Korean J Transplant.  2023 Nov;37(Suppl 1):S241. 10.4285/ATW2023.F-8353.

Flowcytometric xeno-crossmatching: assessment of pig cells (WT, QKO) compatibility with human/non-human primate sera and human leukocyte antigen antibody profiling

• Cho SI
• Park H
• Kang H
• Oh EJ

• KMID: 2551870
• DOI: http://doi.org/10.4285/ATW2023.F-8353

Abstract

Background
Xenotransplantation offers a promising solution to the critical shortage of human donor organs. Genetically modified pigs have emerged as a potential source of organs for transplantation. Swine leukocyte antigen (SLA), the homolog of human leukocyte antigen (HLA), may induce cross-reactivity in HLA-sensitized patients. In this study, we conducted flow cytometric crossmatch experiments using both human and non-human primate (NHP) sera in combination with porcine peripheral blood mononuclear cells (PBMCs).
Methods
Donor PBMCs were isolated from heparinized whole blood collected from pigs (WT and QKO; GGTA1, CMAH, iGb3s, A3galT2 quadruple genes knock-out) by density gradient centrifugation using Ficoll solution. Human sera were obtained from patients who underwent HLA antibody testing using single-antigen Luminex bead assay. NHP sera were also collected. For all tubes, 2.5 ×10⁵ PBMC and 50 mL serum were incubated with fluorochrome-conjugated antihuman immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies. Acquisition and analysis of flow crossmatch results were performed on a Cytek Northern lights spectral flow cytometer. Median fluorescent index (MFI) values were compared across different sera to quantify binding antibody levels.
Results
Analysis using Cytek spectrometry revealed no obvious variation in autofluorescence between WT and QKO pig cells. Notably, both human and NHP serum samples exhibited elevated levels of binding antibodies (IgG/IgM) towards WT pig cells compared to QKO pig cells. Among sensitized patients (positive for anti-HLA antibodies), binding antibody levels were higher for QKO pig cells compared to sera lacking anti-HLA antibodies. Interestingly, human serum samples displayed higher MFI values for IgG than for IgM, whereas NHP serum samples exhibited higher MFI values for IgG compared to IgM. Serum dilution associated with decreased MFI levels in flowcytometric xeno-crossmatching assessments.
Conclusions
Our findings highlight the need for continued research to develop assays capable of identifying antibody specificities (HLA, non-HLA, SLA) and additional antigenic targets.