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Buccal micronucleus cytome assay


Philip Thomas1, Nina Holland2, Claudia Bolognesi3, Micheline Kirsch-Volders4, Stefano Bonassi5, Errol Zeiger6,
Siegfried Knasmueller7 & Michael Fenech1
1CSIRO Human Nutrition, Adelaide, South Australia. 2School of Public Health, University of California, Berkeley, CA, USA. 3Unit of Environmental Carcinogenesis,
National Cancer Research Institute, Genoa, Italy. 4Laboratory for Cell Genetics, Vrije Universiteit, Brussels, Belgium. 5Unit of Molecular Epidemiology, National Cancer
Research Institute, Genoa, Italy. 6Errol Zeiger Consulting, Chapel Hill, NC, USA. 7Institute of Cancer Research, Inner Medicine I, Medical University Vienna, Austria.
Correspondence should be addressed to P.T. (philip.thomas@csiro.au) or M.F. (michael.fenech@csiro.au).

Published online 7 May 2009; doi:10.1038/nprot.2009.53

The Buccal Micronucleus Cytome (BMCyt) assay is a minimally invasive method for studying DNA damage, chromosomal instability,
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

cell death and the regenerative potential of human buccal mucosal tissue. This method is increasingly used in molecular
epidemiological studies for investigating the impact of nutrition, lifestyle factors, genotoxin exposure and genotype on DNA damage,
chromosome malsegregation and cell death. The biomarkers measured in this assay have been associated with increased risk of
accelerated ageing, cancer and neurodegenerative diseases. This protocol describes one of the current established methods for
buccal cell collection using a small-headed toothbrush, the generation of a single-cell suspension, slide preparation using
cytocentrifugation, fixation and staining using Feulgen and Light Green for both bright field and fluorescence microscopic analysis.
The scoring criteria for micronuclei and other nuclear anomalies are also described in detail. The protocol in its current form takes
approximately 4 h to complete from the time of buccal cell collection to the generation of stained slides for microscopic analysis.

INTRODUCTION
The regenerative capacity of tissues and organs in the body is and/or quids, medical treatments, such as radiotherapy as
fundamental to healthy ageing. Regeneration is dependent on the well as occupational exposure to potentially mutagenic and/or
number and division rate of the proliferating (basal) cells, their carcinogenic chemicals, and for studies of chemoprevention of
genomic stability and their propensity for cell death1,2. These events cancer13,18–20. With regard to exposure to radiation it has been
can be studied in the buccal mucosa (BM), which is an easily shown by Moore et al.21 that the BMCyt system can detect a 16-fold
accessible tissue for sampling cells in a minimally invasive manner increase in micronucleus (MN) frequency in oral cancer patients
and does not cause undue stress to study subjects3–13. This method after completion of treatment with photons. The BM also has the
is increasingly being used in molecular epidemiological studies to potential to be utilized to identify inherited genomic instability
investigate the impact of nutrition, lifestyle factors, genotoxin such as Bloom’s Syndrome22.
exposure and genotype on DNA damage and cell death3–8. The In addition, the BMCyt protocol has been used to measure
use of cells from the BM (Figs. 1–3) provides a unique opportunity distinct differences between the cytome profiles associated with
to study the regenerative capacity of the
epithelial tissue, which is of ectodermal
Oral cavity
origin in humans. The assay has been used Stratum
successfully both in our laboratory and in corneum

others to study DNA damage as measured Stratum


granulosum
by micronuclei (MNi) or by the use of
fluorescent probes to detect aneuploidy,
chromosomal breaks and changes in telo- Stratum
spinosum
mere length9–13. The proportion of basal
cells and cells undergoing cell death in BM Stratum
germinativum
is an indication of the regenerative capacity
of this tissue.
Connective tissue
The BM provides a barrier to potential
carcinogens that can be metabolized to
generate potential reactive products14,15. Basal cell Karyorrhectic cell
As up to 90% of all cancers appear to be Binucleated cell Condensed chromatin cell
epithelial in origin, the BM could be used to Basal cell with MN

monitor early genotoxic events as a result of Differentiated cell with NBUD


Differentiated cell Pyknotic cell
potential carcinogens entering the body
through ingestion or inhalation13,16,17. Differentiated cell with MN Karyolytic cell
Exfoliated buccal cells have been used
non-invasively to successfully show the Figure 1 | Diagrammatic representation of a cross section of normal buccal mucosa. The mucosa of
genotoxic effects of lifestyle factors such as healthy individuals illustrating the different cell layers and possible spatial relationships of the various
tobacco smoking, chewing of betel nuts cell types are shown.

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normal ageing relative to that for premature Oral cavity

ageing clinical outcomes such as Down’s


syndrome and Alzheimer’s disease. These
studies highlight the potential diagnostic Karyolytic
cells
value of the cytome approach for determin- Differentiated
ing genome instability events and measur- cell and dying
cell layer
Condensed Karyorrhectic
ing regenerative potential9,23. chromatin cell
Pyknotic
cell cell
7–21
The BM is a stratified squamous epithe-
days
lium consisting of four distinct layers Binucleated Differentiated cell Differentiated cell Differentiated cell
1,2,24,25 cell with MN with NBUD for cells
(Fig. 1) . The various cell types,
to
nuclear anomalies and possible spatial
interrelationships among the various cell migrate
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

types in the BM, which are observed and Binucleated Basal Basal Normal
from
cell cell
scored in a BMCyt assay, are shown dia- Basal cell
with MN with NBUD
Basal cell basal

grammatically in Figures 1–3 (refs. 9,13). Basal cell layer


layer
The stratum corneum, or the keratinized cell Basal cell
to

layer, lines the oral cavity comprising cells oral


that are constantly being shed as a result of cavity
Stem cell
wear and tear of the surface tissue. Below Daughter stem cell

