Epigenetic immune cell counting in human blood samples for immunodiagnostics

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Immune cell profiling is crucial for diagnosing hematologic and immunologic diseases.
• Flow cytometry, the standard method, is limited to liquid blood and requires fresh samples or careful sample preservation.
• Existing methods face challenges with sample accessibility and storage, particularly in resource-limited settings.

Purpose of the Study:

• To develop and validate epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for human leukocyte subpopulation analysis.
• To assess the concordance of epigenetic qPCR with flow cytometry for various immune cell types.
• To evaluate the utility of epigenetic qPCR for immune cell quantification in dried blood spots for diagnosing primary immunodeficiencies.

Main Methods:

• Development of epigenetic real-time quantitative polymerase chain reaction (qPCR) assays targeting human leukocyte subpopulations.
• Analysis of whole blood samples from healthy donors and HIV-positive patients.
• Analysis of dried blood spots from newborns, including those with primary immunodeficiencies.
• Comparative analysis of epigenetic qPCR results against flow cytometry data.

Main Results:

• Substantial equivalence was demonstrated between epigenetic qPCR analysis and flow cytometry for neutrophils, B cells, natural killer cells, CD3+ T cells, CD8+ T cells, CD4+ T cells, and FOXP3+ regulatory T cells.
• Epigenetic qPCR accurately performed both relative and absolute quantification of immune cells.
• The method successfully identified newborns with primary immunodeficiencies using dried blood spots.

Conclusions:

• Epigenetic qPCR provides a precise method for immune cell counting in fresh-frozen blood.
• The application of epigenetic qPCR extends to dried blood spots, enabling immune cell quantification.
• This innovative method can enhance the screening of immune defects and facilitate diagnostics, especially from easily collected, stored, and transported samples like dried blood spots, overcoming logistical barriers in remote or underdeveloped areas.