Assessment of chimerism by next generation sequencing: A comparison to STR/qPCR methods

Area of Science:

• Molecular Biology
• Hematology
• Genetics

Background:

• Chimerism analysis is crucial for monitoring patients post-allogeneic hematopoietic stem cell transplant (allo-HSCT).
• Current methods combining short-tandem repeat (STR) and quantitative polymerase chain reaction (qPCR) are used for engraftment and minimal measurable residual disease (MRD) monitoring.
• There is a need for more sensitive and accurate chimerism analysis methods.

Purpose of the Study:

• To evaluate an insertion/deletion-based multiplex chimerism assay utilizing next-generation sequencing (NGS).
• To assess the sensitivity, accuracy, and workflow efficiency of the NGS assay compared to traditional STR/qPCR methods.
• To determine the assay's utility in engraftment and MRD monitoring for allo-HSCT patients.

Main Methods:

• An insertion/deletion-based multiplex chimerism assay using NGS was developed and tested.
• Simulated unrelated donor-recipient combinations (5) and peripheral blood samples from allo-HSCT recipients (50) were analyzed.
• NGS results were compared with traditional STR/qPCR methods for correlation, bias, and concordance.

Main Results:

• The NGS assay detected a median of 7-8 informative markers per sample.
• The limit of quantitation (LoQ) was determined to be 0.1% recipient, with a total assay time of 19-31 hours.
• NGS demonstrated strong correlation (R² = 0.9852) and 100% concordance with STR/qPCR for detecting recipient increase ≥ 0.1%, indicating high accuracy and reliability.

Conclusions:

• The NGS-based chimerism assay is a sensitive, accurate, and reliable method for post-allo-HSCT monitoring.
• This NGS assay can effectively support engraftment assessment and MRD monitoring in all allo-HSCT settings.
• The NGS assay offers a simplified clinical laboratory workflow compared to conventional STR/qPCR methods.