(P-CB-10) Dithiolthreitol Treatment Eliminates Daratumumab Interference for Detection of Antibodies to Amustaline/Glutathione Pathogen-Reduced RBCs

Background/Case Studies: The amustaline/glutathione pathogen reduction (PR) of red blood cells (RBCs) is designed to reduce the risk of transfusion-transmitted infections and to replace irradiation for the prevention of transfusion associated-graft-versus-host disease. The PR process leaves residual amustaline-derived acridine adducts on the RBC surface.

In clinical trials of PR-RBCs, an indirect antiglobulin test (IAT) with reagent PR-RBCs is used to screen for baseline and treatment-emergent PR-RBC specific antibodies. Each screening panel includes cells with no (control), low and high amounts of surface acridine adducts.

Daratumumab (DARA) used in the treatment of multiple myeloma interferes with blood compatibility screening, including the PR-RBC antibody screen. Dithiothreitol (DTT) treatment of RBCs has been shown to resolve DARA interference in standard blood compatibility testing.

The objective of this study was to determine whether DTT treatment of reagent PR-RBCs resolved DARA interference while still allowing the detection of PR-RBC antibodies.

Study

Design/Methods: Nine PR-RBC reagent panels were treated to remove DARA interference (0 or 0.2 M DTT, 35 min, 37°C). The panels were screened with control anti-acridine antibody in an IAT assay (Ortho ID-Micro Typing System IgG plus C3d gel card) using plasma from DARA-treated patients, with or without added anti-acridine monoclonal antibody (2S-197M1). Surface acridine adducts on panel RBCs were quantitated by flow cytometry using QuantiBrite calibrated beads (BD Biosciences).

Results/Findings: Mock treated cells (no DTT) behaved as expected in 9 panels of reagent PR-RBCs. DARA interference was noted in all cells tested with DARA plasma including the control, while the DARA plasma and control anti acridine antibody combination displayed DARA interference with the negative control showing a positive result (Figure 1). DTT treatment eliminated DARA interference in all panels, including the PR-RBC reagent controls (Figure 1a) and in the DARA plasma and control anti acridine antibody combination, with some modulation of the acridine signal observed (Figure 1b). Surface acridine levels were quantitated by flow of PR-RBC panel cells and showed an average reduction in PE molecules/cell of 52.2% (37.7 - 58.4%).

Conclusions: The standard use of DTT to resolve DARA interference was applied to the PR-RBC antibody screening assay and resulted in the elimination of DARA interference. Although acridine levels were reduced following DTT treatment, the PR-RBC IAT assay was able to detect the presence of positive control anti acridine antibody following DTT treatment, both in the absence and presence of DARA patient plasma.