Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions.
ABSTRACT To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies.
Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN.
Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm.
Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.
- [show abstract] [hide abstract]
ABSTRACT: The use of the micronucleus test on exfoliated cells as an approach to identify genotoxic damage in human tissues which are targets for organ-specific carcinogens and from which carcinomas will develop, is described. Chromosomal damage by carcinogens to dividing basal cells of the epithelium results in the production of micronuclei in the daughter cells which migrate up through the epithelium and are exfoliated. Exfoliated cells can be readily obtained from several tissues, including the oral buccal mucosa (scrapings of oral cells), bronchi (sputum), urinary bladder and ureter (centrifugation of urine), cervix (smears) and esophagus (imprints from biopsies). The micronucleus test on exfoliated cells has been successfully used to: (1) recognize population groups at an elevated risk for cancer of the oral cavity or urinary bladder; (2) estimate synergistic or additive effects of carcinogen exposure (cigarette smokers plus drinkers of alcoholic beverages); (3) pinpoint the site within an organ from which most carcinomas will develop (oral cancers among 'inverted' smokers in the Philippines). The possibility that this assay may also serve as a rapid monitor for chemopreventive agents is suggested by a preliminary trial on the effect of vitamin A/beta--carotene dietary supplementation among 33 betel quid chewers in the Philippines. These individuals received sealed capsules of retinol (100,000 IU/week) and beta-carotene (300,000 IU/week) for a 3-month period. At the end of this time, the frequencies of micronucleated buccal mucosa cells were reduced from an average of 4.2% to 1.4%. No changes were observed in micronucleus frequencies among 11 betel quid chewers not receiving vitamin pills. Non- chewers of betel quid in this population had a micronucleus frequency of 0.5%.Cancer Letters 05/1984; 22(3):241-53. · 4.26 Impact Factor
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ABSTRACT: The micronucleus (MN) assay in human exfoliated cells has been widely used to detect the genotoxic effects of environmental mutagens, infectious agents and hereditary diseases. Substantial variability characterizes the MN frequencies reported by different research groups. One reason for this may be the restricted resolution power of the Feulgen-Fast-Green staining that is routinely used. Here we describe a new version of the MN assay that employs fluorescent propidium iodide staining along with fluorescence in situ hybridization (FISH) with a centromeric probe. Buccal and urothelial cells were collected from 5 healthy unexposed female volunteers and 55,000 cells analyzed for MN frequency and abnormal nuclear events. The Feulgen-Fast-Green and the new fluorescent staining produced very similar results. The frequency of MN in buccal cells was 0.145 +/- 0.118% and in urothelial cells 0.083 +/- 0.074%. No correlation was found between the frequencies of MN in the two types of exfoliated cells. FISH with a centromeric probe allowed MN containing whole chromosomes with a centromere to be differentiated from those containing only acentric fragments. The former appear as a result of chromosome lagging in mitosis, while those without a centromere are due to chromosome breakage. In urothelial cells 43% of MN were centromere-negative and in buccal cells -44%. Fluorescent staining provided more accurate scoring of degenerative cells than standard Feulgen-Fast-Green staining. The combined frequency of pycnotic cells, "broken eggs" and cells with fragmented nuclei did not exceed 2%, while that of karyorrhexis and karyolysis together was as high as 21%. Significant interindividual variability was found in the frequency of cells with karyolysis and karyorrhexis. Thus, the new version of micronucleus assay allows for MN to be scored more precisely, the mechanism of MN formation to be determined and abnormal nuclear events to be readily identified in exfoliated human cells. It is therefore ideal for studying genotoxicity in human populations using exfoliated cells from the mouth, bladder and nose.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 03/1994; 312(1):39-50. · 3.90 Impact Factor
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ABSTRACT: The three-dimensional structure of chemically synthesized CnErg1 (Ergtoxin), which specifically blocks HERG (human ether-a-go-go-related gene) K+ channels, was determined by nuclear magnetic resonance spectroscopy. CnErg1 consists of a triple-stranded beta-sheet and an alpha-helix, as is typical of K+ channel scorpion toxins. The peptide structure differs from the canonical structures in that the first beta-strand is shorter and is nearer to the second beta-strand rather than to the third beta-strand on the C-terminus. There is also a large hydrophobic patch on the surface of the toxin, surrounding a central lysine residue, Lys13. We postulate that this hydrophobic patch is likely to form part of the binding surface of the toxin.FEBS Letters 04/2003; 539(1-3):138-42. · 3.58 Impact Factor
ACTA CYTOLOGICA 0001-5547/06/5004-0398/$19.00/0 © The International Academy of Cytology
To compare Papanicolaou (Pap) and May-Grünwald
Giemsa (MGG) stain as 2 techniques for staining for buc-
cal mucosal cells to detect micronuclei (MN) in field studies.
