Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS)

Abstract

BackgroundSubpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a “just-in-time” design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument.MethodsThe Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data.ResultsThus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel.ConclusionsOur study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters.Trial registrationThis study was registered with ClinicalTrials.gov as NCT01969344.

Multiparameter Single Cell Profiling of Airway Inflammatory Cells

“…The cell yield from induced sputum averaged 6.1 x 10 6 ± x (range 0.8—27.4) with the lowest recovery from healthy controls and higher cell numbers, as expected, from asthmatic and CF subjects (Table 1). To distinguish cell phenotypes and capture the heterogeneity of the cells in sputum, we designed an antibody panel to simultaneously identify multiple cell lineages that accumulate in asthma or CF (15–19) (Table 2, n= 22 antibodies). Gating of live single cells (Figure S1) in CyTOF distinguished lineage specific staining of cytokeratin + non-immune cells which averaged 15.4–18.1% (range 1.7%–48.2%) of cells in asthmatics and healthy controls but were considerably lower in CF subjects (5.6 ± 1.9%; range 0.5%–17.8%).…”

“…Specialized panels can be developed for CF as the CD16 marker to distinguish PMN and eosinophils is often absent in CF (41). Moreover, guides to distinguish monocytes and macrophages--which rely on SSC parameters (17,18) or auto-fluorescence (19) in flow cytometry--can be resolved in future studies by expanding the panel beyond the many shared markers (e.g., CD11c, HLA-DR, CD14).…”

A mucoactive drug carbocisteine ameliorates steroid resistance in rat COPD model

“…Neutrophils are key effector cells in the pathophysiology of COPD, as they release a multitude of mediators and tissue-degrading enzymes such as elastase, which can orchestrate chronic inflammation and tissue destruction [28]. There is a marked increase in the numbers of macrophages in the airways, lung parenchyma, BALF, and sputum of patients with COPD [29], and the number of neutrophils and lymphocytes in the airways is positively correlated with the severity of COPD [30]. Similar events were found in our short-term model: infiltrations with neutrophils and macrophages as well as cytokines and chemokines were found, but no mucus cell hyperplasia occurred in this rat model study with 8 week of CS exposure.…”

Advances in high-dimensional mass cytometry cell and tissue analyses for translational biomarker discovery

“…During the last two decades our knowledge of cellular homeostasis during disease origination and progression has significantly advanced due to the systematic profiling of cellular functions and phenotypes, especially for immune-cell mediated pathologies [1,2]. In fact, many clinical trials now include cellular immunophenotyping as a biomarker component [3,4]. Patient specimens, often as small as a core needle biopsies or superfluous volumes of routinely collected fluids such as blood or sputum [5], are tested by a multitude of single cell profiling methods focusing on gaining a better understating of the cellular milieu.…”

“…In fact, many clinical trials now include cellular immunophenotyping as a biomarker component [3,4]. Patient specimens, often as small as a core needle biopsies or superfluous volumes of routinely collected fluids such as blood or sputum [5], are tested by a multitude of single cell profiling methods focusing on gaining a better understating of the cellular milieu.…”