The Tissue Typing Laboratory tests for compatibility between patients who require an organ or bone marrow transplant and potential donors. In the case of bone marrow transplantation it is important that the tissue type of patient and donor are matched to minimize graft versus host disease. In organ transplantation matching patient and donor tissue types reduces the risk of chronic rejection caused by the development of donor-specific antibodies in the patient against tissue-type mismatches.
A person’s tissue type comprises a set of highly polymorphic proteins called human leukocyte antigens (HLA), which are found on the surface of most cells. HLA plays a central role in adaptive immunity, generating an immune response to pathogens but simultaneously presenting a barrier to marrow and organ transplantation. PCR-based DNA methods have largely replaced serology in tissue typing and, like most laboratories testing large numbers of clinical samples, automation is becoming the norm. In 2006 the New Zealand Tissue Typing Laboratory started using Roche’s MagNA Pure Compact robot to automate DNA extraction.
DNA sequencing has revealed potentially important polymorphisms spread over several exons which, in genomic DNA, can be separated by several kilobases. Tissue typing is becoming an increasingly time-consuming and expensive process using genomic DNA.
We decided to investigate the use of the MagNA Pure robot to isolate total RNA from blood, synthesize cDNA using a Transcriptor cDNA Synthesis kit, and for use as a template for HLA sequencing. RNA is a simpler template than genomic DNA because there are no introns. A random selection of blood samples from transplant patients and donors was selected; these had already been HLA typed using genomic DNA to allow a comparison.