Detection of allo- and autoantibodies in kidney transplantation by flow cytometry (original) (raw)
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Human Immunology, 2009
Pre-sensitizing alloantibodies may represent a grave danger in organ transplantation, increasing the risk of antibody mediated rejection (AMR) and graft loss. However, not all antibodies are harmful to the graft. In our study of a cohort of 325 deceased donor renal allograft recipients, the patients were determined eligible to receive an allograft based on a negative complement dependent cytotoxicity (CDC) crossmatch (XM). Yet at the time of transplantation, many candidates displayed donor specific antibodies (DSA) by more sensitive methods, such as solid phase assays (SPA, Luminex) or flow cytometry crossmatch (FCXM). The majority of the patients who were DSA positive by either SPA (67%) or FCXM (66%) presented an AMR-free clinical course post-transplantation. Among the patients who developed AMR (N=29), 76% proved clinically manageable and did not lose the graft. Analysis of the DSA mean fluorescence intensities (MFI) of Luminex showed no statistically significant difference between patients who experienced AMR episodes and those who did not. Importantly, many of the patients with AMR, did not test positive for DSA by SPA (20/29) or FCXM (14/29). Despite false-positive and false-negative results, the detection of DSA by SPA or FCXM was positively associated with AMR, but not with actuarial graft survival. The field of organ transplantation has always struggled to reconcile two opposing goals: improving transplantation outcome while increasing access to transplantation. SPA and FCXM appear to be over-sensitive and defining patients as "sensitized" according to these methods would block access to transplantation for many candidates who would otherwise benefit greatly from receiving the allograft. Nevertheless, SPA and FCXM are invaluable tools, assisting clinicians to gauge AMR risk and tailor immunosuppression the post-transplantation immunological monitoring accordingly.
Transplantation proceedings, 2012
The complement-dependent lymphocytotoxicity crossmatch (CDC-XM) detects cytotoxic parameters of preformed antibodies. The flow cytometric crossmatch (FCXM) is used to detect the binding of recipient antibodies to donor cells. Because these two assays provide different information, both methods are often performed to assess the compatibility of donor-recipient pairs. The aim of this study was to develop a single assay that can simultaneously detect antibody binding and cytotoxicity. A procedure called cytotoxic flow cytometric crossmatch (cFCXM) that determines cell death and antibody binding simultaneously was developed. The assay was validated in parallel with extended incubation CDC-XM. Receiver operating characteristic analysis was used to determine the cut-off level. Furthermore, pretransplantation sera from seven recipients with pretransplantation donor-specific antibodies (DSA) and negative CDC-XM were retrospectively tested for cFCXM (4 without antibody-mediated rejection (AM...
Transplantation, 2009
Background. Flow cytometric techniques are increasingly used in pretransplant crossmatching, although there remains debate regarding the clinical significance and predictive value of donor-specific antibodies detected by flow cytometry. At least some of the discrepancies between published studies may arise from differences in cutoffs used and lack of standardization of the test. Methods. We selected cutoff values for pretransplant flow cytometric crossmatching (FCXM) based on the correlation of retrospective results with the occurrence of antibody-mediated rejection. The impact on long-term renal graft survival of prospective FCXM was determined by comparing graft survival between patients crossmatched with complementdependent cytotoxicity (CDC) only with those prospectively crossmatched with both CDC and FCXM. Results. Chosen cutoff values gave a positive predictive value of FCXM for antibody-mediated rejection of 83%, and a negative predictive value of 90%. After the introduction of prospective Band T-cell crossmatching by flow cytometry in addition to CDC in our center, there was a significant improvement in renal graft survival in highly sensitized patients (Pϭ0.017). Four-year graft survival in highly sensitized patients after the introduction of FCXM was 89%, which did not differ significantly from that seen in nonsensitized patients (93%; Pϭ0.638). Conclusions. Our data demonstrate that prospective FCXM improves renal transplant outcome in highly sensitized patients, provided that cutoff values are carefully validated and results interpreted in the context of sensitization history and antibody screening results.
Journal of the American Society of Nephrology, 2001
ABSTRACT. Flow cytometric crossmatching (FCXM) and panel reactive antibody (PRA) screening techniques are more sensitive than anti-human globulin enhanced cytotoxicity (AHG-CDC) techniques at detecting anti-HLA antibodies. The clinical significance of a positive FCXM in primary renal transplant recipients with a negative AHG-CDC crossmatch is unclear. We performed retrospective FCXM and flow cytometric panel reactive antibody (FlowPRA) determinations in primary renal transplant recipients with a negative T cell AHG-CDC crossmatch and a negative B cell CDC crossmatch pretransplant. Eighteen (13%) of 143 patients exhibited a positive retrospective T cell FCXM. Of these patients, six (33%) experienced early graft loss with explant histology, demonstrating antibody-mediated rejection in five of six cases. The 12 patients with positive T cell FCXM who maintained their grafts experienced more adverse events posttransplant, including more early, steroid-resistant, and recurrent rejection. ...
Transplant Immunology, 2008
To assess the significance of antibodies detected by complement-dependent cytotoxicity (CDC), solid phase (SPA) and flow cytometry (FC) assays we compared their predictive value in 354 consecutive cases of deceased-donor kidney transplantation. Pre-transplantation screening of anti-HLA class I and class II antibodies was performed by CDC and SPA. The direct crossmatch between recipients' sera and donors' T and B cells was performed by CDC followed by FC and SPA ("virtual cross-match"). The past history of antibodies displayed by the recipient was not considered a contraindication for transplantation even when it showed DSA. A side-by-side comparison of the correlation between graft loss, history of DSA and cross-match results indicated that sensitivity was 5%, 16% and 17% while specificity was 99%, 93% and 86% in CDC, SPA, FC crossmatches respectively. There was no significant difference between the 3 year survival of primary and secondary kidney allografts. We conclude that screening and cross-matching the sera by CDC provides reliable results and optimizes the patient's chances to receive a transplant. SPA and FC, however, are of great importance for identifying patients which require close monitoring by biopsy and serology for early diagnosis and treatment of acute antibody mediated rejection (AAMR).
International Journal of Clinical and Diagnostic Pathology, 2020
Background: The clinical significance of positive results on more sensitive platforms available for antibody screening has been a matter of contention. This study explores the relationship of test results of Complement Dependent Cytotoxicity (CDC) crossmatch, ELISA anti HLA antibody test and Donor Specific Antibody (DSA) assay using donor lysate on the Luminex platform and their impact on graft outcome. Materials and Methods: Participants included patients who underwent renal transplant over four years, in whom pre transplant screening included an initial CDC cross match, followed by final CDC cross match, ELISA and DSA screening by the Luminex on the final pre transplant serum. Relevant clinical data and results of supportive laboratory investigations were taken from HLA laboratory and computerised records. Results: 126 recipients were included in the study. Pre transplant CDC positivity correlated with overall rejection episodes (P=0.03) and the historical crossmatch Ig M/G positivity with biopsy proven rejections (P=0.029). There was no significant correlation between pre-transplant DSA and ELISA results and rejection episodes. A comparison between DSA, ELISA and CDC showed a low association (Kappa value for DSA and CDC:-0.42; ELISA and CDC:-0.63; DSA and ELISA: + 0.159). Conclusion: CDC continues to have relevance in pre transplant screening. In spite of their technical advantages and superior sensitivity, the ELISA pooled antigen assay and the Luminex crossmatch do not appear to show any significant clinical advantage when used with conventional CDC.