Usefulness of ploidy, AgNOR and immunocytochemistry for

differentiating benign and malignant cells in serous effusions

F
L. A. Palaoro*, A. M. Blanco*, M. Gamboni , A. E. Rocherà and R. G. Rotenberg§
*Department of Clinical Biochemistry, UBA, Clinical Hospital, Buenos Aires, Argentina,  Hospital Mater Dei, Buenos Aires,

OO
Argentina, àCytology Laboratory, Department of Clinical Biochemistry, UBA, Buenos Aires, Argentina and §Department of
Clinical Pathology, UBA, Buenos Aires, Argentina

Accepted for publication 24 March 2006

L. A. Palaoro, A. M. Blanco, M. Gamboni, A. E. Rocher and R. G. Rotenberg

Usefulness of ploidy, AgNOR and immunocytochemistry for differentiating benign and malignant cells in

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serous effusions
Objective: The objective of this study was to establish the value of different markers in differentiating reactive
mesothelial cells from metastatic adenocarcinomatous cells in serous effusions (SE).
Methods: Forty-five SE were processed for morphological examination (Papanicolaou stain), assessment of
ploidy, AgNOR counting and immunocytochemical assay of carcinoembryonic antigen (CEA), epithelial
membrane antigens (EMA), Ber-EP4 and Leu-M1. Ploidy was established in an image-analyser in smears stained

D
by the Feulgen stain method. AgNOR dots were counted in the smears stained with the silver nitrate assay for
non-histone proteins present in the nucleolar organizer region. CEA, EMA, Ber-EP4 and Leu-M1 were evaluated
by immunocytochemistry using the streptavidin–biotin complex method.
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Results: All the smears with positive cytology were aneuploid. Three false negatives by morphological studies
were aneuploid, with AgNOR values in two of them corresponding to the neoplastic group. CEA and Leu-M1
showed a low specificity; EMA and Ber-EP4 showed moderate sensitivity.
Conclusions: The assessment of ploidy and the study of AgNOR were better methods than immunocyto-
chemistry for distinguishing between reactive mesothelial cells and adenocarcinomatous cells in serous fluid.
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Keywords: serous effusions, cytology, ploidy, carcinoembryonic antigen, epithelial membrane antigen, Ber-EP4,
Leu-M1, nucleolus organizer region

logical analysis, especially in the differentiation

Introduction
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between adenocarcinoma cells and reactive mesothel-

It is well known that the cytological study of serous
effusions (SE) stained by the Papanicolaou technique
has limitations resulting in large number of false
negative and false positive results. For this reason
morphological examination is complemented by other
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ial cells. However, the use of these additional tech-
niques may not be applicable in routine diagnostic
procedures because they need an additional invest-
ment and trained staff.
Scanning and transmission electron microscopy

methods: classical cytochemistry, immunocytochem-
istry, cytogenetics, molecular biology, DNA cytometry
and ultrastructural studies.1–9
The use of these additional techniques attempts to
add useful diagnostic criteria to the simple morpho-
UN
have been used to evaluate the contribution of the
intermediate filaments and the shape and size of
microvillies to improve the diagnostic sensitivity.10–12
Immunostaining has been frequently used to
investigate different antigens such as carcinoembry-
onic antigen (CEA), epithelial membrane antigen

(EMA), Ber-EP4, B72.3 or proteins from oncogenes

Correspondence:
L. A. Palaoro, PhD, Department of Clinical Biochemistry,
UBA, Clinical Hospital, Buenos Aires, Argentina.
Tel.: xxx; Fax: xxx;
2 E-mail: luispalaoro@yahoo.com.ar
like c-erb-2.2–4,13
The assay of non-histone fibrillar proteins from the
nucleolar organizer regions (AgNOR) with silver
nitrate, called the AgNOR technique, is an interesting

Cytopathology 2006, 17, 1–7 ª 2006 The Authors

Journal compilation ª 2006 Blackwell Publishing Ltd 1

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Journal Name
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Manuscript No.
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Dispatch: 3.8.06
Author Received:
Journal: CYT CE: Balaji
No. of pages: 7 PE: Kavitha R
2 L. A. Palaoro et al.

