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Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols
Published on: October 18, 2016
Simplified quantitation of myeloid dendritic cells in peripheral blood using flow cytometry
J W Upham1, J Lundahl, H Liang
1Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
Insights
A new flow cytometry method reliably counts myeloid dendritic cells (DC) in blood. This technique is reproducible, requires minimal blood, and is suitable for clinical labs, showing DC counts vary with exercise.
Area of Science:
- Immunology
- Cell Biology
Background:
- Dendritic cells (DC) are crucial for initiating T-cell immune responses.
- Accurate enumeration of circulating DC is increasingly important.
- Existing methods for DC identification in blood can be cumbersome.
Purpose of the Study:
- To develop a flow cytometric method for reliable enumeration of absolute myeloid dendritic cell counts.
- To assess the reproducibility and variations in blood DC numbers.
- To compare the new method with an established commercial kit.
Main Methods:
- Utilized three-color staining of whole blood leukocytes to identify myeloid DC (HLA-DR(high), CD33(+), CD14/16(-)).
- Analyzed method reproducibility and DC number variations during clinical hours and post-exercise.
- Compared results with a commercial kit and used four-color FACS analysis for confirmation.
Main Results:
- Identified a distinct myeloid DC population (CD33(+) or CD11c(hi), HLA-DR(+)) in peripheral blood at 17.4 ± 5.4 x 10^6/L.
- Demonstrated high reproducibility (intraclass correlation coefficient = 0.95) and minimal intrasample variability.
- Observed no significant variation in DC numbers during standard lab hours, but a significant increase post-exercise.
Conclusions:
- The developed method is rapid, reproducible, and requires small blood volumes.
- It is suitable for clinical immunology laboratories and uses fewer antibodies than commercial kits.
- This method provides a reliable way to count myeloid dendritic cells in circulation.
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.
