Download - IAEA International Atomic Energy Agency Automatic Analysis of Chromosomal Assays Lecture Module 9
IAEAInternational Atomic Energy Agency
Automatic Analysis of Chromosomal Assays
LectureModule 9
IAEA
Chromosomal aberrations seen in mitosis phase
Dicentrics And rings
Two waytranslocation
Terminal translocation
+ …
Unstable chromosomal aberrations
Stable chromosomal aberrations
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Cytokinesis block micronucleus (CBMN) assay
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Needs for automation
Several steps require operator intervention during the process
•Setting up cultures
•Processing cultures through to making slides
• In case of mass casualty many tubes have to be handled: Difficult; Risk of mistakes
•Most time consuming is scoring
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Methodology for automated sample processing
2 days incubation time
Staining
Blood sampling
Cell culture
Cell division arrest
Red cells lysis
Spreading
Robotic blood handler
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Metaphase harvester
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Metaphase spreader
Slide auto-stainer
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3
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Robotic blood handler1
Tecan Genesis (Hanson et al, 2001)
Tecan Freedom Evo (Martin et al, 2007)
Automatic liquid handling system:
automatic scan of barcodes
pipettor
96 samples per run 6
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Metaphase harvester2
Hanabi PII (Martin et al, 2007)
• Automatic metaphase harvester:
centrifugation
hypotonic treatment with incubation at 37°C
fixative treatment
• 24 samples per run = 2 hours
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• Manual spreading
• Temperature, humidity and airflow controlled
• 5 slides per run = 5 mins
3 Metaphase spreader
Hanabi Metaphase Spreader
(Martin et al, 2007)
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Slide auto-stainer4
Thermo Shandon Varistain
Gemini slide stainer (Martin et al, 2007)
Thermo Shandon Consul coverslipper
(Martin et al, 2007)
• Automatic staining and coverslips
• 150 slides per run = 40 mins9
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Automating the microscopy
• Aberration scoring is time consuming
• Cytogenetic labs only have few technical staff
• Many victims could require dose estimation
• Many cells have to be scored
This lecture will concentrate
on the dicentric assay
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Scoring more cells contributes to reduction of confidence intervals
Number of
cells scored
Number of
dicentrics to
have a
significant dose
Correspondi
ng dose
(Gy)
Low CI
(Gy)
High CI
(Gy)
1000 7 0.11 0.006 0.28
10000 34 0.04 0.001 0.10
100000 259 0.03 0.001 0.06
1000000 2400 0.02 0.001 0.05
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Several options for automation
• Develop your own system:• Customized system
• Not so expensive
• Technically demanding
• Buy a ready to use system (METASYSTEMS, CELLSSCAN, IMSTAR, CYTOVISION…)
• More expensive
• Already validated
• Build with available components (Furukawa 2010)
• Less expensive
• Depends on previous developments
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Validation process
• Compare efficiency with manual processing (reference)
• Evaluating sources of variations
• Construct calibration curves under identical conditions used for dose estimation
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Methodology for Automatic Detection of Dicentrics
From lymphocytes metaphases spread over microscopy
slide
Search and acquisition of
metaphases by a microscope
Analysis of metaphase Images by DCScore
software
Estimation of the dose with a
dose-effect curve
Estimation of the yield of dicentrics
per cell
Validation of detected
dicentrics by an operator
Deletion of non analyzable
metaphases
1 2 3
4
567
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History
• First metaphase finder • Developed in 1960s for conventional staining (Wald, 1967)
• Developed in 1990s for fluorescence staining (Vrolijk, 1994)
• Aberration scoring systems• For dicentrics: Bayley, 1991 and Lörch 1989
• For translocation by FISH: and Piper 1994
• For micronuclei: Castelain, 1993 and Verhaegen, 2994
• In 2000s development of machines for cell culture and samples management
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1. Search and acquisition of metaphases by microscope
Search for metaphases on slide (objectivex10)
1Acquisition of metaphases of gallery (objectivex63)
2
Microscope drive by Metafer 4 software
(MetaSystems)
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2. Deletion of non-analyzable metaphases
What is “non-analyzable metaphase”?
Second division metaphase
Unscorable metaphase
Image with 2 metaphases
Why?
