Cytophatologi 2

• The major serous cavities

are the peritoneal cavity,
the pericardial cavity, and
the two pleural cavities.
• Effusion/ascites refers to
an excessive amount of
fluid in a serous cavity.
HISTOLOGY
GENERAL CYTOLOGY (with
Papanicolaou and Diff-Quik stain)
• Serous effusions may
contain a variety of non-
neoplastic cells, including
mesothelial cells,
macrophages, and other
bloodderived cells (Table
1.1),
• together with other entities
such as psammoma bodies
and various incidental
cellular and noncellular
elements (Table 1.2).
☞The morphology of mesothelial cells can be
evaluated in Papanicolaou (PAP) and Diff-Quik (DQ)
stained smears. In general, the PAP stain allows
better evaluation of nuclear details, while the DQ
stain highlights cytoplasmic details
Feature Romanowsky stains (RWS)* Papanicolaou stain

Cell size Size and shape of cells: the cells are Size and shape of cells:
and shape fl at as they collapse during air- slightly shrunken. The
drying, making them slightly larger cell thickness is greater
in dimension along the plane of the in wet-fi xed smears
slide due to its fi xation in
three dimensions closer
to its natural form
Cytoplasmic The cytoplasm is well demonstrated Cytoplasm: is rendered
details by RWS—thus highlighting even the transparent which
scant amount of cytoplasm (such as improves nuclear
in lymphocytes, small cell details In general
carcinoma, etc.), cytoplasmic cytoplasmic details are
vacuoles (renal cell carcinoma, diminished. However,
macrophages, etc.), cytoplasmic this improves the
blebs (mesothelial
• ☞The morphology cells), different
of mesothelial cells canmorphologic
be evaluated evaluation
in
zones in the cytoplasm (mesothelial of cell
Papanicolaou (PAP) and Diff-Quik (DQ) stained smears. In groups, including
general,cells), etc.stain allows better evaluation
the PAP threedimensional
of nuclear
The details
details, while the DQof cell groups
stain are poorly
highlights clusters details
cytoplasmic
visualized
(Table 1.3).
Feature Romanowsky stains (RWS)* Papanicolaou stain
Nuclear The details of nuclear chromatin to Nuclear details are
details evaluate chromatin clumping and excellent with crisp
parachromatin clearing are not clear chromatin staining
However, RWS are excellent for evaluating facilitating evaluation of
nuclear details of hematopoietic cells, as chromatin clumping and
chromatin clumping and parachromatin parachromatin clearing,
clearing are not that significant for which are some of the
evaluating hematopoietic malignancies most important features
Nucleoli: are not as crisp as with the evaluated for
Papanicolaou stain, but they can be seen interpretation of
as pale structures malignancy.
Thus in brief, RWS does not allow Nucleoli: well discerned
evaluation of chromatin clumping and
parachromatin clearing, but it allows
evaluation of N/C ratio, nuclear size,
shapes, nuclear pseudoinclusions, and
nucleoli. Most of these are adequate for
interpretation of hematopoietic lesions
Extracell Excellent staining of extracellular materials These extracellular
ular such as mucin, colloid, materials are poorly
material pseudocartilagenous and cartilagenous stained
matrix, lymphoglandular bodies in *
lymphoproliferative processes, etc.
 Reactive
mesothelial cell
(RM) with
infl ammatory
cells:
lymphocyte (L),
neutrophil (N),
and eosinophil
(E) (pleural fl
uid). [a, PAP-
stained SurePath
Prep; b, DQ-
stained Cytospin
smear (a,b,
100μ; L, N, E,
100μ zoomed).]
 Mesothelial cells
(peritoneal fl uid): show
outer faintly stained
ectoplasm (1) with inner
denser endoplasm (2)
rich in intermediate fi
laments. The nucleus is
usually central or near
central (b), but may be
eccentric (c). Nucleoli are
readily observed. The
vacuolation generally
begins at the periphery
in ectoplasm (1). [b,c,
PAP-stained Autocyte
Prep smear (b,c, 100μ
zoomed).]
Mesothelial cells
Mesothelial cells (a–c) versus adenocarcinoma cells (d–f) with eccentric
nuclei. A thin rim between nuclear border and cell border (1) is seen in
mesothelial cells. In comparison, the nuclear border in adenocarcinoma
cells touches the cell border without a significant cytoplasmic rim (2).
[PAP-stained SurePath Prep smear (b, c, e, f, 100μ zoomed).]