Eighty cytologic smears (2 per
individual) were taken from
the buccal mucosa of 40 ciga-
rette smokers recruited at a
rural village in Egypt. Forty
smears were stained with Pap
stain and 40 with MGG
stain. All were assessed for cel-
lularity and scored for MN.
Pap stain was faster and easier to process and transport in
the field study than was MGG stain. Regarding MGG
smears, bacteria and cell debris masked the MN as compared
to Pap smears, in which the fixative destroyed the bacteria
and made the cell boundaries clearly demarcated. Using Pap
stain, MN were seen easily in transparent cytoplasm.
Pap stain is the preferred method in field studies for scoring
and detecting MN in cells of buccal mucosa. (Acta Cytol
Keywords: buccal mucosa,
Papanicolaou smear, tissue
stains, micronucleoli, Pa-
xposure of a tissue to
leads to an increase in chromosomal aberrations.1-4
Consistent with this hypothesis, karyotypic anomalies
and elevated DNA content have been observed in var-
ious preneoplastic lesions. Before particular karyotyp-
ic anomalies can be established, a long period of
breakage and translocation of chromatids must occur.
Pap stain is the preferred method
of detecting MN in oral cells from
subjects in a field study....
Evaluation of Papanicolaou Stain for Studying
Micronuclei in Buccal Cells Under Field
Sohair B. A. Ayyad, M.D., Ebenezer Israel, M.D., Maged El-Setouhy, M.D.,
Ghada Radwan Nasr, M.D., Mostafa K. Mohamed, Ph.D., and
Christopher A. Loffredo, Ph.D.
From the Cytopathology and Early Cancer Detection Unit, Ain Shams University Hospitals, and Department of Community, Environmental
and Occupational Medicine, College of Medicine, Ain Shams University, Ain Shams, and Department of Community Health, Cairo Univer-
sity, Cairo, Egypt, and the Department of Epidemiology and Preventive Medicine, University of Maryland, Baltimore, and Cancer Genetics
and Epidemiology Program, Department of Oncology, Georgetown University, Washington, D.C., U.S.A.
Dr. Ayyad is Assistant Professor, Cytopathology and Early Cancer Detection Unit, Ain Shams University Hospitals, Ain Shams University.
Dr. Israel is Professor, Department of Epidemiology and Preventive Medicine, University of Maryland.
Drs. El-Setouhy and Mohamed are Professors, Department of Community, Environmental and Occupational Medicine, College of Medicine,
Ain Shams University.
Dr. Nasr is Assistant Lecturer, Department of Community Health, Cairo University.
Dr. Loffredo is Assistant Professor, Cancer Genetics and Epidemiology Program, Department of Oncology, Georgetown University.
Supported by grant R10TW05944 from the Fogarty International Center, U.S. National Institutes of Health.
Address correspondence to: Christopher A. Loffredo, Ph.D., Cancer Genetics and Epidemiology Program, Department of Oncology, George-
town University, Box 571472, Washington, DC 20007-1472 (firstname.lastname@example.org).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.
Received for publication April 12, 2005.
Accepted for publication July 5, 2005.
