Factor Deficiency and Inhibitors By Mixing Studies

1:1 Mixing Study Results

Not incubated Incubated

Factor deficiency Correction Correction

Immediate acting inhibitor No correction No correction

Time/temperature Correction
No correction
dependent inhibitor (Falsely)
Table adapted from McKenzie, S.,, Clinical l Laboratory Hematology, 2004, p. 790.
Possible Interpretations
Coagulation Screen Results:
PT mixing study results:
Most likely interpretation:
Probable cause(s):
Rare cause:
Coagulation Screen Results:
PTT mixing study results:
Most likely interpretation:
Probable cause(s):
Possible cause
Coagulation Screen Results:
PTT mixing study results:
Most likely interpretation:
Probable cause(s):
Coagulation Screen Results:
PT & PTT mixing study results:
Most likely interpretation:
Probable cause(s):
Possible cause:
Coagulation Screen Results:
PTT mixing study results:
Most likely interpretation:
Probable cause(s):
PT prolonged
PT corrects
Factor VII deficiency
Early response to warfarin, early vitamin K deficiency
Congenital factor VII deficit
PTT prolonged
PTT corrects
Factor deficit
Factor VIII or IX (male) deficiency, or von Willebrand Disease (female)
Factor inhibitor
PTT markedly prolonged (>200 seconds)
PTT corrects
Severe Contact Factor deficit
Factor Prekallikrein, HMWK, XI, or XII
PT and PTT prolonged
PT and PTT correct
Acquired, multiple factor deficiency
DIC, Liver Disease, Vitamin K deficiency
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.
Notes:
Be careful when thawing the pooled plasma because
prolonged incubation at 37C 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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Next Lecture: Coagulation-instruments