biological dosimetry for retrospective dose-assessment in humans · 2015-11-09 · there is need...
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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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
Chromosome mutations in human lymphocytes (special!)
Chromosomal Alterations
Typical for ionising
radiation
- Chromosome – type -
Typical for chemicals
- Chromatid – type -
11
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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.
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
Visualisation of the radiation effect with different
cytogenetic methods
Chromosomes
Binucleated cells + MN
Culture time 48 h Culture time 72 h
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
+ 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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
Impact of the number of scored cells on the
95 % Confidence limits of the dose estimation
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
<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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
<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
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)
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
<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
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)
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
Semi-automatic scoring of dicentrics
What´s about the yield of False Negatives?
False Positive are detected
rapidly:
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
• 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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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)
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
Visualisation of the radiation effect with different
cytogenetic methods
Chromosomes
Binucleated cells + MN
Culture time 48 h Culture time 72 h
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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)
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
Dose response curves, manual / automated MN scoring
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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.
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
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
DoReMi Lecture Series “Molecular Radiation Carcinogenisis”, U. Oestreicher
Thank you for your attention