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#64
CONFIRMATION OF ELISA DETECTED DE NOVO ANTIBODIES BY FLOW CYTOMETRY.
Medhat Askar MD, PhD , Alin Girnita MD , Robert Bray PhD , Howard Gebel PhD , Ron Shapiro MD , Rene Duquesnoy PhD and Adriana Zeevi PhD . Pittsburgh PA, University of Pittsburgh Medical Center, 15213, Pathology ; Atlanta GA, Emory University Hospital, 30322, Pathology and Pittsburgh PA, University of Pittsburgh Medical Center, 15213, Nephrology .
BACKGROUND: Earlier studies attempting to correlate de novo anti-HLA antibody production with acute renal allograft rejection using complement-dependant cytotoxicity (CDC) assays was limited by the sensitivity to differentiate between de novo antibodies vs. low titer pre-existing antibodies.
PURPOSE: In this report, we verify ELISA detection of de novo antibodies and specificity by flow cytometry in cases with moderate to severe (greater than Banff IB grade) biopsy proven acute renal allograft rejection.
METHODS: Four hundred serum samples from 79 kidney transplant recipients were screened by ELISA of which 10 patients with rejection exhibited de novo anti-HLA class I and/or II antibodies. Fourteen samples (pre- and post-transplant samples from 7 cases with rejection and exhibiting antibodies by ELISA) were further evaluated by flow cytometry (Screening, Specificity, and Single Antigen). Retesting was performed blindly after coding samples in a different laboratory. Results of flow testing were analyzed independently from those obtained with ELISA before deciphering the code of the samples.
RESULTS: We detected de novo antibodies in post-transplant samples from this group with acute rejection by both methods. Results of pre- and post-transplant samples were concordant by both methods in tested samples. Flow was able to pick up additional antibody specificities especially when compiling the results of both specificity and single antigen flow PRA.
CONCLUSION: Our data demonstrate that both flow cytometry and ELISA are useful methods for detection of de novo antibody production. For a comprehensive antibody specificity determination multiple approaches may be required in terms of ELISA vs. flow as well as specificity vs. single antigen.