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 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.

A general method for concentrating blood samples in preparation for counting very low numbers of white cells.

BACKGROUND: To count extremely low levels of white cells (WBCs) in WBC-reduced blood components, a larger volume of sample must be processed. The goal was to develop an all-purpose method for concentrating the samples obtained from WBC-reduced red cells or platelets. The method was designed to be compatible with a variety of counting techniques. STUDY DESIGN AND METHODS: Coded samples of red cell concentrates with an expected WBC concentration of 200, 100, 50, and 10 per mL and of the diluent (undetectable WBCs/mL) were sent to three sites on five occasions and counted by the use of the concentration method, crystal violet stain, and a Nageotte counting chamber. Additional samples were tested by flow cytometry, polymerase chain reaction, and volumetric capillary cytometry. RESULTS: The results from the three test sites showed good linearity, with an overall r2 = 0.9994. The lower limit of accurate detection of the assay was 10 WBCs per mL. The results were biased toward underestimation, particularly at one of the test sites (Site A). There were no significantly different results in Sites B and C. The intra-assay CV was acceptable. Precision (reproducibility) at the three test sites varied. CONCLUSION: This method allows reliable determination of WBC concentrations as low as 0.01 per microL in blood. Despite the use of technologists trained in Nageotte chamber counting, validation testing demonstrated that one test site's performance was significantly different from that of the other two sites, because of both underestimation bias and variation in count results. The sample concentration method, when used in conjunction with an automated assay for WBC identification, should permit larger volume analysis with a greater degree of precision and a lower limit of detection than is found in assays that do not concentrate the sample before counting.

HLA antigens on leukocyte fragments and plasma proteins: prestorage leukoreduction by filtration.

HLA alloimmunization following blood transfusion results from recipient exposure to donor alloantigens. Numerous studies have documented that intact donor leukocytes are capable of provoking primary alloimmunization and that leukoreduction can decrease the incidence of primary HLA alloimmunization. HLA antigens also exist in soluble form and are present on leukocyte cell fragments. We measured the concentration of soluble HLA class I antigen in both standard and leukoreduced blood components during storage. Although the concentration of soluble class I HLA protein varied widely among different individuals, the concentration was stable during refrigerated storage of red cell concentrates and was not affected by leukocyte reduction by filtration. We also investigated whether or not HLA antigens present on leukocyte fragments were capable of stimulating either resting or in-vitro-primed lymphocytes in the mixed lymphocyte reaction (MLR).(ABSTRACT TRUNCATED AT 250 WORDS)