Introduction : The most common treatment-associated complication in patients with Hemophilia A is the development of inhibitors. Inhibitors are associated with increased morbidity, greater disability, and a reduced health-related quality of life. For the measurement of functional activity, the Bethesda assay is the most widely used method. One Bethesda Unit is defined as the amount of Inhibitor that results in 50% residual FVIII:C activity in a defined test mixture. The Bethesda assay is referred to as the gold standard, despite having its own limitations. The procedure requires a significant amount of time, a large volume of reagents, and technical expertise. In this study, we describe a center-devised method, the Mini Bethesda Assay, for screening and diagnosing inhibitors in Hemophilia A patients. This is specifically designed for resource-limited settings, such as in India. The aim is to enable better detection of inhibitors through a sensitive approach that optimally utilizes available laboratory resources, thereby serving individuals with hemophilia.

Objective: To diagnose the low-titer inhibitors in Hemophilia A patients by performing a comparative analysis between the inhibitor screen and the Mini-Bethesda Assay.

Method: A retrospective, comparative observational study was conducted from December 2019 to December 2021, including a total of 95 patients with severe Hemophilia A who were registered and admitted for regular prophylaxis. Written informed consent was obtained, and detailed histories and clinical examination findings were recorded for all patients. Study-related samples were processed in the institutional laboratory. The screening for inhibitors was performed using an APTT-based mixing test. For this, in Tube 1, normal plasma, in Tube 2, test plasma, and Tube 3, equal volumes of normal and test plasma were mixed, and all tubes were incubated at 37 °C for 120 minutes and then placed on ice. Additionally, Tuve 4 was prepared by mixing equal volumes of normal and test plasma in Tube 1 and Tube 2. Finally, APTT was performed on samples from Tube 1, Tube 2, Tube 3, and Tube 4. In Mini Bethesda Assay, the standard Nijmegen-Bethesda Assay was followed using only four dilutions as Neat (Undiluted), 1:2, 1:4, 1:8 (dilutions of the test plasma with Imidazole buffered bovine serum albumin) instead of the ten dilutions to bring down the reaction time, cost, reagents, and man-hours.

Results: Out of 95 cases studied using the Inhibitor Screen, 6 cases (6.3%) tested positive for inhibitors, and 89 cases (93.7%) tested negative. With the Mini Bethesda Assay, 20 cases had low titer inhibitors and 5 cases had high titer inhibitors, and 70 cases tested negative. All high-titer inhibitor cases reported concordant screen positivity. Of the 25 inhibitor-positive cases detected by the Mini Bethesda assay, approximately 14 patients (56%) yielded results in neat dilution. Seven cases required a 1:2 dilution, three cases yielded results in a 1:4 dilution, and a single case required a 1:8 dilution. Thus, reducing the number of dilutions did not impact the sensitivity of the test for screening purposes. All high-titer inhibitors were detected, and only one case with a low-titer inhibitor was identified by the Inhibitor screen. The Mini Bethesda Assay demonstrated 100% sensitivity and specificity. It is therefore a better alternative than the Inhibitor Screen for diagnosing low-titer inhibitors.

Conclusion: Sensitivity of Inhibitor screen to diagnose less than 2 BU Inhibitor is only 24% in our study which is improved to 100% by doing Mini Bethesda Assay. By using only four dilutions (neat, 1:2, 1:4, 1:8), it reduced the cost. However, this did not compromise on the sensitivity and specificity of the test, it will be an extremely beneficial test to pick up low titre inhibitors in low income countries as it dramatically reduced the cost. Additionally, the study underscores the limitations of using the Inhibitor Screen independently, particularly in detecting low-titer cases creating a productive impact on one of the largest hemophilia community in the world.

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2025
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