Chapter 1 Introduction
PREAMBLE TO THE BOOK
Effusion in this book refers to an excessive amount of fluid in a serous cavity. As reflected in the title, this book embarks on a diagnostic approach to cytopathologic evaluation of serous cavity effusions and washings. It is intended to introduce the beginner to this field with a simplified algorithmic approach for interpretation of cells in effusions (see Figures 3.1 and 3.2). The discussion will be primarily directed towards the detection, by routine cytologic methods, of neoplastic cells based on morphology alone, or with the help of various ancillary tests, including commonly applied immunocytochemistry (see Chapters 5 and 15). Since a picture is worth a thousand words, an attempt is made, at the risk of moderate repetition, to include as many illustrations as possible.
The major serous cavities are (Figure 1.1) the peritoneal cavity, the pericardial cavity, and the two pleural cavities. Effusions from these cavities and related cytology specimens will be the subject of this book. Cytopathologic evaluation of peritoneal washings is referred to periodically and a chapter is dedicated to it (see Chapter 7).
The book is predominantly focused on diagnostic application of cytomorphology with or without ancillary support by other methods such as immunocytochemistry. A separate chapter is dedicated to an overview of molecular and other special techniques in relation to effusions (see Chapter 13).
It is expected that some readers may not read the book from beginning to end, and choose to refer to the individual chapters sporadically during their clinical practice. Keeping this in mind, some of the themes and features will be repeated now and then in brief throughout the book. To emphasize their significance, some of these important themes will be highlighted as italicized blue text throughout for quick reference.Technical and other reference material are included as appendices in Chapters 14 and 15, with a section on abbreviations used. The concepts and information are compiled in tables, algorithms, sketches, and combination pictures as a quick reference guide for readers after initial reading. Most of the illustrations are labeled with arrows and other indicators to avoid equivocation, especially for beginners in the field.
This introductory chapter describes general details under the following three headings:
HISTOLOGY AND GENERAL CYTOLOGY OF SEROUS CAVITY LINING
HISTOLOGY
The mesothelium forms a parietal and visceral layer in each cavity, where it is reflected over the organs therein. It consists of a flat monolayer of mesothelial cells, which have a tendency to undergo hypertrophy secondary to various stimuli, usually resulting in a somewhat cuboidal appearance (Figure 1.2). Such ‘reactive’ mesothelial cells frequently exfoliate into serous effusions. Although derived from mesoderm, mesothelial cells possess many of the morphologic and biologic features of epithelial cells (see Figure 1.2).
Underlying the mesothelial cells of each serous cavity is a thin layer of fibrous connective tissue with a varying amount of adipose tissue, small blood vessels, and lymphatics. The lymphatic vessels open on to the surface lining of the serous cavities through gaps (stoma) between the mesothelial cells, which provides continuity between the lymphatic system and the serous cavities.1,2 The lymphatics are a significant component of the system for absorption of fluid in serous cavities. Any imbalance in the homeostatic forces in this system results in accumulation of fluid in serous cavities and leads to effusions. It is of interest to note that the recently reported immunomarkers for lymphatic endothelium, such as D2-40 and podoplanin, have been reported to be immunoreactive for mesothelial cells also.3–5 This suggests a relationship between mesothelial cells and lymphatic endothelial cells.
The serous cavities may be affected by a variety of processes, including inflammation, hepatic cirrhosis, congestive heart failure, and metastatic neoplasms. These processes often stimulate reactive changes in mesothelial cells. The damaged mesothelium can be replaced by differentiation of the mesenchymal cells from the underlying stroma (see Figure 1.2). The reactive mesothelial cells are hypertrophied and appear somewhat cuboidal with enlarged nuclei and conspicuous nucleoli (see Figure 1.2). They may show variation in nuclear size and shape, multinucleation, and increased nucleocytoplasmic ratios. All these features are best observed in cytologic preparations.
GENERAL CYTOLOGY (with Papanicolaou and Diff-Quik stain)
Serous effusions may contain a variety of non-neoplastic cells, including mesothelial cells, macrophages, and other blood-derived cells (Table 1.1), together with other entities such as psammoma bodies and various incidental cellular and non-cellular elements (Table 1.2).
