EFFUSION CYTOLOGY

Dr. Adokorach Gladys

Introduction
• The pleural, pericardial and peritoneal cavities are lined by a
single layer of flat mesothelial cells called the serosa.
• These are normally collapsed and contain only a small
amount of fluid.
• In disease states, a greater amount of effusion accumulates.
• Classified as transudative or exudative.
Transudate
• Result from imbalance of hydrostatic and oncotic pressures
• Common causes: Congestive heart failure, cirrhosis and nephrotic
syndrome.
• They have low LDH
• Low protein concentration <3.0g/dL.
• Low specific gravity <1.015
• Low cellular count
• Cellular content usually consists of mesothelial cells and macrophages
with an occasional lymphocytes or neutrophilic leukocyte
Exudates
• Result from injury to the mesothelium
• Causes: Malignancy, pneumonia, Lupus, rheumatoid pleuritis,
pulmonary infarction or trauma.
• Have high LDH and total protein concentration.
• Higher cellular count
• The distinction between transudate and exudate is important because
pleural involvement by a malignancy causes formation of an exudate
and therefore cytologic examination is not needed for a transudate.
Specimen collection
• Specimens obtained by inserting a needle into the pleural space,
pericardial space or peritoneal cavity.
• Care should be taken when withdrawing large amounts of pleural fluid
because of the rare but life threatening complication of re-expansion
pulmonary edema.
• Ascitic fluid in women can also be obtained through cul-de-sac
through the vagina (Culdocentesis).
• Fluid is collected in clean containers and sent unfixed to the
laboratory.
• Fluid is refrigerated at 4°C until the time of slide preparation.
• Effusion specimen can be refrigerated for 2 weeks or longer
without compromising cellular morphology or antigenicity
for immunostains.
Gross appearance of Serous effusions
• Sometimes reveals clues about the cause of the effusion and the
nature of its cellular contents.
• Note should be made of its volume, color, clarity and unusual physical
features such as malodor, opalescence or high viscosity.
• Fluids containing numerous pigmented melanoma cells may be
chocolate brown.
• Old hemorrhagic fluid in the serous cavity appear much lighter brown
due to many hemosiderophages.
• Dark brown-orange or greenish fluid may be due to jaundice
• Effusions containing numerous cholesterol crystals are yellow and
turbid and have a swirling, shimmering, gold paint appearance when
agitated.
• Pseudomyxoma peritonei- Heavy and mucoid. Making it difficult to
aspirate them.
• Chylous effusions have a milky white appearance with a creamy
topmost layer due to their high concentration of emulsified lipid.
Preparatory technique
• A toluidine blue-stained wet film
• Liquid-based preparation (Widely used)
• Cell-blocks
• Immunocytochemistry and molecular studies done on cell block
material
Uses of Wet films
• Extremely simple and quick
• Reveals a distinctly diagnostic cellular picture enabling a report to be
issued within 10-15minutes after receiving the specimen in the
laboratory
• Enables one to identify the super-positive serous effusions, those
teeming with cancer cells.
• May enable one to immediately identify unusual or interesting
cytologic specimens thereby providing an opportunity to prepare
additional smears before the sediment is clotted for preparation of
the cell block.
Usefulness of cell blocks
• Spontaneous clot may enmesh virtually all the cells
• Increases the percentage of positive results
• May reveal histologic aspects of neoplasm
• May reveal entities not visible in smears
Normal cells
• Erythrocytes
• Leukocytes
• Histiocytes
• Mesothelial cells
• The proportion of the different types of these cells varies considerably
depending on certain circumstances such as the cause and duration of
the effusion and the presence or absence of inflammation.
Mesothelial cells
• Undergo hypertrophy and hyperplasia in response to a wide variety of
stimuli such as;
• Inflammation, necrosis of parenchyma, foreign substances such as air,
blood, sterile effusion.
• In presence of an effusion, whatever its cause, mesothelial cells
exfoliate often in large numbers either as isolated cells or as cohesive
clusters of cells or both.
Cytology
• Round to oval nucleus with smooth contours
• Great size variation
• Usually one nucleus
• The chromatin is uniformly granular and nucleoli are readily
identified.
• Giant mesothelial cells are always multinucleated often with >10
nuclei.
• Cytoplasm gray-green to slightly eosinophilic
• Mosaic clusters, monolayer strips and collagen balls in washings
Mesothelial cells in a background of inflammatory cells. Note the dense, lightly cyanophilic
cytoplasm of the mesothelial cells, which tends to fade at the periphery of the cell, their articulation
at flattened apposing cell surfaces, and the smoothly contoured central and eccentric nuclei with
prominent nucleoli. Smear of pleural effusion (Papanicolaou, ×MP).
Giant multinucleated mesothelial cell
It is not uncommon to find a mesothelial cell with a large, solitary
vacuole and an eccentric nucleus joined to a typical non-vacuolated
mesothelial cell, clearly denoting the mesothelial lineage and benign
nature of the vacuolated cell
Mesothelial cells articulate with each other
in characteristic manner
1. Little chains of cells joined at flattened or curved apposing surfaces

