Part No...., Module No....Lesson No. Biological Dosimetry Lecture

Part No...., Module No....Lesson No. Biological Dosimetry Lecture
Part title IAEA Post Graduate Educational Course in Radiation Protection and Safety of Radiation Sources Biological Effects of Ionizing Radiation Effects of Radiation at the Molecular and the Cellular Level Part III: Biological Effects of Ionizing Radiation Module III.1: Effects of Radiation at the Molecular and the Cellular Level Lesson III.1.4: Biological Dosimetry Learning objectives: Upon completion of this lesson, the students will be able to: know chromosome aberrations and their types understand chromosome dosimetry – methodology, calibration, dose assessment, uncertainties know cytogenetic dosimetry – advantages and inconveniencies describe recent development in biodosimetry (MN assay, FISH, PCC technique, ESR) – explanation, method, advantages and inconveniencies know new trends in biological dosimetry Activity: Lecture Duration: 2 hours Materials and equipment needed: none References: 1. UNSCEAR Report: Sources and effects of ionizing radiation. Annex G: Early effects in man of high dose of radiation. Chapter III: Prognostic indicators and biological dosimetry. 1988; 2. IAEA Training Course at IPSN. Medical Emergencies in Case of Radiological Accidents. November Training materials. 3. Muller WU and Streffer C. Biological indicators for radiation damage, J Radiat Biol 1991; 59: 4. Lloyd DC. Chromosome analysis to assess Radiation dose. Radiation Injury and the Chernobyl Catastrophe, Stem Cells, Alpha Med Press 1997; 15 (Suppi 2): 5. IAEA. Biological dosimetry: chromosomal aberration analysis for doses assessment. Technical Report Series n°260. International Atomic Energy Agency, Vienna, Austria 1986. 6. Bauchinger M. Retrospective dose reconstruction of human radiation exposure by FISH / chromosome painting. MutatRes 1998; 404: 7. Natarajan A.T., Vyas R.C., Wiegant J. and Curado M.P., A cytogenetic follow-up study of the victims of radiation accident in Goiania (Brazil), Mutation Res.,247, 8. Lloyd D.C. and Edward A.A. Biological dosimetry after radiations in Chromosome Aberration Basic and Applied Aspects Eds. G.Obe and A.T.Natarajan,(Springer Veriag), ,1990. Biological Dosimetry Lecture IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Part No...., Module No....Lesson No.
Part title Introduction Biological dosimetry plays an important role both in radiation protection and in medical management of radiation accidents: Possibility of rapid triage (some techniques) Provision of reassurance of the workers Provides information on the dose but also on the distribution of the dose Lecture notes: In most of the radiation accidents physical dosimetric information is rarely available. In such situations, biological dosimetry can play an important role in assessment of the absorbed dose. This dosimetry is based on the estimation of changes in the biological system as a result of exposure to ionising radiation. Biological dosimetry plays an important role both in radiation protection and in medical management of radiation accidents. In cases of suspected over exposures and where physical dosimetric information is not available or unreliable, chromosomal aberration analysis (CAA) provides very useful information. In many cases where the physical dosimeters are exposed when being not worn, biological dosimetry provides the much-needed reassurance to the worker. In accidental exposures involving doses in the lethal region, CAA not only provides information on the dose but also on the distribution of the dose. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Part title Content Chromosome aberrations and their types Chromosome dosimetry – methodology, calibration curve, dose assessment and uncertainties Interpretation of CAA data Cytogenetic dosimetry – advantages and inconveniencies Recent development in biodosimetry (MN assay, FISH, PCC technique, ESR) – explanation, method, advantages and inconveniencies New trends in biological dosimetry Lecture notes: This lecture presents the major aspects of the radiological knowledge in the field of cytogenetic biomarkers and some new trends in the fields of biochemical and biophysical indicators: chromosome aberrations and their types; chromosome dosimetry – methodology, calibration curve, dose assessment and uncertainties; interpretation of CAA data; cytogenetic dosimetry – advantages and inconveniencies; recent development in biodosimetry (MN assay, FISH, PCC technique, ESR) – explanation, method, advantages and inconveniencies; new trends in biological dosimetry. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Part title Overview Lecture notes: It is well known that ionising radiation interactions with the cells of living organisms induce complex and inter-connected events. The excitation and ionisation of atoms and molecules along particle tracks result in physico-chemical reactions in the cellular organisation and could thus induce physiological consequences. These various modifications on the status of a normal cell are generally a consequence of the initially induced damages and may be considered as biomarkers of radiation exposure. Even though a large number of biological indicators of radiation such as haematological, physiological, biochemical, neurophysiological and biophysical changes have been identified, the cytogenetic changes in peripheral lymphocytes have served as the most reliable indicator of absorbed radiation. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Chromosome aberrations
Part No...., Module No....Lesson No. Part title Chromosome aberrations Are evident in the chromosome pattern at metaphases or anaphases of the first cell cycle following irradiation Are usually breaks in the chromosomes or they may consist of illegitimate rejoining of breaks, e.g. dicentric chromosomes or centric rings Aberration yields can be used as quantitative indicators of radiation exposure Lecture notes: Radiation induced damage to cellular DNA results in chromosomal breaks, which in turn interact with each other resulting in structural alterations in chromosomes. The type of aberrations produced depends on the proliferative phase of cell cycle during irradiation. If cells are irradiated in G0 /G1 phases of the cell cycle, chromosome type aberrations are produced. When cells are irradiated in the G2 or S phase of the cell cycle, chromatid type of aberrations are produced. Chromosomal aberrations can be visualized easily in cultured peripheral blood cells arrested in metaphase. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Chromosome aberrations (Cont’d)