To obtain realistic distribution of dicentrics per cell
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3. Image of metaphase analyzed by DCScore software
On all metaphase images, detection of:
Chromosomes, Dicentrics (red square)
Criteria:
Contrast, Object size, Form
Classifier:
Configurable (different according to laboratory)
Microscope driven by Metafer 4 software (MetaSystems)
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4. Validation step
• Each dicentric candidate is confirmed or rejected
False positive dicentrics
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5. Estimation of yield of dicentric
• Validated dicentrics/number of cells evaluated (whatever number of chromosomes identified)
• Result is used either to construct calibration curves or to estimate dose
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Dose-effect Curves (Cesium 137)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0 0.5 1 1.5 2 2.5 3Doses (Gy)
Yie
ld o
f d
icen
tric
s
Manual Scoring
Automatic Detection of Dicentrics
12 doses 0 to 3Gy 75 000cells scored
11 doses 0 to 2.5Gy 10 000cells scored
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Application to population triage
• Objectives• Analyse large number of samples quickly
• First step :• Discriminate individuals in 3 classes:
• Exposed
• Potentially exposed
• Unexposed
• Second step : • Dose estimation with best accuracy possible
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Application to population triage
Methodology currently used First step: Manual scoring on 50 metaphases Second step: Manual scoring on 500 metaphases
Response First step: Quick but low accuracy Second step: Very long and good accuracy
What is response of automatic detection of dicentrics? Experimental model
• Dakar accident - 63 individuals potentially exposed
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0
5
10
15
20
25
500metaphases 50metaphases 1000cells 3000cells
Tim
e (d
ay)
per
6 o
per
ator
s
Timing
Manual Scoring Automatic Detection
of Dicentrics of Dicentrics
0
5
10
15
20
25
500metaphases 50metaphases 1000cells 3000cells
Analysis of metaphase
Search/Acquisition of metaphases
Slide preparation
Culture of lymphocytes20.4 days
5.9 days
8.6 days
15.1 days
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First step: victims classification according to first dose estimation
Manual Scoring 50 metaphases
17.4%
10.9%
71.7%
Automatic Detection of Dicentrics
26.1%
54.3%
19.6%
Manual Scoring 500 metaphases
class 0
class 1
class 2
28.3%
54.3%
17.4%
= the reference
50% under-estimation 4.3% under-estimation
Better results with automatic system25
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First conclusion on population triage
Automatic detection of dicentrics performance:
• Timing quite similar to manual scoring on 50 metaphases but slightly longer
• Classification similar to manual scoring on 500 metaphases
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Second step: dose estimation
• Dose obtained with automatic dicentric scoring close to dose obtained with manual scoring of 500 metaphases
(Vaurijoux et al, 2009)
0
0.5
1
1.5
2
2.5
3
3.5
1 2 3 4 5 7 9 10 13 14 16 17 20 21 22 23 24 25 26 27 30 31 33 34 35 36 37 38 39 40 42 43 45 46
Individuals
Dos
e (G
y)
500MS
ADS
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Conclusion of second step
• Automatic detection of dicentrics is
• 3 times faster than manual scoring on 500 metaphases
• Dose estimation close to manual scoring on 500 metaphases
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Application to individual biological dosimetry
• Question• Can automatic detection of dicentrics detect
heterogeneity of exposure?
• Experimental models• In vitro simulations with blood irradiated to 2Gy
and diluted with unexposed blood
• Real cases of accidental exposure previously analysed manually
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In vitro simulations of exposure eterogeneity
With automatic detection of dicentrics:
• Range of heterogeneity detection - from 5% to 75% irradiated blood to 2Gy
0% -0.085% 5.7015% 6.9125% 8.2450% 7.0660% 3.8475% 2.6090% 0.092Gy 0.61
Proportion of blood irradiated at 2 Gy
u-test
0% -0.085% 5.7015% 6.9125% 8.2450% 7.0660% 3.8475% 2.6090% 0.092Gy 0.61
Proportion of blood irradiated at 2 Gy
u-test
With manual scoring of 500 metaphases: (Barquinero, 1997):
• Range of heterogeneity detection - from 12.5% to 75% irradiated blood to 2Gy
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Real cases of accidental exposure (1)
• Heterogeneity was detected similarly with automatic and manual scoring
• One exception - case 6
Manual Scoring 500 metaphases
Automatic Detection of Dicentrics
1 3.42 6.332 4.73 2.853 4.68 2.804 2.47 3.295 21.2 11.86 5.81 -0.717 3.12 3.458 1.93 0.459 -0.10 -0.1110 -0.28 -0.33
u-test
Individuals
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Doses obtained are similar by both methods
(Vaurijoux, Gruel et al, in submission)
Real cases of accidental exposure (2)
0
1
2
3
4
5
6
7
8
1 2 3 4 5 7Individuals
Dos
es C
alcu
late
s by
Dol
phin
(Gy)
MS
ADS
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Real cases of accidental exposure (3)
Fraction of irradiated blood are similar by both methods
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1 2 3 4 5 7Individuals
Fra
ctio
ns
of ir
radia
ted b
lood
MS
ADS
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Telescoring
• Acquired images can be shared electronically between laboratories
• Sent via the Internet
• Requires homogeneous scoring criteria• Several networks are working on this
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Conclusion for automatic detection of dicentrics
Applications• population triage
• individual cases
Automatic detection of dicentrics can• estimate doses with results close to those obtained
by manual scoring on 500 metaphases
• detect heterogeneous exposure
• allow dose reconstitution of irradiated fraction using Dolphin mathematical model
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Other assays
Micronucleus (CBMN)
This is covered separately in another lecture
Translocation• DAPI stained metaphase finder is well developed
and validated
• No commercial software yet for translocation scoring
• Digitally captured images do not fade
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