P148 Uncovering antibodies to cryptic epitopes: Use of flow cytometry crossmatch to differentiate between functional and denatured epitopes

Abstract

HLA antibody (Ab) detection and characterization is a critical step in transplantation. With high sensitivity and allele level Ab identification, the Luminex-based single beads assay is an essential tool in all HLA labs. One of the challenges with the current testing is the potential detection of cryptic or not-physiologically expressed epitopes. Here we report an interesting case of cryptic epitopes and describe the decision making process in preparation for virtual crossmatch requests. A 2 y/o female patient being evaluated for small bowel transplant was tested for presence of HLA Ab. Her class II profile was positive for all DR except DR10, with strongest reactions to alleles with epitopes 98E (DR4,7,9) and 48YQ6 (DR53). The patient typed as DR7,10,53 and the sample was immediately flagged as a possible false pattern of reactivity. Single antigen beads from 2 different vendors showed similar profiles; the same reactivity pattern was also detected by multi-antigen beads. Upon dilution the Ab profile was weaker but similar to the undiluted sample (figure). The pattern “all DR but DR10” could be explained by the patient carrying a rare DR7 allele or a non-expressed DR7 variant. However, no patient cells were available for serological DR7 detection. Molecular typing by SSO and/or SSP may detect a rare DR7 but could not exclude the possibility of a SNP or frameshift mutations creating a premature stop codon behind exon 2. Therefore, the sample was tested by NGS. By sequencing past exon 2 we confirmed that the recipient expresses DRB1∗07:01:01 and no premature stop codons were present in exons 3 or 4. To determine the functional characteristics of the Ab, surrogate donors were tested by flow cytometry crossmatch (FCXM). One donor was DR7 homozygous and the other was DR4, DR7. No reactivity was detected against these donors’ DR antigens, despite the presence of 4 potential targets (figure). This case demonstrates the usefulness of additional testing by FCXM to confirm the presence of false pan-DR reactivity, probably due to a denatured epitopes. Download : Download high-res image (533KB) Download : Download full-size image