A short chain of
mesothelial cells joined
at apposed surfaces.
Windows are forming
between the cells.
2. Larger sheets of mesothelial cells with a mosaic
appearance composed of about ≥10 cells

A tissue fragment
composed of a mosaic of
hypertrophied
mesothelial cells. Note
the partly scalloped
contour of the fragment.
3. Cytoplasm of one cell may appear to be
clasping another cell. Common in
adenocarcinoma cells in serous fluids
Pair of mesothelial cells
showing the type of
articulation where one
cell seems to be clasping
another. Smear of pleural
effusion (Papanicolaou,
×OI).
Mesothelial cells in a cell ball formation with a
3-dimensional appearance.
A collagen ball in a peritoneal washing. It is 3-dimensional with a
collagen core covered by mesothelial cells, which are perceived to be on
different levels by focusing up and down.
Atypical and Reactive Mesothelial cells

A pair of hypertrophied
mesothelial cells from a
patient with hepatic
cirrhosis. Benign
mesothelial cells such as
these are frequently
reported as “atypical” or
“reactive.”
Non-neoplastic conditions
Acute serositis
• Acute pleuritis, pericarditis and peritonitis are usually the result of a
bacterial infection.
• Bacterial infection of the pleura occurs in the setting of pneumonia
which secondarily involves the overlying pleura and results in a
pleural empyema.
• Acute infection of the peritoneal cavity is often secondary to
inflammation of or injury to the bowel as in spontaneous bacterial
peritonitis
• The fluid is a creamy pale yellow (purulent) and often foul-smelling.
• Cytologic preparations are highly cellular and composed almost
exclusively of polymorphonuclear leukocytes.
• Bacteria are demonstrated with special stains in some cases.
 Eosinophilic effusions
• A pleural effusion is considered “eosinophilic” when
eosinophils account for 10% or more of the nucleated cells
present.

• Between 5% and 16% of exudative effusions are eosinophilic

effusions.
• The most common causes are pneumothorax and hemothorax.
• Others include drug reaction, parasitic infections, pulmonary
infarctions and the Churg Strauss syndrome.
• Eosinophilic pericardial and peritoneal effusions are less common
than eosinophilic pleural effusions.
• Cytologic preparations are usually cellular and remarkable for a high
concentration of eosinophils.
• On alcohol-fixed Papanicolaou-stained slides, the defining eosinophilic
cytoplasmic granules are either Oran geophilic or pale-green and
inconspicuous, and the cells are identified more on the basis of their
bilobed nuclei
Eosinophilic pleural
effusion. Numerous
eosinophils in pleural
fluid are more commonly
associated with benign
conditions, like a
pneumothorax (as in this
case) or hemothorax.
Lymphocytic effusion
• Highly cellular
• Composed almost exclusively of dispersed, small
lymphocytes
• Mesothelial cells and histiocytes are either
conspicuously absent or present in small numbers
Smear of pleural
effusion containing
numerous small
mature lymphocytes
(Papanicolaou, ×HP).
Differential Dx

• Malignancy
• Tuberculosis
• Status post coronary artery by-pass
Rheumatoid Pleuritis- Cytomorphology
• Cytologic picture is so characteristic that it has been
termed pathognomonic
• Cytologic preparations are sparsely or moderately
cellular.
• Abundant clumps of granular debris
• Macrophages
Scattered multinucleated histiocytes and clumped granular debris in the
background are characteristic of pleural fluids in patients with
rheumatoid pleuritis.
Rheumatoid pleuritis. A
field dominated by
amorphous granular
material in which there
is a solitary giant
multinucleated
histiocyte.
(Papanicolaou, ×LP).
Lupus Pleuritis
• The characteristic cell is the lupus erythematosus (LE) cell, a
neutrophil or macrophage that contains an ingested cytoplasmic
particle called a hematoxylin body.
• The hematoxylin body may be green, blue, or purple with the
Papanicolaou stain, and magenta with Romanowsky type stains, and
has a glassy, homogeneous appearance.
• Filling the cytoplasm of the neutrophil or macrophage, it often pushes
the nucleus to one side, indenting it into a crescent-like shape.
Hematoxylin body (lupus pleuritis)

