Dicentric Assay Lecture Module 4 Lecture: Dicentric assay Purpose: To present in detail how the dicentric assay is performed. Learning objectives: Upon completion of this lecture the participants will understand: In vitro dose response curves The procedure for lymphocyte culture (also applicable to most of the other assays) Slide staining Manual and automation-assisted scoring for dicentrics Data recording How to calculate and present dose estimates and the sources of uncertainty in the values Duration: 1 hour

Dose response curves (1) 250 kVp X-rays 2.0 Neutron mean energies, MeV 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 The essential points are that the dose response fits best to the linear quadratic model for low LET radiation, Y = αD + βD2, and linear for high LET radiations, Y = αD. There is a difference between gamma and x-rays. This is despite the assumption used in radiation protection that all low LET radiations are weighted identically ( WR = 1 ) when expressed as equivalent dose in Sv. For example the initial slopes of the acute dose response curves, i.e., the alpha coefficients of the linear/quadratic curves show that 250 kVp x-rays are 2-3 times more effective in inducing dicentrics than Co-60 gamma rays. There is also a clear difference between acute and protracted irradiations for the low LET radiations where the β coefficient is reduced. There is no dose rate effect observed with high LET linear response curves. 60Co g-rays 0.0 1 2 3 4 5 Dose, Gy 15772

Dose response curves (2) Which radiations to use ? The dicentric dose response varies with radiation quality. In other words there is an RBE- LET effect. Therefore several curves will need to be prepared. The laboratory should construct calibration curves relevant to the likely radiations encountered in accidents. Most accidents occur with gamma ray sources, followed by orthovoltage x-rays and these are the two basic calibration radiations needed. With time one would expect other dose response curves: with electrons, low energy x-rays, fission neutrons, tritium betas, alpha particles to be added. Incidents with industrial gamma radiography sources are the most frequent type of accident and several isotopes may be used: cobalt-60, caesium-137 and iridium-192 are frequently encountered. Pragmatically, the isotope most likely to be available to research laboratories is Co-60 and a calibration curve with this will be adequate for all industrial gamma source accidents. A separate calibration for 200-250 kVp x-rays is also needed.

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? The background frequency of dicentrics in control subjects is low but not zero. Published surveys have indicated values ranging from 1 dicentric per 1000 – 2000 cells and about 1 in 1000 cells is typical of many studies. When calibration curve fitting, a zero dose point should be included and ideally a laboratory should generate its own background data. The IAEA Manual gives more guidance on how to treat the background. For low LET radiations the dose range for calibration should be 0.25 – 5.0 Gy. Above ~5.0 Gy the data may show evidence of flattening off due to saturation which will distort the quadratic β coefficient in the yield equation. For high LET it is sufficient to calibrate to a maximum of 2.0 Gy. A well fitted calibration curve requires a sufficient number of degrees of freedom. Ideally 10 doses, including zero, should provide this but more are desirable. Most radiation accidents, fortunately, involve low doses and at low doses the initial slope – the α term in the yield equation predominates. Therefore to interpret low dose accident data properly there needs to be an accurate α coefficient with reasonably tight statistical uncertainty. This means producing a lot of low dose calibration data which entails a lot of effort. Although it is said that calibration should start at 0.25 Gy, this is enough to produce a working basic curve. As time permits, it is highly desirable to extend the data to lower doses, say 50, 100 and 150 mGy. At the higher part of the curve the calibration scoring should aim to have 100 dicentrics at each data point. However this is quite impractical at the lower end. Here one should aim at several thousand cells, 3000-5000, per data point. A small panel of normal healthy blood donors, ~5 persons, should be selected with no exceptional irradiation history beyond normal background. Blood from each person should contribute to each data point and appropriate statistical difference tests undertaken to ensure that it is acceptable to pool their data into a single curve.

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 It is essential that dicentrics are scored in M1 metaphases. Therefore a standardized culture method should be established in the laboratory to minimise the contamination with M2 or later cells. This method should be used for culturing irradiated blood for dose response curves and for blood samples from overdose cases. The culture method outlined here is for dicentrics. It is very similar for the other assays but the essential differences are: Two day cultures for dicentrics are extended to 72h for the micronucleus assay. PCC by mitotic fusion requires no culturing but chemically induced PCC does. The dicentric and translocation assays require mitotic arrest with Colcemid but the micronucleus does not. Instead the micronucleus requires cell division blocking with cytochalasin B. The IAEA Manual gives full details of the culture variations.

