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Automatic Analysis of Chromosomal Assays. Lecture Module 9. Chromosomal aberrations seen in mitosis phase. + …. Two way translocation. Dicentrics And rings. Terminal translocation. Unstable chromosomal aberrations. Stable chromosomal aberrations.

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chromosomal aberrations seen in mitosis phase
Chromosomal aberrations seen in mitosis phase

+ …

Two way

translocation

Dicentrics And rings

Terminal translocation

Unstable chromosomal aberrations

Stable chromosomal aberrations

needs for automation
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
slide5

Methodology for automated sample processing

Blood sampling

Cell culture

Cell division arrest

Robotic blood handler

1

2

3

2 days incubation time

Red cells lysis

Metaphase harvester

Spreading

4

Staining

Metaphase spreader

Slide auto-stainer

robotic blood handler

1

Robotic blood handler

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
metaphase harvester

2

Metaphase harvester
  • Automatic metaphase harvester:
    • centrifugation
    • hypotonic treatment with incubation at 37°C
    • fixative treatment
  • 24 samples per run = 2 hours

Hanabi PII(Martin et al, 2007)

metaphase spreader

3

Metaphase spreader
  • Manual spreading
  • Temperature, humidity and airflow controlled
  • 5 slides per run = 5 mins

Hanabi Metaphase Spreader

(Martin et al, 2007)

slide auto stainer

4

Slide auto-stainer

Thermo Shandon Consul coverslipper

(Martin et al, 2007)

Thermo Shandon Varistain Gemini slide stainer

(Martin et al, 2007)

  • Automatic staining and coverslips
  • 150 slides per run = 40 mins
automating the microscopy
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

several options for automation
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
validation process
Validation process
  • Compare efficiency with manual processing (reference)
  • Evaluating sources of variations
  • Construct calibration curves under identical conditions used for dose estimation
slide14

Methodology for Automatic Detection of Dicentrics

1

2

3

From lymphocytes metaphases spread over microscopy slide

Search and acquisition of metaphases by a microscope

Deletion of non analyzable metaphases

4

Analysis of metaphase Images by DCScore software

Validation of detected dicentrics by an operator

7

5

6

Estimation of the dose with a dose-effect curve

Estimation of the yield of dicentrics per cell

history
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
slide16

1. Search and acquisition of metaphases by microscope

1

2

Microscope drive by Metafer 4 software (MetaSystems)

Acquisition of metaphases of gallery (objectivex63)

Search for metaphases on slide (objectivex10)

slide17

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
slide18

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)

4 validation step
4. Validation step
  • Each dicentric candidate is confirmed or rejected

False positive dicentrics

5 estimation of yield of dicentric
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
dose effect curves cesium 137

11 doses

  • 0 to 2.5Gy
  • 10 000cells scored

Manual Scoring

Automatic Detection of Dicentrics

  • 12 doses
  • 0 to 3Gy
  • 75 000cells scored
Dose-effect Curves (Cesium 137)
application to population triage
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
application to population triage1
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
timing
Timing

20.4 days

15.1 days

8.6 days

5.9 days

Manual ScoringAutomatic Detection

of Dicentricsof Dicentrics

first step victims classification according to first dose estimation
First step: victims classification according to first dose estimation

50% under-estimation4.3% under-estimation

Better results with automatic system

= the reference

first conclusion on population triage
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
second step dose estimation
Second step: dose estimation
  • Dose obtained with automatic dicentric scoring close to dose obtained with manual scoring of 500 metaphases

(Vaurijoux et al, 2009)

conclusion of second step
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
application to individual biological dosimetry
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
in vitro simulations of exposure eterogeneity
In vitro simulations of exposure eterogeneity

With automatic detection of dicentrics:

  • Range of heterogeneity detection - from 5% to 75% irradiated blood to 2Gy
  • With manual scoring of 500 metaphases: (Barquinero, 1997):
  • Range of heterogeneity detection - from 12.5% to 75% irradiated blood to 2Gy
real cases of accidental exposure 1
Real cases of accidental exposure (1)
  • Heterogeneity was detected similarly with automatic and manual scoring
  • One exception - case 6
real cases of accidental exposure 2
Real cases of accidental exposure (2)

Doses obtained are similar by both methods

(Vaurijoux, Gruel et al, in submission)

real cases of accidental exposure 3
Real cases of accidental exposure (3)

Fraction of irradiated blood are similar by both methods

telescoring
Telescoring
  • Acquired images can be shared electronically between laboratories
  • Sent via the Internet
  • Requires homogeneous scoring criteria
    • Several networks are working on this
conclusion for automatic detection of dicentrics
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
other assays
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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