Biological dosimetry for retrospective dose-assessment in humans · 2015-11-09 · There is need...

Biological dosimetry for retrospective dose-assessment in humans

Ursula Oestreicher

Federal Office for Radiation Protection

DoReMi Lecture Series “Molecular Mechanisms of Radiation Carcinogenesis”

13.04.2015 – 24. 04.2015, Helmholtz – Center, Munich

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.

Berlin Department

Radiation Protection and the Environment Salzgitter Headquater

Administration

Department Safety of Nuclear Waste Management Department Nuclear Safety

BfS Locations

Freiburg

Department Protection and the Environment

Oberschleißheim/

Neuherberg

bei München

SG - Department Radiation Protection

and Health

BfS staff: about 766

• 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

Department Radiation Protection and Health (SG)

SG Division 1: Effects and risks of ionising and non-ionising radiation

Irradiation exposure

workplace medical application

everyday life

DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher

2.1 mSv / year -

Germany

Dose limit 20 mSv / year

CT scan 8 – 20 mSv

(abdomen)

cell DNA

Organism

6


Molecular level: DNA

Damage

1 Gy photon radiation causes

3000 base damage

1000 single strand breaks

30 – 40 double strand breaks

DNA double strain

base pair


Biological indicators for an exposure

cell chromosome (genetic

information)

incorrect

repair

successful

repair

repair not possible organism

healthy

damaged

dead

Factors influencing Radiation Effects

Radiation quality

Ex

am

ple

dic

/ c

ell

Dose [Gy]

Endpoint: Cell survival

Endpoint: dic / cell

Course of curve is dependent on radiation quality

Low LET radiation: shoulder curve

Ex

am

ple

Su

rviv

al

frac

tio

n

Dose [rads]

Factors influencing Radiation Effects

Cell cycle dependent radiation sensitivity

Cell

su

rviv

al (r

ela

tive)

G1 S G2 M G1 …..

Cell cycle


Chromosome mutations in human lymphocytes (special!)

Chromosomal Alterations

Typical for ionising

radiation

- Chromosome – type -

Typical for chemicals

- Chromatid – type -

11


Biological Dosimetry Def.:

The use of biomarkers to verfiy exposure

to radiation and to estimate absorbed dose

-

Main Task:

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.


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

Established methods to detect radiation exposure on the cytogenetic level

Chromosome analysis

Dicentric chomosome (dic)

Micronucleus (MN) in binucleated cell (BN)

Translocation analysis

FISH

14


Unstable cell

Unstable

cell

Stable cell

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


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


Visualisation of the radiation effect with different

cytogenetic methods

Chromosomes

Binucleated cells + MN

Culture time 48 h Culture time 72 h


+ BrdU

48h culture

+ Cytochalasin b

72h culture

Fluoreszence in situ

hybridization

FISH analysis

FPG staining Giemsa/DAPI staining

„staining“

endpoints

Dicentric analysis Micronucleus analysis

Laboratory routine: cell culture and preparation

„Gold Standard“ - acute exposure -

Formation of Dicentric Chromosomes (Dic)

Dicentric-analysis

dic

ace

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

1.15 0.15 53 subjects, 54 689 cells

0.96 0.14 46 subjects, 47 593 cells

without heavily smokers

20


Comparison of in vivo data of Ra-224 therapy patients

with data after in vitro exposure with -Particles

10 injections of 1MBq [224Ra] Radiumchlorid

Half-life of Ra-224: 3.64 days

In vivo

21


Impact of the number of scored cells on the

95 % Confidence limits of the dose estimation


Desperas J, Szluinska M, Edwards A, Lloyd DC,

Lindholm C, Romm H, Roy L Moss R, Morand J, Wojcik A,

Radiat Prot Dosim 2007;124:115-23

Special statistic software tools for biological dosimetry

23

.

Dose Estimate

Cytogenetics Dose Estimation Software

Created by Liz Ainsbury

Ainsbury EA, Lloyd DC., Health Phys 98:290-5; 2010


0

100

200

300

400

500

600

0 1 2

Zell

en

dic / zelle

Calculation the number of irradiated cells after partial body exposure

dic / cell irradiated unirradiated

0 44 442

1 12

2 2


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.

