DTT Treated Reagent Red Cells for use in Resolving DARA Interference. More Than Just Kell?? Marilyn Stewart MT(ASCP)SBB

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1 DTT Treated Reagent Red Cells for use in Resolving DARA Interference More Than Just Kell?? Marilyn Stewart MT(ASCP)SBB

2 Daratumumab Anti-myeloma and anti-lymphoma agent known to interfere with routine Blood Bank antibody screening tests. IgG monoclonal antibody that binds CD38 that is present on the red cell surface. CD38 is a disulfide-linked molecule and its immune epitopes are disrupted by reducing agents as DTT.

3 Validation Plan: To prove that DTT treated red cells could be used to screen patients receiving DARA and still detect clinically significant allo-antibodies. Screening cells and panel cells were selected for treatment that were homozygous for clinically significant antigens, allowing for rule outs of antibodies.

4 Validation Plan: Reagent screening cells were treated with 0.2M DTT for 30min at 37C, then washed at least four times with PBS Per Judd's Methods in Immunohematology, 3rd edition The treated cells were tested with anti-k after treatment to make sure the DTT treatment was successful. The treated cells were preserved with Alsever s between testing.

5 Validation Plan: Several DARA patients were selected as well as other patients that had significant allo/auto antibodies. Unlike Chapuy CI et al. (Transfusion :2964), we used patient samples with alloantibodies rather than plasma spiked with reagent antisera Gel methodology was used to test.

6 Testing Results: Thirty eight patient samples, including six DARA patients were tested with DTT treated reagent red cells. Of the six patients who had DARA interference in their plasma, all samples had negative reactions with the DTT treated cells except for one patient This patient had weak reactions in one cell (false positive-arc reference lab came back negative).

7 Table of Tested Antibodies Antibody # of patients # of patients with correct detection with DTT treated cells # of patients with unexpected results DARA (false positive) Anti-E Anti-Fya Anti-K (expected negativity) Anti-D Anti-Jka Anti-M Warm Auto Anti-S Anti-e Warm Auto C Anti-C Multiple Ab

8 Testing results: Patients with alloantibodies in their plasma did react with the DTT treated red cells as expected, with the exception of newly formed anti E antibodies in four patients. These four patients showed no reactivity with the E positive DTT treated red cells. Plasma from patients with a long history of anti E (greater than 6 months) did react with the E positive DTT treated red cells (13 pts).

9 Non-Reactive DTT-treated Cells in Four Patients with anti-e Patient Initials Strength of anti-e using standard methods New anti-e less than or equal to 6 months Ab screen comments AM 4+ Yes Pos for 2 wks, subsequently disappeared CR 1+ Identified 3 yr earlier, but had disappeared on most recent prior screen AGR 4+ Yes 1 st screen at UCM was 5mo prior, possibly part of DHTR LH 3+ Yes Newly positive since only prior screen at UCM (negative) 4 mo earlier Possible IgM anti- E? Yes???? Yes Possible lowavidity IgG anti-e? Yes Yes

10 Conclusion: DTT treatment of reagent red cells eliminates DARA interference as described. However, unexpectedly it also lessens the ability of treated cells to react with nascent anti-e from real patient samples. This has not previously been described in the literature, as people use reagent anti-sera. Because of this inability to detect some anti-e alloantibodies, DARA-treated patients at UCM will be given both Kell and E negative blood.

11 Future directions RhCE is not a disulfide linked molecule Mechanism of DTT-mediated E antigen destruction is not clear Given the numerous washes of DTT treated cells before they were incubated with plasma, we do not think that the false negativity in these nascent anti-e patients was due to destructive reduction of IgM antibodies

12 Hypothesis During DTT treatment there may be a slight spatial re-alignment of the RBC antigens. We know that DTT treatment affects the RBC membrane by removing the Kell system antigens. Could the spatial arrangement of the antigens change slightly due to DTT treatment, so that IgM or low avidity IgG antibodies can no longer bridge the gap between antigens?? resulting in negative reactions.

13 Questions? THANK YOU!