N. Clancey, BSc, DVM, MVSc, DACVP
Department of Pathology and Microbiology, Atlantic Veterinary College, Charlottetown, PEI, Canada
A variety of disorders cause pericardial, pleural, and peritoneal effusions. However, just five underlying pathological mechanisms alone, or in combination, create effusions. Understanding these mechanisms helps elucidate how fluid analysis can contribute to diagnosis and case management.
Small amounts of clear serous fluid are normally present in the pericardial, pleural, and peritoneal spaces. These spaces are enclosed by visceral and parietal surfaces lined by mesothelial cells. This fluid acts as a lubricant and transport medium for electrolytes and solutes. Both the protein concentration and cellularity of the fluid are low. Expected nucleated cells include mesothelial cells, macrophages, lymphocytes, and neutrophils.
In health, fluid forms from processes involving anatomic features and Starling’s forces. Anatomic features include capillary permeability, the surface area for fluid movement, and lymphatic openings within the mesothelium responsible for fluid drainage. According to Starling’s law, net capillary to interstitium fluid movement is directly related to the difference between hydraulic and oncotic pressure gradients across the capillary wall, capillary permeability, and the surface area for fluid movement. Plasma filtrate leaves capillaries, enters the interstitial space, diffuses into body cavities, is removed by lymphatics, and returned to plasma. Thus, vascular permeability, mesothelium permeability, lymphatic drainage, and Starling’s forces all play key roles in effusion formation.
Pathologic Mechanisms of Effusion Formation1
Transudates occur because of excess diffusion of plasma water from vessels due to increased hydraulic pressure with or without decreased oncotic pressure. Transudates may also form when lymphatic drainage is impaired. Transudate formation solely due to hypoalbuminemia is rare, as compensatory processes occur in this state. These include decreased intravascular hydraulic pressure, reduced interstitial oncotic pressure, globulin synthesis, and increased lymphatic drainage. A marked hypoproteinemia occurring acutely may result in transudation because insufficient time has occurred for effective compensation. Transudates are typically low in cellularity and protein-poor (<25 g/L), but protein-rich (>25 g/L) transudates may also form.
- Protein-poor transudates
Protein-losing nephropathies and hepatic disease resulting in pre-hepatic (mesenteric lymphatics), presinusoidal (portal), or early sinusoidal hypertension are the most common reasons. Occasionally, protein-losing enteropathies may create protein-poor transudates. If an enteropathy causes marked hypoproteinemia, part of the transudate formation may be due to decreased oncotic pressure. Additionally, some protein-losing enteropathies such as lymphangiectasia may impair drainage of intestinal lymph.
- Protein-rich transudates
Common causes are sinusoidal or post-sinusoidal hypertensive hepatic disease, neoplasia, and congestive cardiac disease. Portal hypertension caused by congestive cardiac disease is post-sinusoidal and post-hepatic. Increased hydraulic pressure occurs in the hepatic sinusoids, leading to a protein-rich effusion.
Exudates form due to increased vascular permeability secondary to inflammatory mediators released from inflamed tissue, allowing plasma and proteins to leak from vessels. Chemotactic substances cause leukocytes to migrate from vessels, contributing to the increased cellularity of exudates. However, other factors may contribute to exudates:
- Inflammatory mediators may cause vasodilation such that increased blood volume enters inflamed tissues. Therefore, increased capillary hydraulic pressure may contribute to the fluid accumulation.
- If blood vessels are directly inflamed, loss of plasma, proteins, and leukocytes into the fluid readily occurs.
- Loss of plasma proteins to the interstitium lowers the osmotic pressure gradient between plasma and interstitium, decreasing the ability of plasma to retain water.
- Mesothelial inflammation creates damage, so protein-rich interstitial fluid more easily moves into cavities.
Vascular trauma enables blood to escape, creating a hemorrhagic effusion. Minor hemorrhage often occurs during sampling, and minor hemorrhage may be present in exudates due to vascular damage associated with inflammation. Acutely formed or collected pathological hemorrhagic effusions have similar features to blood. However, effusion volume and composition will change with time as lymphatic vessels work to resorb effusion water, solutes, and erythrocytes.
This term means leakage of lymph from lymphatic vessels. Lymphorrhage may occur with trauma, lymphatic hypertension, lymph stasis, increased permeability of lymphatic vessels, and dilated lymphatic vessels resulting in defective lymphatic valve function. Occlusion of lymphatic vessels due to compression by a mass, or luminal obstruction by metastatic cells within lymphatic vessels may also contribute to lymphorrhage.
5. Hollow Organ/Tissue Rupture
Damage to the urinary, biliary, or gastrointestinal tract can allow leakage of urine, bile, or gastrointestinal contents, respectively, into the peritoneal cavity. A septic or non-septic inflammatory reaction is commonly associated with these effusions. Fluid findings will vary with time.
