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S. De Sanctis, A. De Amicis, S. Di Cristofaro, E. Regalbuto, V. Franchini, F. Lista and G. Mammana Army Medical and Veterinary Research Center - Rome (ITALY) Army Medical and Veterinary Research Center - Rome (ITALY) Cytokinesis-Block MicroNucleus assay Cytokinesis-Block MicroNucleus assay by manual and automated scoring: by manual and automated scoring: calibration curves and dose prediction calibration curves and dose prediction ConRad 2013 Munich 13-16 May 2013

Transcript of Cytokinesis-Block MicroNucleus assay by manual …media.bsbb.de/Conrad/DE SANCTIS.pdf ·...

S. De Sanctis, A. De Amicis, S. Di Cristofaro,

E. Regalbuto, V. Franchini, F. Lista and G. Mammana

Army Medical and Veterinary Research Center - Rome (ITALY)Army Medical and Veterinary Research Center - Rome (ITALY)

Cytokinesis-Block MicroNucleus assay Cytokinesis-Block MicroNucleus assay

by manual and automated scoring:by manual and automated scoring:

calibration curves and dose prediction calibration curves and dose prediction

ConRad 2013 Munich 13-16 May 2013

INTRODUCTION

Cytokinesis-Block Micronucleus Assay (CBMN)

Standardised and validated technique for dose assessment

As alternative to Dicentric Chromosome Assay (DCA)Advantages

much faster

less expertise

Limits Not radiation specific (various genotoxic agents)

Variability in baseline MN frequency exposures > 0.2-0.3 Gy

INTRODUCTION

RADIATION EMERGENCY

Strategies to enhance the capacity of cytogenetic biodosimetry

Biodosimetry laboratory Network

New scoring approaches Triage mode of scoring

Automated scoring

50 metaphases DCA

200 BNcells CBMN

Image analysis sistems

Metafer4 (MetaSystems)

Slide feeder

(80 slides)

CCD camera

Microscope

motorised scanning stage

(8 slides)

Software MNScore for BNcells and MN detection

Classifier set of parameters (morphology and measurements)

Computer

Metafer4 (MetaSystems)Visual Validation

a) Reject unsuitable cells

b) Correct MN count

CBMN ASSAY Manual and automated scoring Manual and automated scoring

STUDY DESIGN

1) Establishement of manual and automated laboratory

dose- response curves Blood samples: 1 not irradiated and X-ray irradiated with 7

increasing doses (0.25-0.5-0.75-1-2-3-4 Gy) of a healthy male donor

2) Evaluation of dose prediction accuracy 10 blind samples X-ray irradiated at unknown doses (0 - 6.4 Gy)

of the same donor to estimate biological dose in manual and

automated mode

Cytokinesis block

Cytochalasin B 28h

MATERIALS AND METHODS

Scoring (HUMN project)

CBMN Assay Protocol

Dose-response curve

4 slides

4000 BNcells/dose

Dose prediction

2 slides

2000 BNcells/blind

Dose-response curve

4 slides

565 - 5276BNcells/dose

Reject 1,5 - 6%

Dose prediction

2 slides

155 - 3314 BNcells/blind

Reject 1,5 – 9% (40% blind 6.4 Gy)

MATERIALS AND METHODS

Dose Estimate Dose estimation for calibration curves and dose prediction

Dose Estimate Software (Health Protection Agency, UK)

Dose- response calibration curves

Linear-quadratic model: Y= c+αD+βD2

Statistical Analysis Dose-response calibration curves

Comparison of results of manual and automatic: Paired t test

Dose prediction accuracy

- Mean of the absolute differences between physical and estimated

doses (MAD)

- Correlation physical - estimated doses: Sperman's rank corr. test

RESULTSRESULTSDose response curvesDose response curves

RESULTSRESULTS

Dose Dose PredictionPrediction

Physical Dose (Gy)0 0.1 0.7 1.4 2 2.2 2.6 3 4.2 6.4 MAD (Gy) MAD w/o 6.4 Gy r Spearman P

Estimated dose

Manual MN/2000 BN 0.0 0.0 0.6 1.2 1.9 2.0 2.2 2.7 3.6 4.7 0.37 0.22 0.997 <0.0001

Automated MN/*BN 0.1 0.0 0.6 1.4 2.0 2.1 2.5 2.6 3.8 3.7 0.40 0.15 0.976 <0.0001

* 155-3314

Good dose prediction accuracy for both scoring mode

excluding 6.4 Gy dose

CONCLUSIONCONCLUSION

Good agreement between manual and automated CBMN dose

response curves but a visual validation is required

Good dose prediction in both modes for all the doses except

6.4 Gy dose

Without 6.4 Gy dose estimation, dose prediction accuracy is

improved as MAD values indicate

Automated CBMN assay must be considered a sensitive and

reliable tool for triage in large scale accidents

Visual validation: limit to overcome

FUTURE PERSPECTIVESFUTURE PERSPECTIVES

Automated CBMN assay

Improvement of CBMN protocols for autometed scoring

Optimisation of classifier setting

Exercise of dose prediction by automatic scoring in triage mode

Interlaboratory comparison on autometed CBMN assay

International cooperation with exchange of classifier

Development of new software

CBMN intercomparison exercise by automated scoring

• Validate an automatic model for biodosimetry in emergency

New software to speed up automatic analysis• Ulteriori analisi, ampliamento del n.di campioni

Più dati per la valutazione

Biodosimetry laboratory - AMVRC, Rome

Florigio Lista

Andrea De Amicis

Sara Di Cristofaro

Giovanni Faggioni

Valeria Franchini

Elisa Regalbuto

NATO 2011 exercise

Organizers and participants

Environmental Carcinogenesis Unit - IST, Genoa

Claudia Bolognesi

ACKNOWLEDGES