Slide 1

BIODOSIMETRY
AVAILABLE METHODS AND ROLE IN
DOSE ASSESMENT AND PROGNOSIS

Module X


Slide 2

Accidental dosimetry
PHYSICAL
DOSIMETRY

DOSE
RECONSTRUCTION,
Personal Dosimeters

BIOLOGICAL
DOSIMETRY

CYTOGENETIC
DOSIMETRY
Dicentrics, FISH,
PCC, MNA

CLINICAL
DOSIMETRY

NAUSEA,
VOMITING,

BLOOD CELLS
COUNTS,
SKIN REACTIONS...

OTHER
BIOINDICATORS
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Slide 3

Physical dosimetry

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Slide 4

Clinical dosimetry
Crude estimate of absorbed dose
obtainable from clinical presentation

Vomiting
Onset: 2 h after
exposure or later

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MILD ARS (1-2 Gy)

Onset: 1-2 h after
exposure or later

MODERATE ARS
(2-4 Gy)

Onset: earlier than 1 h
after exposure
Onset: Earlier than 30
min after exposure

SEVERE ARS (4-6 Gy)
VERY SEVERE ARS
(6-8 Gy)

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Slide 5

Clinical dosimetry using early
changes in lymphocyte counts

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Slide 6

Clinical dosimetry using
granulocyte counts

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Slide 7

Cytogenetic dosimetry
Analysis of chromosomal aberrations in
peripheral blood lymphocytes - widely used
biological dosimetry method for assessing
radiation dose, especially useful


in persons not wearing dosimeters while exposed
to radiation
 in cases of claims for compensation for radiation
injuries not supported by unequivocal dosimetric
evidence
 for validation of occupational radioprotection
cases involving suspected low-dose exposures


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Slide 8

Biophysical background to
chromosome damage

High LET

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Low LET

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Slide 9

DNA damage

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Slide 10

Chromosomal structure

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Slide 11

Human lymphocytes
 Dose assessment predominantly based on
data obtained from lymphocytes
 Easily obtained in large quantities from
peripheral blood

 Vast majority of peripheral lymphocytes
reside in Go phase of e cell cycle
 Phytohaemagglutinin (PHA) converts resting
lymphocytes into dividing cells allowing
visualization of possible DNA lesions in
methaphase chromosomes

Module Medical X.

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Slide 12

Human karyotype

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Slide 13

Classification of chromosomal
aberrations
Symmetrical Breaks Asymmetrical
(STABLE)
(UNSTABLE)

Centric
Inversion Intrachange Ring

Interchange
Translocation
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Dicentric
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Slide 14

Biological dose assessment
using standard dicentric analysis
• Introduced by M. Bender in 1964
• Isolated lymphocytes stimulated by phytohaemagglutin (PHA)
into mitosis
• Arrest of metaphase using colchicine
• Scoring of dicentric chromosome aberrations in metaphase
spreads

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Slide 15

Dicentric chromosome
aberrations in metaphase spreads

dic

f
dic

f
f
f

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Slide 16

Dose response curves

Y = A+aD + bD2

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Slide 17

RBE

Relationship between
RBE and LET

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LET (keV/m)

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Slide 18

Calibration curves

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Slide 19

a particles
Fast neutrons
(High LET)

Effect

Dicentric yield

Dose estimation of acute
vs chronic exposure

Y = c + aD

Gamma rays,
X-rays acute
exposure
(Low LET)

Y = c + aD + bD2
Y = c + aD
Gamma rays
X-rays chronic exposure
(Low LET)

Dose
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Slide 20

Methods for estimating radiation
doses in partial body exposure:
Sasaki-method

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Slide 21

Dicentric assay
• Most accurate method for dose estimation with
sensitivity threshold of about 0.1 Gy for whole body
low LET radiation
• Especially useful
• in cases where dosimeter not used, e.g.
radiation accident
• to support physical dosimetry results in
radiation protection and safety practice
• to determine partial body exposure not
detected by locally placed dosimeter

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Slide 22

Limitations of dicentric
analysis for dose estimation
• Dicentrics are unstable and lymphocytes
carrying aberration elimininated with time
(average lifetime 150-220 days, depending
on
dose),
hence
can
underestimate
magnitude of dose

• Method useful only within few
irradiation

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months of

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Slide 23

Translocation assay






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In retrospective dosimetry and
chronic exposure reciprocal
translocations used for dose
assessment
Translocations considered stable
in cell division so yield should not
fall with time
Typically detected using specific
whole chromosome DNA
hybridization probes and FISH
methodology

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Slide 24

Stable chromosome aberration
analysis with G-banding

A normal G banded male
karyotype

Module Medical X.

