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

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.

Digital droplet PCR‐based chimerism analysis for monitoring of hematopoietic engraftment after allogeneic stem cell transplantation

“…Problems occurred because of the lower accuracy in mixed chimerism . Recently, the ddPCR was introduced, which uses the InDel markers as well, but the high sensitivity of InDel‐based detection is linked with a high accuracy in mixed chimerisms by partitioning the reaction in multiple droplets (Table ). We confirmed the high correlation of observed and estimated percentages in artificial chimerism mixtures over a broad spectrum of chimerism.…”

“…Problems occurred because of the lower accuracy in mixed chimerism. 8,10,12,15 Recently, the ddPCR was introduced, which uses the InDel markers as well, but the high sensitivity of InDel-based detection is linked with a high accuracy in mixed chimerisms by partitioning the reaction in multiple droplets 8,9,14,17 ( 9,11,18,19 to analyze chimerism, to date, ddPCR characterizes a modern, rapid alternative to STR analysis with outstanding accuracy and sensitivity (routinely≤0.1%). 8 In contrast to ddPCR, the STR analysis is more time consuming and complex.…”

“…It has been evaluated for chimerism analysis after alloHSCT and demonstrated high sensitivity, but there are concerns about its inherent limit of accuracy in mixed chimerism . As a new approach, InDel analysis by digital droplet PCR (ddPCR) showed high sensitivity, accuracy, and reproducibility in chimerism monitoring . However, few reports exist about its clinical feasibility.…”

“…[8][9][10][11][12] As a new approach, InDel analysis by digital droplet PCR (ddPCR) showed high sensitivity, accuracy, and reproducibility in chimerism monitoring. 8,9,13,14 However, few reports exist about its clinical feasibility. Moreover, it is unclear whether the higher sensitivity of qPCR and ddPCR has clinical impact on the outcome of patients or not.…”

Chimerism in health and potential implications on behavior: A systematic review

“…Researchers must address current challenges in studying mixed chimerism samples by developing techniques with adequate sensitivity and versatility to provide confidence in defining cell sources. A relatively new technology that has shown promise for application in the field of microchimerism is droplet‐digital PCR (ddPCR), which one group demonstrated an assay limit of detection and limit of quantification of 0.008% and 0.023%, respectively (Kliman et al, 2018). Future studies will require a comprehensive assessment of subject samples within pedigrees to define the source of microchimerism within the proband.…”