**2. Effusion fluid as a first-hand cytologic diagnostic material**

#### **2.1 Clinical conditions of differential significance**

Mesothelioma is often but not always represented with effusion, the sampled fluid is typically exudate, yellowish, and often bloody [12]. It is reported to be thick and mucoid owing to hyaluronic acid or hyaluronan content. Notably hyaluronan and

*Mesothelioma: Overview of Technical, Immunochemical and Pathomorphological Diagnosing… DOI: http://dx.doi.org/10.5772/intechopen.106570*

N-ERC/mesothelin increase in effusion fluid predict mesothelioma with high specificity, prior to pathological examination. Pleural CEA increase can rule out mesothelioma with a high degree of certainty. Other soluble mesothelioma biomarkers such as C-ERC/mesothelin, osteopontin, fibulin-3, syndecan-1, syndecan-2, and thioredoxin are lacking sufficient accuracy for clinical use [13–15].

The diagnostic difficulty arises since there is a large diversity of other diseases, which can manifest with pleural or peritoneal effusions, creating an abundance of differential diagnoses to navigate in the cytological study. From a pathologist's perspective, benign infective, inflammatory, or other diseases are causing reactive changes in the mesothelial cells. Such reactive conditions manifesting predominantly with exudation can be related to tuberculous pleuritis or empyema or parapneumonic effusion caused by other bacteria, and collagen vascular diseases. Additionally, effusion can also be transudative because of hypoalbuminemia and heart or renal failure [16]. Among benign conditions causing peritoneal exudative effusion are infections such as tuberculosis or spontaneous bacterial peritonitis, whereas predominantly transudative effusion or ascites can be caused by portal hypertension due to liver cirrhosis, alcoholic hepatitis, or hepatic congestion, but also pancreatitis, hypoalbuminemia, or renal failure [17]. Reactive mesothelial cell changes can be extremely hard to distinguish from malignancy (see later). Therefore, another crucial question pathologist face is to confirm malignancy in the effusion cytology and to differentiate mesothelioma from other malignancies such as lung cancer and pleural metastasis from other organs, especially the breast [16]. In peritoneal effusions, other malignancies except mesothelioma to bear in mind are primary peritoneal papillary serous carcinoma, but more often hepatocellular carcinoma, metastatic liver disease, lymphoma with peritoneal involvement or the spread of other intra-abdominal malignancies such as pancreatic, gastric, colorectal, ovarian, or renal carcinomas [17–19]. Pathological differential diagnosis can help to identify the primary site of malignancy in a patient with a history of multiple malignancies or an unknown primary site.

#### **2.2 Handling of material**

Accuracy of pathological diagnosis heavily relies on high quality of material, which depends on its proper handling. The removed effusion is preferably sent to the laboratory fresh if possible with anticoagulants (heparin ethylenediaminetetraacetic acid or sodium citrate) present, but without added fixatives, and it should be refrigerated at 4°C until processing. When longer transportation times are needed, a volume of 50% ethanol can be added as a preservative [9].

Upon arrival in the laboratory, the fluid should be processed without delay. Refrigerated samples should be brought to room temperature, particularly when using preparation techniques associated with liquid-based cytology (LBC). To prepare a cell pellet, the material is centrifuged at 1000 g or more for 10 min. For the cytomorphological evaluation, smears are prepared from centrifuged deposits (preferably by cytospin method) and routinely stained with one of the Giemsa modifications (Romanowsky-Giemsa, Leishman-Giemsa or May-Grünewald-Giemsa kits), which enables well to examine cytoplasmic characteristics. Many labs are splitting the sample and use also Papanicolaou (PAP) stain preferably in liquid-based cytology to facilitate for nuclear evaluation [20].

The recent guidelines of mesothelioma diagnosis require additional IC studies (see later), which can be applied on smears, but the most popular technique is the cell block, obtained after the sediments from cytological specimens are processed, formalin-fixed and embedded into paraffin blocks that can be serial sectioned and stained by the same methods used for histopathology [21].
