Sequence-Specific Analysis of Microchimerism by Real-Time Quantitative Polymerase Chain Reaction in Same-Sex Nonhuman Primates After Islet and Bone Marrow Transplantation

Abstract

Accurate and sensitive detection of microchimerism in nonhuman primates (NHPs) after hematopoietic cell transplantation is essential for monitoring cell engraftment, for evaluating the success of transplant protocols, and for expanding the utility of NHP in transplantation studies. Because limited sequences are available for NHP major histocompatibility complex polymorphic loci, methods that can accurately determine low levels of donor cells in recipients with same-sex bone marrow transplantation are essential. Thirty-seven pairs of primers, 16 from monkey and 21 from human, were screened with cynomolgus DNA samples. Real-time quantitative polymerase chain reaction was developed for accurately determining low levels of donor-specific DNA in the peripheral blood of islet/bone marrow transplant recipients of same sex cynomolgus monkeys. A total of six sets of primer and Taqman(R) probe combinations were included in this study, which are the most informative primer and probe sets ever reported for cynomolgus monkeys. Three pairs of primers were chosen from exon 2 of the Macaca DRB1 gene and another three pairs were chosen from human HLA DRB1 and DRB3 loci. Three of the six primer-probe sets were also found to work well for baboon (Papio hamadryas) and rhesus monkeys (Macaca mulatta). Sensitivity of the assay ranged from 0.03% to 0.1%, depending on the primer-probe set and donor-recipient pair. The methods are reproducible with relatively low standard error and coefficient of variation. This method is an informative, practical and sensitive method for the determination of donor-specific cells in the peripheral blood of NHP recipients of bone marrow transplant.