Analysis of fluids: cells and more (Proceedings)

The fluids most frequently sampled for cytology are peritoneal, pleural, synovial, cerebrospinal and pericardial fluids and washes of the respiratory tract. Some of these fluids are more easily obtained than others. All may potentially yield general, or sometimes more specific information about a disease process.

What to do with the sample?

Fluid samples for cytology should be put into EDTA tubes to best preserve the cell morphology. If you're going to mail samples to an outside laboratory, it's a good idea to make smears of the fresh sample and send these along with the fluid sample to the laboratory. If you think that you may culture the fluid, place at least part of the sample in a red-top tube or on a culture swab, as EDTA is bacteriostatic.

Fluid smears can be made like a blood smear, using a small drop of fluid instead of blood, or a drop of fluid can be placed on a slide and the "squash" technique can be used. If the cell count of the fluid is high, make direct smears. If the fluid has a low cell count, "line" smears can be made or some of the fluid should be spun at a low speed for 3-5 minutes and smears should be made from the sediment. Samples should be allowed to air-dry before staining. No other fixation is needed and can actually damage the sample.

What do we evaluate?

• Physical properties of the fluid including: volume, color, transparency, and odor

• The nucleated cell count should be determined (except for washes) usually by hemacytometer, using a dilution system if the cell count looks moderate to high (i.e. if the fluid is cloudier than water).

• Total protein should be measured (except for washes) using the plasma protein scale of a refractometer.

• PCV or RBC count may also be determined if the fluid looks very bloody.

• Cytologic evaluation

Body cavity effusions

A small amount of fluid is normally present in the body cavities to allow the visceral and parietal surfaces of organs and the body wall to interact freely without friction. The fluid is essentially an ultrafiltrate of blood, and is continually being added to and removed from the body cavities.

Normal body cavity fluid usually has:

• <2.5 g/dl protein (typically around 1 g/dl)

• <5000 cells/μl

• <10% neutrophils in small animals

An effusion is an increase in the amount of body cavity fluid. Fluid is difficult to obtain in small animals if an effusion is not present, but can usually be obtained from normal horses. Classifying the type of effusion helps narrow down the list of possible causes and helps direct other useful diagnostic steps.

Transudates

These are non-inflammatory effusions that result from impeded blood flow, impeded lymph drainage, or low osmotic pressure in blood vessels (i.e. hypoalbuminemia). They are roughly divided into:

Pure Transudates

• Transudates containing very low protein (<1 g/dl) and cell numbers (<1000 cells/μl)

• Low osmotic pressure is a common cause of pure transudates, typically due to hypoalbuminemia.

• Pure transudates are less commonly associated with heart and liver disease than are modified transudates.

Modified Tranudates

• Higher protein content than pure transudates

• Cell counts frequently higher than pure transudates – but typically within the normal reference range or only mildly increased.

• Can occur secondarily to a number of different causes, most associated with some type of impaired lymphatic drainage or venous stasis (increased hydrostatic pressure). The most common causes of a modified transudate are cardiovascular disease and neoplasia. Other possible causes include chronic hepatic disease, tissue inflammation, lung torsion, and diaphragmatic hernia.

• Chylous effusions may fall into this category and have similar causes.

Exudates

• Inflammatory effusions due to increased capillary permeability resulting in a fluid with high protein and cellularity.

• Exudates can be further classified as septic (bacteria present) or non-septic.

Hemorrhagic effusions

• Usually result from neoplasia, trauma, surgery, or hemostatic defects.

• Should be differentiated from blood contamination., but this isn't always easy

• Macrophages containing erythrocytes and hemosiderin are consistent with previous hemorrhage.

• Platelets should be absent unless very recent or active hemorrhage. Their presence suggests probable blood contamination at the time of sampling.

• Erythrocytes may become pale and/or crenated and neutrophils may become hypersegmented over time.

Chylous effusions

• Result from increased lymphatic pressure causing small chyle "leaks" or from rupture of the thoracic duct.

• Typically opaque, usually milky, and white to light pink.