Papanicolaou Stain for Micronuclei
Volume 50Number 4July–August 2006
Such chromatid anomalies or chromosome deletions
can lead to formation of micronuclei (MN), which are
DNA-containing bodies in the cytoplasm without any
structural connection to the main nucleus.5MN are
small, extranuclear bodies separated from the main
one, generated during cellular division by late chro-
mosomes or by chromosome fragments because of
their association with chromosomal aberrations.4,6
The MN test is one of the current rapid, efficient and
economical techniques used as an indicator of geno-
toxicity, as it provides a quantitative measure of the
genotoxic action of carcinogens and mutagens.7-12
MN analysis of exfoliated cells is a convenient tech-
nique to study genotoxic changes directly in target or-
gans affected by the toxicant.2,4,13-30For example, in-
creased MN frequency was found in exfoliated oral
(buccal) cells among smokers and tobacco chewers
with oral carcinomas as compared to cancer-free sub-
jects.14-16Epidemiologic studies have reported smok-
ing habits to be associated with an increased occur-
rence of different kinds of oral carcinoma.17-19
Comparisons between studies are difficult, however,
because of differences in methodologies, especially
with regard to staining techniques, microscopic mag-
nification, number of cells counted and instruments
used for sampling cells. Several investigators have
used Feulgen stain and Fast green for scoring
MN,7,16,20-23while others have used Papanicolaou
(Pap) stain in scoring MN.24-26Others used May-
Grünwald-Giemsa (MGG) stain in scoring MN in
lymphocytes and buccal cells.27,28
Some of these staining methods require strict ad-
herence to controlled laboratory conditions and are
therefore difficult to apply in field studies, in which
such conditions may be difficult to maintain. In our
ongoing studies of tobacco health effects in rural vil-
lages in Egypt, for example, it is necessary to select a
robust method of MN analysis that can be performed
under field conditions of specimen sampling and
preparation. The aim of this study, therefore, was to
compare the Pap and MGG stain as 2 techniques for
evaluating MN in buccal mucosa cells.
A total of 40 cigarette smokers were selected from a
rural village in Qaloubiya Governorate, Egypt; all met
the eligibility criteria of being smokers for at least 5
years, smoking 10 or more cigarettes per day and
smoking the same amount of cigarettes for the previ-
ous year. Exfoliated cells were obtained by scraping
the tongue and buccal mucosa with a wooden spatula
with a rolling motion.4,29The material obtained was
immediately smeared onto the center of a clean glass
slide. For each individual, 2 slides were prepared by
smearing the oral scrapings on the slides for a total of
80 slides (40 slides stained with Pap stain and 40 with
(Pap stain, ×1,000).
Buccal smear showing normal epithelial squamous cells
cell (Pap stain, ×1,000).
Buccal smear showing anucleated epithelial squamous
stain, ×1,000, with phase contrast).
Buccal smear showing binucleated squamous cell (Pap
Ayyad et al
Volume 50 Number 4July–August 2006
For MGG stain, the smears were received in the labo-
ratory, allowed to air dry and then fixed in methanol
(80%) at 0°C for 20 minutes.28,30For Pap stain, we
used the wet fixation method in the field: immediate
submersion of wet cell samples in 95% ethyl alcohol
MGG Method. For this method we prepared fresh
MGG stain (25 g/100 mL methanol) and then stained
the slides in May-Grünwald stain for 3 minutes.
(Staining time may vary depending on the concentra-
tion of stain.) We determined the optimal staining
time with a trial slide before proceeding. Next we
placed slides in dH2O for 1 minute and stained with
(10%) Giemsa stain (stock solution diluted 1:10 in
Sorenson’s buffer) for 7–10 minutes. (It is critical to
determine the optimal staining time before proceed-
ing.) The slides were washed 2 times in dH2O, 3 min-
utes each, and then were air dried. We applied a cov-
erslip with Permount.
