Cytology of effusions (Proceedings)

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Cytology of effusions (Proceedings)

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Oct 01, 2008

An effusion is defined as the escape of fluid into a part. Effusive processes can occur in essentially any tissue in the body. Obtaining this fluid for cytologic evaluation limits the types of samples that are amenable to examination. Synovial fluid, aqueous humor, cerebrospinal fluid and serous body cavity fluids are all routinely evaluated in veterinary medicine. This review will only include pleural, peritoneal and pericardial fluids. As is the case with any information derived from any test, the ultimate interpretation and application requires full knowledge of the case and findings from other sources such as diagnostic imaging.

The peritoneal cavity is covered by a serous membrane of mesothelial cells overlying a connective tissue stroma. The cavity is essentially a potential space, with a small amount (<1 ml/kg) of fluid present to lubricate the viscera and allow their unhindered motions. The equine abdomen is real and fluid can be obtained from healthy animals. The esophageal hiatus, vena caval hiatus and the aortic hiatus allow passage of the corresponding structures through the diaphragm. These coverings are covered by a thin layer of fused endothoracic and transverse fascia. The peritoneal lining is extremely absorbent. Stoma are present that lead to the lymphatic system. Four fifths of the lymph from the peritoneum drains into the sternal lymph nodes, primarily through the diaphragmatic peritoneum. Mesenteric lymph nodes drain the intestine, not the peritoneum, which is drained by the omental lymph nodes. The parietal layer covers the inner walls of the abdominal, pelvic and scrotal cavities, while the visceral portion envelops the abdominal viscera. The pressure within the capillaries of the visceral peritoneum is higher than elsewhere, making the peritoneal cavity particularly prone to developing accumulations.

The entire pleural cavity is lined by a serous membrane known as the pleura. In health this is a potential space that contains only enough fluid to facilitate movement of the organs (e.g. excursion of the lungs during inspiration and expiration) and transmission of forces. It is composed of a single layer of mesothelial cells supported by a delicate network of connective tissue rich in elastic fibers. The visceral pleura covers, and is adherent to, the lungs. Its capillary network is derived from the pulmonary circulation. The parietal pleura covers the remaining thoracic cavity and is sub-divided into the costal, diaphragmatic and mediastinal portions, depending on the part of the cavity involved. Its capillary network is derived from the systemic circulation; however, unlike the visceral pleura, there is a rich lymphatic network for drainage of the pleural space. The mediastinum is a cleft or wall between the right and left mediastinal pleura. It is complete in bovid, goats, pigs or in any young animal. A fenestrated mediastinum can be found in the dog, cat, horse and in sheep; however, with certain diseases (e.g. exudate with fibrin formation), these fenestrae can become occluded, effectively forming a complete mediastinum.

The pericardium is a fibroserous envelope around the heart. It is made up of an outer fibrous and inner serous part, which is composed of parietal and visceral layers. Approximately 0.3 to 1.0 milliliters of fluid resides in the space. The function of the pericardium is speculative; however, whatever the function is, it is not vital, as removal produces no untoward effects. Effusions that develop quickly here have the potential for serious and acutely deleterious clinical affects, as cardiac tamponade can rapidly be fatal.

Normal serous cavity fluid is essentially a filtrate of plasma. Normal effusions are due to a combination of factors including Starlings forces, pressure differential across the visceral pleura that favors absorption, lymphatic drainage, mesothelial cell activity and stoma between mesothelial cells. The different cavities can have a large, ongoing exchange of fluid and solute, yet a low, stable volume. Crudely, effusions form when there is an imbalance between formation and resorption. Forces that can impact the formation-resorption balance include:

Hypoproteinemia, increased hydrostatic pressure, increased vascular permeability and decreased lymphatic drainage. There are also multiple sources outside of Starlings forces including: ruptured urinary bladder, ruptured viscous, fractured liver, neoplastic cells producing fluid, iatrogenic, etc.

