Single human leukocyte antigen flow cytometry beads for accurate identification of human leukocyte antigen antibody specificities

TLDR

Assigning antibody specificity in highly sensitized patients is challenging with cell panels that contain multiple antigens per reaction. The study presents a single antigen bead panel for precise identification of HLA antibody specificities via flow cytometry. The panel uses 110 recombinant single HLAs coated on eight‑color microbeads, combined in a single tube to detect antibodies against eight antigens simultaneously, with each antigen reacting specifically with its monoclonal antibody. The single antigen bead panel achieved higher resolution and sensitivity than conventional panels, revealing masked specificities, detecting 31 of 35 mismatched antigens in transplant rejection sera, and showed no reactivity against recipient antigens, making it valuable for predicting negative crossmatches in highly sensitized patients.

Abstract

It is difficult to assign antibody specificity for highly sensitized patients using a cell panel with multiple antigens per reaction. We describe here a single antigen bead panel for accurate identification of human leukocyte antigen (HLA) antibody specificities by flow cytometry.A total of 110 single recombinant HLAs, including 34 A locus alleles, 57 B locus alleles, and 19 C locus alleles, were produced by a mammalian expression system. These single antigens were coated onto eight different colored microbeads, which were mixed together in one tube for simultaneous detection of HLA antibodies against eight different antigens per flow cytometry test.Single HLA reacted specifically with the serologically defined monoclonal antibodies. The single antigen panel provided higher resolution than the regular cell panel for antibody detection by uncovering the masked specificities. Single antigens also provided higher sensitivity than the multiple antigens coated onto beads for HLA antibody detection as demonstrated by serum dilution studies. In 10 sera from patients who had rejected a kidney transplant, single antigen beads identified antibodies to 31 of 35 antigens that were mismatched in the donor. Most important, none of the reactions were against antigens present in the recipient.An accurate and sensitive HLA antibody detection method is described using flow cytometry beads coated with single HLAs produced by recombinant technology. The single antigen beads should be useful in predicting negative crossmatch in highly sensitized organ recipients and highly sensitized patients requiring platelets.