Validation of DTT treated RBCs to resolve daratumumab interference in antibody screening using Ortho BioVue Cassettes

Background:
Daratumumab (DARA), a monoclonal antibody targeting CD38, is used to treat Multiple Myeloma. It interferes with serological antibody testing, hindering the detection of clinically significant alloantibodies before blood transfusion. The interference is resolved using dithiothreitol (DTT)-treated reagent red blood cells (RBCs), validated for Column Agglutination Technology (CAT) with Coombs anti-IgG ID Cards (Bio-Rad Laboratories) and traditional tube testing. However, many laboratories utilize CAT BioVue cassettes (Ortho Clinical Diagnostics) for automated antibody screening on the Ortho Vision Swift Analyzer. This underscores the potential to adapt the validated DTT-treated reagent RBCs for use with Ortho BioVue cassettes, aligning with the equipment already in use in clinical laboratories. However, the current standard method of DTT-treatment of reagent RBCs by the Association for the Advancement of Blood and Biotherapies (AABB) has been shown to be ineffective in completely eliminating false positive results in the Ortho BioVue cassettes. We have validated a modified DTT treatment protocol to enhance the long-term stability of DTT-treated reagent RBCs and eliminate DARA interference. This approach used a reduced volume of 0.2 M DTT, achieving an RBC:DTT ratio of 30:25, as opposed to the 1:4 ratio recommended by the AABB technical manual.

Aims:
The aim is to validate the feasibility of using Ortho BioVue cassettes with DTT-treated red cells for antibody screening, comparing this method to the BioRad Cards.

Methods:
DTT treatment of reagent RBCs were performed using a 0.2 M DTT solution with an RBC:DTT ratio of 30:25. Antibody screening tests, utilizing DTT-treated reagent RBCs were conducted using Column Agglutination Technology with Coombs anti-IgG ID Cards (BioRad Laboratories) and BioVue Cassettes (Ortho Clinical Diagnostics). These antibody screening tests were carried out on plasma samples from 90 patients undergoing treatment with DARA. Additionally, antibody screening was performed on 10 plasma samples with DARA, each spiked with a single clinically significant alloantibody (RH, Kell, Kidd, Duffy, or MNS). The BioRad cards were manually analyzed, while the Ortho BioVue cassettes were automatically processed using the Ortho Vision Swift Analyzer.

Results:
The DARA interference was eliminated in all 90 samples from DARA-treated patients in both BioVue cassettes and BioRad gel cards. However, three samples required manual reevaluation, and upon further analysis, they were confirmed to be negative. All alloantibodies (anti-D, anti-C, anti-E, anti-Cw, anti-M, anti-S, anti-Fya, anti-Fyb and anti-Jka) were detected except for the sample with anti-K.

Summary / Conclusions:
The present study demonstrates that by lowering the DTT concentration used to treat reagent RBCs, it becomes possible to perform antibody screening using Ortho BioVue Cassettes. This adjustment allows for the use of DTT-treated RBCs in automated systems, making the antibody screening process more efficient while maintaining reliable results, including the identification of alloantibodies previously masked by DARA interference.