Serous Fluid Cytopathology

Vol. 21 • Issue 1 • Page 32


Serous fluids, also known as “effusion fluids,” are commonly received by pathology laboratories for cytopathologic evaluation. However, cytopathologic evaluation of these specimens is relatively complex and requires a special approach-from specimen collection to final interpretation by the pathologist/cytopathologist. All laboratory personnel involved in the process, irrespective of their cytopathology subspecialization status, should be conversant with the challenges along various stages.1,2 Specimens from all four major serous cavities (peritoneal, pericardial and two pleural cavities) are relatively similar and applicable, to some extent, to the specimens from other comparable body cavity fluids such as hydrocele fluid.

In general, the cytomorphologic features of cancer cells in effusions are different than those of exfoliative, brushing and fine-needle aspiration cytology. As a result, the routine cytologic criteria of malignancy applicable to morphology of single cells are usually not of significant help in cytologic evaluation of effusions. A wide spectrum of reactive mesothelial cells along with “rounding” due to surface tension phenomenon may introduce significant pitfalls. Nutrient-rich effusion fluids allow the constituent cells to survive longer and even proliferate (in vivo and in vitro). These features related to the effusions may appear ordinary, but they are important for understanding the strategy to prevent false positive and false negative interpretations.

Cytology of Serous Fluids

Effusion fluids are composed of mesothelial cells and non-neoplastic cells, which are predominantly inflammatory cells with other components, such as psammoma bodies and incidental cellular/non-cellular elements.

Mesothelial cells (1.5-2 times the size of neutrophils: about 15-30 µm, but may be up to 50 m) presenting as solitary or small cohesive clusters appear larger in Differential Quik Stain (Diff-Quik)-stained, air-dried smears than in Pap stained, wet-fixed smears. They evoke significant reactive changes in response to various insults with a remarkably wide cytomorphological spectrum, which overlaps with malignant cells.1,2 Familiarity with this cytomorphologic spectrum in various cytologic preparations, especially in Pap and Diff-Quik stains, is very important.1

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Specific objectives should be addressed to yield appropriate processing and staining. And depending on special requirements, such as specimens with excess blood, the process may have to be modified to address the specific issues.1 It is important to have appropriate clinical details. Additionally, the histotechnologist-immunochemistry technologist should be conversant with the subtractive coordinate immunoreactivity pattern (SCIP) approaches, beginning with section cutting stage.


Unique features applicable to effusion fluid cytology should be considered during final interpretation.

Wide cytomorphological spectrum of reactive mesothelial cells is relatively more challenging in association with some clinical scenarios.

Lack of a second foreign population is consistent with mesothelial cells (either reactive or neoplastic) except when metastatic cancer cells outnumber the mesothelial cells and appear as a single predominant population.

Second foreign population of non-mesothelial and non-inflammatory cells is usually consistent with metastatic neoplasm.

Rarer cases, such as sarcoma and pediatric effusions with mostly blue round cell tumors,3 usually have previous history.

Second population is relatively easily detected in Romanowsky-stained preparations (e.g., Diff-Quik) than in Pap-stained preparations (Figure).

Immunocytochemistry may be applied for objective confirmation of second population. The SCIP approach with identical orientation of serial sections allows precise evaluation of immunostained cell block sections.

Mesothelioma usually lack overt atypia. Quantitative (relatively hypercellular with many mesothelial cells) and qualitative (many large three-dimensional groups of mesothelial cells) findings are important clues.


General cytomorphological features should be modified with special considerations.

Cell groups with intercellular cohesion are similar to general cytology, while proliferation spheres (tissue fragments) are seen typically in metastatic carcinoma.

Other arrangements of neoplastic cells include papillary configurations, which may not represent true papillary tumors. True papillae with cores in pleural and pericardial effusions represent mesothelial structures; if neoplastic, these favor mesothelioma over adenocarcinoma.

Cytoplasm of neoplastic cells lack typical two-zone pattern (easily seen in Diff-Quik-stained preparations), with eccentric nuclei touching the cell membrane without any zone along the nuclear margins due to lack of microvilli and favor adenocarcinoma (if histocyte is excluded by studying nuclear details in Pap stained preparations).

Interpretation of other structures and cytological features is similar to general cytology.


