Mutagenesis vol. 26 no. 1 pp. 147–152, 2011
1093/mutage/geq058
REVIEW
Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and
future
Svetlana Avlasevich, Steven Bryce, Marlies De Boeck1,
Azeddine Elhajouji2, Freddy Van Goethem1,
Anthony Lynch3, John Nicolette4, Jing Shi5 and
Stephen Dertinger*
Litron Laboratories, 200 Canal View Boulevard, Rochester, NY 14623, USA,
1
Genetic & Exploratory Toxicology, Johnson and Johnson Pharmaceutical
R&D, Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse,
Belgium, 2Preclinical Safety, Novartis Institutes for Biomedical Research,
CH-4002 Basel, Switzerland, 3GlaxoSmithKline R&D (3F02), Park Road, Ware,
Hertfordshire, UK, 4Abbott Laboratories, R45M/AP13A, 100 Abbott Park Raod,
Abbott Park, IL 60064, USA and 5BioReliance Corp., 14920 Broschart Raod,
Rockville, MD 20850, USA
*To whom correspondence should be addressed. Tel: þ585 442 0930;
Fax: þ585 442 0934; Email: sdertinger@litronlabs.com
Received on May 25, 2010; revised on July 15, 2010;
accepted on August 23, 2010
The relative simplicity of the in vitro micronucleus (MNvit)
endpoint has made it amenable to several automated
scoring approaches. Flow cytometry is one such scoring
platform that has been successfully employed. This review
describes the origins of the MNvit assay, as well as the
evolution and properties of flow cytometry-based scoring
systems. While the current state-of-the-art methods ac-
quire micronucleus (MN) frequency data very efficiently, it
is becoming clear that they also endow the assay with high
information content. For instance, simultaneous with MN
frequency determinations, several additional endpoints are
acquired that provide insights into cytotoxicity, cell cycle
perturbations and, in the event of MN induction, in-
formation about genotoxic mode of action. This review
concludes with a discussion regarding data gaps and also
recommendations for additional work that is needed to
more fully realise the potential of flow cytometric MNvit
scoring.
Significant achievements
The in vitro micronucleus (MNvit) test is a simple cytogenetic
assay based on the scoring of extranuclear chromatin-containing
structures in actively dividing cell populations. These so-called
micronuclei (MN) result from acentric chromatid or chromo-
some fragments (i.e. those lacking a centromere) or whole
chromosomes that are unable to migrate to the spindle poles
during mitosis and subsequently are not incorporated into
either of the daughter nuclei. Therefore, MN can only arise in
cells that have undergone cell division. The induction of these
extranuclear bodies was first recorded in Vicia faba root cells
by Thoday (1) and their use as a quantitative measure of
cytogenetic damage was originally proposed by Evans et al.
(2). Several years later, the idea of scoring MN as a biological
dosimeter of radiation exposure in humans was proposed by
Fliedner et al. (3).
Ó The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.
doi:10.
The MN test became a routine assay in environmental
mutagenesis following the development of the rodent bone
marrow MN test (4,5). A mammalian cell culture version of the
MN test was originally proposed by Heddle (6) and later
described by Countryman and Heddle (7) in human peripheral
blood lymphocytes. The presence of whole chromosomes in
MN can be ascertained using suitable molecular cytogenetic
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methods, e.g. immunochemical labelling of kinetochore proteins
(8) or fluorescence in situ hybridisation (FISH) using a combi-
nation of centromeric and telomeric probes (9). This permits
chromosome loss and/or non-disjunction (FISH only) to be
ascertained and therefore the MNvit assay can provide evidence
for the underlying mode of action (MOA)—clastogenicity
(structural chromosome aberrations) or aneugenicity (chromo-
some loss or gain). The predominant induction of either type of
MN can be used to classify chemical activity and therefore the
information is useful for risk assessment purposes. As many
compounds that are positive in the MNvit test are mammalian
genotoxic carcinogens, the endpoint is often used as a simple
screen to identify agents that may be tumorigenic (for reviews
see refs 10–12).
