title: Comparison of cell counting methods in rodent pulmonary toxicity studies: automated and manual protocols and considerations for experimental design.
abstract: Pulmonary toxicity studies often use bronchoalveolar lavage (BAL) to investigate potential adverse lung responses to a particulate exposure. The BAL cellular fraction is counted, using automated (i.e. Coulter Counter®), flow cytometry or manual (i.e. hemocytometer) methods, to determine inflammatory cell influx. The goal of the study was to compare the different counting methods to determine which is optimal for examining BAL cell influx after exposure by inhalation or intratracheal instillation (ITI) to different particles with varying inherent pulmonary toxicities in both rat and mouse models. General findings indicate that total BAL cell counts using the automated and manual methods tended to agree after inhalation or ITI exposure to particle samples that are relatively nontoxic or at later time points after exposure to a pneumotoxic particle when the response resolves. However, when the initial lung inflammation and cytotoxicity was high after exposure to a pneumotoxic particle, significant differences were observed when comparing cell counts from the automated, flow cytometry and manual methods. When using total BAL cell count for differential calculations from the automated method, depending on the cell diameter size range cutoff, the data suggest that the number of lung polymorphonuclear leukocytes (PMN) varies. Importantly, the automated counts, regardless of the size cutoff, still indicated a greater number of total lung PMN when compared with the manual method, which agreed more closely with flow cytometry. The results suggest that either the manual method or flow cytometry would be better suited for BAL studies where cytotoxicity is an unknown variable.
authors.affiliation: a Health Effects Laboratory Division, National Institute for Occupational Safety and Health , Morgantown , WV , USA and.
authors.forename: Patti C
authors.initials: PC
authors.lastname: Zeidler-Erdely
authors.affiliation: a Health Effects Laboratory Division, National Institute for Occupational Safety and Health , Morgantown , WV , USA and.
authors.forename: James M
authors.initials: JM
authors.lastname: Antonini
authors.affiliation: a Health Effects Laboratory Division, National Institute for Occupational Safety and Health , Morgantown , WV , USA and.
authors.forename: Terence G
authors.initials: TG
authors.lastname: Meighan
authors.affiliation: b Army Public Health Center (Provisional) , Aberdeen Proving Ground , MD , USA.
authors.forename: Shih-Houng
authors.initials: SH
authors.lastname: Young
authors.affiliation: a Health Effects Laboratory Division, National Institute for Occupational Safety and Health , Morgantown , WV , USA and.
authors.forename: Tracy J
authors.initials: TJ
authors.lastname: Eye
authors.affiliation: a Health Effects Laboratory Division, National Institute for Occupational Safety and Health , Morgantown , WV , USA and.
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Hammer
authors.affiliation: a Health Effects Laboratory Division, National Institute for Occupational Safety and Health , Morgantown , WV , USA and.
authors.forename: Aaron
authors.initials: A
authors.lastname: Erdely
mesh_terms: D000393:Air Pollutants / Q000633:toxicity; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology*; D002452:Cell Count / Q000379:methods*; D005434:Flow Cytometry; D019570:Inhalation Exposure / Q000009:adverse effects*; D016014:Linear Models; D008168:Lung / Q000187:drug effects* / Q000473:pathology; D008297:Male; D008670:Metals / Q000633:toxicity; D051379:Mice; D008810:Mice, Inbred C57BL; D052638:Particulate Matter / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D012107:Research Design / Q000592:standards*; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D000393:Air Pollutants; D008670:Metals; D052638:Particulate Matter
keywords: Bronchoalveolar lavage; carbon nanotubes; cytotoxicity; flow cytometry; hemocytometer; metals; welding fume