A Modified Dithiothreitol Protocol for Eliminating Daratumumab Interference

Background:
Daratumumab (DARA) is a monoclonal antibody directed against the glycoprotein CD38, used as treatment for
patients with Multiple Myeloma. DARA causes interference in serological antibody testing, complicating the detection of clinical
significant alloantibodies prior to blood transfusion. The interference is eliminated by using dithiothreitol (DTT)-treated reagent red
blood cells (RBCs). However, a disadvantage of the DTT-method is the time-consuming procedure and the hemolysis observed
during long-term storage of DTT-treated reagent RBCs.
To overcome this challenge, the Department of Clinical Immunology, Herlev University Hospital, has developed a modified protocol
for DTT treatment that improves stability of reagent RBCs by 33 days without any visual hemolysis and ensures detection of
alloantibodies. This protocol is a modification of AABB´s technical manual due to a reduced amount of DTT used for the treatment
of reagent RBCs.
The purpose of the modified protocol was to enable preparation of DTT-treated reagent RBCs in conjunction with regular reagent
RBCs in order to improve laboratory efficiency and to reduce turnaround time for antibody detection.

Aims:
The aim of this study is to validate the modified protocol for DTT treatment of reagent RBCs, and to measure the reduced time
spent on detection of alloantibodies.

Methods:
DTT treatment of reagent RBCs was performed, with DTT supplied by the manufacturer Sigma Aldrich, according to
AABB´s technical manual except for a RBC:DTT ratio of 30:25 (vol:vol). Multiple batches of DTT-treated reagent RBCs were
continuously prepared and distributed to three transfusion services in The Capital Region and Region Zealand of Denmark from
February 2017 to February 2018. Antibody screening tests were performed, at all three study sites, with untreated and DTT-treated
reagent RBCs on plasma samples from DARA-treated patients (n=50) and on plasma samples (n=60) from patients with known
clinically significant alloantibodies (n=70). Antibody screening tests were performed using Column Agglutination Technology with
Coombs anti-IgG ID Cards (BioRad Laboratories).
Analysis time was measured and compared using both immediately and in advance prepared DTT-treated reagent RBCs.

Results:
The outcome of the study showed that DARA interference was eliminated in all 50 samples from DARA-treated patients.
55 of 70 alloantibodies were detected and the remaining 15 alloantibodies within the Kell system were negative, using the modified
protocol for DTT-treated reagent RBCs.
DTT-treated reagent RBCs prepared in advance provided antibody screening test results within 37 minutes compared to 90 minutes
with immediately prepared DTT-treated reagent RBCs.

Summary / Conclusions:
Validation results show, that the modified protocol for DTT-treatment of reagent RBCs eliminates
DARA interference and detects alloantibodies examined except for alloantibodies within the Kell system.
DTT-treated reagent RBCs, prepared in advance, reduce antibody detection time by 53 minutes.
The present study show that a central preparation of DTT-treated reagent RBCs and distribution to transfusion services ensures
quality, improves laboratory efficiency and therefore prevents delayed blood transfusions.