PURPOSE: We developed a protocol for assessing human kidney biopsies by flow cytometry. Using this technique we asked whether or not antibody bound to microvascular endothelial cells of transplanted kidneys could be measured and if it correlated with rejection and clinical course.

METHODS: A third of a core of a standard 16 gauge transplant biopsy was reduced to a single cell suspension using a collagenase digest. In multicolor flow cytometry analyses, renal microvascular endothelial cells (RMEC) are identified as CD45-/HLA-DR+/CD31+/CD34+. RMEC- bound antibody is determined by the binding of anti-kappa and anti-lamda to these cells. In our analyses anti- lambda and -kappa antibodies are conjugated to the same fluorophore so total antibody bound to RMEC is assessed.

RESULTS: Kidneys affected with transplant glomerulopathy and vascular rejection have higher levels of antibody binding to RMEC than kidneys with non-specific inflammation. Antibody binding to RMEC varies among kidneys judged to have cellular rejection. For an individual patient, RMEC antibody binding correlated with clinical course and response to treatment. It did not uniformly correlate with C4d deposition or the presence of donor specific antibodies (DSA).

CONCLUSION: Antibody binding to RMEC can be measured using multicolor flow cytometry and in cases of rejection indicates a vascular injury. It can be used to monitor clinical course during rejection and may add to current methodologies of monitoring DSAs and C4d deposition.

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