CHAPTER 3 Serous effusions
Introduction
Serous cavity effusions are relatively simple to drain and collect for therapeutic and diagnostic purposes. For this reason, they comprise a significant proportion of general laboratory specimens. Paradoxically, the cytopathological evaluation of these specimens is relatively complex. Proper handling of specimens from the initial stage of collection to final interpretation is important. For optimal results with effusions, all the personnel involved, including clinicians, cytotechnologists and pathologists, should be familiar with the intricacies of specimen collecting, processing and interpreting.1
Anatomy, histology and cytology
Anatomy
The major serous cavities include the two pleural cavities, the peritoneal cavity and the pericardial sac (Fig. 3.1). These cavities are lined by parietal and visceral mesothelium. The visceral mesothelium is reflected over the organs therein. The serous cavity histology and serous fluid cytology of individual cavities are significantly identical without any site specific differences.
Histology
The serous cavities are lined by a flat monolayer of mesothelial cells, which have a tendency to undergo reactive changes to various stimuli leading to a somewhat cuboidal appearance (Fig. 3.2). Serous cavity effusions contain these ‘reactive’ mesothelial cells as one of the major components. Although mesothelial cells are derived from mesoderm, they demonstrate many morphological and biological features of epithelial cells (Fig. 3.2).
A thin layer of fibrous connective tissue with a varying amount of adipose tissue, small blood vessels and lymphatics supports the mesothelial cells. The lymphatic vessels open through gaps (stoma) between the mesothelial cells on to the surface of the serous cavities2,3 and are a significant component of the system for absorption of fluid in serous cavities. Imbalance of homeostatic forces in this system results in accumulation of fluid in serous cavities, leading to effusions.
General cytology of serous fluids
Mesothelial cells
Mesothelial cells round-up and appear polyhedral after exfoliation due to the surface tension of the surrounding fluid. The morphology of mesothelial cells (Box 3.1) can be evaluated in Papanicolaou (PAP) and Diff-Quik (DQ)-stained smears. In general, the PAP stain allows better evaluation of nuclear details, while the DQ stain highlights cytoplasmic details.
Cytological findings: mesothelial cells
In cytological preparations, mesothelial cells are usually about 15–30 μm in diameter (1.5 to 2 times the size of neutrophils), but they may vary significantly, ranging up to 50 m in diameter. They may be present as solitary cells (Fig. 3.3) or in small cohesive clusters (Figs 3.4, 3.5). They appear larger in Diff-Quik-stained air-dried smears than the wet-fixed shrunken cells in Papanicolaou-stained smears (Fig. 3.6).
The nuclear details are better seen in PAP-stained smears. The nuclei are usually central or slightly off centre, but may be distinctly eccentric (Figs 3.7, 3.8). When eccentric, the nuclear membrane does not touch the cell border. Careful examination shows this narrow rim is due to microvilli on the surface adjacent to the eccentric nucleus (Fig. 3.8). This feature may be applied to distinguish mesothelial cells from histiocytic macrophages and adenocarcinoma cells, which characteristically show peripherally located nuclei touching the cell membrane (Fig. 3.9). They are best appreciated in DQ-stained preparations (Fig. 3.10). The nuclei are typically round to oval with smooth contours. Even malignant mesothelial cells may have a perfectly round nucleus with smooth contours.
Fig. 3.7 Mesothelial cell (pleural fluid). This mesothelial cell shows a relatively eccentric nucleus. The swollen microvilli are obvious as ruffled borders with peripheral blebs (arrow 1). Although subtle and difficult to perceive, there is a rim (arrow 2) separating the nuclei from the cell border. This feature helps to distinguish eccentric cells in adenocarcinoma, which may lack this rim (compare with Figure 3.9A) (DQ Cytospin).
