One Stage Factor V Assay General Approach in Investigation of Haemostasis Lecture 10: One Stage Factor V Assay

One Stage Assay When Prothrombin time (PT) and activated partial Thromboplastins time (APTT) are prolonged, a one- stage assay is used to detect a deficiency of factor II, factor V, or factor X. If PT is abnormal but APTT is normal, factor VII may be deficient. 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.

Principle: The assay of a clotting factor relies upon measuring the degree of correction of the Prothrombin time (PT) when plasma is added to a plasma sample specifically deficient in the factor to be measured.

Reagents and Equipment PT / PTT Reagent Specific factor-deficient plasma (II, V, VII, and X) Imidazole buffered saline, pH 7.3 ± 0.1 Normal reference plasma (commercial reference plasma with known factor levels) Note: It is recommended that the factor-deficient plasma utilized be verified as having less than 1% activity for the specific factor being measured and close to 100% activity of all other factors.

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. 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).

% of Factor Dilution Buffered Saline (mL) Amount of plasma (mL) Tube No. 100 1:10 0.9 0.1 1 50 1:20 0.5 0.5 of tube no. 1 2 25 1:40 0.5 of tube no. 2 3 12.5 1:80 0.5 of tube no. 3 4 6.25 1:160 0.5 of tube no. 4 5 3.13 1:320 0.5 of tube no. 5 6 1.56 1:640 0.5 of tube no. 6 7 0.78 1:1280 0.5 of tube no. 8 8

Preparation of the activity curve Warm Thromboplastins to 37°C. Perform the following test procedure on each dilution. 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 2 min. Add 0.2 mL of commercial Thromboplastins to the sample and determine the clotting time. Preparation of the activity curve

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. Preparation of the activity curve

A graph shows the results of a 1-stage PT-based factor X assay with varying factor X levels - note the parallel lines. The reference plasma is shown in red. The axes are both logarithmic and the dilutions are plotted on the X-axis and the clotting time in seconds on the Y axis.

Procedure for testing patient plasma Warm Thromboplastins to 37°C. Prepare 1:10 and 1:20 dilutions of citrated patient plasma with Imidazole-buffered saline. If a third dilution is desired, Prepare 1:5 Dilution for test plasma expected to have reduced level and 1:40 dilution for test plasma expected to be normal Add 0.05 mL of specific factor-deficient plasma to 0.05 mL of diluted patient plasma. Add 0.2 mL of Thromboplastins to the sample and determine the clotting time.

Procedure for testing patient plasma 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. Read the percent activity directly from the activity curve. A “Blank” should also be tested as follows: 0.1 ml buffer , 0.1 ml Factor V deficient plasma The clotting time for the blank reflects the quality of the deficient plasma and should be equivalent to less than 1%. 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. Specific volumes required for adding factor-deficient plasma, diluted patient plasma, and Thromboplastins reagent may vary depending on the automated analyzer used. Procedure for testing patient plasma

Reference Ranges The reference ranges for the factors are as follows: factor II: 80% to 120% of normal factor V: 50% to 150% of normal factor VII: 65% to 140% of normal factor X: 45% to 155% of normal. Laboratories should establish their own reference ranges although it is probable that many do not and choose to use either published ranges or those provided with a commercial standard.

Discussion If you find a low FV result (and similarly if you find a low FVIII result) you should request a FVIII (or FV) assay to exclude these rare autosomally inherited disorders. If you find a low FVII result make sure that the test was performed using human TF in the PT. Some FVII gene mutations e.g. FVII Padua can give rise to varying factor levels depending upon the source of TF used in the PT. Wherever possible, human recombinant TF should be employed as this gives a result that more closely relates to the situation found in vivo. If you find a low level of a vitamin K dependent clotting factor - consider the possibility that that the patient is on warfarin, has true vitamin K deficiency or may have a mutation within the genes involved in encoding the proteins involved in the vitamin K cycle. Remember - PT-based factor assays will be reduced in patients with vitamin K deficiency and in patients on oral vitamin K antagonists such as warfarin.

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