Introduction: Clinical xenotransplantation is now a reality, with advances in genetic engineering and xeno-immunobiology enabling FDA clearance of a first-in-human kidney xenotransplantation multi-center trial including Johns Hopkins. Blood-derived products such as plasma, intravenous immunoglobulin (IVIG), and red blood cells (RBCs) are frequently administered to transplant recipients and may harbor xeno-antibodies capable of triggering complement activation and graft injury. Robust pre-transfusion screening strategies are needed to ensure compatibility of blood products with xeno-organs.
Methods: Ninety plasma units and 24 RBC units were obtained from the local blood supplier, along with 8 IVIG lots (Gamunex and Gammaguard). Samples were incubated with porcine peripheral blood mononuclear cells (PBMCs) from GGTA1 knockout (KO), multi-KO (GGTA1, CMAH, B4GALNT2; for CDC), and multi-KO + human transgenes (txg; for antibody binding assay) pigs. IgM and IgG binding and CDC were quantified by flow cytometry as median fluorescence intensity (MFI) and % cell death, background-subtracted and analyzed using Wilcoxon signed-rank test. IgM MFIs against GGTA1 KO cells were indexed to a control, and ROC analysis with Youden index was used to identify a cutoff predictive of CDC ≥20%.
Results: IgM and IgG binding were significantly lower to multi-KO/txg cells vs. GGTA1 KO (p < 0.0001). CDC showed a similar trend, with greater lysis against GGTA1 KO targets (p = 0.0004). IgM binding to GGTA1 KO strongly correlated with CDC (R = 0.65, p < 0.0001), and an IgM index cutoff of 1.52 predicted CDC ≥20% with high sensitivity/specificity. IVIG lots showed uniformly low IgM binding to all targets but higher IgG binding to GGTA1 KO than to multi-KO/txg. RBCs showed low IgM but high IgG binding to GGTA1 KO, indicating potential to deliver xeno-reactive antibodies despite being considered serologically inert.
Conclusions: Using a 20% cell lysis threshold for CDC, 4% of plasma samples would be excluded for multi-KO/txg compared to 23% for GGTA1. Given strong correlation with CDC, IgM index-based screening may help preselect compatible products. Multi-KO/txg cells showed reduced immunoreactivity compared to GGTA1 KO across a large panel of blood products. However, despite overall reduced reactivity, outliers with high immunoreactivity remain a concern even in more extensively genetically modified strategies, underscoring the importance of blood product screening in clinical xenotransplantation.