Flow cytometric analysis of urine lymphocytes isolated from patients with renal transplants—purification of urine lymphocytes

Abstract

Early diagnosis of rejection is a pivotal problem in renal transplantation. Recent advances in urinary cell analysis using flow cytometry are still burdened with difficulties concerning urine lymphocyte (UL) isolation. The analysis of lymphocytes washed out with the urine from the kidney transplant offers a tool to monitor noninvasively the intragraft immune response. However, the demand for optimal isolation of UL with high viability and good separation of other cell types has not, as yet, been met. The present study was undertaken to evaluate the optimal conditions for harvesting UL in order to perform adequate UL analysis by flow cytometry. We found that UL viability is mainly dependent on the time of urine harvesting. Low UL viability was caused by high urine osmolality due to high concentrations of urea and glucose. In contrast, high protein concentrations protected UL viability. Hence, the following algorithm of adequate UL isolation for flow cytometric analysis was established: (1) Collection of morning urine directly onto foetal calf serum (FCS: 30% v/v); (2) UL isolation within 2 h; (3) Erythrocyte lysis with subsequent two-step density gradient isolation of UL from residual erythrocytes, granulocytes (Ficoll-Isopaque, 1.077 g/cm 3) and from uroepithelial cells (30% methylglucamine 3,5-diacetomido-2,4,6-triiodobenzoicum, 1.085 g/cm 3); (4) Flow cytometric analysis of UL using the `live gate' setting in the area of blood lymphocyte cluster. Adequate UL isolation and special settings of the flow cytometer may provide a useful tool for early diagnosis and the noninvasive monitoring of renal transplant rejection.