Fig. 3.10 Metastatic adenocarcinoma with a two-cell population (ascitic fluid). (A) Reactive mesothelial cells (arrow 1) with central nuclei, peripheral vacuolation, and knobby borders formed by the cell membrane, contrast better in Diff-Quik-stained preparation (B) from adenocarcinoma cells (arrow 2) with eccentric nuclei touching the cell membranes without any rim of cytoplasm between the nucleus and cell membrane. Compare the community border of adenocarcinoma cell groups formed mostly by nuclear contours. (A, PAP ThinPrep; B, DQ Cytospin). (A, from Shidham and Atkinson 2007.1)
Mesothelial cell cytoplasm does not always show two zones in DQ-stained preparations (Fig 3.11). In some cells it may be finely granular with a variable degree of basophilia, without the two-zone pattern. As the mesothelial cells imbibe water from the surrounding fluid, their cytoplasm may acquire a foamy macrophage phenotype with pale vacuolated cytoplasm. The degree of vacuolation is directly proportional to the duration for which the cells were in the fluid. When the effusion becomes chronic, the cytoplasmic vacuoles become larger but the vacuoles of mesothelial cells are usually small. They occur at the periphery of the cells (Fig. 3.11), but may be randomly distributed (Fig. 3.13) or even central with nuclear overlap. The nuclei in some mesothelial cells may be displaced to the periphery by the vacuolated cytoplasm secondary to phagocytic activity or degenerative changes. A single, large cytoplasmic vacuole displacing the nucleus may resemble that in signet ring cells of adenocarcinoma.Figs. 3.12 and 3.13
Fig. 3.12 Malignant epithelioid mesothelioma (pleural fluid). The mesothelioma cells are numerous with more mesothelial cells in individual groups than in reactive conditions and with three-dimensional, papillary-like groups (B), showing knobbly borders formed predominantly by cytoplasm. Individual mesothelioma cells do not show significant variation from reactive mesothelial cells and lack overt features of malignancy. As with reactive mesothelial cells, the mesothelioma cells show two-zone staining with peripheral vacuolation (arrow in C) (PAP ThinPrep). (Shidham and Atkinson 2007.1)
The microvilli of mesothelial cells may prevent adjacent cells from completely opposing each other, thereby creating a gap between two cells. This space between two adjacent mesothelial cells is referred to as a mesothelial window (Fig. 3.14), which may be subtle or very wide in cytology smears. Such spaces between adjacent cells in effusions are not specific for mesothelial cells and may be seen in other types of cell groups including those of metastatic cancers.4
The surface of mesothelial cells has numerous long slender microvilli, which impart a peripheral rim of pallor in PAP-stained preparations. This characteristic feature of mesothelial cells has been applied to distinguish them from other cells such as carcinoma cells.5–7 Although the microvilli cannot be seen directly under the light microscope, their presence may be inferred by a thin rim of cytoplasm along the side of the eccentric nucleus in some mesothelial cells (Figs 3.7, 3.8). This feature, although observed in both types of staining, is more easily recognised with DQ stain (Fig. 3.7). The swollen microvilli impart ruffled borders and peripheral blebs in DQ smears (Fig. 3.7). Microvilli are best seen by electron microscopy (EM).
Although mesothelial cells do not proliferate in effusions after exfoliation, they may complete an already started mitotic division. The presence of mitotic figures (Fig. 3.15) in effusion cytology suggests a process that is capable of causing significant proliferative activity in response to whatever is causing the effusion. As with cytopathological evaluations in general, the presence of nucleoli and mitotic figures should not lead to a false interpretation of malignancy.1 Other general morphological features of malignancy should be applied to arrive at such conclusion. Once a specimen is correctly interpreted as malignant, nucleoli and mitotic figures may then be considered for further categorisation and grading of a neoplasm.
Mesothelial cells produce hyaluronic acid, which, if present, may be seen as magenta coloured intracytoplasmic or extracellular material in DQ-stained smears. Although less distinct, this may be seen as light grey streaks in the background in PAP-stained smears. Hyaluronic acid in the centre of small groups of mesothelial cells may be misleading as it superficially resembles mucin in adenocarcinoma acini. Hyaluronic acid is positive with periodic acid-Schiff (PAS) and Alcian blue stains. This is lost if the sections are treated with hyaluronidase prior to staining. Hyaluronic acid is not a substrate for diastase, and is not digested by it.
