Ultra-Sensitive Droplet Digital PCR for the Assessment of Microchimerism in Cellular Therapies.
Biol Blood Marrow Transplant
; 24(5): 1069-1078, 2018 05.
Article
em En
| MEDLINE
| ID: mdl-29305193
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.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Bioensaio
/
Reação em Cadeia da Polimerase
/
Quimeras de Transplante
/
Quimerismo
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Biol Blood Marrow Transplant
Assunto da revista:
HEMATOLOGIA
/
TRANSPLANTE
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Austrália