In a nomenclature proposal published in 2010 monocytes were subdivided into classical and non-classical cells and in addition an intermediate monocyte subset was proposed. Over the last couple of years many studies have analyzed these intermediate cells, their characteristics have been described, and their expansion has been documented in many clinical settings. While these cells appear to be in transition from classical to non-classical monocytes and hence may not form a distinct cell population in a strict sense, their separate analysis and enumeration is warranted in health and disease.

Increased EVs function as carriers for signaling mediators, such as WNT-5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.

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We present a novel single-platform assay for determination of the absolute number of human blood monocyte subpopulations, i.e., the CD14 11 CD16 2 and the CD14 1 CD16 11 monocytes. A four-color combination of antibodies to CD14, CD16, CD45, and HLA-DR reduces the spill-over of natural killer cells and of granulocytes into the CD14 1 CD16 11 monocyte gate. For these CD14 1 CD16 11 monocytes, the intra-assay coefficient of variation (CV) was 4.1% and the inter-assay CV was 8.5%. Looking at a cohort of 40 donors aged 18-60 years, we found no age dependence. There was however an effect of gender in that females had lower CD14 1 CD16 11 monocytes (45.4 AE 13.5 cells/ll) compared with males (59.1 AE 20.3 cells/ll) (P < 0.02). Using this novel approach, we can confirm that exercise will lead to more than three-fold increase of the CD14 1 CD16 11 monocytes. Also, we show that therapy with low doses of glucocorticoids will deplete these cells. This robust single-platform assay may be a useful tool for monitoring the absolute number of monocyte subpopulations in health and disease. ' International Society for Advancement of CytometryKey terms monocyte heterogeneity; gender; exercise; glucocorticoids MYELOMONOCYTIC stem cells in bone marrow give rise to monocytes and these are released into blood where they circulate for a few days and then migrate into tissue where they develop into different types of macrophages (1). Monocytes were initially defined based on morphology and cytochemistry. With the advent of monoclonal antibodies and flow cytometry, human monocytes were characterized with antibodies like CD14. This technology also allowed for the definition of two monocyte subsets, i.e., the classical CD14 11 CD162 and the CD14 1 CD16 11 nonclassical monocytes (2) with the latter cells characterized by higher MHC class II expression and by higher production of the cytokine tumor necrosis factor (3). With the recognition of the potential role of the nonclassical CD14