Comparative analysis of chromogenic vs clot.based one stage APTT assay for determination of factor VIII level

In the laboratory, factor VIII can be measured by three different methodologies, such as one-stage clotting assay, two-stage clotting assay, and chromogenic assay. These assays differ in ease of use, variety of reagents available, sensitivity to mild hemophilia A, and interference from lupus anticoagulants (LACs). Certain factor VIII gene mutations can cause discrepancy in results between one-stage activated partial thromboplastin time (APTT) and chromogenic assays.

The coagulometer for factor VIII assay is Sysmex CS-5100. All data were expressed as mean ± standard deviation (SD).

A total of 135 cases were studied. Of these, 100 cases were of mild hemophilia A diagnosed by molecular genetics and, 15 cases were positive for LAC, which were confirmed by dilute Russell Viper venom test. Clot-based one-stage APTT assay showed 65% sensitivity and 80% specificity in diagnosing mild hemophilia A cases and out of 15 LAC cases, it showed false positivity in five cases. Chromogenic assay showed 85% sensitivity and 90% specificity in diagnosing mild hemophilia cases and was 100% specific in excluding LAC cases.

One-stage APTT assay is the most commonly used test for determining factor VIII levels but chromogenic assay are considered as the gold standard and recommended as the reference method by European Pharmacopoeia and ISTH subcommittee. Mild hemophilia A patients with missense mutations show discrepancy between the one-stage clot-based APTT assay and chromogenic assays for determination of factor VIII level and this can lead to misdiagnosis or misclassification of mild hemophilia A. Therefore, it is recommended that both the assays should be used in the evaluation of mild hemophilia cases.