Cell culture (2) Two basic methods: Whole blood culture Separated lymphocyte culture Whole blood culture: This is the simplest and quickest method. An aliquot of the blood sample is added to a mixture of culture medium, serum, antibiotics, bromodeoxuridine and phytohaemagglutinin. Separated lymphocyte culture: The technique is identical to the method above except that instead of placing whole blood into culture an enriched suspension of lymphocytes is used. Again full details are in the IAEA Manual. There are two ways to enrich the lymphocytes. 1) Allow the blood sample to settle by gravity, or more speedily by adding PHA and serum to the blood and gentle centrifuge. Remove the buffy coat layer that occurs at the top of the red cell layer. 2) Use commercial blood separation Ficoll Hypaque tubes that, after centrifugation, produce a lymphocyte rich layer which can be removed. Important points: 1. The separated lymphocyte method tends to result in more metaphases on the slides but involves more effort which may not be ideal, especially if large numbers of cultures have to be processed concurrently. The whole blood method is usually quite satisfactory. The Ficoll Hypaque separation produces particularly clean preparations, which is an advantage for computer driven metaphase finder microscopes, but is unnecessarily complicated for conventional by-eye scoring. 2. There are several culture media available. It is important to decide on one and use just that for all the biodosimetry work. The medium should routinely give minimal numbers of M2 cells at 48h. It is suggested to avoid fast growth media such as RPMI 1640 or F-10 and instead use a slower medium such as MEM or TC-199. (Notes continued on next slide)

Cell culture (3) A rack of cultures in a 5% CO2 in air incubator. They are held for 48h at 37oC in 10 mL loose-lidded, round bottom tubes culture vessel at a slope angle of 45o. The exact details of the relative volumes of the culture constituents, stock solutions etc are given in the IAEA Manual. At 45h Colcemid is added to the cultures and 3h later the cultures are terminated and the cells fixed. Important points (continued from previous slide notes) 3. Depending on the manufacturer’s data sheets it is often necessary to add L-glutamine to the medium shortly before use because this amino acid has a short storage life in purchased media. 4. Antibiotics and bromodeoxyuridine (BudR) should also be added to the medium shortly before use and because BudR, which is required so that slides can be stained by the FPG method (see later), is UV light sensitive, it should be kept in the dark. Vessels containing BudR can be wrapped in aluminium foil. Cultures should be prepared in subdued lighting, such as a yellow safe-light, and incubated in the dark. 5. Foetal calf serum is the best to use. Human AB serum is also acceptable. Because of considerable batch variation, heat inactivated serum should be used. This is available commercially, alternatively details for inactivation are in the Manual. Various mitogens are available but PHA is universally used for biodosimetry because it stimulates more types of T lymphocytes. Colcemid can be added earlier than at 45h and some laboratories routinely add it at 24h or even at the start of culture. This ensures that there are no M2 cells present. However prolonged use of Colcemid can cause excessive contraction of the metaphase chromosomes so that analysis is difficult. This can be overcome by reducing the Colcemid concentration-details in the Manual. 8. Colcemid, a synthetic version of colchicine, is the preferred arresting agent to use because colchicine is more cytotoxic.

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 Again full details of the procedure are given in the Manual.

Slide making Essential points Clean, grease-free slides Cold, wet slides Laboratory ambient temperature and humidity Adjust concentration of metaphases The quality of the slide is very important; too much débris, too much residual cytoplasm, too few metaphases and tightly packed chromosomes are all problems that can arise and contribute to making slides very difficult to analyse or even unscorable. Laboratories should experiment and find a technique that routinely gives good results. There are many techniques described in the literature. Some tips: Do not always trust manufacturers who advertise that their slides are clean Store slides in a mixture of 1:1 methanol : ether as a degreasing fluid 3. Dry and polish slides just before use using clean tissue paper. Make sure that the tissues, like paper handkerchiefs, do not have lanolin added to make them soft. 4. Place dry clean slides in a freezer for a few minutes, then melt frost with one’s breath and drop the cell suspension onto the slide 5. 1 or 2 drops is sufficient per slide 5. An alternative technique is to dip the clean slide in iced water which has a layer of methanol on its surface, shake off surplus liquid and drop the cells onto the slide. 6. Sometimes the ambient air temperature and humidity in the laboratory may be wrong and the metaphases will not spread well. There are controlled environment cabinets available designed especially for slide making in cytogenetics labs. 7. Before making the slides, check the concentration of the cell suspension. Place 1 drop on a slide, allow to dry and check with a phase contrast microscope. If necessary concentrate or dilute the suspension before continuing to make the slides.

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 As well as the metaphases, the slide may have a lot of cellular débris and cytoplasmic material that gives a background cloudiness. Removing this can considerably clean-up the slides and make scoring easier. Removing the background in particular can make the space between sister chromatids much clearer and so improve aberration identification.

