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Digital PCR to assess platelet recovery after stem cell transplantation

Bone Marrow Transplantation volume 53, pages 1586–1588 (2018)Cite this article

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Engraftment of platelets in patients after human hematopoietic stem cell (HSC) transplantation serves as an early and relevant sign of the success of the transplantation. The time to platelet engraftment appears to be a valid predictor of the probability of complications in transplanted patients and especially for allogeneic HSC transplantation [1]. At least two different criteria of platelet engraftment, i.e. definitions according to the CIBMTR and EBMT, have been widely applied. Both criteria utilize daily platelet counts to assess the time to a threshold count of more than 20,000/µL platelets in peripheral blood, provided that platelet transfusions have been terminated for either at least three [2] (EBMT) or at least seven [3] (CIBMTR) consecutive days. Here, we describe the development of a digital PCR assay to specifically quantify graft-derived platelets versus transfused single-donor platelets. The performance of this assay was then tested with samples from allogeneic HSC transplant patients.

Samples of 2 mL EDTA anticoagulated blood from patients were mixed with an equal volume of 0.9% NaCl and centrifuged at 100×g for 10 min without brake (Hettich Rotina 46S, Hettich, Tuttlingen, Germany). A final centrifugation step at 1569×g for 5 min followed. The pellet was resuspended in 500 µL phosphate buffered saline (PBS, Invitrogen Karlsruhe, Germany). The number of platelets in the PRP was adjusted with PBS to 5 × 106–2.5 × 107 to extract the mt DNA from these platelet suspensions (500 μL). The MagnaPure Large Volume protocol (MagnaPure compact, Roche Diagnostics GmbH, Mannheim, Germany) was used to extract the mt DNA. Elution volume was 50 µL. The PCR strategy was based on generic amplification primers and allele-specific probes. The probes were designed to specifically detect alleles carrying SNPs at positions 73, 195, 295, 310, 16069, 16399 and 16,516 of the non-coding regions HVR 1 and HVR 2 of the mt genome. Droplet generation, amplification and detection of PCR products were performed according to the manufacturer’s instructions (QX200 ddPCR System, Bio-Rad Laboratories GmbH, München, Germany) using 5 µL extracted DNA.

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Authors and Affiliations

  1. DRK-Blutspendedienst NSTOB, Institut Bremen-Oldenburg, Oldenburg, Germany

    Andrea Doescher, Hans-Hermann Kapels & Eduard K Petershofen

  2. Universitätsklinik für Innere Medizin II – Onkologie und Hämatologie, Oldenburg, Germany

    Jochen Casper & Doris Kraemer

  3. DRK-Blutspendedienst NSTOB, Institut Springe, Springe, Germany

    Thomas H. Müller

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  1. Andrea Doescher
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  2. Jochen Casper
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Correspondence to Andrea Doescher.

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Doescher, A., Casper, J., Kraemer, D. et al. Digital PCR to assess platelet recovery after stem cell transplantation. Bone Marrow Transplant 53, 1586–1588 (2018). https://doi.org/10.1038/s41409-018-0238-x

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