1. Basics of Biodosimetry Part 1
Lecture
Module 2
Lecture: Basics of biodosimetry - Part 1
Purpose: To present an overview of biological dosimetry as an introduction to the more detailed course lectures that follow
Learning objectives: Upon completion of this part 1 lecture the participants will have been introduced to:
The requirements for a biological endpoint to function as a dosemeter
The early clinical changes that give some indication of severity of exposure
Why biological dosimetry is needed in radiological protection programmes
An outline of the history of biodosimetry
A consideration of the dose that is specified by biodosimetry
Some basic points about peripheral blood lymphocytes
Duration: 1 hour

2. What is Biodosimetry?
Biodosimetry is the use of any biological change in an irradiated person that can be sufficiently quantified to indicate their radiation dose
Biological measurements to arrive at a dose estimate are totally independent of physical measurements such as from a film, TLD or OSL badge, dose reconstructions with a phantom or calculations based on time and distance from the source.
However it must be appreciated that all sources of information, biological, physical, calculational and peoples’ recollection of an incident need to be integrated to arrive at the best estimate of dose.

3. The ideal specification for a biological dosemeter
Specific to radiation
Low background
Low donor variability
Low doubling dose
Dose response calibration
Persistent effect
Easy sampling
Rapid result
Cost
One needs to be measuring an effect that is not confounded by other insults such as chemical contamination.
One needs an effect that has a low natural background incidence in control subjects.
It needs to have a similar dose response among all people so that one can calibrate the system with measurements from a small panel of subjects rather than from the patients themselves.
A low dose that doubles the background frequency means that the biological dosemeter is sensitive at low doses.
The method has to be capable of being calibrated against known doses from specified radiations. Ideally this is done by irradiating cells in vitro. Otherwise one has to resort to in vivo animal experiments or persons given medical irradiations.
The effect has to be persistent because there may be a delay of days, weeks or months, between irradiation and the discovery that an exposure has occurred.
Appropriate measurements on the patient must be easy, relatively pain free and ethically acceptable.
Because patients, family etc are frequently worried and stressed, the results are required as soon as technically possible.
The cost of the analysis should be reasonable.

4. What are our options?
For high doses: >2 Gy Prodromal nausea and vomiting Differential white blood cell counts Localised > 3 Gy Erythema Conjunctivitis
Vomiting and nausea are very crude indicators with considerable variability between persons and of course are not exclusive to a radiation exposure.
Blood cell counts are more quantitative but also variable between persons.
Localised high doses cause skin reddening, and conjunctivitis if the eyes are in the field. The time course and magnitude of these changes after irradiation is also variable between people.

5. Erythema and blistering
Skin reactions from a localised high dose

6. Changes in neutrophil numbers after irradiationGy
Infections, fever, death
<1
1 - 2
2 - 5
Normal
>5 - 6
100
% of
normal 50
If frequent and repeated blood cell counts are possible then more dramatic changes in cell counts are observed with increasing dose. This technique is more quantitative than observing nausea, vomiting and erythema but nevertheless there is a marked degree of variability between people in their normal background counts and also their radiation response. Often there is a delay before blood sampling starts and the early and most informative dramatic changes are missed. In the absence of a pre-exposure background count for that patient the practice is to use the first sample as the patient’s base-line value and monitor subsequent changes.
Neutrophils (sometimes called granulocytes) are particularly useful because there is an early transient rise to above normal levels and then a steep fall in their numbers. Sometimes, as shown in the blue curve, there is a transient rise noted after days. This is due to a speeding up and release into the blood stream of a reserve of near mature cells. Lymphocytes also fall rapidly after a high dose, but without the initial marked rise.
Critical period,infections,fever 15 30 45
180
Time, d

7. Other possible endpoints
Altered levels of biochemicals in body fluids
Gene mutations
Genes up- or down-regulated
Increased numbers of DNA double strand breaks
These are examples of biological endpoints that are currently under active investigation as possible biodosemeters with varying degrees of promise. Currently none are worked up sufficiently for routine deployment. They will not be covered in this training course.

8. Our best options Cytogenetic indicators: Dicentrics Translocations
Micronuclei
Aberrations in prematurely condensed chromosomes
These are the end points that can be measured in lymphocytes from a small blood sample from an irradiated person. They form the basis of this training course.

