Post on 29-Jan-2016
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IAEAInternational Atomic Energy Agency
Dicentric Assay
LectureModule 4
IAEA15772
Dicentricsper cell
Dose, Gy0
2.0
1.5
1.0
0.5
0.03 41 2 5
Neutron meanenergies, MeV
0.7 7.6 14.7 250 kVp X-rays
60Co -rays
1 Gy min-1
0.5 Gy min-1
0.2 Gy h-1
0.18 Gy h-1
Dose response curves (1)
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Dose response curves (2)
Which radiations to use ?
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Dose response curves (3)
• Background control
• What doses to use ?
• How many data points ?
• How many cells to score ?
• How many donors to use?
Each lab should produce its own calibration curves; do not use other’s
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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
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Cell culture (2)
Two basic methods:
• Whole blood culture
• Separated lymphocyte culture
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Cell culture (3)
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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
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Slide making
Essential points
• Clean, grease-free slides
• Cold, wet slides
• Laboratory ambient temperature and humidity
• Adjust concentration of metaphases
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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
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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
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FPG Harlequin staining - an M2 cell
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Slide scoring
Two methods:
• Conventional by-eye
• Automated microscope assistance
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Scoring at the microscope
The old way The new way
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How many cells to score for overdose case ?
• 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
Essential points
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95% confidence limits on 500 or 1000 cells scored
Dose estimate Confidence No of cells scored
(mGy) limits 500 1000
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
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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- centrics etc, are converted to dicentric equivalents
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Data recording (2)
Use a standard score sheet
• The sheet should include scorer and case ID
• Full retrieval of the observations on each cell
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Information storage
Securely preserve
• Data sheets
• Slides
• Surplus fixed cells
• Case file notes
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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?
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How to present the dose estimate (2)
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How to present the dose estimate (3)
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How to present the dose estimate (4)
Which method to use?
In practice, in most cases the first and simpler
method is sufficient
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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!
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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
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How to present the dose estimate (7)
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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?
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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
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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
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