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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