Biological dosimetry for retrospective dose-assessment in humans Ursula Oestreicher Federal Office for Radiation Protection

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1 Biological dosimetry for retrospective dose-assessment in humans Ursula Oestreicher Federal Office for Radiation Protection DoReMi Lecture Series Molecular Mechanisms of Radiation Carcinogenesis , Helmholtz Center, Munich

2 Federal Office for Radiation Protection Scientific-technical Superior Federal Authority in the portfolio of the BMUB (Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety). Bundesamt für Strahlenschutz (BfS) was founded in 1989 (Chernobyl!) BfS works for the safety and protection of man and the environment against damages due to ionising and non-ionising radiation.

3 Salzgitter Headquater Administration Berlin Department Radiation Protection and the Environment Department Safety of Nuclear Waste Management Department Nuclear Safety BfS Locations BfS staff: about 766 Freiburg Oberschleißheim/ Neuherberg bei München Department Protection and the Environment SG - Department Radiation Protection and Health

4 Department Radiation Protection and Health (SG) SG Division 1: Effects and risks of ionising and non-ionising radiation WG1.2: Biological radiation effects, Biological dosimetry Radiation sensitivity (individual, age, gender) Low dose effects Effects of different radiation qualities Effects of different dose rates Biomarkers of exposure Biological dosimetry

5 Dose limit 20 msv / year Irradiation exposure CT scan 8 20 msv (abdomen) workplace 2.1 msv / year - Germany medical application everyday life

6 cell DNA Organism 6

7 Molecular level: DNA Damage 1 Gy photon radiation causes 3000 base damage 1000 single strand breaks double strand breaks DNA double strain base pair

8 Biological indicators for an exposure successful repair healthy incorrect repair damaged organism cell chromosome (genetic information) repair not possible dead

9 Example Survival fraction Example dic / cell Factors influencing Radiation Effects Radiation quality Endpoint: dic / cell Endpoint: Cell survival Dose [rads] Course of curve is dependent on radiation quality Low LET radiation: shoulder curve Dose [Gy]

10 Cell survival (relative) Factors influencing Radiation Effects Cell cycle dependent radiation sensitivity G1 S G2 M G1.. Cell cycle

11 Chromosomal Alterations Chromosome mutations in human lymphocytes (special!) Typical for ionising radiation - Chromosome type - Typical for chemicals - Chromatid type - 11

12 Main Task: Biological Dosimetry Def.: The use of biomarkers to verfiy exposure to radiation and to estimate absorbed dose Cytogenetic analysis of persons in case of assumed overexposure with ionising radiation. Biological dosimetry provides an individual and independent information. - The laboratory of the Federal Office for Radiation Protection (Bundesamt für Strahlenschutz, BfS) is the official laboratory charged with the performance of biological dosimetry in Germany since 1982.

13 Essential requirements for biological indicators as meaningful dosimeter Low background level Clear dose effect relationship for different radiation qualities and dose rates Specific to ionising radiation Non invasive Fast availability of dose estimation Good reproducibility Comparabiliy of in vitro and in vivo results DoReMi Lecture Series Molecular Radiation Carcinogenisis, U. Oestreicher

14 Established methods to detect radiation exposure on the cytogenetic level Chromosome analysis Dicentric chomosome (dic) Translocation analysis FISH Unstable Unstable cell cell Stable cell Micronucleus (MN) in binucleated cell (BN) DoReMi Lecture Series Molecular Radiation Carcinogenisis, U. Oestreicher 14

15 Human peripheral Lymphocytes Lymphocytes circulate in the whole body synchron, in a DNA presynthetic stage of cell cycle ( G0 phase) (only few < 0, 2 % are in the autosynthetic cell cycle) 15

16 Material and Method Blood sampling with lithium - heparinized vacutainers Set up of lymphocyte cultures using RPMI, PHA, BrdU Incubation for 48 h, 37 C Fixation and slide preparation DoReMi Lecture Series Molecular Radiation Carcinogenisis, U. Oestreicher