The MNvit test has been used in a variety of established
rodent cell lines (CHO, V79, CHL and L5178Y) and in
primary human lymphocytes. Protocols have been described
with and without the use of cytochalasin B, an agent that
blocks cytokinesis (13). The cytokinesis-block method permits
MN to be scored only in cells that have completed a single
mitosis after genotoxicant exposure because such cells are
easily recognised as binucleated cells (14,15). Limiting scoring
of MN to once-divided binucleated cells guards against false-
negative results stemming from cytostatic effects of genotox-
icants, suboptimal cell culture conditions and, in the case of
primary lymphocytes, differences in mitogenic response (16).
The MNvit test has been used in academia and industry for
hazard identification as an alternative/replacement of the
in vitro chromosome aberration test, a time-consuming method
that requires considerable expertise to visually identify struc-
tural anomalies in metaphase chromosomes. MNvit is also
a valuable follow-up test to elucidate MOA for risk assessment
purposes. The test has already gained widespread international
use as it offers significant advantages over the assessment of
chromosome aberrations since analysis is more objective and
quicker, resulting in a much higher throughput.
Over the past decade, several studies have been performed to
support the validity of the MNvit test for incorporation into the
standard regulatory genotoxicity test battery as an alternative to
the in vitro structural chromosome aberration test. These
studies include, in part, the international validation efforts
coordinated by the Société Francxaise de Toxicologie Génétique
(17–21), the work performed by Homiski et al. (22) and studies
by Miller et al. (23,24). Miller et al. reported .85% correlation
between the in vitro structural chromosome aberration test and
the MNvit test. More recently, formal validation for the MNvit
147
S. Avlasevich et al.
test has been carried out by the European Centre for the
Validation of Alternative Methods (25). Based on this
retrospective validation, it has been concluded that the MNvit
test is reliable and a relevant genotoxicity assay that can be
used as an alternative to the in vitro structural chromosome
aberration test. Indeed, recent revisions of international
regulatory guidances such as the new European Chemicals
Regulation REACH (The Registration, Evaluation, Author-
isation and Restriction of Chemicals) and International
Conference on Harmonisation (ICH) S2 R guideline for
pharmaceuticals have recommended the adoption of the
MNvit test as an alternative to the structural chromosome
aberration assay and/or mouse lymphoma assay. Furthermore,
the Organisation for Economic Co-operation and Development
(OECD) guideline for the conduct of the MNvit test (TG 487)
for regulatory purposes was recently approved.
It was realised early on that the simplicity of the MN
endpoint should facilitate automation (26), and several
different scoring platforms have been described. There were
attempts at image analysis and DNA densitometry (27–30), as
well as flow cytometry (31–36). The majority of the flow
cytometric work was based on the approach of Nüsse and
Kramer (31) that involved lysis of outer membranes. In
conjunction with one or more nucleic acid dyes, it was possible
to discriminate the liberated nuclei and MN based on their
DNA-dye associated fluorescence intensities. Furthermore,
beyond MN scoring, flow cytometry allowed sorting of MN
to combine with FISH for centromeric probes or chromosome-
specific paints to identify MN content and its genomic origin
(37–39).
While flow cytometry was clearly shown to be a high-
throughput platform with great potential, the early methods
experienced problems discriminating MN from cellular debris
in the cell preparations. Later modifications to those of Nüsse
and Kramer attempted to overcome these problems, taking
advantage of the higher resolution of later machines and using
improved gating based on a combination of light scatter and
fluorescence signals to distinguish MN signals from debris
(35,40). Although these modifications improved the reliability
of the assay, it remained difficult to discriminate MN from
cellular debris, especially chromatin associated with apoptotic
cells.
The reliability of flow cytometry-based MN scoring was
improved when the procedures of Nüsse et al. were modified
(41,42) and subsequently configured into a commercially
available kit (In Vitro MicroFlowÒ; Litron Laboratories). The
most significant of these modifications were (i) incorporation
of a fluorescent dye to differentiate MN from chromatin
associated with dead and dying cells and (ii) incorporation of
concurrent assessments of cell/culture health that identify
overly cyotoxic treatment conditions that tend to lead to
unreliable MN measurements.
