Residency · Residency · Pathology

Body Cavity Fluids and Effusion Cytology

Overview

Serous effusions from the pleural, peritoneal (ascitic), and pericardial cavities are among the most common cytology specimens encountered in clinical practice. The primary diagnostic challenge is distinguishing reactive mesothelial cells from malignant cells, whether metastatic carcinoma or primary mesothelioma. A systematic approach integrating morphology, cell block immunohistochemistry, and clinical context is essential for accurate diagnosis.

Specimen Processing

Collection and Preparation

Fresh fluid should be processed promptly to preserve cellular morphology; heparin or EDTA may be added to prevent clotting. Cytospin preparations concentrate cells for direct examination and are particularly valuable for paucicellular specimens. Direct smears are prepared in both air-dried (for Diff-Quik staining) and alcohol-fixed (for Papanicolaou staining) formats. Liquid-based preparations (ThinPrep or SurePath) produce thin monolayer slides for optimal visualization.

The cell block is the single most critical preparation in effusion cytology. It is made by processing clotted sediment as formalin-fixed paraffin-embedded tissue, enabling immunohistochemistry, special stains, and molecular testing. The cell block is essential for definitive identification of the malignancy type and primary site. When lymphoma is suspected, fresh fluid should be submitted in a heparinized container for flow cytometry before any fixation.

Cell Block Preparation Methods

Several techniques exist for cell block preparation, including the thrombin-plasma clot method (most commonly used), the HistoGel method, the collodion bag technique, and automated cell block systems. The choice depends on institutional preference and specimen volume.

Normal and Reactive Mesothelial Cells

Morphology

Normal mesothelial cells are round to oval, occurring singly, in flat sheets, or in small clusters. They have a central round nucleus with a smooth nuclear membrane, fine chromatin, and a small nucleolus. A distinctive feature is the "two-tone" cytoplasm, consisting of a dense peripheral (ectoplasmic) rim with a lighter central endoplasm. Characteristic "windows" or intercellular spaces are visible between adjacent cells in clusters. Binucleation is common, and mitotic figures may be present in reactive states. In benign effusions, mesothelial cells are often the dominant cell population.

Reactive Mesothelial Proliferation

Reactive mesothelial cells show increased cellularity, nuclear enlargement, prominent nucleoli, and mitotic figures. They may form papillary clusters or three-dimensional groups that closely mimic adenocarcinoma or mesothelioma. Key features favoring a reactive process include uniform nuclear enlargement, smooth nuclear membranes, and maintained cell-to-cell spacing. Clinical context is critical: recent surgery, infection, pulmonary embolism, and radiation therapy all cause reactive mesothelial proliferation.

Malignant Effusions

Metastatic Adenocarcinoma

Metastatic adenocarcinoma is the most common malignancy found in effusions. Morphologically, it presents as three-dimensional cell clusters (morulae, papillary groups, or acinar formations), cell-in-cell arrangements indicating cellular cannibalism, large cells with high nuclear-to-cytoplasmic ratios, irregular nuclear membranes, macronucleoli, and cytoplasmic vacuoles containing mucin. Unlike mesothelial clusters, adenocarcinoma clusters lack the characteristic "windows" pattern and instead show community borders with a smooth outer edge.

Common Primary Sites

Lung carcinoma is the most common cause of malignant pleural effusions, identified by TTF-1 and napsin A positivity. Breast carcinoma is common in women, expressing GATA3, mammaglobin, and ER. Ovarian and peritoneal carcinomas are the most common cause of malignant ascites in women, positive for PAX8, ER, and WT1. Gastrointestinal tract carcinomas cause malignant ascites and express CDX2, CK20, and villin. Renal cell carcinoma is identified by PAX8, RCC marker, and CD10.

Mesothelioma vs. Reactive Mesothelial Cells

Distinguishing mesothelioma from reactive mesothelial proliferation on cytology alone is extremely challenging. Cytologic features favoring mesothelioma include markedly increased cellularity with numerous single and clustered mesothelial cells, cell-in-cell (engulfment) patterns, large papillary clusters with scalloped "berry-like" borders, nuclear atypia with enlarged irregular nuclei and coarse chromatin with prominent nucleoli, and absence of two distinct populations (no inflammatory background). Definitive distinction often requires cell block immunohistochemistry and clinical-radiological correlation. Loss of nuclear BAP1 staining supports mesothelioma over reactive proliferation with approximately 60-70% sensitivity. Loss of MTAP by immunohistochemistry serves as a surrogate for homozygous CDKN2A deletion and supports mesothelioma. FISH for homozygous CDKN2A deletion (p16) was previously the gold standard and is being gradually replaced by MTAP immunohistochemistry.

Mesothelioma vs. Adenocarcinoma: IHC Panel

Mesothelial markers (positive in mesothelioma) include calretinin (nuclear and cytoplasmic), WT1 (nuclear), CK5/6, D2-40 (podoplanin), and HBME-1 (less specific). Carcinoma markers (positive in adenocarcinoma) include MOC-31, BerEP4, claudin-4, CEA (monoclonal), and B72.3/TAG-72. The recommended approach uses at least 2 mesothelial markers and 2 carcinoma markers, recognizing that no single marker is 100% sensitive or specific. BerEP4 is the most sensitive carcinoma marker, while calretinin is the most sensitive mesothelial marker. Once carcinoma is confirmed, organ-specific markers (TTF-1, PAX8, GATA3, CDX2) identify the primary site.

