Principles of Virtual Crossmatch Testing for Kidney Transplantation

Abstract

Human leukocyte antigens (HLAs) are the primary determinants of alloimmunity. A crossmatch test is a test that determines the immunologic risk of a recipient with a potential donor by ensuring that there are no transplant-relevant circulating antibodies in the recipient directed against donor antigens. Physical crossmatch (PXM) tests, such as complement-dependent cytotoxicity crossmatch (CDCXM) and flow cytometry crossmatch (FCXM), require mixing of patient serum and donor cells, are labor intensive, and are logistically challenging. Virtual crossmatch (VXM) test assesses immunologic compatibility between recipient and potential donor by analyzing the results of 2 independently done physical laboratory tests-patient anti-HLA antibody and donor HLA typing. The goal of VXM is pretransplant risk stratification-though there is no consensus on whether such risk assessment involves predicting the PXM result or the posttransplant outcome. Although the concept of VXM is not new, the advent of solid-phase assays for detecting circulating antibodies in the recipient directed against individual HLA and DNA-based methods for typing donor HLA specificities at a higher resolution makes the routine use of VXM a reality. Accordingly, VXM may be applied at different scenarios-both for sensitized and nonsensitized patients. Implementation of VXM-based approach has resulted in statistically significant reduction in cold ischemia time without an increase in hyperacute rejection episodes. Though there are considerable challenges, VXM is expected to be used more often in the future, depending on the transplant center's tolerance of immunologic risk.

Keywords: calculated panel reactive antibody; donor specific antibody; human leukocyte antigen typing; kidney allocation; single antigen bead assay; unacceptable antigen.

Figure 1

Potential applications of virtual crossmatch test at the recipient center for kidney transplantation. The application of VXM may be envisioned for different scenarios. Figure depicts 6 potential candidates awaiting kidney transplantation. HLA p, q, r, s, and t, are representative examples reflecting the spectrum of HLA phenotype in the community. Candidate 1 is not sensitized and has no circulating antibodies against any HLA; VXM result is reported as negative and this candidate’s PXM result is likely to be negative against any donor. Candidate 2 has HLA-specific antibodies but does not meet the individual center’s criteria for listing those HLA as unacceptable antigens. Once an organ is allocated for this candidate, though this individual has DSA, after careful analysis of the donor HLA and recipient antibody profiles, the laboratory may report the VXM result as negative and the center may decide to proceed with the transplant. Candidate 3 is highly sensitized (e.g., CPRA 99%). Based on the candidate’s reported unacceptable antigens, organ allocation is personalized. Accordingly, an offer for this individual—after taking the reported unacceptable antigens into consideration—should mean a high probability of a negative PXM result, provided there are no other DSA of concern. Candidates 4 and 5 are identical in terms of their HLA phenotype, antibody profile, and listing of unacceptable antigens. After organ allocation, the local laboratory does a careful analysis of donor HLA and recipient antibody profile and may report the VXM result as negative for candidate 4 and as positive for candidate 5. Accordingly, the center may procced with the transplant for the former but may request a PXM test and delay the transplant pending the results of PXM for the latter. In the United States, when to call an HLA as unacceptable for a given patient is left to the discretion of the transplant center. Thus, for an individual patient, there may be a discrepancy in VXM results during allocation (kidney offer taking into account the unacceptable antigens are equivalent to a negative VXM result) versus after allocation (VXM result may be positive taking into account other DSA that were not reported as unacceptable). It may not be possible to finalize VXM result under certain circumstances. Candidate 6 is such an example, where a kidney is offered from a donor who has a HLA phenotype that is not represented in the SAB assay. Because HLA q+ antigens of donor 6 are not present in the beads in the SAB assay, antibodies directed against HLA q+ in candidate 6—if present—would not be detected in the SAB assay. Accordingly, failure to detect antibodies in candidate 6 directed against HLA q+ is not a proof of absence of that antibody in circulation. Also, when DSA is positive, decision to proceed to transplant based on VXM results depends on the transplant center’s willingness to assume the long-term risks associated with antibodies against HLA. Nevertheless, option to do a PXM for ambiguous results or for predetermined center or patient-specific criteria should always be available. Also, decisions based on patient anti-HLA antibody profile assume—currently in most laboratories—that circulating IgG antibodies against HLA are the only antibodies of relevance in transplantation. CPRA, calculated panel reactive antibody; DSA, donor- specific antibody; HLA, human leukocyte antigen; PXM, physical crossmatch; SAB, single-antigen bead; VXM, virtual crossmatch.