9. Why do we need biodosimetry ?
Doctors treat symptoms, not doses
Biodosimetry can detect false positives and false negatives
Biodosimetry can forewarn to expect later clinical developments
Biodosimetry can indicate partial body or inhomogeneous exposure
Early clinical indications like nausea may be due to stress, food poisoning etc. and be wrongly diagnosed as irradiation. Alternatively some people can receive doses well above the nausea threshold and still experience no sickness. These anomalies can be resolved by the presence or absence of chromosome aberrations in their blood lymphocytes.
Cytogenetics can indicate high, but possibly only localised doses, well before clinical signs are present and so warn the doctors to expect later developing problems due to tissue injury.

10. Why do we need biodosimetry?
Dose information can inform doctors in the dose range where medical intervention is needed
Doses <1 Gy will need no treatment, only counselling
False alarms, i.e., no dose, need reassurance
Large events result in epidemiology follow-up
If treatment is needed for large doses then knowledge of the dose is useful to the doctors.
With lower doses patients need counselling and in particular information on their possible increased risk of developing cancer. This can only be quantified by knowing the dose.
In many radiation incidents people wrongly suspect that they have been irradiated. Particularly in the absence of physics information, people fear the worst. Cytogenetics can often resolve these fears by finding no detectable increase in aberration frequencies. Because of sampling statistical considerations that will be covered later in the training course, it is impossible to categorically say ‘no dose’. This is because an estimation of zero dose carries an upper confidence limit. Therefore when advising patients where no increase in aberrations is detected the statistical uncertainties have to be clearly explained.
Very large events such as the Japanese A- bombings, Chernobyl etc. that involve large numbers of people initiate long term scientific studies and in particular cancer incidence epidemiology. This requires knowledge of individuals’ doses and biodosimetry can contribute to the data bases.

11. Why do we need biodosimetry?
Physical dosemeter badges do not necessarily reflect the wearer’s dose
People, sometimes members of the public, who are not routinely monitored can be involved in radiation events
In industrial settings, the nuclear power industry, hospitals, research institutes etc people who work with radiation are routinely issued with dosemeter badges. These very accurately record the dose that they receive, with a low dose sensitivity far better than that of any biological technique. However there are a number of ways that this may not be a true reflection of the dose to the radiation worker. In non- homogeneous fields, narrow beams for example, the badge may or may not be on the part of the body in the field. Badges can be irradiated, sometimes malevolently, when they are not worn. Radiation workers sometimes forget to wear their badge. Also non-badged persons can be involved in an incident.
Biological dosimetry can often resolve such situations.

12. Overall place of biodosimetry in radiation dose monitoring
It supplements, but does not replace, physical monitoring
It works for overdoses, it does not have the sensitivity of a badge
It resolves anomalies
It helps quantify dose in the absence of reliable physics
It relieves anxiety
These points summarise the previous slides on why we need biodosimetry. The important point to get across is that biodosimetry is not trying to make physical monitoring redundant.
It is important that the biological dosimetry laboratory is integrated into the national radiological protection infrastructure of any country and there is a good liaison with physical dosimetry services.

13. Brief early history of cytogenetic dosimetry
Early in the 20th century it was known that radiation damages chromosomes
1960 breakthrough; method to visualize human chromosomes in white blood cells
Calibrations at Oak Ridge, USA
Biodosimetry performed on mid-1960s criticality accidents in USA
Long before it became possible to see human chromosomes clearly, experiments with broad beans and fruit flies had shown that radiation damages chromosomes and causes structural rearrangements of chromosomes called aberrations. Also with higher doses more damage was induced; a dose response effect.
The breakthrough for human studies was a paper by Nowell, Cancer Research 20: , ‘Phytohaemagglutinin: an initiator of mitosis in cultures of normal human leukocytes’. Quite soon afterwards it was determined that phytohaemagglutinin (PHA) which is a drug extracted from a bean plant was stimulating the small lymphocytes in the blood to enter a division cycle. The cells were cultured until they reached mitosis and the chromosomes could be seen in metaphase. Studies with radiation soon followed and it was realised that the same types of aberrations were seen as had been described earlier with beans and flies. In Oak Ridge blood was irradiated in vitro to produce dose response curves. These curves were applied to aberration frequencies observed in lymphocytes from victims of criticality accidents; the Recuplex and Y-12 accidents. These gave credible results and, particularly because fission neutrons were involved which posed many problems for physical dosimetry, the Oak Ridge experience received much attention in the radiation protection community.
Other laboratories were set up, research begun, and many years of steady improvement followed. Further opportunities to carry out biodosimetry soon arose and it became clear that this was a viable and useful technique.

14. The dicentric assay The first type of aberration to be used
Most frequently still used
Therefore a large body of experience
Now a routine procedure
Has been described as the biodosimetry ‘gold standard’
For many years the dicentric assay was the method used for biological dosimetry. It is still much used and latterly other assays; translocations, micronuclei and fragments in prematurely condensed chromosome have been added. With experience of using the dicentric much has been learned on where the assay works well and, in contrast, situations where it is difficult to apply or inappropriate. In many instances, which will be described later in the course, these newer techniques have helped with the shortcomings of the dicentric assay.

