In vivo tracking of transfused platelets by next-generation sequencing of mitochondrial DNA

Abstract

Tracking transfused platelets is important to evaluate platelet transfusion efficiency. Traditionally, corrected count increments were used; however, quantitative polymerase chain reaction-based methods have recently been developed. As both these methods have some limitations, we developed a new method based on next-generation sequencing (NGS) of platelet mitochondrial DNA (mtDNA). We identified several single nucleotide variant markers by sequencing the entire mtDNA region of platelets, and used NGS to estimate the proportion of each platelet unit. This method was validated using mixed platelets obtained from different donors at various ratios. We confirmed the applicability of this method in patients who received platelet transfusions using pre- and posttransfusion samples from 12 patients with hematological malignancies. The method showed good linearity (r2 >0.99 in the range of mixing ratios from 1:1 to 1:50) in the platelet-mixing experiment. In addition, platelet tracking in patients who received transfusions was feasible using this method. Furthermore, it was possible to track individual platelets in patients who received a single platelet transfusion and in those who received multiple transfusions, including a patient who received 5 platelet transfusions. Hence, this NGS-based platelet-tracking method can be used for patients with various conditions.