Fluid cytology in serous cavity effusions

Fluid Cytology
SEROUS CAVITY EFFUSIONS
Part II
By
Moderator : Dr B P Nag
Student : Dr Tashi Agarwal

Closed cavities of the body
1. Pleural Cavity
2. Pericardial Cavity
3. Peritoneal Cavity

Pleural fluid – Lung – Thoracocentesis.

Pericardial fluid – Heart
Pericardiocentesis.

Peritoneal fluid – Abdominal cavity –
Paracentesis/Peritoneal washing.

• The intrathoracic and intraperitoneal organs are
covered by a single layer of mesothelial cells, which is
continuous with the lining of the thoracic and peritoneal
cavities. The potential space between the two layers of
epithelium contains a small amount of lubricating fluid.
•Serous fluid lies between the membranes lining the
body cavities(parietal) and those covering the organs
within the cavities(visceral).
COMPOSITION AND FORMATION

• Production and reabsorption are normally at a
constant rate. They are influenced by
– Changes in osmotic and hydrostatic pressure in the
blood.
– Concentration of chemical constituents in the plasma
– Permeability of blood vessels and membranes.
• An accumulation of fluid, called an effusion,
results from an imbalance of fluid production
and reabsorption. This fluid accumulation in the
pleural, pericardial, and peritoneal cavities is
known as serous effusion.

Causes:
1. Increased Hydrostatic Pressure
• Congestive heart failure
2. Decreased Colloid Pressure
• Hypoproteinemia
• Increased capillary permeability (inflammation
and infection)
• Lymphatic obstruction (tumors)

Histology of the Epithelium
• Serous membranes consist
of connective tissue that is
normally lined by a single
layer of mesothelial cells
• The mesothelial cells lining
the fibrous tissue are flat(1)
• Focal reactive changes are
seen as hypertrophy of
some cells which assume a
cuboidal contour.(2)(3)
mesothelium
Image courtesy of Boston University
peritoneal cavity
Connective tissue of serosa

Biologic nature of effusion
• Effusions are classified into four categories
– Hydrostatic
– Infectious
– Noninfectious inflammatory
– Malignant
• Each category can be one of two types
– Transudate
– Exudate

Hydrostatic Effusions
– Due to imbalance of intravascular pressure
resulting in increased flow of plasma into body
cavities
– Higher fluid to cell ratio
– Low protein level
– Cardiac failure is common cause

Infectious Effusions
– Due to direct effects of organism or as by-
product of inflammation
– Varying types of inflammatory cells can be
present
– Cultures and/or gram and acid fast stains may
be useful to identify an organism

Noninfectious Inflammatory
Effusions
– Due to autoimmune conditions or reaction to a
stimulus
– Autoimmune conditions include rheumatoid
arthritis and systemic lupus
– Reactive conditions include tissue necrosis or
radiation therapy

Malignant Effusions
– Due to a primary tumor (such as
mesothelioma) or a metastatic tumor
– Patient history is extremely helpful in
rendering a diagnosis
– Knowledge of the appearance of the fluid is
also helpful (cloudy, bloody, partially clotted)

Classification of serous fluids
Transudates & Exudates
Transudative
• Hepatic cirrhosis
• Hypoproteinemia eg. Nephrotic Syndrome

Exudative
• Bacterial pneumonia/peritonoitis.
• Tuberculosis, other granulomatous diseases
• Viral or mycoplasma infection
• Pancreatitis, bile peritonitis.
• Neoplasms: Bronchogenic carcinoma, metastatic
carcinoma, lymphoma, mesothelioma, pulmonary
infarct, hepatoma, ovarian cancer, prostate cancer.
• Non Infectious inflammatory disease: rheumatoid
disease, SLE

FEATURE TRANSUDATE EXUDATE
Definition Filterate of blood plasma
without changes in endothelial
permeability.
Due to physiomechanical
factors.
Oedema of inflamed tissue
associated with increased
vascular permeability,
damage to serous
membranes.
Character Non-inflammatory oedema Inflammatory oedema
Grossly Typically clear, pale yellow fluid Usually cloudy, yellow or
bloody
Protein content Low, no tendency to coagulate
as mainly albumin, no
fibrinogen.
High, readily coagulates due
to high content of fibrinogen.
Glucose content Same as plasma Low
Specific gravity Low High
pH >7.23 <7.23
LDH Low High
Effusion LDH/Serum LDH ratio <0.6 >0.6
Cells Few cells, mainly mesothelial
cells and cellular debris
Many cells, inflammatory as
well as parenchymal.

