Increased accuracy of renal allograft rejection diagnosis using combined perforin, granzyme B, and fas ligand fine-needle aspiration immunocytology

Background. Two major routes by which cytotoxic T lymphocytes induce apoptosis in target cells are the perforin-granzyme and the Fas ligand/Fas pathways. Intragraft expression of message for these immune activation genes has been shown to correlate very closely with clinical rejection. We have immunolabeled fine-needle aspiration biopsy samples using a panel of cytotoxic T-cell activation markers to evaluate the immunocytochemical identification of the protein products of these genes in the verification of human renal allograft rejection. Methods. In this retrospective pilot study, 140 fineneedle aspiration biopsy samples from 50 human renal allografts were labeled using alkaline phosphatase/ anti-alkaline phosphatase immunocytochemistry incorporating monoclonal antibodies to perforin, granzyme B, and Fas ligand. Levels of positive labeling for these markers were compared with the original clinical diagnosis of rejection. Results. An excellent correlation with clinical rejection was obtained when all three antibodies were positive. The false positive rate for each antibody was sufficient to make any one alone or in combination with one other unreliable for diagnosing rejection. When all three antibodies gave positive labeling, agreement with clinical rejection status was superior to using conventional morphological cytology. Conclusions. In addition to providing valuable morphological information regarding the composition of inflammatory leukocyte populations and the preservation status of renal parenchymal cells, fine-needle aspiration biopsy samples may be labeled using combined perforin, granzyme B, and Fas ligand immunocytochemistry to offer a safe and reliable method for diagnosing rejection with an excellent level of accuracy. Acute and chronic rejection are still the major causes of renal allograft loss despite major advances in immunosuppression and transplant management. Acute rejection has been shown to be the strongest predictive factor of subsequent chronic rejection (1). Furthermore, a recent study has shown that kidneys undergoing an early acute rejection episode have poorer long-term survival even if the serum creatinine returns to the preacute episode level; the likelihood of chronic rejection is even greater if the serum creatinine fails to return to the baseline (2). Thus, the early diagnosis and treatment of acute cellular rejection remains of critical importance. The current gold standard for the diagnosis of rejection is needle-core biopsy (NCB) histopathology used in conjunction with clinical findings. Fine-needle aspiration cytology (FNAC) is used as a less-invasive method than NCB for investigating causes of suboptimal graft function including rejection and acute tubular necrosis. Fine-needle aspiration biopsy may be practiced safely on a daily basis to study changes in the composition and function of intragraft cell populations before, during, and after acute rejection episodes (3).