alternative for the differentiation between reactive
and neoplastic cells in effusions.14
Recently, image-analysis systems and flow cytom-
etry have allowed the accurate determination of DNA
content and the study of the cell cycle.15–19
patients (over almost 18 months). In some cases
pleural or peritoneal biopsies were performed, and
in two cases the diagnosis was established by autopsy,
although in some patients an initial diagnosis of

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malignancy could be made by cytological examination
While some malignant tumours are diploid (e.g. (five cases of pleural effusion and six cases of
some malignant mesotheliomas), most SE originate in ascites).20

metastatic carcinomas containing aneuploid cells.
The objective of this study was to analyse the
relative usefulness of three different techniques (im-
munocytochemistry, AgNOR and image-analysis) in
the differential diagnosis of reactive mesothelial cells
and neoplastic cells exfoliated from metastatic adeno-
OO
All the malignant effusions were a consequence of
metastatic adenocarcinomas; there were no meso-
theliomas in our series.
Tables 1 and 2 show the causes of the malignant
and benign effusions.
1. Immunoassay for CEA, EMA, Ber-EP4 and Leu-M1:

carcinomas.
Study population
Forty-five effusions (20 peritoneal and 25 pleural)
were studied. The SE were collected from patients
from the Department of Pathology, Mater Dei Clinical
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The immunocytochemistry was performed by the
labelled streptavidin–biotin complex method13:
after washing with phosphate-buffered saline
(PBS), the smears were covered with 3% H2O2
for 5 minutes to block endogenous peroxidase
activity. After a rinse the slides were incubated
for 30 minutes with the primary antibodies: CEA

D
and the Department of Biochemistry, Cytology Area, 3 monoclonal (DAKO), EMA monoclonal (DAKO),
Clinical Hospital (University of Buenos Aires), Argen- Ber-EP4 monoclonal (DAKO) and Leu-M1
tina. 4 monoclonal (Becton-Dickinson). The smears

Materials and methods

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Table 1. Causes of malignant effusions

The fluids were centrifuged at 1000 g for 10 minutes. Malignant effusions n

From the cell deposits smears were made and fixed in Ovary adenocarcinoma 8
96% ethanol. At least two smears from each sample
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Lung adenocarcinoma 4
were stained by the Papanicolaou method. The rests Colon adenocarcinoma 1
were stored for (1) immunocytochemical assay with Gastric adenocarcinoma 1
maximal fixation time of 60 days at )20 C, (2) Endometrium adenocarcinoma 1
staining with the AgNOR technique, and (3) process- Breast carcinoma 4
ing for the study of ploidy. Rectum carcinoma 1
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Carcinomas from unknown primary sites 5

Total 25
Cytological criteria
The cytological diagnoses were recorded as benign on Table 2. Causes of benign effusions
malignant. The reactive changes in mesothelial cells
are characterized by enlargement of nuclei (often Benign effusions n
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multiple) and nucleoli, clumped chromatin and cel- Rheumatoid arthritis 2

lular grouping in sheet, balls or rosettes. Ovarian fibroid 2
The normal mesothelial cells present cytoplasm Peritonitis 3
with small vacuoles, ruffled borders and scanty, small Lung infarct 1
nucleoli. There are ÔwindowsÕ between adjacent cells. Bronchial asthma 2
Typical adenocarcinomatous cells usually show Cirrhosis 2
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coarser and denser chromatin than mesothelial cells Congestive heart failure 3
Systemic lupus erythematosus 1
and bigger nucleoli. The groups are three-dimen-
Unknown origin 2
sional, without ÔwindowsÕ, and have peripheral nuclei.
Cystadenoma of ovary 2
The final diagnosis was established through evalu- Total 20
ation of the clinical history and follow up of the

Cytopathology 2006, 17, 1–7 ª 2006 The Authors

Journal compilation ª 2006 Blackwell Publishing Ltd
1 Ploidy, AgNOR and immunomarkers in effusions 3

were rinsed with PBS and incubated with biot- than 1.1, even when this peak is not a stemline
inylated antimouse antibody (1 : 100 DAKO) for (dominant lineage). When the DNA index was
15 minutes. After a further rinse, peroxidase- 1 ± 0.1, the population was classified as diploid.23
labelled streptavidin was applied for 15 minutes

