Alan H. Rebar1; Rose E. Raskin2, DVM, PhD, DACVP
Accumulations of excessive fluid in the pleural, peritoneal, or pericardial space represent edema of these body cavities. As such, the dynamics of fluid accumulation are governed by Starling's law. In general, pleural, peritoneal, and pericardial effusions can result from either inflammatory or non-inflammatory causes. Non-inflammatory edema results from arterial pressure (a rare cause in veterinary medicine), venous stasis, hypoalbuminemia (reduces plasma oncotic pressure), or lymphatic obstruction. Inflammation causes fluid accumulation through vascular damage and leakage.
Evaluation of abnormal fluid accumulation is based upon integrated assessment of both physical and cytologic characteristics.
Physical assessment of effusions includes estimation of total solids (total protein) by refractometry and determination of total nucleated cell count with either an automated particle or manual methods. On the basis of physical characteristics, effusions are classified as transudates, modifies transudates, or exudates. As a general rule (there are exceptions which will be discussed later), transudates are non-inflammatory in origin while exudates are the result of inflammation. Thus, physical characteristics of effusions allow classification according to pathophysiologic mechanism of fluid accumulation but reveal little about specific disease diagnosis.
Cytologic evaluation of effusions augments physical evaluation by allowing the diagnostician to identify specific etiologic agents, to follow the progression of a disease process, and to monitor response to therapy. The decision of whether or not to culture effusion fluid is based upon cytologic findings. Through cytologic examination, inflammatory reactions are classified as to type, hyperplastic responses of the mesothelial lining can be identified, and neoplasia is diagnosed.
Cytologic Evaluation of Effusions
1. Normal Findings
Cells present in normal pleural, pericardial, and peritoneal fluids include low numbers of mesothelial cells and occasional inflammatory cells. Mesothelial cells are present in small clusters or as individuals. If knocked loose from the cavity lining during the collection process, mesothelial cells resemble squamous cells with a low N/C ratio and abundant faintly basophilic cytoplasm. Cells with this morphology have historically been referred to as "pale" mesothelial cells. Normal mesothelial cells which are present in the fluid prior to collection have rounded up and are quite basophilic ("dark" mesothelial cells). They measure between 25 and 35µ in diameter. Nuclei are centrally located, round, and uniformly granular. Cytoplasm is abundant. The most striking characteristic of these cells is the presence of an eosinophilic peripheral brush border or "skirt".
The inflammatory cells present in normal fluids have the morphology of normal peripheral blood leukocytes. The predominant leukocyte seen varies with the species. In dogs and horses, neutrophils are prevalent. In cats and cattle, lymphocytes predominate.
2. Transudates and Modified Transudates
Cellular morphology in true transudates is similar to that in normal fluid; as emphasized previously, the principal alteration in transudation is increased fluid volume. The principle cellular constituent of the modified transudate (even the pseudochylous effusion) is the reactive mesothelial cell.
Because of the ability of mesothelial cells to respond to irritation by proliferation, the presence of increased numbers of mesothelial cell clusters and rafts is a common finding in reactivity. Mitoses are increased and occasional multinucleated reactive mesothelial cells are seen. Reactive mesothelial cells in clusters are capable of imbibing lipid from the effusion fluid and when they do, they take on the characteristics of secretory cells. In this form they must be differentiated from metastatic adenocarcinoma or primary mesothelioma. This is done by critically evaluating the cell populations for criteria of malignancy.
Reactive mesothelial cells also may assume a variety of other morphologies. Individual mesothelial cells sloughed into effusions gradually lose their brush border and may develop phagocytic capabilities, accumulate intracellular debris, and become indistinguishable from macrophages.
As modified transudates mature, the proportion of inflammatory cells they contain increases. In most cases the principle inflammatory cell is the non-degenerate neutrophil, but neutrophils rarely account for more than 30% of the total cell population.
