Coagulation%20Factor%20Assays - PowerPoint PPT Presentation

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Coagulation%20Factor%20Assays

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COAGULATION FACTOR ASSAYS ONE-STAGE QUANTITATIVE ASSAY METHOD FOR FACTORS II, V, VII, AND X Mr. Mohammed A. Jaber Principle The prothrombin time(PT) is the basis of ... – PowerPoint PPT presentation

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Title: Coagulation%20Factor%20Assays


1
Coagulation Factor Assays
One-Stage Quantitative Assay Method for Factors
II, V, VII, and X
  • Mr. Mohammed A. Jaber

2
Principle
  • The prothrombin time(PT) is the basis of this
    test system, with specific factor deficient
    plasmas -one stage- being added to the patient
    plasma.
  • The percentage of factor activity is determined
    by the amount of correction of the PT when
    specific dilutions of patient plasma are added to
    the factor-deficient plasma.
  • These results are obtained from an activity curve
    made using clotting times of dilutions of normal
    reference plasma and specific factor-deficient
    plasma.

3
Reagents and Equipment
  • Commercial thromboplastin
  • Specific factor-deficient plasma (II, V, VII, and
    X)
  • Note It is recommended that the factor-deficient
    plasma utilized be verified as having less than I
    activity for the specific factor being measured
    and close to 100 activity of alI other factors.
  • Imidazole buffered saline, pH 7.3 0.1 or
    Owren's buffer
  • Normal reference plasma (commercial reference
    plasma with known factor levels)
  • Instrument Same as that used for PT assay

4
Procedure
  1. Preparation of the activity curve.
  2. Procedure for testing patient plasma.

5
Preparation of the activity curve
  • Prepare 110, 120, 1 40, 180, 1160, 1320,
    1640, and 1 1280 serial dilutions of the normal
    reference plasma with imidazole-buffered saline
    or Owren' s buffer.
  • The 1 10 dilution is considered 100 factor
    activity.
  • It is recommended that at least five dilutions be
    used to prepare the factor activity curve,
    although it is common to use seven or eight
    dilutions (Table).

6
of Factor Dilution Buffered Saline (mL) Amount of plasma (mL) Tube No.
100 110 0.9 0.1 1
50 120 0.5 0.5 of tube no. 1 2
25 140 0.5 0.5 of tube no. 2 3
12.5 180 0.5 0.5 of tube no. 3 4
6.25 1160 0.5 0.5 of tube no. 4 5
3.13 1320 0.5 0.5 of tube no. 5 6
1.56 1640 0.5 0.5 of tube no. 6 7
0.78 11280 0.5 0.5 of tube no. 8 8
7
Preparation of the activity curve
  • Warm thromboplastin to 37C.
  • Perform the following test procedure on each
    dilution.
  • Note These steps are commonly performed by an
    automated coagulation analyzer.
  • Add 0.05 mL of specific factor-deficient plasma
    to 0.05 mL of the diluted normal reference plasma
    and warm to 37C for the allotted time based on
    the manufacturer's specifications.
  • Add 0.1 mL of commercial thromboplastin to the
    sample and determine the clotting time.
  • Testing may be performed either singly or in
    duplicate. If performing duplicate testing,
    repeat steps 1 and 2 on the duplicate sample and
    average results.

1. 50 µL of specific factor-deficient plasma
2. 50 µL of diluted normal reference plasma
3. 100 µL of PT reagent
8
Preparation of the activity curve
  • Plot results on 2 X 3 cycle log graph paper, with
    percent factor activity on the x-axis and seconds
    on the y-axis. Draw a best-fit line.
  • The curve will demonstrate a plateau at the least
    concentrated dilutions and should be plotted as
    such, demonstrating the end of sensitivity for
    the assay.
  • If using an automated analyzer, the curve is
    generally constructed internally and stored for a
    specified length of time.

9
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10
Procedure for testing patient plasma
  • Warm thromboplastin to 37C.
  • Prepare I 10 and I 20 dilutions of citrated
    patient plasma with imidazole-buffered saline or
    Owren's buffer. If a third dilution is desired,
    prepare a 1 40. It is important to keep samples
    and dilutions refrigerated until they are to be
    tested.
  • Add 0.05 mL of specific factor-deficient plasma
    to 0.05 mL of diluted patient plasma.

11
Procedure for testing patient plasma
  1. Add 0.1 mL of thromboplastin to the sample and
    determine the clotting time.
  2. Testing may be performed either singly or in
    duplicate. If performing duplicate testing,
    repeat steps 3) and 4) on the duplicate sample
    and average the results.
  3. Repeat steps 3), 4), and 5) on the 1 20 and
    .140 dilution of patient plasma, multiplying the
    measured result by 2 or 4 respectively to correct
    for the dilution ratio when compared with the I
    10 dilution. The results of the 1 10, 1 20 and
    140 dilutions should agree within 15. Report
    the average of the results.

1. 50 µL of specific factor-deficient plasma
2. 50 µL of diluted patient plasma
3. 100 µL of PT reagent
12
Procedure for testing patient plasma
  • Note
  • Inhibitors will often have a "dilutional" effect,
    demonstrating nonparallel curves with increasing
    dilutions.
  • This should be considered if the results of the
    1 10, 1 20 and 140 dilutions do not agree
    within 15. In this case, results should not be
    averaged, but further dilutions such as 1 80,
    and I 160 performed until results of two
    consecutive dilutions match within 15 and
    measure within linearity of the calibration
    curve.

13
Procedure for testing patient plasma
  • Read the percent activity directly from the
    activity curve (Fig.). From this curve, a result
    of 35 seconds on a 1 10 dilution of patient
    plasma would be interpreted as 8.3 activity. If
    the curve was generated using an automated
    coagulation analyzer, the results will
    automatically be read from the curve and printed
    out.
  • Note Specific volumes required for adding
    factor-deficient plasma, diluted patient plasma,
    and thromboplastin reagent may vary depending on
    the automated analyzer used.

14
Interpretation
  • An approximate range of 50 to 150 is considered
    normal, Each laboratory should define its own
    reference range based on instrument, reagent, and
    patient population.

15
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