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(1 : 100 DAKO). The activity of peroxidase was
Results
detected with 0.01% H2O2 and diaminobenzidine
for 15 minutes. The slides were dehydrated, The ploidy study was carried out using 45 smears. The

clarified with xylene and mounted with Canada
balsam. Positive histological sections served as
controls for the monoclonal antibodies: colon
adenocarcinoma for CEA, breast lobular carci-
noma for EMA, collecting renal tubules of kidney
biopsies for Ber-EP4 and Reed-Stemberg cells
OO
22 SE positives by cytology were aneuploid, but three
SE considered negative by morphological criteria
(from two breast carcinomas and one lung carcinoma)
were aneuploids too (false negatives) (Figures 1 and
2).
There was only one false positive according to

from Hodgkin lymphoma for Leu-M1. Omitting
the primary antibody in the above reaction
provided a negative control. Immunostainings
were evaluated without previous knowledge of
the cytology. The development of a brown-stain
in the cytoplasm was taken as a positive result. In
most of the assays for CEA and Ber-EP4, the
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AgNOR values. EMA gave negative results in three
cases with positive cytology, confirmed by other
markers and by the clinical history (one gastric
carcinoma, one ovarian carcinoma and one unknown
origin).
Three cases belonging to the negative group by
conventional cytological analysis of the Papanicolaou

staining was stronger in the cell membranes. In
many smears a cross-staining with haematoxylin
was carried out.
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2. AgNOR technique: The smears previously fixed in
96% ethanol were incubated in the dark for
25 minutes with a mixture of silver nitrate 5% P/
V and gelatine 1% P/V in formic acid 1% V/V
(2 : 1). After washing with deionized water and
D
stained slides were false negatives, with AgNOR
values corresponding to the neoplastic group in two
cases and CEA positivity in the three cases (one breast
carcinoma and two lung carcinomas). EMA was
positive in one of these cases.

BAD QUALITY FIG.

sodium thiosulphate 1% P/V, the slides were
EC

dehydrated and mounted with Canada balsam.21

We calculated the mean value of individual dots
per nucleus, counting 100 nuclei. The AgNORs
were counted only when it was possible to
distinguish each individual dot, free or in clusters.
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Our reference values are: 4.88, s ¼ 1.50 for SE

with reactive mesothelial cells and 13.78, s ¼
3.89 for SE with neoplastic cells.14
3. Assessment of ploidy: The study of ploidy was
carried out with an image-analyser (CAS 200;
Becton-Dickinson). The smears were stained with
CO

the Feulgen technique for CAS. Rat hepatocytes

were used as an external control and human
lymphocytes as an internal control. The DNA
content from each smear was quantified in 150–
200 cells using a quantitative DNA programme
QDA 3.0. The results were presented as histo-
UN

grams.22. By means of the DNA index we com-

pared the position of the histogram peak of a
sample in reference to the position of the diploid
peak. We considered a population as aneuploid 6 Figure 1. DNA histogram of a diploid tumour in an effusion
when it showed a peak with DNA index greater as determined by image analysis.

Cytopathology 2006, 17, 1–7 ª 2006 The Authors

Journal compilation ª 2006 Blackwell Publishing Ltd
 4 L. A. Palaoro et al.

BAD QUALITY FIG.

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6 Figure 2. DNA histogram of an aneuploid tumour in an Figure 3. Neoplastic cells from adenocarcinoma in pleural
effusion as determined by image analysis. fluid showing a high number of AgNOR particles (AgNOR
technique 1250·).

Two cases from the true negative group showed

COLOUR FIG.
slight reactivity for CEA. Ber-EP4 was positive for all
the ovarian, breast and lung carcinomas, but it was

D
negative in the remainder of the malignancies. Only
one benign effusion (cystadenoma of ovary) showed
reactivity for this marker. A false-negative smear TE
(lung carcinoma) was slightly positive for Ber-EP4.
Fourteen of the malignant effusions showed reac-
tivity for Leu-M1 (this marker was investigated in 17
of all positive smears). Of the true negative smears,
one ovarian fibroid and two with peritonitis were
positive for Leu-M1 (Table 3) (Figures 3–6).
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Discussion
Figure 4. CEA positivity in adenocarcinomatous cells from
The predominant task of cytological body fluid study is
pleural fluid (immunocytochemistry – cross-staining with
the confirmation of a benign or malignant effusion. haematoxylin 500·).
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The study of ploidy can be useful to improve

diagnostic efficiency, especially in hypercellular spec-
imens which contain numerous reactive mesothelial
cells which can mimic malignancy, but may also
contain non-apparent malignant cells. These cells can
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be overlooked by the cytopathologist. The assessment
of ploidy in these cases can differentiate diploid cells
(both benign and malignant) from malignant meta-
static cells, which are predominant aneuploid.15