Connective tissue
this layer, lies the stratum granulosum, or
the granular cell layer, and the stratum Figure 2 | Sequential origins and spatio-temporal sequence of the various cell types in the BMCyt assay.
spinosum, or the prickle cell layer, contain- Shown is the time frame of events from a stem cell to a basal cell and subsequent differentiation and cell
ing populations of differentiated, apoptotic death and the different types of genetic damage that can occur.
and necrotic cells. Beneath these layers are
the rete pegs or stratum germinativum,
containing actively dividing basal cells and basal stem cells, which used to measure aneuploidy by determining the frequency of nuclei
produce progeny that differentiate and maintain the profile, with abnormal chromosome number10. Tandem probes have been
structure and integrity of the BM1,13,26,27. Figure 2 shows the successfully applied to measure chromosome breaks in specific
sequential spatio–temporal origins of the various cell types and important regions of the genome10,31,32. The use of such molecular
the time frame from a daughter basal cell originating in the basal probes to gain further information on the genomic damage in
layer to its eventual differentiation, migration and exfoliation at the buccal cells is shown in Figure 4.
BM surface layer. The time frame for cellular migration from the The methodology and concepts described in this protocol may be
basal layer to the keratinized surface layer is thought to range from applied to other types of exfoliated cells such as those of the
7 to 21 d, although experimental data investigating such migration bladder, nose and cervix but the morphological characteristics,
rates are limited2,26,28.
In this assay cells derived from the BM are harvested from the
inside of a patient’s mouth using a small-headed toothbrush. The Buccal Cytome Model—2008
cells are washed to remove the debris and bacteria, and a single-cell
suspension is prepared and applied to a clean slide using a Normal genome
cytocentrifuge. The cells are stained with Feulgen and Light Basal cell
Green stain allowing both bright field and permanent fluorescence Normal
analysis that can be undertaken microscopically. Healthy normal Chromosome
breakage or loss
cells can then be distinguished from those considered abnormal
Apoptotic cell death
based on the nuclear-to-cytoplasmic ratio and nuclear morphology (morphogenetic Gene amplification
and texture (Fig. 3). or induced by
Micronucleated
genome damage)
The BMCyt assay has been used to measure biomarkers of DNA
Cytokinesis
damage (micronuclei and/or nuclear buds), cytokinetic defects defect
(binucleated cells) and proliferative potential (basal cell frequency)
and/or cell death (condensed chromatin, karyorrhexis, pyknotic Necrotic
Cell death? Nuclear bud
and karyolytic cells). The following BMCyt protocol describes one
of the current established methods for buccal cell collection, slide Condensed
chromatin
preparation, cellular and nuclear staining and scoring criteria. The
various cell types and aberrations that are scored in the BMCyt
assay are illustrated in Figure 3. The protocol can also make use of Karyolysis Binucleated
Karyorrhexis Pyknosis
molecular probes for DNA adduct, aneuploidy and chromosome
break measures within the nuclei of buccal cells29–31. It is possible to Figure 3 | Diagrammatic representation of the various cell types scored in
use antibodies to measure DNA adducts such as those induced by the BMCyt assay. Analyses of various cell types is done on the basis of the
polycyclic aromatic hydrocarbons in the nuclei of buccal cells29. scheme proposed by Tolbert et al.27 and recent observations in studies of the
Furthermore, chromosome-specific centromeric probes have been effect of ageing9.

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sampling and scoring methods are neither properly described nor


Quantify using image cytometry
standardized for cells from these tissues. of spots by measuring area
Other methods currently available for the BMCyt use tongue Fluorescent antibody
and optical density of spots

depressors, tooth picks and metal spatulas to collect buccal cells; to DNA adducts
Monosomic
however, the number of cells collected is usually less than those Normal
collected using a toothbrush33–36. Buccal smears can be applied diploid
Score
directly to a clean slide but invariably the cells are clumped and Chromosome specific visually or
by
contain bacteria and debris that can hamper accurate cytological centromeric probes automated
analysis upon staining (P.T. unpublished observation). Several spot
counting
different staining techniques for buccal cells have been
reported9,33,37,38. However, DNA-specific stains such as acridine Tandem probe Single break trisomic
labelling
orange or propidium iodide are recommended in comparison with
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

Romanowsky stains such as Giemsa or Leishmann’s, as the former Score


visually or
stains minimize the possibility of scoring false positives for DNA by
damage events such as micronuclei, which often occur with automated
spot
Romanowsky stains13. No counting
breaks

Experimental design Two breaks

When considering the study design for biomonitoring using cells of


Figure 4 | Molecular probe methods for measuring DNA adducts, aneuploidy
the BM, it is most important to outline a clear hypothesis and
and chromosome breaks.
ensure that the study is sufficiently powered to measure a biologi-
cally meaningful effect size. The important considerations for study
design are detailed below. and to avoid continual erosion in a single region of the BM. This is
performed on the inside of both cheeks using a different brush
Important demographic and exposure variables. It is important (for sampling left and right areas of the mouth) to maximize cell
to establish normal range values for the different buccal cytome sampling and to eliminate any unknown biases that may be caused
biomarkers from healthy control subjects that have not been by sampling one cheek only. It is important to keep the sampling
exposed to abnormal doses of genotoxins and cytotoxins. This method constant as distribution of cell types could vary slightly
allows an investigator to identify key variables affecting the depending on the method used. It is important to note that
observed frequency of the biomarkers such as age, gender, vitamin repeated vigorous brushing of the same area can lead to increased
B status (particularly folate and vitamin B12), genotype and collection of cells from the less differentiated basal layer23. Table 2
smoking status. Detailed information on genotoxin exposure shows the effect of repeated sampling on the results of the BMCyt
(e.g., type of toxin, duration of exposure, commencing date of assay. The results in Table 2 show that the frequency of basal
exposure relative to sampling date of buccal cells) is also required in cells and karyorrhectic cells tends to increase with repeated sam-
order to achieve a meaningful interpretation of data. This may be pling (P trend o 0.001 and P ¼ 0.048 respectively), but no change
useful in identifying a threshold dose and the time frame of is observed for other biomarkers in the BMCyt assay.
exposure required for a particular genotoxin to produce a measure-
able change in BMCyt biomarkers. It is important in case–control Transport and storage. In some investigations buccal cells may
studies that the groups are matched properly for these variables. For have to be collected from a distant site. This could result in a delay
further information on the use of controls and study design in of several days before samples are delivered to the laboratory for
biomonitoring studies the reader is referred to Albertini et al.39.
Table 1 provides a list of important variables to be recorded when
using the BMCyt assay in epidemiological studies. TABLE 1 | Important variables to record when using the BMCyt assay in
epidemiological studies.
Sampling time in acute and chronic exposure. As the buccal cells  Date of Birth
turn over every 7–21 d, it is theoretically possible to observe the  Gender
genotoxic effects of an acute exposure approximately 7–21 d  Body mass index
later13,21,40. However, additional sampling at later time points  Smoking status and history
may be required depending on the extent to which cell division  Alcohol consumption status and history
has been inhibited by the genotoxin. Ideally, repeat sampling, at  History of cancer, cardiovascular disease and neurodegenerative
least once every 7 d after acute exposure, should be performed for disease
28 d or more so that the kinetics and extent of biomarker induction  Medication or recent illness (disease history)
can be thoroughly investigated. In the case of chronic exposure  Inherited mutations that predispose to degenerative diseases
(e.g., due to habitual diet or alcohol consumption or smoking) it is listed above as well as accelerated ageing syndromes (e.g., BRCA1
recommended that multiple samples are taken at least once every 3 and ATM mutations)
months to take into account seasonal variation.  Dietary habits measured using a validated food frequency
questionnaire
Uniformity of sampling. A circular expanding motion is used  Exposure to chemical carcinogens and radiation e.g., X-ray
with toothbrush sampling to enhance sampling over a greater area  Genotype

