Download Physical properties of the fluid should be noted, such as volume

CYTOPATHOLOGY OF BODY CAVITY EFFUSIONS
Mary Anna Thrall
Donald J Meuten
Physical properties of the fluid should be noted, such as volume, color, transparency, clot
formation if not in EDTA, and odor. Total protein by refractometry should be determined by using the
plasma protein scale of the refractometer. While specific gravity may be measured on the urine specific
gravity scale, it does not add to information other than as an index of protein content. Total nucleated cell
counts can be determined by the same methodology as leukocyte counts for whole blood, using either a
hemocytometer (Unopette system) or an electronic cell counter.
A fresh, direct film of the fluid should be made by placing a drop of fluid on a glass slide, placing
a spreader slide directly on top of the drop, allowing the drop to spread, and pulling the two glass slides
apart. If the cell count is less than 5000/µl, one should examine films of fluid sediment. Cells may be
concentrated by centrifuging the fluid, pouring off most of the supernatant, then re-suspending the cells in a
drop of the effusion by “flicking” the tube with one’s finger. Sediment films can then be prepared in the
same manner as are direct films. Alternately, cytocentrifuges are commercially available that sediment the
cells in a small area in the center of the slide. The film is allowed to air dry, then stained with a
Romanowsky stain such as Wrights, Wrights-Giemsa, Diff-Quik, etc.
Microscopic examination
The slide is examined at low power (10x), then with a 50x or 100x oil objective lens. The
predominant cell type present should be noted, as well as the distribution of other cell types, and the
presence or absence of microorganisms. Cells seen in effusions are those encountered on blood films or
other types of cytologic specimens; additionally, mesothelial cells that line the body cavities are often
present.
Neutrophils: Neutrophils are usually well preserved (non-degenerate) in non-septic inflammatory
lesions, appearing much as they do in peripheral blood. As the neutrophils age, the nuclei become
hypersegmented and eventually pyknotic . In septic inflammatory lesions, the neutrophils undergo rapid
degeneration and eventual rupture. This lytic process is called karyolysis and is characterized by nuclear
swelling in which the nucleus becomes pink staining and smudged in appearance. Cytoplasm may become
more basophilic and vacuolated, but cytoplasmic appearance is relatively unimportant in determining if
cells are degenerate
Lymphocytes: Normal lymphocytes appear much as they do in peripheral blood. Immature
lymphoid cells (lymphoblasts) are characterized by their large size and the presence of a nucleolus.
Plasma cells are similar in size to small lymphocytes, but the nuclear chromatin is more dense, the
cytoplasm is blue and abundant, and a perinuclear clear area (Golgi apparatus) is usually apparent.
Macrophages in tissue are derived from blood monocytes. These cells phagocytize cellular
debris, foreign material and certain microorganisms, primarily mycobacteria. Other types of bacteria are
rarely seen within phagocytic vacuoles of macrophages. Neutrophils, cellular debris, erythrocytes, red
blood cell pigments, lipids, and phospholipids, however, are commonly encountered within the cytoplasm
of these phagocytic cells. Macrophages range in size from 12 µ to 100 µ, have a round to oval nucleus that
may contain an apparent nucleolus, and have light blue, usually vacuolated cytoplasm.
Eosinophils appear similar to eosinophils in peripheral blood and are associated with various
types of lesions, including allergic inflammation, parasitic inflammation, eosinophilic granulomas, collagen
necrosis, and mast cell tumors.
Mast cells may be present in low concentration in many types of inflammatory disorders, but if
present in high concentrations, mast cell neoplasia should be suspected. Mast cells are round cells with
round to oval nucleus and cytoplasm that contains purple granules. The granules occasionally do not stain
well with Diff-Quik.
