Mixing Studies-aPTT or PT 1:1 Mix

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General Approach of Haemostasis
Lecture 7 Mixing Studies
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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

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Test method

• The patient plasma is mixed 11 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.

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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).

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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.

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Procedure

• Prepare a 12 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.

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• 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.
• Mix patient plasma with pooled normal plasma in
  equal volumes in a plastic test tube. In two
  separate tubes, pipette a volume of patient
  plasma and a volume of pooled normal plasma.
• Incubate all 3 tubes for 1 to 2 hours at 37C.
• Combine the incubated patient plasma tube and the
  incubated pooled normal plasma and use as the
  control tube.
• Measure the APTT or PT for the mixed and
  incubated tube, and the control tube.

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Values Expected
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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).

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Interpretation
Table A Differentiation of Factor Deficiency and
Inhibitors By Mixing Studies
11 Mixing Study Results 11 Mixing Study Results
Not incubated Incubated
Factor deficiency Correction Correction
Immediate acting inhibitor No correction No correction
Time/temperature dependent inhibitor Correction (Falsely) No correction
Table adapted from McKenzie, S.,, Clinical l
Laboratory Hematology, 2004, p. 790.
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Possible Interpretations
Coagulation Screen Results PT prolonged
PT mixing study results PT corrects
Most likely interpretation Factor VII deficiency
Probable cause(s) Early response to warfarin, early vitamin K deficiency
Rare cause Congenital factor VII deficit

Coagulation Screen Results PTT prolonged
PTT mixing study results PTT corrects
Most likely interpretation Factor deficit
Probable cause(s) Factor VIII or IX (male) deficiency, or von Willebrand Disease (female)
Possible cause Factor inhibitor

Coagulation Screen Results PTT markedly prolonged (gt200 seconds)
PTT mixing study results PTT corrects
Most likely interpretation Severe Contact Factor deficit
Probable cause(s) Factor Prekallikrein, HMWK, XI, or XII

Coagulation Screen Results PT and PTT prolonged
PT PTT mixing study results PT and PTT correct
Most likely interpretation Acquired, multiple factor deficiency
Probable cause(s) DIC, Liver Disease, Vitamin K deficiency
Possible cause Warfarin therapy

Coagulation Screen Results PTT slightly moderately prolonged
PTT mixing study results No correction
Most likely interpretation Immediately reacting antibody inhibitor
Probable cause(s) Lupus anticoagulant

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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.

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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 11 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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