Other Malignancies in Effusions

Lymphoma

Lymphoma in effusions presents as a dispersed single-cell pattern without the clustering seen in carcinoma. Small cell lymphomas (CLL/SLL, follicular lymphoma) show monotonous small lymphoid cells, while large cell lymphomas (DLBCL) demonstrate large cells with prominent nucleoli and basophilic cytoplasm. Flow cytometry is essential for demonstrating light chain restriction and aberrant antigen expression, and cell block enables immunohistochemical confirmation.

Squamous Cell Carcinoma

Squamous cell carcinoma in effusions appears as orangeophilic keratinized cells on Papanicolaou stain or dense blue cytoplasm on Diff-Quik, with tadpole and fiber cell shapes and tumor diathesis. It is relatively uncommon in effusions and usually derives from a lung or cervical primary.

Melanoma

Metastatic melanoma presents as dispersed large cells with eccentric nuclei, prominent cherry-red nucleoli, dusty melanin pigment, and intranuclear pseudoinclusions. Immunohistochemistry confirms the diagnosis with S100, HMB-45, Melan-A, and SOX10 positivity.

Special Situations

Peritoneal Washings in Gynecologic Oncology

Peritoneal washings are obtained at the time of staging surgery for ovarian, endometrial, and fallopian tube carcinoma. Positive peritoneal cytology upstages certain tumors, including endometrial carcinoma. The key diagnostic challenge is distinguishing malignant cells from reactive mesothelium and endosalpingiosis -- benign Mullerian inclusions consisting of ciliated tubal-type epithelium that can closely mimic low-grade serous carcinoma.

Pericardial Effusions

Pericardial effusions are less common but clinically urgent, as large-volume effusions can cause cardiac tamponade. Lung and breast carcinoma are the most frequent malignant causes. Processing and interpretation are identical to pleural and peritoneal fluids.

Cerebrospinal Fluid (CSF)

CSF normally has very low cellularity (fewer than 5 cells per milliliter, all lymphocytes or monocytes), making cytospin preparation essential for adequate evaluation. Malignancies encountered in CSF include lymphoma and leukemia, metastatic carcinoma, medulloblastoma/PNET, and meningeal carcinomatosis. Reactive conditions include viral meningitis (lymphocytes), bacterial meningitis (neutrophils), and post-surgical changes.

<image>A medical illustration comparing the cytomorphologic features of reactive mesothelial cells, mesothelioma, and metastatic adenocarcinoma in pleural effusion specimens. Panel A (Reactive mesothelial cells, Pap stain): A flat sheet of uniform mesothelial cells with round nuclei, smooth nuclear membranes, characteristic intercellular windows, and the two-tone cytoplasm pattern with a dense peripheral rim. Panel B (Mesothelioma, Diff-Quik stain): A large three-dimensional morula of atypical mesothelial cells with scalloped berry-like borders, cell-in-cell engulfment pattern, nuclear enlargement, and prominent nucleoli. Panel C (Metastatic adenocarcinoma, Pap stain): A tight three-dimensional cluster of malignant cells with community borders, high N:C ratio, macronucleoli, cytoplasmic vacuoles, and absence of intercellular windows.</image>

<image>A medical illustration showing the immunohistochemistry panel approach for distinguishing mesothelioma from metastatic adenocarcinoma on cell block sections. Two columns of paired IHC images. Left column (Mesothelioma): Calretinin showing strong nuclear and cytoplasmic positivity, WT1 showing nuclear positivity, CK5/6 showing cytoplasmic positivity, and BerEP4 showing negativity. Right column (Adenocarcinoma, lung primary): Calretinin negative, WT1 negative, TTF-1 showing nuclear positivity, and BerEP4 showing strong membranous positivity. A summary table below lists recommended panel with expected staining patterns and sensitivity/specificity for each marker.</image>

Clinical Pearls

Cell block preparation is the single most important step in effusion cytology, and material should always be prioritized for cell block to enable immunohistochemistry and molecular testing. The characteristic "windows" between mesothelial cells in clusters help distinguish benign mesothelial proliferations from adenocarcinoma, which typically shows tight community borders without intercellular spaces. BAP1 loss by immunohistochemistry on cell block is a powerful tool for distinguishing mesothelioma from reactive mesothelial proliferation, but 30-40% of mesotheliomas retain BAP1, so a retained (positive) result does not exclude mesothelioma. Mesothelioma should never be diagnosed on cytology alone without radiologic correlation, as reactive mesothelial proliferation can closely mimic mesothelioma cytologically. When lymphoma is suspected, fresh fluid must be submitted for flow cytometry before fixation, as this analysis cannot be performed after formalin fixation. Endosalpingiosis (benign ciliated tubal-type epithelium) in peritoneal washings can mimic low-grade serous carcinoma; the presence of cilia and bland nuclear features favors a benign diagnosis. A negative effusion cytology does not exclude malignancy, as sensitivity is approximately 60-70% for pleural malignancy and lower for mesothelioma.

References

• Cibas ES, Ducatman BS. Cytology: Diagnostic Principles and Clinical Correlates. 5th ed. Elsevier; 2021.
• Churg A, et al. Tumors of the Serous Membranes. AFIP Atlas of Tumor Pathology, 4th Series. ARP Press; 2012.
• Hjerpe A, et al. Guidelines for the cytopathologic diagnosis of epithelioid and mixed-type malignant mesothelioma. Acta Cytol. 2015;59(1):2-16.
• Berg KB, et al. Loss of BAP1 expression to separate benign from malignant mesothelial proliferations. Mod Pathol. 2021;34(7):1271-1278.
• Monaco SE, et al. Cytopathology of malignant effusions and lavage specimens. Surg Pathol Clin. 2020;13(1):137-162.

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