Light's Criteria
• If at least one of the following three criteria is
present, the fluid is virtually always an
exudate
• If none is present, the fluid is virtually always a
transudate
1. Pleural fluid protein/serum protein ratio greater than 0.5.
2. Pleural fluid LDH/serum LDH ratio greater than 0.6.
• Pleural fluid LDH greater than two thirds the upper limits
of normal of the serum LDH

Pleural Fluid
• Sources of benign conditions
– Primary pulmonary infection or infarction
– Secondary due to abdominal disease, i.e.
cirrhosis
• Sources of malignant conditions
– Most common sources are lung, breast,
lymphoid neoplasms, GI and mesothelioma

Pericardial Fluid
– Congestive heart failure, infection,
radiation, hypothyroidism, uremia,
hypoproteinemia
– Commonly associated with lung or breast,
followed by lymphoma, sarcoma and
melanoma

Peritoneal Fluid
– Portal venous hypertension,
hypoproteinemia and salt retention
– Commonly associated with ovary, breast,
GI, lymphoid neoplasms and mesothelioma
– In malignancies of unknown origin,
consider genital tract for women and GI
tract for men

COLLECTION
• Fluid is collected by needle aspiration (100
mL) from the respective cavities.
• Serous fluids – EDTA for cell counts and
morphology. For other tests in Heparin or
blood culture tubes.
• Fresh specimens for cytology may be stored
upto 48 hours in the refrigerator with
satisfactory results.

RECOMMENDED TESTS
ROUTINE
• Gross examination
• Pleural fluid/serum protein ratio
• Pleural fluid/serum LD ratio
• Examination of Romanowski-stained
smear(malignant cells, LE cells)
Useful tests in most patients
• Stains and cultures for microorganisms
• Cytology

Additional testing may aide in
diagnosis
• Glucose – Low in TB, rheumatoid disease and bacterial
infections
• Amylase – High in pancreatitis, esophageal perforation
• pH – Low in rheumatoid disease, hemothorax, acidosis
• Ammonia –Intestinal necrosis, perforation, effusion(-) vs.
urine(+)
• Creatinine – Negative in effusion, positive in urinothorax
• Bile – Included in the differential dx of green appearing
effusion
• Cell Block and Immunochemistry*– Tumor typing

• Electron Microscopy – Differentiate adenoca vs.
mesothelioma
• Flow Cytometry – Useful in hematopoietic malignancies.
• ADA - >40 U/L rapid evidence of tuberculous effusions.
• INF-Gamma – increased in tuberculous pleuritis.
• Lipids – chylous from pseudochylous.
• CRP – screening test for organ disease, parapneumonic
infections.
• TSA (Tuberculostearic acid) – Tuberculosis.
• Tumour markers – CEA, PSA, CA-125.
• Fibronectin – in malignant ascites.

SAAG
Diagnosis of
ascites due to
portal
hypertension is
established by
measurement of
serum ascites
albumin
gradient(SAAG).

•Quantity
• Colour
• Appearance
• Clot
• Turbid fluids - Supernatant Clear - Cellular elements
Hazy - Chylous (obst. of Thoracic duct)
- Pseudochylous(Rh pleuritis, TB, Myxedema)
MACROSCOPIC
EXAMINATION

Feature Chylous Pseudochylous
Onset Sudden Gradual
Appearance Milky-white, or yellow
to bloody
Milky or greenish,
metallic sheen
Microscopic
Examination
Lymphocytosis Mixed cellular
reaction, cholestrol
crystals
Triglycerides >110 mg/dL <50 mg/dL
Lipoprotein
electrophoresis
Chylomicrons present Chylomicrons absent