17 Visualisation of the radiation effect with different cytogenetic methods Culture time 48 h Culture time 72 h Chromosomes Binucleated cells + MN

18 Laboratory routine: cell culture and preparation + BrdU 48h culture + Cytochalasin b 72h culture staining Fluoreszence in situ hybridization FPG staining Giemsa/DAPI staining endpoints FISH analysis Dicentric analysis Micronucleus analysis

19 Formation of Dicentric Chromosomes (Dic) Gold Standard - acute exposure - Dicentric-analysis dic ace

20 Biological Dosimetry after acute exposure Dic - assay In vitro curves for dicentric yields plotted against dose for several qualities of radiation The spontaneous frequency of dicentric is very low (1 in 1000 cells) dicentrics per 1000 cells SEM subjects, cells subjects, cells without heavily smokers 20

21 Comparison of in vivo data of Ra-224 therapy patients with data after in vitro exposure with -Particles In vivo 10 injections of 1MBq [224Ra] Radiumchlorid Half-life of Ra-224: 3.64 days 21

22 Impact of the number of scored cells on the 95 % Confidence limits of the dose estimation

23 Special statistic software tools for biological dosimetry Dose Estimate Cytogenetics Dose Estimation Software Created by Liz Ainsbury. Desperas J, Szluinska M, Edwards A, Lloyd DC, Lindholm C, Romm H, Roy L Moss R, Morand J, Wojcik A, Radiat Prot Dosim 2007;124: Ainsbury EA, Lloyd DC., Health Phys 98:290-5;

24 Zellen Calculation the number of irradiated cells after partial body exposure dic / cell irradiated unirradiated dic / zelle

25 Getting prepared for a large scale radiation accident There are a lot of activities on the international level and in many different fields to get prepared in a radiation emergency situation There is need for new methods in biodosimetry, which allow a high troughput of samples in a short time period Some assays can be improved and be automated. The existing assays are complementary and will be combined as a multi parameter approach. In a large scale accident, single labs will be overwhelmed. There is need for mutual assistance and networking.

26 Establishing of biodosimetry networks Europe: Trilaterales Network: D - F UK RENEB (EU Project) Global: WHO: BioDoseNet IAEA: RANET (Response and Assistance Network ) Quality Assurance: Intercomparisions

27 Harmonizing and standardising of the methode ISO 19238:2004, 2014 Radiation protection -- Performance criteria for service laboratories performing biological dosimetry by cytogenetics ISO 21243:2008 Radiation protection -- Performance criteria for laboratories performing cytogenetic triage for assessment of mass casualties in radiological or nuclear emergencies -- General principles and application to dicentric assay 27

28 How much cell are to score? (ISO 21243) Number of samples < >151 Spreads to score Dicentrics to score Maximum Total Spreads ( ) ( ) ( ) ( ) ( ) ( ) (>3020) Total Hours (scoring time) hours hours hours hours hours >202 hours Total Days * (scoring time) 0,6-1,5 days 1,5-3 days 2,5-3,7 days 2,9-3,9 days 2,8-4,2 days >4,3 days Assumptions: 1) > 10 Samples is a Triage scenario 2) Analysis Time / Metaphase = 3 min 3) Scanning + Set-up Time per slide: 20 min 4) * Total Days Working = 24 hour working days, 3 shifts of 8 hours, 2 persons / shift, 2 metaphase finders

29 How much cell are to score? (ISO 21243) Number of samples < >151 Spreads to score Dicentrics to score Maximum Total Spreads ( ) ( ) ( ) ( ) ( ) ( ) (>3020) Total Hours (scoring time) hours hours hours hours hours >202 hours Total Days * (scoring time) 0,6-1,5 days 1,5-3 days 2,5-3,7 days 2,9-3,9 days 2,8-4,2 days >4,3 days Assumptions: 1) > 10 Samples is a Triage scenario 2) Analysis Time / Metaphase = 3 min 3) Scanning + Set-up Time per slide: 20 min 4) * Total Days Working = 24 hour working days, 3 shifts of 8 hours, 2 persons / shift, 2 metaphase finders

