Fluid cytology in serous cavity effusions

Fluid Cytology SEROUS CAVITY EFFUSIONS

Part II

ByModerator : 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

• Congestive heart failure

• 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 physiomechanicalfactors.

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 mesothelialcells 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 transudate1. 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, parapneumonicinfections.

• TSA (Tuberculostearic acid) – Tuberculosis.

• Tumour markers – CEA, PSA, CA-125.

• Fibronectin – in malignant ascites.

Diagnosis of ascites due to portal hypertension is established by measurement of serum ascitesalbumin 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, cholestrolcrystals

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

Fluid Cytology

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

Mesothelial cells

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

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

Normal Components

• Mesothelial cells in peritoneal washings

– Broad and flat sheets

– Slightly rounded up

– Cuboidal

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.

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

Pelvic wash: Benign mesothelial cells. Mesothelial cells can also have a cuboidalappearance 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

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: dormantmesothelial cell (or serosal cover cell) (1), phagocytic macrophage with vacuoles(2), and monocytes (3), in addition to segmented neutrophilicgranulocytes(4).

Abnormal Findings

Lymphocytosis (>50%)

• Tuberculosis

• Viral infection

• Malignancy (lymphoma, other neoplasms)

• True chylothorax

• Rheumatoid pleuritis

• Systemic lupus erythematosus

• Uremic effusions

• Transudates (≈30%)

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

NEUTROPHILIA(50%)

• Bacterial pneumonia (parapneumoniceffusion)

• Pulmonary infarction

• Pancreatitis

• Subphrenic abscess

• Early tuberculosis

• Transudates (>10%)

Eosinophilia (>10%)

• Pneumothorax (air in pleural space)• Trauma• Pulmonary infarction• Congestive heart failure• Infection (especially parasitic, fungal)• Hypersensitivity syndromes• Drug reaction• Rheumatologic diseases• Hodgkin disease• Idiopathic

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 crystalsPeritoneal wash

Starch granulesPeritoneal wash

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

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

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

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”.

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

– NeuroendocrineTumors

– 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

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.

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

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

Suggested Immunochemistry Markers

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-

• 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

Primary Effusion Lymphoma

• 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

Pleural fluid 40x. Cells from this tumor range from immunoblastic (round nucleus with a prominent nucleolus) to anaplastic (large, irregular, multilobulated).

Suggested Immunochemistry Markers –Primary Effusion Lymphoma

• 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

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.

Peritoneal effusion (breast ca) –Note the large intracytoplasmicvacuoles with mucin droplets.

Suggested Immunochemistry Markers –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

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

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

Suggested Immunochemistry Markers –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

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

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.

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

Suggested Immunochemistry Markers – 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 secretoryvacuole, a “signet ring” cell.

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

Suggested Immunochemistry Markers –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

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

Suggested Immunochemistry Markers –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

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.

Suggested Immunochemistry Markers –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

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

Suggested Immunochemistry Markers –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

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.

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

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

Suggested Immunochemistry Markers –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.

THANK YOU !

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