Characterizing the antibody response to amustaline/glutathione pathogen-reduced red blood cells

Abstract

Background: The clinical significance of natural and treatment-emergent antibodies specific for amustaline/glutathione pathogen-reduced red blood cells (PRRBCs) is not known.

Study design and methods: A Phase 3, randomized clinical trial of PRRBCs (ReCePI) compared PRRBCs with conventional RBCs in cardiac or thoracic-aorta surgery. Subjects transfused during and for 7 days after surgery were screened for PRRBC-specific antibodies at baseline, 28 and 75 days post-surgery. Subjects with treatment-emergent antibodies were assessed for evidence of hemolysis. Cryopreserved subject RBC samples were assayed by flow cytometry for circulating PRRBCs using an acridine-specific (2S197-2M1) monoclonal antibody, and for human IgG-coated RBCs. RBC-surface acridine density was quantitated using a commercial calibrated phycoerythrin (PE)-bead panel.

Results: Five of 159 (3.1%) PRRBC and zero of 162 conventional RBC recipients developed treatment-emergent PRRBC-specific IgG, low titer antibodies detected 26-80 days post-surgery after exposure to 1-3 PRRBC units, without clinical evidence of hemolysis. DAT and eluate were weak (w+) positive and PRRBC-specific in one subject. A monocyte monolayer assay (MMA) was non-reactive in the three subjects with an interpretable result. Flow cytometry demonstrated circulating PRRBCs in all five subjects expressing surface acridine concentrations at the limit of detection (approximately 150-301 PE molecules/RBC) compared with freshly transfused PRRBCs (approximately 7500 PE molecules/RBC). In some samples, loss of surface acridine expression could not be distinguished from clearance of the PRRBCs.

Discussion: Treatment-emergent PRRBC-specific antibodies with the characteristics of nonclinically significant antibodies were detected in five subjects transfused with PRRBCs. Flow cytometry demonstrated persistent circulating PRRBCs with minimal surface acridine expression. (www.

Clinicaltrials: gov Identifier NCT03459287).

Keywords: RBCs; antibodies; pathogen reduction.

FIGURE 1

Serial hemoglobin assessments in subjects with PRRBC‐specific treatment‐emergent antibodies. Surgery occurred on Day 0. Solid large circles represent timing of study RBC transfusions and open diamonds represent timing of positive antibody screen results with specificity for PRRBCs.

FIGURE 2

Day 39 sample from subject 011‐011 labeled for RBC‐surface acridine or human IG, compared with a negative control of unstained RBCs and positive controls of reagent pathogen‐reduced RBCs (anti‐acridine control) and human IgG1 anti‐acridine antibody coated reagent pathogen‐reduced RBCs (anti‐human IgG control). RBC surface acridine was quantified as 198 PE molecules/RBC on the negative control, 7500 PE molecules/RBC on the positive control representative of the transfused RBCs, and 301 PE molecules/RBC on the 011‐011 subject RBCs. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Day 91 and 107 samples from subject 016‐012 and Day 32, 43 and 75 samples from subject 002‐029 labeled for RBC‐surface acridine or human IG. RBC surface acridine was quantified in subject 016‐012 as 204 PE molecules/RBC on the Day 91 sample and 201 PE molecules/RBC on the Day 107 sample; and on subject 002‐029 as 192 PE molecules/RBC on the Day 32 sample, 170 PE molecules/RBC on the Day 43 sample and 153 PE molecules/RBC on the Day 75 sample. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Day 39, 52 and 88 samples from subject 010‐049 and Day 55, 76 and 103 samples from subject 008‐014 labeled to detect RBC‐surface acridine or human IG. RBC surface acridine for subject 010‐049 was quantified as 292 PE molecules/RBC on the Day 32 sample, 219 PE molecules/RBC on the Day 43 sample and 203 PE molecules/RBC on the Day 75 sample: and for subject 008‐014 as 198 PE molecules/RBC on the Day 55 sample, 199 PE molecules/RBC on the Day 76 sample and 199 PE molecules/RBC on the Day 103 sample. [Color figure can be viewed at wileyonlinelibrary.com]