Immunostaining of cell-block sections with the SCIP approach may be applied for objective confirmation of a second foreign population with further immunocharacterization of the detected second foreign population to evaluate the primary site as indicated.

Vimentin will “immunostain” the usual constituent (mesothelial and inflammatory) cells of effusions for a basic topographic map of cell block sections. Any unstained cells, if present, can be easily subtracted to identify and separate them out as a second population from the basic map by this approach.1

The second population may further be interpreted by studying the coordinate immunoreactivity pattern and evaluating complimentary immunomarkers-at least two mesothelial immunomarkers, such as lCalretinin and D2-40, with at least two non-mesothelial, adenocarcinoma immunomarkers, such as BerEP4, B72.3, MOC31, monoclonalCEA, etc. Additional appropriate immunomarkers, including two-color immunostaining,4 may be added to evaluate a primary source of the metastatic disease, as used in general immunocytochemistry.5

Mesothelioma Diagnosis

Immuohistochemistry cannot distinguish between reactive and neoplastic mesothelioma for diagnosis of mesothelioma. This differential diagnosis is largely based on the quantitative (numerous versus a few) and qualitative (large groups versus small groups) features in concert with appropriate clinical and radiological correlation.1

A special approach is recommended for preparation of formalin-fixed, paraffin- embedded cell blocks to achieve alignment of most diagnostic cells in effusion specimens.6 Although cytology smears may show good immunostaining pattern,7 they are not appropriate for routine immunocytochemical evaluation in general and for effusion fluids.

Immunocytochemical evaluation of effusion fluids performed with the above basic approach would allow improved interpretation outcomes.

Effusion cytology, with its significant role in patient care and disease management, is a relatively frequent test confronted by pathologists and pathology laboratories in many settings. Although this is a challenging area, the methodical approach mentioned above makes evaluation and interpretation relatively simple.1,2,8,9

In general, if cytological features do not support the malignant interpretation, it is prudent to issue an equivocal communication (with clear recommendations and instructions) to repeat the cytopathologic examination, especially if the effusion recurs or persists. Usually, the morphology and number of diagnostic cells improve in re-accumulated effusion specimens.1

Dr. Shidham is co-editor-in-chief and executive editor, CytoJournal; professor, vice-chair, AP; director of Cytopathology, Cytopathology Fellowship; director of Pathology Residency Training Program, Department of Pathology, Wayne State University School of Medicine, Karmanos Cancer Center and Detroit Medical Center. The author thanks Anushree Shidham and Anjani Shidham for their copy editing support.


1. Shidham VB and Atkinson BF. Editors and contributors ‘Cytopathologic Diagnosis of Serous Fluids’ First edition, Elsevier (W. B. Saunders Company) 2007.

2. Shidham VB, Falzon M. Serous effusions: reactive, benign and malignant. In Gray & Kocjan, ed. Diagnostic Cytopathology, Elsevier, 3rd edition, Chapter 3.

3. Hallman JR, Geisinger KR. Cytology of fluids from pleural, peritoneal and pericardial cavities in children. A comprehensive survey. Acta Cytol 1994;38:209-17.

4. Shidham V.B., Varsegi G, D’Amore K. Two-color immunocytochemistry for evaluation of effusion fluids for metastatic adenocarcinoma. CytoJournal 2010 Feb 10;7:1. Available for free at: (last accessed Dec. 12, 2011).

5. Shidham VB, Kajdacsy-Balla AA. Immunohistochemistry: Diagnostic and Prognostic Applications. In Detrick B, Hamilton RG, Folds JD eds. Manual Molecular and Clinical Laboratory Immunology. American Society of Microbiology Press. 7th ed, Chapter 47. p 408-413.

6. Varsegi G.M., Shidham V. (2009). Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells. J Vis Exp 2009 Jul 21;(29). pii: 1316. Video article is available free on web as open access at

7. Shidham VB, Chang CC, Rao RN, Komorowski R, Chivukula M. Immunostaining of cytology smears: A comparative study to identify the most suitable method of smear preparation and fixation with reference to commonly used immunomarkers. Diagn Cytopathol 2003 Oct;29(4):217-21.

8. Shidham VB. Effusion Fluid Evaluation Made Simple: A brief review of cytomorphologic and SCIP approach. CytoJournal (In press).

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