Table 3. Ploidy, AgNOR and immunomarkers in effusions

Cytological diagnosis Ploidy CEA EMA Ber-EP4 Leu-M1 AgNOR True diagnosis

22 Positive 22 Aneuploid and 0 diploid +18 +16 +16 +14 +18 22 Malignant and 0 benign
)1 )3 )4 )3 )1
3NR 3NR 2 NR 5 NR 3NR
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23 Negative 20 Diploid and 3 aneuploid +4 +2 +1 +3 +2 20 Benign and 3 malignant

)14 )15 )18 )14 )17
5 NR 6 NR 4 NR 6 NR 4 NR

CEA, carcinoembryonic antigen; EMA, epithelial membrane antigen; NR, not recorded. AgNOR +: >13.78 particles/nucleus;
AgNOR ): <4.88 particles/nucleus.

Cytopathology 2006, 17, 1–7 ª 2006 The Authors

Journal compilation ª 2006 Blackwell Publishing Ltd
 1 Ploidy, AgNOR and immunomarkers in effusions
COLOUR FIG.
Figure 5. EMA positive in adenocarcinomatous cells from
pleural fluid (immunocytochemistry – cross-staining with
haematoxylin 500·).
COLOUR FIG.
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Figure 6. Neoplastic cells from adenocarcinoma in ascitic
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fluid expressing Ber-EP4 (immunocytochemistry – cross-
staining with haematoxylin 500·).
Euploidy has been reported in some cases of pleural
malignancy.8,24 The percentage of euploid malignant
effusions is higher in malignant mesothelioma.7 As
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there were no malignant mesotheliomas in our
clinical samples, the diploid population was consid-
ered in principle as benign.
Final diagnoses were correlated with cytological
criteria, histological examination, clinical and radio-
logical studies and follow up. In our series, all benign
UN
effusions had euploid DNA values. This fact is in
agreement with many reports.15,16
All the smears with positive cytology were aneup-
loid. Several papers reported aneuploidy in malignant
effusions.15,16 Two cases that were found to be false
Cytopathology 2006, 17, 1–7 ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd
5
negatives by morphological studies showed an aneup-
loid DNA content.
The selective use of a small panel of markers such
as MOC-31, Ber-EP4 and EMA,25 CEA, Ber-EP4 and
Leu-M126 or MOC-31, Ber-EP4 and BG827 has
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been proposed to help in the differentiation
between reactive cells and adenocarcinomatous cells
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in SE.
Anti-EMA is an antibody raised against the mem-
brane of breast epithelial cells. The patterns of EMA
staining within carcinoma cells may appear as diffuse
cytoplasmic or as a rim of intense staining at the
periphery of the cell. Some authors also describe the
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same pattern in mesothelial cells. In our cases, only
one smear showed slight diffuse reactivity for EMA in
a benign pathology. In three true positive cases, EMA
was negative. Although EMA is considered a good
marker for breast and ovarian carcinoma, one of these
last three cases was a metastasis of breast carcinoma.
In summary, EMA showed a moderate sensitivity
D
because of the non-expression of the antigen by a
number of carcinomas.28
We used a monoclonal antibody against CEA, a
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glycosylated cell surface glycoprotein strongly ex-
pressed in many adenocarcinomas (for instance in
adenocarcinomas of the respiratory and gastrointesti-
nal tracts). The use of a monoclonal antibody decrea-
ses the cross-reactivity with non-specific cross-
reacting antigen, present in leucocytes and some
mesothelial cells.29
In our series, reactivity for CEA correlated with
positive cytology except in one case (sensitivity 95%),
but in two cases of true negative cytology CEA was
expressed. We did not find lack of CEA reactivity in
ovarian adenocarcinomas, as reported in other papers.
CEA has limited value because of its low specificity,
being expressed in benign cells.13,30,31
Ber-EP4 antibody was generated using a mem-
brane-enriched fraction derived from the MDF-7
breast cancer cell line.32 This explains the character-
istic membranous staining of the neoplastic cells. In
some papers Ber-EP4 is reported at least as useful as
CEA and Leu-M1.33 In our series all the neoplastic
cells from tumours of ovary, lung and breast were
positive for this marker, but the remainder of the
