New methods for the quantification of mixed chimerism in transplantation

Abstract

BackgroundQuantification of chimerism showing the proportion of the donor in a recipient is essential for the follow-up of hematopoietic stem cell transplantation but can also be useful to document an immune tolerance situation after solid organ transplantation. Historically, chimerism has been quantified from genomic DNA, but with technological advances, chimerism from donor-derived cell-free DNA seems particularly relevant in solid organ transplantation.MethodsThe reference method was until recently the short tandem repeat technique, but new innovative techniques as digital PCR (dPCR) and NGS, have revolutionized the quantification of chimerism, such as the so-called microchimerism analysis. After a short review of chimerism methods, a comparison of chimerism quantification data for two new digital PCR systems (QIAcuity™ dPCR (Qiagen®) and QuantStudio Absolute Q (ThermoFisher®) and two NGS-based chimerism quantification methods (AlloSeq HCT™ (CareDx®) and NGStrack™ (GenDX®)) was performed.ResultsThese new methods were correlated and concordant to routinely methods (r²=0.9978 and r²=0.9974 for dPCR methods, r²=0.9978 and r²=0.9988 for NGS methods), and had similar high performance (sensitivity, reproductibility, linearity).ConclusionFinally, the choice of the innovative method of chimerism within the laboratory does not depend on the analytical performances because they are similar but mainly on the amount of activity and the access to instruments and computer services.

Utility of Next-Generation Sequencing-Based Chimerism Analysis for Early Relapse Prediction following Allogenic Hematopoietic Cell Transplantation

“…Liacini et al [ 2 ] calculated the overall CV as 5.08% (ranging from 0% to 11.21% at the expected chimerism value of 0.1–10%), and Janakiraman et al [ 13 ] demonstrated within- and across-run variability of 4.7% and 4.1%, respectively. Additionally, Picard et al documented a lower LOQ (0.22%) than that reported by the manufacturer ( Table 5 ) [ 8 ].…”

“…Various methods can be employed for chimerism analysis utilizing polymorphic markers to differentiate donor cells from recipient cells. These methods encompass chromosome analysis, fluorescence in situ hybridization (FISH), short tandem repeat (STR) PCR, quantitative PCR (qPCR), digital PCR (dPCR), and next-generation sequencing (NGS) [ 2 , 7 , 8 ]. Each method has its strengths and weaknesses in terms of sensitivity, precision, applicability, and cost-effectiveness.…”

Chimerism Testing by Next Generation Sequencing for Detection of Engraftment and Early Disease Relapse in Allogeneic Hematopoietic Cell Transplantation and an Overview of NGS Chimerism Studies

“…Recently, a paper published by Picard et al [26] using a total of 38 samples, a comparison of chimerism quantification data for two new digital PCR systems and two NGS-based chimerism quantification methods was performed. They found that all three existing NGS kits, Devysr ® (Devyser Chimerism), CareDX (AlloSeq HCT), and GenDx (NGStrack) are similar in terms of analytical performance.…”

Seventy-five years of service: an overview of the College of American Pathologists’ proficiency testing program in histocompatibility and identity testing

“…ME evaluation is also challenging when the sample only contains component “A” or “B”, due to limitations in analytic sensitivity [STR sensitivity is around 1%–5% ( Picard et al, 2023 )]. Of note, a small number of laboratories use methods like NGS, real-time PCR or digital PCR, which can reach a limit of detection down to 0.01%–0.1% ( Picard et al, 2023 ).…”

“…ME evaluation is also challenging when the sample only contains component “A” or “B”, due to limitations in analytic sensitivity [STR sensitivity is around 1%–5% ( Picard et al, 2023 )]. Of note, a small number of laboratories use methods like NGS, real-time PCR or digital PCR, which can reach a limit of detection down to 0.01%–0.1% ( Picard et al, 2023 ). Discrepant results are usually due to wrong interpretation of “stutter” bands, which are PCR errors due to strand slippage during primer extension ( Levinson and Gutman, 1987 ), resulting in 1 less STR.…”