Human Blood Monocyte Subsets: A New Gating Strategy Defined Using Cell Surface Markers Identified by Mass Cytometry

Area of Science:

• Immunology
• Hematology
• Cardiovascular Research

Background:

• Human monocytes are classified into classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16+) subsets.
• Monocyte subset alterations are linked to clinical outcomes, particularly cardiovascular disease, where intermediate monocytes predict events.
• Current methods for defining monocyte subsets yield inconsistent results, hindering mechanistic studies.

Purpose of the Study:

• To develop a more accurate method for identifying and purifying human monocyte subsets.
• To improve the reliability of monocyte subset analysis in the context of cardiovascular disease research.

Main Methods:

• Utilized cytometry by time-of-flight mass cytometry with 36 cell surface markers.
• Employed viSNE (visual interactive stochastic neighbor embedding) for high-dimensional analysis of monocyte populations.
• Developed and validated a revised gating scheme incorporating CCR2, CD36, HLA-DR, and CD11c markers.

Main Results:

• Standard CD14 and CD16 gating results in significant contamination of intermediate (≈86.0%) and nonclassical (≈87.2%) monocyte subsets.
• The novel gating scheme, using additional markers, increased intermediate and nonclassical monocyte purity to 98.8% and 99.1%, respectively.
• Demonstrated the applicability of the revised gating scheme using conventional flow cytometry in patients with cardiovascular disease.

Conclusions:

• A refined panel of surface markers and a new gating strategy significantly enhance monocyte subset identification and purity.
• This improved method is crucial for accurate monocyte function studies in clinical settings, especially for cardiovascular disease.
• The findings provide a more robust approach for immunological research involving human monocyte subsets.