Idiopathic pulmonary fibrosis (IPF) is a rare but devastating lung disease in which the pulmonary vasculature and other tissues undergo a profound remodeling that produces a severe decline in breathing efficiency. IPF usually occurs in adults with a history of cigarette smoking, an activity that damages lung tissue. Individuals with IPF have fibrotic lung regions with very few blood vessels, adjacent to unaffected tissue that is highly vascularized. Evidence suggests that when re-endothelization fails to occur after alveolar injury, the resulting loss of alveolar–capillary integrity may trigger the process of fibrosis.

It is suspected that circulating endothelial cells (CECs), endothelial progenitor cells (EPCs), and fibrocytes may be correlated with response to vascular injury and to tissue repair that occurs in the lungs, even though each of these populations makes up less than 1% of circulating nucleated cells in blood. If so, endothelial cells may potentially be used as biomarkers of IPF disease progression and prognosis. However, data on CECs, EPCs, and fibrocytes from individuals with IPF have been very difficult to acquire. These cells are exceedingly rare, so their detection requires the sensitive analysis of a large number of cells.

De Biasi and coworkers recently published methods to measure CEC and EPC populations, as well as fibrocytes, from individuals with IPF. To identify the rare cells in freshly isolated blood samples, they designed multiparametric labeling schemes for the target populations and took advantage of the speed and precision of the Invitrogen™ Attune™ NxT Flow Cytometer (Figure 1). The Attune NxT cytometer permitted the researchers to acquire phenotypic data on 35,000 cells per second, which enabled them to analyze the more than 10 million cells per sample needed for a meaningful statistical analysis of such small numbers of target events. Additional research will be needed to determine whether characteristics of these rare circulating cells can be used as biomarkers of IPF progression and prognosis, but De Biasi and coworkers have demonstrated that such research is possible with tools available today.

Figure 1. Gating strategy for the identification of circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs). Debris, monocytes, and dead cells were excluded by use of an electronic gate and dump channel, containing cells identified by an anti-CD14 monoclonal antibody and a viability marker (LIVE/DEAD™ Fixable Far Red Dead Cell Stain). CECs and EPCs were identified on the basis of expression of CD45, CD34, and CD133: CECs were defined as CD45dim, CD34+, and CD133−, while EPCs were defined as CD45−, CD34+, and CD133+. The expression of CD309 (VEGFR-2, KDR) was detected among EPCs and CECs. For this phenotype analysis, cells were acquired using a 14-color, 4-laser, high-speed Attune™ NxT Flow Cytometer. Reprinted with permission from De Biasi S, Cerri S, Bianchini E et al. (2015) BMC Med 13:277, and under the Creative Commons Attribution 4.0 International License (

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