An idiogram showing the
banding patterns of individual
chromosomes by fluorescent
and Giemsa staining

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Slide 25

Stable chromosome
aberration analysis with FISH
Translocation

Deletion

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Slide 26

Painting chromosomes
Pancentromeric and telomeric probes

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Slide 27

Applicability of stable
chromosome aberration analysis
for biological dosimetry
• Method based on scoring stable
chromosome aberrations (translocations
and insertions) detected with fluorescent
in-situ
hybridization
of
whole
chromosomes
• Requires complex
technical equipment

procedures

and

• May be use decades after exposure
• Sensitivity threshold a few cGy but
method not feasible for doses less than
0.2 Gy because of expense and time
needed for analysis

• Spontaneous level of stable chromosome
aberrations not well established

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Slide 28

Premature chromosome condensation
(PCC) assay
•Initially introduced by Johnson and Rao (1970)
•Mitotic-inducer cells (i.e. CHO) isolated using
chemical (colcemid) and physical (rapid shaking of
flask) technique
•Test cells (i.e. human lymphocytes) fused with CHO
cells using polyethylene glycol (PEG)
•Interphase DNA of test cells condense into
chromatid/chromosome-like structures (46 for nonirradiated human cells)

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Slide 29

PCC technique
CHINESE HAMSTER
OVARY (CHO) CELLS
(Grown in BrdU)

FUSE IN PEG
PERIPHERAL BLOOD

COLCEMID
MITOTIC SHAKE OFF
(METAPHASE CELLS)

LYMPHOCYTES

CHO

FICOL SEPARATION

Incubate 1 h
(Medium+PHA+Colcemid)

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PCC

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Slide 30

Evaluation criteria for
scoring PCCs

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Slide 31

PCCs and FISH

Unirradiated control

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Irradiated cells with
excess break

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Slide 32

Estimation of irradiated
body fractions

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Slide 33

Applicability of PCC assay for
biological dosimetry
 Dose estimates obtainable within 48 hours
of receipt of blood in laboratory
 Radiation induced mitotic delay does not
interfere with assay since performed on
interphase nuclei and does not require cell
division
 Method envisioned applicable after partialbody/supra-lethal exposure & improves
detection level of lower doses
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Slide 34

Micronucleus (MN) assay

Cytochalasin B

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Slide 35

MN and nucleoplasmic bridges in
binucleated cells
(Giemsa stained)

A

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B

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Slide 36

MN assay with
pancentromeric probe

A

B

centromere negative

centromere positive

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Slide 37

Application of MN assay for
biological dosimetry





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Micronuclei not specific to radiation
exposure
Discrimination between total and
partial body exposure more difficult
High doses of radiation interfere with
cell division
High baseline frequency and age
dependency make reliability of assay
questionable
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Slide 38

Glycophorin A (GPA) somatic
cell mutation assay
• Performed by two-color immunofluorescence flow
cytometry on peripheral blood erythrocytes
• Based of measuring N/0 variants of erythrocytes,
which display phenotype consistent with loss of
expression of GPA (M) allele
• Can be performed only on individuals heterozygous
at this locus that codes for the N/M blood group
antigens (approximately half of population)
• Prompt but requires complex and expensive
equipment
• Sensitivity threshold about 0.2-0.25 Gy
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Slide 39

Application of GPA assay for
biological dosimetry

Relationship between glycophorin A mutant frequency in red blood
Module Medical X.cells and radiation dose for about 1200 A-bomb survivors

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Slide 40

Biophysical assays - ESR
(electron spin resonance)






Module Medical X.

Persistent free radicals formed in solid matrix
biomaterial (e.g. dental enamel, nail clippings,
hair) from accidentally exposed victim can be
detected via ESR
Measurements provide reliable biophysical
dose estimates & partial body exposure
information
In some circumstances, certain clothing
material, particularly hard plastics and
buttons, may be measured and absorbed
dose estimated
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Slide 41

Characterization of
biological dosimetry methods

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Slide 42

Review points
• In radiation accidents, important to estimate the
absorbed doses in victims to plan appropriate
medical treatment
• In most accidents, physical dosimetry of
absorbed dose is not possible. Even where
possible, important to confirm the estimates by
other methods
• Most commonly used method cytogenetic
analysis of chromosomal aberration in peripheral
blood
lymphocytes
using
dicentrics,
translocations, PCC and micronuclei assays
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