Mesothelial cells tend to proliferate and exfoliate when fluid accumulates in a body cavity. They
may appear singly or in clusters of 2,4, 8, or 16 cells. They are large (12 - 30 µ), have light to dark
basophilic cytoplasm, and have single or multiple, round to oval nuclei with one or more nucleoli . Cells in
mitosis may be seen. The cytoplasmic border may appear to have a pink "fringe" around it. Most helpful
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in differentiating mesothelial cells from carcinoma cells are the sheer numbers of cells present, as well as
the size of the clusters. When carcinoma cells are exfoliating, large numbers are usually observed, and
clusters of up to 100 cells may be observed. Mesothelial cells are usually in small clusters, often consisting
of no more than 8 cells.
Microorganisms: Bacteria stain blue with Romanowsky stains, and must be distinguished from
background protein and stain sediment. They are usually somewhat uniform in size, present within the
cytoplasm of neutrophils, and if present in large numbers, may be both free and phagocytized. Even the
presence of small numbers of bacteria is significant. If different types of bacteria are seen, GI rupture
should be suspected. Large GI ruptures can be difficult to distinguish cytologically from GI aspirates .
Usually the leukograms or clinical condition of the animal are helpful. If only Clostridial organisms are
seen, ruptured liver abscess should be suspected. If large numbers of non-phagocytized bacteria are
present, and almost all neutrophils contain phagocytized bacteria, the prognosis is usually poor.
Other types of microorganisms, such as fungal organisms and protozoa, may also be infrequently
seen in body cavity effusions. Four yeast-type organisms, Coccidioides, Blastomyces, Histoplasma, and
Cryptococcus, are pathogenic with a tendency to become systemic, and easy to distinguish cytologically.
All fungi and yeast stain blue-black with Gram stain, and blue with Wrights stain. Toxoplasma gondii
tachyzoites may be seen in various tissues when systemic disease is present. They may be seen free or
within various cell types, especially macrophages. They elicit a mixed inflammatory response. The
organisms are blue with a purple nucleus and are elongated and curved, approximately 6 µm in length and
1 µm in width.
Transudates
Impeded flow causes an increased capillary or lymphatic hydrostatic pressure, and low osmotic
pressure results in leakage of fluid from capillaries. When either of these conditions occur, the result is a
fluid accumulation of a capillary filtrate that is low in protein and cells. Transudates that form as a result of
low osmotic pressure usually have a very low protein content (less than 1 g/dl), and are referred to as pure
transudates. Low osmotic pressure is caused by hypoalbuminemia, which may be a result of inadequate
protein intake (starvation, malabsorption, maldigestion, parasitism), inadequate albumin synthesis due to
severe liver disease, or excessive protein loss, such as may be seen with protein losing nephropathy,
protein-losing enteropathy, hemorrhage, or massive exudative lesions. Dependent edema may also be
present in these patients.
Transudates that form as a result of impeded flow are often modified somewhat by the addition of
a small amount of protein and a few cells, and are commonly referred to as modified transudates.
Disorders that cause venous stasis or impaired lymphatic drainage include congestive heart failure,
neoplasia, liver cirrhosis, diaphragmatic hernia, etc.
Exudates
Inflammatory effusions (exudates) are formed in response to inflammatory mediators that result in
increased capillary permeability. The result is fluid accumulation that contains a large amount of protein
and many cells. Hemorrhagic effusions are usually caused by trauma, neoplasia, or surgery. Although
clear-cut differentiation between transudates and exudates is often difficult, they are classified by the
following criteria:
Property
Transudate
Exudate
Appearance
Total Protein (g/dl)
Specific gravity
Cell count (/µl)
Clot
Clear
<3
<1.018
< 10,000
No
Cloudy
>3
>1.018
>10,000
Yes
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Interpretation
It is diagnostically worthwhile to classify fluids as either inflammatory or non-inflammatory.
Fluids with low protein and cell counts are considered transudates, or non-inflammatory effusions. When
protein is approximately 2.0 to 3.5 g, the transudate is considered to be a "modified" transudate. If the cell
count and protein content are high, and cells present are various types of blood cells that migrate to a site of
inflammation, the fluid is considered to be inflammatory, or resulting from inflammation . Fluid is then
further classified according to cell types.