The current state-of-the-art utilises a dual dye sequential
staining procedure. More specifically, at the time of harvest,
intact cells are incubated with ethidium monoazide bromide
(EMA). EMA is a nucleic acid that enters the compromised
membranes of necrotic and late-stage apoptotic cells (43). A
unique characteristic of EMA is that it can be covalently bound
to DNA following a photoactivation step. This makes it
possible to label dead/dying cells with EMA without loss of
signal as cells are further processed. This EMA labelling step is
followed by exposure to a detergent-containing lysis solution
that includes the nucleic acid dye SYTOXÒ Green, which
148
provides pan-DNA labelling. In this manner, the sequential dye
protocol results in differential staining of healthy cells’
chromatin (EMA
/SYTOXþ) relative to necrotic and late-
stage apoptotic cells’ EMAþ/SYTOXþ profile. By excluding
EMAþ chromatin from analysis, flow cytometric scoring
of nuclei and MN is accomplished with significantly reduced
interference from the presence of dead/dying cells (see
Figure 1). This methodology has been shown to provide good
agreement between flow- and microscopy-generated data
(41,42), although as described below, to achieve adequate
assay specificity, it is important to limit the highest concen-
trations of test article to those that are moderately, not overly,
cytotoxic.
Because cytotoxicity limits are an important element of
mammalian cell culture genetic toxicology assays (44), one
important advance to the flow-based methodology was the
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addition of fluorescent latex microspheres. The use of these so-
called ‘counting beads’ is common in flow cytometry
applications that require absolute counts (45). The premise of
counting beads is that when they are present at a known
consistent density, they can supply information about the
volume that has passed through the flow cytometer. Thus, by
adding counting beads to the MN assay cultures (for instance to
suspension cultures at time of seeding), it becomes possible to
determine nuclei-to-bead ratios for each sample, and these
values can be used to calculate relative survival. Efficiency
remains high since these measurements are acquired concur-
rently with flow cytometric MN counts. As reported by Bryce
et al. (45), the flow-based relative survival measurement often
reveals cytotoxicity that alternate methods, including Coulter
counter-based relative survival and population doubling times,
as well as Annexin–propidium iodide staining, often under-
represent. We hypothesise that flow cytometry-based assess-
ment tends to be more sensitive because of the multiparametric
nature of these measurements. For instance, to be included in
a relative survival calculation, the flow-based analyses require
liberated nuclei to exhibit forward and side scatter properties of
healthy cells and also to exhibit characteristic EMA and
SYTOX fluorescence profiles. By restricting MN scoring to
test article concentrations that do not demonstrate excessive
cytotoxic profiles based on this nuclei-to-bead ratio approach,
assay specificity is improved (42,46).
A second endpoint that reflects the health of a culture is the
percentage of EMAþ events. As apoptotic cells contribute
multiple EMAþ events, whereas necrotic nuclei contribute
one, this statistic is particularly sensitive to programmed cell
death. In some cell lines, especially those grown in suspension,
this measurement can be useful for determining the validity of
an assay (e.g. vehicle controls should have a characteristic low
percentage of EMAþ events) or to identify test article
concentrations that are too cytotoxic for reliable MN
enumeration (41,46).
Each of the authors has worked with the sequential labelling
method described above for at least 3 years. Based on this
collective experience, we have found that there are several
compelling characteristics of flow cytometric scoring.
