Pleural, Peritoneal, and Pericardial Effusions

Tinted Formalin

The formalin used to fix the cell block material can be colored pink-orange by adding a small amount of powdered eosin. The embedded specimen then stands out tinted in the paraffin wax, and the histotechnologists will find it much easier to cut the sections. Adding eosin to the formalin does not alter the appearance of the stained sections.

Thrombin Solution

This is prepared by adding 10 mL of distilled water to a vial containing 5000 units of powdered thrombin.

Toluidine Blue Stain

Prepare the stain as follows:

• Toluidine blue (0.5 g)

• 95% ethanol (20.0 mL)

• Distilled water (80.0 mL).

To prevent fungal growth, store the stain in a refrigerator.

Cytology

The prototypic mesothelial cell exfoliated into a serous effusion ( Fig. 19-19 ) is round, about 25 µm in diameter, and has a single central or eccentric nucleus. With the Papanicolaou stain, the cytoplasm is dense and gray-green or slightly eosinophilic and tends to become less dense at the periphery of the cell, imparting an indistinct foamy or even scalloped appearance to the cell membrane. The nucleus is round or oval and possesses a well-defined, smoothly contoured membrane. The chromatin is uniformly granular, and nucleoli are readily identified.

Mesothelial cells vary considerably in size, from small cells about 9 µm in diameter with relatively little cytoplasm to giant cells more than 60 µm in diameter. Large, hypertrophied and much smaller mesothelial cells are frequently seen joined to each other. Giant mesothelial cells are almost always multinucleated, often with ≥10 nuclei ( Fig. 19-20 ).

Cytoplasmic vacuolation, when present, may take several forms:

• 1.

  One or more tiny, unobtrusive vacuoles scattered throughout the cytoplasm but more likely to be near the nucleus

• 2.

  A large, solitary vacuole that seems to displace the nucleus to the periphery of the cell

• 3.

  An elongated, perinuclear vacuole that curves around a segment of the nuclear membrane like a tiny sausage.

It is not uncommon to find a mesothelial cell with a large, solitary vacuole and an eccentric nucleus joined to a typical non-vacuolated mesothelial cell, clearly denoting the mesothelial lineage and benign nature of the vacuolated cell ( Fig. 19-21 ).

Mesothelial cells articulate with each other in a characteristic manner. One form of articulation consists of little chains of cells joined at flattened or curved apposing surfaces ( Fig. 19-22 ). Groups composed of two to four mesothelial cells are frequently seen to have this type of articulation. Between the apposing cellular surfaces, clefts or “windows” may develop, suggesting that the conjoined cells are about to become detached from each other. Less commonly seen, although with the same type of articulation, are larger sheets of mesothelial cells with a mosaic appearance composed of about ≥10 cells ( Fig. 19-23 ). Another articulation characteristic of mesothelial cells is illustrated in Fig. 19-24 , in which the cytoplasm of one cell appears to be clasping another cell. It should be mentioned that all of these types of articulation can be seen, although much less frequently, in adenocarcinoma cells in serous fluids.

Tissue fragments composed of mesothelial cells characteristically have knobby or scalloped contours due to individual cells protruding from the periphery ( Fig. 19-25 ). In smears, it is the presence of these more visible protruding cells that allows the mass of cells to be identified as mesothelial. Occasionally, however, large, round, smoothly contoured clusters of mesothelial cells are found, a situation in which it is more difficult to determine the mesothelial nature of the cells. It should be possible, however, to detect at the edge of the cluster a fine rim of cytoplasm showing the peripheral cytoplasmic fading characteristic of mesothelial cells. Cell block preparations can be extremely useful in this situation, because they may more readily reveal the benign nature of these fragments. Should any doubt remain about whether the cells are mesothelial or adenocarcinomatous, immunocytochemistry applied to cell block material usually enables one to distinguish between mesothelial and adenocarcinoma cells (see “ Mesothelioma ,” below).

