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. Pooled plasma must be used to ensure

approximately 100% of all factors are present.

DO NOT Use a single-sourced normal plasma.
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.

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

Mixing Study Results 1:1

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.

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Next Lecture: Coagulation-instruments