Increased objectivity
Automation is inherently a more objective means of acquiring
data relative to visual inspection of slides. Furthermore,
strategies for controlling intra- and inter-laboratory variation
are readily incorporated when a flow cytometric platform is
employed. For instance, in the case of MN scoring, the
 Flow cytometric analysis of MNvit

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Fig. 1. Bivariate plots of nuclei, MN and other subcellular particles from bleomycin (genotoxicant)- or sodium dodecyl sulphate (SDS) (cytotoxicant)-treated TK6
cells as analysed by flow cytometry. Panels (A and D): these plots illustrate that a fraction of chromatin associated with apoptotic and necrotic cells is double-
positive, that is it exhibits both SYTOX-Green and EMA fluorescence. Through the use of software ‘gating logic’, it is possible to exclude events that exhibit an
EMA-positive staining characteristic. Panels (B and E): these plots were used to score nuclei and MN values, and in these cases, gating logic was not configured to
exclude EMA-positive events. By not considering EMA fluorescence characteristics, the cytotoxicant SDS exhibits artificially high MN frequencies. Panels (C and
F): these plots were used to score nuclei and MN values, but in these cases, gating logic was configured so that only events that were SYTOX-Green-positive and
EMA-negative were considered. By eliminating EMA-positive events from the analysis, the genotoxicant bleomycin but not the cytotoxicant SDS exhibits an
elevated micronucleus frequency.

standard practice is to adjust photomultiplier tube voltage
settings so that G1 nuclei in vehicle control cultures appear in
the same fluorescent channels each day analyses are performed.
Furthermore, it is also possible to maintain very consistent
definitions of the MN scoring region, for instance 1/100th to
1/10th of the G1 nuclei’s fluorescence intensity.
Number of cells scored
As MN are relatively rare events, an accurate determination of
their frequency clearly benefits from interrogating more cells
per replicate. Based on practical considerations, microscopy
and image analysis tend to limit analyses to 1000–2000 cells
per replicate, whereas flow cytometry provides the opportunity
to readily acquire MN frequency information based on 5000
cells, usually in timeframes of 2–3 min. This potential to
increase the number of cells evaluated and thereby enhance
statistical power may be especially important for work with
weak genotoxicants or even potent MN inducers when one is
interested in carefully defining the low end of the dose–
response curve.
Efficiency and throughput
Beyond the high rate of data acquisition, there are other
features of the MN scoring application that makes conduct of
the assay very efficient. For instance, 96-well plate versions of
the assay have been described for both suspension and
attachment cell lines (47,48). In both cases, all processing
steps, from treatment, staining and lysis, occur in the same
plate. Furthermore, for those flow cytometers that are equipped
with a robotic sampler, it is also possible to acquire data from
these same plates, a situation that clearly streamlines the
conduct of the entire assay. When attachment cell lines are
used, further efficiencies are realised because all washing steps
can take place with simple aspirations without the need for
centrifugation steps. Taken together, these characteristics can
lead to new experimental designs that were not previously
feasible with microscopic inspection. For example, a recent
report described MNvit experiments that utilised 22 concen-
trations of test article studied in quadruplicate (49). Experi-
ments of this type can be useful in several contexts, including
screening programmes that wish to avoid the time and
resources associated with preliminary dose-range finding
assays. This type of design may also be valuable when one
is interested in carefully defining the low end of a dose–
response curve, for instance to determine no observable effect
levels and lowest observable effect levels.
High information content
As alluded to previously, flow cytometry provides a rich data
set that conveys information about test article cytotoxicity,
which is achieved concurrently with MN counts. This
information can be extremely valuable when interpreting MN

149
S. Avlasevich et al.

results. Thus, while nuclei-to-bead measurements can be
readily converted to informative relative survival values,
%EMAþ events are sensitive to necrosis/apoptosis, and the
SYTOX fluorescence distribution of nuclei can detect test
article-induced perturbations to the cell cycle.
MOA signatures
Related to the high information content nature of flow
cytometric measurements, work with certain cell lines, most
convincingly with CHO-K1 to date, suggest that clastogenic
and aneugenic signatures are evident from flow cytometric data
(48,50,51). For instance, while both clastogens and aneugens
increase %MN, only aneugens are observed to markedly
increase the frequency of hypodiploid nuclei and the median
SYTOX fluorescence intensity of MN events (see Figure 2).
Compatibility with diverse cell types
beyond the capacity to survey large numbers of cells, the
multiparametric platform permits one to simultaneously collect
cytotoxicity and cell cycle data, as well as clues as to the
genotoxic MOA. Indeed, the promise of an automated
mammalian cell assay that provides quantitative data and
MOA information may allow the MNvit test to move from
a simple hazard identification screen to one that may also
supply weight of evidence information that is useful for risk
assessment purposes.
