PB0507 - Comparisons among functional inhibitor assays to detect anti-factor VIII alloantibodies in congenital hemophilia A

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Chatphatai Moonla, MD, MSc

Hematologist
Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; UNC Blood Research Center, University of North Carolina at Chapel Hill, NC, USA

Co-Author(s)

AS

Autcharaporn Sukperm

Research scientist
Center of Excellence in Translational Hematology, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand
Krung Thep, Krung Thep, Thailand
BA

Benjaporn Akkawat

Research scientist
Center of Excellence in Translational Hematology, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand
Krung Thep, Krung Thep, Thailand
Ponlapat Rojnuckarin, MD, PhD

Professor
Excellence Center in Translational Hematology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Bangkok, Krung Thep, Thailand

Background: Nijmegen-Bethesda assay (NBA) using buffered normal pooled plasma and factor VIII (FVIII)-deficient plasma (FVIIIDP) is recommended to increase accuracy of widely-used conventional Bethesda assay (BA) for FVIII inhibitor detection in people with congenital hemophilia A (PwHA). Subsequently, the US Center of Disease Control and Prevention (CDC) suggests preanalytical heat inactivation at 56°C for 30 minutes to improve assay sensitivity by dissociating FVIII from alloantibodies, while 4% bovine serum albumin solution (BSA) may substitute for costly FVIIIDP.

Aims: To compare performances of various clot-based FVIII inhibitor assays among Thai PwHA.

Methods: During 2017-2021, platelet-poor plasma from PwHA were tested by conventional BA, NBA using FVIIIDP or BSA, and CDC-modified NBA (CDC-NBA) using FVIIIDP or BSA. Inhibitor negativity was defined as a level < 0.4 Nijmegen-Bethesda unit [NBU]/mL on NBA using FVIIIDP.

Results: Of 176 samples (80 PwHA; mean age 25.5 years), all with negative inhibitors on conventional BA ( < 0.4 Bethesda unit [BU]/mL; n=68) were true negative, while 73.3% of the low-titer inhibitor group on conventional BA (≥0.4 but < 5.0 BU/mL; n=86) were false positive and one in the high-titer inhibitor group on conventional BA (≥5.0 BU/mL; n=22) was reclassified into the low-titer group on NBA. Among those with NBA-based inhibitors < 5.0 NBU/mL (n=155), replacing FVIIIDP with BSA and CDC modifications slightly increased the inhibitor titers (Table 1). By receiver-operating characteristic curves, the optimal cutoffs for inhibitor negativity on NBA using BSA, CDC-NBA using FVIIIDP, and CDC-NBA using BSA were < 0.48, < 0.44, and < 0.51 NBU/mL, respectively, while that on conventional BA was as high as < 0.90 BU/mL (Table 2).

Conclusion(s): Nijmegen modifications, rather than conventional BA, should be used to quantify anti-FVIII alloantibodies, particularly for the low titers, in PwHA. Due to modest shifts in the inhibitor levels, alternative cutoffs may be considered for NBA using BSA and CDC-NBAs.