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TABLE 2 | Changes in the ratios of buccal cell types (mean, standard deviation and P values) following sequential buccal cell sampling.
Micronuclei Nuclear buds Basal cells Binucleated Condensed Karyorrhectic Pyknotic Karyolytic
per 1,000 per 1,000 per 1,000 cells per 1,000 chromatin cells cells per 1,000 cells per 1,000 cells per 1,000
Sequential samples cells cells cells cells per 1,000 cells cells cells cells
Sample 1 (0 min) 0.50 ± 0.57 0.50 ± 0.57 28.75 ± 12.79 9.25 ± 4.78 25.75 ± 15.59 20.3 ± 13.1 0.50 ± 0.57 156.5 ± 118.4
Sample 2 (90 mins) 0.50 ± 0.57 0.25 ± 0.50 51.5 ± 7.9a 12.0 ± 4.16 22.5 ± 11.47 29.0 ± 16.79 0.7 ± 1.5 111.8 ± 49.17
Sample 3 (270 mins) 0.0 ± 0.0 0.25 ± 0.50 59.5 ± 6.75a 9.75 ± 3.78 30.0 ± 5.1 31.0 ± 18.31 0.0 ± 0.0 135.5 ± 49.17
Sample 4 (360 mins) 0.50 ± 0.57 0.25 ± 0.50 65.5 ± 5.19a 11.5 ± 5.82 21.25 ± 2.06 29.3 ± 19.21 0.0 ± 0.0 102.0 ± 32.30
Sample 5 (450 mins) 0.25 ± 0.5 0.0 ± 0.0 84.75 ± 6.0a,b 9.0 ± 1.83 31.0 ± 13.29 34.3 ± 23.16 0.0 ± 0.0 129.3 ± 25.25
ANOVA P values 0.6272 0.6363 o0.001 0.7968 0.6749 0.2320 0.5032 0.6696
P value for linear trend 0.5744 0.1742 o0.001 0.8841 0.6146 0.0489 0.1672 0.4729
Buccal cells were collected from volunteers (n ¼ 4, 2 male, 2 female, age range 40–50 yrs) over five different time points, 0, 90, 270, 360 and 450 mins. Buccal cells were collected as outlined in the cell sampling
and preparation part of this manuscript. Slides were stained as outlined in the Feulgen staining steps of this protocol. Slides were classified and scored as outlined under the scoring criteria detailed in this protocol.
Statistical analysis for mean, standard deviation and one-way ANOVA and linear P trends were performed using Graphpad PRISM (Graphpad inc., San Diego, CA). Significance was accepted at P o 0.05. aDenotes a
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

significant difference from sampling time at 0 min. bDenotes a significant difference from time 0, 90, 270 and 360 mins.

analysis, which may cause sample deterioration. In this case, a stains have been shown to increase the number of false positives as
reliable method for preserving samples before delivery is required they positively stain keratin bodies that are often mistaken for
and an appropriate procedure for this purpose is described in the micronuclei and are therefore not appropriate for this type of
Buccal Cell Collection option B in the PROCEDURE (Buccal cell analysis41. For these reasons, it is advisable to avoid Romanowsky
collection). stains in favor of DNA-specific fluorescent-based stains such as
propidium iodide, DAPI, Feulgen, Hoechst 33258 or Acridine
Effects of filtration. We have performed experiments to investi- Orange42. It is recommended that Feulgen be used because perma-
gate whether the filtration process in the preparation of the single- nent slides can be obtained that can be viewed under both trans-
cell suspension has adverse effects on cell population ratios. The mitted and/or fluorescent light conditions.
larger cell aggregates that are filtered out show the same cellular
population ratios as the eventual single-cell suspension used in the Criteria for identifying and scoring cell types in the buccal
slide preparation for analysis. This means that removal of cell micronucleus cytome assay. The scoring criteria for the various
clumps by filtration, which facilitates slide scoring, does not select distinct cell types and nuclear anomalies in the BMCyt assay are
against a particular cell type, which would otherwise have had mainly based on those originally described by Tolbert et al.27. These
significant effects on both data analysis and interpretation. criteria are intended for classifying buccal cells into categories that
distinguish between ‘normal’ cells and cells that are considered
Cell fixation and nuclear staining. It is possible to use alternative ‘abnormal’ on the basis of cytological and nuclear features, which
fixative solutions such as methanol:glacial acetic acid (3:1) or are indicative of DNA damage, cytokinetic failure or cell death.
80% methanol, but the staining of both cytoplasm and nuclei These criteria are summarized in Table 3 and photomicrographs of
may be altered slightly relative to the use of ethanol:glacial acetic cell types and nuclear anomalies are shown in Figure 5.
acid (3:1). One of the unique features of the Feulgen staining A more detailed description of the scoring criteria for the BMCyt
technique used in this protocol is that the DNA material appears as assay cell types is outlined below.
bright red in color when viewed under fluorescence with a far-red Normal basal cells have a larger nucleus-to-cytoplasm ratio than
filter (emission wavelength range of 580–620 nm). This is impor- the differentiated buccal cells (Fig. 5a). Basal cells have a uniformly
tant because cells identified as containing MNi or other anomalies stained nucleus and are smaller in size and more oval in shape when
(e.g., nuclear buds) on the bright field can be confirmed as being compared to the more angular and flat differentiated buccal cells.
positive by examining the cells under fluorescence. Furthermore, No DNA-containing structures apart from the nucleus are observed
the nuclear texture, which is essential in classifying condensed in these cells. The cytoplasm is typically stained a darker shade of
chromatin and karyorrhectic cells, is usually easier to discern using green with Light Green compared to the differentiated cells.
fluorescence microscopy. This minimizes the incidence of false Normal ‘differentiated’ cells (Fig. 5b) have a uniformly stained
positives or false negatives, thereby giving a more accurate assess- nucleus, which is oval or round in shape. They are distinguished
ment of DNA damage and nuclear anomaly events. from basal cells by their larger size and by a smaller nucleus-to-
cytoplasm ratio. No other DNA-containing structures apart from
Staining artifacts. Earlier studies have shown that false-positive the nucleus are observed in these cells. These cells are considered to
results in the MN frequency can be obtained as a result of using be terminally differentiated relative to basal cells, as no mitotic cells
Romanowsky-type stains such as Giemsa, May-Grunwald Giemsa are observed in this population2.
and/or Leishmann’s, which leads to inaccurate assessment of DNA Cells with micronuclei (Fig. 5c,d) are characterized by the
damage13,41. In a study investigating MN frequency in relation to presence of both a main nucleus and one or more smaller nuclear
the staining techniques in the BM of smokers against non-smokers, structures called micronuclei (MNi). The micronuclei are round or
a 4- to 5-fold increase in MN frequency in smokers was found using oval in shape and their diameter should range between 1/3 and 1/16
Romanowsky stains, which are not DNA-specific41,42. However, of the main nucleus. MNi have the same staining intensity and
when a specific DNA fluorescent dye (e.g., 4, 6-diamidino-2- texture as the main nucleus. Most cells with MNi will contain only
phenylindole (DAPI), Feulgen or Acridine orange) was used there one MN but it is possible to find cells with two or more MNi.
were no significant differences between these groups41. Romanowsky Baseline frequencies for micronucleated cells in the BM are usually

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TABLE 3 | Criteria for classification of BMCyt cell cytome assay cell types based on morphological features of cells stained with Feulgen/Light Green
with reference to photomicrographs shown in Figure 5a–j.
Buccal cell type Morphological features Figure
Basal Large nucleus: cytoplasm ratio relative to differentiated cell 5a
Smaller and more oval than differentiated cells
Uniformly stained nucleus
Darker green cytoplasm relative to differentiated cell when viewed
under transmitted light
Differentiated Smaller nuclear: cytoplasmic ratio 5b
More angular and flatter than basal cells
Uniformly stained round nucleus
Micronucleated Contains both main nucleus and micronucleus 5c,d
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