While enthusiasm for such a promising technology is
warranted, it needs to be tempered by the fact that the latest
protocols for flow cytometry analysis are still relatively new.
Whereas it has been a relatively straight-forward process to
compare automated MN scoring against microscopic counts,
for other evaluations such as aneugenic versus clastogenic
MOA signatures, validation strategies will be more complex.

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The dual-staining technique and cell lysis procedures are
compatible with several cell lines, a characteristic that provides
investigators with a great deal of flexibility. Cell lines that have
been studied and found to be compatible to date are L5178Y,
TK6, WIL-2-NS, CHO-K1, V79 and HepG2 (41,42,50–52).
While the advantageous characteristics of flow cytometry-
based MN scoring described above have energised the genetic
toxicology community to explore ways to incorporate this
technology into their safety assessment work, it is also evident
that the method imposes certain limitations. For instance, the
use of cytochalasin B in microscopy-based methods restricts
analysis to once-divided cells. Visual inspection is also able to
detect effects such as nucleoplasmic bridges and nuclear buds,
endpoints that are not apparent with flow cytometry. So as
microscopy-based scoring continues to develop into a compre-
hensive ‘cytome assay’ (12), progress with flow cytometric
analysis of in vitro MN also continues, presenting considerable
opportunities/challenges of its own. The authors’ perspectives
on the future of MN scoring by flow cytometry are provided
below.
Knowledge gaps and recommendations for future research
Flow cytometric analysis offers an opportunity for efficient
MN scoring, as well as higher information content. That is,
Assays for aneugenicity or non-disjunction events using anti-
kinetochore probes or FISH have well-established historical
data and criteria for judging a chemical aneugenic. The current
flow cytometric state-of-the-art provides some characteristic
MOA signatures, but to date, this has really only been
demonstrated in two cell lines, CHO-K1 and V79 (47,49,50).
Indeed, there is some evidence that these signatures are not as
strong or specific in several commonly used cell
lines—L5178Y and TK6. More work is necessary to establish
a better understanding of the cell lines that are capable of
providing aneugenic versus clastogenic signatures, and there is
a need to more definitively formulate quantitative criteria that
are useful for making these MOA determinations.
As described previously, a major challenge of flow
cytometric systems has been the detection of bona fide MN
resulting from a genotoxic effect as opposed to DNA fragments
produced during apoptosis. While EMA labelling segregates
out most DNA originating from dying cells, it is not effective at
labelling those chromatin fragments generated before plasma
membrane integrity has become compromised. For example,
when Collins et al. (53) studied the cytotoxicant dexametha-
sone, they reported increased MN counts by flow cytometry
that were not observed by microscopy, even though EMA was
used to exclude chromatin from dead/dying cells. Only later
when it became clear that induction of EMA-positive events
and also nuclei-to-bead ratio measurements should be used to

Fig. 2. Bivariate plots of nuclei, MN and other subcellular particles from bleomycin (clastogen)- or griseofulvin (aneugen)-treated CHO cells analysed by flow
cytometry. This cell line exhibits flow cytometric profiles (‘signatures’) that provide insights into genotoxic mode of action. Panel (A): solvent control, with a low
(baseline) MN frequency. Panel (B): clastogenic signature, where a significant increase in MN frequency is not accompanied by marked induction of hypodiploid
nuclei or an upward shift in MN fluorescence intensity (see histogram, insert, where counts are plotted versus SYTOX-Green fluorescence). Panel (C): aneugen
signature, where a significant increase in MN frequency is accompanied by marked induction of hypodiploid nuclei and an upward shift in MN fluorescence
intensity (see histogram, insert, where counts are plotted versus SYTOX-Green fluorescence).

150

set the top analysable concentration of test article was
agreement between microscopy and flow cytometry observed
(46). The message from work with this cytotoxicant and others
is clear—although EMA labelling markedly diminishes the
influence that dead cells have on MN counts, it is not absolute.
