The Clinical Study Of Urine Flow Cytometry On Diagnosing The Acute Renal Allograft Rejection

Objective:Acute rejection is a common event to renal allograft and is a major factor in determining both short-term and long-term outcomes for the transplant recipients. Despite the growing array of immunsuppressive therapies available to transplant recipients, it is necessary to predict acute rejection early. Currently, acute renal allograft rejection is suspected only when the serum creatinine level rises, after the adverse effect of immunological and inflammatory processes on graft function. Surveillance allograft biopsies may prove to be beneficial in predicting rejection, but clinical application is limited by the invasive nature of this procedure. The rapid developments of immunology and urine flow cytometry make it possible to monitor acute rejection by measurement of cell surface markers of urine, which is the center step of acute rejection. So the aims of this research were: (1) to explore the use of urine flow cytometry as a noninvasive tool on diagnosing the acute renal allograft rejection; (2) to study the expression of cell surface markers in urine and evaluate its role as a markers of acute renal allograft rejection;Materials and methods:â…  . In the clinical study urine flow cytometry was performed on the urine of 63 kidney transplant recipients admitted to the hospital for evaluation of acute graft dysfunction blinded to the clinical evaluation performed by the patient's primary physician. All initial evaluations included complete serum chemistries, cyclosporineor tacrolimus trough levels, urinalysis, urine culture, and renal Doppler sonogram. The subsequent evaluation was conducted by the physician in charge of the case while he/she was unaware of the results of urine flow cytometry. The results were compared with the discharge diagnoses. Acute graft dysfunction was defined as an increase in the serum creatinine above baseline (confirmed twice before admission).II. Freshly voided urine (40ml) was centrifuged for 10 min, the supernatant discarded, and the sediment resuspended and washed three times in phosphate-buffered saline. NH4CL was added for lysis of red blood cells. The cell suspension was then aliquoted and stained with the following fluorescinated monoclonal antibodies:anti-CD14, anti-CD25, anti-CD54, anti-CD103, anti-HLA-DR.Cells were stained with nonspecific murine IgG as negative controls. The antibody-treated cells were fixed in formaldehyde and stored until analysis by flow cytometry.III. Analysis was carried out on a FACScan. Ten-thousand cells were counted in each sample when feasible. Cell-poor samples were run for 2 min and the analysis was done on all the cells counted in that time.IV. The results of urine flow cytometry by clinical diagnoses are categorized in two groups. Statistical analysis was done using the chi-square test as indicated. P<0.05 was considered significant for the comparison of the findings among the distinct clinical categories. P<0.001 was considered significant for the comparison of the mean fluorescence of each antibody with the control during the analysis.Results:I . The clinical diagnoses were as follows: acute rejection, n=33; chronic rejection, n=17; drug toxicity, n=8; acute tubular necrosis, n=l; others, n=4.II. The specificity value for the diagnosis of AR was: 91% for the presence of HLA-DR-positive cells, 82% for the presence of CD54 and 58% for the presence of CD 103. The samples associated with CR had 53% for the presence of CD14-positive cells with a specificity value for the diagnosis. The other samples were remarkable for the lack of expression of the antigens studied.Conclusion:These results suggest that UFC may be useful in monitoring renal transplant patients for acute rejection and as a discriminator from other causes of renal allograft dysfunction.