Micronuclei are round or oval with similar stain intensity as main nucleus
Micronuclei usually have 1/3–1/16 diameter of main nucleus
Micronuclei must be located in cellular cytoplasm
Scored in basal and differentiated cells only
Nuclear bud Main nucleus has a sharp constriction forming a bud 5e
Bud is attached to main nucleus
Bud has similar staining intensity as main nucleus
Bud diameter can be quarter to half nuclear diameter
Binucleated Cells contain two main nuclei 5f
Nuclei are of similar size and staining intensity
Condensed chromatin Nucleus shows areas of aggregated chromatin 5g
Distinct areas of nucleus are more intensely stained
Nucleus exhibits striated pattern
Karyorrhectic Nucleus has extensive aggregated chromatin 5h
Nuclear fragmentation may be evident
Pyknotic Cell has small shrunken nucleus 5i
Nucleus is uniformly and intensely stained
Nucleus diameter is 1/3–2/3 diameter of normal nucleus
Karyolytic Nucleus is depleted of DNA 5j
Nucleus is not stained by Feulgen

within the 0.5–2.5 MNi/1,000 cells range13. Cells with multiple MNi other and usually have the same morphology as that observed in
are rare in healthy subjects but become more common in indivi- normal cells. The significance of these cells is unknown, but they are
duals exposed to radiation or other genotoxic agents. The nuclei in probably indicative of failed cytokinesis following the last nuclear
micronucleated cells have the morphology of nuclei in normal cells. division in the basal cell layer. It has recently been shown that
The MNi must be located within the cytoplasm of the cells. The chromosomal non-disjunction occurs with a higher frequency in
presence of MNi is indicative of chromosome loss or fragmentation binucleated cells that fail to complete cytokinesis, rather than in
occurring during earlier nuclear division13,43. MNi are scored only cells that have completed cytokinesis48. This mechanism identified
in differentiated cells with uniformly stained nuclei. It is possible to recently is thought to be a cytokinesis checkpoint for aneuploid
score MNi in basal cells, but this is impractical owing to the low binucleated cells48. The binucleate:mononucleate cell ratio may
frequency of this cell type. Cells, which are pyknotic (i.e., shrunken therefore prove to be an important biomarker for identifying
nuclei), and have condensed chromatin or karyorrhectic nuclei (see individuals with a cytokinesis failure caused by higher-than-normal
below), are not scored for MNi. rates of aneuploidy, such as that observed in Down’s syndrome9,10.
Cells with nuclear buds (Fig. 5e) contain nuclei with an apparent Buccal cells with condensed chromatin (Fig. 5g) show a roughly
sharp constriction at one end of the nucleus suggestive of a budding striated nuclear pattern in which the aggregated chromatin is
process, i.e., elimination of nuclear material by budding. In the intensely stained. Similar nuclear morphologies have also been
original Tolbert et al. publication27 these were referred to as ‘broken shown in other cell types49,50. In these cells it is apparent that
egg’ cells. The nuclear bud (NBUD) and the nucleus are usually in chromatin is aggregating in some regions of the nucleus while being
very close proximity and appear to be attached to each other. The lost in other areas. When chromatin aggregation is extensive the
nuclear bud has the same morphology and staining properties as nucleus may appear to be fragmenting51. These cells may be
the nucleus; however, its diameter may range from a half to a undergoing early stages of apoptosis, although this has not been
quarter of that of the main nucleus. The mechanism leading to shown conclusively. These cells as well as karyorrhectic cells
nuclear bud formation is not known but it may be related to the invariably result in fragmented nuclei, leading to eventual disin-
elimination of amplified DNA or DNA repair44–47. tegration, and sometimes appear to contain bodies similar to MNi,
Binucleated cells (Fig. 5f) are cells containing two main nuclei but these are not scored as MNi in the assay as their origin cannot
instead of one. The nuclei are usually very close and may touch each be accurately determined.

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Figure 5 | Images of the different cell types c


stained using Feulgen and Light Green scored in
a b d e
the BMCyt assay viewed by transmitted light or
under fluorescence with a far red filter; (a) basal
cell; (b) differentiated cell; (c) early differentiated
cell with micronucleus (arrow); (d) late
differentiated cell with micronucleus (arrow);
(e) differentiated cell with nuclear bud (arrow);
(f) binucleated cell; (g) condensed chromatin cell;
(h) karyorrhectic cell; (i) pyknotic cell;
(j) Karyolytic cell. Upper panels light microscopy,
lower panels fluorescence microscopy. All images
were taken at 1,000 magnification. g j
f h i
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

Karyorrhectic cells (Fig. 5h) have nuclei


that are characterized by more extensive
nuclear chromatin aggregation relative to
condensed chromatin cells. They have a
densely speckled nuclear pattern indicative
of nuclear fragmentation leading to the
eventual disintegration of the nucleus51,52.
These cells may be undergoing a late stage
of apoptosis, but this has not been conclu-
From each slide, the following information should therefore
sively proven. These cells should not be scored for MNi in the assay.
be recorded:
Pyknotic cells (Fig. 5i) are characterized by a small shrunken
1. The number of basal and differentiated cells per 1,000
nucleus, with a high density of nuclear material that is uniformly
cells scored.
but intensely stained51,52. The nuclear diameter is usually one- to
2. The number of pyknotic, condensed chromatin, karyorrhectic
two-thirds of a nucleus in normal differentiated cells. The biological
and karyolytic cells per 1,000 cells.
significance of the pyknotic cells and the mechanism leading to
3. The number of binucleates per 1,000 cells scored.
their formation are unknown, but it is thought that these cells may
4. The number of MNi and NBUDs in at least 2,000 differentiated
be undergoing a unique form of cell death; however, the precise
cells.
mechanism remains unknown. They may represent an alternative
Preferably, cells are scored using both bright field and fluores-
mechanism of nuclear disintegration that is distinct from the
cence microscopy. Cells containing MNi or NBUDS on bright
process leading to the condensed chromatin and karyorrhectic
field should be confirmed as being positive for these biomarkers
cell death stages13,53.
by examining the cell under fluorescence. The incidence of false
Karyolytic cells (Fig. 5j) are cells in which the nucleus is
positives can be minimized as DNA materials such as nuclei and
completely depleted of DNA and is apparent as a ghost-like
MNi fluoresce brightly red when viewed under fluorescence
image that has no Feulgen staining51,52. Therefore, these cells
with a far-red filter (emission, wavelength range of 580–620
appear to have no nucleus and represent a very late stage in the
nm). Cell inclusions or debris that do not contain DNA are not
cell death process.
expected to fluoresce under these conditions.
5. The frequencies of the various cell types in the assay are
Scoring method. Slides should be coded before scoring by a
represented either as the number of cells in a 1,000 cells or as
person not involved in the experiment so that the person who
a percentage. For example, should 187 basal cells be scored
scores the slides is not aware of the treatment conditions, individual
in 1,000 cells, then the frequency may be reported either as
or groups to which the cells on the slides belong. Slides are best
187% or 18.7%.
examined at 1,000 magnification using a good-quality bright field
and/or fluorescence microscope (e.g., Leica DMLB, Nikon Eclipse
E 600). The BMCyt assay is amenable to automated scoring by SCORING PROCEDURE FLOW CHART

image cytometry, but these systems, which could potentially


improve applicability in population studies, have yet to be devel- STEP 1 STEP 2

oped and validated54. Score 1,000 cells to Score 2,000


determine the frequency of
The optimal way to score slides in the BMCyt assay is to first these cell types:
differentiated
cells for
determine the frequency of all the various cell types in a minimum presence of MNi
• Basal and NBUD
of 1,000 cells. Following this step, the frequency of DNA damage • Differentiated
biomarkers (MNi and NBUDs) is scored in a minimum of 2,000 • Binucleated
• Condensed chromatin
differentiated cells. A flow diagram of the scoring procedure is • Karyorrhectic
• Pyknotic
shown in Figure 6. The frequency of MNi and NBUDS could be • Karyolytic
scored in basal cells, but this is impractical because of the very low
frequency of these cells. Figure 6 | Flow diagram describing scoring method.

