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One-Stage Quantitative

One-Stage Quantitative. Assay Method for Factors VIII, IX, XI, and XII. Principle. This method is based on the ability of patient plasma to correct specific factor-deficient plasma as determined by the APTT test. Results in percent activity are obtained from an activity curve.

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One-Stage Quantitative

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  1. One-Stage Quantitative Assay Method for Factors VIII, IX, XI, and XII

  2. Principle This method is based on the ability of patient plasma to correct specific factor-deficient plasma as determined by the APTT test. Results in percent activity are obtained from an activity curve.

  3. Reagents and Equipment • APTT reagent • 0.02 M CaCl2 (0.025 M for some systems) • Specific factor-deficient plasma (VIII, IX, XI, and XII) • Normal reference plasma (commercial reference plasma with known factor levels) • Imidazole-buffered saline, pH 7.3 ± 0.1 • Instrument for APTT assay

  4. Procedure Preparation of activity curve Procedure for testing patient plasma

  5. Preparation of activity curve: • Prepare 1: 10, 1:20, 1 :40, 1:80, 1 :60, 1:320, I :640, and 1: 1280 serial dilutions of the normal reference plasma with imidazole-buffered saline (see Table). The1: 10 dilution is considered 100% factor activity. The calibration curve may be prepared automatically by a coagulation analyzer. • Prewarmthe CaCl2 and APTT reagent to 37°C. Note: These steps are commonly performed by an automated coagulation analyzer.

  6. Perform the following test procedure on each dilution. • Add 0.05 mL of specific factor-deficient plasma and 0.05 mL of diluted normal reference plasma to 0.05 mL of APTT reagent. Mix well and incubate for the specified time according to reagent manufacturer's instructions. • Add 0.05 mL of CaCl2 into the mixture at the end of the incubation time 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 the results.

  7. Results are plotted on a log-log graph paper • Results are plotted on a log-log graph, with percent factor on the x-axis and seconds on the y-axis, and a regression line determined. The curve will demonstrate a plateau at the least concentrated dilutions and should be plotted as such, demonstrating the limit of sensitivity for the assay.

  8. Procedure for testing patient plasma: • These steps are commonly performed automatically via a coagulation analyzer. • Prewarm CaCl2and APTT reagent to 37°C. • Prepare 1: 10 and 1:20 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 and 0.05 mL of diluted patient plasma to 0.05 ml; APTT reagent. Mix well and incubate for the recommended time.

  9. Procedure • Add 0.05 mL of CaC12 into the mixture at the end of specified time and determine the clotting time. • Testing may be performed either singly or in duplicate. If performing duplicate testing, repeat steps c and d on the duplicate sample and average the results. • Repeat steps c, d, and e on the I :20 and 1 :40 dilution of patient plasma, multiplying the measured result by 2 or 4

  10. Procedure • respectively to correct for the dilution ratio when com-pared with the 1:10 dilution . The results of the 1:10, 1:20 and 1:40 dilutions should agree within 15%. Report the average of the results. 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 1:160 performed until two consecutive dilutions match each other within 15% and are within the measurable linearity of the curve.

  11. Procedure • Read the percent activity directly from the activity curve. If using an automated analyzer, the results may be automatically read from the curve and printed out. Note: Specific volumes required for adding factor-deficient plasma, diluted patient plasma, and APTT reagent may vary depending on the automated analyzer used. The volumes previously recommended may be used if performing the test manually.

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

  13. Comment • If the result from the 1 :20 dilution is greater than the highest curve point (i.e., 1: 10), prepare additional dilutions of the patient sample with buffered saline until results fall within the linearity range of the curve. If the result from the 1: 10 dilution is lower than the lowest curve point (i.e., 1: 1280), report the factor activity as less than the lowest calibrator value. • To calculate the percent activity for additional dilutions tested, multiply the measured result by the dilution ratio of the sample to the 1:10 dilution (e.g., 1:40/1:10 = dilution factor of 4) to determine the percent activity of the patient sample.

  14. Comment • These tests require the same considerations as the APTT and PT assay in regard to quality control, specimen handling, reagent preparation, and points of procedural importance.

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