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

Abstract

Introduction Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative approach for multiple hematologic diseases. The success of alloHSCT is evaluated by analyzing the proportion of living donor cells in blood and bone marrow samples of the recipient (chimerism analysis). To monitor the engrafted cells, donor's individual genetic markers are analyzed in peripheral blood and bone marrow samples, usually by using short tandem repeat (STR) analysis. An alternative method to measure chimerism is based on insertion and deletion markers (InDels) analyzed by digital droplet PCR (ddPCR); however, this approach is rarely evaluated in clinical practice. Methods In this study, we examined the usefulness of ddPCR‐based chimerism analysis against the standard STR analysis in samples around day+30 after alloHSCT in clinical practice using peripheral blood and bone marrow samples. Results The median absolute difference between ddPCR and STR analysis was 0.55% points for bone marrow chimerisms and 0.25% points for peripheral blood chimerisms, respectively, including variation in the range of maximum 2% for both methods. The results of every single sample gave the same clinical message. Conclusion According to our data, chimerism analysis by ddPCR has an excellent correlation with STR‐based analyses. Due to its fast and easy applicability, the ddPCR technique is suitable for chimerism monitoring in clinical practice.

Digital-Droplet PCR for Quantification of CD19-Directed CAR T-Cells

“…Preceding clinical trials have confirmed the strength of ddPCR to detect low-levels of target sequences in various applications, such as micro-chimerism analysis, minimalresidual disease (MRD) detection in hematologic malignancies, and liquid-biopsy (Klein-Scory et al, 2018;Link-Lenczowska et al, 2018;Mika et al, 2019;Franke et al, 2020). Real-time qPCR is technically challenging and complex, since qPCR depends on calculation of copies/µgDNA via standard-curves.…”

“…This is avoided in our approach to quantify CD19-CARs by means of ddPCR. Here, we have used two primer-probe pairs and pooled the results from both assays, considering our previous results demonstrating increased sensitivity and retest-reliability of a ddPCR approach in chimerism analyses following hematopoietic stem cell transplantation (Mika et al, 2019).…”

“…Digital PCR was performed as previously described by our group (Mika et al, 2019). The reaction was set up in 20 µl sample volume containing 10 µl of 2 × ddPCR Supermix (no dUTP, BioRad TM ), 1 µl FAM-labeled FMC63 primer-probe-assay, 1 µl Hex-labeled housekeeper primer-probe assay, 0.4 µl EcoRI restriction enzyme (Fast digest, NEB TM ) and 10-75 ng gDNA (volume 5 µl), and nuclease-free water to adjust the sample to the final volume.…”

“…Previously, we explored a ddPCR approach for chimerism analyses following allogeneic hematopoietic stem cell transplantation. ddPCR compared favorably to qRT-PCR as to robustness and routine applicability in this setting (Mika et al, 2019).…”

Digital PCR: A Reliable Tool for Analyzing and Monitoring Hematologic Malignancies

“…dPCR was employed for chimerism analysis in transplanted patients, the term "mixed chimerism" (MC) meaning the condition of a mixture of genetic profiles resulting after allo-HSCT. A series of studies demonstrated the power of dPCR as a tool for clinical chimerism analysis as compared to established methods based upon Indel and short tandem repeats (STRs) detection [123][124][125][126][127][128]. George et al, in 2013, were the first to develop a dPCR-based method for quantifying chimerism.…”