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

• Elevated blood fibrocyte count is an emerging prognostic biomarker in asthma.
• The lack of reliable, clinically-applicable assays precludes further evaluation.
• An optimized whole-blood single-platform flow cytometry assay is described here.
• The assay is analytically and clinically valid and provides reproducible measures.
• Stabilized blood samples can be stored for 96 h at 4 °C before processing.

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.