Modified Pap Method. For this method we fixed the
smears in 95% ethyl alcohol. Then we performed hy-
dration with a running tap water wash. We applied
nuclear stain (DNA related), Mayer’s hematoxylin for
4 minutes, followed by a rinse in a stream of gently
running water for 15 minutes. Dehydration by 70%
and 95% ethyl alcohol was then performed with 10
dips each. Cytoplasmic stain (Orange green) was ap-
plied for 1 minute, followed by a rinse with 95% ethyl
alcohol for 5 minutes. Cytoplasm and nuclear stain
(RNA specific) by EA polychrome was applied for 1.5
minutes, followed by a rinse with 95% ethyl alcohol (2
times), 10 dips each. The next step was dehydration
opaque with phase contrast.
Buccal smear showing micronuclei (Pap stain, ×1,000,
(karyorrhexis) (Pap stain, ×1,000).
Buccal smear showing fragmented nucleus
and inflammatory cell on its lower border (MGG stain, ×1,000).
Buccal smear showing squamous cell with micronucleus
cell debris (MGG stain, ×1,000).
Buccal smear showing broken egg nucleus, bacteria and
with absolute ethyl alcohol (2 times), 10 dips each, and
then clearing by absolute ethyl alcohol and xylene
(1:1) for 1 minute. The preparation was mounted with
MN Test. Screening for MN was performed under an
oil immersion lens (×1,000) followed by phase con-
trast microscopy for confirmation of MN, according
to established methods.21,26,24The opaque extranu-
clear-intracytoplasmic bodies were counted as MN,
whereas binucleated cells, karyorrehexis, karyolysis
and broken eggs were not considered MN.6,22,29At
least 1,000 intact epithelial cells per individual were
scored, and for each person we calculated the average
percent micronucleated cells. In addition, we calculat-
ed the total number of MN (TMN) and the number of
cells with MN (CMN) per individual since some cells
had multiple MN.
Pap stain was easier to read than the MGG stain and
easier to process and transport in the field. Of 40 spec-
imens (80 slides), 2 (4 slides) were not adequate (had
<1,000 cells per individual). Two specimens (4 slides)
that were stained with MGG had bacterial colonies
and cell debris masking the oral cells completely, so
MN could not be detected. Figures 1–5 illustrate Pap
smears, while 6–7 illustrate MGG smears.
This study showed that under field conditions Pap
stain is more practical than MGG stain. MGG stain
requires slides to be air dried and then placed in
methanol (80%) at 0°C. That was difficult to apply in
the field and resulted in difficulties in transport. The
methanol required by that method is not the substance
of choice for fixation of cytologic specimens. Further-
more, the slides stained with MGG stain were full of
bacteria, which often masked MN. They make the
scoring of MN difficult and largely inaccurate.
Regarding Pap stain, the 95% ethyl alcohol used for
fixation is the best fixative because it has a bactericidal
effect. The alcohol allows the permeability of dyes
across cell boundaries, permits cell adhesion to the
glass surface and replaces cellular water. It also pene-
trates the cell rapidly and maintains morphologic in-
tegrity. Pap stain is preferred also as it consists of nu-
clear stain (hematoxylin) that stains all nuclear DNA,
both intranuclear and extranuclear. It consists also of
2 counterstains that make the cytoplasm transparent
and the cell well demarcated.
Many previous studies used other stains, such as
Feulgen–fast green, for MN study.21,7,23This method
of staining takes a long time (>4 hours), and the
method of fixation is more difficult to use under field
Papanicolaou Stain for Micronuclei
Volume 50Number 4 July–August 2006
conditions. Its components are more expensive than
those of Pap stain. Feulgen stain alone cannot delin-
eate the cytoplasm: it needs 1% Fast green to demar-
cate the cytoplasm of cells. If the cytoplasm is too dark
and the nucleus not clear and distinct, it needs destain-
ing in ethanol until the optimal contrast is
In summary, comparisons between 40 smears of
buccal mucosa stained with Pap stain and 40 smears
stained with MGG stain demonstrated that, and this
conclusion is consistent with studies by Tsu,26
Roberts24and Guzman et al.25
The authors thank Drs. Fatma Abdel Aziz, Eman
Mafouz and Nabiel Mikhail for their support of this
study, including field work, supervision of staff, col-
lection of specimens and data management.
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