Part No...., Module No....Lesson No. Part title Chromosome aberrations (Cont’d) Types: Unstable dicentrics, centric rings, and fragments Stable translocations, Insertions, and inversions Lecture notes: The nature and the number of radio-induced chromosome aberrations depend on the density of the ionisation along the track of the particle when traversing the cell. Gamma rays, for example, penetrate tissues deeply but cause less damages to the encountered cells than the less penetrating alpha particles. In biological dosimetry, two kinds of structural chromosome-type aberrations are usually taken into account: unstable chromosome aberrations as dicentrics, centric rings and fragments; stable chromosome aberrations such as translocations, insertions and inversions. A good quantitative correlation has been established between the frequency of dicentric chromosomes and radiation dose. Hence, chromosomal aberration analysis (CAA) provides a reasonable estimate of equivalent uniform whole body exposure. In 1962, Bender et Gooch suggested the use of chromosome aberration analysis in peripheral blood lymphocytes to detect and quantify radiation exposure.The radio-induced yield of dicentrics and centric rings in the peripheral blood lymphocytes is still considered as the only medico-legal technique for a biological dose assessment. In case of accidental overexposure, the yield of dicentrics is calibrated against in vitro dose-effect relationships which are established in vitro for different types of radiation and at different dose rates. An advantage of CAA is that the dicentric aberrations are almost radiation specific and caused by very few clastogenic chemotherapeutic agents. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Radiation-induced dicentric chromosome
Part No...., Module No....Lesson No. Part title Radiation-induced dicentric chromosome Lecture notes: Formation of a radiation-induced dicentric chromosome illustrated in a diagrammatic form. This aberration involves an interchange between two separate chromosomes. The primary lesions can be induced by one-track or two-track events. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Chromosome dosimetry - Methodology
Part No...., Module No....Lesson No. Part title Chromosome dosimetry - Methodology Blood sample – 5 mL 10% foetal calf serum, BrdU, and phytohaemagglutinin as a mitogen Colcemid for arresting the cells in metaphase Incubation and fixation Microscope at x 1000 magnification Criterion for scoring – presence of 46 centromeres Lecture notes: At least 24 hours must lapse between radiation exposure and blood collection to allow for the uniform distribution of lymphocytes in the body pools. Since the peripheral blood lymphocytes (PBL) are long – lived, samples collected even after a lapse of 6-8 weeks provide a reliable estimate of the dose. About 5 mL of blood sample is drawn from the exposed individual and collected in a heparinized vial. Whole blood cultures are set up with 0.5 mL of blood in 4 mL of culture medium supplemented with 10% foetal calf serum, BrdU at a final concentration of 15 M and phytohaemagglutinin as a mitogen. After 45 h of culturing at 37oC the cells are arrested in metaphase by adding colcemid (0.05 g/mL). After further incubation for 3 h the cells are spun down and incubated in hypotonic solution (0.075 M KCl) for minutes at 37oC. Cells are then fixed in 3:1 Methanol Acetic acid. Cell concentrate in 0.5 mL fresh fixative are spread on precleaned glass slides and stained with Giemsa and the metaphase spreads are scored under the microscope at x 1000 magnification. The criterion for scoring is the presence of 46 centromeres. Generally induced dicentrics and centric rings are accompanied by an acentric fragments. Generally, 500 metaphase cells are scored per sample. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Normal and aberrant metaphases
Part No...., Module No....Lesson No. Part title Normal and aberrant metaphases a b c d Lecture notes: Figure shows photomicrographs of normal and aberrant metaphases. a - normal metaphase spread with 46 chromosomes; b - cell containing dicentrics and associated fragments; c, d - cells containing translocations. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Dose assessment–calibration curve
Part No...., Module No....Lesson No. Part title Dose assessment–calibration curve Necessary for each laboratory because of variations in radiation response of lymphocytes Method: Irradiation in vitro blood samples from healthy individuals to graded radiation doses Plotting the frequency of dicentric against dose Lecture notes: Radiation response of lymphocytes may vary among laboratories due to the differences in culture conditions. Hence it is necessary for each laboratory to establish its own calibration curves for a variety of radiations. Blood samples from healthy individuals are irradiated in vitro to graded radiation doses in the range of Gy for low LET radiations (X-rays, gamma-rays and -rays) and Gy for fast neutrons. It is ideal to score 100 dicentrics per dose point. Since it is very difficult to do so at lower doses at least a few thousand metaphases are scored for each data point. Calibration curves are obtained by plotting the frequency of dicentric against dose. The reliability of dose response curves can be improved by obtaining blood samples from at least three different individuals. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Types of calibration curves
