title: Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood.
abstract: Experiments described herein were designed to evaluate the performance characteristics of a flow cytometry-based system that scores the incidence of peripheral blood micronucleated reticulocytes (MN-RETs). These procedures represent the continued refinement of a previously reported anti-CD71-based method (Dertinger et al. [1996]: Mutat Res 371:283-292), with the following modifications: incorporation of a third fluorescent label to exclude platelets from the MN-RET region, and use of a CD71-associated fluorescence thresholding technique to increase data acquisition rates. Mouse, rat, and human blood samples were analyzed using both the previously described two-color procedure (anti-CD71-FITC and propidium iodide) and a newly developed three-color technique (which adds an antiplatelet-PE antibody). The rodent specimens were also evaluated by standard microscopy procedures (acridine orange staining). Mouse blood was collected via heart puncture of vehicle- and 5-fluorouracil-treated CD-1 mice; blood samples from saline-treated Sprague-Dawley rats were collected from the tail vein and via heart puncture. Rodent blood samples were analyzed by both the two- and three-color methods. Human blood specimens, obtained via arm venipuncture from cancer patients undergoing radiation therapy, were analyzed for MN-RETs using the two-color method. Subsequently, blood samples from a single chemotherapy patient were analyzed by both the two- and three-color methods. Finally, the chemotherapy patient blood samples and blood samples from 15 healthy volunteers were evaluated at very high densities in conjunction with a CD71-associated fluorescence thresholding technique. Results of these investigations showed that data from mouse blood analyzed by the two- and three-color procedures correlated well with microscopy data (r values = 0.917 and 0.937 for the two- and three-color methods, respectively); all three methods confirmed the genotoxicity of 5-FU. Data from rat tail vein samples showed improved reproducibility with the three-color technique, but no significant difference between the two techniques was seen with the heart puncture specimens. Human blood analyzed according to the two-color procedure produced unreliable results, as platelets and platelet aggregates impacted the rare MN-RET scoring region. The three-color technique effectively overcame this problem and produced reproducible measurements that fell within expected ranges. For human blood analyses, the high cell density/CD71-thresholding technique provided significant improvements over the low-density technique, as it allowed data acquisition to occur approximately six times faster with no loss of sensitivity.
authors.affiliation: Litron Laboratories, Rochester, New York 14620, USA. sdertinger@litronlabs.com
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Dertinger
authors.forename: Kevin
authors.initials: K
authors.lastname: Camphausen
authors.forename: James T
authors.initials: JT
authors.lastname: Macgregor
authors.forename: Michelle E
authors.initials: ME
authors.lastname: Bishop
authors.forename: Dorothea K
authors.initials: DK
authors.lastname: Torous
authors.forename: Svetlana
authors.initials: S
authors.lastname: Avlasevich
authors.forename: Siân
authors.initials: S
authors.lastname: Cairns
authors.forename: Carol R
authors.initials: CR
authors.lastname: Tometsko
authors.forename: Cynthia
authors.initials: C
authors.lastname: Menard
authors.forename: Thierry
authors.initials: T
authors.lastname: Muanza
authors.forename: Yuhchyau
authors.initials: Y
authors.lastname: Chen
authors.forename: Richard K
authors.initials: RK
authors.lastname: Miller
authors.forename: Karin
authors.initials: K
authors.lastname: Cederbrant
authors.forename: Kerstin
authors.initials: K
authors.lastname: Sandelin
authors.forename: Ingrid
authors.initials: I
authors.lastname: Pontén
authors.forename: George
authors.initials: G
authors.lastname: Bolcsfoldi
mesh_terms: D000328:Adult; D000818:Animals; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D002869:Chromosome Aberrations; D020732:Cytogenetic Analysis; D004249:DNA Damage; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D008297:Male; D051379:Mice; D015162:Micronucleus Tests; D008875:Middle Aged; D009369:Neoplasms / Q000097:blood* / Q000532:radiotherapy; D011419:Propidium; D051381:Rats; D017207:Rats, Sprague-Dawley; D011990:Receptors, Transferrin; D012156:Reticulocytes / Q000378:metabolism / Q000473:pathology*
chemical_list: D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D000964:Antimetabolites, Antineoplastic; C068126:CD71 antigen; D011990:Receptors, Transferrin; D011419:Propidium; D005472:Fluorouracil