MICROSCOPIC EXAMINATION
Cell counts :
• Total cell counts may be performed using
manual hemocytometer methods, however,
automated cell counts are increasingly used
with serous fluid specimens.
• Differential counts

N X Dilution factor
Area of total squares counted X Depth
Total cell count =
*Correlation of cell count with cytomorphological findings is essential.
Calculation of Cell count

CELL COUNT – PLEURAL FLUID
Total WBC count is almost never diagnostic
• >10,000/uL : Inflammation
• >50,000/uL : Parapneumonic effusions, usually
empyema.
• <5,000/uL : Chronic exudates(malignancy, TB)
• <1,000/uL : Transudates

CELL COUNT – PERITONEAL FLUID
WBC count > 250/uL
and Neutrophils > 50%
Bacterial peritonitis

Normal Components
1. Mesothelial cells
2. Histiocytes
3. Neutrophils
4. Lymphocytes
5. Eosinophils

Mesothelial cells
Bland cells forming a monolayer covering serous surfaces of body
cavities. (20 to 40 microns in diameter).
- Nuclei
• Single or binucleated
• Centrally located but can be eccentric
• Round to oval with well-defined, smooth nuclear borders
• Fine chromatin
• Inconspicuous nucleoli
– Cytoplasm
• Dense center with pale periphery
• Lacy “skirt” cell borders
• Blunt cytoplasmic processes due to degeneration

Sheets of benign mesothelial can contain upwards of 50 cells. 20x

40x
Copyright © 2012 Hologic, All rights reserved.

20x
Pericardial effusion: Pericardiocentesis may yield sheets of benign mesothelial cells
similar to those found in other body cavity effusions.

60X

Normal Components
• Mesothelial cells in peritoneal washings
– Broad and flat sheets
– Slightly rounded up
– Cuboidal

20x
Specimen type: peritoneal wash
Sometimes sheets fold so that overlapping of cells is evident. The benign nature of these
mesothelial cells is obvious based on their uniform arrangement and appearance.

40x
Peritoneal wash.
Benign mesothelial cells from a peritoneal wash can appear to be rounded up into a
more 3D group with some overlap.

40x
Pelvic wash: Benign mesothelial cells. Mesothelial cells can also have a cuboidal
appearance with more condensed cytoplasm. Again, note the cohesive sheet of cells
with uniformity of nuclei throughout.

Normal Components
• Histiocytes
– Usually present in effusion. Prominent in cancer, TB and
embolism
• Lymphocytes
– Some amount usually present. Can be numerous in conditions
ranging from congestive heart failure and infections to carcinoma
and lymphoma
• Eosinophils
– Usually a good prognosis. Most eosinophilic effusions are pleural
and due to allergic reactions to dust. Other causes include
pulmonary infarct, pneumonia, trauma, hydatid disease
• Neutrophils
– Presence can have many causes, particularly infection. Malignant
effusions are seldom associated with acute inflammation

20x
Histiocytes have smaller nuclei than mesothelial cells with granular or vacuolated
cytoplasm. Compare these histiocytes with the single mesothelial cell at the arrow.

Pleural cytology,
nonspecific exudate:
dormant
mesothelial cell (or
serosal cover cell) (1),
phagocytic macrophage
with vacuoles
(2), and monocytes (3),
in addition to
segmented neutrophilic
granulocytes
(4).

60x
Abnormal Findings

Lymphocytosis (>50%)
• Tuberculosis
• Viral infection
• Malignancy (lymphoma, other neoplasms)
• True chylothorax
• Rheumatoid pleuritis
• Systemic lupus erythematosus
• Uremic effusions
• Transudates (≈30%)

40x
Pleural fluid, 40x. Benign lymphocytes mixed in a proteinaceous background with
benign mesothelial cells.

