2214
Detection of Donor T and B Cells Specific C4d Fixing Alloantibodies using Flow Cytometry: A Novel Diagnostic Approach
Dharmendra Jain, PranavDorwal, NeetuTyagi, AmitPande, Simmi, VimarshRaina
Molecular Genetics and Immunology Laboratory, Medanta -The Medicity, Gurgaon, India
ABSTRACT:
Introduction &Aim: Donor-specific complement-fixing alloantibody identification is known to play a major role in antibody-mediated renal transplantation rejection and management. Specifically, the deposition of C4d in the peritubular capillaries in a kidney biopsy is a valuable marker of antibody-mediated rejection (AMR). Various methods have been reported for the detection of allo-antibodies in recipient sera. These antibodies can be HLA or non-HLA, complement-fixing or non-complement fixing, donor T and/or B cell specific. C4d Flow-PRA is one of the screening methods to identify HLA specific complement fixing antibodies. However, results are limited by lack of donor specificity in method.
Method: We hereby report a novel method christened Donor-specific-Flow-C4d (DFC) of identifying donor-specific (T and/or B cell), C4d-fixing alloantibodies. The test was validated using pooled reference serums and comparing the results with immunohistochemistry findings. The schematic protocol is described in Figure-1
Results:In present report we have discussed a series of cases representing a variety of cases in renal transplants wherein the newer method i.e. DFC was beneficial. Some of the prototype cases citedwas of AMR, where initially there was a dilemma of AMR versus ACR as the result of histopathological finding of allograft biopsy and FCXM were not sufficient to confirm it as complement mediated rejections. The positivity on the DFC method confirmed the diagnosis of AMR due to donor B cell specific, complement fixing, alloantibodies for both of these cases. An another case was of a deferred pre-transplant donor, wherein the recipient serum was confirmed to be positive for donor T and B cell specific; complement fixing, HLA alloantibodies without using an invasive procedure of allograft biopsy. This was helpful in pre- transplant prediction of a possibility of an AMR. There were also a few cases with a clear indication of correlation of the DFC and the histopathological findings. The patients were managed with the therapeutic protocols used for an ACR.
Conclusion: With the added advantage of being non-invasive and Donor T and B cell specificity, this newer method provides information pre-transplant; whereas kidney biopsy based C4d evaluation can only be done post-transplant. It helps in decision of donor deferral, which is important in Asian countries like India, where most of the transplants are live related. We postulate that this method incorporates the best features of all the available modalities (i.e. FCXM, C4d-FlowPRA and NIH-CDC).
Figure-1: Pictorial representation of C4d activation and detection protocol in DS-Flow-C4d Protocol. Abbreviation: (DC-Donor cells)