GREEN FIEND OneStage Quantitative Assay Method for Factors

GREEN FIEND One-Stage Quantitative Assay Method for Factors VIII, IX, XI, and XII 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND 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. 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND Reagents and Equipment APTT reagent 0. 02 M Ca. Cl 2 (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, p. H 7. 3 ± 0. 1 • Instrument for APTT assay • • 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND PROCEDURE 1. Preparation of activity curve 2. Procedure for testing patient plasma 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND 1. Preparation of activity curve: A. 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). The 1: 10 dilution is considered 100% factor activity. The calibration curve may be prepared automatically by a coagulation analyzer. B. Prewarm the Ca. Cl 2 and APTT reagent to 37°C. Note: These steps are commonly performed by an automated coagulation analyzer. 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND C. Perform the following test procedure on each dilution. 1) Add 0. 05 m. L of specific factor-deficient plasma and 0. 05 m. L of diluted normal reference plasma to 0. 05 m. L of APTT reagent. Mix well and incubate for the specified time according to reagent manufacturer's instructions. 2) Add 0. 05 m. L of Ca. Cl 2 into the mixture at the end of the incubation time and determine the clotting time. 3) 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. 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND Results are plotted on a log-log graph paper D. 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. 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND 2. Procedure for testing patient plasma: – These steps are commonly performed automatically via a coagulation analyzer. 1. Prewarm Ca. Cl 2 and APTT reagent to 37°C. 2. 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. 3. Add 0. 05 m. L of specific factor-deficient plasma and 0. 05 m. L of diluted patient plasma to 0. 05 ml; APTT reagent. Mix well and incubate for the recommended time. 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND Procedure 4. Add 0. 05 m. L of Ca. C 12 into the mixture at the end of specified time and determine the clotting time. 5. 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. 6. 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 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND 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. 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND Procedure G. 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 factordeficient 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. 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND 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. 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND Comment 1. 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. 2. 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. 1234 Sample Street, Anytown, St. 12345 GREENFIEND

GREEN FIEND Comment 3. 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. 1234 Sample Street, Anytown, St. 12345 GREENFIEND