Comparative Analysis of Assays for Detection of Cellular Immunity Towards CMV and M. Tuberculosis in Samples from Deceased Donors

Assays for analysis of cell-mediated immunity are increasingly recognised as a valuable diagnostic tool to facilitate determination of the infection status with CMV and M. tuberculosis in patients before and after transplantation. Up to now, although of clinical interest, no data exist on the potential feasibility of these assays in deceased donors. This study was carried out to comparatively evaluate the performance of an ELISA, an ELISPOT and a flow-cytometric assay (FACS) for the determination of cell-mediated immunity towards M. tuberculosis and CMV in samples from deceased donors. Blood-samples from 64 donors (51.7±17.1 years) were recruited at the time of organ procurement. A CMV-ELISA was used as a gold standard for prior CMV infection. ELISA and FACS were carried out from whole blood, ELISPOT was performed from PBMC isolated with T-cell extend reagent (Oxford Immunotec). Specific stimulation was performed using a CMV lysate, PPD, and soluble ESAT-6/CFP-10 proteins in combination with commercial assay formats (Cellestis QuantiFERON-CMV/TB for ELISA, T-SPOT. TB by Oxford Immunotec for ELISPOT). PHA or SEB were used as positive controls. Indeterminate results were defined as positive control reactions below the cut-off in the absence of detectable immunity towards specific antigens. Indeterminate results were observed in 47% of ELISA, 19% of FACS and 0% for ELISPOT assays. A total of 56.3% of samples were CMV-seropositive. CMV-specific immunity was detected in 50.0%, 54.0%, and 60.1% of ELISA (QuantiFERON-CMV), FACS, and ELISPOT-samples, respectively. Agreement with serology was highest for FACS (92%, κ=0.84), followed by ELISPOT (82%, κ=0.64), and ELISA (80%, κ=0.60). Agreement between ELISA and serology increased if the CMV lysate was used as stimulus (97%, κ=0.92), although the rate of indeterminate results remained high (41.8%). Agreement between ELISPOT and FACS was substantial (κ=0.79), and moderate between QuantiFERON-CMV and ELISPOT (κ=0.41) or QuantiFERON-CMV and FACS (κ=0.46). Again, agreement among assays increased, if ELISA was performed with the CMV lysate (κ=0.81). The percentage of PPD-positive results differed between assays (25% for ELISA, 28.8% for FACS, and 53.7% for ELISPOT). Among PPD-positive samples, 16.7% were QuantiFERON-TB positive, 26.7% were positive in an ESAT-6/CFP-10-specific FACS-assay, and 37.9% were positive in the T-SPOT. TB assay. In the routine setting of this study, 61.8% of ELISA samples, 95.3% of FACS samples and 88.2% of ELISPOT samples could be processed within a time frame that is recommended to ensure cell-viability, although this time did not have any measurable effect on results. In conclusion, cellular immunity may be analysed from samples of deceased donors, although the rate of indeterminate results is higher than that of healthy individuals. Among valid results, agreement between serology and the different assay formats is almost perfect for CMV-specific analyses, especially if the same antigen is used for stimulation. Likewise, cellular immunity towards M. tuberculosis is detectable. However, the percentage of positive results was in general lower and showed more pronounced differences among assay formats which warrants further study. In general practice, the time frame until processing may frequently be prolonged in deceased donors, as sampling and shipment often occurs outside routine working-hours.