Although these typical features are not specific for mesothelial cells, they help to distinguish them from other cells in effusions including metastatic malignant cells. Some mesothelial cells may show a morphological spectrum overlapping with that of certain neoplasms, including malignant melanoma and adenocarcinoma, especially of the breast and ovary (Figs 3.16, 3.17).
Reactive mesothelial cells
Various pathological processes such as inflammation, neoplasia, and trauma lead to reactive changes in the extremely sensitive mesothelial cells lining the serosal cavities. Mesothelial cell hypertrophy and proliferation in response to an altered environment may lead to a remarkably wide morphological spectrum, which may even overlap with malignant cells (Figs 3.18, 3.19).2,8,9 Such exfoliated mesothelial cells with a wide range of appearances are usually referred to as ‘reactive mesothelial cells’. The resolution of the underlying pathological process may reverse these changes.
Cytological findings: reactive mesothelial cells
Reactive mesothelial cells range in size from 15 to 30 μm but may be larger than 50 μm in diameter with variable amounts of cytoplasm (Figs 3.3, 3.18, 3.19). The enlarged nuclei show some variation in their size and shape, usually with conspicuous nucleoli. Binucleation and multinucleation are frequent (Fig. 3.20). They may be present as cohesive clusters including papillary configurations. Some cells may show high nuclear/cytoplasmic ratios with scant cytoplasm and slightly hyperchromatic nuclei with prominent nucleoli (Fig. 3.19). This astonishingly wide morphological spectrum may overlap remarkably with that of some malignant cells in effusions (Figs 3.18, 3.19) and may lead to diagnostic pitfalls.
Binucleation and multinucleation: Two or more nuclei may be present in reactive mesothelial cells as in vivo change within the serous cavity (Fig. 3.20) and are frequent in peritoneal dialysis fluids.10 However, this may also be due to in vitro changes. Formation of small aggregates secondary to processing such as filtration, Cytospin® preparation, centrifugation, or liquid-based cytology preparations may lead to fusion of the cytoplasmic borders of the ‘sticky’ reactive mesothelial cells with degenerative change and may appear multinucleated. This is not uncommon in specimens left at room temperature for several hours.
Phagocytic activity: Phagocytic activity of reactive mesothelial cells transforms them into foamy macrophages (Figs 3.11, 3.13) with pale vacuolated cytoplasm as they imbibe water from the effusion fluid. Distinguishing foamy reactive mesothelial cells from foamy histiocytic macrophages is usually of little clinical significance. But morphological features of some mesothelial cells with vacuoles may overlap with malignant cells and mislead one to interpret these cells as adenocarcinoma.
Although of little clinical significance, morphological differentiation between macrophage-like mesothelial cells and histiocytic macrophages is difficult. Some nuclear features may assist. A round to oval nucleus with smooth contours favours a mesothelial cell origin and a bean-shaped (kidney-shaped, reniform) nucleus with slightly irregular contours favours a histiocytic macrophage.
Cell-in-cell configuration: One cell may wrap around an adjacent cell, leading to the appearance of a cell-in-cell arrangement. Although this is commonly associated with reactive mesothelial cells, it is highly non-specific and may also be seen in malignant cells in serous fluids.9
Cohesive clusters and/or papillary structures: Reactive changes in the mesothelial lining of serous cavities may exfoliate some cohesive cell groups. Some of these may demonstrate papillary configurations (Fig. 3.21). As a quantitative feature, there are fewer cohesive clusters but more solitary mesothelial cells in reactive effusions (Fig. 3.22) than those associated with mesothelioma, which show a cellular specimen with relatively more and larger cohesive groups and/or papillary clusters (Fig. 3.12).
