Fluid lesions: Cytology of effusions (Proceedings)

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Normally, only a small amount (milliliters) of fluid are present in the thorax and abdomen.

Normally, only a small amount (milliliters) of fluid are present in the thorax and abdomen. Effusions, defined as an accumulation of fluid in one or more body cavities result from multiple causes including increased hydrostatic pressure, decreased oncotic pressure, increased vascular permeability, decreased lymphatic drainage, infection, neoplastic infiltration, or rupture of organs or structures within the cavity.

Sample collection is done following aseptic preparation of the site. Local anesthetic or sedation may be required depending on the site of aspiration and the state of the patient.

Abdominocentesis: ultrasound guided aspiration may be necessary if there is only a small amount of fluid present, however in most cases non-guided aspiration is adequate. To obtain a cytologic sample, a 3-way stopcock is not needed, however to remove larger volumes of fluid, a needle with attached stopcock is helpful. Typically a 22g or occasionally a 20g needle attached to a 12-20 ml syringe is used. The mid-ventral region is most commonly sampled to avoid aspiration of the spleen or bladder.

Thoracocentesis is usually performed from the 6th, 7th, or 8th intercostal space, just below the costochondral junction. The needle is inserted through the middle of the intercostal space to avoid vessels on the caudal edge of the rib. Often, a 22g needle, 12-20 ml syringe, and 3-way stopcock is used for aspiration. Alternatively, a needle may be attached to extension tubing and then to the syringe or stopcock. The patient should be checked frequently for the first few hours following aspiration to assess respiratory and cardiac function. A thoracic radiograph is indicated if the patient's respiration worsens, to evaluate for the presence of pneumothorax, particularly tension pneumothorax.

The fluid should be placed in an EDTA tube. If any biochemical analysis is anticipated, it is best to also save some of the fluid in clot tube. Fluid for culture should be placed in a sterile container, culturette or inoculation broth. Fluid sample can be sent to a reference laboratory for further processing and interpretation or can be analyzed in-house.

Fluid analysis should include gross evaluation of color and turbidity, nucleated cell count (NCC), determination of total solids, and cytologic evaluation. The first is to examine the fluid grossly. The appearance and clarity can provide clues as to the diagnosis and also guide slide preparation and additional sample analysis. Normal fluid and transudates are clear and colorless. Increased cellularity due to either inflammatory or neoplastic cells produce a cloudy white or opaque color. Fresh blood or hemorrhage will induce a red-pink coloration; icterus a yellow color, bile will be greenish, and lipid (chyle) will be milky white. If the specimen is bloody or turbid, it is helpful to centrifuge a small portion (e.g. via microhematocrit) and repeat the observations on the supernatant.

Next, the total solids (TS) is measured using a refractometer. Total solids is often called total protein however as a refractometer measures light interference, other solutes will contribute to the final reading. Specific gravity can also be measured using a refractometer.

Cellularity can be quantified using an automated instrument (e.g. a hematology analyzer) or manually using a hemocytometer. Cellularity can also be estimated from a direct smear, however an estimation of cellularity cannot be determined if the sample has been sedimented or concentrated by other mechanisms. If there are 'clumps' or other particulate matter in the sample, a cell count on the fluid portion will typically underestimate the overall cellularity.

Biochemical analysis of the fluid can provide useful information in some cases. Commonly analyzed are triglyceride & cholesterol (chylous effusion), creatinine (uroabdomen), bilirubin (biliary or gall bladder rupture). Increased fluid LDH may be associated with septic or neoplastic effusions, while low glucose (< 10 mg/dl) and pH (< 6.9 ) may be associated with septic effusions.

There are several options for preparing slides from effusions. The optimal choice depends on the cellularity of the sample and the presence/absence of clumps. If the fluid is cloudy to opaque, it is best to make direct smears (like a blood film) or squash preps (see the session entitled Setting up Cytology in Your Practice for further details). Any clumps in the fluid should be extracted and used to make squash preps. If the fluid is clear to slightly turbid, a sediment smear is recommended. This is performed by centrifuging the sample at approximately 1000 rpm for 5-10 minutes, removing the majority of the supernatant, and then resuspending the cells in the remaining supernatant before making the slide preparations. Slides should be air-dried. If slides are to be evaluated in-house, they should be stained with Romanowsky-type stains. If they are to be sent to a reference laboratory, it is best to send at least one unstained slide. It is always best to make slides and send these along with the fluid as the character of the fluid may change substantially if there are bacterial or other organisms in the fluid.

Effusions can be classified into transudates, modified transudates, or exudates based on cell count and protein concentration. Neoplastic effusions typically are modified transudates or exudates, but can be seen in any category.