• Protein content measured on a refractometer may be markedly erroneously high due to the lipid content of the sample.

• Cell count may reach into the exudate range (typically 5000-30,000 cells/μl).

• Small lymphocytes are predominant early, but increasing numbers of neutrophils and macrophages occur with an inflammatory response over time.

• Triglyceride concentration is higher in chyle than in serum and chyle is low in cholesterol. The cholesterol:triglyceride ratio of the fluid is <1.

Neoplastic effusions

• Can be diagnosed if neoplastic cells are exfoliated into the fluid.

• Types include: lymphoma, mast cell sarcoma, carcinoma, and mesothelioma.

• Inflammation may be present, more commonly with carcinoma.

• Total protein and cell counts can be variable, TP is usually 3.0-4.5 g/dl and cells counts are usually 3-30,000 cells/μl.

• Differentiating carcinoma from mesothelioma can be extremely difficult cytologically.

• Differentiating reactive mesothelial cells from neoplasia can also be challenging.

Cytology of the respiratory tract

Normal cell types:

For tracheal washes, the most common cell type should be cuboidal to tall columnar, often ciliated respiratory epithelial cells. Goblet cells, which are epithelial cells that produce mucus, may also be seen, although if they are noticeably increased in number, they indicate chronic irritation or inflammation. For BALs, macrophages are the predominant normal cell type.

In both types of samples, neutrophils should be <5% of the cell population. Low number of lymphocytes and a few mast cells may be seen. Eosinophil numbers are normally low in dogs and horses, but are extremely variable in cats.

A small amount of mucus is normally present and looks like strands of purple material. Increased mucous production typically occurs with inflammation. Curschmann's spirals are spirally-twisted mucous casts that indicate chronic excessive mucous production.

Synovial fluid

Synovial fluid has a unique viscosity. Grossly, a subjective assessment can be made by noting the stringiness of fluid between fingers. Viscosity can also be estimated by examining the smear, as cells tend to row in a highly viscous fluid.

Normal joint fluid contains few erythrocytes. Increased numbers of erythrocytes may be due to contamination or previous hemorrhage into the joint. A large amount of blood contamination makes evaluation of the joint fluid difficult.

The nucleated cell count should be <3,000/μl (<500/μl in horses). Neutrophils should comprise <10% of the cell population. Large mononuclear cells may consist of macrophages, blood monocytes, or synovial membrane cells. Occasional chondrocytes, osteoblasts, and osteoclasts may be seen.

If the cell count is > 3,000/μl (> 500 if equine) or the neutrophil percent is >10%, the joint is considered inflamed (suppurative, mononuclear, or mixed).

If the inflammation is suppurative, the neutrophils should be examined carefully for evidence of sepsis (degenerative changes or the presence of bacteria).

Hemarthrosis should be distinguished from blood contamination. Erythrophagocytosis is typically noted with hemarthrosis. Xanthochromia (yellow color) may be present if the bleeding is not acute. This can be seen with vitamin K antagonist toxicity, hemophilia, and possibly trauma. The presence of platelets suggests blood contamination.

Cerebrospinal fluid (CSF)

After you get CSF, the analysis should be done as soon as possible (preferably within 30 minutes), as cells deteriorate rapidly in the low protein content of CSF. Normal CSF should be clear and colorless. Yellow coloration, or xanthochromia, is typically indicative of old hemorrhage, but may also be due to a marked increase in protein content (>400 mg/dl) or severe icterus.

Normal CSF has very low cell count and protein. Cell counts should be <8 cells/μl and the cells should primarily be mononuclear cells. Neutrophils are rare in normal CSF, but they are frequently introduced with blood contamination. Eosinophils are also rare.

The total protein content of CSF in dogs is <35 mg/dl, in cats <48 mg/dl, and in horses <80mg/dl. It will vary some depending on which site it's taken from. Protein content is typically increased with inflammation/ pleocytosis. Increased protein without pleocytosis can also be seen with degenerative disease or malacia.

Pleocytosis = increase in cell count