Cytokinesis Block Micronucleus
(CBMN) Assay
Lecture
Module 7
IAEA
International Atomic Energy Agency
Contents
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What are micronculei and how are they formed
The CBMN assay for radiation dose assessment
Micronucleus dose response
Micronucleus background frequencies
Applications of the CBMN assay for biological dosimetry
Improvements of the CBMN assay
1) CBMN centromere assay
2) automated CBMN assay
3) CBMN cytome assay
4) semi-automated CBMN-centromere assay
• Limitations of the CBMN assay
• Ongoing developments
• Practical issues: NDI, Scoring criteria, protocols
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What are micronuclei (MN) and how
are they formed
Micronuclei are:
• small extranuclear fragments found in interphase
cells
Micronuclei represent:
• acentric chromosome fragments (MN-ac) or whole
chromosomes (MN-wc) that are not incorporated in
the daughter nuclei during cell division
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What are micronuclei (MN) and how are
they formed
Micronuclei arise during exposure to
• clastogenic agents (e.g.,ionizing radiation) (MN-ac)
• aneugenic agents (MN-wc)
A small number of micronuclei (esp. MN-wc) appear
spontaneously
MN are not radiation specific!
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What are micronuclei (MN) and how
are they formed
MN-ac are the result of :
• Non- or misrepaired DNA double strand breaks
(DSB)
MN-wc are the result of
• whole chromosomes that do not attach to the
mitotic spindle
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The cytokinesis block micronucleus
(CBMN) assay
• CBMN assay was developed by Fenech and Morley in 1985
• In CBMN assay Cytochalasin B, a cytoplasmic division
inhibitor, is added to cell culture to identify cells that
underwent one division
• These cells are identified as binucleate (BN) cells
• CBMN assay is mostly applied to peripheral blood
lymphocytes (PBL), but any cell that can divide can be used
• CBMN assay is often used as a general toxicology test
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The CBMN assay for radiation dose
assessment
• Ionizing radiation is a strong clastogenic agent and thus a
potent inducer of DSB and by consequence also of MN
• In CBMN assay for radiation dose assessment, MN are
typically scored in 1000 binucleate PBL
• Radiation-induced MN in PBL contain mainly acentric
chromosome fragments (MN-ac) resulting from non-repaired
or misrepaired DNA dsb by non-homologous end joining
(NHEJ) repair pathway
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Human Lymphocytes
Advantages:
• Easily obtained from the peripheral blood
• The majority of peripheral blood lymphocytes are in the G0
phase of the cell cycle
• Phytohaemagglutinin (PHA) converts these resting
lymphocytes into dividing cells by which it is possible to
visualize DNA lesions in metaphase chromosomes or
interphase cells after division
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The CBMN assay for radiation dose
assessment
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Micronuclei in binucleate lymphocytes
A
B
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The MN dose response curve
• Radiation-induced MN are strongly correlated with radiation
dose and quality
• The in vitro MN dose-response calibration curves follow the
same shape as described for the standard dicentric assay:
• for low LET radiation, MN follow a linear-quadratic dose
response : y = c + αD + βD2
• for high LET radiation a linear dependence y = c + αD is
observed, with high LET radiation being more effective
in generating MN at the same dose levels
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The MN dose response curve
1200
Micronuclei/1000 BN cells
1000
800
600
400
200
0
0
1
2
3
4
5
Dose (Gy)
MN frequency /1000 cells plotted against gamma ray dose
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MN background frequencies
• Drawback of the CBMN assay for applications in
the low dose range:
• High and variable spontaneous frequency
(2-36 MN/1000 BN cells)
 unreliable for low dose assessment below
~ 0.2- 0.3 Gy X-or -rays
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MN background frequencies
• Factors influencing the MN background levels:
• age, gender, diet, exposure to environmental mutagens
• age: systematic increase with age
• gender: systematically higher in females vs. males
 males: 0.24 - 0.44 MN/1000BN/year
 females: 0.52 - 0.54 MN/1000BN/year
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MN background frequencies
• What do spontaneous MN contain?