NEUTROPHILIA(50%)
• Bacterial pneumonia (parapneumonic
effusion)
• Pulmonary infarction
• Pancreatitis
• Subphrenic abscess
• Early tuberculosis
• Transudates (>10%)

Eosinophilia (>10%)
• Pneumothorax (air in pleural space)
• Trauma
• Pulmonary infarction
• Infection (especially parasitic, fungal)
• Hypersensitivity syndromes
• Drug reaction
• Rheumatologic diseases
• Hodgkin disease
• Idiopathic

60x

Abnormal Components
• Microorganisms
• LE cells
• Sickle cells
• Charcot-Leyden crystals
• Psammoma bodies
• Collagen balls

LE Cells
LE cell: Wright-stained smear of the pleural fluid shows a typical single LE cell
consisting of a neutrophil with flattened nucleus engulfing a central paler
homogenous mass.

Non-cellular Components
Cholesterol crystals
Peritoneal wash
Starch granules
Peritoneal wash
20x 60xCopyright © 2012 Hologic, All rights reserved. Copyright © 2012 Hologic, All rights reserved.

Reactive changes
• Reactive changes are common and may be due
to:
– Pulmonary embolism or infarct
– Active cirrhosis or hepatitis
– Uremia
– Pancreatitis
– Long-term dialysis
– Radiation and chemotherapy
*usually accompanied with marked inflammation.

Reactive changes
• Features of reactive mesothelial cells include:
– Varying cell sizes
– Increased numbers of mesothelial cells
– Enlarged central or paracentral nuclei
– Binucleation or multinucleation
– Coarsening of chromatin that remains evenly
distributed
– Chromocenters and nucleoli may become prominent
– “Sibling images” are a clue

60x
Pleural effusion : Reactive mesothelial cells can have a wide range of sizes.

60x
Pleural effusion: Enlarged nuclei, small multiple nucleoli and spaces “windows” between
adjacent cells. Inflammatory cells are present in the background.

60x
Pleural fluid: Reactive mesothelial cells can exhibit coarse chromatin, irregular nuclear
outlines and nucleoli. It is important to look for a spectrum of changes and to correlate
the cytology with clinical history. Notice the “windows”.

60x
Reactive mesothelial cells tend to present in a more monolayer arrangement and not
in the large 3-dimensional clusters of malignant mesothelioma. Again notice the
“windows”.

Suggested Immunochemistry Markers
Reactive mesothelial cells
• Calretinin +
• CK 5/6 +
• p53 -
• Desmin +
• EMA -
Mesothelioma
• Calretinin +
• CK 5/6 +
• p53 +
• Desmin -
• EMA +

• Metastatic:
– Adenocarcinoma
– Squamous Cell
Carcinoma
– Neuroendocrine
Tumors
– Lymphoma/leukemia
– Melanoma
– Sarcoma
– Other Neoplasms
• Primary:
– Malignant
Mesothelioma
– Effusion Lymphoma
Tumors causing malignant effusions:

Malignant Mesothelioma
• Very rare, usually associated with asbestos
exposure
• Most cases occur in men aged 50 to 70
years, particularly with a background in
auto mechanics or construction
• Commonly found in the pleural cavity but
can occur in the peritoneal cavity as well

Malignant Mesothelioma
• Cytologic features:
– One cell population of malignant mesothelial cells
– Larger clusters with irregular, knobby, flower-like
borders
– Dense endoplasm with delicate, lacy ectoplasm
– Central enlarged nucleus with macronucleoli and
irregular borders
– Abnormal mitotic figures may be seen but are not
diagnostic of mesothelioma

10x
Pleural effusion (Mesothelioma) - Various magnifications show “cannon-ball” mass of
tumor cells. Key to diagnosis is the single cell population. Individual malignant
mesothelial cells exhibit rim of ruffled, less dense cytoplasm, surrounding dense
cytoplasm around the nucleus.

20x
Pleural effusion - Numerous large clusters of tumor cells may be seen in a background
of blood and proteinaceous debris.

60x
Pleural Fluid – Abnormal mitotic figures may be noted but can also be associated with
other malignancies, as well as reactive mesothelial cells.