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Information that should be included on a score sheet for the measuring the frequency of micronucleated cells is more stable
BMCyt assay is shown below: and should be preferred.
1. Name of the person scoring the slides. 2. The frequency of MN and all the other endpoints must be
2. Code number of each slide. expressed as the number of events per 1,000 cells, independent
3. Total number of buccal cells scored. of the number of cells scored.
4. The number of basal cells and differentiated cells per 1,000 3. The size of the study groups must be evaluated in advance
cells scored. according to the results of statistical power calculations. Unless
5. The number of pyknotic, condensed chromatin, karyorrhectic the presence of NBUDs or other endpoints is a specific target of
and karyolytic cells per 1,000 cells. the study, power estimates should be based on MN frequency.
6. The number of binucleated cells per 1,000 cells scored. 4. Given the common report of a skewed distribution of damaged
7. The number of MNi and NBUDs in 2,000 differentiated cells. cells, the normal distribution of all biomarkers should be tested
before statistical analysis. In the case of significant departure
This information is then used to calculate (a) the frequency of the
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

from normality, necessary data transformation should be


various cell types per 1,000 cells and (b) the frequency of MNi and
applied before analysis.
NBUDs in differentiated cells per 1,000 cells. A typical score sheet is
5. The presence of statistically significant differences in the occur-
shown in Supplementary Table 1 online.
rence of damaged cells between the study populations should
Statistical methods and power analysis always be tested with parametric or non-parametric univariate
All international guidelines for genotoxicity tests recommend analyses. A threshold value of P o 0.05 is generally recom-
evaluating the biological significance of a result followed by the mended.
use of statistics, when appropriate. The biological significance of an 6. The control for a confounding variable and the evaluation of
effect should be critically assessed, considering its size, dose effect modifiers should be performed using multivariate statis-
response, reproducibility and plausibility. As regards the statistical tical modeling. Given the count nature of these biomarkers,
analysis of these buccal cytome biomarkers, a few basic issues Poisson (in absence of overdispersion), negative binomial or
should always be considered and properly addressed. lognormal models can be applied. The point estimate of effect is
1. The frequency of micronucleated cells should be used as the generally a mean ratio, and an appropriate confidence interval
main endpoint; however, the scoring of the number of micro- (usually 95%) should be provided.
nuclei may also be used as a separate measure to show whether 7. The presence of several possible endpoints poses a problem of
cells with multiple micronuclei were present. Given the rarity of multiple comparisons. If the study is oriented toward hypothesis
cells bearing more than a single micronucleus there are no testing an appropriate correction for multiple comparisons
substantial differences between the two approaches, although should be considered.

MATERIALS
REAGENTS EQUIPMENT
. Tris-HCl (Sigma, cat. no. T-3253) . Small-headed toothbrushes (2-cm head length) (Supply SA, cat. no.
. Ethylenediaminetetraacetic acid tetra (EDTA) sodium salt 85300012)
(Sigma, cat. no. E5391) . 30 ml yellow-topped polystyrene containers (Sarstedt, cat. no. 60.9922.918)
. Sodium chloride (Sigma, cat. no. S5886) . 10 ml graduated sterile pipettes (Falcon, cat. no. 7551)
. Isoton II (Coulter Electronics, cat. no. 8546719) . Milli-Q water purification system (Millipore Milli-Q, Adelab Scientific)
. Sodium hypochlorite Solution (125 ml per liter) (Chemwell Products, . Swinnex filter holders 25 mm (Millipore, cat. no. MILSX0002500)
cat. no. 1791) . Nylon net filters 100 mm (Millipore, cat. no. MILNYH02500)
. Dimethyl sulfoxide (DMSO), hybrimax, sterile–filtered (Sigma, cat. no. . Cell counter (e.g., Coulter Electronics model ZB1). A hemocytometer
D2650) can be used if an electronic cell counter is not available.
. Ethanol (Ajax Finechem, cat. no. 214-2.5L) . TV-10 polystyrene tubes (Sarstedt, cat. no. 60.9921.829)
. Glacial acetic acid (Ajax Finechem, cat. no. A1-2.5L) ! CAUTION Acetic acid . Sterile plugged Pasteur pipettes 900 (22–23 cm) (Chase, cat. no. 93P)
is corrosive, a respiratory irritant and can cause serious burns. Fixative . Syringes 10 ml (Crown Scientific, cat. no. SS+10S)
should be prepared in a fume hood or similar extraction cabinet and the . Needles 18G (Crown Scientific, cat. no. 2525RA)
following personal protection used: Tyvek gown, double nitrile gloves, . Counting vials 15 ml (Johns Diluent vials, cat. no. DSV002)
P2 dust mask and safety glasses. . Cytocentrifuge (e.g., Shandon Cytocentrifuge, Thermo Electron Corporation)
. 50% Ethanol made up with milli-Q deionized water (18.2 O resistivity) . Cytocentrifuge cups—supplied with instrument from Thermo Electron
. 20% Ethanol made up with milli-Q deionized water (18.2 O resistivity) Corporation
. 5 M Hydrochloric acid (HCl) (Merck, cat. no. 101255Y) ! CAUTION HCl is . Filter cards—Shandon 300  100 , thick, white, boxes of 200 (Thermo Electron
corrosive, a respiratory irritant and can cause serious burns. The acid Corporation)
should be handled in a fume hood or similar extraction cabinet and the . Microscope slides, frosted end, 76 mm  26 mm (300  100 ), 1 mm thick
following personal protection used: Tyvek gown, double nitrile gloves, P2 (HD Scientific MZO2 110, HD Scientific)—wiped with alcohol and allowed
dust mask and safety glasses. to dry before use.
. Schiff’s reagent (Sigma, cat. no. S-5133) ! CAUTION Schiff’s reagent is a skin, . Coplin jars, unit holds 5 single 300  100 (75 mm  25 mm) slides vertically
eye and respiratory irritant and should be handled in a fume hood or similar or 10 slides back-to-back. Screw cap is white linerless polypropylene, which
extraction cabinet and the following personal protection used: Tyvek gown, reduces solvent evaporation (Biolab, cat. no. 355)
double nitrile gloves, P2 dust mask and safety glasses. . Polypropylene slide staining rack, accommodates up to 20 slides with
. 0.2% (wt/vol) aqueous Light Green (Gurr’s, cat. no. 06477) removable snap-on handle (70 mm  86 mm  21 mm), (Pro Sci Tech)
. DePex (or DPX) mounting medium (Merck, cat. no. 3197) . Coverslips, no. 1, 22 mm  50 mm (HD Scientific, cat. no. MZ LD2250)
. Polyethylene glycol (Merck, cat. no. 29577) . Hand homogenizer (Wheaton Scientific, 0.1 mm–0.15 mm gauge)

