General Approach of Haemostasis
MIXING STUDIES
Khoa Xet Nghiem Huyet Hoc
Mixing studies:

Mixing studies are tests performed on blood
plasma used to distinguish factor deficiencies
from factor inhibitors

Ex: lupus anticoagulant, or specific factor
inhibitors, such as antibodies directed against
factor VIII.
Mixing studies:


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 37°C will show prolongation.
Test method
• 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).
• Nonspecific inhibitors like the lupus
anticoagulant usually are not time dependent;
the immediate mixture will show
prolongation.
Reagents and Equipment
Pooled Plasma - platelet-poor plasma from 20
or more healthy, male and female adult donors.
o DO NOT use a single-sourced normal
plasma.
o Pooled plasma must be used to ensure
approximately 100% of all factors are
present.
o Do Not Use Lyophilized Normal Control.
 Other reagents required to perform the screen
test(s) (i.e., PT or PTT).
Reagents and Equipment

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.
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
37°C for 1 to 2 hours ( factor VIII inhibitor).
Table A Differentiation of Factor Deficiency and
Inhibitors By Mixing Studies
1:1 Mixing Study Results
Factor deficiency
Immediate acting inhibitor
Time/temperature
dependent inhibitor
Not incubated
Incubated
Correction
Correction
No correction
No correction
Correction
(Falsely)
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 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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