A flow cytometric protocol for enumeration of endothelial progenitor cells and monocyte subsets in human blood

Accumulating evidence intensively advises circulating endothelial progenitor cells (EPCs) and monocyte subsets as surrogate cellular biomarkers in cardiovascular and cancer disease. However, a general standard on their quantification is still elusive, thus precluding a routine monitoring and comparative interpretation of clinical studies.

We intend to develop an advanced and express flow cytometric protocol for proper ex vivo quantification of monocyte subsets and EPCs in human blood.

We employ now lyse/no-wash procedure and bead-based determination of absolute cell counts. We use three-color antibody panels at appropriate compensation. Analysis of rare events and low antigen expression in the EPC experiment is strengthening by sequential gating with exclusion of dead cells, as well as by matching high-intensity fluorochromes to low-density markers and by implementing the fluorescence-minus-one control.

Analysis of peripheral blood of ten healthy donors revealed median (IQR) value of 1.88 (1.35-2.85) viable CD45(dim)CD34)VEGFR2+ EPCs per microliter. Analysis of monocytes revealed 329.5 (264.5-374.8), 16.0 (8.0-22.2) and 26.5 (19.8-36.3) cells per microliter for classical CD14++(high))CD16⁻, intermediate CD14++CD16+(mid) and non-classical CD14+(low))CD16++ monocytes.

Our current protocol provides quantitative information under a simple gating logic while using commonly accepted fluorochromes. This assay is therefore highly adapted for routine use.