Download Presentation
## Dicentric Assay

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -

**Dicentric Assay**Lecture Module 4**Dose response curves (1)**250 kVp X-rays Neutron mean energies, MeV 2.0 0.7 7.6 14.7 1 Gy min-1 1.5 Dicentrics per cell 0.5 Gy min-1 1.0 0.2 Gy h-1 0.18 Gy h-1 0.5 60Co g-rays 0.0 0 1 2 3 4 5 Dose, Gy 15772**Dose response curves (2)**Which radiations to use ?**Dose response curves (3)**Each lab should produce its own calibration curves; do not use other’s • Background control • What doses to use ? • How many data points ? • How many cells to score ? • How many donors to use?**Cell culture (1)**• Need to score first metaphases (M1) • 48h to complete first cell cycle • Choose a medium that is not too fast growth • Use PHA for mitogenic stimulation • Monitor cell cycling with FPG staining**Cell culture (2)**Two basic methods: • Whole blood culture • Separated lymphocyte culture**Cell culture (4)**Fixation This is a simple procedure Basic steps: • Harvest cells from culture • Swell cells by hypotonic salt solution • Fix cells in alcohol : acetic acid mixture • Wash several times in fixative to remove non-metaphase cell debris**Slide making**Essential points • Clean, grease-free slides • Cold, wet slides • Laboratory ambient temperature and humidity • Adjust concentration of metaphases**Pre-treatment**Prior to staining • Clean up the background cloudiness • Brief wash in an RNAse A solution – full protocol is given in the IAEA Manual**Slide staining**Fluorescence plus Giemsa stain (FPG) • Essential to ensure M1 scoring Giemsa stain • If confirmed that there are no M2 cells present • If using early Colcemid culture method**Slide scoring**Two methods: • Conventional by-eye • Automated microscope assistance**Scoring at the microscope**The old way The new way**How many cells to score for overdose case ?**Essential points • Depends on dicentric frequency • Depends on the statistical uncertainties needed • Depends on urgency for result • Depends on available skilled scorers • Depends on availability of a metaphase finder**95% confidence limits on 500 or 1000 cells scored**Dose estimate Confidence No of cells scored (mGy) limits 5001000 100 Upper 320 245 Lower <0 16 250 Upper 448 380 Lower 111 141 500 Upper 677 627 Lower 333 383 1000 Upper 1178 1127 Lower 830 881**Data recording (1)**The count of objects must balance A metaphase with: A dicentric plus its fragment still = 46 A centric ring plus its fragment = 47 Each excess acentric increases the count above 46 by +1 Tri-, quadri- centricsetc, are converted to dicentric equivalents**Data recording (2)**Use a standard score sheet • The sheet should include scorer and case ID • Full retrieval of the observations on each cell**Information storage**Securely preserve • Data sheets • Slides • Surplus fixed cells • Case file notes**How to present the dose estimate (1)**• Read the dose off the appropriate dose response curve • This is easy • Calculate the statistical uncertainties on the dose estimate • This is a little more difficult • Report the result to the doctor or patient • This can be difficult: do they understand statistics?**How to present the dose estimate (4)**Which method to use? In practice, in most cases the first and simpler method is sufficient**How to present the dose estimate (5)**• Does the ‘customer’ – understand what is a confidence limit? • The biodosimetry laboratory has to be prepared to explain the result and put it into simple comprehensible terms. Not easy!**How to present the dose estimate (6)**At low doses • If the lower 95% confidence limit is negative it can be ignored and only the upper limit is of concern • Consider presenting the result graphically as a probability distribution**How to present the dose estimate (8)**Presenting the dose as an odds ratio • This also applies to low, possibly zero, dose events • It needs two sources of information: A dose estimate from e.g., a badge A dose estimate from dicentrics • Which is correct?**How to present the dose estimate (9)**Odds ratio • Badge = 66 mSv • Dicentrics = 1 in 1000 cells (background?) • Dicentrics = zero dose with UCL = 100 mGy • Consider 2 possibilities: zero or 66 mSv • Odds ratio 4.5 : 1.0 in favour of zero dose • Easy for a patient to understand**Conclusions**This lecture has covered the dicentric assay from the aspects of: • Producing dose response curves • Processing the lymphocytes • Making and scoring the slides • Presenting the dose estimates in a way that can be understood by non-scientists