Inflammatory effusions are classified according to the standard rules for inflammation as neutrophilic, mixed, or histiocytic. In neutrophilic reactions, neutrophils (either non-degenerate or degenerate) comprise >70% of the inflammatory cells seen. Mixed reactions are characterized by a mixture of neutrophils and macrophages, and in histiocytic inflammation, macrophages are the prevalent cell seen.
Inflammatory effusions are by their nature irritative to the lining of involved body cavities. Consequently, virtually all inflammatory effusions are characterized by some degree of reactive mesothelial cell hyperplasia. Because of the tendency of reactive mesothelial cells to cytologically merge with the monocyte/macrophage continuum, they are considered to be a part of the macrophagic component of any inflammatory effusion.
Most inflammatory effusions are cytologically nonspecific in terms of etiologic diagnosis. However, as with inflammatory responses elsewhere, cytologic morphology provides significant clues as to underlying cause. Neutrophilic inflammatory effusions indicate severe irritation. If neutrophils are degenerate, then an effort should be made to identify bacterial organisms within phagocytes. This is generally easiest at the featheredge of the smear. If organisms are not seen, the fluid should still be cultured. Mixed inflammatory and macrophagic effusions reflect less severe irritation and are found with resolving neutrophilic effusions or in association with less irritating etiologic agents than bacteria (e.g., fungal organisms or foreign bodies).
a) Chylous effusions: Chylous effusions are the result of leakage of lymph into the body cavity and may involve either the pleural or peritoneal space. Most commonly, the thoracic cavity is involved (chylothorax) as a result of trauma to the thoracic duct.
Cytologically, chylous effusions are characterized by the presence of large numbers of morphologically normal small lymphocytes. Lesser numbers of reactive lymphocytes are also present. Because these fluids are mildly irritating, long-standing chylous effusions also may contain moderate numbers of reactive mesothelial cells and other inflammatory cells. We have seen several cases of chronic chylothorax where significant numbers of eosinophils were seen. The presence of lipid in the background of the slide, visualized as small unsustained droplets at the periphery of the nucleated cells, is variable.
It is important to note that in the cat, cardiac disease results in pleural effusions which are indistinguishable from chylothorax. The mechanisms behind these effusions have not been clarified; however, it is well established that heart failure causes venous and lymphatic stasis with increases pressure. In the cat it appears that these circumstances predispose to lymphatic leakage and result in a secondary chylous effusion.
b) Hemorrhagic effusions: True hemorrhagic exudates can occur in any of the major body cavities. Grossly, these effusions are red to serosanguinous depending upon the age of the exudate and the extent of the hemorrhage. Physical evaluation reveals a protein level reflective of but somewhat less than that of peripheral blood. Both nucleated cell counts and red blood cell counts are elevated.
Cytology is needed to differentiate true hemorrhagic exudates from sample contamination at the time of collection. Hemorrhagic exudates contain predominantly red blood cells with lesser numbers of nucleated cells. The most significant indicator of true hemorrhage is the presence of activated macrophages containing phagocytized red cells (erythrophagocytosis) or hemosiderin. These cells are best observed at the featheredge of sediment smears. Erythrophagocytosis is not seen if hemorrhage is strictly a collection artifact. A second significant observation is whether or not platelets are seen. True hemorrhagic exudates are devoid of platelets but they are commonly observed in contaminated samples.
c) Neoplastic effusions: Neoplastic processes, both primary and metastatic, are relatively common causes of both abdominal and thoracic effusions in dogs and cats. They are less frequent but still represent an important cause of effusion in the horse.
Neoplastic effusions may be accompanied by significant hemorrhage and/or inflammation but generally they are non-inflammatory. Grossly, the fluid may be clear to cloudy and hemorrhagic. Total protein levels are elevated but nucleated cell counts are highly variable.
In dogs and cats the common causes of neoplastic effusions are thymic lymphosarcoma (pleural), and adenocarcinoma or carcinoma (either pleural or peritoneal). In horses, squamous cell carcinoma of the stomach (peritoneal) is by far the most frequent cause, with lymphosarcoma occurring only occasionally. Mesothelioma can be a rare cause of effusion in any species.