Part No...., Module No....Lesson No. Part title Types of calibration curves Lecture notes: Calibration curves for low LET radiations are generally linear quadratic; and nearly linear for fission neutrons. Statistical analysis of this curve provides the coefficients necessary for dose assessment. For low LET radiations the relationship is described by: Y= D + D2 where - Y is the frequency of the dicentrics, is the spontaneous frequency of the dicentrics (obtained from Bhabha Atomic Research Centre, Trombay, Mumbai, India for the healthy unexposed individuals), - ,  are linear and quadratic coefficients respectively, - D = absorbed dose in Gy. For fast fission neutrons the linear response is described by: Y= n D, where - n is the linear coefficient for neutrons. Each laboratory has to estimate the dose based on its own calibration curve parameters. Figure: As an example, linear, quadratic dose-response curves for dicentrics (conventional analysis) for the various radiation qualities indicated. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Uncertainties in dose estimation
Part No...., Module No....Lesson No. Part title Uncertainties in dose estimation Sources of errors: From the Poison distribution of dicentrics From individual variation in response The combined error is: S. E. = ± [c 2 + (V a /b)2] 1/2 Lecture notes: There are two important sources of error in the dose estimate. First, the error arises from the Poisson distribution of dicentrics. Second, the calibration curve error arising from individual variations in response. Generally, the calibration error is of the order of ±10% and does not vary significantly depending upon the dose level. If the yield of dicentrics is ‘Y’, the doses corresponding to Y + (S. E.) x 1.96 and Y - (S. E.) x 1.96 provide 95% confidence limits for the estimates. It must be stressed that the Poisson error dominates in the low dose region (<0.5 Gy) yielding very low aberration frequencies. At higher doses the contribution from both the sources of error are comparable. Table 1 shows 95% confidence limits associated with the dose estimates in the range of Gy. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Confidence limits for four estimates of acute gamma radiation dose
Part No...., Module No....Lesson No. Part title Confidence limits for four estimates of acute gamma radiation dose Dose estimate, Gy Confidence limits Number of cells examined 200 500 1000 0.10 Upper - 0.334 0.25 Lower <0.005 0.61 0.50 0.40 0.03 0.12 0.87 0.71 0.64 0.19 0.30 0.36 1.00 1.35 1.21 1.13 0.69 0.81 0.85 Lecture notes: Table shows lower and upper 95% confidence limits associated with the dose estimates in the range of Gy for four estimates of acute gamma radiation dose. Chromosomal aberration analysis has provided the upper limit of the possible equivalent dose when the physical dosimeter has recorded a very high dose, e.g. a radiation worker’s TLD showed a dose of 1395 mSv but CAA 4 dicentrics in 500 metaphases which corresponds to an equivalent whole body dose of 200 mGy ( mGy, 95% confidence interval). For life threatening exposures in excess of 1 Gy, biological dosimetry provides quantitative dose estimate with errors less than 10-20%. In accidents involving lethal doses, suffice it to score 25 metaphases to get preliminary information. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Interpretation of CAA data
Part No...., Module No....Lesson No. Part title Interpretation of CAA data Detection threshold for CAA - ~100 mGy 1 dicentric in 500 cells is interpreted as an absorbed dose of 100 mGy 50% possibility that the observed dicentrics may not be due radiation exposure no dicentrics in 500 cells scored is interpreted as 0 dose in reality it may correspond to a dose less than 100 mGy In low dose region CAA is qualitative but combination with other investigations is needed Lecture notes: Since the reported values of spontaneous background frequency of dicentrics is in the range of 1 in in 1000 cells, the detection threshold for CAA is around 100 mGy. Hence, the presence of 1 dicentric in 500 cells is interpreted as an absorbed dose of 100 mGy, even though there is a 50% possibility that the observed dicentrics may not be due radiation exposure. Similarly, when no dicentrics are observed among the 500 cells scored, it is interpreted as 0 dose but in reality it may correspond to a dose less than 100 mGy. In this low dose region CAA is qualitative but helpful in detecting the genuinity of the suspected exposure when combined with other investigations. Since the probability of finding 2-3 dicentrics in 500 metaphases in unexposed individuals is very small (0.05), the reliability of the assay is considerably improved when 2 or more dicentrics are scored. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Cytogenetic dosimetry
Part No...., Module No....Lesson No. Part title Cytogenetic dosimetry Advantages Easy to assess biomarker (dicentrics) Biomarker is sensitive ( Gy) Biomarker id considered to be radio-induced Dose dependency of dicentrics up to 4 Gy Method is simple Lecture notes: One of the main advantages of cytogenetic dosimetry based on dicentric yield in peripheral blood lymphocytes is that this biomarker is easy to assess, is sensitive ( Gy) and is considered to be specifically radio-induced. Dicentrics are almost dose-dependant (Poisson law) up to 4 Gy. The technique used is simple and reflects a biological cell damage. This biomarker could be assessed at any moment, whereas a personal physical dosimeter is not always worn by the victim. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Cytogenetic dosimetry (Cont’d)
Part No...., Module No....Lesson No. Part title Cytogenetic dosimetry (Cont’d) Inconveniences Method is time consuming and sophisticated Observation speed depends on the quality of cell spreading and on the number of dicentrics per metaphase Only PHA-stimulated lymphocytes can be analysed Lymphocytes bearing dicentrics decline with time Lecture notes: One inconvenient of dicentric scoring is that the recognition of their morphology requires skilled people and is time-consuming. Moreover, the observation speed largely depends on the quality of cell spreading and on the number of dicentrics per metaphase. Two other inconvenients are that: (i) only PHA-stimulated lymphocytes can be analysed; (ii) lymphocytes bearing dicentrics decline with time. According to the literature, the mean half-time for the disappearance of dicentrics is supposed to be about three years and the persistence of lymphocytes bearing these unstable chromosome aberrations is still subject of controversy. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

