Dicentric Assay
Lecture
Module 4
IAEA
International Atomic Energy Agency
Dose response curves (1)
2.0 Neutron mean 0.7 7.6 14.7 250 kVp X-rays
energies, MeV
1 Gy min-1
1.5
Dicentrics
per cell 1.0
0.5 Gy min-1
0.2 Gy h-1
0.18 Gy h-1
0.5
0.0
0
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60Co
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2
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Dose, Gy
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g-rays
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Dose response curves (2)
Which radiations to use ?
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Dose response curves (3)
Each lab should produce its own
calibration curves; do not use other’s
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Background control
What doses to use ?
How many data points ?
How many cells to score ?
How many donors to use?
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Cell culture (1)
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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
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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
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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
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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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