A novel flow-cytometric based method to assess post-HSCT donor chimerism exploiting RNA hybridization

Area of Science:

• Immunology
• Hematology
• Transplantation Science

Background:

• Donor-recipient chimerism analysis is crucial for managing hematopoietic stem cell transplantation (HSCT) patients, particularly those with mixed chimerism.
• Current methods involving sorted immune cell subsets are time-consuming and technically demanding.
• Accurate monitoring of chimerism in specific immune cell subsets is essential for understanding immune reconstitution post-HSCT.

Purpose of the Study:

• To develop and validate a novel, flow cytometry-based method for detecting donor cell chimerism in sex-mismatched HSCT.
• To assess chimerism in specific immune cell subsets without the need for prior fluorescence-activated cell sorting (FACS).
• To evaluate the utility of this method in understanding immune reconstitution patterns in HSCT patients.

Main Methods:

• Utilized the RNA PrimeFlow™ system for RNA hybridization to detect Y chromosome-encoded KDM5D mRNA, distinguishing male and female cells.
• Coupled this RNA detection with multiparametric immunophenotyping for simultaneous chimerism assessment and immune cell subset identification.
• Applied the method to a cohort of 10 HSCT patients and compared results with standard PCR-based methods.

Main Results:

• The novel flow cytometry approach demonstrated high sensitivity (around 1%) in distinguishing male and female cells.
• The method showed consistency with established PCR-based chimerism analysis.
• Variable donor chimerism was observed across different T lymphocyte subsets, with a notable enrichment of recipient chimerism in CD8+ central memory T cells.

Conclusions:

• The developed RNA PrimeFlow™-based flow cytometry method offers a sensitive and efficient way to assess donor chimerism in specific immune cell subsets post-HSCT.
• This technique eliminates the need for laborious FACS sorting, streamlining the analysis of immune reconstitution.
• The findings highlight subset-specific immune reconstitution dynamics, particularly in T cell populations, advancing knowledge in the field.