1. IAEA International Atomic Energy Agency Automatic Analysis of Chromosomal Assays Lecture Module 9

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

3. IAEA Cytokinesis block micronucleus (CBMN) assay 3

4. IAEA 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 4

5. IAEA 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 4 3 5

6. IAEA Robotic blood handler 1 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

7. IAEA Metaphase harvester 2 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 7

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

9. IAEA Slide auto-stainer 4 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 mins 9

10. IAEA 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 10

11. IAEA Scoring more cells contributes to reduction of confidence intervals Number of cells scored Number of dicentrics to have a significant dose Corresponding dose (Gy) Low CI (Gy) High CI (Gy) 100070.110.0060.28 10000340.040.0010.10 1000002590.030.0010.06 100000024000.020.0010.05 11

12. IAEA 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 12

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

14. IAEA 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 123 4 5 6 7 14

15. IAEA 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 15

16. IAEA 1. Search and acquisition of metaphases by microscope Search for metaphases on slide (objectivex10) 1 Acquisition of metaphases of gallery (objectivex63) 2 Microscope drive by Metafer 4 software (MetaSystems) 16

17. IAEA 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 17

18. IAEA 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) 18

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

20. IAEA 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 20

21. IAEA Dose-effect Curves (Cesium 137) Manual Scoring Automatic Detection of Dicentrics 12 doses 0 to 3Gy 75 000cells scored 11 doses 0 to 2.5Gy 10 000cells scored 21

22. IAEA 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 22

23. IAEA 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 23

24. IAEA Timing Manual Scoring Automatic Detection of Dicentricsof Dicentrics 20.4 days 5.9 days 8.6 days 15.1 days 24

25. IAEA First step: victims classification according to first dose estimation = the reference 50% under-estimation 4.3% under-estimation Better results with automatic system 25

26. IAEA 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 26

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

28. IAEA 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 28

29. IAEA 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 29

30. IAEA 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 30

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

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

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

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

35. IAEA 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 35

36. IAEA 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 36