Retrospective Dosimetry
by
Translocation Analysis
Lecture
Module 5
IAEA
International Atomic Energy Agency
Introduction
• Recognized drawback of dicentric and cytokinesis
block micronucleus (CBMN) assays is that damage
is unstable and therefore is eliminated from
peripheral blood lymphocyte pool at rate that cell
renewal occurs
• It has long been recognized that analysis for more
persistent types of damage, e.g. stable
translocations, is needed to address biodosimetry
for old or long term exposures
• FISH techniques has made this possible
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Approach before FISH
• To calculate corrected dose taking into account the mean
life of lymphocytes and exponential loss of cells with
unstable aberrations
• There are few data which enable reliable correction factor
to be deduced
• depending on factors such as infections, depression of aberration yield
probably cannot be expressed simply as function of time alone
• Nevertheless, an exponential disappearance rate with a
half-time of about three years is accepted. As a general
approximation this seems suitable when the sampling delay
is long, say five or more years
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Initial dose x exp(-0.693 x i/3). i = elapsed time in years
years
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Dose (Gy)
2
4
2
4
1,59
3,17
1,26
2,52
1,00
2,00
0,79
1,59
0,63
1,26
0,50
1,00
0,40
0,79
0,32
0,63
0,25
0,50
0,20
0,40
0,16
0,32
0,13
0,25
0,10
0,20
0,08
0,16
0,06
0,13
0,05
0,10
0,04
0,08
0,03
0,06
0,02
0,05
0,02
0,04
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4.5
Dose (Gy)
4
3.5
3
2.5
2
1.5
1
0.5
0
0
5
years
10
15
20
After 2 and 4 Gy irradiation: expected decrease in the
estimated dose using dicentrics. This simulation considers
an exponential disappearance rate of lymphocytes carrying
unstable aberrations with a half-time of three years.
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How valid is 3 years half life value?
This seems to apply only to people with normal hematology. There is
evidence that following high acute exposure reduction of dicentrics is
biphasic: initially fast and then slower. Persistence of each phase seems to
depend on initial frequency of aberrations
2
1.8
1.6
Dose range 4.3 - 12.3 Gy
1.4
1.2
1
0.8
0.6
Dose range 1.1 - 2.2 Gy
0.4
0.2
0
0
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2
4
6
8
10
12
14
16
Data from highly irradiated victims of the Chernobyl accident.
Sevan’kaev et al., Radiat. Prot. Dosim. 113: 152-161 (2005)
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Another approach is to consider the dilution with time of
undamaged cells entering the circulation, and to estimate the
initial dose using the method applied for partial irradiations. This
is not feasible after long periods.
This
approximation
gives realistic
results only in
some cases
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Data from highly irradiated victims of the Chernobyl accident.
Sevan’kaev et al., Radiat. Prot. Dosim. 113: 152-161 (2005)
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After DNA damage by ionizing radiations, misrepair of broken pieces
can produce a dicentric chromosome, this type of aberration can easily
be visualized using solid stain.
In theory there is the same probability for radiation to form a dicentric or a
reciprocal translocation. There is experimental evidence supporting a 1:1 ratio.
Translocations can be visualized after laborious banded karyotyping or more
easily by FISH
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FISH
Fluorescence in situ Hybridization (FISH) techniques using DNA probes
that hybridize with the entire chromosome length produces
multicoloured “Chromosome painting”
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Example: accident in Estonia (1)
After the accident the frequency of dicentrics decreases clearly as
time passes
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Data from: Lindholm, Int.J. Radiat. Biol. 70: 647-656 (1996)
Lindholm et al., Int.J. Radiat. Biol. 74: 565-571 (1998)
Lindholm and Edwards, Int.J. Radiat. Biol. 80: 559-566 (2004)
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Example: accident in Estonia (2)
But the frequency of translocations remained relatively constant
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Data from: Lindholm, Int.J. Radiat. Biol. 70: 647-656 (1996)
Lindholm et al., Int.J. Radiat. Biol. 74: 565-571 (1998)
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Lindholm and Edwards, Int.J. Radiat. Biol. 80: 559-566 (2004)
Cocktail of DNA probes
labelled with fluorochromes
Main steps of the
FISH technique
Fixed cells on slides
Denaturation of
probe and target
Hybridization
and post-hybridization
washes
Counterstain with a
fluorescent dye, and
analysis using
fluorescence microscopy
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FISH techniques have many applications in medicine and in fundamental
cytogenetics and can be applied to metaphases and nuclei
Human metaphase with monocoloured painted
chromosomes #1, #4 and #11 labeled with Cy3
(red), centromeres highlighted with a
pancetromeric probe labeled with FITC (green),
and the rest counterstained with DAPI.
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Binucleated lymphocytes hybridized with
centrometric specific probes for chromosomes
4 (green and red labelled DNA probes,
resulting in a yellow signal), chromosome 7
(green labelled) and 18 (red labelled).
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With range of probes and fluorochrome combinations now commercially available it is
possible to highlight all chromosomes by method known as multicolour FISH (mFISH).
This permits full karyotyping and thus scoring all inter-chromosomal translocations.
