British Journal of Haematology , 2001, 112, 995±1003 Polymorphisms in the non-coding region of the human mitochondrial genome in unrelated plateletapheresis donors H. S. P. Garritsen, 1 A. Hoerning, 1 F. Hellenkamp, 1 U. Cassens, 1 K. Mittmann 2 and W. Sibrowski 1 1 Department of Transfusion Medicine and Transplantation Immunology, University Hospital Mu Ènster, and 2 Department of Medical Biotechnology, Fachhochschule Mu Ènster, Steinfurt, Germany Received 5 June 2000; accepted for publication 17 October 2000 Summary. Human mitochondrial DNA polymorphisms are unique targets to discriminate nucleated cells and platelets between donor and recipient in the setting of transplanta- tion or transfusion. We have previously used this approach to discriminate allogeneic platelets from autologous platelets after transfusion. In the present study, we used DNA sequencing to investigate polymorphisms present in two of the hypervariable segments (HVR1 and HVR2) found within the non-coding region of the mitochondrial genome among 100 plateletapheresis donors. Alignments were made with the Cambridge Reference Sequence (CRS) for human mitochondrial DNA (mtDNA). Combining the sequencing information of HVR1 and HVR2 we could demonstrate that, of the 100 investigated mtDNA samples, none was identical to the CRS. We found a total of 2±17 polymorphisms per donor in the investigated regions, most of them were basepair substitutions (563) and insertions (151). No deletions were found. Sixty-six of the 110 detected polymorphisms were detected in more than one sample. Seven polymorphisms are newly described and have not been published in the Mitomap database. Our results demonstrate that polymerase chain reaction analysis of the many polymorphisms found in the hypervariable region of mitochondrial DNA represents a more informative target than previously described mitochondrial polymorphisms for discriminating donor±recipient cells after transfusion or transplantation. Keywords: mitochondrial DNA, plateletapheresis, D-loop region, automated sequencing, platelet transfusions. Methods to evaluate post-transfusion platelet recovery and survival are limited and consist of either radioactive- or biotin-labelling (Kelton, 1986; Stohlawetz et al, 1999). Apart from reported adverse effects such as antibody formation (Cordle et al, 1999), these methods require processing steps that may influence the quality of the blood product and, subsequently, platelet survival. We therefore decided to focus on a molecular biology-based approach using mitochondrial DNA (mtDNA) polymorph- isms. This system requires no additional labelling of platelets because it uses existing mitochondrial DNA polymorphisms to discriminate donor from recipient platelets. Mitochondria are eukaryotic cytoplasmatic organelles in which oxidative phosphorylation takes place. They contain mitochondrial DNA (mtDNA) that encodes 13 protein subunits of the complexes in the oxidative phosphorylation pathway, the 12S and 16S rRNA and 22 tRNAs required for mitochondrial protein synthesis (Wallace et al, 1995). Human platelets contain a mean of four mitochondria (Shuster et al, 1988), each with one copy of mitochondrial DNA. This circular 16´6 kb human mtDNA was completely sequenced in 1981 (Anderson et al, 1981). Using two published mtDNA polymorphisms, we pre- viously reported a method using sequence specific primer- polymerase chain reaction (SSP-PCR) for the detection of transfused allogeneic platelets (Garritsen et al, 1997). However, our original method relied upon continent-specific polymorphisms (Asian, African and Caucasian) and was therefore limited to donor±recipient pairs of different races. In order to apply mitochondrial PCR analysis to a greater number of donor±recipient pairs, we designed primers to amplify two hypervariable segments of the control region of mtDNA: hypervariable region 1 (HVR1) and hypervariable region 2 (HVR2). Both are hotspots for basepair substitu- tions (Wallace et al, 1995). Because sequencing would provide the highest resolution for screening for suitable polymorphisms, we sequenced the amplicons using auto- mated DNA sequencing. The results of mtDNA sequencing of HVR1 and HVR2 of 100 unrelated plateletapheresis donors are presented and discussed here. q 2001 Blackwell Science Ltd 995 Correspondence: Dr H. S. P. Garritsen, Tissue Typing Laboratory, Department of Transfusion Medicine and Transplantation Immun- ology, University Hospital Mu Ènster, Domagkstr. 11, 48149 Mu È nster, Germany. E-mail: Garrits@uni-muenster.de