If neutrophils predominate, the inflammation is considered suppurative (purulent). If neutrophils
are degenerate, sepsis should be considered, and the slide examined carefully for the presence of
microorganisms. If both neutrophils and macrophages are present, the inflammation is considered mixed.
If macrophages predominate, the inflammation is considered mononuclear, or granulomatous. If
eosinophils predominate, the inflammation is eosinophilic, etc.
Non-septic causes of inflammatory effusions include gallbladder or bile duct rupture, urinary
bladder or ureter rupture , sterile foreign bodies, neoplasia, etc. Fluid from patients with bile peritonitis can
be easily recognized by the cytologic appearance. Free gold-colored pigment is usually present, and when
phagocytized by macrophages, the bile pigment is black. The presence of bile can be confirmed by
determining bilirubin concentration on the fluid, which is consistently at least two-fold higher than the
serum bilirubin concentraton.
Effusions caused by Feline Infectious Peritonitis do not usually fit into the transudate/exudate
classification, as they are typically very high in protein (4-9 g/dl; mean, 6 g/dl) and quite low in cell
numbers (500-20,000/µl; mean, 5,000/µl). The fluid is usually yellow and viscous and forms partial clots
on standing. The inflammation is usually mixed, neutrophils are non-degenerate, and a large amount of fine
stippled background material is present that should not be confused with bacteria, since it is small and
amorphous.
Effusions in which lymphocytes predominate are usually due to either lymphoma, or presence of
chyle as a result of a ruptured or leaking thoracic duct . These can usually be differentiated, as those
resulting from lymphoma usually contain lymphoblasts, while chylous effusions usually contain normal
appearing lymphocytes. As chylous effusions become chronic, increasingly greater numbers of
neutrophils and macrophages are usually present. The nucleated cell count (NCC) may range from 5000/µl
to 30,000/µl. The total protein by refractometry is usually erroneously high (3-6 g/dl), due to the lipid
content of the fluid. Triglyceride concentration of chylous fluid is usually determined, in order to confirm
the presence of chyle; triglyceride concentration is greater in chyle than in serum or other types of
effusions.
Neoplastic effusions may be modified transudates or inflammatory exudates. The total protein is
variable (usually ranges from 3 to 4.5 g/dl, and the cell count is also variable (usually ranges from 3000/µl
to 30,000/µl). They are characterized by the presence of neoplastic cells, usually either epithelial
(carcinoma), mesothelial (mesothelioma) or lymphoid (lymphoma). Connective tissue tumors (sarcomas)
are relatively rare in body cavities, and also do not readily exfoliate cells. Neoplastic epithelial cells may
be very difficult to distinguish from reactive mesothelial cells that are shed from the peritoneal lining of
body cavities. Epithelial cells are quite variable in size, but are usually larger than mesothelial cells If
present in clusters, they tend to have cell to cell association, or bridging. Non-glandular epithelial cells
tend to be round with a moderate amount of pale to medium blue cytoplasm. They have a single round to
oval purple staining nucleus with smooth chromatin, and often contain a small round nucleolus. Glandular
(exocrine) epithelial cells usually have abundant vacuolated cytoplasm.
Hemorrhagic effusions usually result from neoplasia or trauma, and have properties very similar to
peripheral blood. The PCV should be measured. Films made from the buffy coat are useful in looking for
neoplastic cells. Macrophages with phagocytized RBC and hemosiderin are usually present. Platelets are
not usually present; one should suspect peripheral blood contamination of the sample if platelets are
observed.
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JOINT FLUID
INDICATIONS. Joint fluid aspirates are indicated whenever joint inflammation or hemorrhage is suspected.
Aspirates from the carpal joint are also sometimes diagnostically useful even when specific joint pain is not
apparent.
EQUIPMENT. A 22-25 gauge needle and a 3 to 6 ml syringe are needed. Other equipment and supples
include clippers, soap and skin disinfectant, microscope slides, and anticoagulant (EDTA).