CAA under complex exposure conditions
Part No...., Module No....Lesson No. Part title CAA under complex exposure conditions Could be used to assess non-uniform or protracted exposures Peculiarities of CAA: Distribution of the dicentrics deviates from Poisson statistics (number of cells containing more than 1 dicentric are seen frequently) Degree of overdispersion can provide useful information regarding the fraction of the body exposed and dose to the part of the body Lecture notes: CAA has been useful in the assessment of the dose even under complex exposure conditions such as non-uniform or protracted exposures. In case of accidents involving non-uniform and partial – body exposures the distribution of the dicentrics deviates from Poisson statistics. In other words, damage is overdispersed i.e. number of cells containing more than 1 dicentric are seen frequently. The degree of overdispersion (deviation from Poisson) can provide useful information regarding the fraction of the body exposed and dose to the part of the body. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

CAA for acute and protracted exposure
Part No...., Module No....Lesson No. Part title CAA for acute and protracted exposure Dose estimation for acute and protracted exposure: Dlower = [-a+ V(a 2 + 4b Y)] / 2 b (acute) Dupper = Y/a (protracted) where Y corresponds to the dicentric frequency Lecture notes: In many accidents the exact duration over which the exposure has occurred may not be known. The yield of dicentrics may significantly reduce at lower dose rates as a result of the repair processes, which tend to reduce the chromosomal damage. If the duration of exposure is exactly known, the b coefficient has to be suitably reduced. For protracted exposures received over different durations b coefficient (the quadratic component) reduces as follows: approximately 55% for 6 h; 75% for 12 h and 85% for 24 h. For chronic accumulated radiation exposures, and exposures received over a duration exceeding 10 h for doses less than 1 Gy; 24 h for doses greater than 1 Gy, the estimates may be entirely based on the relationship Y = D. In accidents where the total doses are greater than 1 Gy and the duration of the total exposure is unknown, dose estimates should be provided for both acute and protracted exposures. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Recent developments in biodosimetry
Part No...., Module No....Lesson No. Part title Recent developments in biodosimetry Micronucleus assay (MN assay) Fluorescent In Situ Hybridisation (FISH) and stable chromosome aberrations Premature chromosome consideration (PCC) Electron spin resonance (ESR) measurements Lecture notes: Efforts are in progress to develop alternate methods for biological dosimetry. These include micronucleus assay (MN assay) in cytochalasin blocked binucleated lymphocytes, assay of chromosome breaks in interphase cells by fusing the peripheral blood lymphocytes with mitotic Chinese hamster cells (PCC technique), assay of stable chromosomal exchanges (translocations) in lymphocytes by fluorescence in-situ hybridization (FISH) technique and electron spin resonance (ESR) measurements of the ionizing radiation induced free radicals trapped in tissues like tooth enamel and bone. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Part title MN assay – explanation Mechanisms of MN production Acentric fragments, multicentric chromosomes, damaged kinetochores or damaged spindle fibres Radiation induced MN Originate primarily from acentric chromosome fragments resulting from chromosome breakage Show a dependence on radiation dose and quality Lecture notes: Micronuclei appear as discrete round small masses of DNA found in the cytoplasm outside the main nuclei and can widely vary in size. The appearance of micronuclei necessitates a successful complete mitosis cycle. In the course of time various mechanisms of micronuclei production were proposed: acentric fragments, multicentric chromosomes, damaged kinetochores or damaged spindle fibres. Nowadays as confirmed by FISH technique applied to MN assay, it is accepted that radiation-induced MN originate primarily from acentric chromosome fragments resulting from chromosome breakage. A minor number of radiation-induced MN can also be formed by whole chromosomes that lag behind during mitosis due to some defect at the level of the spindle or the kinetochore protein. As radio-induced MN show a dependence on radiation dose and quality, the scoring of MN in human blood lymphocytes was suggested as an alternative to the dicentric scoring for radiation protection purposes. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Part title MN assay - method Background frequency of MN per 1000 binucleated cells Doses less than 250 mSv can not be detected with reliability Different methodological protocols with some modifications exist Lecture notes: Scoring of micronuclei in cultured lymphocytes is done by blocking the cytokinesis using cytochalasin B. Since the background frequency of MN can vary from 5-20 per 1000 binucleated cells, doses less than 250 mSv can not be detected with reliability using this technique. Simplicity and speed of scoring MN as compared to dicentrics makes this technique attractive. As many as binucleated cells can be scored per man-day against the 200 metaphases for CAA. The prospects of automation of scoring MN are very bright. In accidents involving large number of individuals this technique may prove useful. An aliquot of 0.1 ml whole blood is cultured in 12.5 cm2 microtiter wells (Costar) with 1 ml medium (RPMI 1640, 25 % foetal calf serum and 2.5 % PHA). A concentration of 5 g/ml Cytochalasin B is added after 40 hours culture. All cultures are incubated in a humidified atmosphere of 5 % CO; at 37 °C and arrested after 64 h culture. Cultures are then centrifuged into microtubes at 30g for 5 min. The pellet is mixed with 2 ml of a 125 mM K.C1 solution for 8 min and centrifuged once more. The pellet is fixed twice in ethanol/acetic acid (6/1, v/v). Finally, the cell suspension is spread on clean slides. Slides are stained with 3% Giemsa for bright field microscopy studies or with 5 g/ml propidium iodide when fluorescence is used. Whatever the method of scoring used, the number of binucleated cells is counted and related to total cells. MN in mononucleated, trinucleated and tetranucleated cells are excluded from scoring. At least binucleated cells are observed per sample. A brief review of the literature shows that there is more than one methodological protocol. Various modifications were introduced in the course of time and a prerequisite of the MN assay is the preservation of the cellular cytoplasm and a high binucleate cell rate. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