15. A dicentric in a metaphase chromosome spread

16. What is the ‘dose’ that we are measuring?
Absorbed dose
Unit is gray (Gy)
We relate the chromosome aberration yield to dose by reference to a dose response calibration curve in Gy
We are measuring dose to lymphocytes
We do NOT operate in sieverts (Sv)
The absorbed dose is a measure of the energy absorbed in matter; in our case in the human body.
Average dose is the total energy deposited in the total volume divided by the mass of the volume
The primary units are joules per kilogram, (J/kg) and the SI unit for this is the gray (Gy). 1 J/kg = 1 Gy.
Previously the non-SI unit of rad was used and in some countries this is still the official unit for absorbed dose. 1 Gy = 100rads
Calibration curves are produced by exposing blood in vitro to known doses. This is monitored with a physical instrument such as an ionisation chamber held in the same geometry as the blood samples. The calibration of the ionisation chamber, or other type of physical system, should be traceable to a national or international standard.
Physical devices that measure photons (or neutrons) are usually calibrated in terms of air kerma. When determining dose to tissue, or blood samples, correction factors need to be applied. These are standard protocols and some guidance is given in the IAEA Manual.
Because we are assaying peripheral blood lymphocytes, strictly the dose that we are measuring is dose to lymphocytes. In some special circumstances we are measuring dose to lymphocyte nuclei. For example, following an intake of tritiated water the short track lengths of the beta particles mean that the dose delivered to a nucleus, and therefore its chromosomes, comes from the tritium contained within that nucleus.
The dose value obtained by reference to a calibration curve is the average dose to the lymphocytes. In most cases this approximates to an averaged whole body dose because the lymphocytes are mobile and widely distributed around the body.
Effective dose and equivalent dose are derived units used for risk assessment in radiological protection and their SI unit is sieverts (Sv). The Sv is not suitable for determining effects of high doses. Biological dosimetry laboratories should therefore calibrate and report their dose estimates in absorbed dose – in Gy, and specify the radiation type involved.

17. What are the lymphocytes that we use
This is a small lymphocyte seen in a standard blood smear. It has a very large nucleus, about 6-7 microns in diameter, surrounded by a thin outer region of cytoplasm.

18. Lymphocytes PHA stimulates T-lymphocytes
Lymphocytes in the blood are non-dividing
They are a synchronised population of cells that can be stimulated to divide and can be harvested at first metaphase
In non-dividing cells dicentrics are predominantly induced by radiation
The blood contains two broad types called T and B cells and PHA stimulates the T cells to enter their division cycle. Both types have their precursors in the bone marrow, but T cells then undergo a maturation process in the thymus gland (hence the designation ‘T’) They then enter the peripheral lymphocyte pool.
The lymphocyte pool in a healthy young adult is about 500 x 109 cells but at any time only about 2% are in the circulating blood. The remainder are permeating throughout all the other tissues of the body. There is a constant interchange or redistribution of cells between the blood and the rest of the body; the mean residence time for a single transit in the blood is about 30 min. Because the lymphocytes are constantly redistributing between the blood, the other tissues and back to the blood any lymphocytes with chromosome damage induced anywhere in the body will eventually appear in the blood. They are sometimes described as ‘millions of microscopic dosemeters’ that enable an average whole body dose to be estimated.
Mature lymphocytes in the blood are a non-dividing population in a so-called resting or G0 state. In fact they are very active carrying out their various functions in the immunological process. The only thing they are resting from is dividing. Lymphocytes are therefore a very convenient, easily sampled and synchronised population of cells. In the G0 state only a limited number of aberration types, notably the dicentric, is induced and because mature lymphocytes do not divide again in the body this damage is stored in the cell until sampled and stimulated by in vitro culture to metaphase

19. The lymphocyte in vitro cell cycle
From the moment of stimulation in culture with PHA the lymphocyte passes through the various phases of the cycle and reaches its first metaphase at approximately 48h. This is the optimum time to block the cells from progressing further, harvest the metaphases and place them on slides for microscope analysis.

20. Conclusions This lecture has described:
Requirements for biological dosemeter
Clinically observable effects of high doses; poorly quantitative
Best options for more quantitative dosimetry; cytogenetics
Why we need biodosimetry; how it helps
Early beginnings in 1960s with the dicentric
Consideration of dose that we specify
Some basic points about peripheral blood lymphocyte