Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project.
One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.
Fenech
Michael
M
CSIRO Health Sciences and Nutrition, Adelaide, SA, Australia.
Bonassi
Stefano
S
Turner
Julie
J
Lando
Cecilia
C
Ceppi
Marcello
M
Chang
Wushou Peter
WP
Holland
Nina
N
Kirsch-Volders
Micheline
M
Zeiger
Errol
E
Bigatti
Maria Paola
MP
Bolognesi
Claudia
C
Cao
Jia
J
De Luca
Giuseppe
G
Di Giorgio
Marina
M
Ferguson
Lynnette R
LR
Fucic
Aleksandra
A
Lima
Omar Garcia
OG
Hadjidekova
Valeria V
VV
Hrelia
Patrizia
P
Jaworska
Alicja
A
Joksic
Gordana
G
Krishnaja
A P
AP
Lee
Tung-Kwang
TK
Martelli
Antonietta
A
McKay
Michael J
MJ
Migliore
Lucia
L
Mirkova
Ekaterina
E
Müller
Wolfgang-Ulrich
WU
Odagiri
Youichi
Y
Orsiere
Thier
T
Scarfì
Maria Rosaria
MR
Silva
Maria J
MJ
Sofuni
Toshio
T
Surralles
Jordi
J
Trenta
Giorgio
G
Vorobtsova
Irena
I
Vral
Anne
A
Zijno
Andrea
A
Suralles
Jordi
J
D000704:Analysis of Variance; D022003:Cell Nucleus Structures / Q000235:genetics*; D006801:Humans; D007391:International Cooperation; D007753:Laboratories; D008214:Lymphocytes / Q000502:physiology* / Q000528:radiation effects; D008297:Male; D015162:Micronucleus Tests / Q000592:standards*; D015588:Observer Variation; D016012:Poisson Distribution; D012015:Reference Standards