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BACKGROUND: Because mitochondria are abundant in white cells and are also present in platelets, polymorphic sequences in mitochondrial DNA (mtDNA) represent a unique target for polymerase chain reaction (PCR)- based detection of donor material. STUDY DESIGN AND METHODS: A PCR assay was developed that uses sequence-specific primers (SSP) focused on two continent-specific mtDNA polymorphisms. Results were validated by the use of informative restriction endonucleases. Three commercially available methods to extract mtDNA from white cell-reduced human platelets was compared. In preparation for in vivo studies, in vitro mixing studies 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 matched the sequence expected from the published genetic code. Fifteen individuals 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 restriction enzymes. Results with SSP-PCR were nearly perfectly concordant with those of restriction enzyme analysis. Although the use of three DNA extraction methods allowed the preparation of mtDNA that was suitable for PCR, large and consistent differences (ranging from 10- to 1000-fold) in endpoint sensitivity were found. In vitro mixing studies reproducibly documented that the SSP-PCR assay could detect as little as 1 percent of donor platelets mixed with recipient blood. CONCLUSION: PCR-SSP can be reliably used to identify human mtDNA polymorphisms. By optimization of the method of mtDNA extraction, the sensitivity of PCR-SSP assay was greatly increased. This assay should prove useful in investigations of allogeneic platelet transfusions without cell labeling. It may also be applied to studies of the donor cell microchimerism that follows transfusion or transplantation.