Giant multinucleated mesothelial ( Fig. 19-20 ) cells are not uncommon, having been reported in 26% of 396 serous fluids. They may be misinterpreted as giant multinucleated histiocytes, a rarity in serous effusions, found almost exclusively in specimens from patients with rheumatoid pleuritis or pericarditis. Furthermore, the cytoplasm of giant multinucleated mesothelial cells does not exhibit the fine, diffuse vacuolation typical of histiocytes. It may contain several well-defined vacuoles, with the residual non-vacuolated cytoplasm being typical of mesothelial cells in general. In contrast to the nuclei of histiocytes, the nuclei of giant multinucleated mesothelial cells are smoothly contoured with well-defined membranes, and they contain prominent nucleoli. Giant multinucleated mesothelial cells are usually accompanied by numerous smaller cells whose mesothelial lineage is easily recognized.

“Atypical” and “Reactive” Mesothelial Cells

We commonly come across reports of “atypical” or “reactive” mesothelial cells, designations given to mesothelial cells that are hypertrophic ( Fig. 19-26 ) and often hyperplastic. Furthermore, mesothelial cells that are not well spread and well stained, especially if the serous effusion is bloody, may appear dark, thereby creating diagnostic uncertainty – hence, the use of the terms atypical and reactive.

Mesothelial Cells in Wet Films

The morphologic features of mesothelial cells seen in Papanicolaou-stained smears are also visible in toluidine blue-stained wet films, with several important differences. First, mesothelial cells in toluidine blue-stained wet films are noticeably larger than those in permanent, wet-fixed preparations. (This size difference applies to all types of cells examined in wet films.) Another difference is that mesothelial cells in wet films frequently contain tiny, glistening, golden, cytoplasmic lipidic inclusions not seen in the permanent preparations because they are dissolved by solvents in the fixative or staining circuit. Finally, the types of articulation characteristic of mesothelial cells are usually more difficult to discern in wet films, although careful observation reveals that they are present.

Cell Block Preparations

Mesothelial cells in cell block preparations generally do not present any particular diagnostic difficulty. Because these preparations are stained with hematoxylin and eosin, the cytoplasmic staining reaction is one of various shades of pink. Except for this, all of the previously mentioned morphologic features of mesothelial cells seen in Papanicolaou-stained smears are discernible in cell block preparations, although not as readily as in smears.

Diagnostic Pitfalls

Papillary fragments of mesothelium that become detached from the serosa, especially during a washing procedure, may consist of a connective tissue core covered by a layer of mesothelial cells. Such connective tissue cores may become calcified to form psammoma bodies, illustrating that malignancy must not be diagnosed on the presence of psammoma bodies alone; the diagnosis must be based on the accepted nuclear changes of malignancy in any accompanying cells.

Becker and coworkers reported an example of overdiagnosis due to the presence in pericardial fluid of exfoliated micropapillary excrescences covered by mesothelial cells. Benign papillary proliferations may develop on any serous surface and are usually attributed, especially in the female pelvis, to the effects of previous inflammation, including those induced by surgical operation. They do not cause effusion.

With the Papanicolaou stain, such fragments of collagenous connective tissue covered by mesothelium are frequently seen as smoothly contoured, homogeneous, pale green bodies covered by a layer of flattened cells ( Fig. 19-27 ) – hence the term collagen ball that we adopted to describe this entity. Collagen balls are virtually confined to washings of the peritoneal cavity or specimens obtained by culdocentesis. We have rarely seen a collagen ball in a spontaneous pleural, peritoneal, or pericardial effusion; in such specimens, collagen balls are almost always small and unobtrusive and may be overlooked. The 3-dimensional, spheroidal nature of peritoneal collagen balls is realized by focusing up and down on the ball and finding that the covering cells are visible at different planes. An inexperienced observer might regard these characteristic fragments as adenocarcinoma cells distended by mucin. Apart from the fact that their nuclei do not possess the features of malignancy, any doubt about the composition of these fragments can be settled by cell block preparations, which show the characteristic eosinophilic collagenous cores.

