Quantitative detection of T-cell activation markers by real-time PCR in renal transplant rejection and correlation with histopathologic evaluation

Background. The quest for noninvasive methods to diagnose rejection in solid-organ transplants has been rejuvenated by recent observations that specific cytotoxic T-cell markers are up-regulated during rejection. Methods. We developed a one-step real-time polymerase chain reaction (PCR) method allowing reliable detection of the expression of several T-cell genes within a relatively short period of time. The assay is highly sensitive and reproducible with a wide dynamic range allowing accurate quantification of target mRNA in as little as 3 pg total RNA. The utility of this assay in detecting renal allograft rejection was evaluated. Peripheral blood mononuclear cells were collected from 27 patients undergoing kidney allograft biopsies for renal dysfunction after transplantation. Expression of the T-cell activation markers, granzyme B, perforin, and HLA-DRA, was quantified and correlated to the histopathologic changes in the renal biopsies. Results. In cases with allograft rejection (n=8), peripheral lymphocyte expression was increased for granzyme B (P<0.001) and perforin (P<0.08) compared with cases without rejection (n=19). Granzyme B mRNA up-regulation showed the highest specificity for detecting rejection (95%). Moreover, HLA-DRA mRNA was significantly up-regulated (P<0.0016) and had the highest sensitivity (88%) detecting rejection. The up-regulation of both granzyme B and HLA-DRA was most specific in detecting rejection, P<0.001. Conclusions. These data demonstrate that a rapid test of target gene up-regulation using real-time PCR can be used as an aid in the diagnosis of kidney allograft rejection. This is also the first report on the possible utility of HLA-DRA mRNA up-regulation as a marker for kidney transplant rejection.