Mixing

Studies-
aPTT or PT 1:1 Mix
 Principle
• The APTT and/or PT are useful as screening tests for
distinguishing between factor deficiencies and
coagulation inhibitors.
• When patient plasma is mixed with pooled normal
plasma whose factor levels are approximately 100%,
there should be sufficient factor present (at least
50% normal level) to correct a prolongation caused
by a factor deficiency. There is only partial or no
correction in the presence of an inhibitor.
• Mixing studies can help determine the appropriate
next steps to take to diagnose the cause of an
abnormal APTT or PT
 Reagents and Equipment

• APTT or PT reagent
• CaCl2 (0.02 or 0.025 M)-for aPTT mixing
study
• Pooled normal plasma
 Procedure
1. Collect blood by clean venipuncture technique according to
recommended procedures previously described.
2. Process and store the plasma samples following
recommended guidelines.
3. Prepare the reagents according to the manufacturer's
recommendations
4. Perform the APTT or PT on patient plasma according to the
manufacturer's recommendations.
5. Mix the patient plasma 1: 1 with pooled normal plasma.
6. Repeat the APTT or PT on the mixture.
7. Compare the results with the patient's baseline APTT or PT
Procedure
 Notes:

1. Repeating the mixing study with 4 parts patient

sample and 1 part normal pooled plasma may
increase the chance of detecting a weak
inhibitor.
2. If a time-dependent inhibitor is suspected,
incubated mixing studies should also be
performed.
PROCEDURE: Incubated Mixing Studies

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.
Incubated Mixing Studies
 Interpretation

• If the APTT or PT is corrected by normal plasma, a factor

deficiency is indicated. The addition of normal plasma sup-
plies the coagulation factor or factors that are deficient, and
thus corrects the APTT or PT.
• 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. On incubation, a weak inhibitor progressively
inactivates the coagulation factor, thus prolonging the APTT or
PT This time-dependent pattern is most typical of a factor VIII
inhibitor.
 Comment
• The antibody that inhibits factor VIII is
most often a specific IgG antibody.
• These antibodies are often present as
weak inhibitors but are temperature and
time dependent, thus causing only a
slightly prolonged APTT on initial testing.
• Mixing tests may yield APTT results
intermediate between the clotting times of
patient and normal control.
 Comment
• On incubation at 37°C, both the undiluted
patient plasma and plasma mixtures show
prolonged times, but the normal pooled
plasma shows little or no change. Because
of the nature of the factor VIII inhibitor, the
mixture of patient plasma and normal
pooled plasma must be incubated for 60 to
120 minutes to allow for the inhibitor's
progressive activity.
 Comment

• 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