MN assay - advantages and inconvenience
Part No...., Module No....Lesson No. Part title MN assay - advantages and inconvenience Advantages Rapid and easy to perform technique Inconveniencies Less sensitive than dicentric scoring Difficult to interpret for low doses Difficult to analyse total- and partial-body exposure Lecture notes: The major advantage of MN counting for radiobiological purposes is that this technique is rapid and easy to perform. A full automation is soon expected by some laboratories. In the same field, MN detection by flow-sorting is under progress. However, some inconvenients are well-known. First of all, the MN determination is not as sensitive as that of chromosomal aberrations, especially as the dicentric scoring. Moreover, an increase of MN rate could be difficult to interpret particularly at low doses, because of the known variability of individual dose response and the unknown background frequency of the subject to analyse. The mean of the background frequency is high, in the range of MN/ binucleated cell for a healthy population. Finally, a discrimination between total- and partial-body exposure is more difficult than in the case of dicentrics scoring because micronuclei show overdispersion. Another negative point is that MN are formed by exclusion of chromosomal material from the cell nucleus and are thus a secondary-, rather than a primary-effect of cytogenetic radio-induced damage. It is still not known if the incorporation of chromosome material into radiation-induced MN is an unbiased process. Moreover, the culture time is longer than the one used for the chromosome aberrations scoring and some important variables may influence base-line micronucleus frequency in cytokinesis-blocked lymphocytes. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Premature chromosome consideration (PCC) - explanation
Part No...., Module No....Lesson No. Part title Premature chromosome consideration (PCC) - explanation PCC assay was discovered in 1970 Method allows the visualisation of the initial radio-induced damage in the nucleus of the unstimulated peripheral blood lymphocytes Lecture notes: After the discovery of the PCC by Johnson and Rao in 1970, PCC assay was introduced in different studies as, for example: (i) suitability for biological dose assessment in case of total- and partial-body irradiation and (ii) in combination with FISH labeling for the study of formation kinetics of chromosome aberrations in irradiated human lymphocytes. One of the main interest of the PCC technique is that this method allows the visualisation of the initial radio-induced damage in the nucleus of the unstimulated peripheral blood lymphocytes. Indeed, it is well known that radiation induce more initial breaks than the observed chromosome aberrations in metaphases. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Part title PCC - method Interphase cells are fused with mitotic cells in presence of a fusion agent The diffuse chromatin of the interphase nucleus condenses in a prophase-like reaction and rapidly forms "premature chromosomes“ Method used in IPSN (France) is presented as an example Lecture notes: In the PCC assay, interphase cells are fused with mitotic cells (cell lines HeLa or CHO) in presence of a fusion agent (Sendai virus or polyethylene glycol). Due to the mitotic factors in the hybrid cells, the diffuse chromatin of the interphase nucleus condenses in a prophase-like reaction and rapidly forms "premature chromosomes". One of the method used in routine is as follows (IPSN data, France). For the production of mitotic cells, a Chinese Hamster Ovary (CHO) cell line is used (ATCC collection, CHOK1, CCL-61). The CHO cell cultures are carried out in the growth medium [McCoy's 5A-Glutamax 1, 10 % foetal calf serum, penicillin-streptomycin at 100 UI/ml of medium, (Life Technologies, France)]. The CHO cells are grown in tissue-culture flasks (75 cm2. Falcon, France), at 37 °C in an humid atmosphere of 5 % CO;, and are subcultured twice a week. The day before the fusion experiment, a flask of confluent cells is subcultured and divided into two new flasks to obtain exponentially-growing cells. The next day, the culture supernatant containing dead cells is discarded and 14 ml of complete growth medium containing 0.2 g/m1 colcemid™ (KaryoMAX, Life Technologies, France) are added. Mitotic CHO cells are harvested by a shake-off procedure 4h later, scored and used as soon as possible for PCC assay. Mitotic index is found to be greater than 90 %. The whole blood (for the fusion) is collected into vacutainer tubes containing 1 ml of Acid Citrate-Dextrose as anticoagulant (ACD, Polylabo, France). Lymphocytes are isolated from whole peripheral blood using a Ficoll gradient (Lymphoprep™, Phannacia, France) in specially designed tubes (Leucoprep™, Polylabo, France). Separated cells are then washed three times with RPMI 1640 (Life Technologies, France) and counted on an automated hematocymeter (Technicon HI™, Bayer Diagnostic, France). PCC are obtained according to a modified procedure of Pantelias and Maillie. Before fusion, all the cells (CHO and lymphocytes) are washed with RPMI 1640 medium to eliminate colcemid and serum respectively. About, mitotic CHO cells are mixed with Go lymphocytes in round-bottomed culture tubes and washed with PBS. After gentle centrifugation (100 g, 5 min), the supernatant is discarded and the pellet is dried. Polyethylene glycol (PEG1450, 0.1 ml, 50 % w/v, Sigma Chemical Co, France) is injected onto the cell pellet using a 1 ml syringe. After 1 min, PBS is added to dilute PEG, the obtained cell suspension is centrifuged (100 g, 5 min) and the pellet is resolved in complete RPMI 1640 medium. Colcemid is then added and the cell suspension is incubated for 75 min at 37 °C to allow the chromatin condensation. After an hypotonic shock with KC1 (0.075 M, 1 min at 37°C) and two fixations in methanol: acetic acid (3 / 1, v/v), cells are dropped on wet slides, air dried and stained with 4 % Giemsa. Hybrid cells are observed under a bright field microscopy, with a 1000x magnification and PCC excess fragments are scored by subtracting 46 from the observed residual PCC fragments. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

