Automatic analysis of chromosomal assays
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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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Automatic Analysis of Chromosomal Assays

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Automatic analysis of chromosomal assays

Automatic Analysis of Chromosomal Assays

Lecture

Module 9


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


Cytokinesis block micronucleus cbmn assay

Cytokinesis block micronucleus (CBMN) assay


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


Automatic analysis of chromosomal assays

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


Scoring more cells contributes to reduction of confidence intervals

Scoring more cells contributes to reduction of confidence intervals


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


Automatic analysis of chromosomal assays

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


Automatic analysis of chromosomal assays

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)


Automatic analysis of chromosomal assays

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


Automatic analysis of chromosomal assays

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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