NATURE PROTOCOLS | VOL.4 NO.6 | 2009 | 831


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. Microscope with excellent optics for bright-field and fluorescence (desired concentration)/stock solution (12 M). This results in 83.3 ml of acid
examination of stained slides at 1,000 magnification (e.g., Leica DMLB, being required in our 200 ml working solution. The 5 M HCl solution should be
Leica; Nikon Eclipse 600, Nikon) freshly prepared each time before use. ! CAUTION Add 83.3 ml of acid slowly to
. Slide storage boxes (Kartell, cat. no. 278) 116.7 ml of water and not the reverse. This is important to avoid an exothermic
. Parafilm (Sigma, cat. no. P7793-1EA) reaction resulting in the generation of heat and potential splashing. This
REAGENT SETUP procedure should be performed in a well-ventilated fume hood with appropriate
Buccal cell buffer To prepare 1 liter of buccal buffer, weigh 1.6 g Tris–HCl, safety precautions.
1.2 g EDTA and 37.2 g of sodium chloride, and dissolve in 600 ml of Milli-Q Ethanol/glacial acetic acid fixative Ethanol is mixed with glacial acetic acid in
water. Thoroughly dissolve the salts and adjust the volume to 1,000 ml. Adjust the ratio of 3:1. The fixative should be freshly prepared each time. This
pH to 7.0 and autoclave at 121 1C for 30 min. The buffer can be stored for up to procedure should be undertaken in a well-ventilated fume hood and
3 months in sealed bottles at room temperature (approximately 18–22 1C). personal protection should be used. ! CAUTION Acetic acid is corrosive, a
5 M Hydrochloric acid Schiff’s reagent is commonly used to detect aldehydes, respiratory irritant and can cause serious burns. A fixative should be
producing a red coloration. As aldehydes are not usually found free in nuclei prepared in a fume hood or similar extraction cabinet and the following
they are induced by the use of 5 M HCl, which converts some of the deoxyribose personal protection should be used: Tyvek gown, double nitrile gloves,
in the DNA to aldehydes, which can then be detected by Schiff’s reagent. During P2 dust mask and safety glasses.
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

staining a negative control slide is included, which is not treated with 5 M HCl. Sodium hypochlorite solution 80 ml of 12.5% (vol/vol) sodium hypochlorite
Following treatment with Schiff’s reagent, no nuclear magenta coloration is made up to 1 liter using Milli-Q deionised water (18.2 O resistivity) to give a
should be observed in the negative control slide. This control is included to working solution of 1% (vol/vol). Store the solution for 1 week at room
determine the efficacy of the acid treatment, toward the artifactual generation of temperature (18–22 1C).
aldehydes and the relative degree of positive Schiff’s nuclear staining in the test Light Green cytoplasmic stain 500 ml of Light Green cytoplasmic stain is
slides. In order to determine the concentration of laboratory-sourced HCl, the prepared by dissolving 1 g of Gurr Light Green powder in 450 ml of Milli-Q
molarity is derived from the stated specific gravity, which may vary among water. When dissolved make up to 500 ml and filter through Whatman No. 1
suppliers. The specific gravity of the British Drug House (BDH) HCl that is used filter paper. Store in the dark at room temperature, where it should remain active
in our laboratory is 1.18 g ml1, which is the equivalent of 1,180 g liter1. The for a few years.
molecular weight of HCl is 36.5 g mol1. The molarity can therefore be Saccomanno’s fixative The Saccomanno’s fixative consists of 50%
calculated by dividing the specific gravity by the molecular weight, resulting in (vol/vol) ethanol and 2% (vol/vol) polyethylene glycol diluted in water.
an acidic molarity of 32.3 M (1,180/36.5). However, the HCl acid assay Use 20 ml of the fixative per subject. The fixative can be stored for up to
specifications detailed on the accompanying product sheet indicate a 37% 3 months at 4 1C.
(vol/vol) acidic solution. In order to calculate the true molarity one has to adjust EQUIPMENT SETUP
this percentage value. Our stock acid solution is found to have a molarity of Cytocentrifuge cups must be clean, rinsed six times in distilled or deionized
12 M by multiplying the calculated initial value of 32.3 M by 0.37 (37% vol/vol). water and completely dried before assembly.
In order to prepare a 200 ml working solution of 5 M HCl from our stock Microscope slides should be wiped with alcohol and allowed to dry
solution the following calculation is performed: 200 ml (desired volume)  5 M before use.

PROCEDURE

Buccal cell collection TIMING B10 min
1| Before buccal cell collection, the mouth of the subject should be rinsed twice thoroughly with 100 ml of water to remove
excess debris.
! CAUTION Human samples should be considered as infectious and the appropriate safety precautions should be taken.
The appropriate institutional research ethics committees should approve the studies using human participants.

2| Buccal cell samples can either be collected and processed fresh using option A or be stored and fixed in
Saccomanno’s fixative and processed at a later date using option B.
(A) Collection of buccal samples for fresh-cell analysis
(i) For each subject prepare two 30-ml yellow-topped containers labeled LC (left cheek) and RC (right cheek), each
containing 10 ml of buccal cell buffer.
(ii) Gently but firmly rotate a small-headed toothbrush (2-cm head length) 10 times against the inside of the cheek wall in
a circular motion starting from the middle and gradually increasing in circumference to produce an outward spiral effect.
(iii) Place the head of each brush into its respective buffer container and rotate repeatedly such that the cells are
dislodged and released into the buffer, thereby producing a cloudy suspension of buccal cells in the buccal cell buffer.
The brushes are then discarded following sampling and are not reused. Store the cell suspensions in a buffer at 4 1C.
(B) Collection of buccal samples for fixed-cell analysis
(i) For each participant prepare two 30-ml yellow-topped containers labeled LC (left cheek) and RC (right cheek),
each containing 10 ml of Saccomanno’s fixative.
(ii) Gently but firmly rotate a small-headed toothbrush (2-cm head length) 10 times against the inside of the cheek wall in a
circular motion starting from the middle and gradually increasing in circumference to produce an outward spiral effect.
Use a different toothbrush for each cheek.
! CAUTION It is important to remember not to revisit the mouth with the same toothbrush, so as to avoid the
introduction of the fixative to the mucosal lining. Use a new toothbrush for resampling.
(iii) The head of the brush is then placed into the fixative container and rotated such that the cells are dislodged and
released into the suspension.
? TROUBLESHOOTING

832 | VOL.4 NO.6 | 2009 | NATURE PROTOCOLS


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(iv) Tightly seal the tops of the fixative containers and cover in parafilm to prevent leakage during transit from the
remote collection location to the laboratory.
(v) The containers are then returned to the laboratory for analysis by a reputed courier service, and the laboratory should be
informed of their shipment and anticipated arrival date, so that they can be processed as soon as possible after receipt.
When received, the cells are collected in Saccomano’s fixative and are treated in the same manner as fresh buccal cells
that have been collected in buccal cell buffer (starting from step 3 in the ‘Buccal cell harvesting and slide preparation’
section below).
’ PAUSE POINT Buccal cells fixed in Saccomanno’s solution can be stored at 4 1C for up to 7 d before preparing slides.


Buccal cell harvesting and slide preparation TIMING B2 h
3| Transfer the fresh or fixed cells collected from both the right and left cheeks into separate TV-10 centrifuge tubes.
Centrifuge the cells for 10 min at 581g at room temperature.
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

4| Aspirate off the supernatant leaving approximately 1 ml of cell suspension and replace with 5 ml of buccal cell buffer.
Briefly vortex the cells.

