Enumerating regulatory T cells in cryopreserved umbilical cord blood samples using FOXP3 methylation specific...

Area of Science:

• Immunology
• Cell Biology
• Transplantation Science

Background:

• Allogeneic haematopoietic cell transplantation (HCT) offers curative potential for hematological disorders but involves significant risks.
• Optimizing HCT outcomes requires improved graft selection strategies, including HLA matching and analysis of graft cellular content.
• Higher T regulatory (Treg)/T cell ratios in grafts are linked to better outcomes in adult peripheral blood grafts, but this remains unstudied in umbilical cord blood (CB) due to cryopreservation challenges.

Purpose of the Study:

• To compare the efficacy of flow cytometry and an epigenetic, DNA-based methodology for enumerating Treg and CD3+ cells in cryopreserved CB units.
• To determine the most robust and efficient method for assessing Treg content in previously cryopreserved CB samples.
• To evaluate the agreement, consistency, and error susceptibility of both methods with fresh and cryopreserved CB.

Main Methods:

• Comparison of flow cytometry and epigenetic DNA-based methodology for cell enumeration.
• Assessment of Treg and CD3+ cell numbers in both fresh and cryopreserved CB samples.
• Evaluation of method agreement, consistency, and error rates.

Main Results:

• Epigenetic enumeration provided consistent and comparable results for both fresh and frozen CB samples.
• Flow cytometry assessment of Tregs and CD3+ cells was only feasible in fresh samples due to significant cryopreservation-induced cell death.
• Epigenetic analysis yielded similar cell numbers in fresh and frozen CB.

Conclusions:

• Epigenetic assessment is advantageous for analyzing cryopreserved CB compared to flow cytometry.
• This DNA-based method allows for multiple assessments from small CB segments, crucial for clinical studies.
• Epigenetic analysis offers a reliable approach for evaluating graft cellular content in cryopreserved CB for HCT.