The lobes of the nucleus are

pushed against the side of the
neutrophil by a large,
homogeneous,
intracytoplasmic body
(Wright-Giemsa).
Smear of pericardial effusion illustrating two LE cells in a
background of neutrophilic leukocytes (Papanicolaou, ×HP).
Congestive heart failure- cytologic features
• Non-specific cytologic features
• Usually low to moderate number of neutrophils
• Occasional hemosiderophages
Pneumonia- cytologic feature
• Inflammatory cells of various types depending on the nature and
duration of the pneumonia
INFARCT
• Non-specific inflammatory picture
• Mixture of inflammatory cells
Tuberculosis- cytologic features
• High proportion of lymphocytes and few mesothelial
cells
• If secondary infection occurs the effusion becomes
purulent
HEPATIC CIRRHOSIS- CYTOLOGIC
FEATURES
• Longstanding peritoneal effusions are typical
• Mesothelial cells, lymphocytes and histiocytes
• More than a few neutrophils raises the question of spontaneous
bacterial peritonitis.
• For some reason, peritoneal effusions from patients with hepatic
cirrhosis seem to pose a problem in cytodiagnosis owing to the
presence of hypertrophied mesothelial cells, either solitary or in
clusters.
PARASTIC INFECTIONS

Echinococcus granulosus Paragonium westermani

MALIGNANT EFFUSIONS
• Some tumors have a greater tendency than others to spread to the
pleural, pericardium or peritoneum.
• In children, the most common cause of a malignant pleural or
peritoneal effusion is Non-Hodgkin lymphoma.
Tips for detecting malignant cells in
effusion
1. Identify benign mesothelial cells first
2. Search for a second population that is clearly different (Not
counting lymphocytes or histiocytes)
3. Numerous large clusters- exception is mesothelioma
4. Malignant cells in cells block sections are frequently
situated in lacunae.
Cell block lacunae (pleural
fluid). In cell block sections,
malignant cells are often
situated in an empty space
(lacuna); the reason for this
artifact is unknown. It is
commonly seen with
adenocarcinomas, rarely
with lymphomas and
melanoma
(hematoxylin-eosin [H & E]
stain).
PRIMARY TUMORS
• Mesothelioma
• Primary effusion lymphoma
DIFFUSE MALIGNANT
MESOTHELIOMA
• Account for less than 2% of malignant effusions.
• Strongly linked to asbestos exposure.
• It arises most commonly in the pleura and less commonly in
the peritoneum.
• Primary tumors of the pericardium or tunica vaginalis of the
testis are rare.
• Latency period 30 to 40years
Histologic classification
• Epithelioid- Variants (Tubopapillary, adenomatoid, sheetlike,
deciduoid, small cell and clear cell)

• Sarcomatoid

• Desmoplastic

• Biphasic
• Immonoreactive for cytokeratins, desmin, calretinin, Wilms Tumor
protein 1(WT1) and D2-40
• Common symptoms: Chest pain, and shortness of breath
Morular pattern

