Method Description

The Chromogenic Factor VIII assay is performed on the Instrumentation Laboratory ACL TOP 700 using the CRYOcheck Chromogenic Factor VIII kit. In this 2-stage assay, patient plasma is diluted and combined with reagents containing bovine factor X, human factors IXa and IIa, calcium chloride, and phospholipids. The factor VIII in the patient's plasma aids in the activation of factor X to factor Xa. After a specified incubation period, chromogenic substrate is added at which time, the factor Xa, present from the previous step, hydrolyzes the substrate into peptide and p-nitroaniline (pNA). The color produced by the release of pNA is measured photometrically at 405 nm and is proportional to the factor VIII in the sample.(Package insert: CRYOcheck Chromogenic Factor VIII. Precision BioLogic Inc; Rev V04, 06/2020)

In the Bethesda procedure, patient plasma is heat inactivated (HI) at 56° C for 30 minutes. Next using the HI patient plasma, serial dilutions are prepared and mixed in equal volumes with normal pooled plasma. The mixture is incubated 2 hours at 37° C. At the end of the incubation, chromogenic factor VIII (CHF8) activity is measured and compared to a control performed at the same time. The difference between the CHF8 activity of the patient's incubation mixture and that of the control is used to calculate the titer. The residual CHF8 activity is converted to Bethesda units: 50% residual CHF8 is equal to 1 Bethesda unit. Assays using the same basic principle as the Bethesda assay are used to quantitate the inhibitors of the other coagulation factors.(Kasper CK, Aldedort LM, Counts RB, et al. A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh. 1975;34[03]:869-872. doi:10.1055/s-0038-1651378; Miller CH. Laboratory testing for factor VIII and IX inhibitors in haemophilia: A review. Haemophilia. 2018;24[2]:186-197. doi:10.1111/hae.1342)