It was our belief that collagen balls were probably detached from microexcrescences that are normally present on the surface of the ovary. However, Sasaki and colleagues demonstrated that “serosal balls,” their name for the entity, may be found with at least equal frequency in peritoneal washings of men. Consequently, they suggested, probably correctly, that serosal balls in peritoneal washings originate by detachment of minute fragments of serosa and underlying stroma during a surgical operation; these detached fragments then round up to produce balls.

To anyone embarking on cytopathology, it soon becomes obvious that the mesothelial cell in all of its variations offers the greatest potential for being misinterpreted as metastatic adenocarcinoma. The important question is how can one eliminate or at least diminish the chance and frequency of such error?

The first step is to rely on only well-prepared specimens in which the cells are wet fixed (if the Papanicolaou stain is being used), not overstained, and easily visible. With such preparations, the mesothelial nature of individual cells should be obvious. Smears that are too thick or very bloody increase the potential for overdiagnosis. In such smears, the staining reaction of mesothelial cells is darker, with the consequence that their nuclei appear hyperchromatic and suggestive of malignancy.

When the cytologic picture remains ambiguous or when mesothelial cells appear to be hypertrophic or hyperplastic (“atypical” or “reactive”), immunocytochemistry can be helpful.

Cytology

Red blood cells are readily recognized in Papanicolaou-stained smears as neatly round orange-red discoids about 7 µm in diameter, although many may become distorted into elongated forms in the process of spreading the cellular sample. Fixation may cause red blood cells to undergo lysis, leaving residual empty cell membranes with a slightly cyanophilic staining reaction. Hemoglobin released from these cells may remain as part of the proteinaceous background of a smear, thereby imparting a reddish cast to the smeared material, both to the naked eye and under the microscope. In the toluidine blue-stained wet films, red blood cells are unstained, having a light straw yellow color.

Apart from these artifacts, normal red blood cells generally show little morphologic variation.

Intact red blood cells may be phagocytosed by macrophages. When red blood cells disintegrate, their hemoglobin is converted into hematoidin or hemosiderin, which, if present in a large enough amount, imparts a distinctly yellow (xanthochromic) appearance to the supernatant. In stained wet films, hematoidin pigment may occasionally be seen as sheaves of fine, elongated, yellow-brown extracellular crystals, whereas hemosiderin is seen as dark blue granules in the cytoplasm of macrophages. In Papanicolaou-stained smears, hematoidin retains the same appearance, but the staining reaction of hemosiderin is golden brown to olive green. Hemosiderin gives a positive staining reaction for iron (Perls test), whereas hematoidin does not.

Cytology

In wet films, the cytoplasm remains unstained or weakly stained ( Fig. 19-28 ).

Neutrophils in non-infected effusions are generally well preserved. In infected purulent fluids, neutrophils degenerate; in some effusions, virtually every neutrophil is necrotic. Necrotic neutrophils become transformed into ill-defined, light gray to gray-blue particles without any visible nuclear material or cytoplasmic granularity ( Fig. 19-29 ). The nuclei of necrotic cells may also condense into round, solitary, deeply cyanophilic masses, or they may become fragmented.

Eosinophilic Pleural Effusion

Several comprehensive reviews during the early days of cyto­diagnosis attest to the fascination that eosinophilic effusions, especially pleural effusions, have held for clinicians. However, the arbitrary criterion for designating a pleural effusion as eosinophilic has varied from an effusion with as few as 5% to one with at least 50% of the cells being eosinophils; we have adopted the criterion of a concentration of 10% or more as used by Koss.

Eosinophilic pleural effusion has been reported in association with a wide variety of conditions: allergy, autoimmune disorders, pneumonia (including viral pneumonia), pulmonary infarct, fungal infection, parasitic infection, malignant neoplasms, pulmonary tuberculosis, artificial and spontaneous pneumothorax, and hemothorax, including that induced by thoracic trauma. A total of 81 of the 127 (64%) cases of eosinophilic pleural effusion analyzed by Spriggs and Boddington and 8 of 30 (27%) analyzed by Veress and coworkers were associated with some form of thoracic trauma.