Mesothelial cell clusters with scalloped (knobbly) contours generally have fewer cells than those found in mesotheliomas. Scalloped contours may also resemble clusters of cells from other neoplasms (Fig. 3.23). However, the individual mesothelial cells at the periphery show the recognisable two-zone cytoplasmic features of mesothelial cells, and the nuclei do not touch the cell borders of the cells along the periphery of the reactive mesothelial cell groups. The cytoplasmic rim of individual cells in the group forms the outline of such clusters (Fig. 3.12). In contrast, usually the nuclear border forms a significant proportion of this peripheral outline of the groups in adenocarcinoma cell clusters (Fig. 3.24).
Fig. 3.24 Metastatic mammary carcinoma (pleural fluid). The periphery of the group is formed predominantly by the areas where the nucleus touches the cell borders (arrow) without any rim of cytoplasm. Compare this with groups of mesothelial cells- both reactive (Fig. 3.5) and neoplastic (Fig. 3.17). (DQ Cytospin smears).
Diagnostic pitfalls: reactive mesothelial cells
Reactive mesothelial cells (Fig. 3.25) in effusions, especially those associated with some of the conditions mentioned below, may lead to potential pitfalls.
Hepatocellular carcinoma: Cases of hepatocellular carcinoma associated with effusion are usually negative for malignant cells, but frequently show atypia within the reactive mesothelial cells.11
Trauma to organs covered with mesothelium such as spleen, liver and lung.
Pelvic inflammatory diseases: Mesothelial cell clusters (Fig. 3.26) frequently observed in benign reactive conditions such as pelvic inflammatory diseases may lead to a diagnostic pitfall, with erroneous interpretation as ovarian adenocarcinoma. This is significant especially in aspirates of the cul-de-sac, which often contain some clusters in a papillary-like configuration.12
Pelvic or peritoneal washings: Sheets of mesothelial cells are rare in effusions, but they are usual features in pelvic or peritoneal washings. They are the result of forcible mechanical detachment from the serosal membrane during operative incision or intra-operative lavages, or both. Although superficially they may resemble the clusters of squamous cells, they usually are not a diagnostic challenge.13
‘Atypical’ mesothelial cells: Reactive effusions, especially those associated with some clinical conditions may elicit marked changes in mesothelial cells. Some of these reactive mesothelial cells may resemble malignant cells. However, the morphological features and other criteria discussed in this chapter are not sufficient to be interpreted definitively as malignant. Such cells may be categorised as ‘atypical’.14 Such reactive mesothelial cells with atypia may demonstrate: high nuclear/cytoplasmic ratios, nuclear enlargement, nuclear hyperchromasia, coarse clumped chromatin, and prominent macronucleoli. They may be seen as large cohesive groups with scalloped edges and moulding of cells, with extensive morphological variation. The terminology of ‘atypical mesothelial cells’ (Fig. 3.25) should be discouraged strongly and should not be used without an explanatory note/comment in the report.8
Such cells are not uncommon in effusions associated with cirrhosis of the liver, pulmonary infarction, congestive heart failure, collagen vascular diseases, renal failure with uraemia, pancreatitis,15 bile peritonitis, therapeutic radiation, chemotherapy, and large benign intra-abdominal masses with florid reactive mesothelial hyperplasia of the overlying serosa.14
Macrophages
Cytological findings: macrophages
Mesothelial cell macrophages have overlapping morphological features but show some subtle differences such as round to oval nuclei in contrast to reniform nuclei in histiocytic macrophages, and centrally placed nuclei rather than peripheral or eccentric nuclei in histiocytic macrophages (Figs 3.11, 3.13). Some cells may show eccentric nuclei, but the nuclear margin is usually not in close approximation to the cell border. Usually there is a narrow rim of cytoplasm due to numerous long slender microvilli in mesothelial macrophages (Figs 3.7, 3.8, 3.9). In contrast to the ill-defined cell borders in histiocytic macrophages, those in mesothelial cell macrophages are well defined with blebs. They may exfoliate as cohesive groups with distinct knobbly contours, in contrast to the ill-defined loosely cohesive groups with irregular outlines of histiocytic macrophages.
