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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 this test system, with specific factor deficient plasmas -one stage- being added to the patient plasma.
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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 prothrombintime(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.
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'sbuffer • Normal reference plasma (commercial reference plasma with known factor levels) • Instrument: Same as that used for PT assay
Procedure • Preparation of the activity curve. • Procedure for testing patient plasma.
Preparation of the activity curve • Prepare 1:10, 1:20, 1 :40, 1:80, 1:160, 1:320, 1:640, 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).
Preparation of the activity curve • Warm thromboplastin to 37°C. • 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 37°C 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
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.
Procedure for testing patient plasma • Warm thromboplastin to 37°C. • 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.
Procedure for testing patient plasma • Add 0.1 mL of thromboplastin to the sample and determine the clotting time. • 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. • Repeat steps 3), 4), and 5) on the 1 :20 and .1:40 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 1:40 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
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 1:40 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.
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 thromboplastinreagent may vary depending on the automated analyzer used.
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.