SPECIMEN COLLECTION PROCEDURE

For staging purposes, the peritoneal cytologic sample must be obtained as soon as the peritoneal cavity is entered. Occasionally, tumor cells are inadvertently spilled into the peritoneal cavity during the course of the exploratory laparotomy and removal of the primary tumor. The significance of tumor cells identified in washings performed after iatrogenic contamination of the peritoneum is not known.While artificial contamination of the peritoneum with tumor is disquieting, the prognostic value of PWC is predicated on the assumption that the results reflect the natural biology of the patient’s tumor. This dilemma can be avoided when the cytologic samples are collected as an initial step in the exploration.

If there is pre-existing spontaneous fluid in the pelvis, this may be collected and labeled as ‘peritoneal fluid’ or, if excessive, as ‘ascites.’ An 100 mL volume of saline, preferably a balanced salt solution such as peritoneal dialysis fluid, is instilled into the peritoneal cavity, agitated, aspirated, and sent for cytologic evaluation.

In order to save costs, Sharifi et al¹⁶ recommended that peritoneal washings collected during laparotomy for disease presumed to be benign be held and sent for cytology only if malignancy is subsequently diagnosed. However, abrading the mesothelial surfaces is traumatic, and delay in processing an unfixed PW specimen can result in compromised cellular detail. Presumably, a frozen section or other intraoperative pathologic consultation needed by the surgeon to plan the surgical approach would limit the delay in processing to an hour or two in the majority of cases. If this approach is contemplated, it would be wise to utilize balanced salt solution for the washings.

INTERPRETATION APPROACH

Although washing the peritoneal cavity with saline or balanced salt solution may be expected to yield a cell sample similar to ascitic fluid, there are differences that must be appreciated in order to accurately evaluate these samples. Rather than the freely desquamated cells seen in spontaneous ascitic fluid, many of the cells present in peritoneal washings have been mechanically stripped from the underlying connective tissue. In addition, cell types that are not commonly seen in ascites (probably because they are normally shed in very low numbers) may be stripped from the surfaces.^13,14 The differences between ascites and peritoneal washings are shown in Table 7.1. An awareness of these differences between ascites and peritoneal washes will aid in interpretation.

Table 7.1 Comparison of cell patterns in peritoneal washings and ascites

MESOTHELIAL CELL POLYMORPHISM (see also Chapter 2)

Because the mesothelial cells in peritoneal washings are typically stripped from the underlying connective tissue, these cells are commonly present as flat sheets (Figure 7.1) of varying cell numbers.^{12–14,17,18} If large, these sheets are frequently folded or rolled. In general, these sheets are easily recognized as such and create little difficulty. Sometimes the sheets are squeezed together so that the nucleocytoplasmic ratio is artifactually distorted (Figure 7.2). The cells typically are arranged in a uniform honeycomb pattern. Single cells with flattened, polygonal shapes can also be seen in some cases (Figure 7.3). Nuclei are centrally placed with a round-oval, occasionally bilobed appearance (Figure 7.4). The nuclei in normal mesothelial cells can vary considerably in a single sample (Figure 7.5) (see also Chapter 2). The chromatin pattern is finely granular and a single micronucleolus is usually seen in the well-preserved mesothelial cells (Figure 7.6). The cytoplasm is thin with a polygonal shape. The staining quality of the cytoplasm is variable, although usually faintly azurophilic. Occasionally, multinucleated mesothelial cells (Figure 7.7) are present as a part of these sheets, with six or more nuclei forming a ring within the center of a giant cytoplasm. Degeneration of the mesothelial cells in peritoneal washing samples shows as paranuclear vacuoles that impinge on the nucleus (Figure 7.8). This may give the appearance of jagged, angulated nuclear contours.

Figure 7.1 Flat sheet of normal mesothelial cells in a peritoneal washing. This cell pattern reflects the manner of collecting the cells in a peritoneal washing and highlights the difference from spontaneously exfoliated cells in effusions. (Modified PAP stain, 20×.)

Figure 7.2 The mesothelial sheets are subject to distortion so that the nuclear-cytoplasmic ratio can show considerable variation in benign mesothelial cells. (Modified PAP stain, 40×.)

Figure 7.3 In some cases, the mesothelial cells are found as predominantly single cells. (Modified PAP stain, 60×.)

Figure 7.4 Although generally round-oval, mesothelial nuclei in these samples can sometimes appear bi- and multilobated. Some nuclei also show longitudinal grooves. (Modified PAP stain, 60×.)

Figure 7.5 Nuclear size in mesothelial cells can vary considerably in peritoneal washings and it is important not to overcall in such cases. All of the cells in this field are benign. (Modified PAP stain, 60×.)

Figure 7.6 Normal mesothelial cells with well-preserved round-oval nuclei, finely granular chromatin, and single small nucleoli. (Modified PAP stain, 60×.)

Figure 7.7 Multinucleated mesothelial cell sheets can sometimes be seen, generally against a background of previous laparotomy or other mesothelial injury. (Modified PAP stain, 20×.)