Extensive vacuolation with peripheral displacement of nuclei in some macrophages resembles the secretory vacuoles of adenocarcinoma cells. The mucicarmine stain for mucin performed on cell block sections or direct cytology smears may help. Positive mucin staining favours adenocarcinoma (Fig. 3.27); however, negative staining does not confirm a reactive macrophage nature. Using a histochemical stain such as mucicarmine to confirm adenocarcinoma is a relatively simple approach. It is economical, but variation in inter-laboratory reproducibility and improper quality control may compromise the sensitivity and specificity of this test.
Blood-derived cells
Other cells present in effusions include red blood cells, lymphocytes, neutrophils, eosinophils, basophils and megakaryocytes.16 Their proportion depends on the extent of peripheral blood contamination and the cause of the effusion.
Cytological findings and diagnostic pitfalls: blood-derived cells
Megakaryocytes (Fig. 3.28) in effusions are rare. They may be associated with a myeloproliferative disorder or with extramedullary haemopoiesis, secondary to conditions such as extensive bone marrow replacement by metastatic carcinoma. Their interpretation is relatively easy in DQ-stained preparations. They are seen as large cells with a variable amount of cytoplasm and multilobed nuclei. Multilobation may not be distinctly visible in all cells. Their presence in a pleural effusion may be secondary to haemorrhaging pulmonary microvasculature.17 They may be misinterpreted as neoplastic or as a viral cytopathic effect especially in PAP-stained preparations.
Other intrinsic entities16
Cytological findings and diagnostic pitfalls: other intrinsic entities
Psammoma bodies
These concentrically lamellated calcific spherules (Fig. 3.29) are encountered in up to 3.7% of effusions.18 The acellular structures have a tendency to crack in smears. They may be surrounded by benign or malignant cells. In PAP-stained preparations, they appear cyanophilic (blue-green) to acidophilic (pink).
In pleural and pericardial effusions, psammoma bodies are usually associated with malignancies including papillary thyroid carcinoma, ovarian papillary serous carcinoma and others (Fig. 3.29). However, in peritoneal effusions and washings, they may also be associated with benign conditions. One-third of cases with psammoma bodies in peritoneal specimens may be associated with benign processes such as ovarian cystadenoma/cystadenofibroma, endometriosis, endosalpingiosis and papillary mesothelial hyperplasia.18
Collagen balls
These mesothelial cell covered fragments of collagen have been reported in 4–29% of peritoneal washings.13 Their prevalence is relatively higher in specimens submitted as pelvic washings (5.8%) than those submitted as peritoneal washings (1.6%).19,20 They should not to be misinterpreted as a component of a papillary or mucinous gynaecological neoplasm.21 They probably originate from the surface of the ovaries, and are usually restricted to specimens from females,16 but collagen balls have been reported in ascitic fluid from a male with encapsulating peritonitis.22
Detached ciliary tufts
Fluid from the pouch of Douglas and peritoneal washings1 may show detached ciliary tufts from the ciliated epithelium lining the fallopian tubes. They are seen as non-nucleated fragments of cells with cilia (Fig. 3.30). They may demonstrate linear, rotating, jerky motility in wet preparations of fresh specimens, and may be misinterpreted as parasites.23 In PAP-stained smears they are relatively difficult to find. They do not have any pathological significance and probably represent cyclical physiological shedding from the tips of ciliated cells in the fallopian tubes during the luteal phase of the menstrual cycle.24
Curschmann’s spirals
Curschmann’s spirals are similar to those seen in sputum and bronchial washings, although generally smaller in effusions. They have been reported in spontaneous pleural and peritoneal effusions. They are hypothesised to be formed from mucus secreted by mucus-producing adenocarcinoma cells in neoplastic conditions. In non-neoplastic conditions, they are formed from submesothelial connective tissue mucosubstance released into the effusion fluid through the inflamed serosal lining, which has increased permeability.25
Sperm
Rarely sperm may be observed unexpectedly. The possibility of contamination during collection, transportation, and processing should be ruled out. However, the authors recently observed spermatozoa in an effusion with mixed inflammation, mostly acute inflammatory cells and histiocytes. The number of sperm was low, with degenerative changes in many. Some neutrophils showed phagocytosed heads of sperms (personal experience) (Fig. 3.31).