Spriggs concluded that pleural eosinophilia is the normal reaction of the pleura to the introduction of air into the pleural cavity and suggested that the reaction is more likely a result of suspended particles of animal or vegetable origin than of the air itself.

When pneumothorax can be excluded, the commonest causes of eosinophilic pleural effusion seem to be pulmonary infarct, pneumonia, and neoplasm. Hodgkin's disease is rarely associated with eosinophilic pleural effusion. Only a minority of cases can be attributed to recognizable hypersensitivity states, including parasitic infestation. Another cause of pleural eosinophilic effusion is the benign type of effusion associated with the inhalation of asbestos; in a series of 60 cases, 26% showed eosinophilia of various degrees.

When all of the possible causes of eosinophilic pleural effusions have been eliminated, there is a substantial residue of cases in which the cause cannot be identified.

Despite the uncertainty surrounding the pathogenesis of idiopathic eosinophilic pleural effusions, they seem to have a favorable prognosis, even in patients with a previous history of cancer.

Because it has not been possible to identify a single linking pathologic mechanism behind the formation of eosinophilic pleural effusion, it has been suggested that these effusions exist in two forms: an effusion of relatively acute onset related to an allergic reaction or to thoracic trauma and the more chronic form with a longer clinical course.

Eosinophilic Peritoneal Effusion

Eosinophilic peritoneal effusions are rare. Examples of eosinophilic peritoneal effusion associated with malignant neoplasm, various allergic states, parasitic infection, eosinophilic gastroenteritis, and chronic peritoneal dialysis have been reported. It is possible that one or more of the agents used in peritoneal dialysate, such as antiseptic, talc, particles of tubing, and peritoneal catheters, provoke a hypersensitivity reaction characterized by an efflux of eosinophils. In addition to eosinophilia, chronic peritoneal dialysis may stimulate mesothelial hypertrophy and hyperplasia.

Eosinophilic Pericardial Effusion

Eosinophilic pericardial effusion is even rarer than eosinophilic peritoneal effusion. A few examples in association with pulmonary eosinophilia, including one associated with the use of the drug cromolyn sodium, have been reported. Examples of Hodgkin's disease and of lymphocytic lymphoma first being manifested by eosinophilic effusion have been reported.

Cytology

Most eosinophils have bilobed nuclei, although many nuclei have three or occasionally four lobes. The lobes are slightly larger and more neatly round than those of neutrophils. A pyknotic form in which the nucleus is represented by a solitary chromatinic mass with no lobes is occasionally seen, especially in fluids that have stood for some time after aspiration ( Fig. 19-30 ).

In Papanicolaou-stained smears, eosinophils are smaller than in wet films and the cytoplasmic granules are much less obvious, frequently being manifested by only a fine eosinophilic granularity. Instead, the cytoplasmic staining reaction may be light green with little or no granularity, depending on the quality of the stain; such cells are recognized as eosinophils by their size and the bilobation of their nuclei. Cell block preparations demonstrate eosinophils well ( Fig. 19-31 ). Eosinophils may undergo necrosis, although we have not observed the widespread necrosis of eosinophils in smears similar to that seen in neutrophils in empyema fluids.

Cytology

Mast cells and basophils are readily recognized with a Romanowsky stain, but they are not recognizable in Papanicolaou-stained smears or in cell blocks stained with hematoxylin and eosin. They can be recognized in toluidine blue-stained wet films ( Fig. 19-32 ), in which the granules are a delicate purple-red, quite different from the deep blue of the stain itself.

Mast cells, slightly larger than basophils, are about the size of small mesothelial cells. Their nuclei are round or oval and centrally placed, whereas the nuclei of basophils are usually bilobed. The cytoplasm of mast cells is entirely occupied with granules; the cytoplasm of basophils contains far fewer.