Mesothelioma
• Calretinin +
• WT-1 +
• CEA -
• TTF-1 -
• MOC-31 -
• Ber-EP4 -
• B72.3 -
Adenocarcinoma
• Calretinin -
• WT-1 -
• CEA +
• TTF-1* +
• MOC-31 +
• Ber-EP4 +
• B72.3 +
*Lung and thyroid only

Primary Effusion Lymphoma
• Very rare subtype of diffuse large B-cell
lymphoma
• Associated with human herpes virus 8 (HHV-
8)
• Pleural, pericardial or peritoneal effusion
• Most cases occur in HIV positive patients with
rare cases in immunocompromised patients
• Absence of mass lesion and HHV-8 positivity
is essential for diagnosis

• Population of single, large cells
• Nuclei range from round to irregular and
lobulated
• Coarse chromatin
• Prominent, irregular nucleoli
• Cytoplasm is scant to abundant
• Morphologically similar to diffuse large B-
cell lymphoma

20x

40xCopyright © 2012 Hologic, All rights reserved.
Pleural fluid 40x. Cells from this tumor range from immunoblastic (round
nucleus with a prominent nucleolus) to anaplastic (large, irregular,
multilobulated).

Suggested Immunochemistry Markers –
• CD30 +
• CD45 +
• CD5 -
• CD20 -
• S100 -
• CEA -

Adenocarcinoma
– Glandular acini, papillae or cell balls
– Increased N/C ratios
– Irregular nuclear membranes
– Abnormal chromatin
– Large or irregular nucleoli
– Secretory vacuoles with mucin

Specific Patterns of Adenocarcinoma
• Breast
– Cells are usually fairly bland and uniform
– Aggregates or predominantly single
– Diagnostic clues are cannonballs,
intracytoplasmic lumens and single file
chains of cells
– Positive for mucicarmine stain

20x
Peritoneal effusion (Breast ca) – Many small clusters and single malignant cells contrast
with benign mesothelial cells. As in pleural effusions, these cells tend to be present in
large rounded clusters as well as singly.

60x
Peritoneal effusion (breast ca) –
Note the large intracytoplasmic
vacuoles with mucin droplets.

Adenocarcinoma Breast
• CK 7 +
• CK 20 -
• BerEP4 +
• ER/PR +
• Mammaglobin +
• TTF-1 -

• Lung
- Large pleomorphic cells that can range
from bland to bizarre
- Nuclei are often hyperchromatic with fine
to coarsely granular chromatin and
prominent nucleoli
- Cytoplasm is often vacuolated and may
contain mucin

20x
Pleural effusion (Lung ca) – Often it is easy to distinguish adenocarcinoma from
mesothelioma. Note the eccentric nuclei, lacy cytoplasm and community borders.

20x
Pleural effusion (Moderately differentiated lung ca) – Papillary glandular
arrangements of tumor cells, prominent nucleoli, vacuolization and mitotic figures.

Adenocarcinoma Lung
• CK 7 +
• CK 20 -
• TTF-1 +
• BerEP4 +

• Ovarian
– 3 types
• Mucinous
• Serous
• Endometrioid
– Most often characterized by cells with large,
transparent vacuoles
– Presence of psammoma bodies may suggest
ovarian origin but not diagnostic of malignancy

20x
Pleural effusion - Cells of papillary serous ovarian adenocarcinoma in a pleural
effusion. Their cell and nuclear size is variable. Increased nuclear to cytoplasmic
ratio and cytoplasmic vacuoles are features. Cells may exist singly or in small
acinar groups.

60x
Peritoneal effusion - Papillary cluster of glandular serous borderline tumor
forming rounded “cannon-balls” of tumor cells. Note the smooth border,
compared to the scalloped border of reactive mesothelial cells.

40x
Peritoneal wash (Ovarian serous borderline tumor) - Papillary cluster of tumor cells
with prominent cytoplasmic vacuolization.

– Adenocarcinoma Ovary
Mucinous Adenocarcinoma
• CK 7 +
• CK 20 +
• BerEP4 +
• CA 125 +
Non-mucinous Adenocarcinoma
• CK 7 +
• CK 20 -
• ER/PR +
• CEA -

Gastric Carcinoma
• Cells with malignant features are present as
a distinct population. Some may exhibit
nuclear displacement by a large secretory
vacuole, a “signet ring” cell.