Solid, morule-like
spheres, some of them
elongated, are composed
of cells that resemble
normal mesothelial cells.
A fluid composed of
many large clusters is
virtually always
malignant (Papanicolaou
stain).
Mulberry clusters
Branching pattern
A branching pattern
is seen in some
cases. Note the
knobby contours
(Papanicolaou
stain).
In most mesotheliomas, the nuclear-to-cytoplasmic ratio of
normal mesothelial cells is recapitulated (Papanicolaou
stain)
 In other cases, the nuclear to-cytoplasmic ratio is
significantly increased (Papanicolaou stain).
Cytoplasmic vacoulization
Differential diagnosis
• Reactive mesothelial cells
• Metastatic tumor
• Adenocarcinoma
• Squamous cell carcinoma
• Epithelioid hemangioendothelioma
• Epithelioid angiosarcoma
MESOTHELIOMA Vs REACTIVE
MESOTHELIAL CELLS
• Because reactive mesothelial cells of the pleura and peritoneum do
not form numerous large morulae, the diagnosis of mesothelioma is
straightforward when the specimen is highly cellular and contains
many large clusters of enlarged mesothelial cells.
• In almost all cases, mesotheliomas show clonal cytogenetic
aberrations indicative of malignancy, the most common being
deletions of 1p, 3p, 6q, 9p, and 22q.
• With a combination of appropriate probes, some deletions can be
detected by fluorescence in situ hybridization (FISH)
A, Reactive mesothelial cells can show some variation in nuclear size and nuclear membrane irregularity (Papanicolaou
stain). B, Mesotheliomas usually show greater cytomegaly, but this can be difficult to assess on a case-by-case basis
(Papanicolaou stain).
Mesothelioma Vs Adenocarcinoma
• Two distinct population in adenocarcinoma
• Tumor cells separated by slit-like windows and have abundant dense
cytoplasm are more likely to be mesothelial in origin.
• Mesothelioma cells form a morphologic continuum with benign
appearing mesothelial cells at one end.
• On cell block sections, a core of edematous collagen and stromal cells,
surrounded by neoplastic cells is more commonly seen in
mesothelioma than in adenocarcinoma
• Ring-like structures with hallow cores seen in adenocarcinoma are
very uncommon in mesothelioma.
• Clusters with a knobby (Mulberry-like) contour, rather than smooth,
cannonball-like edge of many adenocarcinoma is characteristic of
mesotheliomas
PRIMARY EFFUSION LYMPHOMA
• Rare subtype of diffuse large B-cell lymphoma that is associated with
HHV-8 and manifests with a pleural, pericardial or peritoneal effusion.
• All cases are positive for HHV-8 and its detection is important for
confirming the diagnosis.
• Most cases arise in setting of HIV
• Prognosis is poor; median survival is less than 6months.
Cytomorphology
• Dispersed large cells
• Round or irregular nucleus
• Prominent nucleolus
• Abundant basophilic cytoplasm
Primary effusion lymphoma (PEL). A, The malignant cells are large, with thick nuclear membranes, irregularly distributed
chromatin, and prominent nucleoli. Apoptotic bodies are present (Papanicolaou stain). B, The presence of human
herpesvirus 8 (HHV-8), demonstrated here by immunohistochemistry, is a sine qua non of PELs.
Differential Diagnosis
• Diffuse large B-cell lymphoma other than PEL
• Pyothorax associated lymphoma
• Anaplastic large cell lymphoma
• Post-transplant lymphoproliferative disorder
• Carcinoma
• Melanoma
METASTATIC TUMORS
ADENOCARCINOMA
• Most common metastatic tumor found in effusions.
• Cytomorphology
• Large spheres or isolated cells
• Cytoplasmic vacuolization
• Signet ring cells (Gastric, breast)
Adenocarcinoma of the lung (pleural fluid). Clusters of very large, highly atypical
cells like these are easily spotted and identified as malignant, but in the absence
of a known primary, special stains might be needed for precise classification
(Papanicolaou stain).
Ductal carcinoma of the breast (pleural fluid). A,Ductal breast cancers often
exfoliate as large spheres of malignant cells (Papanicolaou stain). B, The
hollow nature of the spheres is apparent on cell block sections [H & E] stain.
Adenocarcinoma of the stomach (pleural fluid). Large numbers of
isolated signet ring cells are characteristic of many gastric cancers
(Papanicolaou stain).
DDx
• Reactive mesothelial cells
• Mesothelioma
Squamous cell carcinoma- Cytomorphology
• Large clusters or isolated cells
• Keratinized or non-keratinized
• Dense cytoplasm
Squamous cell carcinoma (SQC) of the cervix (pericardial fluid). Nonkeratinizing squamous cell
cancers shed large spheres of malignant cells (A, Papanicolaou stained cytocentrifuge
preparation. B, hematoxylin-eosin [H &E]–stained cell block preparation).
Squamous cell carcinoma (SQC) of the lung (pleural
fluid). The malignant cells have coarsely textured
chromatin and platelike cytoplasm (Papanicolaou stain).
SMALL CELL CARCINOMA
• Small cells (Isolated in chains
and clusters)
• Nuclear molding
• Scant cytoplasm
Malignant melanoma- Cytomorphology
• Effusion possibly straw-colored or light to dark brown
• Cells isolated or in small to medium sized clusters
• Cytoplasm usually pigmented, light to heavy brown but may be
amelanotic
Reporting results- Example
• Positive- consistent with metastatic adenocarcinoma
• Negative- numerous neutrophilic leukocytes
• Negative- cytologic picture compatible with rheumatoid pleuritis
• END