• Analysis of MN content was performed using a FISH
pan-centromeric probe
 the majority of spontaneous MN contain a
centromere (> 70%)  whole chromosome
 the age increase is due to an increase in
centromere-positive MN
*often the X-chromosome is involved  explains the
observed gender difference
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MN background frequencies
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MN background frequencies
Centromere
negative
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positive
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Applications of the CBMN assay for
biological dosimetry (1)
• Chernobyl nuclear power plant and Semipalatinsk nuclear
test site studies
• CBMN assay was applied to assess protracted
exposure, due to the incorporation of long-lived
radionuclides
• Conclusions: MN frequencies measured in a large
number of residents living in the vicinity of the nuclear
sites were increased and significantly associated with
the estimated internal absorbed dose
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Applications of the CBMN assay for
biological dosimetry (2)
For accidents involving few people, only a limited number of
MN studies are available:
(1) The Istanbul accident with an unshielded former radiotherapy Co source (10 scrap metal workers)
(2) Accident (Europe) with a 50 kV contact radiotherapy X ray
device (one hospital worker)
• Blood samples taken between 1 and 6 months after the accident
Conclusions:
• MN-derived dose estimates were in striking agreement with dose
values obtained from dicentric studies
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Applications of the CBMN assay for
biological dosimetry (3)
Application for biomonitoring
• Several studies have been performed on large populations of
nuclear power plant workers and hospital staff
 a dose dependent increase of 0.0175 MN and 0.03 MN per 1000 BN
cells/mSv was found in 2 different studies performed on nuclear power
plant workers (receiving accumulated doses ranging from 10 to 248 mSv)
Application of CBMN-centromere assay in first study resulted in increase
of 0.025 MN per 1000 BN cells/mSv, with dose-dependent increase
completely due to centromere-negative micronuclei
 CBMN assay, especially CBMN-centromere assay allows accumulated
doses exceeding 50 mSv to be detected, at population level
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Improvements of the CBMN assay (1)
• Development of the CBMN-centromere assay
 to increase the low dose sensitivity
• Development of the CBMN cytome assay
 to score more endpoints related to radiation exposure
• Development of the CBMN assay for automated scoring
 to allow rapid analysis of large sample sizes (triage)
• Development of a combined CBMN-centromere assay for
automated scoring
 to combine high speed MN analysis with a more accurate
assessment in the low dose range
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Improvements of the CBMN assay (2)
• The CBMN-centromere assay
• allows discrimination between centromere-positive MN
(MNCM+)(whole chromosomes) and centromere-negative
MN (MNCM-)(acentric chromosome fragments)
• several studies showed that the majority of spontaneous
MN are CM+ (70% and more), while most radiationinduced MN are CM• the number of MNCM+ shows only
a very small increase with dose
• manual scoring of MNCM- lowers
detection limit to 0.05 - 0.1 Gy
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Improvements of the CBMN assay (3)
• Scoring of nucleoplasmic bridges (NPBs) in the
CBMN cytome assay
• In CBMN cytome assay NPBs, joining two nuclei in BN
cell, can be scored
• NPBs originate from dicentric chromosomes
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Improvements of the CBMN assay (4)
• Advantage of scoring NPBs for biological dosimetry
• NPB background frequency is lower than for MN and is not affected
by gender
• NPBs provide a direct method of measuring asymmetrical
chromosome rearrangement (dicentrics, rings) in once divided cells
• NPBs are more radiation specific then MN
• NPBs are increased in a dose-related manner and correlate well with
dicentric and ring chromosome frequencies analyzed in metaphase
cells
• analysis of NPBs is quicker compared to dicentric analysis as in the
CBMN assay a large number of BN cells are accumulated
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Improvements of the CBMN assay (5)
• Automated scoring for the CBMN assay
• among all cytogenetic methods, CBMN assay allows most easy and
rapid scoring of radiation damage
• to further increase the throughput of this technique, automated scoring
procedures have been developed
• automated MN scoring is very attractive for:
1) population triage in case of large scale radiation accidents
2) for large-scale assessment of genetic damage in radiation
workers receiving a high radiation burden (biomonitoring)
• automated MN scoring results in a more reproducible analysis
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Improvements of the CBMN assay (6)
• Automated scoring for the CBMN assay
• different image analysis systems for automated MN
scoring are available:
- MN software module integrated in the metaphase finder
system MSearch of Metasystems
1) identification of 2 adjacent similar nuclei (e.g. DAPI stained)
2) MN scoring in a circular region defined around the 2 nuclei of the BN cell
- MN analysis system running on the PathFinder Cellscan
capture station
1) identification of the cytoplasm of Giemsa-stained cells
2) detection of the number of nuclei in the cell, allowing identification of BN cells
3) automated scoring of MN in the BN cell
