Automatic Analysis of Chromosomal Assays

1. Automatic Analysis of Chromosomal Assays Lecture Module 9

2. Chromosomal aberrations seen in mitosis phase + … Two way translocation Dicentrics And rings Terminal translocation Unstable chromosomal aberrations Stable chromosomal aberrations

3. Cytokinesis block micronucleus (CBMN) assay

4. 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

5. 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

6. 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

7. 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)

8. 3 Metaphase spreader • Manual spreading • Temperature, humidity and airflow controlled • 5 slides per run = 5 mins Hanabi Metaphase Spreader (Martin et al, 2007)

9. 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

10. 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

11. Scoring more cells contributes to reduction of confidence intervals

12. 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

13. Validation process • Compare efficiency with manual processing (reference) • Evaluating sources of variations • Construct calibration curves under identical conditions used for dose estimation

14. 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

15. 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

16. 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)

17. 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

18. 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)

19. 4. Validation step • Each dicentric candidate is confirmed or rejected False positive dicentrics

20. 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

21. 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)

22. 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

23. 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

24. Timing 20.4 days 15.1 days 8.6 days 5.9 days Manual ScoringAutomatic Detection of Dicentricsof Dicentrics

25. First step: victims classification according to first dose estimation 50% under-estimation4.3% under-estimation Better results with automatic system = the reference

26. 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

27. Second step: dose estimation • Dose obtained with automatic dicentric scoring close to dose obtained with manual scoring of 500 metaphases (Vaurijoux et al, 2009)

28. 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

29. 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

30. 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

31. Real cases of accidental exposure (1) • Heterogeneity was detected similarly with automatic and manual scoring • One exception - case 6

32. Real cases of accidental exposure (2) Doses obtained are similar by both methods (Vaurijoux, Gruel et al, in submission)

33. Real cases of accidental exposure (3) Fraction of irradiated blood are similar by both methods

34. Telescoring • Acquired images can be shared electronically between laboratories • Sent via the Internet • Requires homogeneous scoring criteria • Several networks are working on this

35. 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

36. 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