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

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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.

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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

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Procedure

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

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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).

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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
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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
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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.

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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.

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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
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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.

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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.

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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.

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