PCC - advantages and inconvenience
Part No...., Module No....Lesson No. Part title PCC - advantages and inconvenience Advantages The method is applicable at high doses (no need for irradiated cell to reach the mitosis) The method may be required to all cellular types PCC could be useful in case of a localised exposure Technique is quick Inconveniencies Technique is sophisticated and delicate PCC assay needs to have a correct mitotic cell line Lecture notes: The main advantage is that the evaluation of some chromosomal damages using PCC avoids the difficulties arisen by the variability in lymphocytes stimulation. The method is applicable even at high doses because there is no need for the irradiated cell to reach the mitosis. Another advantage of this technique is that since a preliminary culture is not required, this technique potentially may be applied to all cellular types. This could be useful in case of a localised irradiation (for example for skin cells). An important advantage of this method is that, information on the exposure can be derived within 3-4 h of obtaining the blood sample. Further, since the technique does not involve cell division, the artifacts associated with post irradiation stimulation and progression through the cell cycle do not interfere with the analysis. Even in accidents involving cells exposed to doses in excess of 5 Gy, the cells can easily undergo condensation even though they may fail to reach metaphase. PCC technique should be specifically useful in such situations. In spite of the speed of analysis, this technique is being in use in very few centres. Further work needs to be done to have a clearer understanding of the factors, which influence the process of condensation in order to get reproducible results, and good chromosome spreads. The main inconvenience is that the technique is still heavy and delicate, especially for the excess fragment recognition. Moreover, the PCC assay needs to have a correct mitotic cell line and needs to be taken care of. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

FISH assay - explanation
Part No...., Module No....Lesson No. Part title FISH assay - explanation Useful in case when the time lapse between overexposure and chromosome aberration analysis is long FISH assay provides an easy detection of translocations Method is based on the long-term persistence of translocations after exposure Lecture notes: Dose assessment is especially problematic when the time lapse between overexposure and chromosome aberration analysis is long or even sometimes unknown. In this case, the scoring of stable chromosome aberrations as translocations, may be an indication of past-overexposure. The Fluorescence In Situ Hybridisation technique "FISH-painting" provides an easy detection of translocations. The yield of aberrations involving only the subset of chromosomes painted is scaled up to the full genomic frequency by assuming a random break distribution. The retrospective dosimetry using translocation yield is based on the long-term persistence of translocations after exposure. In addition, very few human past-overexposure have a well-defined physical dosimetry. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Part title FISH assay - method Method is based on the common recognition, and hybridisation, of two complementary nucleic acid sequences (single strand DNA) FISH-painting is used for stable translocation detection Painting probes are routinely used in a cocktail of three chromosomes covering about 20% of the genomic DNA content Lecture notes: As for conventional cytogenetics, a two-day whole blood culture is necessary to obtain metaphasic chromosomes and the culture arrest procedure is similar. In a molecular point of view, the Fluorescence In Situ Hybridisation is based on the common recognition, and hybridisation, of two complementary nucleic acid sequences (single strand DNA). The first sequence belongs to a DNA- (or RNA-) probe, usually fluorescently or chemically labeled. The second one belongs to the nucleic target which is generally a chromosome. Among the various FISH techniques, the FISH-painting is used for stable translocation detection. Painting probes are routinely used in a cocktail of three chromosomes covering about 20% of the genomic DNA content. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

FISH assay - advantages and inconvenience
Part No...., Module No....Lesson No. Part title FISH assay - advantages and inconvenience Advantages Retrospective biodosimetry Inconvenience Expensive Lower sensitivity in comparison with other methods Lecture notes: One of the advantages of this technique would be the retrospective biodosimetry. At the present time it is clear that the temporal persistence of translocations is much higher than the dicentric one. Nevertheless, the level of this persistence is still not defined and some data clearly show a decline. The persistence of the translocations depends on the heterogenic lifespan of T-lymphocytes and probably also depends on the simultaneous presence of dicentrics in the same cell. One main inconvenient of FISH-painting is the financial cost (DNA probe, fluorescent microscopy, etc.) and the time spent to get the results from a technical and scoring point of view. Moreover, translocations are less specific of ionising radiation than dicentrics. A translocations background estimation of healthy individuals of various ages and life-styles is necessary. Finally, for biological dosimetry purposes, the choice of the scoring criteria and the DNA probe cocktail is very important and might explain some differences between the results found in different countries. Still now, the translocation yield estimated after FISH-painting is not validated as a medico-legal biomarker but it remains promising for retrospective dosimetry purposes. