BIODOSIMETRY

AVAILABLE METHODS AND ROLE IN

DOSE ASSESMENT AND PROGNOSIS

Module X
Module Medical X. - 2
Accidental dosimetry
PHYSICAL
DOSIMETRY
BIOLOGICAL
DOSIMETRY
CLINICAL
DOSIMETRY
DOSE
RECONSTRUCTION,
Personal Dosimeters

CYTOGENETIC
DOSIMETRY
Dicentrics, FISH,
PCC, MNA
NAUSEA,
VOMITING,
BLOOD CELLS
COUNTS,
SKIN REACTIONS...
OTHER
BIOINDICATORS
Module Medical X. - 3
Physical dosimetry
Module Medical X. - 4
Clinical dosimetry
Crude estimate of absorbed dose
obtainable from clinical presentation
Vomiting
Onset: 2 h after
exposure or later
Onset: 1-2 h after
exposure or later
Onset: earlier than 1 h
after exposure
Onset: Earlier than 30
min after exposure
MILD ARS (1-2 Gy)
MODERATE ARS
(2-4 Gy)
SEVERE ARS (4-6 Gy)
VERY SEVERE ARS
(6-8 Gy)
Module Medical X. - 5
Clinical dosimetry using early
changes in lymphocyte counts
Module Medical X. - 6
Clinical dosimetry using
granulocyte counts
Module Medical X. - 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
Module Medical X. - 8
Biophysical background to
chromosome damage
********************************
High LET
* * * * * * * * *
Low LET
Module Medical X. - 9
DNA damage
Module Medical X. - 10
Chromosomal structure
Module Medical X. - 11
Human lymphocytes
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 G
o
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. - 12
Human karyotype
Module Medical X. - 13
Classification of chromosomal
aberrations
Inversion
Symmetrical
(STABLE)
Breaks
Intrachange
Asymmetrical
(UNSTABLE)
Centric
Ring
Interchange
Translocation
Dicentric
Module Medical X. - 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
Module Medical X. - 15
Dicentric chromosome
aberrations in metaphase spreads
dic
dic
f
f
f
f

Module Medical X. - 16
Dose response curves
Y = A+aD + bD
2

Module Medical X. - 17
Relationship between
RBE and LET
LET (keV/m)
R
B
E

Module Medical X. - 18
Calibration curves
Module Medical X. - 19
Gamma rays,
X-rays acute
exposure
(Low LET)
Gamma rays
X-rays chronic exposure
(Low LET)
a particles
Fast neutrons
(High LET)
Dose
D
i
c
e
n
t
r
i
c

y
i
e
l
d

Y = c + aD + bD
2
Y = c + aD

Y = c + aD

Dose estimation of acute
vs chronic exposure
E
f
f
e
c
t

Module Medical X. - 20
Methods for estimating radiation
doses in partial body exposure:
Sasaki-method
Module Medical X. - 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
Module Medical X. - 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 months of
irradiation
Module Medical X. - 23
Translocation assay
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
Module Medical X. - 24
Stable chromosome aberration
analysis with G-banding
A normal G banded male
karyotype

An idiogram showing the
banding patterns of individual
chromosomes by fluorescent
and Giemsa staining
Module Medical X. - 25
Stable chromosome
aberration analysis with FISH
Translocation
Deletion
Module Medical X. - 26
Painting chromosomes
Pancentromeric and telomeric probes
Module Medical X. - 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 procedures and
technical equipment
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
Module Medical X. - 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 non-
irradiated human cells)
Module Medical X. - 29
PCC technique
PERIPHERAL BLOOD
FICOL SEPARATION
FUSE IN PEG
LYMPHOCYTES
CHO
CHINESE HAMSTER
OVARY (CHO) CELLS
(Grown in BrdU)

COLCEMID

MITOTIC SHAKE OFF
(METAPHASE CELLS)
PCC
Incubate 1 h
(Medium+PHA+Colcemid)
Module Medical X. - 30
Evaluation criteria for
scoring PCCs
Module Medical X. - 31
PCCs and FISH
Irradiated cells with
excess break Unirradiated control
Module Medical X. - 32
Estimation of irradiated
body fractions
Module Medical X. - 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 partial-
body/supra-lethal exposure & improves
detection level of lower doses
Module Medical X. - 34
Micronucleus (MN) assay
Cytochalasin B
Module Medical X. - 35
MN and nucleoplasmic bridges in
binucleated cells
(Giemsa stained)
A
B
Module Medical X. - 36
MN assay with
pancentromeric probe
A
B
centromere negative
centromere positive
Module Medical X. - 37
Application of MN assay for
biological dosimetry
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
Module Medical X. - 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
Module Medical X. - 39
Application of GPA assay for
biological dosimetry
Relationship between glycophorin A mutant frequency in red blood
cells and radiation dose for about 1200 A-bomb survivors
Module Medical X. - 40
Biophysical assays - ESR
(electron spin resonance)
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

Module Medical X. - 41


Characterization of
biological dosimetry methods
Module Medical X. - 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