title: Flow cytometric analysis of micronuclei in rat peripheral blood: An interlaboratory reproducibility study.
abstract: In anticipation of proposed OECD guideline changes that may include increasing the number of reticulocytes scored for micronuclei, an inter-laboratory reproducibility study of the rat peripheral blood micronucleus assay was performed using flow cytometry. In this experiment, male Sprague-Dawley (SD) rats were treated with the model clastogen cyclophosphamide (CP: 5, 10 or 15mg/kg) by a single oral administration. As controls, rats were treated with physiological saline (solvent) in the same manner as for the model clastogen. Peripheral blood was collected from each rat 48h after the treatment. The blood samples were prepared at the Public Interest Incorporated Foundation, BioSafety Research Center (BSRC) in duplicate using the rat MicroFlow(PLUS) Kit. After fixation, one replicate set of samples was shipped to Litron Laboratories, and each sample was analyzed by flow cytometry at the two laboratories. In addition, the frequency of micronucleated reticulocytes (MNRETs) was determined at the BSRC by microscopic analysis using supravital acridine orange (AO) staining. The reproducibility of micronucleated reticulocyte frequencies analyzed by microscopy and flow cytometry showed good correlation (r(2)=0.84). The frequencies of micronucleated reticulocytes analyzed by flow cytometry at the two independent laboratories showed good concordance (r(2)=0.97). The data indicate that the flow cytometric micronucleus analysis method is a good alternative to manual microscopic analysis. Flow cytometry allows groups to readily score 5000 or even 20,000 RETs in a matter of minutes compared to manual analysis. This results in increased reliability of the assay by achieving better statistical power.
authors.affiliation: Public Interest Incorporated Foundation, BioSafety Research Center (BSRC), 582-2, Shioshinden, Iwata, Shizuoka 437-1213, Japan. Electronic address: kasamoto-sawako@anpyo.or.jp.
authors.forename: Sawako
authors.initials: S
authors.lastname: Kasamoto
authors.affiliation: Public Interest Incorporated Foundation, BioSafety Research Center (BSRC), 582-2, Shioshinden, Iwata, Shizuoka 437-1213, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Mukai
authors.affiliation: Public Interest Incorporated Foundation, BioSafety Research Center (BSRC), 582-2, Shioshinden, Iwata, Shizuoka 437-1213, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Masumori
authors.affiliation: Public Interest Incorporated Foundation, BioSafety Research Center (BSRC), 582-2, Shioshinden, Iwata, Shizuoka 437-1213, Japan.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Suzuki
authors.affiliation: Public Interest Incorporated Foundation, BioSafety Research Center (BSRC), 582-2, Shioshinden, Iwata, Shizuoka 437-1213, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Tanaka
authors.affiliation: Litron Laboratories, 3500 Winton Place, Rochester, NY 14623, USA.
authors.forename: Dorothea K
authors.initials: DK
authors.lastname: Torous
authors.affiliation: Osaka Prefecture University, 1-58, Rinku Ourai Kita, Izumisano, Osaka 598-8531, Japan.
authors.forename: Jyoji
authors.initials: J
authors.lastname: Yamate
authors.affiliation: Public Interest Incorporated Foundation, BioSafety Research Center (BSRC), 582-2, Shioshinden, Iwata, Shizuoka 437-1213, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hayashi
mesh_terms: D000818:Animals; D003520:Cyclophosphamide / Q000633:toxicity; D005434:Flow Cytometry; D007753:Laboratories / Q000592:standards*; D008297:Male; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced* / Q000706:statistics & numerical data; D009153:Mutagens / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012156:Reticulocytes / Q000187:drug effects* / Q000473:pathology; D012680:Sensitivity and Specificity; D018675:Toxicity Tests / Q000592:standards*
chemical_list: D009153:Mutagens; D003520:Cyclophosphamide
keywords: Flow cytometry; Micronucleus assay; Rat; Reticulocytes