IAEAInternational Atomic Energy Agency

Automatic Analysis of Chromosomal Assays

LectureModule 9

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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

1

Metaphase harvester

2

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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