adenocarcinomas were negative. One of the three
false-negative fluids was positive for Ber-EP4 (sensi-
tivity 74%). We concluded that the sensitivity for Ber-
EP4 is lower compared with the sensitivity for CEA.
For this reason Ber-EP4 is not used in our routine
immunocytochemical studies.
6 L. A. Palaoro et al.
Leu-M1 (CD-15) antibody reacts with the mem-
brane-related trisaccharide fucosyl-N-acetyllactosa-
mine on myelomonocytic cells, but many adeno-
carcinomas can express this marker.34
There is some controversy over the advantages of
using Leu-M1. While some authors think that a positive
reaction for CEA and/or Leu-M1 is the best indicator of
metastatic adenocarcinoma,35 others report that Leu-
M1 is of little value in screening for carcinomatous
cells.36 In the present paper, the assay for Leu-M1 was
developed in 34 smears: it was positive in 14 of the
adenocarcinomas assayed (70%) and in three negative
smears. The false-positive reactivity could be related to
the release of CD-15 by dying inflammatory cells.34 This
low specificity and relative low sensitivity make it
unsuitable for use as a routine marker in the differen-
tiation between malignant and benign cells.
Immunocytochemistry can be useful in discrimin-
ation between malignant mesothelioma and secondary
malignancies. Calretinin, cytokeratin 5/6 or mesothelin
are considered specific markers for mesotheliomas,25,37
but in our cases there were no malignant mesotheli-
oma. In only three positive smears (data not shown) did
we investigate calretinin expression to completely
exclude a mesothelial origin. As there were no malig-
nant mesotheliomas in our series, we concentrated our
efforts on differentiating reactive mesothelial cells from
adenocarcinomatous cells.
AgNOR is a proliferating marker useful in the
EC
differential diagnosis of reactive mesothelial cells and
adenocarcinomatous cells in SE. In two cases with
negative cytology, the values of AgNOR showed the
presence of metastatic carcinoma cells.
There were no false positives in the morphological
RR
examination of the smears, but cytological analysis
had limited value in the interpretation of the false-
negative group.
Taking only the AgNOR index, the sensitivity
increased from 88% to 95%, whilst taking only the
DNA index, the sensitivity increased from 88% to
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100%.
In summary, the assessment of ploidy and the study
of AgNOR are reliable methods for distinguishing
between reactive mesothelial cells and malignant
(adenocarcinomatous) cells in serous fluid.22
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figures along with your proof corrections to the PE.
 MARKED PROOF
ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

Please correct and return this set

ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

Please use the proof correction marks shown below for all alterations and corrections. If
you wish to return your proof by fax you should ensure that all amendments are written
clearly in dark ink and are made well within the page margins.

Instruction to printer Textual mark Marginal mark

Leave unchanged
Insert in text the matter
indicated in the margin
Delete
Delete and close up
Substitute character or
substitute part of one or
more word(s)
Change to italics
Change to capitals
Change to small capitals
Change to bold type
Change to bold italic
Change to lower case
Change italic to upright type (As above)
Insert `superior' character
Insert `inferior' character
Insert full stop
Insert comma
Insert single quotation marks (As above)
Insert double quotation
marks
Insert hyphen
Start new paragraph
No new paragraph
Transpose
Close up
Insert space between letters
Insert space between words
Reduce space between letters
Reduce space between words
under matter to remain Stet
New matter followed by
through matter to be deleted
through matter to be deleted
through letter or through New letter or new word
word
under matter to be changed
under matter to be changed
under matter to be changed
under matter to be changed
under matter to be changed
Encircle matter to be changed
through character or where under character
required e.g.
(As above) over character e.g.
(As above)
(As above)
and/or
(As above) and/or
(As above)
linking letters
between letters affected
between words affected
between letters affected
between words affected