Getting prepared for a large scale radiation accident


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

Harmonizing and standardising of the methode

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

ISO 19238:2004, 2014

Radiation protection -- Performance criteria for

service laboratories performing biological

dosimetry by cytogenetics

27


<10 10-25 25-50 50-75 75-100 100-150 >151

Spreads

to score 500-1000 50 50 40 30 20 20

Dicentrics

to score 100 30 30 30 30 30 30

Maximum

Total Spreads

(500 -

5000)

(500-

1250)

(1250-

2500)

(2000-

3000)

(2250-

3000)

(2000-

3000) (>3020)

Total Hours

(scoring

time)

29-71

hours

71-142

hours

117-175

hours

134-200

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

Number of samples

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

How much cell are to score? (ISO 21243)

138-184

hours


<10 10-25 25-50 50-75 75-100 100-150 >151

Spreads

to score 500-1000 50 50 40 30 20 20

Dicentrics

to score 100 30 30 30 30 30 30

Maximum

Total Spreads

(500 -

5000)

(500-

1250)

(1250-

2500)

(2000-

3000)

(2250-

3000)

(2000-

3000) (>3020)

Total Hours

(scoring

time)

29-71

hours

71-142

hours

117-175

hours

138-184

hours

134-200

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

Number of samples







<10 10-25 25-50 50-75 75-100 100-150 >151

Spreads

to score 500-1000 50 50 40 30 20 20

Dicentrics

to score 100 30 30 30 30 30 30

Maximum

Total Spreads

(500 -

5000) (500-

1250)

(1250-

2500)

(2000-

3000)

(2250-

3000)

(2000-

3000) (>3020)

Total Hours

(scoring

time)

29-71

hours

71-142

hours

117-175

hours

138-184

hours

134-200

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

Number of samples







Semi-automatic scoring of dicentrics

What´s about the yield of False Negatives?

False Positive are detected

rapidly:


• Standard chromosome preparation

• Metaphasefinder is searching for 150 Metaphases 3 min

• 150 Metaphases will be captured automatically in HR 15 min

• 150 Metaphases will be analyzed automatically <2 min

• Candidates of dicentric chromosomes needs to be evaluated ~ 3 min

• Total scoring time <25 min

Scoring procedure of automated steps and human evaluation can be

performed time independent and separately at different stations

Performance of semi-automatic scoring of dicentrics

Possible throughput per scoring station 50 samples / 24h


Course: “Interdisciplinary Radiation Research Focussing on Radiation Protection” , 02 - 13 May 2011, BfS, Munich, Germany

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)

Retrospective Biological Dosimetry

Fluorescence in situ Hybridisation (FISH)

In vitro curve for translocation yields plotted

against dose for 137Cs gamma radiation

Control N Age

range

Scored

cells Translocations

FG /

1000 Cells

Age

< 40 years 18 21 - 37 47 352 57 3.75 0.63

Age

> 40 years 35 40 - 85 88 934 213 7.50 0.72

The spontaneous frequency of translocations is age dependent


0.0

0.5

1.0

1.5

2.0

2.5

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86

Age in years

To

tal

tran

slo

cati

on

s p

er

100 c

ell

eq

uiv

ale

nts

Linear with loglinear curvature term

Age in categories

Spontaneous translocation frequencies

(Labs: 16, N =1933)

International study of factors affecting human chromosome translocations.

Mutat Res. 2008 Apr 30;652(2):112-21


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

Fading of Dicentrics - Persistence of Translocations

Estonia accident (1996-2003: EU Concerted Action zu FISH)


Visualisation of the radiation effect with different

cytogenetic methods

Chromosomes

Binucleated cells + MN

Culture time 48 h Culture time 72 h


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


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.

1000 BN cells /donor/ dose point were analysed

Gallery of automatic detected MN (sorted)


Dose response curves, manual / automated MN scoring


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.


Tool

Specificity

to

radiation

Exposure

scenario

Sensitivity to

radiation

Signal stability Speed of

performance

Dicentrics Excellent WB and PB 0.1 – ca. 5 Gy Several

weeks

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

Established methods of biological dosimetry


Thank you for your attention

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Biological dosimetry for retrospective dose-assessment in humans · 2015-11-09 · There is need...

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