Table 1.1 Blood-derived constituents of effusions
| Cells | Diff-Quik stain | Papanicolaou stain |
|---|---|---|
| Red blood cells | Eosinophilic, anucleated, round, with central pallor | Eosinophilic Biconcave disks Anucleated |
| Neutrophils | Nucleus: basophilic, multilobed with 2–5 lobes Cytoplasm: faintly eosinophilic with fine granularity | Nucleus: basophilic, multilobed with 2–5 lobes Cytoplasm: faintly cyanophilic, granular |
| Eosinophils | Nucleus: basophilic, bilobed (may have up to four lobes) Cytoplasm: numerous, coarse, eosinophilic granules | Nucleus: basophilic, bilobed (may have up to four lobes) Cytoplasm: light pink to light green with coarse granularity |
| Basophils | Nucleus: central, rounded to irregularly shaped Cytoplasm: coarse, basophilic granules which may overlap the nucleus | Nucleus: central, rounded to irregularly shaped Cytoplasm: coarsely granular, light pink to light green |
| Histiocytes | Nucleus: reniform (kidney or bean shaped), central to eccentric Cytoplasm: vacuolated, foamy, without distinct ectoendoplasmic staining pattern, and without peripheral cytoplasmic blebs | Nucleus: reniform (kidney or bean shaped), central to eccentric Cytoplasm: foamy, may contain phagocytosed material such as hemosiderin pigment |
| Megakaryocytes | Morphology similar to that in bone marrow smears Nucleus: large, multilobed nuclei Multilobation may not be distinct in all cells Cytoplasm: variable amount | Large, multilobed nuclei Cytoplasm: variable amount. Megakaryocytes with high N/C ratio may be misinterpreted as neoplastic or viral cytopathic effect, especially in PAP-stained preparations |
Table 1.2 Other entities observed in serous cavity effusions and washings
Mesothelial cells
After exfoliation, mesothelial cells round up and appear polyhedral due to the surface tension of the surrounding effusion fluid. They may be seen as solitary cells (Figures 1.3, 1.4) or in small cohesive clusters (Figures 1.6, 1.8). The cells are of various sizes and may be round to oval. 
The morphology of mesothelial cells 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 (Table 1.3).
Table 1.3 Complementary roles of the Papanicolaou and Romanowsky stains*
| Feature | Romanowsky stains (RWS)* | Papanicolaou stain |
|---|---|---|
| Cell size and shape | Size and shape of cells: the cells are flat as they collapse during air-drying, making them slightly larger in dimension along the plane of the slide | Size and shape of cells: slightly shrunken. The cell thickness is greater in wet-fixed smears due to its fixation in three dimensions closer to its natural form |
| Cytoplasmic details | The cytoplasm is well demonstrated by RWS—thus highlighting even the scant amount of cytoplasm (such as in lymphocytes, small cell carcinoma, etc.), cytoplasmic vacuoles (renal cell carcinoma, macrophages, etc.), cytoplasmic blebs (mesothelial cells), different zones in the cytoplasm (mesothelial cells), etc. The details of cell groups are poorly visualized | Cytoplasm: is rendered transparent which improves nuclear details In general cytoplasmic details are diminished. However, this improves the morphologic evaluation of cell groups, including three-dimensional clusters |
| Nuclear details | The details of nuclear chromatin to evaluate chromatin clumping and parachromatin clearing are not clear However, RWS are excellent for evaluating nuclear details of hematopoietic cells, as chromatin clumping and parachromatin clearing are not that significant for evaluating hematopoietic malignancies Nucleoli: are not as crisp as with the Papanicolaou stain, but they can be seen as pale structures Thus in brief, RWS does not allow evaluation of chromatin clumping and parachromatin clearing, but it allows evaluation of N/C ratio, nuclear size, shapes, nuclear pseudoinclusions, and nucleoli. Most of these are adequate for interpretation of hematopoietic lesions | Nuclear details are excellent with crisp chromatin staining facilitating evaluation of chromatin clumping and parachromatin clearing, which are some of the most important features evaluated for interpretation of malignancy. Nucleoli: well discerned |
| Extracellular material | Excellent staining of extracellular materials such as mucin, colloid, pseudocartilagenous and cartilagenous matrix, lymphoglandular bodies in lymphoproliferative processes, etc. | These extracellular materials are poorly stained |
* There are many variants popular in different countries and institutions. Some examples are May-Grünwald-Giemsa (MGG), Wright, Leishman, Giemsa, and Diff-Quik stains (Table 14.7). In this book, the Diff-Quik stain is referred to as being synonymous with RWS, as it is used in our laboratory and in most laboratories in the USA. However, there are many different manufacturers of similar kits consisting of the first reagent methanol for fixation, followed by eosin solution (pink), and last Azure B solution (blue) (Tables 14.7A, 14.10F). Depending on the general trend and choice of any particular institution, other types of Romanowsky stain may be used (Table 14.7)
In cytologic preparations, mesothelial cells are usually about 15–30 mm in diameter (1.5–2 times the size of neutrophils), but they may vary significantly and may range up to 50 mm in diameter. 