Rather, for reliable analyses and interpretation of flow
cytometry-based data, a more holistic approach of evaluating
test article-induced cytotoxicity is required.
Gaining deeper insights into test article-induced toxicities
can only benefit interpretation of cytogenetic endpoints such
as MN. Thus, beyond scoring %EMA-positive events and
performing sensitive flow-based relative survival measure-
ments, a potentially productive line of investigation will be the
evaluation of other indices of cytotoxicity that can be
multiplexed onto flow cytometry-based MN scoring protocols.
For instance, experiments are currently underway that are
assessing the merits of collecting pretreatment and post-
treatment cell-to-bead ratios (that is ‘healthy’ cells based on
light scatter properties prior to lysis/staining steps). Unlike
nuclei-to-bead ratios at the time of harvest, these pretreatment
and post-treatment cell-to-bead measurements can be used to
calculate relative increased cell counts and population
doublings. These cytotoxicity indices are considered useful
because they are more responsive to cell death and cytostasis
as compared to relative survival measurements (54). Other
endpoints of cytotoxicity made in parallel or concurrently with
MN scoring should be investigated for their ability to identify
treatment conditions that lead to spurious MN counts or bona
fide MN that are the result of secondary effect(s) stemming
from excessive toxicity. Fluorescence-based assessment of
mitochondrial membrane potential is one example.
While the EMA/SYTOX sequential staining strategy works
well with several mammalian cell lines, its compatibility with
primary cells is less clear. Microscopically scored MN assays
can include cytochalasin B to ensure interrogated cells have
gone through one mitosis. This especially benefits assays that
utilise primary cells such as human lymphocytes since only
a relatively minor portion of the cell population undergoes
division upon mitogen stimulation. Since the flow cytometric
assay employs a lysing technique that does not distinguish
between once-divided and quiescent cells, one would expect
assay sensitivity to be negatively affected. To relate to this
concern, two lines of investigation should be pursued as
follows: (i) addition of an additional fluorescent reagent that
labels chromatin from once-divided cells that is compatible
with EMA and SYTOX Green and (ii) the use of parallel
cultures that could be used to derive a cell division statistic. In
either case, for instance, as described by Schreiber et al. (55),
one can use this information to normalise flow-based MN
counts to the proportion of cells that divided in culture. Success
with either approach could have important implications, as it
may allow one to extend the so-called in vitro MN scoring
approach to human biomonitoring applications that are directed
at assessing in vivo exposure situations.
Finally, the bulk of work with flow cytometric MNvit assays
has been in the areas of assay development, basic research and
early hazard identification screening. With recent acceptance of
OECD guidelines for the MNvit, there will be a natural desire
to transition flow-based MN analyses from a basic research tool
to one that can support the registration of new chemicals. This
effort would obviously benefit from additional studies that
further characterise sensitivity and specificity of flow-based
approaches, as well as assessments of inter- and intra-
Flow cytometric analysis of MNvit
laboratory reproducibility. Data that addresses these concerns
have been published with encouraging results (46,50), but wide
adoption as a regulatory assay will require further evaluations
with a broader variety of chemicals. Once assay validity,
significant MN responses, preferred cytotoxicity assessments
and MOA information are more clearly defined, we believe the
assay could be used in a number of areas, including the
regulatory environment. As with other assays being developed
for broad dissemination and regulatory use, especially those
that are endowed with high information content, a task for the
developers and early adopters of the system will include
education. This phase of work, in conjunction with continued
assay validation efforts, is important so that the wealth of
data generated by flow-based MN assays is interpreted
appropriately.
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Acknowledgements
The authors thank Michael Fenech for providing the opportunity to contribute
to this special issue of Mutagenesis.
Conflict of interest statement: S.A., S.B. and S.D. are employed by Litron
Laboratories, a company that holds patents related to flow cytometry-based
micronucleus scoring. Litron sells kits based on this technology that are
commercially known as In Vitro MicroFlow Kits.
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