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 Including BudR in the culture medium allows one to stain the slides by FPG. The full method is in the IAEA Manual. Important points: 1. FPG staining is a more complicated procedure than conventional Giemsa staining and sometimes does not work well. 2. It can be difficult to see the harlequin effect and this may be misinterpreted as all metaphases being in M1. Most should be in M1 but there can be considerable donor variability. Some persons appear to have inherently faster cycling lymphocytes and so more contamination with M2 cells after 48h in culture. 3. To check that the FPG staining has worked properly one should have a stock of slides in the lab made from 72h cultures as these will have plentiful M2 cells. One of these slides can be included in the staining batch as a positive control. 4. FPG processing can cause the chromatids to swell and make the metaphase more difficult to score. Conventional Giemsa staining does not produce this problem. Therefore an acceptable procedure is to check one slide from a patient with FPG. If the proportion of M1 cells is acceptable; 95% or higher, remaining replicate slides can be stained and scored with conventional Giemsa.

FPG Harlequin staining - an M2 cell This is an M2 metaphase. Each chromosome shows the differential staining with one chromatid stained lightly and the other darkly. This metaphase should not be scored. By contrast an M1 metaphase would have both chromatids darkly stained. 12

Slide scoring Two methods: Conventional by-eye Automated microscope assistance By-eye: Code the slides to minimise bias Scan the slide methodically from left to right or up and down Scanning should be done at a low magnification (~ x100 – x200) and at this level, with experience, it is possible to evaluate the quality of the chromosomes spreading and approximate completeness, i.e., close to 46 objects, and so select those metaphases that appear worthy of examination at high magnification. The crucial point is that at x100 the quality selection is made without being able to see whether aberrations are present and this minimises observer bias. Turn to high magnification (x1000 oil immersion) and make a snap judgement on whether the spread is scorable – rejection criteria would be too many twisted, overlapping, chromosomes, chromatids pulling apart so that centromeres are difficult to see, chromosomes too contracted or bloated, staining not good enough to resolve chromosome structure. Again, to minimise bias, this should be done while trying to avoid noting the presence of any aberrations. Then, having decided to score, there are two possible ways to proceed: 1. Look through the metaphase, counting the number of objects and note any aberrations. If the count is <46 the cell should be rejected at this point but it is useful to make a marginal note of cells with <46 but containing a dicentric minus its fragment. Metaphases with 46 or more objects should be scored and the numbers of objects should balance with any aberrations – see later. Record the data, including the x,y coordinates, return to x100 magnification, continue the methodical scan and look for the next scorable metaphase. (Notes continued on the next slide)

Scoring at the microscope The old way The new way (Notes continued from previous slide) 2. Do not bother to spend time counting but decide whether the spread is more-or-less complete, inspect the chromosomes and note the presence or absence of aberrations. This latter method is termed ‘Quickscan’ and has been adopted to save time if there is a surge of work such as a large multi-casualties event. It will be discussed later in the training course. The preferred scoring procedure is the former albeit slower object counting method. Some intercomparisons have shown that, with experience, both methods produce comparable dose estimates. It should be stressed that the scoring procedure in a case investigation should be the same as that used for making the in vitro calibration curve. Therefore if Quickscan is to be used on cases there should also calibration curves with Quickscan. Automated scoring: Currently there is no fully automated system; all need operator intervention. Metaphase finder microscopes will find and capture candidate spreads that fulfil set quality criteria, present them in focus at high magnification to the operator who then can score by-eye using a computer screen or the microscope optics. Software exists for automatic dicentric (and micronucleus) recognition and currently this is under evaluation. In general, automated metaphase finding reduces the operators workload and fatigue by about two-thirds.

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 Many of these points are discussed later in the course when considering statistical treatment of data and the ways to respond to a major multi-casualties event. Here the essentials are summarised. A high dose accident will result in a high frequency of aberrations. For example a whole body acute exposure to 4 Gy of Co-60 gamma rays will produce an average dicentric frequency of about 1 per cell. This will soon become apparent and scoring just 50-100 cells is sufficient to provide a good dose estimate with narrow confidence limits. Lower doses require more cells to be scored. The uncertainty is normally given as 95% confidence limits – see the next table. For doses up to 1 Gy a minimum of 500 cells should be scored and often a case can be made to extend the scoring to 1000 cells to improve the statistics. However scoring 500 cells by-eye requires about 1-2 person-days seated at the microscope and so there are cost implications. Scoring with assistance from a metaphase finder allows more cells to be analysed and the lower limit of dose detection can be improved. Eventually however one runs into the problem of distinguishing a low frequency of dicentrics from the assumed background level. Calculations have shown that automated scoring of 10,000 cells is about the practical limit of usefulness of automation and, with this, one could discriminate a dose of 70 mGy of gamma radiation. (Obe et al, Chromosomal aberrations in human populations and cancer, pp 139-161 in Cancer Risk Evaluation: Methods and Trends. Pub: Wiley, 2011) Usually a result is needed quickly and particularly when a lot of patients require investigation the laboratory can work in a triage mode and produce a preliminary dose estimate by scoring perhaps just 50 cells. For a low dose case this carries wide uncertainties but can be enough to assist with the early clinical triage of casualties and can then be followed up later with more cells scored.