Transudates are characterized by minimal cell counts comprised primarily of mesothelial cells and macrophages, a specific gravity < 1.017, and a protein concentration less than 2.5 g/dl resulting from hypoalbuminemia or leakage of low protein intestinal lymph due to portal hypertension. Differentials include hypoalbuminemia due to severe hepatic disease, protein losing enteropathy or protein losing nephropathy, as well as some cases of congestive heart failure, diaphragmatic hernia, and lung lobe torsion (although these may also fall into the modified transudate category).

Modified transudates have similar cell numbers and distribution to transudates but have protein concentration greater than 2.5 g/dl resulting from leakage of high protein hepatic lymph or occasionally inflammatory proteins. Modified transudates have a variety of causes, one of the most common is right sided heart failure, however low cellularity neoplastic effusions often fall in this category.

Exudates are characterized by an increased protein concentration (> 3 g/dl) and marked cellularity - usually comprised of neutrophils if the exudate is inflammatory. Intracellular bacteria are found in septic exudates. Bacterial toxins are responsible for presence of degenerate neutrophils characterized by swollen nuclei and indistinct, smudged chromatin patterns. Streams of lightly eosinophilic nuclear debris may be encountered if these cells rupture during the slide-making process. The exudate associated with FIP is somewhat different than the traditional exudates and is distinguished by a remarkably high protein concentration (often greater than 5 g/dL) with a relatively low number of neutrophils and macrophages. Neoplastic effusions often fall into the exudate category.

Some effusions have diagnostic characteristics. Those are described below.

Hemorrhage is particularly common in pericardial effusions but may be seen in any effusion. Hemorrhage must be differentiated from blood contamination. Rapid clotting of the specimen and the presence of platelets are typical of blood contamination. However, platelets may be present in peracute hemorrhage. Erythrophagocytosis and hemosiderin laden macrophages indicate true hemorrhage.

Chylous effusions are milky in color due to an increased triglyceride concentration. Small lymphocytes predominate early in the pathogenesis, however the percentage of neutrophils and macrophages will increase over time. In a chylous effusion, fluid triglycerides are typically greater than serum triglycerides while fluid cholesterol is lower than serum cholesterol. Triglycerides of >300 mg/dl is considered diagnostic

Bile peritonitis is characterized by clouds of greenish material free in the background of the sample as well as macrophages containing yellow-green pigment. The fluid itself may also be greenish. Fluid bilirubin will be increased (greater than that seen in the serum). Rarely, bilirubin crystals may be seen in the fluid.

Uroabdomen initially results in a fluid with low protein and increased creatinine. Over time, the process will typically become inflammatory and the protein will increase. Urea nitrogen is a less reliable indicator of uroabdomen as it equilibrates more rapidly.

Neoplastic effusions generally have a high cell count and high protein concentration, however sometimes only low numbers of cells are seen. Occasionally this is due to the presence of large clumps that are mistaken for a clot and excluded from the counting and evaluation process. Neoplastic effusions most often contain those cells that exfoliate well: lymphomas, carcinomas, histiocytic neoplasia, mast cell tumors, and mesothelial cell tumors although mesenchymal populations are occasionally been seen on cytology. Mesothelial cells must be interpreted with caution as these cells, which can be found singly and in clumps, may demonstrate marked reactive changes that mimic neoplastic changes, particularly in mesothelial cells from the pericardial cavity.

FIP is characterized by a fluid with a high protein concentration and a minimal cell count typically comprised of macrophages and non-degenerate neutrophils. The effusion is most often seen in the abdominal cavity but may also be detected as a pleural effusion.

Cytologic examination includes examining the cellularity of specimen, identifying the predominant cell type, identifying the type and distribution of other cell types, searching for microorganisms, and finally making a final impression based on all of the data at hand (including the history, physical exam, other diagnostic data, and the full characterization of the effusion).

The types of cells seen in effusions differ based on the underlying process. Typically however, these fall into several categories – inflammatory cells, mesothelial cells, and neoplastic cells.

Large mononuclear cells predominate in normal fluid, transudates, and modified transudates. These are comprised of macrophages and mesothelial cells. In many cases, these are readily differentiated but activated macrophages and reactive mesothelial cells may form a morphologic continuum. Therefore these cells are often grouped on a differential.

Macrophages are 2-3x the size of neutrophils and have a oval to bean shaped nucleus and a moderate amount of basophilic cytoplasm that is often foamy or vacuolated. These cells will be found individually. Rarely, multinucleated macrophages may be seen. Phagocytosis of cellular debris (leukocytophagia or erythrophagia) as well as other particulate matter or organisms may be present.