Centromeres and telomeres of all chromosomes can be separately highlighted too.
Left picture: using pan-telomeric (red signals) and pan-centromeric probes (green signals), white arrows
indicate centric-chromosome fragments lacking telomeric signals at one end (indicating an incomplete
aberration); yellow arrows indicate two acentric fragments, and the red arrow a tricentric.
Right picture an mFISH karyotype where each chromosome pair can be analyzed individually; white arrows
indicate an exchange between chromosomes 6 and 11, and the yellow arrows between 11 and 12.
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For biological dosimetry purposes most common technique is to “paint” several
chromosomes with one or more fluorochromes, and to detect aberrations
between “painted” and “unpainted” genomic material
Human metaphase with coloured
painted chromosomes #2 (FITC,
green), #4 (Texas Red) and #8
(FITC+Texas Red, yellow), and the
rest counterstained with DAPI. An
apparently simple translocation, or
two-way translocation [t(Ba),t(Ab)]
involving chromosome # 2 is
observed.
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Because the aberrations detected are those between painted and
unpainted material, efficiency in detecting translocations depends
on number of painted chromosomes
Using a mono-coloured cocktail of chromosomes, and according to the
Lucas’ formula, the efficiency in detecting bicoloured aberrations is
2.05·fp·(1-fp). The maximum efficiency will be with painting 50% of the
genome.
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Routine FISH analysis
• Generally, painting three of larger chromosomes (i.e. #1 to #12), representing
about 20% of genome, leads to about 33% efficiency in detecting translocations
when single colour is used. Percentage of the genome that each cocktail ‘paints’ is
related to total genome considering the physical lengths of chromosomes
• It is advisable not to include chromosomes 7 or 14 in probe combinations, as
translocations and other aberrations involving these chromosomes can arise in vivo
during immunological development and may thus confound quantification of
radiation effect
• For retrospective biological dosimetry single colour FISH for triple cocktail of target
chromosomes appears to be sufficient. Multiple colour painting of triplet increases
detection efficiency (if chromosomes #1, #4 and #12 are highlighted from about 31%
to about 34%) and gives better detection of complex translocations that can be
encountered following high recent exposures
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Scoring criteria
There is consensus on which metaphases should be scored. Well-spread metaphases are
considered suitable for scoring if cells appear to be intact, centromeres are morphologically
detectable and present in all painted chromosomes, and fluorochrome labelling is sufficiently
bright to detect exchanges between chromosomes.
Human metaphase with coloured painted chromosomes #1 (FITC, green), #4 (FITC+Cy3 yellow)
and #11 (Cy2, red), centromeres highlighted with pancentromeric probe labelled with Cy3 (red),
and rest counterstained with DAPI. Simple translocation, or two-way translocation [t(Bc),t(Cb)]
involving chromosomes # 1 and 11 is observed.
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Nomenclature
To describe the chromosome aberrations detected by painting two specific nomenclature
systems were developed independently and descriptions based on the conventional
terminology of routine cytogenetic scoring were also used.
Protocol for Aberration Identification and
Nomenclature Terminology (PAINT) was
developed to be purely descriptive of each
aberrant painted object in the metaphase,
without cross-reference to other aberrant
objects in the cell. Each colour is designated
by a letter, starting alphabetically with the
counterstain. Capital letters designate the
component that bears a centromere, and
multiple coloured painting is accommodated
by including further letters in the
nomenclature.
Tucker et al., Cytogenet Cell Genet. 68:211-221
(1995).
Counterstained
a
A
b
B
c
C
Painted 1 colour
Painted 2 colour
Painted 3 colour
d
D
Examples
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t(Ba)
dic(BA)
t(Ab)
t-ins(Abab)
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Savage and Simpson (S&S) proposed a terminology comprising numerals and letters
describing each exchange in its entirety. The exchanges were classified according to
the number of chromosomes (C), the number of arms (A) and the number breaks (B)
involved (CAB families).
This so-called S&S system applies only to single paint patterns. However, it can be used with dual and triple
paint patterns but each painted chromosome has to be scored in isolation irrespective of the colours of
partners. This nomenclature has considerable uses in mechanistic studies, particularly, for example, in
understanding complex rearrangements.
Examples
Number of breaks needed
2
2
2B
3
2A
2G
Name of the exchange
3
The nomenclature contains
aberrations from CAB 2/2/2 to 5/5/5,
and it is difficult to handle. So, for
retrospective studies is not suitable.
But has a lot of interest for
mechanistic studies.
2F
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A more conventional terminology may be employed that names translocations as
reciprocal or terminal. Reciprocal has also been called complete or two-way, and
terminal is also called incomplete or one-way.
Reciprocal
Complete, or two-way translocation
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Terminal
Incomplete, or one-way translocations
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Nomenclature used in practice
• Nowadays most widely used method for recording data is to describe each
abnormal metaphase as a unit using PAINT nomenclature but in slightly
modified way that considers underlying mechanisms of formation of
aberrations
• Abbreviations of PAINT system are used but a note is made of
associations between objects in metaphase, thereby incorporating aspects of
the conventional terminology too
• Chromosome aberrations are classified as simple or complex, latter being
when three or more breaks in two or more chromosomes are needed to
produce observed abnormality. Aberrations are considered complete when all
broken pieces are rejoined and as incomplete when one or more pieces
appear unrejoined
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This metaphase contains a dicentric between two painted
chromosomes and a dicentric between two unpainted chromosomes.