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Improvements of the CBMN assay (7)
• Automated scoring for the CBMN assay
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Improvements of the CBMN assay (8)
• Automated scoring for the CBMN assay
Example of BN cells with and
without MN captured by the
MN module for automated MN
scoring of Metasystems
• on 1 slide automated scoring
of MN in about 2000 BN cells
can be performed in less
than 8 minutes
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Gallery of captured BN cells with MN
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Improvements of the CBMN assay (9)
• The CBMN assay for automated scoring
• the figure shows a dose response curve obtained for
automated MN scoring (by MNScore of Metasystems),
based on MN data of 10 individuals
• Results
• dose of 1 Gy can be
detected with accuracy
of 0.2 Gy
• 95% CI of 0 Gy and
1 Gy doses do not overlap
• accurate dose estimations
are also achieved at
higher doses of 2 and 3 Gy
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Taken from Willems et al., IJRB 86(1),2-11, 2010
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Improvements of the CBMN assay (10)
• Development of a combined CBMN-centromere
assay for automated scoring
• to enhance sensitivity, reliability and processing time of
MN assay by combining automated MN assay with
FISH pan-centromere scoring
• This will allow :
 systematic biomonitoring of radiation workers
exposed to low doses
 more accurate assessment of exposure in second
phase of mass radiation casualty event - after early
triage - when the time constraint will be less strict
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Improvements of the CBMN assay (11)
• Development of a combined CBMN-centromere
assay for automated scoring
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number of MN/1000BN
20
15
MN CM+
10
MNCM-
MN total
5
0
0
0.05
0.1
0.2
0.3
0.5
Dose (Gy)
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This figure shows the average of the total number of MN (MNtotal), MNCM+ and
MNCM- for 10 donors as a function of dose (up to 0.5Gy). The error bars are SEM.
(modified from Baeyens et al., IJRB, 2011)
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Improvements of the CBMN assay (12)
• Development of a combined CBMN-centromere
assay for automated scoring
Conclusion
• Semi-automated CBMN-centromere assay combines high
speed MN analysis with a more accurate assessment in
the low dose range, down to 0.05 Gy which makes it of
special interest for large-scale radiation applications
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Limitations of the CBMN assay (1)
• Major limitations of CBMN assay are related to:
• retrospective dosimetry
• accidents involving partial body irradiation
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Limitations of the CBMN assay (2)
• Retrospective dosimetry
• In some accident studies MN data were compared with
data obtained by the FISH translocation test, which is the
endpoint of choice for retrospective dosimetry
• Istanbul accident (10 scrap metal workers) and
accident of hospital worker with a radiotherapy X ray
device (slide 19)
• blood samples were taken at different time points after
the accident (1month -1year)
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Limitations of the CBMN assay (3)
• Retrospective dosimetry
• Results and conclusions
 dose estimates obtained by scoring MN (comparable to dicentrics)
are lower (20 to 30%) than dose estimates obtained by scoring
translocations
 correction of the MN values for the delayed blood sampling in the
hospital worker (MN disappear with a half-life of 342 days) result in a
dose estimate comparable to that obtained with FISH translocations
 underestimation of radiation doses because MN (and dicentrics) are
unstable chromosome aberrations which have a limited in vivo
persistence
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Limitations of the CBMN assay (4)
• Accidents involving partial body irradiation
• in practice, most accidents involve partial body exposures (PBE)
 problem with PBE: underestimation of dose because of presence of
undamaged lymphocytes present outside the irradiation field
For dicentric assay mathematical methods exist to calculate PBE
• The contaminated Poisson method (Dolphin 1970) based on the
finding that after whole body (WB) irradiation dicentrics are Poisson
distributed
• The Qdr method (Sasaki and Miyata 1968)
For CBMN assay as MN are already slightly overdispersed after WB
irradiation the applicability of a MN frequency distribution analysis with
respect to a PBE is questionable and still needs further investigation
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The CBMN assay: practical issues
• Scoring criteria for the CBMN assay
• Nuclear division index (NDI)
• Protocols
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Scoring criteria for the CBMN assay (1)
Criteria for manual scoring of BN cells
• Cytokinesis-blocked cells should have following characteristics:
(a) cells should be binucleated (BN)
(b) two nuclei in a BN cell should be situated within same cytoplasm
(c) two nuclei in a BN cell should be approximately equal in size,
staining pattern and intensity
(d) two nuclei within BN cell may be unconnected or may be
attached by one or more fine NPB
(e) two main nuclei in BN cell may
touch but ideally should not overlap
each other
(f) BN cells should not overlap each other
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Scoring criteria for the CBMN assay (2)
Criteria for manual scoring of MN
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MN are morphologically identical to but smaller than the main nuclei.