Mesothelial will be found both individually and in clusters. When first exfoliated, these cells have a polygonal shape and very angular borders giving sheets of mesothelial cells a cobblestone pattern. As the cells split off from each other, they round up and often have a distinctive eosinophilic cytoplasmic fringe. These cells have a round nucleus, are occasionally binucleate, have visible nucleoli, and contain basophilic cytoplasm. As these cells become reactive, the cytoplasm can become deeply basophilic, nucleoli can become multiple and prominent, and binucleate cells increase. Reactive mesothelial cells can be difficult to differentiate from neoplastic epithelial cells. As a rule of thumb, reactive mesothelial cells from the abdominal cavity can have moderate atypical, those from the thoracic cavity even more atypia, and those from the pericardial sac often have marked atypia.

Neutrophils usually comprise < 60% of the total nucleated count in small animals. These should be non-degenerate and resemble the neutrophils seen in blood. The presence of even small numbers of degenerate neutrophils in the effusion should prompt a search for microorganisms and/or culture of the fluid. Some organisms live in pockets within the cavity and only those cells coming in contact with the toxins in the pocket will be degenerate.

Eosinophils are uncommonly seen and, when present in significant numbers, can be associated with parasitic, inflammatory (hypereosinophilic), infectious (especially fungal), and neoplastic processes.

Lymphocytes may be seen in small numbers and these should be present as small well-differentiated appearing lymphocytes. However, plasma cells and a reactive lymphoid population can be seen, particularly if there is leakage of chyle.

Mast cells may be seen in small numbers from animals that do not have mast cell neoplasia. However the presence of large numbers or atypical mast cells raises concern for mast cell neoplasia.

Neoplasia can arise from the cells in the abdominal or thoracic cavities, leak into the cavities through the lymphatics or vasculature, rupture through an organ wall, or metastasis into the cavity structures. Thus, all types of neoplasia can be found in effusions.

Lymphoma is one of the most common neoplasm found in effusions. Typically these are lymphoblastic in appearance however lymphoma of the small and intermediate cell type may also be detected in effusions. Lymphoblasts are round cells that are 1-3x the size of neutrophils. They contain a round nucleus with finely granular or stippled chromatin, single to multiple nucleoli, and scant amounts of deeply basophilic cytoplasm. These cells may have pink or purple cytoplasmic granules (LGL). In cats, it is not uncommon for lymphoblasts in effusions to have a few small punctate vacuoles. Occasionally, the nuclei of both canine and feline lymphoma may take on bizarre shapes including flower like shapes, cleaved and indented nuclei, and the presence of micronuclei. These can be very difficult to differentiate from histiocytic neoplasia.

Histiocytic sarcoma (malignant histiocytosis) may resemble lymphoma although in the more classical presentations, these cells are large to very large (2-5x the size of neutrophils) and frequently contain numerous multinucleated cells within the population. These cells often have more abundant cytoplasm than do cells from lymphoma and cytophagia, especially erythrophagia, may be seen.

Mast cell neoplasia is another commonly seen round cell tumor in effusions, particularly abdominal effusions. These cells may be normal in morphology but present in large numbers or may be atypical with variable granulation as well as moderate to marked anisocytosis and anisokaryosis. They may be accompanied by eosinophils, however this does not occur in all cases.

Carcinomas are often characterized by the presence of large numbers of cells, often appearing in variable sized clusters. Occasionally clusters of these cells have a acinar or tubular appearance. These cells may be rounded (particularly when individualized in the fluid), polygonal, or columnar in shape. Cell to cell cohesion is evident in the cell clusters. Neoplastic epithelial cells in effusions tend to have coarse, sometimes even ropy chromatin with prominent, large, multiple, angular, and sometimes bizarre nucleoli. Marked anisocytosis and anisokaryosis may be seen. Perinuclear punctate vacuolation is common. However, despite the marked atypia that is often present in some carcinomas, it can be difficult to differentiate reactive mesothelial cells from neoplastic epithelial cells.

Mesothelial cell neoplasia can be well-differentiated or poorly differentiated in appearance. They are often characterized by large numbers of cells. In some cases, except for the very large numbers of cells, the cells have no specific criteria of malignancy. Cytologically (and histologically), these can be very difficult to diagnose. Poorly differentiated mesothelial cell neoplasia has features similar to those described above for carcinoma. Binucleates and multinucleates are commonly seen.

Spindle cell neoplasia is uncommonly diagnosed by examination of an effusion despite the fact that hemangiosarcoma is a very common cause of a hemorrhage effusion in the abdominal, thoracic, and pericardial spaces. Neoplastic cells, when seen, will be present in very low numbers. These cells will be spindle shaped, stellate or polygonal in shape with oval nuclei, stippled chromatin, single to multiple nucleoli, and moderately basophilic cytoplasm.

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