A six-color flow cytometric assay for the analysis of peripheral blood dendritic cells

Background: Flow cytometric analysis of peripheral blood dendritic cells (PBDCs) and their myeloid (mDCs) and plasmacytoid (pDCs) subsets is a less invasive procedure that is acquiring growing clinical relevance. Because dendritic cells (DCs) lack unique lineage markers, current methods that are based on 3- or 4-color assays do not allow multiparametric analysis of DC subsets. In this study a dedicated 6-color assay was developed. Methods: mDCs and pDCs were counted and characterized for the expression of activation/maturation markers by using a single-platform 6-color assay. Whole-blood samples from 20 healthy controls were directly stained with either CD80-FITC/CD40-PE/lineage-PerCP-Cy5.5/CD123-PE-Cy7/ CD11c-APC/HLA-DRAPC-Cy7 or CD86-FITC/CD83-PE/lineage-PerCP-Cy5.5/ CD123-PE-Cy7/CD11c-APC/HLA-DR-APC-Cy7 combination, in the presence of commercial fluorospheres. A dual-platform 3-color assay currently in use was run in parallel for comparison. Results: The 6-color assay provided mDCs and pDCs counts similar to counts obtained by the 3-color assay. Only the 6-color assay could show differential expression of activation markers by mDCs and pDCs, with pDCs expressing lower levels of costimulatory molecules and HLA-DR, but higher levels of CD83. Conclusions: The 6-color assay described here may be a sensitive tool for assessing possible variations in the number and features of mDCs and pDCs whose reciprocal balance is critical in understanding the more detailed orchestration of immune responses.