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.

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 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
Immediate acting
inhibitor
Time/temperature
dependent inhibitor

Correction
No correction
Correction
(Falsely)

Correction
No
correction
No
correction

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:

PT prolonged
PT corrects
Factor VII deficiency
Early response to warfarin, early vitamin K deficiency
Congenital factor VII deficit

Coagulation Screen Results:

PTT mixing study results:
Most likely interpretation:

PTT prolonged
PTT corrects
Factor deficit

Probable cause(s):
Possible cause

Factor VIII or IX (male) deficiency, or von Willebrand Disease (female)

Factor inhibitor

Coagulation Screen Results:

PTT mixing study results:
Most likely interpretation:
Probable cause(s):

PTT markedly prolonged (>200 seconds)

PTT corrects
Severe Contact Factor deficit
Factor Prekallikrein, HMWK, XI, or XII

Coagulation Screen Results:

PT & PTT mixing study results:
Most likely interpretation:
Probable cause(s):
Possible cause:

PT and PTT prolonged

PT and PTT correct
Acquired, multiple factor deficiency
DIC, Liver Disease, Vitamin K deficiency
Warfarin therapy

Coagulation Screen Results:

PTT mixing study results:
Most likely interpretation:
Probable cause(s):

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.