Figure 7.8 Paranuclear vacuoles develop in degenerating mesothelial cells that may alter nuclear contours (arrow). (Modified PAP stain, 60×.)

It is important to recognize that mesothelial cells in washings can have a variety of appearances in the same sample, particularly in cases in which there is an inflammatory component such as pelvic inflammatory disease,^12,13 preoperative rupture of an ovarian cyst,^13,14 or cystic neoplasm.^13,14 Inflammatory lesions of the gastrointestinal tract such as diverticulitis or appendicitis can have a similar effect. Healing of intraperitoneal inflammatory conditions is accompanied by adhesion formation in which a fibrinous exudate is replaced by mesothelial-lined fibrovascular connective tissue. Vascular tangles (Figure 7.9) composed of longitudinal capillaries wrapped in tangled fibrillar material can be seen in peritoneal washings as the cytologic residua of lysed adhesions.¹³

Figure 7.9 Structures composed of tangled fibrillary material (arrows) appear to represent capillaries and associated connective tissue material that reflect lysed adhesions. This pattern can be seen associated with healed inflammation or endometriosis. (Modified PAP stain, 60×.)

In some cases with large benign tumors, such as uterine leiomyomas or ovarian fibrothecomas (Figure 7.10), the benign mesothelial cells can be highly reactive and raise concerns for malignancy. This is particularly true in cases with Meigs’ syndrome¹³ in which ovarian fibrothecomas are associated with benign ascites. In such cases, there is a rather uniform distribution of reactive changes in the mesothelial cells, so that there is a spectrum of reactive changes in a single population rather than two discrete patterns (see Chapters 2 and 3). Correlation with the surgical pathology specimen will generally help to clarify the nature of the process. Rarely, we have seen malignant cells from extragenital malignancies in peritoneal washings from women undergoing surgery for benign female genital tract disease.^13,14 In these cases, the malignant cells were unequivocal and did not merge into the spectrum of reactive mesothelial cells.

Figure 7.10 Mesothelial sheets associated with large ovarian fibromas and spontaneous peritoneal fluid can have a rolled-up, pseudopapillary appearance (arrow). (Modified PAP stain, 40×.)

Epithelial cells from ruptured cystic endometriosis, benign cystic ovarian tumors, and bowel mucosa have been described in peritoneal washings.¹³ Although cytologically bland, these cells are clearly foreign to the peritoneal cavity and, therefore, can cause concern. Rupture of benign cystic tumors can also spill three-dimensional epithelial cell groups into the peritoneal washings.¹³ Discussion with the surgeon or review of the operative note can clarify this issue in problematic cases.

MESOTHELIAL SURFACE REACTIONS

The peritoneal surface in women has been referred to as the ‘secondary müllerian system’ because of its embryologic relationship with the müllerian ducts and because of the müllerian-derived lesions that appear to develop de novo in the peritoneum.¹⁹ It is important that anyone who examines peritoneal washings be familiar with these lesions so as not to interpret cells from these lesions as malignant.

Endosalpingiosis

Endosalpingiosis can be described as ectopic fallopian tube epithelium^20–23 involving pelvic structures and lymph nodes. It is seen as cystic invaginations or papillary formations on the surfaces of the ovaries, paratubal tissues, and omentum as well as within pelvic and para-aortic lymph nodes. Microcalcifications and psammoma bodies are frequent (Figure 7.11). Some authors attribute this entity to mesothelial metaplasia,²³ although others have suggested that it results from the implantation of inflamed epithelium from the fallopian tubes.²¹ The major significance of endosalpingiosis is the need to distinguish this benign process from metastatic carcinoma.^{13,14,20,22,24–29}

Figure 7.11 Psammoma bodies are surrounded by cuboidal epithelium (arrow). This pattern is typical of endosalpingiosis, although it can be seen with low-grade serous tumors. Typically in the latter instance, multiple abnormal cell groups will be present, with or without psammoma bodies. If only one or two groups (such as those pictured here) are present in a case, they are unlikely to represent peritoneal extension of a serous carcinoma, although review of the histology is often helpful. (Modified PAP stain, 60×.)

Cytologically, endosalpingiosis has been described as small aggregates and papillary fragments of cells characterized by cylindrical cell shapes with oval nuclei set in scant basophilic cytoplasm.^{12,13,24,25,27,28} In some cases, cilia and psammoma bodies can be seen.^13,19–29 The epithelial cells surrounding psammoma bodies often have a scalloped appearance (see Figure 7.11). Nuclear features are usually bland, with round-oval nuclei, finely distributed chromatin and single round micronucleoli.^27–29 The necessity and occasional difficulty in distinguishing endosalpingiosis from serous neoplasia has been described in several reports.^{13,14,22,25,27–30} This problem is compounded by the fact that endosalpingiosis and benign and malignant serous neoplasms will coexist in some patients.^{13,14,22,28–30} In endosalpingiosis, cell groups with the typical features are present only sparsely in the peritoneal washing. When there are many cell groups and/or large cell clusters that suggest endosalpingiosis, the possibility that they represent a serous neoplasm should be considered. Correlation with histology can be very helpful in these instances.

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