60x
Peritoneal effusion (gastric adenoca) – “signet ring” cell seen.

Adenocarcinoma Gastric
• CK 7 -
• CK 20 +
• MUC5AC +
• BerEP4 +

Squamous Cell Carcinoma
- Large fragments and/or single cells
- Cytoplasm may be dense and
occasionally orangeophilic
- Hyperchromatic nuclei
- Coarse chromatin
- Nucleoli not prominent

40x
Pleural fluid - Characteristic features of squamous cell carcinoma are seen:
orangeophilic cytoplasm and hyperchromatic nuclei.

Squamous Carcinoma
• CK 5/6 +
• CEA +
• p63* +
* p63 staining is especially useful in
non-keratinizing SCC

Malignant Melanoma
– Usually single, large cells
– Large, eccentric nuclei
– Frequent intranuclear cytoplasmic
invaginations
– Binucleation is common
– Very prominent nucleoli
– Melanin pigment presence is diagnostic

20x
Peritoneal effusion (Malignant melanoma) – May present in effusions with or
without an antecedent skin tumor. They often metastasize to unusual places
such as small bowel serosa.

Melanoma
• S100 +
• HMB45 +
• Melan-A +
• MART-1 +

Small Cell Carcinoma
- Tissue aggregates with molding
- Single file arrangements can be seen
- Small, rounded cells
- Coarse chromatin
- Inconspicuous nucleoli

20x
Pleural effusion – Small dark cells of small cell undifferentiated carcinoma can
appear singly and in small groups. Note the nuclear molding.

Small Cell Carcinoma
• CK 7 +
• CK 20 -
• TTF-1 +
• Chromogranin +
• Synaptophysin +
• CD56 +

Lymphoma/Leukemia
• Non-Hodgkin Lymphoma is most common and
accounts for 10-15% of malignant effusions
• In children, hematopoietic neoplasms are the
primary common cause of a malignant effusion
• Majority of patients will have biopsy proven
disease before effusion develops
• Reed-Sternberg cells in Hodgkin disease are
rarely found in effusions

Lymphoma/Leukemia
• Classification of the cells is possibly based on:
- Size of cells
- Degree of nuclear abnormalities (cleaved, non-cleaved)
- Resemblance of cells to lymphoblasts or Burkitt cells
– Single cell, monomorphic cell pattern
– Small cells with scant, delicate cytoplasm
– High N/C ratios
– Irregular nuclear membranes (cleaves, nuclear knobs)
– Prominent nucleoli

20x
Pleural effusion (Non-Hodgkin’s lymphoma) - These neoplastic lymphocytes
appear small and monotonous, but compared to the background
lymphocytes, they are considerably larger. Nuclei are vesicular with nuclear
clefting and lobation and variably prominent nucleoli.

60x
Reed-Sternberg cells are much larger than normal cells. Reed Sternberg cells -
binucleated giant cell with prominent inclusion-like nucleoli.

60x
Pleural effusion (Chronic lymphocytic leukemia) - An example of small, round
lymphocytes in a pleural effusion.

Basic Lymphoid Markers
B Cell Markers
• CD5 +/-
• CD20 +
• LCA (CD45) +
• BCL-2 +
T Cell Markers
• CD3 +
• CD5 +
• LCA (CD45) +
Hodgkin Lymphoma Markers
• CD15 +
• CD30 +
• LCA (CD45) -

References
1. Henry’s clinical diagnosis and management by laboratory
methods. In Joseph A, Knight MD, Carl R. Kjeldsberg MD,
editors. Cerebrospinal, synovial and serous body fluid.
Twenty first edition. Richard A. Mcpherson, Matthew R
Pincus.
2. Winifred Gray, Gabriella Kocjan, Diagnostic cytopathology. In
malcom Galloway, Maria Thorn, editors. Brain and
cerebrospinal fluid. Third Edition.
3. ThinPrep®
Non-Gyn Lecture Series. Body Fluid Cytology.

Fluid cytology in serous cavity effusions

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