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 These values have been calculated from an acute cobalt-60 dose response curve: Y = 0.02103D + 0.06307D2. The table illustrates how the uncertainty on a dose estimate can be reduced by scoring more cells.

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 The total number of objects in the metaphase should balance. An undamaged metaphase will of course have 46 chromosomes However a dicentric plus its fragment will also have 46 Several dicentrics, each with a fragment will still = 46 A ring with a fragment will = 47 It is important that acentric fragments associated with dicentrics or rings are considered as part of the dicentric and ring and are not recorded separately. Acentrics can also be formed independently by a terminal or interstitial deletion from a single chromosome, i.e., not linked to a dicentric or ring. These are termed excess acentrics and are recorded as such. Each increases the overall object count by +1. They must not be combined with the acentrics of dicentrics and rings. Acentrics, whether excess or part of an exchange, vary in size from double dots (termed minutes), larger dots that sometimes appear to be hollow (acentric rings) and varying length linear structures (fragments). In practice for biological dosimetry purposes their discrimination into the different types is not necessary and they can be simply referred to as an acentric or a fragment. A tricentric will be accompanied with 2 acentrics and is scored as being equivalent to 2 dicentrics; a quadricentric will have 3 fragments and is equivalent to 3 dicentrics, and so on for higher levels of polycentrics. As well as recording them as dicentric equivalents it is important to make a marginal note where tricentrics etc occur.

Data recording (2) Use a standard score sheet The sheet should include scorer and case ID Full retrieval of the observations on each cell It is important for a laboratory to use a standard format score sheet for recording the data. This applies both to hand written recording from by-eye scoring and from metaphase finder assisted scoring. Automated systems have the facility of keyboard data entry, automatic recording of some data such as x,y coordinates and many facilities of electronic data storage and handling. The score sheet should include the case code number, slide number, scorer ID, date and which microscope was used. Most important, whether hand written or electronic, the full details of the primary data on each cell should be retrievable. This allows for all possible compilations and aggregations of data to be made at a later date when the overall result is being interpreted and analysed statistically.

Information storage Securely preserve Data sheets Slides Surplus fixed cells Case file notes Research data sheets, such as the primary scoring data from calibration curves and the output from curve fitting computer programs, need to be filed and stored for future reference. For overdose case investigations paperwork and slides or cells should also be preserved. Given the long latency for some cancers, it is possible that an old overexposure investigation may be reopened many years later to resolve a claim for compensation. Laboratories must establish what are their national legal requirements concerning data protection for the preservation of paperwork and materials. Stained slides tend to fade with time but should a re-evaluation be necessary it is possible to remove coverslips and restain with Giemsa, but not FPG. It is also a good practice to store any unused and unstained slides and these are best kept at -20oC. Surplus fixed cells suspensions can also be reduced down by centrifugation into a manageable small volume of fixative, say 2 mL and stored at -20oC in sealed ampoules. Fixed cell suspensions give far better results when examined decades later than cells already on slides.

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? The IAEA Manual gives full details, with worked examples, of how to calculate the uncertainties on the dose estimate.

How to present the dose estimate (2) Uncertainties are expressed as upper and lower 95% confidence limits and an example is shown here. The simplest method is: The observed dicentric yield is 0.05 dicentrics/cell. Calculate (see the Manual), or look up in standard tables, the 95% confidence limits on 0.05 assuming the Poisson distribution. These are upper: 0.072 and lower: 0.034 dicentrics / cell Observe where these 3 values intercept the dose response curve and read off the dose and its 95% confidence limits. Thus The dose estimate is 0.73 Gy with lower and upper confidence limits of 0.57 and 0.91 respectively. This method therefore has considered that the only contributor to the uncertainty is the statistical sampling error on the scoring of the patient’s dicentric frequency.