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Part title ESR technique Study subjects: Bone Teeth Hair Skin Method is: Dose dependent Simple Lecture notes: ESR studies on biological samples such as bone, teeth, hair and skin, following irradiation can detect doses as low as 0. 3 Gy. The intensity of the ESR signal is proportional to the dose and can be used to detect lethal as well as sub-lethal doses. The intensity of the ESR signal is greater for photons of lower energy and very poor for neutrons. Since the signal persists very long, it can be a useful method in accidents. Among the various biological materials, ESR study with tooth enamel has provided reliable dose estimates for the A-bomb survivors. Simplicity of this assay makes this technique promising for retrospective biological dosimetry, particularly in cases involving whole body exposures. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

New trends in biological dosimetry
Part No...., Module No....Lesson No. Part title New trends in biological dosimetry Physico-chemical modification of the membrane as a potential bio-indicator of radiation exposure Multiparametric approach of biochemical determinations Lecture notes: Radiological accidents of groups of individuals often require a rapid evaluation of the absorbed dose in order to identify those who must receive an adequate therapy. Physical, clinical and biological dosimetry are usually combined for the best dose assessment. Conventional cytogenetic dosimetry currently provides the best assessment of low doses (< 0.5 Gy) and is the only one which has actually a medico-legal value, but this technique is not as suitable as expected for large numbers of exposed individuals, or in case of a partial-body irradiation. Because the majority of the radiation accident cases result from an heterogeneous exposition, new bio-indicators of radiation exposure have to be proposed which give some information on the seriousness of the lesion and the extend of the irradiated area. Since twenty years, different biochemical parameters have been studied extensively (amylase, taurine, thymidine, and others). Unfortunately, all these parameters taken alone are not sensitive enough for dose assessment. However, a multiparametric approach of biochemical and haematological indicators seems possible for a preliminary dose prediction. In radiobiology, the essential target of ionising radiation in the cell is considered to be the DNA molecule. Cytogenetics determinations, as describe above are based on these radio-induced DNA damages. So, research in radiobiology field was focalised this 20 past years on this point of view. Because the deposited energy could occur in the complete volume of the cell, other cellular components could be affected by the deleterious effects of ionising radiation (membrane, mitochondria) and this offers new perspectives to develop new bio-indicators of radiation exposure. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Physico-chemical modification of the membrane - explanation
Part No...., Module No....Lesson No. Part title Physico-chemical modification of the membrane - explanation Membrane as the first structure, which ionising particles go throughout To induce similar cellular damage, the absorbed dose for the membrane must be 100 times higher than the absorbed dose for DNA Studies with doses of up to 1000 Gy Development of sophisticated technology – decrease the range of doses to assess (below 10 Gy) Lecture notes: Geometrically, the first structure, which ionising particles go throughout, is the membrane. In the past years, experiments related to membrane radio-sensitivity were focused on the consequences of energy deposit on cell death. In order to induce similar cellular damage, the absorbed dose received by the membrane must be 100 times higher than the absorbed dose received by DNA. This was presumably the reason for which further studies were performed by using extremely high radiation doses of up to 1,000 Gy. However, it was later suggested that, because of the high polyunsaturated fatty acid level of membranes, radio-induced lipid peroxidation could induce important structural and functional alterations at lower dose range. More recently, several studies on the various types of cellular death (apoptosis and necrosis), occurring after irradiation doses of a few Grays, showed that not only the membrane integrity in the cellular survival signal transmission was involved, but also that specific membrane damage played a crucial role in programmed cell death. Development of new sophisticated technology provides now the opportunity to reappraise, for radiation doses below 10 Gy, the ionising radiation effects on cell membrane status. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Physico-chemical modification of the membrane
Part No...., Module No....Lesson No. Part title Physico-chemical modification of the membrane Advantages Sensitivity Rapidity Technique could be used in the case of local irradiation Inconvenience Technique is useful only 24 to 48 hours after exposure Lecture notes: The sensitivity and the rapidity of assessment of this technique are the main advantages. The assessment of fluidity index seems to be a good complement to cytogenetic analysis in case of patients selection after a massive radiation overexposure. Because the assessment of fluidity index is possible on every isolated type of cells, application of this technique on skin biopsy could give some information in case of a local irradiation. This possibility is also under investigation. The observed effect disappear quiet quickly after irradiation, thus this technique seems to be useful only 24 to 48 hours post-irradiation. The applicability of this method to in vivo models still to be proved. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Multiparametric approach of biochemical determinations
