Hi, Using the ACL 9000, we have seen some low results for the factor VIII activity test, as low as 0.3%. In those instances we were told to dilute the sample 1:2, and retest. On those retests we get the same results, should we report the result as it is or with a less than something value? Miguel A. Vélez MT (ASCP) Immuno Reference Laboratory.
Hello, again, Mr. Vélez, and thank you for your question. Operators typically perform the factor VIII assay on three or four dilutions. They prepare a primary dilution of 1:10 in Veronal or Owren phosphate buffer and further dilute by (for example) 1:2, 1:4, and 1:8, making the final dilutions 1:10, 1:20, 1:40, and 1:80. They assay all four dilutions and multiply the initial assay results by the dilution factors, 1, 2, 4, and 8, and check for parallel results. Most operators consider results that match within ±10% to be parallel. Non-parallel results indicate a plasma inhibitor, which requires follow-up. Most automated coagulometers are programmed to perform factor VIII (and other factor) assays at these or similar dilutions and to provide the necessary computations.
Further, most operators re-assay plasmas that yield results <10% or perhaps <5% for greater sensitivity, using established laboratory protocol. For the re-assay, they typically prepare a 1:5 dilution in place of the standard 1:10, then divide the initial assay result by 2 for a final result. Many instruments are programmed to do this reflexively, providing accurate results in severe hemophilia A; such results help to guide therapy. They could also conceivably perform the assay at 1:2 and divide the initial assay result by 5, though the accuracy suffers as the dilution factor rises.
Clinically, a factor VIII activity level <1% is classified as severe hemophilia A. Levels from 1–5% are moderate, and 5–30% are mild. These categories help define the treatment approach to hemophilia symptoms. There is little clinical reason for providing greater sensitivity when the result is <1%, as the treatment would be unchanged, thus most operators report 1% or <1%, but not fractional results.
For additional information, factor assay methods are provided in greater detail in Audio Module 24, “Factor Assays.” I hope this helps! Geo.