Enumeration of circulating fibrocytes for clinical use in asthma by an optimized single-platform flow cytometry assay

Abstract

BackgroundElevated numbers of circulating fibrocytes are associated with inadequately controlled asthma, poor response to available therapies, and increased risk of adverse outcomes. The lack of reliable and clinically-applicable assays precludes a proper evaluation of blood fibrocyte count as a prognostic biomarker in asthma. This report concerns the use of a multiparameter flow cytometry assay for the enumeration of fibrocytes in the whole blood.MethodsConsenting fibrocyte donors were 19 patients with asthma well controlled by current treatment, 16 patients with treatment-resistant asthma, 9 patients with transiently uncontrolled asthma and 14 age-matched normal individuals. Blood sampling was performed once in patients with transiently uncontrolled asthma and twice, at an interval of one week, in the other subjects. The assay was performed in 100 μl of whole blood and involved a sequential gating strategy and absolute fibrocyte counting with a single instrument (single-platform assay).ResultsThe quantification of circulating fibrocytes by this assay was analytically and clinically valid. In individuals with stable clinical conditions, the repeatability of blood fibrocyte counts over one week was good. The intraclass correlation coefficient was 0.939 and 96.88% of the total variability reflected on-average differences among the tested subjects. Stabilized blood samples could be stored at 4 °C for up to 96 h before processing.ConclusionsThe novel assay for the enumeration of fibrocytes in the whole blood is reliable and clinically applicable.General significanceThis report demonstrates the validity and reliability of the first optimized assay for the enumeration of circulating fibrocytes in multicenter clinical trials.

Pathogenetic and prognostic roles of bloodborne fibrocytes in asthma

“…They circulate as a part of the peripheral blood mononuclear cell (PBMC) subpopulation of total leukocytes (Bucala et al, 1994;Chesney et al, 1998;Abe et al, 2001;Bianchetti et al, 2014) and have light scatter features on flow cytometry analysis similar to those of circulating myeloid dendritic cells (mDCs) and some myeloid progenitor cells (mPCs) (Bianchetti et al, 2014). They constitutively express some of the receptors that mediate the trafficking of other BMderived CD34 + PCs, mDCs, and eosinophils (Els) in asthma (Bellini et al, 2013;Mattoli, 2015), such as the C-X-C motif chemokine receptor 4 (CXCR4) (Abe et al, 2001), the C-C motif chemokine receptors (CCRs) CCR3, CCR5, and CCR7 (Abe et al, 2001;Isgrò et al, 2013a), and the receptor for interleukin (IL)-33 suppressor of tumorigenicity 2 (ST2) (Bianchetti et al, 2012a).…”

“…Its major limitation is that it precludes the isolation of a viable population of Fcs for functional analyses, because intracellular staining for COL1 requires prior cell fixation and permeabilization. To address this issue, novel multiple parameter approaches have been developed that allow for the identification and sorting of a pure population of circulating and tissue Fcs by flow cytometry on the basis of the coexpression at high levels on the cell surface of CD45/CD45RO, CD34, CD11b, and CD13 (Bianchetti et al, 2012a;Isgrò et al, 2013a;2013b), which is a distinctive feature of Fcs (Bucala et al, 1994;Chesney et al, 1998;Isgrò et al, 2013b;Bianchetti et al, 2014). Taking advantage of these technical improvements, it has been possible to uncover many functional properties of isolated Fcs and improve our understanding of the molecular mechanisms and signaling pathways involved in their trafficking and activation in asthma (Bellini et al, 2012;Bianchetti et al, 2012a;Isgrò et al, 2013a;2013b), as discussed below.…”

“…In spite of the many promising observations, a proper evaluation of blood Fc count as a prognostic biomarker in asthma has long been precluded by the absence of a reliable assay with clinical applicability (Bianchetti et al, 2014). Most of the studies listed in Table 1 actually used methods that require PBMC isolation and further manipulation of isolated cells.…”

“…Because of the unpredictable cell loss during blood sample processing, these methods do not allow for an accurate and reproducible enumeration of circulating Fcs and cannot be used at large scale. However, the recently developed multiparameter flow cytometry assay for direct Fc enumeration in the whole blood with a single instrument (Bianchetti et al, 2014) (Table 1) has been standardized and optimized for possible use in clinical practice and in multicenter clinical trials. It provides accurate and reproducible measures (Bianchetti et al, 2014) and can be employed to evaluate in larger clinical studies the validity and utility of blood Fc count as a prognostic biomarker and new outcome measure in asthma (Mattoli, 2015).…”

“…However, the recently developed multiparameter flow cytometry assay for direct Fc enumeration in the whole blood with a single instrument (Bianchetti et al, 2014) (Table 1) has been standardized and optimized for possible use in clinical practice and in multicenter clinical trials. It provides accurate and reproducible measures (Bianchetti et al, 2014) and can be employed to evaluate in larger clinical studies the validity and utility of blood Fc count as a prognostic biomarker and new outcome measure in asthma (Mattoli, 2015). * Features of partly controlled/uncontrolled asthma over 2 months and emergency room visit for asthma exacerbation in the past year in spite of intensive treatment with high doses inhaled corticosteroids and/or oral corticosteroids in combination with bronchodilators: prebronchodilator forced expiratory volume in 1 s <80% of predicted normal; daytime symptoms >2 d per week; any limitations of activities because of symptoms; night-time symptoms or on awakening at least once per week; reliever use >2 d per week; ≥3 of the above features per week.…”