The power of the clincopathologic evaluation of effusions is undeniable. Using widely available tools and instruments, samples can be obtained with minimal risk to the patients, yet the diagnostic yield can be tremendous, depending on the specific disease entity present. Abdominocentesis is indicated when ascites is grossly evident or when fluid is identified via diagnostic imaging. A non-productive, "dry", tap does not rule out intra-abdominal pathology, as fluids and processes can compartmentalize and/or not produce a large quantity of fluid. A Diagnostic peritoneal lavage (DPL) can be performed when a sample cannot be obtained from the abdominal cavity using a straight abdominocentesis. Thoracocentesis typically will be performed when auscultation or radiographs indicate the location and extent of an effusion. Pericardiocentesis can be a diagnostic and therapeutic procedure that is performed when clinical signs, electrocardiographic, radiographic or echocardiographic findings indicate the presence of an effusion.

The proper handling of fluid can dramatically affect the diagnostic yield from samples properly obtained. If in doubt, contact your reference lab for details concerning submissions. Ethylenediaminetetraacetic acid (EDTA) is the ideal additive for cytologic analysis, as it provides the best morphology, with minimal distortion if smears are made relatively quickly. This tube can also be used for determination of the total nucleated cell count (TNCC) / white blood cell (WBC) count and total protein (TP), assessed by refractometry. If cells are left in EDTA tubes, they will age naturally and artifactually become vacuolated. EDTA is bacteriostatic and therefore not appropriate for culture. Tubes without additives can be utilized if any biochemical assays are to be performed on the sample. Although not ideal, they can be used for culture, mainly aerobic. Serum separator tubes (SST) should be avoided, as the gel can entrap cells and compromise cytologic evaluation.

If a delay is anticipated before processing, several air-dried direct smears (high cellularity) or line / concentrated smears (low cellularity) should be made, with the remainder being refrigerated. These smears will serve as a reference point for the cytopathologist to compare to the aged sample. Labeling can be very important, as is shipping overnight on ice and sending slides in slide transporters.

Long delays allow the macrophages to become vacuolated and begin to phagocytize erythrocytes, confusing the differentiation between in vitro vs. in vivo erythrophagia. In addition, hypersegmentation and pyknosis all begin to occur in neutrophils and other nucleated cells due to aging, respectively. In addition, any bacteria (pathologic or contaminant) can overgrow, making interpretation difficult.

Sample preparation is just as important as the microscopic evaluation. Direct smears can be made exactly like a peripheral blood smear technique (Figure 1). This is n extremely simple, fast technique to visualize nucleated cells in an effusion that has moderately high to high TNCC (>10,000/(l). This is the preparation that should be consulted if there are concerns about true, in vivo morphologic changes to cells and/or structures in the background (e.g. plant material, alleged ingesta, etc.). A drop of the fluid is placed on one end of a clean, new slide. The spreader slide is backed up into the drop, which then spreads towards the edges, but not to the edge. The spreader slide is advanced off the slide in one firm, smooth motion, producing a bullet shaped smear


Figure 1
Figure 1- Line diagram of a smear made from an effusion

The line technique is useful for making smears from samples with moderate nucleated cellularity. The technique is identical to the blood smear technique; however, the spreader slide is stopped short of the edge and lifted straight up, producing a line that contains a concentration of nucleated cells. In samples with TNCC's <5,000 / (l, there are two methods which can be employed to evaluate the nucleated cells. The first involves centrifuging a sample (1,000 - 1,500 rpm for 5 minutes), pouring off the supernatant and re-suspending the remaining pellet with 0.5 ml of fluid. A smear is made from this concentrated solution similar to making a blood smear. A second method is to take a portion of fluid and spin it in a micro-hematocrit tube. The tube is broken at the level of the buffy coat, which is then applied to a slide and streaked out as a blood smear. These preparations are useful in hemodilute samples. Samples with very low cellularity can be cytocentrifuged or prepared using gravity and a home made device.