Multinucleated mesothelial cell: a
mesothelial cell with three nuclei
(arrows), which may vary in size.
[PAP-stained SurePath Prep smear
(100μ).]
Monolayered fl at sheet of mesothelial
cells: may resemble squamous metaplastic
cells. The spaces between mesothelial cells,
mesothelial windows, are common (red
arrows). Microvilli prevent the adjacent cells
from apposing their cell borders with each
other. Depending on many variables, the
mesothelial windows may be subtle to very
wide (peritoneal washing). [PAP-stained
Cytospin smear (100μ).]
 Mesothelial windows (arrows): reactive mesothelial cells, pleural fluid. [DQ-
stained Cytospin smear (a–d 100μ; inset of b, 100μ zoomed).]
 Mesothelial windows (arrows): reactive mesothelial cells, pleural fluid.
[PAP-stained SurePath Prep (100μ; inset, 100μ zoomed).]
 Macrophages:
mesothelial and
histiocytic
macrophages show
morphological
overlap. a–d are
morphologically
suggestive of
mesothelial
macrophages; e and f
favor histiocytic
macrophages (pleural
fl uid). [a–f, DQstained
Cytospin smear (100μ
zoomed).]
 Vacuolated mesothelial cells with macrophage features (pleural fl uid).
Panorama of cytomorphologic features with central to slightly eccentric
nuclei. [a–l, DQ-stained cytospin smears (a–l, 100μ zoomed).]
 Mesothelial cells with central to slightly eccentric nuclei (ascitic fluid).
The cytoplasm shows a two-zone staining pattern with peripheral
vacuolation (red arrow 1). For additional ranges see also Figures 2.7, 2.8,
and 2.10. [a–c, PAP-stained ThinPrep smear (a–c, 100μ zoomed).]
GROUPS OF REACTIVE MESOTHELIAL CELLS
• Groups of reactive mesothelial cells in effusions are likely to be the
most important diagnostic pitfall. The conditions associated with
groups of reactive mesothelial cells in sheets in effusions are as
follows:
• Hepatomegaly related to congestive heart failure, leading to
peritoneal effusion with exfoliation of sheets of reactive mesothelial
cells from the surface of the congested liver.
• Ischemic conditions such as pulmonary infarction, ischemic colitis,
and occlusion of mesenteric blood vessels frequently show reactive
changes in the serosal membranes surrounding the ischemic areas.
These reactive mesothelial cells may exfoliate in sheets into the
effusions and may contain intracytoplasmic hemosiderin granules or
red blood cells or both.
• Trauma to organs covered with mesothelium such as spleen, liver,
and lung, etc.
• Large retroperitoneal masses—slowly growing retroperitoneal
masses, including benign retroperitoneal neoplasms growing close
to the peritoneum, may elicit reactive changes in the overlying
mesothelium. This mesothelium tends to exfoliate in sheets into the
peritoneal cavity and may be seen in smears of peritoneal effusions.
• Postoperative—following laparotomy and thoracotomy. Artifactual
desquamation in this situation is secondary to surgical trauma.
Conditions associated with significant
reactive changes in mesothelial cells
• Liver disease (cirrhosis)
• Underlying neoplasia (hamartoma, subserosal implants,
fibroids)
• Foreign substance (talc, asbestos)
• Traumatic irritation (hemodialysis, operative
procedures)
• Chemoradiation therapy Pancreatic disease
(pancreatitis)
• Inflammation and infection (pleuritis, pericarditis)
• Infarction (lung infarct, pulmonary embolism)
• Collagen disorders (rheumatic fever, lupus
erythematosus)
• Renal disease (uremia)
• Chronic inflammation (pelvic inflammatory disease)
 Reactive mesothelial cells in clusters (ascitic fluid), mixed with chronic
inflammatory cells within the groups and between the mesothelial cells in
the background [PAP-stained ThinPrep smear (100μ zoomed).]
Reactive mesothelial cells mixed with chronic infl ammatory cells (ascitic
fluid). Mesothelial cells are present as isolated cells and as small groups
[PAP-stained ThinPrep smear (100μ zoomed).]
 Degenerative intracytoplasmic vacuoles
• Degenerative intracytoplasmic
vacuoles usually
▫ do not occupy the entire
cytoplasm of a cell and
▫ do not show ballooning.
▫ The borders of such
degenerative vacuoles are
usually ill-defi ned (see Figure
4.5b,c).
