From Complement-dependent Cytotoxicity to Virtual Crossmatch: The Path Travelled in Renal Transplant Immunology

On December 23, 1954, a historic milestone in medicine was achieved in Boston with the first successful human kidney transplant.[1] Ronald Herrick donated one of his kidneys to his identical twin brother, Richard, who was suffering from end-stage renal disease. Owing to their genetic identity, the transplant was performed without the need for immunosuppression, and Richard went on to live another 8 years. This event heralded a new era in organ transplantation and laid the groundwork for future immunological advancements. In the same decade, Jean Dausset made a seminal discovery by identifying the Human Leukocyte Antigen (HLA) system, observing alloantibody responses following blood transfusions. By 1965, his work established that HLA expression is controlled by a single genetic locus.[2] While the HLA system plays a vital role in immune defense through antigen recognition, it has also become evident that mismatched HLA molecules pose a major challenge to transplant success, triggering immunomediated graft rejection. The importance of HLA antibodies in transplantation was further highlighted in 1969 by Patel and Terasaki, who demonstrated the utility of complement-dependent cytotoxicity (CDC) crossmatching. The landmark paper published in NEJM reported that 24 of 30 graft failures occurred with a positive CDC crossmatch, compared to only 8 of 195 with a negative one. They concluded that “the ethics of transplanting kidneys without the prior knowledge of the results of the crossmatch test… can reasonably be expected to be questioned in the face of this evidence.”[3] This discovery firmly established the CDC crossmatch as a standard tool in pretransplant evaluation, helping identify patients at risk for hyperacute rejection and determining transplant contraindications. Subsequent modifications, including DTT (dithiothreitol) treatment, prolonged incubation, triple-wash techniques, the use of antihuman globulin, and serial dilution protocols, aimed to improve the sensitivity of CDC assays. Nevertheless, the method remained limited in detecting noncomplement-fixing donor-specific antibodies (DSAs). A significant leap forward came in 1983 when Dr. Garovoy et al. introduced the flow cytometry-based crossmatch (FCXM), offering a more sensitive and reliable method for DSA detection.[4] This assay involves incubating donor lymphocytes with recipient serum, followed by fluorescein-labeled antihuman immunoglobulin (Ig) G antibodies. Although FCXM detects noncomplement-fixing antibodies, it is not without pitfalls. False negatives can result from the prozone effect, low-titer DSAs, or suboptimal sample handling, while false positives may arise from prior treatment with intravenous Ig or rituximab. Modifications such as the Halifax protocol and pronase digestion (digests FcR, CD20 in B cells), which are used particularly to enhance B-cell FCXM sensitivity, have further refined the test. The flow crossmatch test is interpreted by a shift in fluorescence by channel, the ratio of fluorescence of the patient to that of the negative control. Given the fallacies of cell-based crossmatches, bead-based assays (solid phase assays) were developed in the early 2000s. Despite different bead assays, such as Single antigen bead assay (SAB), phenotype bead assay, and mixed bead assay, SAB is more sensitive and specific for DSA detection. A pivotal study by Amico et al., published in Transplantation (2009), underscored the importance of integrating solid-phase assays. Among 334 patients with negative CDC crossmatches, SAB testing revealed that 20% had anti-HLA antibodies. Among those with detectable antibodies, approximately 55% developed antibody-mediated rejection within 200 days posttransplantation.[5] The clinical relevance of DSAs detected via SAB in patients with a negative FCXM was further demonstrated by Adebiyi et al. in a study published in the American Journal of Transplantation. They reported a 25% prevalence of pretransplant DSAs in FCXM-negative patients, notably higher than previously reported (f). The elevated incidence was partly attributed to using a lower mean fluorescence intensity (MFI) cutoff (>500). Importantly, 1-year acute rejection rates did not significantly differ between DSA-positive and DSA-negative groups (15.4% vs. 11.4%, P = 0.18) in FCXM-negative patients. The study concluded that, in the absence of desensitization, pretransplant DSA poses minimal immunological risk if FCXM is negative and should not constitute a barrier to transplantation.[6] In the Indian context, the lysate-based Luminex crossmatch (a bead-based assay) was widely used due to accessibility and cost considerations. However, this method is neither validated nor recommended by international guidelines for decision-making regarding transplant eligibility. Studies from India have reported limitations, including false negatives owing to the absence of HLA-C, DQ, and DP antigens on the bead array and false positives due to nonspecific binding that inflates MFI values.[7] The virtual crossmatch (VXM) is now regarded as the most robust strategy. It integrates high-resolution HLA typing from donors and DSA profiling from recipients using SAB assays on the Luminex platform. Nevertheless, SAB assays come with their own limitations, most notably, the incomplete representation of donor HLA antigens on commercial-bead panels. This limitation holds particular significance in the Indian context, as demonstrated by Daniel and Kumar et al. from CMC Vellore,[8] who reported that over 50% of HLA alleles common in the Indian population are not represented in the commercially available SAB panels, raising the likelihood of false-negative VXMs. Recognizing the complexity of antibody detection, both the European Federation for Immunogenetics and the American Society for Histocompatibility and Immunogenetics, currently recommend a comprehensive pretransplant assessment using a combination of CDC, flow cytometry crossmatch (FCXM), and Luminex SAB assays. Advancements in tissue-typing techniques have introduced innovative approaches to HLA matching at the epitope level. Despite significant progress in characterizing and understanding epitopes, the clinical advantage of epitope-based matching over traditional antigen-level matching and identifying truly immunogenic epitopes remains to be clearly established. Tools such as HLAMatchmaker, EMMA, and PIRCHE II enable the assessment of eplet mismatch load. However, widespread adoption of these technologies in India is still far off. In this issue of IJT, Shruti et al.[9] present a detailed review of cell-based HLA crossmatching, outlining its methodology, interpretation, and limitations. Developed countries have transitioned mainly to flow cytometry and virtual crossmatching, relegating the CDC to historical use. This, however, requires a stringent and well-standardized FCXM and single antigen bead assay available 24 h a day, which is not uniformly available in our country. Although CDC crossmatch remains a cost-effective option in India, it cannot be phased out until advanced assays like SAB become widely available, affordable, and standardized for the Indian context.

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