Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood.
An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.
Torous
Dorothea K
DK
Litron Laboratories, Rochester, New York 14620, USA. dtorous@litronlabs.com
Hall
Nikki E
NE
Illi-Love
Anne H
AH
Diehl
Marilyn S
MS
Cederbrant
Karin
K
Sandelin
Kerstin
K
Pontén
Ingrid
I
Bolcsfoldi
George
G
Ferguson
Lynnette R
LR
Pearson
Amira
A
Majeska
Jenness B
JB
Tarca
James P
JP
Hynes
Geoffrey M
GM
Lynch
Anthony M
AM
McNamee
James P
JP
Bellier
Pascale V
PV
Parenteau
Monique
M
Blakey
David
D
Bayley
Janet
J
van der Leede
Bas-Jan M
BJ
Vanparys
Philippe
P
Harbach
Philip R
PR
Zhao
Shuou
S
Filipunas
Anthony L
AL
Johnson
Carol W
CW
Tometsko
Carol R
CR
Dertinger
Stephen D
SD
D000818:Animals; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D001564:Benzo(a)pyrene / Q000633:toxicity; D003520:Cyclophosphamide / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D008297:Male; D008727:Methotrexate / Q000633:toxicity; D051379:Mice; D015162:Micronucleus Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity; D011990:Receptors, Transferrin; D012156:Reticulocytes; D014750:Vincristine / Q000633:toxicity
D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C068126:CD71 antigen; D009153:Mutagens; D011990:Receptors, Transferrin; D001564:Benzo(a)pyrene; D014750:Vincristine; D003520:Cyclophosphamide; D008727:Methotrexate