So a dic (BB)+ace (b) plus and dic (AA)+ace(a)
dic(BB) ace(b)
dic(AA) ace(a)
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More examples
Ins(Bab)
Ins(Aba)
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What is stable?
This metaphase contains dicentric between unpainted chromosomes and translocation between
painted and unpainted chromosomes. So, dic(AA)+ace(a) plus and t(Ba)+t(Ab). It is important to
consider that translocation is stable aberration, but this cell is unstable due to presence of
dicentric and its acentric
t(Ba) t(Ba)
Dic(AA) ace(a)
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Dose-Effect Curves
Similar to dicentrics, for dose estimations with translocations detected by FISH,
each laboratory needs to establish its own curves. Curve should be made with
same FISH probe cocktail that is routinely used for case investigations. Doing this
removes need to convert to genome equivalence which could introduce some extra
uncertainty
It is recommended to score all aberrations
detected in entire chromosome set, not
just those affecting the painted material,
but also those affecting unpainted
material. This allows one to establish
dose-effect curves for translocations in
stable cells (cells without dicentrics, rings
or acentrics)
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Dose-Effect Curves
However, it should be pointed that for low-LET radiations, when calibration
curves for translocations have been constructed taking into account stable or
total cells, there were no differences in the fitted coefficients if only apparently
simple translocations were considered
Dose effect curves for all simple translocations
observed in all cells, or those only in stable cells
t(Ba)+t(Ab)
two-way translocation
t(Ab)
+
one-way translocations
t(Ba)
All simple translocations
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Data Handling
When only translocations between painted and unpainted chromosomes
material are scored it may be necessary to convert observed frequency to full
genome equivalence
• Conversion is recommended procedure to use when data have to be combined or intercomparisons
have to be made between results from various studies where different combinations of whole
chromosome painting probes have been used
• Assumption, sometimes referred to as Lucas formula, is that probability of the involvement of
particular painted chromosome in aberration is proportional to its DNA content. This assumption
gives reasonable approximation. However, there is consensus that when using the (DNA content) in
Lucas formula, larger chromosomes may tend to be overestimated in their participation in simple
exchange aberrations compared to smaller ones
• Therefore, use in Lucas formula of (DNA content)2/3 rather than (DNA content) gives more accurate
results. Some authors have argued that this kind of proportionality could be symptomatic of
interchanges involving primarily chromatin near boundary of chromosome territories
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Genomic equivalence- painting with one colour
fp is the fraction of the genome covered by the DNA probes, and 1-fp the remainder unpainted genome
All possible exchanges are between:
painted and painted
f p  f p  f p2
painted and unpainted
f p  (1  f p )
unpainted and painted
f p  (1  f p )
unpainted and unpainted
(1  f p )  (1  f p )  (1  f p ) 2
Can not be detected
2  f p  (1  f p )
Can be detected
Can not be detected
Intrachromosomal exchanges, that can not be detected = 0.026. This number depends on the number of
human chromosomes and the relative DNA content of them.
If the total exchanges is FG, and the the fraction of all exchanges than can be detected is Fp then :
Fp
FG

2 f p (1  f p )
1  0.026
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
2 f p (1  f p )
0.974
 2.05 f p (1  f p ) 
Fp
FG
 2.05 f p (1  f p )  FG 
Fp
2.05 f p (1  f p )
The genomic equivalent frequency
of translocation is calculated using
this formula
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Example
If chromosome pairs 1, 2 and 4 are painted. Their respective
DNA contents (male) are 0.0828, 0.0804 and 0.0639. 2743.
Therefore, fp = 0.2271, so that FP/FG = 0.360
This combination of chromosomes painted is 36% efficient in
measuring bicoloured translocations.
Therefore, to obtain the full genome translocation yield the
observed yield is divided by 0.36
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Control Level of Translocations (1)
• Control levels of translocations are higher than for
dicentrics, and to some extent this is due to translocations
being a persisting type of aberration
• It is therefore important to take the translocation
background into account, particularly after low doses, when
attempting retrospective biodosimetry
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Control Level of Translocations (2)
• Comprehensive meta-analysis currently provides best
international database, broken down by age, gender, race
and smoking habits (Sigurdson et al. 2008)
• It is clear that age is major factor that determines
background frequency of translocations
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Age-control relationship
This is genomic
equivalent frequency
Translocation frequency per 100 cells
From number of translocations observed in individual, it is important to
subtract the generic background frequency expected for his or her age
2.5
2
1.5
1
0.5
0
0
20
40
60
80
Years


Translocat ion frequency per 100 cells  100  e 7.925  e 9.284  age  e( 0.01062 age)
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Data from Sigurdson et al., Mutat. Res 652:112-121 (2008)
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