title: 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: BACKGROUND
  Subpopulations 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.
  
  
  METHODS
  The 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.
  
  
  RESULTS
  Thus 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.
  
  
  CONCLUSIONS
  Our 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.
  
  
  BACKGROUND
  This study was registered with ClinicalTrials.gov as NCT01969344 .
authors.affiliation: Research Service, VA Ann Arbor Healthcare System, Ann Arbor, MI, 48105, USA. cmfreema@umich.edu.|Pulmonary & Critical Care Medicine Division, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI, 48109, USA. cmfreema@umich.edu.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Freeman
authors.affiliation: Pulmonary & Critical Care Medicine Division, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI, 48109, USA. scrudgin@umich.edu.
authors.forename: Sean
authors.initials: S
authors.lastname: Crudgington
authors.affiliation: Research Service, VA Ann Arbor Healthcare System, Ann Arbor, MI, 48105, USA. valerie.stolberg@va.gov.
authors.forename: Valerie R
authors.initials: VR
authors.lastname: Stolberg
authors.affiliation: Pulmonary & Critical Care Medicine Division, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI, 48109, USA. browjean@umich.edu.
authors.forename: Jeanette P
authors.initials: JP
authors.lastname: Brown
authors.affiliation: Pulmonary & Critical Care Medicine Division, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI, 48109, USA. jsonstei@umich.edu.
authors.forename: Joanne
authors.initials: J
authors.lastname: Sonstein
authors.affiliation: Center for Environmental Medicine, Asthma, and Lung Biology, Chapel Hill, NC, 27599, USA. Neil_Alexis@med.unc.edu.
authors.forename: Neil E
authors.initials: NE
authors.lastname: Alexis
authors.affiliation: Center for Airways Disease, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. cmd@med.unc.edu.
authors.forename: Claire M
authors.initials: CM
authors.lastname: Doerschuk
authors.affiliation: Marsico Lung Institute/University of North Carolina Cystic Fibrosis Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. basta@email.unc.edu.
authors.forename: Patricia V
authors.initials: PV
authors.lastname: Basta
authors.affiliation: Marsico Lung Institute/University of North Carolina Cystic Fibrosis Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. carretta@email.unc.edu.
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Carretta
authors.affiliation: Marsico Lung Institute/University of North Carolina Cystic Fibrosis Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. david_couper@unc.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Couper
authors.affiliation: Center for Genomics and Personalized Medicine, Wake Forest University, Winston-Salem, NC, 27157, USA. ahastie@wakehealth.edu.
authors.forename: Annette T
authors.initials: AT
authors.lastname: Hastie
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Departments of Medicine and Genetic Medicine, Weill Cornell Medical College, New York, NY, 10021, USA. rkaner@med.cornell.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kaner
authors.affiliation: Marsico Lung Institute/University of North Carolina Cystic Fibrosis Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. woneal@med.unc.edu.
authors.forename: Wanda K
authors.initials: WK
authors.lastname: O'Neal
authors.affiliation: Division of Pulmonary, Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City, UT, 84112, USA. robert.paine@hsc.utah.edu.
authors.forename: Robert
authors.initials: R
authors.lastname: Paine
authors.affiliation: Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198, USA. srennard@unmc.edu.
authors.forename: Stephen I
authors.initials: SI
authors.lastname: Rennard
authors.affiliation: Department of Medicine, Columbia University Medical Center, New York, NY, 10032, USA. ds2231@mail.cumc.columbia.edu.
authors.forename: Daichi
authors.initials: D
authors.lastname: Shimbo
authors.affiliation: Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of California at San Francisco, San Francisco, CA, 94143, USA. Prescott.woodruff@ucsf.edu.
authors.forename: Prescott G
authors.initials: PG
authors.lastname: Woodruff
authors.affiliation: Division of Pulmonary, Critical Care, and Sleep Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, 90095, USA. MZeidler@mednet.ucla.edu.
authors.forename: Michelle
authors.initials: M
authors.lastname: Zeidler
authors.affiliation: Pulmonary & Critical Care Medicine Section, Medicine Service, VA Ann Arbor Healthcare System, Ann Arbor, MI, 48105, USA. jlcurtis@umich.edu.|Pulmonary & Critical Care Medicine Division, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI, 48109, USA. jlcurtis@umich.edu.|Pulmonary and Critical Care Medicine Section (506/111G), Department of Veterans Affairs Healthsystem, 2215 Fuller Road, Ann Arbor, MI, 48105-2303, USA. jlcurtis@umich.edu.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Curtis
mesh_terms: D015415:Biomarkers; D001992:Bronchoalveolar Lavage Fluid; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology; D002469:Cell Separation; D003713:Dendritic Cells / Q000166:cytology; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007962:Leukocytes / Q000166:cytology; D008137:Longitudinal Studies; D008264:Macrophages / Q000166:cytology; D010641:Phenotype; D029424:Pulmonary Disease, Chronic Obstructive / Q000097:blood* / Q000175:diagnosis / Q000378:metabolism*; D012107:Research Design; D018401:Sample Size; D012907:Smoking; D013147:Spirometry; D013183:Sputum / Q000378:metabolism*
chemical_list: D015415:Biomarkers