5| Centrifuge the cells for 10 min at 581g, room temperature. Aspirate the supernatant and resuspend the cells in another
5 ml of buccal buffer.
m CRITICAL STEP The best results are achieved after two washes in buccal cell buffer. This buffer helps to inactivate endogenous
DNAases present in the oral cavity and to remove bacteria and cell debris that could complicate scoring.
6| Remove the supernatant and replace with 5 ml of fresh buccal cell buffer.

7| Vortex the cell suspension and then homogenize for 2–3 min using a hand-held tissue homogenizer to increase the number
of single cells in suspension.
8| Pool the cells from the left and right cheek tubes into a 30 ml container before drawing the cells into a syringe
using an 18G needle.

9| To remove large aggregates of unseparated cells, pass the cells into a TV-10 tube through a 100 mm nylon filter held in a
Swinex holder.

10| Centrifuge the cells for 10 min at 581g at room temperature and remove the supernatant. Resuspend the cells in 1 ml of
buccal cell buffer.
11| To count the cells using a Coulter Counter, set the instrument settings for counting human buccal cells (e.g., Coulter
Counter Model ZB1; threshold: 8, attenuation: 1, aperture: 1/4, manometer: 0.5 ml).

12| Dilute 300 ml of the cell suspension into 15 ml of Isoton.


13| Perform the count in duplicate to determine the mean cell number.
? TROUBLESHOOTING
14| Dilute the cell suspension to 80,000 cells ml1 with buccal cell buffer.

15| To further aid in cellular disaggregation and obtain slide preparations with clearly separated cells, add 50 ml of DMSO per
ml of cell suspension.
16| Preparation of cells on microscope slides can be carried out in option A using a cytocentrifuge, or option B, manually.
? TROUBLESHOOTING
(A) Preparation and transfer of cells onto the microscope slides using a cytocentrifuge
(i) Follow the cytocentrifuge manufacturer’s instructions for the assembly of the microscope slides, filter cards and
cytocentrifuge cups within the cytocentrifuge rotor. Ensure that the microscope slides are labeled with a code that
matches the donor cells.
(ii) Resuspend the cells thoroughly using a Pasteur or Gilson pipette to disaggregate them.
(iii) Add 120 ml of the cell suspension into the well of each sample cup.
! CAUTION Loading and cytocentrifugation of the cell culture sample must be carried out in an approved cytoguard
safety cabinet to avoid the possibility of infectious disease transfer from the buccal cells. Appropriate safety protection
including gloves must be worn.
m CRITICAL STEP The required volume may need a slight adjustment depending on the concentration of cells in
suspension and the optimal cell density for slide scoring; this needs to be determined empirically.

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(iv) Replace and lock the rotor lid and centrifuge the cells for 5 min, at 600 r.p.m., between 18 and 20 1C.
(v) Upon completion of the spin place the rotor in a safety cabinet and follow the manufacturer’s instructions for opening
each slide holder.
(vi) Air-dry the microscope slides for exactly 10 min at room temperature.
(vii) Fix the cells in a slide-staining rack containing 200 ml of ethanol: glacial acetic acid mix (3:1) for 10 min followed
by further air-drying for 10 min at room temperature.
(viii) Disinfect the cytocentrifuge cups in 1% (vol/vol) sodium hypochlorite solution for 30 min and then rinse thoroughly
(six times) in Milli-Q water and allow to dry completely before additional use.
(B) Manual preparation and transfer of cells onto the microscope slides
(i) Fix the cells obtained in step 15 using the required volume of ethanol: glacial acetic acid, 3:1, to give a concentration
of 80,000 cells ml1.
(ii) Using a pipette, drop 120–150 ml of cell suspension onto the clean, dry and appropriately labeled microscope
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

slides, and allow to air-dry for 10 min before staining.


Buccal cell staining for microscopy TIMING B1 h 45 min
17| Immerse the microscope slides with the fixed cells for 1 min each in Coplin jars containing 50% (vol/vol) and
20% (vol/vol) ethanol. Wash the cells for 2 min in a Coplin jar containing Milli-Q water.

18| Place the slides in a Coplin jar containing 5 M HCl for 30 min and then rinse in running tap water for 3 min.

19| Place one slide in Milli-Q water instead of 5 M HCl for 30 min as a negative control to check for the efficacy of the
5 M HCl treatment.

20| Drain the slides, but do not allow them to dry out, and place them in a Coplin jar containing Schiff ’s reagent for 60 min in
the dark at room temperature.

21| Rinse the slides in running tap water for 5 min and then rinse well in Milli-Q water.

22| Counter stain the cells by immersing in Coplin jars containing 0.2% (wt/vol) Light Green for 20–30 s and rinse well in
Milli-Q water.

23| To blot away any residual moisture, immediately place the slides facedown onto Whatman no. 1 filter paper.
m CRITICAL STEP Do not apply any pressure or rub on the cell spots as this will dislodge cells.

24| Place the slides on a slide tray and allow them to dry for about 10–15 min.

25| Examine the efficiency of staining and the density of the cells at 100 and 400 magnification.
? TROUBLESHOOTING

26| Leave the slides to dry completely for at least 30 min before applying coverslips.
! CAUTION Apply the coverslips in a fume hood to avoid inhalation of organic solvent in DePex and leave the slides in the
hood until completely dry. Wear nitrile gloves when applying DePex medium.
’ PAUSE POINT Slides can be left overnight at room temperature to dry but should be covered to prevent dust from settling on
them.
27| Place the slides to be coverslipped on tissue paper and set out one coverslip alongside each.
28| Put two large drops of DePex (use a plastic dropper) on each of the coverslips in the approximate area corresponding to
the cell spots.

29| Invert the slide and place on the coverslip. Allow the DePex to spread. Turn the slide so that the coverslip is on top, and
press the coverslip gently to expel any excess DePex and air bubbles.
m CRITICAL STEP Press the coverslip gently, as it may break. Ensure that the spots do not have air bubbles over them.

30| Wipe excess DePex from the edges of the slide and ensure that the medium or glass does not cover any of the frosted
label area, as a coding label will not stick on the DePex.

31| Place the slides on a tray and leave overnight in the fume hood to dry.

32| Store the slides in slide boxes at room temperature.

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33| Observe the slides using transmitted light microscopy; the nuclei and the micronuclei are magenta in color, whereas the
cytoplasm will be pale blue/green (see Fig. 5). In negative controls (i.e., no 5 M HCl treatment) the nuclei will not be stained
with magenta color.
? TROUBLESHOOTING
34| The cells can also be viewed under fluorescence with a far-red filter because Feulgen stained DNA appears bright red in
color under these conditions.


Microscopy TIMING B60–80 min/slide
35| Using the scoring criteria described in Table 3 count a minimum of 1,000 cells, and determine the frequency of each cell
type in the sample, e.g., basal and differentiated cells (see Table 3 and Fig. 5).
? TROUBLESHOOTING
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

36| Using the scoring criteria described above (Table 3), record the number of cells with MNi and NBUDs in a minimum of
2,000 differentiated cells (see Fig. 5c–e).
? TROUBLESHOOTING

 TIMING
Steps 1 and 2, buccal cell collection: B10 min
Steps 3–16, buccal cell harvesting and slide preparation: B2 h
Steps 17–34, buccal cell staining for microscopic analysis: B1 h 45 min
Steps 35 and 36, scoring of stained slides: B60–80 min/slide

? TROUBLESHOOTING
Troubleshooting advice can be found in Table 4.

