title: Microfluidics-based capture of human neutrophils for expression analysis in blood and bronchoalveolar lavage.
abstract: Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying patients who may benefit from targeted therapies. However, isolating human blood polymorphonuclear cells (PMNs) for genomic analysis has been challenging. We used a novel microfluidic technique that isolates PMNs by capturing CD66b(+) cells and compared it with dextran-Ficoll gradient isolation. We also used microfluidic isolation techniques for blood and bronchoalveolar lavage (BAL) samples of patients with acute respiratory distress syndrome (ARDS) to evaluate PMN genomic alterations secondary to pulmonary sequestration. PMNs obtained from ex vivo lipopolysaccharide (LPS)-stimulated or -unstimulated whole blood from five healthy volunteers were isolated by either dextran-Ficoll gradient, microfluidics capture, or a combination of the two techniques. Blood and BAL fluid PMNs were also isolated using microfluidics from seven hospitalized patients with ARDS. Gene expression was inferred from extracted RNA using Affymetrix U133 Plus 2.0 GeneChips. All methods of PMN isolation produced similar quantities of high-quality RNA, when adjusted for recovered cell number. Unsupervised analysis and hierarchical clustering indicated that LPS stimulation was the primary factor affecting gene expression patterns among all ex vivo samples. Patterns of gene expression from blood and BAL PMNs differed significantly from each other in the patients with ARDS. Isolation of PMNs by microfluidics can be applied to both blood and BAL specimens from critically ill, hospitalized patients. Unique genomic expression patterns are obtained from the blood and BAL fluid of critically ill patients with ARDS, and these differ significantly from genomic patterns seen after ex vivo LPS stimulation.
authors.affiliation: Department of Surgery, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Warner
authors.forename: Kenneth T
authors.initials: KT
authors.lastname: Kotz
authors.forename: Ricardo F
authors.initials: RF
authors.lastname: Ungaro
authors.forename: Amer S
authors.initials: AS
authors.lastname: Abouhamze
authors.forename: M Cecilia
authors.initials: MC
authors.lastname: Lopez
authors.forename: Alex G
authors.initials: AG
authors.lastname: Cuenca
authors.forename: Kindra M
authors.initials: KM
authors.lastname: Kelly-Scumpia
authors.forename: Claudia
authors.initials: C
authors.lastname: Moreno
authors.forename: Kerri A
authors.initials: KA
authors.lastname: O'Malley
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Lanz
authors.forename: Henry V
authors.initials: HV
authors.lastname: Baker
authors.forename: Larry C
authors.initials: LC
authors.lastname: Martin
authors.forename: Mehmet
authors.initials: M
authors.lastname: Toner
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Tompkins
authors.forename: Philip A
authors.initials: PA
authors.lastname: Efron
authors.forename: Lyle L
authors.initials: LL
authors.lastname: Moldawer
mesh_terms: D055371:Acute Lung Injury / Q000097:blood / Q000473:pathology*; D015703:Antigens, CD / Q000032:analysis*; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology*; D016022:Case-Control Studies; D015815:Cell Adhesion Molecules / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D058851:GPI-Linked Proteins / Q000032:analysis; D020869:Gene Expression Profiling; D006801:Humans; D046210:Microfluidic Analytical Techniques; D009504:Neutrophils / Q000473:pathology*; D012313:RNA / Q000302:isolation & purification; D012128:Respiratory Distress Syndrome / Q000097:blood / Q000473:pathology*
chemical_list: D015703:Antigens, CD; C504120:CEACAM8 protein, human; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D012313:RNA