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Title: Mixing Studies-aPTT or PT 1:1 Mix Author: mohammed h. jaber Last modified by: AVENIS Created Date: 8/16/2006 12:00:00 AM Document presentation format – PowerPoint PPT presentation

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Title: Mixing%20Studies-aPTT%20or%20PT%201:1%20Mix


1
General Approach of Haemostasis MIXING
STUDIES
Khoa Xet Nghiem Huyet Hoc
2
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.

3
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

4
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.

5
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6
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.

7
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.

8
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).

9
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.

10
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11
Values Expected
12
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13
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).

14
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.
15
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

16
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.

17
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.

18
Thank you
http//site.iugaza.edu.ps/ialaswad/ http//site.i
ugaza.edu.ps/wael
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