Initial Fluid Evaluation
Fluid evaluation begins at collection, noting the colour, clarity, and ease of sampling. If initially clear fluid turns bloody during collection, or vice versa, blood contamination likely occurred. Collection of frank blood supports a hemorrhagic effusion or incidental sampling of a highly vascular organ such as the spleen. A cloudy, white fluid is typical of chylous effusions. Dark green fluid is typical of direct gall bladder sampling. Depending on the degree of leakage and time, fluid from bilious effusions may vary from dark green to light green-yellow. Colourless, clear fluids are typically protein-poor and hypocellular, suggesting a transudate. Yellow, opaque fluids are typically protein-rich and highly cellular, supporting an exudate.
These are generalizations and can be useful, although in one study fluid color did not have strong associations with any particular disease process.2 The total nucleated cell count (TNCC) and protein concentration are rarely known prior to smear preparation. However, recognizing fluid transparency at initial collection can help guide smear preparation.3 When in doubt, submission of both direct and sediment smears is ideal.
Preparing Fluid Samples for Submission
This preparation is identical to that used for blood smears. At very minimum, at least one direct smear should be submitted. Fluids with low TNCCs yield hypocellular direct smears. Thus, clear and colourless fluids warrant preparation of smears using techniques that aid in cell concentration.
These are created similarly to a blood smear technique, but the spreading slide is stopped and lifted vertically off the underlying slide such that cells concentrate along a line. Line preparations are ideal for protein-poor, low-cellular fluids. High-protein fluids may result in an area too thick for microscopic evaluation.
These are prepared similarly to urine sediment preparations and are ideal for protein-poor, low-cellular fluids. Fluid is gently centrifuged for 5 minutes, most of the supernatant is removed, the sediment is re-suspended, and a small amount is transferred to a slide for creating a direct smear or slide-over-slide preparation.
These preparations require special commercial cytocentrifuges typically limited to larger laboratories. They are highly valuable for low-cellular samples.
Assessing Fluids at Diagnostic Laboratories
Laboratories routinely provide gross fluid characteristics, TNCCs, red blood cell (RBC) counts, total protein concentration, and cytological analysis for effusion samples. Additional diagnostic tests can be performed as desired to aid diagnosis with select effusions.
These can be performed manually using hemocytometers or automatically using electronic analyzers. Electronic analyzers vary in sensitivity, and potential exists for cells and debris to obstruct these machines. Additionally, non-nucleated cells or fragments, debris, and organisms can falsely contribute to automated TNCCs. Hence, assessing cellularity is one reason cytological evaluation is required. When effusions are pink to red, the erythrocyte count or hematocrit should be assessed. The fluid RBC count may be similar to the patient’s blood RBC count in early hemorrhagic effusions, but declines as erythrocytes are absorbed by the lymphatics and altered oncotic gradients cause fluid to shift into the body cavity.
Total Protein Concentrations
Estimates can be determined using refractometers. This should ideally be performed on post-centrifugation fluid supernatant, especially with turbid fluid samples, to reduce artifactual increases. Additive in some EDTA tubes can refract light causing falsely increased values, particularly with low sample volumes.4 More accurate protein concentrations are obtained using chemistry analyzers.
Often the most important part of fluid analysis, as relying only on cell counts and protein concentrations may produce incorrect diagnoses. Smears are evaluated for relative nucleated cell proportions, neutrophil degeneration, Cytophagia, infectious organisms, neoplastic cells, foreign material, frank or previous hemorrhage, and any other features. Several excellent textbooks are suggested for further details.5-8
Not routinely performed but desired in select cases. These may include measurement of concentrations of urea, creatinine, triglycerides, bilirubin, lipase, lactate, glucose, and pH. Suspected bilious effusion samples should be protected from ultraviolet light because it degrades bilirubin. Lactate, glucose, and pH assessment should be performed immediately because storage yields erroneous values. Fresh samples from neoplastic effusions may be further evaluated using advanced modalities, such as clonality testing or immunophenotyping using flow cytometry.
1. Stockham SL, Scott MA. Fundamentals of Veterinary Clinical Pathology. 2nd ed. Ames, IA: Blackwell Publishing; 2008.
2. Bohn AA. Analysis of canine peritoneal fluid analysis. Vet Clin North Am Small Anim Pract. 2017;47(1):123–133.
3. Stokol T. Fluid analysis: thoracic, abdominal, joint. In: Ettinger SJ, Feldman EC, Côté E, eds. Textbook of Veterinary Internal Medicine: Disease of the Dog and Cat. 8th ed. St. Louis, MO: Elsevier; 2017.
4. Estepa JC, Lopez I, Mayer-Valor R, et al. The influence of anticoagulants on the measurement of total protein concentration in equine peritoneal fluid. Res Vet Sci. 2006;80(1):5–10.
5. Barger AM, MacNeill A. Small Animal Cytologic Diagnosis. 1st ed. Boca Raton, FL: CRC Press; 2017.
6. Cian F, Freeman K. Veterinary Cytology. 2nd ed. Boca Raton, FL: CRC Press; 2017.
7. Raskin RE, Meyer D. Canine and Feline Cytology: A Color Atlas and Interpretation Guide. 3rd ed. St. Louis, MO: Elsevier; 2016.
8. Valenciano AC, Cowell RL. Cowell and Tyler’s Diagnostic Cytology and Hematology of the Dog and Cat. 4th ed. St. Louis, MO: Elsevier; 2013.