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Updated: Apr 11, 2026
Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis
Published on: October 17, 2018
Comparison of two different strategies for human monocyte subsets gating within the large-scale prospective CARE FOR
Adam M Zawada1, Lisa H Fell1, Kathrin Untersteller1
1Department of Internal Medicine IV - Nephrology and Hypertension, Saarland University Medical Center, Homburg, Germany.
Insights
Intermediate monocyte counts predict cardiovascular events in chronic kidney disease (CKD) patients, regardless of the gating strategy used for cell enumeration. This finding holds true even when comparing rectangular and trapezoid gating methods.
Area of Science:
- Immunology
- Cardiovascular Medicine
- Nephrology
Background:
- Monocytes are key immune cells with distinct subsets: classical, intermediate, and nonclassical.
- Accurate enumeration of monocyte subsets relies on specific gating strategies based on CD14 and CD16 expression.
- Rectangular gating (RG) and trapezoid gating (TG) strategies have been proposed for differentiating intermediate and nonclassical monocytes.
Purpose of the Study:
- To compare the efficacy of RG and TG strategies in enumerating monocyte subsets.
- To evaluate the predictive value of monocyte subset counts for cardiovascular events in patients with chronic kidney disease (CKD).
- To assess whether intermediate monocyte counts predict cardiovascular outcomes independently of the gating strategy.
Main Methods:
- Analysis of monocyte subsets in 416 CKD patients from the CARE FOR HOMe study.
- Application of both RG and TG strategies for monocyte subset gating.
- Kaplan-Meier and Cox-Regression analyses to determine the predictive role of monocyte subset counts for cardiovascular events.
- Surface marker expression analysis to characterize monocyte subsets.
Main Results:
- Higher intermediate monocyte counts, identified by both RG and TG strategies, significantly predicted cardiovascular events (P < 0.001).
- Intermediate monocyte counts remained independent predictors of cardiovascular events after adjusting for confounders (HR = 1.013 with RG, HR = 1.015 with TG).
- Monocytes classified as intermediate by RG and nonclassical by TG exhibited characteristics of both subsets, suggesting potential overlap or definitional ambiguity.
Conclusions:
- Intermediate monocytes are robust independent predictors of cardiovascular outcomes in CKD patients, irrespective of whether RG or TG gating strategies are employed.
- The findings highlight the clinical relevance of intermediate monocytes in cardiovascular risk assessment within the CKD population.
- Further research is needed to identify specific markers for an unequivocal definition of intermediate monocytes, potentially enhancing their predictive power for cardiovascular events.
Abstract:
Monocytes are heterogeneous cells consisting of (at least) three subsets: classical, intermediate, and nonclassical monocytes. Correct enumeration of cell counts necessitates well-defined gating strategies, which are essentially based upon CD14 and CD16 expression. For the delineation of intermediate from nonclassical monocytes, a "rectangular gating (RG) strategy" and a "trapezoid gating (TG) strategy" have been proposed. We compared the two gating strategies in a well-defined clinical cohort of patients with chronic kidney disease (CKD). Within the ongoing CARE FOR HOMe study, monocyte subsets were reanalyzed in 416 CKD patients, who were followed 3.6 ± 1.6 years for the occurrence of a cardiovascular event. Gating was performed by either RG or TG. We analyzed the expression of surface markers, and compared the predictive role of cell counts of monocyte subsets, as defined by RG and TG, respectively. With both gating strategies, higher intermediate monocyte counts predicted the cardiovascular endpoint in Kaplan-Meier analyses (P < 0.001 with RG; P < 0.001 with TG). After correction for confounders, intermediate monocyte counts remained independent predictors in Cox-Regression analyses (HR = 1.013 [95% CI: 1.006-1.020; P < 0.001] with RG; HR = 1.015 [95% CI: 1.006-1.024; P = 0.001] with TG). NRI was 3.9% when reclassifying patients from quartiles of intermediate monocyte counts with RG strategy toward quartiles of intermediate monocytes counts with TG strategy. In expression analysis, those monocytes which are defined as intermediate monocytes by the RG strategy and as nonclassical monocytes by the TG strategy share characteristics of both subsets. In conclusion, intermediate monocytes were independent predictors of cardiovascular outcome irrespective of the applied gating strategy. Future studies should aim to identify markers that allow for an unequivocal definition of intermediate monocytes, which may further improve their power to predict cardiovascular events.
