Simplified quantitation of myeloid dendritic cells in peripheral blood using flow cytometry
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BACKGROUND: Recognition of the importance of dendritic cells (DC) in the initiation of T-cell-dependent immune responses has led to increasing interest in methods for the identification of DC within the circulation. We sought to develop a flow cytometric method that would allow the reliable enumeration of absolute myeloid DC counts in minimally manipulated blood samples. METHODS: Myeloid DC were identified by three-color staining of whole blood leukocytes as a discrete population of mononuclear cells expressing high levels of HLA-DR and CD33, yet having little or no expression of CD14 and CD16. This method was analyzed for reproducibility and variation in blood DC number during typical clinical day hours and after exercise. The new method was compared to an established commercial kit method. RESULTS: FACS sorting of the CD33(+) DC showed that they morphologically resembled immature DC, and developed cytoplasmic projections typical of mature DC following overnight culture in granulocyte macrophage-colony stimulating factor (GM-CSF). Within peripheral blood, these DC were found at a mean concentration of 17. 4 +/- 5.4 x 10(6) per liter, corresponding to 0.93 +/- 0.27% of mononuclear cells. Comparison of duplicate samples stained and analyzed in parallel showed that the intrasample variability was very low, with an intraclass correlation coefficient of 0.95. The frequency of CD33(+) myeloid DC and their light scatter characteristics were similar to that of CD11c(+) myeloid cells. Four-color FACS analysis revealed complete identity of CD11c(hi), HLA-DR(+) DC with CD33(+), HLA-DR(+) DC. Only rare CD33(+) DC coexpressed CD123 and HLA-DR. Numbers of blood myeloid DC, identified by CD33 staining, showed no significant variation during standard laboratory hours. However, their numbers rose significantly during vigorous exercise, in parallel to other blood cells. CONCLUSIONS: The method described herein is rapid, reproducible, requires only small volumes of blood, can be readily used by a clinical immunology laboratory, and requires fewer antibodies than a currently available commercial method.
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