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Assay of Factor VIII coagulant activity (VIII: C) in Factor VIII concentrates has since long met difficulties, such as l) non-paralleility of dose-response curves of plasma standard and Factor VIII concentrate, 2) spuriously low values of VIII: C in concentrates as revealed by abnormally high in vivo recoveries after transfusion, and 3) large interlaboratory variation in assay results. In an attempt to analyze the cause of these problems several parameters of the one-stage assay system were varied systematically and their effect on the parallellity of dose-response curves and on the final VIII: C value was analyzed. Nonparallel1ity was partially corrected with a protein-rich dilution medium, and almost always completely with undiluted instead of 1:1 diluted hemophilic substrate plasma. In both conditions apparently higher VIII:C values were found.  A number of assay systems used by different producers of Factor VIII concentrates were compared. The standard and, in some cases, the phospholipid reagent seemed to contribute for the largest past to the inter1aboratory variation, but also other, as yet unidentified, factors exerted some influence. These findings initiated a cooperative study by five Red Cross Blood Transfusion Services in Europe on standardization of the one-stage assay for VIII:C. This resulted in a better correspondence between these institutes (CV 13%) compared to the previous situation (CV 23%).  It is concluded that 1) substrate plasma should not be diluted, especially when Factor VIII concentrate is to be tested against a plasma standard, 2) the standard should be of the same type as the testmaterial, and 3) this standard should be properly calibrated against the International Standard for Factor VIII.

A Methodological Investigation On The One-Stage Factor VIII Assay