Comparison of two different strategies for human monocyte subsets gating within the large‐scale prospective CARE FOR HOMe Study

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 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. Key terms cd14; cd16; monocytes; gating strategy; cardiovascular disease; chronic kidney disease MONOCYTE heterogeneity was first described in 1989 based on the differential expression of the lipopolysaccharide coreceptor CD14 and the FccRIII receptor CD16 (1). Initially two monocyte subpopulations have been distinguished by flowcytometry: a population, which expresses high levels of CD14 and no CD16, and a population which coexpresses both markers (CD161 monocytes).Subsequent research characterized CD161 monocytes as proinflammatory cells, as they were found to express high levels of TNFa and IL12p40/IL12p70 (2,3) and as their cell counts were found to be elevated in many inflammatory conditions [reviewed in (4,5)].

slan-defined subsets of CD16-positive monocytes: impact of granulomatous inflammation and M-CSF receptor mutation

“…The intermediate cells are defined within the CD14 vs CD16 dot plot, in that they are separated from the nonclassical monocytes using a vertical line and from the classical monocytes using a horizontal line (rectangular gating strategy). 21 These intermediate monocytes account for 16.8 cells per microliter in this example, whereas the nonclassical monocytes account for 39.3 cells per microliter. When we added the anti-slan antibody to the staining panel, we showed that this reagent can clearly separate slan-positive and slannegative monocytes (Figure 1, center).…”

“…In this plot, the classical, nonclassical, and intermediate monocytes were separated by a vertical line to the left of the classical monocytes. 20,21 Alternatively, slan expression on the DR-positive cells was displayed in a 1-color histogram. The slan-positive and slan-negative cells were then displayed in a CD14/CD16 histogram, and the slan-positive CD16-positive nonclassical monocytes and the slan-negative CD16-positive intermediate monocytes were determined.…”

Intra-Hepatic Depletion of Mucosal-Associated Invariant T Cells in Hepatitis C Virus-Induced Liver Inflammation

“…Liver biopsies with less than 41 events in the MAIT cell gate were excluded from analysis for activation and degranulation markers. CD14 ++ CD16 − , CD14 ++ CD16 + and CD14 + CD16 ++ monocyte subsets were identified as described 31 and analyzed for activation marker expression if their event counts exceeded the 25 th percentile of the respective subset in all liver biopsies (n=33, 33, 23 and 23 events, respectively).…”

Human Blood Monocyte Subsets

“…Yet, there is still uncertainty concerning the relative contribution of each subset in disease, in part due to conflicting reports concerning the functions of each subset. Monocytes are typically gated in flow cytometry using either a quadrant-based or trapezoid-based gating scheme using CD14 and CD16 to distinguish among the three monocyte subsets 10,11 . Regardless of the approach, the placement of gates discriminating among the monocyte subpopulations is an inherently subjective exercise.…”