30 How much cell are to score? (ISO 21243) Number of samples < >151 Spreads to score Dicentrics to score Maximum Total Spreads ( ) ( ) ( ) ( ) ( ) ( ) (>3020) Total Hours (scoring time) hours hours hours hours hours >202 hours Total Days * (scoring time) 0,6-1,5 days 1,5-3 days 2,5-3,7 days 2,9-3,9 days 2,8-4,2 days >4,3 days Assumptions: 1) > 10 Samples is a Triage scenario 2) Analysis Time / Metaphase = 3 min 3) Scanning + Set-up Time per slide: 20 min 4) * Total Days Working = 24 hour working days, 3 shifts of 8 hours, 2 persons / shift, 2 metaphase finders

31 Semi-automatic scoring of dicentrics False Positive are detected rapidly: What s about the yield of False Negatives?

32 Performance of semi-automatic scoring of dicentrics Standard chromosome preparation Metaphasefinder is searching for 150 Metaphases 150 Metaphases will be captured automatically in HR 150 Metaphases will be analyzed automatically Candidates of dicentric chromosomes needs to be evaluated Total scoring time 3 min 15 min <2 min ~ 3 min <25 min Possible throughput per scoring station 50 samples / 24h Scoring procedure of automated steps and human evaluation can be performed time independent and separately at different stations

33 Biological Dosimetry after chronic exposure or exposure in the past Fluorescence in situ (FISH) assay Reciprocal translocations FISH-analysis: chromosome 2,4 und 8 t(ab) + t(ba) Course: Interdisciplinary Radiation Research Focussing on Radiation Protection, May 2011, BfS, Munich, Germany 33

34 Retrospective Biological Dosimetry Fluorescence in situ Hybridisation (FISH) In vitro curve for translocation yields plotted against dose for 137 Cs gamma radiation The spontaneous frequency of translocations is age dependent Control Age < 40 years Age > 40 years N Age range Scored cells Translocations F G / 1000 Cells

35 Total translocations per 100 cell equivalents Spontaneous translocation frequencies (Labs: 16, N =1933) Linear with loglinear curvature term Age in categories Age in years International study of factors affecting human chromosome translocations. Mutat Res Apr 30;652(2):112-21

36 Fading of Dicentrics - Persistence of Translocations Estonia accident ( : EU Concerted Action zu FISH) Intercomparison of translocation and dicentric frequencies between laboratories in a follow-up of the radiological accident in Estonia, int. j. radiat. biol 2002, vol. 78, no. 10, 883± 890

37 Visualisation of the radiation effect with different cytogenetic methods Culture time 48 h Culture time 72 h Chromosomes Binucleated cells + MN

38 Formation of micronuclei and nuclear plasmatic bridges Mikronuclei results from missegregation of acentric fragments or Chromosomes during mitosis dizentric chromosomes may results in nukleoplasmatic bridges

39 Method Cytokinesis-block (CB) micronucleus (MN) assay DAPI + vectrashield stained slides Automated Scoring MNScore software developed by MetaSystems (Altlussheim, Germany) combined with a microscope and a motorised scanning station BN cells /donor/ dose point were analysed Gallery of automatic detected MN (sorted)

40 Dose response curves, manual / automated MN scoring

41 Fitted automated dose response curve Bars: The 95% CIs based on the data of 10 donors. The black arrow demonstrates a dose estimation of 1 ± 0.2 Gy, based on the fitted dose response curve.

42 Established methods of biological dosimetry Tool Specificity to radiation Exposure scenario Sensitivity to radiation Signal stability Dicentrics Excellent WB and PB 0.1 ca. 5 Gy Several weeks Speed of performance Few days FISH Good WB and PB 0.5 ca 5 Gy Several years Few days Micro nuclei Good WB and PB 0.3 ca 5 Gy Several weeks Few days

43 Thank you for your attention

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