Comparative evaluation of 'TaqMan' RT-PCR and RT-PCR ELISA for immunological monitoring of renal transplant recipients.
By sequentially monitoring cytokine gene expression (using RT-PCR ELISA technology) in peripheral blood cells of renal transplant recipients in the early post-operatively period we have shown that expression patterns correlate with clinical events, namely acute allograft rejection. This strategy may have the potential of predicting acute rejection prior to clinical detection. Unfortunately, the technique used was time consuming and only semi-quantitative and, therefore, not suitable for clinical application. In this study, we have sought to confirm the results of the early work using a real time quantitative RT-PCR technique ('TaqMan'), which may be applicable in the clinical laboratory. 'TaqMan' primers and probes were designed for Interleukin (IL)-4 and IL-10 using Primer Express software. Cytokine gene expression for both cytokines was re-measured in stored cDNA samples from 27 non-rejectors and 14 patients experiencing an episode of biopsy proven acute rejection. Compared to pre-transplant levels, IL-4 gene expression fell significantly on post-operative days 2 and 7 before returning to baseline values by day 14 in the non-rejectors. In the rejectors, the initial significant fall was again seen, but with an earlier return to pre-transplant levels at the time of rejection diagnosis. This was followed by a further significant fall in levels 48 h after the initiation of anti-rejection therapy. These different patterns for rejectors and non-rejectors were seen using both techniques. For IL-10, gene expression increased significantly following transplantation throughout the study period when compared to baseline values. This pattern was seen using both techniques. In the rejectors, there were different patterns seen depending on the technique used. When using RT-PCR ELISA, the initial rise was again seen followed by a return to baseline values at the time of rejection diagnosis followed by a further significant rise in gene expression after the start of anti-rejection treatment. The pattern resembled those of the non-rejectors when expression was measured using 'TaqMan'. This study has confirmed that sequential monitoring of cytokine gene expression, measured in peripheral blood mononuclear cells, detects significant changes that correlate with clinical events in renal transplant recipients, including acute rejection, although not all changes detected with RT-PCR ELISA were confirmed. Therefore, real time quantitative RT-PCR technology may be useful in monitoring the immunological status of these patients in the early post-operative period.
Wessex and Renal Transplant Unit, St Mary's Hospital, Milton Road, Portsmouth, UK. paulgibbs@torre.demon.co.uk
Paul J
PJ
Gibbs
Lam Chin
LC
Tan
Sami A
SA
Sadek
W Martin
WM
Howell
D004797:Enzyme-Linked Immunosorbent Assay; D006084:Graft Rejection / Q000276:immunology; D006085:Graft Survival / Q000276:immunology; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis / Q000235:genetics; D015847:Interleukin-4 / Q000096:biosynthesis / Q000235:genetics; D016030:Kidney Transplantation / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013053:Spectrophotometry; D013601:T-Lymphocytes / Q000378:metabolism
D016753:Interleukin-10; D015847:Interleukin-4