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Updated: Mar 18, 2026
Quality-Controlled Sputum Analysis by Flow Cytometry
Published on: August 9, 2021
A novel flow cytometric-based method to measure kinase inhibition in sputum from COPD subjects
G C Nicholson1, R A Holloway2, B R Leaker1
1Respiratory Clinical Trials Ltd , London , UK.
Introduction:
Janus kinases (JAKs) regulate inflammatory gene expression through phosphorylation of signal transducer and activator of transcription (STAT) proteins. Expression of STAT proteins is increased in chronic obstructive pulmonary disease (COPD), and may be involved in driving chronic inflammation. Oral JAK inhibitors are effective as anti-inflammatory therapy but exhibit dose-limiting adverse effects. Development of inhaled compounds would be enhanced by robust biomarkers that directly reflect the anti-inflammatory and pharmacological activity in the lung.
Methods:
A novel flow cytometry assay was developed to measure STAT1 phosphorylation in sputum inflammatory cells. The standard sputum processing method was refined to improve sputum cell viability. The flow cytometric assay was used to assess the reproducibility of the measurement of STAT1 phosphorylation and the in vitro activity of a pan JAK-inhibitor on three separate visits in patients with COPD.
Results:
Upregulation of STAT1 phosphorylation was measured following in vitro IFNγ stimulation of sputum macrophages (stimulated/unstimulated ratio 1.57; p<0.00001). Upregulation was inhibited following in vitro preincubation with a pan JAK-inhibitor (inhibited+stimulated/unstimulated ratio 0.97). STAT1 phosphorylation activity could only be measured in macrophages.
Conclusions:
Sputum from patients with COPD can be used to reproducibly measure phospho-STAT expression in sputum macrophages. The flow cytometry-based method can be used to evaluate kinase inhibitors in vitro and subsequently in ex vivo studies. The assay is particularly useful for the assessment of inhaled compounds where whole blood assays may not be relevant.
Insights
A new flow cytometry assay accurately measures STAT1 phosphorylation in sputum macrophages from COPD patients. This method aids in evaluating inhaled anti-inflammatory drugs targeting Janus kinases (JAKs).
Area of Science:
- Pulmonary Medicine
- Immunology
- Biomarker Development
Background:
- Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins are key regulators of inflammatory gene expression.
- Increased STAT protein expression in chronic obstructive pulmonary disease (COPD) may drive chronic lung inflammation.
- Oral JAK inhibitors show anti-inflammatory effects but have dose-limiting side effects, necessitating inhaled alternatives.
Purpose of the Study:
- To develop and validate a novel flow cytometry assay for measuring STAT1 phosphorylation in sputum inflammatory cells.
- To establish a reproducible biomarker reflecting lung-specific anti-inflammatory and pharmacological activity for inhaled JAK inhibitors in COPD patients.
Main Methods:
- Refinement of standard sputum processing to enhance inflammatory cell viability.
- Development of a flow cytometry assay to quantify STAT1 phosphorylation in sputum cells.
- Assessment of assay reproducibility and in vitro activity of a pan JAK-inhibitor in COPD patients over three visits.
Main Results:
- STAT1 phosphorylation was significantly upregulated in sputum macrophages upon IFNγ stimulation (stimulated/unstimulated ratio 1.57; p<0.00001).
- In vitro preincubation with a pan JAK-inhibitor effectively inhibited STAT1 phosphorylation (inhibited+stimulated/unstimulated ratio 0.97).
- STAT1 phosphorylation activity was specifically measurable in sputum macrophages.
Conclusions:
- Sputum macrophages provide a reproducible source for measuring phospho-STAT expression in COPD patients.
- The developed flow cytometry assay is suitable for in vitro and ex vivo evaluation of kinase inhibitors.
- This assay is particularly valuable for assessing inhaled compounds, offering a relevant alternative to whole blood assays for lung-targeted therapies.
