A SEQUENCE-SPECIFIC POLYMERASE CHAIN-REACTION ASSAY FOR MITOCHONDRIAL-DNA POLYMORPHISMS IN HUMAN PLATELETS AND WHITE CELLS

BACKGROUND: Because mitochondria are abundant in white cells and are a lso present in platelets, polymorphic sequences in mitochondrial DNA ( mtDNA) represent a unique target for polymerase chain reaction (PCR)-b ased detection of donor material. STUDY DESIGN AND METHODS: A PCR assa y was developed that uses sequence-specific primers (SSP) focused on t wo continent-specific mtDNA polymorphisms. Results were validated by t he use of informative restriction endonucleases. Three commercially av ailable methods to extract mtDNA from white cell-reduced human platele ts was compared. In preparation for in vivo studies, in vitro mixing s tudies designed to mimic transfusion were conducted to investigate the performance of the SSP-PCR assay. RESULTS: The gene sequences of two representative examples of amplicons obtained with the new SSP-PCR mat ched the sequence expected from the published genetic code. Fifteen in dividuals were classified as either positive (n = 6) or negative (n = 9) for the Asian polymorphism by the use of published primers known to flank the polymorphic site followed by digestion with appropriate res triction enzymes. Results with SSP-PCR were nearly perfectly concordan t with those of restriction enzyme analysis. Although the use of three DNA extraction methods allowed the preparation of mtDNA that was suit able for PCR, large and consistent differences (ranging from 10- to 10 00-fold) in endpoint sensitivity were found. In vitro mixing studies r eproducibly documented that the SSP-PCR assay could detect as little a s 1 percent of donor platelets mixed with recipient blood. CONCLUSION: PCR-SSP can be reliably used to identify human mtDNA polymorphisms. B y optimization of the method of mtDNA extraction, the sensitivity of P CR-SSP assay was greatly increased. This assay should prove useful in investigations of allogeneic platelet transfusions without cell labeli ng. It may also be applied to studies of the donor cell microchimerism that follows transfusion or transplantation.