Extraneous entities and non-cellular material
A variety of extraneous structures may be present in effusion specimens.16 Physiological mechanisms may result in spontaneous exfoliation of cells from such structures in some effusions. Depending on the path of the needle through neighbouring structures while aspirating the specimen, these can be a challenge, especially for the inexperienced interpreter and in cases with inadequate clinical details.
Cytological findings and diagnostic pitfalls: extraneous entities
Cells from the female genital tract with degenerative changes may accumulate in the cul-de-sac in longstanding effusions as a result of reflux via the fallopian tubes secondary to menstruation. This phenomenon occurs particularly in the presence of an intrauterine contraceptive device. Peritoneal effusions may show implants of endometriosis and endosalpingiosis. Some may demonstrate degenerative nuclear atypia with hyperchromasia and may lead to the pitfall of malignant misinterpretation. The latter pitfall is also applicable to müllerian inclusions encountered in peritoneal washings.26,27
Ectopic pancreas has been misinterpreted as malignant in a patient with a history of ovarian adenocarcinoma, the cells having been aspirated from the small bowel wall.28
Longstanding effusions may show cholesterol crystals, especially in rheumatoid pleuritis.
Serosanguinous effusions may show haematoidin crystals with haemosiderin-laden macrophages.
Specimen types, collection and processing
Effusions
The narrow gap of 5–10 μm that separates the parietal and visceral layers of the serosa under physiological conditions is widened in the presence of an effusion of fluid into the serous cavity. Irrespective of their cellular composition, all effusions are pathological. The causative factors include increased vascular permeability, vasodilatation, blockage of lymphatics, breakdown of small blood vessels and haemodynamic imbalance of the microcirculation. An association between vascular endothelial growth factor (VEGF) and malignant effusions due to increased vascular permeability has been reported.29
Even in tertiary care institutions reactive effusions are more common than malignant effusions. Malignant effusions usually show diagnostic malignant cells on cytological evaluation. Some benign effusions, however, may also show diagnostic cytomorphological features.30
Types of effusions
With reference to diagnostic cytopathology, effusions may be reactive (secondary to conditions such as collagen diseases, circulatory system disorders, trauma, inflammation and infection) or malignant (that is, positive for malignant cells).1
Pathophysiologically, effusions may be: transudates, exudates, or chylous (Table 3.1). Although transudative effusions usually do not require diagnostic evaluation for malignant cells, exudative effusions generally need cytological evaluation to determine their cause.
Malignant effusions
Effusions secondary to cancer are usually recurrent and haemorrhagic. Non-traumatic massive haemorrhagic effusions are almost always due to cancer. Perhaps with the exception of central nervous system tumours, malignant neoplasms from almost any site can metastasise to serous cavities and present as an effusion. However, a rare case of diffuse leptomeningeal gliomatosis involving the peritoneal cavity has been reported in a patient with a ventriculo-peritoneal shunt.31
A malignant effusion re-accumulates rapidly in comparison to reactive effusions. With this information in mind, it is prudent to recommend a repeat specimen if the initial specimen is suspicious but not conclusive for malignancy as it is easy to obtain a repeat sample when it re-accumulates. This can lead to a definitive diagnosis without the danger of false positive interpretation.1
The predominant cause of an initially indeterminate interpretation is artefact secondary to degenerative changes both in vivo and in vitro. A repeat specimen should be recommended to be submitted immediately after collection, to avoid in vitro degenerative artefacts.32 The significance of relevant clinical details cannot be overstated. The cytological interpretation of a repeat specimen is usually easier, because the number of neoplastic cells in recurrent malignant effusions often increases, with many cohesive clusters and cell balls.
Both epithelial and non-epithelial neoplasms may cause malignant effusions. Epithelial neoplasms include metastatic carcinoma4 and malignant mesothelioma.33 Haematological neoplasms,34 melanoma and sarcomas35 are the non-epithelial neoplasms and, apart from the haematological malignancies such as lymphoma, these are rare cause of effusions.
