| Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood. | |
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MedLine Citation:
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PMID: 15517570 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Stephen D Dertinger; Kevin Camphausen; James T Macgregor; Michelle E Bishop; Dorothea K Torous; Svetlana Avlasevich; Siân Cairns; Carol R Tometsko; Cynthia Menard; Thierry Muanza; Yuhchyau Chen; Richard K Miller; Karin Cederbrant; Kerstin Sandelin; Ingrid Pontén; George Bolcsfoldi |
Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Environmental and molecular mutagenesis Volume: 44 ISSN: 0893-6692 ISO Abbreviation: Environ. Mol. Mutagen. Publication Date: 2004 |
Date Detail:
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Created Date: 2004-12-14 Completed Date: 2005-03-08 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 8800109 Medline TA: Environ Mol Mutagen Country: United States |
Other Details:
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Languages: eng Pagination: 427-35 Citation Subset: IM |
Affiliation:
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Litron Laboratories, Rochester, New York 14620, USA. sdertinger@litronlabs.com |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Adult Animals Antigens, CD / immunology, metabolism Antigens, Differentiation, B-Lymphocyte / immunology, metabolism Antimetabolites, Antineoplastic / pharmacology Chromosome Aberrations* Cytogenetic Analysis DNA Damage Female Flow Cytometry / methods* Fluorouracil / pharmacology Humans Male Mice Micronucleus Tests Middle Aged Neoplasms / blood*, radiotherapy Propidium / diagnostic use Rats Rats, Sprague-Dawley Receptors, Transferrin Reticulocytes / metabolism, pathology* |
| Grant Support | |
ID/Acronym/Agency:
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R44ES010752-02/ES/NIEHS NIH HHS; R44ES011244-03/ES/NIEHS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Antigens, CD; 0/Antigens, Differentiation, B-Lymphocyte; 0/Antimetabolites, Antineoplastic; 0/CD71 antigen; 0/Receptors, Transferrin; 36015-30-2/Propidium; 51-21-8/Fluorouracil |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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