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

Lorenza Bianchetti; Mirko Isgrò; Maurizio A Marini; Alberto Bellini; Matthias Schmidt; Sabrina Mattoli

doi:10.1016/j.bbacli.2014.06.002

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

BBA Clin. 2014 Jun 20:1:52-8. doi: 10.1016/j.bbacli.2014.06.002. eCollection 2014 Jun.

Authors

Lorenza Bianchetti¹, Mirko Isgrò¹, Maurizio A Marini², Alberto Bellini³, Matthias Schmidt³, Sabrina Mattoli³

Affiliations

¹ Avail Biomedical Research Institute (ABRI), Basel, Switzerland ; Laboratory of Cytopathology and Cytogenetics, Italian ABRI Operative Unit, Milan, Italy.
² Multispecialty Outpatient Clinic and Diagnostic Center, Milan, Italy.
³ Avail Biomedical Research Institute (ABRI), Basel, Switzerland.

Abstract

Background: Elevated 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.

Methods: Consenting 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).

Results: The 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.

Conclusions: The novel assay for the enumeration of fibrocytes in the whole blood is reliable and clinically applicable.

General significance: This report demonstrates the validity and reliability of the first optimized assay for the enumeration of circulating fibrocytes in multicenter clinical trials.

Keywords: 7-AAD, 7-amino-actinomycin-D; AF, Alexa Fluor; Asthma; BMC, blood mononuclear cell; Biomarker; CCC, concordance correlation coefficient; CI, confidence interval; COL1, type I collagen; CT, threshold cycle; Clinical outcome; ET-1, endothelin-1; FSC, forward scatter; Fibrocyte enumeration; Flow cytometry; ICC, intraclass correlation coefficient; MFI, mean fluorescence intensity; PB, Pacific Blue; SD, standard deviation; SS, sum of squares; SSC, side scatter; Single-platform assay; α-SMA, α-smooth muscle actin.