PMID- 16888079
OWN - NLM
STAT- MEDLINE
DCOM- 20070313
LR  - 20191210
IS  - 1096-6080 (Print)
IS  - 1096-0929 (Linking)
VI  - 94
IP  - 1
DP  - 2006 Nov
TI  - Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An 
      efficient method of monitoring chromosomal damage in the rat.
PG  - 92-107
AB  - We have evaluated a flow cytometric method that allows assessment of 
      micronucleated reticulocytes (MN-RETs) in microliter quantities of peripheral 
      blood and compared results using this assay with those of established microscopic 
      methods of scoring bone marrow and peripheral blood from rats treated with 
      well-characterized genotoxic agents. Young reticulocytes (RETs) are labeled with 
      FITC-anti-CD71 (transferrin receptor) and micronuclei with propidium iodide (with 
      RNase treatment). Red blood cells parasitized with Plasmodia serve as a 
      calibration standard for DNA content. Microscopic scoring used acridine orange 
      (AO) staining of methanol-fixed slides or supravital AO staining. The effect of 
      the rat spleen on the parameters evaluated was determined by comparing age- and 
      sex-matched normal and splenectomized rats treated with cyclophosphamide, 
      cis-platin, or vinblastine under treatment conditions that established a 
      steady-state frequency of MN-RETs in the bone marrow and peripheral blood 
      compartments. The data demonstrate the sensitivity and reproducibility of the 
      flow cytometric assay in the Sprague-Dawley rat, and comparative studies using 
      identical blinded samples at multiple laboratories show that inter- and 
      intra-laboratory reproducibility is much higher with the flow method than with 
      the microscopic methods currently employed for regulatory studies. A significant 
      effect of splenic selection against genotoxicant-induced MN-RETs was observed 
      with each of the three scoring methodologies, despite the fact that the flow and 
      supravital AO techniques restrict analysis to the youngest fraction of RETs. The 
      high precision of flow-based measurements also demonstrated a slight but 
      statistically significant level of selection against spontaneously arising 
      MN-RET. Despite these spleen effects, assay sensitivity for blood-based analyses 
      was maintained by the flow method as it was shown to have superior counting 
      statistics, lower variability, and higher sensitivity than manual scoring. The 
      data suggest that flow cytometric assessment of micronucleus induction can be 
      integrated into routine toxicity testing, eliminating the need for a separate 
      bioassay.
FAU - MacGregor, James T
AU  - MacGregor JT
AD  - FDA National Center for Toxicological Research, Rockville, Maryland 21012, USA. 
      jtmacgregor@earthlink.net
FAU - Bishop, Michelle E
AU  - Bishop ME
FAU - McNamee, James P
AU  - McNamee JP
FAU - Hayashi, Makoto
AU  - Hayashi M
FAU - Asano, Norhide
AU  - Asano N
FAU - Wakata, Akihiro
AU  - Wakata A
FAU - Nakajima, Madoka
AU  - Nakajima M
FAU - Saito, Junichiro
AU  - Saito J
FAU - Aidoo, Anane
AU  - Aidoo A
FAU - Moore, Martha M
AU  - Moore MM
FAU - Dertinger, Stephen D
AU  - Dertinger SD
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Validation Study
DEP - 20060803
PL  - United States
TA  - Toxicol Sci
JT  - Toxicological sciences : an official journal of the Society of Toxicology
JID - 9805461
RN  - 0 (Antineoplastic Agents)
RN  - 5V9KLZ54CY (Vinblastine)
RN  - 8N3DW7272P (Cyclophosphamide)
RN  - F30N4O6XVV (Acridine Orange)
RN  - Q20Q21Q62J (Cisplatin)
SB  - IM
MH  - Acridine Orange/chemistry
MH  - Animals
MH  - Antineoplastic Agents/blood/toxicity
MH  - Bone Marrow Cells/drug effects/metabolism
MH  - Chromosome Aberrations/chemically induced
MH  - Cisplatin/blood/toxicity
MH  - Clinical Laboratory Techniques/standards
MH  - Cyclophosphamide/blood/toxicity
MH  - Female
MH  - Flow Cytometry/*methods
MH  - Male
MH  - Micronuclei, Chromosome-Defective/*drug effects
MH  - Micronucleus Tests/methods/standards
MH  - Microscopy, Fluorescence/methods
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Reproducibility of Results
MH  - Reticulocytes/*drug effects/metabolism
MH  - Splenectomy/methods
MH  - Staining and Labeling/methods
MH  - Time Factors
MH  - Vinblastine/blood/toxicity
EDAT- 2006/08/05 09:00
MHDA- 2007/03/14 09:00
CRDT- 2006/08/05 09:00
PHST- 2006/08/05 09:00 [pubmed]
PHST- 2007/03/14 09:00 [medline]
PHST- 2006/08/05 09:00 [entrez]
AID - kfl076 [pii]
AID - 10.1093/toxsci/kfl076 [doi]
PST - ppublish
SO  - Toxicol Sci. 2006 Nov;94(1):92-107. doi: 10.1093/toxsci/kfl076. Epub 2006 Aug 3.