Cytology

Histiocytes are found in various proportions in almost every serous effusion. Their size varies considerably, from about 15 to 100 µm in diameter, with most within the range of 20–40 µm. In smears, the typical histiocyte is easily identified by its size, eccentric round or bean-shaped nucleus, and lightly stained lacy cytoplasm ( Fig. 19-33 ). Because histiocytes are phagocytic, their cytoplasm may contain leukocytes, nuclear particles ( Fig. 19-34 ), red blood cells, carbon particles, lipid droplets, melanin, or hemosiderin.

Histiocytes tend to be isolated but may coalesce, presumably because of their long microvilli becoming entangled. This usually gives a loose, sheet-like appearance to a group of macrophages. Such sheets do not have the tight cohesiveness and sharp definition of sheets of mesothelial cells; they tend to straggle at the periphery, and the overall cohesiveness of the group has a loose quality, with spaces between individual cells. Furthermore, the lacy, porous appearance of the cytoplasm contrasts sharply with the dense cytoplasm of mesothelial cells. Fig. 19-35 exemplifies in a smear of pericardial effusion the cohesive property of histiocytes, in this case to form balls; the patient had fibrinous pericarditis.

Histiocytes may contain large solitary or loculate cytoplasmic vacuoles that appear to displace the nucleus to the periphery of the cell ( Fig. 19-36 ), a picture suggestive of signet ring adenocarcinoma. In cell block preparations of a spontaneously formed clot, groups of macrophages that have become tightly packed against each other as the clot contracted may be seen; these compact groups of histiocytes may be mistaken for fragments of adenocarcinoma.

Histiocytes usually have a single nucleus, although binucleation is not uncommon. The finding of giant multinucleated histiocytes formed by fusion of a number of macrophages or by mitotic division of a nucleus is a rarity, virtually confined to effusions caused by rheumatoid pleuritis or rheumatoid pericarditis (see “ Rheumatoid Disease ,” below). Giant multinucleated mesothelial cells are seen with much more frequency, and whenever a cell is regarded as a giant multinucleated histiocyte, the question should be raised about whether it is really a mesothelial cell.

Cytology

Almost every serous fluid contains at least a few lymphoid cells, readily recognized in Papanicolaou-stained smears as small cells having little or no cytoplasm and being dominated by round, deeply staining nuclei ( Fig. 19-37 ). Their size varies according to the amount of drying occurring during preparation of the smear and before fixation. In the thicker parts of a smear, where air drying is minimal, the lymphoid cells are smaller, whereas at the periphery of a smear, where some air drying is almost inevitable, lymphoid cells have a larger diameter and appear flatter and less darkly stained. This variation in size and staining reaction is not seen in cell block preparations.

The nuclei of mature lymphocytes are neatly rounded, although some may be slightly indented or notched. Nucleoli are difficult to find in the Papanicolaou smears, although they are frequently visible in wet films. When lymphocytes are present in large numbers, it is customary to find a few less mature, slightly larger lymphoid cells. In these less mature forms, the chromatin is paler and nucleoli are visible. Cytoplasm, although scanty, is most readily seen in stained wet films and in Papanicolaou-stained smears which had undergone momentary drying before fixation.

Mature plasma cells similar to those in bone marrow may be found, usually in effusions containing many lymphoid cells ( Fig. 19-38 ). Larger, less mature-appearing plasmacytoid cells are also frequently seen in such effusions; they may contain two or more nuclei.

Apart from their size and shape, one of the most important features distinguishing lymphoid cells from those of small-cell carcinoma is their property of remaining separated from each other. In contrast, the cells of small-cell carcinomas form cohesive clusters. This property of lymphoid cells being separated from each other is obvious even in cell block preparations.

In contrast to purulent effusions, those dominated by non-neoplastic lymphoid cells contain only a very small percent of necrotic cells. A prolonged search under high power is usually needed to find what seems to be a necrotic lymphocyte. Such a necrotic lymphocyte may show the type of karyorrhexis consisting of tiny round particles reminiscent of a dispersed drop of mercury (mercury drop karyorrhexis). This type of karyorrhexis, difficult to find in benign lymphocytic effusions, is frequently seen in effusions containing lymphoma cells.