Determination of unacceptable antigens by summation of anti-HLA eplet antibody strength (MFI) based on single antigen bead assays: Excellent correlation with negative cell based cross matches

• Published in: Human immunology 2022-06, Vol.83 (6), p.482-493
• Main Authors: Norin, Allen J., Das, Ballabh, Mondragon-Escorpizo, Mary O., Bajaj, Harsha, Sumrani, Nabil, John, Devon, Salifu, Moro O.
• Format: Article
• Language: English
• Subjects: Antibodies; Antilymphocyte Serum; Epitopes; Eplets; Flow cytometry cross matches; Graft Rejection; Histocompatibility Testing - methods; HLA; HLA Antigens; Human leukocyte antigens; Humans; Isoantibodies; Kidney Transplantation; Prospective Studies; Reproducibility of Results
• ISSN: 0198-8859; 1879-1166
• DOI: 10.1016/j.humimm.2022.04.004

The reliability of single antigen bead (SAB) assays and their use in predicting a negative cell based cross match (CBXM) is essential in the era of expanded organ sharing. A wide range of accuracy (80–95%) in predicting negative CBXM has been reported. We hypothesized that in SAB assays an antibody against an HLA eplet that was common among a number of different HLA alleles would be distributed among all of the shared eplet positive SABs. This would reduce binding to the donor specific SAB resulting in an under-estimate of antibody strength. We tested this proposal in adsorption studies using, instead of lymphocytes, a novel reagent, single-SAB (sSAB). Properties of SAB assays were examined that provided a basis for conducting adsorption – elution experiments with the sSABs. We found that incubation of sera with sA*02:01 or sB*42:01 not only depleted reactivity to these alleles but also depleted reactivity to beads that shared the reactive eplet. Anti-eplet strength from SAB data (sum of the MFI of eplet positive SABs (MFI-s) was compared with CBXM out comes in two case studies and with 99 proficiency testing sera. In these validation studies, an MFI-s above 11,000 was associated with a positive FCXM. This approach was placed into clinical practice for listing unacceptable antigens that shared a common eplet. CDCXMs (n = 3261) and FCXMs (n = 1012) were performed on patients listed in UNOS for deceased donor kidneys. All CDCXMs were negative and all FCXMs except one were negative. We conclude that summation of eplet strength provides a highly reliable method of predicting prospective negative CBXMs resulting in substantial savings of time and effort. Based on shared eplet summation data, CMS/NYSDOH has accepted our bead based XM (BBXM) method (aka, virtual XM) performed prior to transplant as fulfilling the regulation that XM results be available before kidney transplantation.