Quantitative polymerase chain reaction assessment of chimerism in non-human primates after sex-mismatched islet and bone marrow transplantation

Background. Accurate assessment of chimerism in recipients of islet and bon e marrow transplantation (BMT) may allow for a clearer assessment of the ro le of chimerism in islet engraftment or rejection. A quantitative polymeras e chain reaction (PCR) assay was developed for the detection of the sex-det ermining region of the Y chromosome (SRY) in peripheral blood samples from female non-human primate recipients of allogeneic male islets and vertebral body marrow (VBM) from the same donor. Methods. The assay incorporates a synthetic internal standard (IS) containi ng the same primer template sequences as the target to compete for primer a nnealing and amplification. Each DNA sample was coamplified with a constant amount of IS. The concentration of male DNA in the test samples was calcul ated from the regression equation of a standard curve that was generated by plotting the logarithm of the ratio of the intensities of SRY to IS PCR pr oducts versus the logarithm of known percentages of input male DNA. Results. This method allows for a correction of the variability of efficien cy of the PCR technique and also overcomes the drawback of time-consuming c ompetitive PCR. Using this assay, we quantitated the amount of male DNA in samples taken from female baboon recipients of male islets and VBM. There w as detectable male donor DNA in the samples taken one day after BMT; pre-BM T samples were negative. This technique works well for samples obtained fro m rhesus and cynomogus monkeys as well. Conclusions. It is a practical method for accurately evaluation of chimeris m after sex-mismatched allogeneic BMT in non-human primate models.