They also have the following characteristics:
(a) diameter of MN in human lymphocytes should be smaller then 1/3rd
of the mean diameter of the main nuclei
(b) MN are non-refractile and can therefore be readily distinguished
from artefacts such as staining particles
(c) MN are not linked or connected to the main nuclei
(d) MN may touch but not overlap the main nuclei and the micronuclear
boundary should be distinguishable
(e) MN usually have the same staining intensity as the main nuclei but
occasionally staining
may be more or less
intense
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Scoring criteria for the CBMN assay (3)
Criteria for scoring NPB in the CBMN-cytome assay
•
Description is given in:
- The IAEA Manual and
- Cytokinesis-block micronucleus cytome assay.
M. Fenech, Nature Protocols 2(5), 1084-1104, 2007
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Nuclear division index (NDI)
•
In the CBMN assay, the relative frequencies of cells with 1,
2, 3, etc nuclei (NDI) can be used to define cell cycle
progression and how this is affected by radiation exposure
•
Calculation of the NDI
NDI= M1 + M2 + M3 + M4 / N
- 500 cells are scored
- M1 to M4 represent the number of cells with one to four nuclei and N is
the total number of viable cells scored
•
More details about the calculation of the NDI and its
uncertainty are described in the IAEA Manual
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Protocols (1)
Standard Cytokinesis-block Micronucleus protocol
- a heparinized blood sample is taken and complete culture medium
(RPMI+ supplements + 10% FCS ) is prepared
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Protocols (2)
Standard Cytokinesis-block Micronucleus protocol
- 0.5 ml of blood is added to complete culture medium
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Protocols (3)
Standard Cytokinesis-block Micronucleus protocol
- Phytohaemagglutinin (PHA) is added as the mitogen
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Protocols (4)
Standard Cytokinesis-block Micronucleus protocol
- Blood culture is incubated at 37°C and with 5%CO2
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Protocols (5)
Standard Cytokinesis-blocked Micronucleus protocol
- after 24 hours incubation cytochalasin B is added to block cytokinesis
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Protocols (6)
Standard Cytokinesis-block Micronucleus protocol
- Cells are harvested 3 days after culture start (between 68-72h):
- Harvest protocol:
- Blood cultures are centrifuged
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Protocols (7)
Standard Cytokinesis-block Micronucleus protocol
- Harvest protocol:
Cells are hypotonically treated with cold KCl (0.075 M)
KCl
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Protocols (8)
Standard Cytokinesis-block Micronucleus protocol
- Harvest protocol:
After centrifugation cells are fixed with fixative solution (10 methanol / 1
acetic acid / 11 Ringer salt solution). For the automated scoring 4/1/5
ratio is used.
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Protocols (9)
Standard Cytokinesis-block Micronucleus protocol
- Harvest protocol:
After centrifugation cells are washed another 2 or 3 times with a fix
solution containing 10 methanol / 1 acetic acid (for automation: 4/1)
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Protocols (10)
Standard Cytokinesis-block Micronucleus protocol
- Harvest protocol:
After the last fixation step, the supernatant is removed and cells
suspended in about 1 ml of fixative are dropped on clean slides, air dried
and stained with Giemsa (for manual scoring) or DAPI (for automated
analysis).
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Protocols (11)
Standard Cytokinesis-block Micronucleus protocol
Giemsa stained slide
DAPI stained slide
Illustrations produced in the laboratory of Prof. Vral, Ghent University, Belgium
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