• In comparison, the true
intracytoplasmic vacuoles with
secretion usually
▫ balloon the entire cell and
▫ occupy most of the cytoplasm,
and
▫ may show secretion in the
lumen (targetoid vacuole)
(Figure 4.7a).
▫ These vacuoles usually have
well-defined borders (Figure
4.7b).
• Ancillary tests, including
histochemistry, such as a periodic
acid–Schiff (PAS) stain with diastase
digestion and a mucicarmine stain,
may help to discriminate between
these entities (Figure 4.8).
• Similar changes may also be
produced when effusion specimens
are left at room temperature for a
long time. These artifacts are
frequent in effusions collected
during the weekend and not
processed immediately
Figure 4.8 Metastatic
mucinous adenocarcinoma
(pleural fluid).
Intracytoplasmic
mucicarmine-positive
mucin (red arrow). [Cell
block section, mucicarmine
stain (100μ).]
 Figure 4.7 Secretory cytoplasmic
vacuoles. a. Metastatic papillary
carcinoma of thyroid (pleural fl uid).
Targetoid secretory vacuole with
colloid in neoplastic cell (blue arrow).
b. Metastatic colonic adenocarcinoma
(ascitic fl uid): intracytoplasmic vacuole
with welldefi ned margin (red arrow).
[a, DQ-stained SurePath smear; b,
PAP-stained Cytospin smear (a,b, 100μ
zoomed).]

Figure 4.5 Degenerative vacuoles in reactive

mesothelial cells (ascitic fl uid). Note relatively fuzzy
boundaries of vacuoles (arrowheads in b,c) without
any secretions (compare with Figure 4.7b). The
secretory vacuoles containing mucin in neoplastic
cells usually show secretion with a targetoid
appearance (compare with Figure 4.7a). Some of
these cells may have nuclear features overlapping
with cancer cells (c) and may be misinterpreted as
cancer cells, especially in patients with clinical
history of adenocarcinoma. RM, reactive mesothelial
cells. [a–c, PAP-stained SurePath preparation (a,
100μ F1 (Focus 1) and F2 (Focus 2); b,c, 100μ
zoomed).]
Figure 4.6 Metastatic ovarian serous papillary carcinoma (ascitic fluid).
Adenocarcinoma cells with degenerative cytoplasmic vacuoles (arrows),
which may resemble adenocarcinoma cells with true secretory vacuoles,
such as those seen in ovarian mucinous cystadenocarcinoma. [a–c,
PAPstained SurePath preparation (a–c, 100μ; insets, 100μ zoomed).]
Figure 4.9 Chronic infl ammatory cells with a few reactive mesothelial cells (pleural fl uid). a.
With DQ stain, the typical nuclear morphology helps to interpret them as polymorphic
lymphocytes, which is consistent with chronic infl ammatory cells. b. With PAP stain, these
chronic infl ammatory cells may resemble cells of lymphoma (compare with Figure 4.12d) and
round blue cell tumors, especially in children. RM, reactive mesothelial cell. [a, DQ-stained
Cytospin smear; b, PAP-stained SurePath smear (a, 100μ; b, 100μ).]
Figure 4.12 Follicular lymphoma (peritoneal fl uid). The lymphoid population with rare reactive mesothelial cells
(arrowheads RM in a,d) resemble chronic infl ammatory cells (see also Figure 4.9) and cells of round blue cell
tumors, especially in PAP-stained preparations (d). The typical nuclear morphology in a DQ-stained preparation
(a–c) helps to interpret the round cells as atypical lymphocytes (arrows b,c,e,f in a,d). The fl ow cytometry
demonstrated a monoclonal lymphoid population. The patient had follicular lymphoma with a colon mass (g).
RM, reactive mesothelial cells. [a–c, DQ-stained Cytospin preparation; d–f, PAP-stained SurePath preparation; g,
hematoxylin and eosin (HE)-stained paraffi n-embedded tissue section of colon mass (a, 100μ; b,c, 100μ
zoomed; d, 100μ, e,f, 100μ zoomed; g, 100μ).]
Atypical mesothelial cells
• Reactive mesothelial cells with
cytomorphology overlapping that of
malignant cells. The cells show:
▫ enlarged nuclei
▫ high nucleocytoplasmic ratios
▫ nuclear hyperchromasia
▫ clumped chromatin
▫ macronucleoli
▫ extensive morphologic variability
▫ the cells may be in large groups with
scalloped borders
Figure 2.12 Malignant epithelioid mesothelioma (pleural fl uid). Mesothelioma cells show numerous large three-
dimensional groups of cells. The individual mesothelioma cells do not show any significant variation from reactive
mesothelial cells without remarkable features of malignancy. The mesothelioma cells, like reactive mesothelial cells, show
two-zone staining (red arrow 1) with peripheral vacuolation (blue arrow 2). [a–h, PAP-stained ThinPrep smear (a,b, 20μ; c,
100μ; d–h, 100μ zoomed).]
