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