Comparative analysis of chromogenic vs clot based CDC modified, Nijmegen-Bethesda assay for detection of factor viii inhibitor titre

Abstract

Background:-Inhibitors to infused factor VIII are the most significant complication of hemophilia treatment. These inhibitors are usually IgG antibodies, that react with FVIII in a time and temperature dependent manner. Coagulation factor VIII inhibitors can be detected by Chromogenic, clot based and immunological assays. However, there is lack of consensus as to what constitutes a positive inhibitor, including the appropriate cut-off for inhibitor measurement The main objective of this study is to compare the sensitivity and specificity of chromogenic Nijmegen Bethesda assay (CNBA) with Centre for disease control modified Nijmegen Bethesda (CDC-NBA) assay against the Reference control method (RCM).

Materials and Methods: The Coagulometer used for inhibitor titre  quantification is Sysmex CS-5100. APTT reagent used isPathromtin SL supplied by seimensSeimens. All data were expressed as Mean ± SD. Statistical formulae were used for sensitivity and specificity calculations. Unpaired students t test was used whereever necessary and a P value of <0.05 is considered as statistical significance

Results: A total of 150 cases were tested for inhibitor titre using CNBA vs CDC-NBA. For low titre Inhibitor (<2 NBU), CNBA has 92% and 86% and CDC-NBA has 80 and 60% sensitivity and specificity respectively. These results show that CDC-NBA shows false positive results at low inhibitor titre. For High titre Inhibitor ( >2 NBU) CNBA has 88% and 80% and CDC-NBA has 85 and 70 % sensitivity and specificity respectively.

Conclusion :- These results shows that CNBA is more sensitive and specific than CDC-NBA at both low and high inhibitor titre. Moreover chromogenic assays can differentiate factor specific inhibitor from nonspecific inhibitors like lupus anticoagulant and unfractionated heparin therapy.

Keywords: Hemophilia, Bethesda assay, ELISA, Factor VIII, Inhibitor, Mixing study

Abbrevations: APLA- Antiphospholipid antibody syndrome, CDC:NBA- Centers for Disease Control and Prevention - Nijmegen-Bethesda
Assay, CNBA:- chromogenic Nijmegen Bethesda assay