Ultra-Sensitive Droplet Digital PCR for the Assessment of Microchimerism in Cellular Therapies

David Kliman; Gloria Castellano-Gonzalez; Barbara Withers; Janine Street; Elizabeth Tegg; Oksana Mirochnik; Joey Lai; Leighton Clancy; David Gottlieb; Emily Blyth

doi:10.1016/j.bbmt.2017.12.802

Ultra-Sensitive Droplet Digital PCR for the Assessment of Microchimerism in Cellular Therapies

Biol Blood Marrow Transplant. 2018 May;24(5):1069-1078. doi: 10.1016/j.bbmt.2017.12.802. Epub 2018 Jan 2.

Authors

Affiliations

¹ Westmead Institute of Medical Research, University of Sydney, Westmead, Australia.
² Westmead Institute of Medical Research, University of Sydney, Westmead, Australia; Sydney Medical School, University of Sydney, Camperdown, Australia.
³ Sydney Medical School, University of Sydney, Camperdown, Australia; Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead, Australia.
⁴ Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead, Australia.
⁵ Westmead Institute of Medical Research, University of Sydney, Westmead, Australia; Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead, Australia; Sydney Cellular Therapies Laboratory, Westmead, Australia.
⁶ Westmead Institute of Medical Research, University of Sydney, Westmead, Australia; Sydney Medical School, University of Sydney, Camperdown, Australia; Sydney Cellular Therapies Laboratory, Westmead, Australia; Blood and Marrow Transplant Unit, Department of Haematology, Westmead Hospital, Westmead, Australia.
⁷ Westmead Institute of Medical Research, University of Sydney, Westmead, Australia; Sydney Medical School, University of Sydney, Camperdown, Australia; Sydney Cellular Therapies Laboratory, Westmead, Australia; Blood and Marrow Transplant Unit, Department of Haematology, Westmead Hospital, Westmead, Australia. Electronic address: emily.blyth@sydney.edu.au.

PMID: 29305193
DOI: 10.1016/j.bbmt.2017.12.802

Abstract

Current techniques to assess chimerism after hematopoietic stem cell transplantation (HSCT) are limited in both sensitivity and precision. These drawbacks are problematic in the context of cellular therapies that frequently result in microchimerism (donor chimerism <1%). We have developed a highly sensitive droplet digital PCR (ddPCR) assay using commercially available regents with good performance throughout the range of clinically relevant chimerism measurements, including microchimerism. We tested the assay using spiked samples of known donor-recipient ratios and in clinical samples from HSCT recipients and patients enrolled on clinical trials of microtransplantation and third-party virus-specific T cells (VSTs). The levels of detection and quantification of the assay were .008% and .023%, with high levels of precision with samples of DNA content ranging from 1 to 300 ng DNA. From the panel of 29 insertion-deletion probes multiple informative markers were found for each of 43 HSCT donor-recipient pairs. In the case of third-party cellular therapies in which there were 3 DNA contributors (recipient, HSCT donor, and T-cell donor), a marker to detect the cellular product in a background of recipient and donor cells was available for 11 of 12 cases (92%). Chimerism by ddPCR was able to quantify chimerism in HSCT recipients and comparison against standard STR analysis in 8 HSCT patients demonstrated similar results, with the advantage of fast turnaround time. Persistence of donor microchimerism in patients undergoing microtransplantation for acute myeloid leukemia was detectable for up to 57 days in peripheral blood and bone marrow. The presence of microtransplant product DNA in bone marrow T cells after cell sorting was seen in the 1 patient tested. In patients receiving third-party VSTs for treatment of refractory viral infections, VST donor DNA was detected at low levels in 7 of 9 cases. ddPCR offers advantages over currently available methods for assessment of chimerism in standard HSCT and cellular therapies.

Keywords: Adoptive T-cell therapy; Chimerism; Microtransplantation; Virus-specific T cells; ddPCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biological Assay / methods*
Cell- and Tissue-Based Therapy
Chimerism*
DNA / analysis
Hematopoietic Stem Cell Transplantation / methods
Hematopoietic Stem Cell Transplantation / standards
Humans
Methods
Polymerase Chain Reaction / methods*
Transplantation Chimera / genetics*

Substances

DNA