They appear larger in DQ-stained air-dried smears than the wet-fixed shrunken cells in PAP-stained smears (see Figure 1.10). In PAP-stained smears, the perinuclear zone, with its higher density of intermediate filaments, shows relatively dense staining endoplasm with a surrounding narrow zone of pale ectoplasm associated with microvilli (see Figure 1.3). In DQ-stained smears, the endoplasm is lightly stained with peripheral darker ectoplasm (see Figure 1.4). The cell borders are round with smooth contours but have ruffled surfaces with blebs. 
In general, the two-zone staining characteristic of mesothelial cells is better seen in DQ-stained preparations (see Figure 2.1).
Although this typical appearance of mesothelial cells helps to distinguish them from other cells, including malignant cells, in effusions, it is not specific for mesothelial cells. Non-mesothelial neoplasms such as malignant melanoma and adenocarcinomas of the breast and ovary may demonstrate some morphologic overlap with mesothelial cells (Figures 1.5, 2.4, 4.5, 4.6).
The nuclei of mesothelial cells are usually centrally placed or slightly off center, but may be distinctly eccentric (see Figure 2.3). Even when they are eccentric, their nuclear membranes do not touch the cell border. Careful examination shows a narrow rim of cytoplasm adjacent to the eccentric nucleus (see Figure 1.5). This narrow rim is due to the microvilli on the surface of mesothelial cells.
Binucleation and multinucleation of mesothelial cells is frequent, especially in non-malignant effusions. It is not uncommon to see variation in the sizes of multiple nuclei in the same cell (Figure 1.7). Nucleoli are usually seen and may be prominent (see Figure 1.3). However, huge macronucleoli equal to one-third the size of the nuclear diameter associated with some malignancies such as melanoma, hepatocellular carcinoma, germ cell tumors, and prostatic adenocarcinoma are rarely found in reactive mesothelial cells. The chromatin is usually finely granular (powdery) with various degrees of chromasia. Many of these nuclear details can also be seen in DQ-stained smears, but malignancy-related hyperchromasia and chromatin details cannot be evaluated properly in DQ-stained smears (see Figure 1.4).
With DQ staining, mesothelial cell cytoplasm may not always show the two zones (see Figure 2.2). Instead, the cytoplasm may be finely granular throughout and show a variable degree of basophilia. As the mesothelial cells imbibe water from the effusion fluid, their cytoplasm may acquire a foamy macrophage phenotype with pale vacuolated cytoplasm (Figures 1.9, 2.5). The degree of vacuolization is directly proportional to the duration that the cells remain in the fluid medium. As the effusion becomes chronic, the cytoplasmic vacuoles become larger. The cytoplasmic vacuoles of mesothelial cells are usually small and occur at the periphery of the cells, but they may be randomly distributed or even be central with nuclear overlap (see Figure 1.9). A single, large, cytoplasmic vacuole displacing the nucleus may cause a mesothelial cell to resemble a signet ring cell of adenocarcinoma (see Figure 4.5).
Differences between macrophage-like mesothelial cells and histiocytic macrophages, although not of great clinical significance, are difficult to identify by morphology alone. Nuclear morphology may help to make some distinction. Mesothelial cells have round to oval nuclei with smooth contours, whereas histiocytic macrophages typically show bean-shaped (kidney-shaped, reniform) nuclei with slightly irregular contours (see Figure 1.9).
The surfaces of mesothelial cells have numerous, long, slender microvilli, which impart a peripheral rim of pallor in PAP-stained preparations. Microvilli are best seen by electron microscopy (EM). This characteristic feature of mesothelial cells has been applied to distinguish them from other cells such as carcinoma cells.6–8 Although the microvilli cannot be seen directly under the light microscope, their presence may be inferred from a thin rim of cytoplasm by the side of an eccentric nucleus (see Figure 1.5). This feature, although observed in both types of staining, is more easily recognized with DQ stain (see Figure 3.7). 
The swollen microvilli impart ruffled borders and peripheral blebs in DQ smears (see Figures 1.4, 2.2).
The microvilli of mesothelial cells may prevent adjacent cells from completely apposing each other, thereby creating a gap between the adjacent cell membranes, which gives rise to the appearance of a space referred to as a mesothelial window (see Figures 1.8, 1.15, 1.16). Depending on many variables, these windows may be subtle or very wide in cytology smears. Such spaces caused by microvilli of adjacent cells are not specific for mesothelial cells; they may be seen in cell groups of some metastatic cancers in effusions (see Figure 9.2c).
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