TABLE 4 | Troubleshooting advice.


Step Problem Possible reason Solution
2B(iii) Leaking sample in transit Cracked lid or not tightened Ensure lid is tightened and seal well with parafilm

13,16 Insufficient cell count Insufficient pressure applied with Obtain a second sample or concentrate the cell
toothbrush suspension into 240 ml. Apply 120 ml to each
well producing 1 slide with 2 spots if
using cytocentrifuge. This consists of two
consecutive steps

25 Low or excess cell density on Inaccurate cell count If the cell density is too high or sparse, concentrate
cytocentrifuge slide or dilute the cell suspension as necessary by
centrifuging the cells and resuspend in a larger
or smaller volume of buffer and repeat the cell
harvesting and staining steps. Check settings on
coulter counter

33 Buccal cell deterioration evident Use of inappropriate buffer to Use Saccomanno’s fixative if cells cannot be
when viewing slides store cells and/or high storage processed on the same day and/or if collected in
temperature remote location from laboratory. Storage
temperature in buffer or Saccomanno’s should
not exceed 4 1C

35,36 Uncertainty in classification of Poor Feulgen staining on slide Examine cell under both transmitted light and
cell types on presence of MNi preparation under fluorescence because nuclear texture
and NBUD and DNA staining intensity is better under
fluorescence. Otherwise, make new slide
preparations from residual cells stored in buccal
cell buffer or Saccomanno’s fixative.
Make a fresh batch of Schiff’s reagent and check
concentration of HCl
Abbreviations: MNi, Micronuclei; NBUD, Nuclear bud.

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TABLE 5 | Typical BMCyt results for cells collected from healthy young and old subjects.
Basal cell no. Diff cell no. BN cell no. PYK cell no. CC cell no. KYL cell no. KHC cell no. NBUD no. MN frequency
(a) Young controls n ¼ 30 (mean age 22.47 ± 2.2 yrs, range 18–26 yrs)
Minimum 5.00 460.00 2.00 0.00 13.00 43.00 1.00 0.00 0.00
25% percentile 12.00 629.00 8.00 2.00 30.50 109.00 4.00 0.00 0.00
Median 22.00 715.00 10.00 2.00 45.50 181.00 6.50 1.00 0.00
75% percentile 37.00 788.50 14.00 5.00 60.50 256.50 12.00 1.00 0.00
Maximum 91.00 895.00 27.00 10.00 103.00 441.00 44.00 4.00 4.00
Mean 27.40 712.90 11.63 3.23 46.67 187.90 9.36 0.93 0.30
s.d. 21.36 112.70 5.49 2.35 20.53 108.60 8.71 1.11 0.83
s.e. 3.89 20.58 1.00 0.43 3.74 19.83 1.59 0.20 0.15
Lower 95% CI of mean 19.43 670.80 9.58 2.35 39.00 147.30 6.11 0.51 0.01
Upper 95% CI of mean 35.37 755.00 13.69 4.11 54.33 228.50 12.62 1.34 0.61
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

(b) Older controls n ¼ 30 (mean age 68.13 ± 2.8 yrs, range 64–75 yrs)
Minimum 15.00 373.00 2.00 0.00 11.00 14.00 2.00 0.00 0.00
25% percentile 57.50 570.50 8.50 0.00 32.50 74.50 30.00 0.00 0.00
Median 92.50 651.50 14.00 1.00 64.50 104.50 48.50 1.00 1.00
75% percentile 128.50 737.50 21.00 3.50 84.00 135.00 68.00 2.00 3.00
Maximum 187.00 902.00 42.00 8.00 155.00 270.00 137.00 4.00 6.00
Mean 93.53 655.90 15.80 1.96 65.10 113.60 51.50 1.16 1.43
s.d. 44.97 116.20 9.01 2.10 34.76 56.44 33.29 1.34 1.56
s.e. 8.21 21.22 1.64 0.38 6.34 10.30 6.07 0.24 0.28
Lower 95% CI of mean 76.74 612.50 12.43 1.17 52.12 92.56 39.07 0.66 0.84
Upper 95% CI of mean 110.30 699.30 19.17 2.75 78.08 134.70 63.93 1.66 2.01
Results shown are ‘per 1,000 cells’, i.e., %. Diff, Differentiated; BN, Binucleated; PYK, Pyknotic; CC, Condensed Chromatin; KYL, Karyolytic; KHC, Karyorrhectic; MN, Micronucleus; NBUD, Nuclear bud; s.d., standard
deviation; s.e., standard error; CI, confidence interval.

ANTICIPATED RESULTS
The anticipated results with the buccal micronucleus cytome assay (BMCyt) are dependent on the level of exposure and potency
of genotoxic or cytotoxic agents, genetic background and the age and gender of the donor cells being tested. Detailed typical
results for healthy young and old participants, who are not abnormally exposed to genotoxins, are shown in Table 5. The table
includes the minimum and maximum value, 25th and 75th percentiles, the median, mean, standard deviation (s.d.), standard
error, and lower 95% and upper 95% confidence interval for each BMCyt biomarker in young and old controls. The BMCyt assay
has as yet not been approved for diagnostic use and is applied only for research purposes at this stage. However, it is
reasonable to consider individual values that are beyond two s.d of the mean for the biomarker measured as likely to be
abnormal when comparing results within the same age group. For example, on the basis of the data in Table 5, a young adult
with a micronucleus frequency value of 4% would be considered to have an abnormally high micronucleus frequency, given that
the mean for the group is 0.3 and the s.d. is 0.8.
The frequency of the buccal cytome biomarkers in the assay may vary with respect to their relative frequencies depending on
the rate of ageing, as has been shown in recent studies on Down’s syndrome and Alzheimer’s disease, in which the frequency of
basal cells was significantly lower as compared with the controls9,23. Most buccal cell studies have only evaluated changes in
micronucleus frequencies in relation to biomonitoring, lifestyle and dietary factors and have not scored the remaining
cytome biomarkers to show a more comprehensive evaluation. Anticipated changes in micronucleus frequency in the
buccal cells may be large in the case of ionizing radiation exposure. Following radiotherapy, MNi frequency increased 16-fold
as compared with the baseline levels21.

Note: Supplementary information is available via the HTML version of this article. 3. Sarto, F., Tomanin, R., Giacomelli, L., Iannini, G. & Cupiraggi, A.R. The
micronucleus assay in human exfoliated cells of the nose and mouth: application
ACKNOWLEDGMENTS We are grateful to the numerous volunteers whose donation to occupational exposures to chromic acid and ethylene oxide. Mutat. Res. 244,
of buccal cell samples over the many years has enabled us to achieve the 345–351 (1990).
refinement of this protocol. We are particularly indebted to Professor Hans Stich 4. Machado-Santelli, G.M., Cerqueira, E.M., Oliveira, C.T. & Pereira, C.A.
who pioneered this field of research and whose vision enabled the evolution of this Biomonitoring of nurses handling antineoplastic drugs. Mutat. Res. 322,
protocol. 203–208 (1994).
5. Burgaz, S. et al. Urinary cyclophosphamide excretion and micronuclei frequencies
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