Part No...., Module No....Lesson No. Part title Multiparametric approach of biochemical determinations Method is simple Biological material is easily accessible Method could be useful for a first screening and as a compliment to cytogenetic methods Biochemical indicators are not so specific to radiation exposure Lecture notes: Biochemical and haematological parameters are accessible using routine automate analysers (RA-XT and H-l, Technicon, Bayer Diagnostics, USA). The pig model is a good one to test the possibility of a multiparametric approach of biochemical and haematological indicators for dose prediction. The biological material necessary for the technique (blood, urine, etc.) is easily accessible. Simple routine methods are available for quantitative analysis. Generally, the time required for the determinations is relatively short. A multiparametric approach of biochemical indicators could thus be useful for a first screening in case of a mass radiological accident and a good complement to cytogenetic methods. None of the biochemical indicators is a bio-indicator of radiation exposure when taken alone. They are not so specific to radiation exposure as dicentrics. Many factors influence biochemical parameters levels (nutrition, disease, medication). Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Criteria for good biomarker
Part No...., Module No....Lesson No. Part title Criteria for good biomarker a good dose-dependence over a relevant dose range of doses a large specificity to ionising radiation; a short availability after irradiation and a persistence of the effect all the different radiation qualities should be covered by the method fractionated, chronic and partial body exposure should be detectable the biological material that contain it, or is at the origin of the biomarker must be easily accessible evaluation should be either easy and rapid or transferable to machines Lecture notes: According to Muller and Streffer (1991), the ideal biomarker would possess the next criteria; - a good dose-dependence over a relevant dose range of doses; - a large specificity to ionising radiation; - a short availability after irradiation and a persistence of the effect. If it is not permanent, the time-dependence of the decay of the effect must be known; - all the different radiation qualities should be covered by the method; - fractionated, chronic and partial body exposure should be detectable; - the biological material that contain it, or is at the origin of the biomarker must be easily accessible; - evaluation should be either easy and rapid or transferable to machines. This situation is still an Utopia, but the simultaneous using of various biomarkers might provide the information needed. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Biomarkers for biological dosimetry
Part No...., Module No....Lesson No. Part title Biomarkers for biological dosimetry Method Conditions of exposure Triage in an accident Homoge-neous Heteroge-neous Past overexposure Unstable CA (dicentrics, etc) + - MN PCC Stable CA (translocation) Biochemical indicators ? Biophysical indicators Lecture notes: Nowadays, the radiation dose after an external, acute, whole body exposure can be determined relative precisely using biological indicators. The yield of chromosome aberrations in human peripheral blood lymphocytes is still the most useful biodosimeter for accidental whole-body exposure estimation. So, conventional cytogenetic technique is still the only reference technique which has a medico-legal value for accidental dose assessment. However, dose assessment has to be improved by the development of other new techniques, more specific to problems encountered in different cases of radiation overexposure i.e. heterogeneous- or past-overexposure. Problems arise however with internal contamination or with chronic low dose exposure. A multiparametric approach of biological dosimetry, greatly depending on the involved irradiation situation would be the optimum process for dose assessment. In case of triage in situation of radiological emergency, some techniques seem more appropriate). Thus, the best advantage of the detection of micronuclei in human blood lymphocytes is the speed of the analyse and the possibility to do an easy automation. A multiparametric approach of biochemical parameters seems elsewhere to be a good complement to the reference technique in case of emergency which is the dicentric determination. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Part title Summary This lecture presented materials about biological dosimetry Background Techniques Interpretation of the results Recent development Comments are welcomed Let’s summarize the main subjects we did cover in this session. In the lecture were presented the following aspects: chromosome aberrations and their types; chromosome dosimetry – methodology, calibration curve, dose assessment and uncertainties; interpretation of CAA data; cytogenetic dosimetry – advantages and inconveniencies; recent development in biodosimetry (MN assay, FISH, PCC technique, ESR) – explanation, method, advantages and inconveniencies; new trends in biological dosimetry. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Where to Get More Information
Part No...., Module No....Lesson No. Part title Where to Get More Information UNSCEAR Report Annex G, Chapter III: Prognostic indicators and biological dosimetry (New York: United Nations) 1988; IAEA Training Course at IPSN. Medical Emergencies in Case of Radiological Accidents. November Training materials. Natarajan A.T., Vyas R.C., Wiegant J. and Curado M.P., A cytogenetic follow-up study of the victims of radiation accident in Goiania (Brazil), Mutation Res.,247, Lloyd D.C. and Edward A.A. Biological dosimetry after radiations in Chromosome Aberration Basic and Applied Aspects Eds. G.Obe and A.T.Natarajan,(Springer Veriag), ,1990. Add module code number and lesson title IAEA Post Graduate Educational Course in Radiation Protection and Safty of Radiation Sources

Part No...., Module No....Lesson No. Biological Dosimetry Lecture

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Part No...., Module No....Lesson No. Biological Dosimetry Lecture

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