 Metastatic adenocarcinoma with a two-cell population (ascitic fl uid). Blue arrow RM
highlights reactive mesothelial cells (central nuclei, peripheral vacuolation, community
borders of cell groups formed by cell membrane) and red arrow NC highlights
adenocarcinoma cells (eccentric nuclei touching the cell membranes without any rim
of cytoplasm between the nucleus and cell membrane; the community border of cell
groups is formed by mostly nuclear contours). [a, DQ-stained Cytospin smear; b, PAP-
stained ThinPrep smear (a,b, 100μ zoomed).]
Figure 8.1 Hypercellular
specimen in a case of
mesothelioma with three-
dimensional clusters and two-
dimensional sheets. [Autocyte
Prep, PAP stain, 10μ.]
Figure 8.2 Moderate cellular
specimen of pleural fl uid with
reactive mesothelial cells with
two-dimensional groups. Infl
ammatory cells are present in
the background. [DQ, 20μ.]
Three-dimensional papillary groups with
knobby outlines (arrow) in a case of
mesothelioma. [Autocyte Prep, PAP stain,
20μ.]
A case of metastatic pancreatic carcinoma in
peritoneal fl uid, highlighting a cohesive
group of neoplastic cells with smooth
community borders (arrow). Compare with
knobby outlines in mesothelioma (Figure
8.3). [DQ stain, 20μ.]
Figure 8.5 Two-dimensional mesothelial cell groups in a case of
mesothelioma with intercellular windows (arrow). Also note a
multinucleate atypical mesothelial cell at the periphery of the group
(arrowhead). [Thinprep, PAP stain, 40μ.]
Figure 8.6 Mesothelial cell with prominent microvilli
(arrow). [Autocyte Prep, PAP stain, 60μ.]
Figure 8.4 Mesothelial cells with two-tone cytoplasm (arrow) and
peripheral cytoplasmic blebs (arrowhead). [Autocyte Prep, PAP stain 40μ.]
Figure 8.7 Cell block from a case of mesothelioma showing
hypercellular atypical mesothelial cell groups. Extracellular
mucinous material is present in the background (arrow).
[Hematoxylin and eosin (HE), 40μ.]
Comparative cytologic evaluation of reactive mesothelial
cells & mesothelioma in effusion fluids
Reactive mesothelial cells
Moderately cellular specimens
Mainly mono-layered sheets
Cell groups (relatively smaller) with
knobbly outlines
Intercellular windows present
No acinus formation
Mild size variability
Giant mesothelial cells and multinucleate
cells usually absent
Peripheral cytoplasmic blebs and
microvilli may be present, but not very
prominent
Nuclear features of malignancy—
pleomorphic and enlarged nuclei,
prominent nucleoli, and atypical mitoses—
are not prominent
Mesothelioma
Hypercellular specimens
Two-dimensional sheets and
three-dimensional cell groups
Cell groups (relatively larger)
with knobbly outlines
Intercellular windows present
No acinus formation
Greater variation in size
May be present
Usually prominent
May be present
 Comparative cytologic evaluation of mesothelioma and
adenocarcinoma in effusion fluids
Mesothelioma
Hypercellular specimens
Two & three dimensional cell groups
Knobbly outlines to cell groups
Acinus formation usually not present
Cellular variability present
Giant mesothelial cells present
Nuclear features of malignancy—
pleomorphic & enlarged nuclei,
prominent nucleoli, & atypical mitoses,
may be subtle but usually present
Two-tone cytoplasmic appearance
present
Intercellular windows present
Peripheral cytoplasmic blebs with
microvilli present
Spectrum of changes without a distinct
‘foreign’ population
Adenocarcinoma
Hypercellular specimens
Two & three dimensional cell groups
Smooth contours (‘community borders’)
Usually present
Cellular variability present
Bizarre malignant cells present
Nuclear features of malignancy—
pleomorphic and enlarged nuclei,
prominent nucleoli, and atypical
mitoses—are present
Absent
Absent
Absent
Usually identifi able as a ‘foreign’ cell
population