COLLECTION TECHNIQUE AND SAMPLE HANDLING. The area over the joint is clipped and prepared as for
surgery, and the leg is alternately flexed and extended while the joint surface is palpated. The needle is
inserted into a soft part and synovial fluid is gently aspirated; negative pressure is then released and the
needle removed. The needle should be advanced gently through the joint capsule and redirected if bone is
encountered. Detailed instructions for approaches to various joints are well reviewed elsewhere . Films
are made as for other body fluids, placing a drop of fluid on a slide, placing a spreader slide over the drop,
allowing the dorp to spread, then pulling the two slides apart. The slide is then air dried and stained with a
Romanowsky stain. Remaining synovial fluid should be placed in an EDTA blood tube (purple top).
While normal joint fluid does not clot, fluids modified by inflammation may contain fibrinogen, and thus
clot. Color, turbidity and viscosity should be recorded. Cell counts are desirable if adequate quantity of
fluid is collected, because cell counts are difficult to estimate accurately on joint fluid films. These may be
performed manually, using a hemacytometer, or electronically, using an electronic particle counter. If
performing manual counts, a Unopette system using diluting fluid other than acetic acid should be used, as
acetic acid causes the mucin to clot, invalidating results. Normal synovial fluid contains less than 3000
cells/µl. If an adequate quantity of fluid is collected, a mucin clot test can be performed to assess the
quality and quantity of mucin (glycosaminoglycans (GAG)). Fluid for the mucin clot test must be collected
in heparin (green top), as EDTA degrades glycosaminoglycan. Following centrifugation, synovial fluid is
added to 3% glacial acetic acid at a ratio of 1:4. Normally, a ropey clot will form. When hyaluronic acid
(GAG) is decreased. a clot does not form. Hyaluronic acid can also be measured directly. Joint fluid is a
dyalysate of plasma which is modified by the addition of GAG by synovial membrane cells. Normally,
joint fluid contains relatively little protein. If adequate quantity of fluid is collected, protein concentration
can be measured. Normal protein concentration as measured by refractometry is approximately 2 to 4.5
g/dl. If sepsis is suspected after cytologic examination, joint fluid should be submitted to the lab for
bacterial culture.
EVALUATION OF SYNOVIAL FLUID FILMS. The film should be approached as any other cytologic specimen
and scanned using the low power (10x objective) to subjectively evaluate the degree of cellularity and to
find an appropriately thin area in which the cells are spread and can be identified. Viscosity can also be
subjectively assessed as fluids with high (normal) viscosity tend to have cells aligned in rows. The film is
then examined using the oil immersion lens. Glycosaminoglycan (hyaluronic acid) concentration can be
roughly estimated by the density of the eosinophilic granular background which is similar to the
background seen in effusions from cats with Feline Infectious Peritonitis. Normal synovial fluid contains
very few erythrocytes; increased numbers of RBCs may be due to contamination or hemorrhage within the
joint. Nucleated cells should be classified as neutrophils, eosinophils, lymphocytes, and large mononuclear
cells, which may be blood monocytes, macrophages, or synovial membrane cells. I do not attempt to
distinguish these, although if cytophagia is present, it should be recorded. Chondrocytes, osteoblasts, and
osteoclasts may also be observed on occasion. A 100 cell count differential should be performed. Relative
neutrophil count should not exceed 10 % of the total. The remainder of the cells in normal fluid are
lymphocytes and large mononuclear cells, with large mononuclear cells predominating. If the total
nucleated cell count exceeds 3000/µl or the relative neutrophil count exceeds 10%, the fluid is
characterized as inflammatory (suppurative, mixed, or mononuclear). If suppurative inflammation is
present, the neutrophils should be examined carefully for evidence of sepsis (karyolysis or the presence of
bacteria), although sepsis is a relatively rare cause of suppurative inflammation in joints of dogs.
INTERPRETATION. Hemarthrosis. True hemarthrosis must be distinguished from blood contamination at
the time of sampling. With hemarthrosis, erythrophagocytosis is usually present, and if not very acute,
supernatant will be xanthochromic. Hemarthrosis is seen in young hemophiliac dogs and in dogs with
warfarin toxicosis. Hemorrhage associated with inflammation is usually mild when compared to true
hemarthrosis.
