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

Michael Fenech, Stefano Bonassi, Julie Turner, Cecilia Lando, Marcello Ceppi, Wushou Peter Chang, Nina Holland, Micheline Kirsch-Volders, Errol Zeiger, Maria Paola Bigatti, Claudia Bolognesi, Jia Cao, Giuseppe De Luca, Marina Di Giorgio, Lynnette R. Ferguson, Aleksandra Fucic, Omar Garcia Lima, Valeria V. Hadjidekova, Patrizia Hrelia, Alicja JaworskaGordana Joksic, A. P. Krishnaja, Tung Kwang Lee, Antonietta Martelli, Michael J. McKay, Lucia Migliore, Ekaterina Mirkova, Wolfgang Ulrich Müller, Youichi Odagiri, Thierry Orsiere, Maria Rosaria Scarfì, Maria J. Silva, Toshio Sofuni, Jordi Suralles, Giorgio Trenta, Irena Vorobtsova, Anne Vral, Andrea Zijno

Research output: Contribution to journalArticle

123 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)45-64
Number of pages20
JournalMutation Research - Genetic Toxicology and Environmental Mutagenesis
Volume534
Issue number1-2
DOIs
Publication statusPublished - Jan 10 2003
Externally publishedYes

Fingerprint

Lymphocytes
Cell Culture Techniques
Micronucleus Tests
Cytokinesis
Gamma Rays
Chromosomes
Biomarkers
Radiation
Staining and Labeling
Population

Keywords

  • DNA damage
  • Human lymphocytes
  • HUMN project
  • Micronucleus
  • Nucleoplasmic bridges
  • Protocol
  • Scoring
  • Variability

ASJC Scopus subject areas

  • Health, Toxicology and Mutagenesis
  • Genetics

Cite this

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. / Fenech, Michael; Bonassi, Stefano; Turner, Julie; Lando, Cecilia; Ceppi, Marcello; Chang, Wushou Peter; Holland, Nina; Kirsch-Volders, Micheline; Zeiger, Errol; Bigatti, Maria Paola; Bolognesi, Claudia; Cao, Jia; De Luca, Giuseppe; Di Giorgio, Marina; Ferguson, Lynnette R.; Fucic, Aleksandra; Lima, Omar Garcia; Hadjidekova, Valeria V.; Hrelia, Patrizia; Jaworska, Alicja; Joksic, Gordana; Krishnaja, A. P.; Lee, Tung Kwang; Martelli, Antonietta; McKay, Michael J.; Migliore, Lucia; Mirkova, Ekaterina; Müller, Wolfgang Ulrich; Odagiri, Youichi; Orsiere, Thierry; Scarfì, Maria Rosaria; Silva, Maria J.; Sofuni, Toshio; Suralles, Jordi; Trenta, Giorgio; Vorobtsova, Irena; Vral, Anne; Zijno, Andrea.

In: Mutation Research - Genetic Toxicology and Environmental Mutagenesis, Vol. 534, No. 1-2, 10.01.2003, p. 45-64.

Research output: Contribution to journalArticle

Fenech, M, Bonassi, S, Turner, J, Lando, C, Ceppi, M, Chang, WP, Holland, N, Kirsch-Volders, M, Zeiger, E, Bigatti, MP, Bolognesi, C, Cao, J, De Luca, G, Di Giorgio, M, Ferguson, LR, Fucic, A, Lima, OG, Hadjidekova, VV, Hrelia, P, Jaworska, A, Joksic, G, Krishnaja, AP, Lee, TK, Martelli, A, McKay, MJ, Migliore, L, Mirkova, E, Müller, WU, Odagiri, Y, Orsiere, T, Scarfì, MR, Silva, MJ, Sofuni, T, Suralles, J, Trenta, G, Vorobtsova, I, Vral, A & Zijno, A 2003, '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', Mutation Research - Genetic Toxicology and Environmental Mutagenesis, vol. 534, no. 1-2, pp. 45-64. https://doi.org/10.1016/S1383-5718(02)00248-6
Fenech, Michael ; Bonassi, Stefano ; Turner, Julie ; Lando, Cecilia ; Ceppi, Marcello ; Chang, Wushou Peter ; Holland, Nina ; Kirsch-Volders, Micheline ; Zeiger, Errol ; Bigatti, Maria Paola ; Bolognesi, Claudia ; Cao, Jia ; De Luca, Giuseppe ; Di Giorgio, Marina ; Ferguson, Lynnette R. ; Fucic, Aleksandra ; Lima, Omar Garcia ; Hadjidekova, Valeria V. ; Hrelia, Patrizia ; Jaworska, Alicja ; Joksic, Gordana ; Krishnaja, A. P. ; Lee, Tung Kwang ; Martelli, Antonietta ; McKay, Michael J. ; Migliore, Lucia ; Mirkova, Ekaterina ; Müller, Wolfgang Ulrich ; Odagiri, Youichi ; Orsiere, Thierry ; Scarfì, Maria Rosaria ; Silva, Maria J. ; Sofuni, Toshio ; Suralles, Jordi ; Trenta, Giorgio ; Vorobtsova, Irena ; Vral, Anne ; Zijno, Andrea. / 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. In: Mutation Research - Genetic Toxicology and Environmental Mutagenesis. 2003 ; Vol. 534, No. 1-2. pp. 45-64.
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abstract = "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.",
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T1 - Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes

T2 - Results of an international slide-scoring exercise by the HUMN project

AU - Fenech, Michael

AU - Bonassi, Stefano

AU - Turner, Julie

AU - Lando, Cecilia

AU - Ceppi, Marcello

AU - Chang, Wushou Peter

AU - Holland, Nina

AU - Kirsch-Volders, Micheline

AU - Zeiger, Errol

AU - Bigatti, Maria Paola

AU - Bolognesi, Claudia

AU - Cao, Jia

AU - De Luca, Giuseppe

AU - Di Giorgio, Marina

AU - Ferguson, Lynnette R.

AU - Fucic, Aleksandra

AU - Lima, Omar Garcia

AU - Hadjidekova, Valeria V.

AU - Hrelia, Patrizia

AU - Jaworska, Alicja

AU - Joksic, Gordana

AU - Krishnaja, A. P.

AU - Lee, Tung Kwang

AU - Martelli, Antonietta

AU - McKay, Michael J.

AU - Migliore, Lucia

AU - Mirkova, Ekaterina

AU - Müller, Wolfgang Ulrich

AU - Odagiri, Youichi

AU - Orsiere, Thierry

AU - Scarfì, Maria Rosaria

AU - Silva, Maria J.

AU - Sofuni, Toshio

AU - Suralles, Jordi

AU - Trenta, Giorgio

AU - Vorobtsova, Irena

AU - Vral, Anne

AU - Zijno, Andrea

PY - 2003/1/10

Y1 - 2003/1/10

N2 - 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.

AB - 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.

KW - DNA damage

KW - Human lymphocytes

KW - HUMN project

KW - Micronucleus

KW - Nucleoplasmic bridges

KW - Protocol

KW - Scoring

KW - Variability

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