How to present the dose estimate (3) In this example the uncertainties on the dose estimate also include the uncertainties on the dose response curve. The curve is drawn with its uncertainty derived from the standard errors on the fitted α and β coefficients. The observed dicentric frequency is again read off the central curve to provide the dose estimate and again gives 0.73 Gy. However the upper confidence level on the dicentric frequency (YU) is read off the lower confidence curve and this gives the upper confidence limit on the dose estimate (DU) of 0.97 Gy Similarly the lower confidence level on the dicentrics (YL) is read off the upper curve to produce the lower confidence limit on the dose (DL) of 0.53 Gy

How to present the dose estimate (4) Which method to use? In practice, in most cases the first and simpler method is sufficient If the laboratory has a good dose response curve, based on the scoring of many thousands of cells, its uncertainty contributes very little to the overall uncertainty on the dose estimate and in practice can be ignored. The much greater contributor is the observation on the patient which is usually based on 1000 or fewer cells.

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! Usually one reports the results of a biological dosimetry investigation to a medical doctor. The doctor then has to inform the patient and possibly the employer, a regulator, a safety official an even a lawyer. Do these people really understand the uncertainty? For example, there have been instances where the upper confidence limit has been entered on the worker’s official dose record, ‘just to be cautious’. One can quote the statistical definition “ The 95% confidence limits define an interval that embraces the true dose on 95% of occasions” Does this help? Probably not. One needs to explain in lay- person terms which may not be strictly statistically rigid. So, taking the previous example of a dose estimate of 0.73 (0.57; 0.91) Gy, it is acceptable to say that our best estimate of the dose is 0.73 Gy but this carries statistical uncertainties because we only scored 500 cells. There is a 2.5% chance that the dose could be as low as 0.57 Gy but equally a 2.5% chance that it could be as high as 0.91Gy.

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 A negative lower limit, indicating a negative dose is of course nonsense and one only has to present the dose estimate with its upper 95% limit. Szluinska et al ( Radn Prot Dosim 123, 443-449, 2007) have suggested that low doses can be presented as probability distributions. Their paper gives the procedure which results in a graph that for some people can be very helpful in understanding the situation.

How to present the dose estimate (7) This example shows the probability distributions for low dicentric frequencies of 0, 1 or 2 in 1000 cells. These are so close to the generic background frequency of 1/1000 that the probability is that the dose was zero but the statistical limitations on the dicentric assay mean that one can not state ‘zero dose’ categorically. The probability that the patient was unexposed is proportional to the area beneath the left side of the curve. This is about 80% for 0 dicentrics, 25% for 1 dicentric and 5% for 2 dicentrics scored in 1000 cells. Thus in the case of 0 /1000 the patient can see easily that there is an 80% chance that he was not exposed and it is only the tail of the distribution that lies above zero dose. From the upper 95% confidence limit the patient can see that the chance of the dose being equal to or higher than 0.1 Gy is 2.5%. Similarly for the case of 2/1000 the patient can see that there is some chance (5%) of having received no dose but it is much more likely that there was an exposure. The upper 95% confidence limit is quite high at 0.2 Gy but the distribution peaks at a much lower dose. It is also worth noting that the biodosimetry statistical package ‘Dose Estimate’ available for free from the cytogenetics group at UK Health Protection Agency includes a similar facility based on a Bayesian statistics method. It gives the actual probablility of having received a certain dose, or of having received a dose higher than or lower than a chosen value - quite easier to understand.

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? This idea is elaborated with worked examples in the IAEA Manual.

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 Suppose, for example, a radiation worker’s badge gave a reading of 66mSv, a dicentric analysis is undertaken and 1 dicentric is seen in 1000 cells. This is consistent with normal background. The best biodosimetery estimate is therefore zero dose but it carries an upper 95% confidence limit of 100 mGy and so 66mSv can not be rejected. (The laboratory’s calibration curve equation is Y = 0.001 + 0.04D + 0.06D2). So considering just the two possibilities; the dose was either zero or 66mSv, an odds ratio of 4.5 : 1.0 can be calculated in favour of zero dose. This is derived from the relative probabilities of observing 1 dicentric from the two doses. If the dose is zero then the expected number of dicentrics seen is the normal background- 1/1000 cells. If the dose was 66 mGy, from the dose response curve this equates to 3.9 dicentrics / 1000 cells. The chances of observing 1 when 1 and 3.9 are expected are derived from the Poisson distribution, e-1 and 3.9e-3.9 which are 0.368 and 0.08 respectively. The ratio of the two values is 4.5. Presenting the chances of the badge dose being real as an odds ratio (rather like the language of betting on a horse race!) is probably more comprehensible to the lay person.

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