Degenerative Arthropathies. This category of disease usually results from trauma and includes
degenerative joint disease, osteochondritis dissecans, elbow and hip dysplasia, ruptured cruciate ligament,
etc. The volume of joint fluid is usually increased, and the total nucleated cell count is only slightly
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increased, and may be within the normal range. Differential nucleated cell count is usually within the
normal range. Some authors report that large mononuclear cells are more vacuolated or phagocytic.
Osteoclasts may be seen in samples taken from animals with severe disease.
Inflammatory arthropathies. Both infectious and noninfectious causes of inflammation result in
increased total nucleated cell count and increased numbers of neutrophils. Increased numbers of
erythrocytes are usually observed. Infectious arthritides are rare in small animals. Bacterial arthritis is
usually confined to one joint and is commonly a result of a penetrating wound. Bacterial endocarditis and
naval ill may result in bacterial polyarthritis, but both are quite rare. Almost all polyarthritides are noninfectious. Cytology associated with septic arthritis is similar to septic inflammation of any tissue.
Neutrophils are greatly increased and some degree of karyolysis is often present. Bacteria may be observed
in the cytoplasm of neutrophils, but are usually few in number. Romanowsky stains, rather than Gram
stain, should be used when looking for bacteria, as gram negative bacteria are difficult to see in Gram
stained material. If bacteria are suspected, joint fluid should be cultured. (Recent studies have shown that
culture of fluid is more successful than synovial biopsy culture.)
Organisms most commonly cultured are coliforms, Corynebacterium, Erysipilothrix, Pasteurella,
Salmonella, staphylococci and streptococci. Borellia infections (Lyme Disease) may be associated with
polyarthritis. The inflammatory response is suppurative, neutrophils are non-degenerate, and organisms are
not observed in the film. The diagnosis is confirmed by serology. Mycoplasma arthritis is rare in small
animals but has been reported. Organisms appear as small magenta-colored inclusions in mononuclear
cells. The inflammatory response is purulent and neutrophils are typically non-degenerate. Fungal
arthritides are occasionally seen; the causative organism can be observed on fluid films. Blastomyces
dermatitidis, Coccidiodes immitis, Cryptococcus neoformans and Sporothrix schenckii have been reported.
The inflammatory response is usually a mixture of neutrophils and macrophages, and organisms can be
seen usually within macrophages, except for Coccidiodes, which is very large and not phagocytized.
Protozoal arthritides are also rare. Leishmania has been demonstrated in macrophages in synovial fluid of
dogs with disseminated leishmaniasis. Total cell counts and differentials were normal in these joint fluids.
Ehrlichia infections are occasionally associated with polyarthritis in dogs. The cellular response is typical
of purulent inflammation, and Ehrlichia morulae are observed in 1 to 2 % of neutrophils in the joint fluid.
Noninfectious arthritides are classified as either immune-mediated or non-immune-mediated. The vast
majority are immune-mediated, polyarticular, and are further subclassified clinically and radiographically
as erosive (rheumatoid, polyarthritis of Greyhounds), non-erosive (Systemic lupus erythematosus,
polyarthritis/polymyositis syndrome, idiopathic, and drug associated), and proliferative. Synovial fluid
changes in this group of diseases are very similar. The total nucleated cell counts are markedly increased
and non-degenerate neutrophils are the predominant cell type. Neutrophils with phagocytized cellular
debris (presumably nuclear material) may be observed. Idiopathic eosinophilic polyarthritis has also been
observed in the dog and horse; the predominant cell type is the eosinophil and the pathogenesis is presumed
to be immune-mediated.
Neoplasia. Neoplasia involving joint spaces can occasionally be diagnosed cytologically.
Exfoliated spindle shaped tumor cells may be observed in joint fluids affected with synovial-cell sarcomas,
chondrosarcomas, fibrosarcomas, osteosarcomas, and hemangiosarcomas. Other tumor types such as
carcinomas and lymphosarcoma have been reported rarely. Mixed inflammation usually accompanies
neoplastic joint disease.
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