1001-LB: Interpretation and Meaning of Anti-HLA Antibodies Detected by Fluorescence Techniques

Aim Determination of anti-HLA antibodies by fluorescence techniques is now the standard of practice. The antibodies are typically detected via Luminex® technology with a detected anti-HLA antibody assigned a mean fluorescence intensity (MFI) based on its abundance and binding capacity in the patient serum. The goal is to use the anti-HLA antibodies for a “virtual crossmatch” for (1) better patient-donor matching in organ transplantation, (2) for determination of appropriate platelet donors for those with chemotherapy-associated or poststem cell transplantation-associated thrombocytopenia, and (3) for determining whether allelespecific antibodies may be present which can interfere with hematopoietic stem cell transplantation (HSCT). The assay is more sensitive than the previously utilized CDC (complement-dependent-cell-cytotoxicity) assay, with the unintended risk that a patient may be inappropriately predicted as incompatible. The overall aim of the study was to compare the results of a CDC crossmatch with the donor-specific anti-HLA antibody (DSA) MFI values in order to determine which DSA (anti-HLA-A, anti-HLA-B, anti-HLA-Cw, anti-HLA-DR, anti-HLADR51, anti-HLA-DR52, anti-HLA-DR53, and anti-HLA-DQ) are likely meaningful at which MFI values. Method Patients and potential donors underwent HLA-typing by routine CDC assay with OneLambda® typing trays and by reverse SSO using OneLambda® reagents. Donor T and B lymphocytes underwent crossmatches with patient serum utilizing standard CDC assays in microtiter trays. Patient anti-HLA antibodies were determined using OneLambda® reagents via Luminex® analysis. CDC crossmatches are reported as negative (1 and 2) or positive (4, 6, and 8). DSA MFI were plotted versus the CDC crossmatch results. Statistical analysis was performed utilizing Student t test and Microsoft Excel. Data were also analyzed via contingency table for determining specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV). Results In the organ transplant setting, we compared the MFI of DSA with crossmatch results determined by the CDC technique. MFI values less than 1000 correlated well with having a negative CDC crossmatch, but values >1000 correlated poorly (specificity 51%, PPV 38%). For > 3000 MFI, specificity = 63%, PPV = 43%. For >10,000 MFI, specificity = 84%, PPV = 63%. NPV for: 1000 = 100%; 3000 = 97%; 10,000 = 98%. These data indicate the use of 1000 MFI as the cutoff for positive detection of DSA will result in prediction of an unsuitable donor-patient virtual match ∼62% of the time, improving to ∼ 37% for MFI of >10,000, but there is the risk for allowing for positive crossmatches in those proceeding forward. Additionally, the newer fluorescence-based assays allow for detection of anti-HLA antibodies at the allele-level of discrimination. Summarizing the presented data with data not shown: anti-HLA antibodies can be divided into (1) those which bind to a cell surface and activate complement, and thereby may be detrimental to endothelial cells in transplanted organs and (2) those which can bind to a cell surface, e.g., platelets, and directly exert a detrimental effect (without need for complement activation), likely by targeting the cells for removal by the reticuloendothelial system. These results indicate that better techniques need to be developed for determination of truly detrimental DSA, in order to better discriminate appropriate donor-recipient pairing for organ transplantation. Finally, since anti-HLA antibodies have been shown to result in “resistance to engraftment” in HSCT, the specific identification of anti-HLA antibodies directed against specific HLA allele products can allow for better HSCT donor selection, or the need for anti-HLA antibody reduction treatment prior to transplantation.