Physical Characteristics

Normal equine synovial fluid is light yellow and clear, with no suspended particulate material. The volume of fluid that can be collected depends on the joint being sampled and the health of the joint. In general, the ability to collect synovial fluid samples of adequate volume for testing does not pose the problem in horses that it does in dogs and cats. At least 1 ml of fluid usually can be collected from the major limb joints of healthy horses.

Viscosity: Though techniques for quantifying viscosity have been described, viscosity can be quickly estimated by placing a drop between the thumb and forefinger, slowly pulling the fingers apart, and observing the strand of synovial fluid that forms between the fingers. Synovial fluid of normal viscosity forms a strand at least 2.5 cm long before it breaks. Another simple technique is to observe the length of the strand of synovial fluid that forms on the end of a needle before it drips from the needle. Again, normal synovial fluid forms a strand at least 2.5 cm long.

Thixotropism: Normal synovial fluid does not clot upon standing but often becomes gelatinous. This phenomenon is called thixotropism; the specimen can be returned to its fluid state by agitation.

Cellular Composition

Nucleated Cell Count: Total nucleated cell counts of normal equine synovial fluid have been reported to vary widely from one joint to another. Though counts over 2,000 cells/μl have been reported for fluid from the temporomandibular joints of clinically normal animals, fluid from the more commonly sampled limb joints of healthy horses usually contains less than 500 nucleated cells/μl.^2,3

Erythrocytes: Normal synovial fluid is almost completely free of erythrocytes, and erythrocyte counts are seldom performed on synovial fluid specimens. Nevertheless, almost all specimens contain some erythrocytes due to contamination with blood during sample collection. Erythrocytes may also enter synovial fluid in a variety of diseases involving the joint. Physical and cytologic characteristics of synovial fluid help differentiate erythrocytes associated with sample contamination during collection from erythrocytes associated with joint disease. These characteristics are discussed later in this chapter.

Differential Cell Count: Published values regarding the relative proportions of the various cell types that occur in normal synovial fluid vary widely. One useful general guideline is that the proportion of neutrophils in normal synovial fluid should not exceed 10%, with the possible exceptions of some samples with very low total cell counts and samples with marked blood contamination.

The most obvious differences in reported values are in the proportions of mononuclear cells. Most mononuclear cells in normal synovial fluid are lymphocytes and large mononuclear cells. The latter include monocytes/macrophages and, probably, some synovial lining cells. It may be possible to distinguish synovial lining cells from macrophages in cytologic preparations. Some cells that have been classified as “clasmatocytes” are probably synovial lining cells. Intact clumps of synovial lining cells are occasionally aspirated (Fig. 10-1). In our laboratory, mononuclear cells in synovial fluid are routinely classified as lymphocytes or macrophages. Macrophages usually predominate, though the proportions of macrophages and lymphocytes vary from one joint to another.⁴ The relative proportions of lymphocytes and macrophages seldom provide useful diagnostic information. Some laboratories routinely report all lymphocytes, macrophages, and synovial lining cells under the heading of “mononuclear cells.” Eosinophils occur infrequently (<1% of total cells) in normal equine synovial fluid.

Fig. 10-1 Synovial lining fragment in synovial fluid from a horse. (Wright’s stain)

The size of macrophages and degree of cytoplasmic vacuolation in synovial fluid vary widely. Some macrophages have relatively dark blue cytoplasm with few or no vacuoles. Other macrophages, often the larger ones, have numerous clear cytoplasmic vacuoles. Some of this vacuolation is an artifact caused by delayed processing of the fluid after collection. Another morphologic feature of some synovial fluid macrophages is the presence of few to many metachromatic cytoplasmic granules (Fig. 10-2).

Fig. 10-2 Three neutrophils and a macrophage-containing metachromatic cytoplasmic granule in synovial fluid from a horse with degenerative joint disease.

Throughout the background of most synovial fluid smears is mucin, which appears as finely granular pink material. This mucin should not be confused with bacteria, which stain dark blue with Wright’s stain.

Synovial Fluid Collection

Synovial fluid can be aspirated from most joints with a 1-inch, 18- to 20-gauge needle. The skin should be prepared by clipping the hair and performing a surgical scrub to avoid introducing bacteria into the joint. Ethyl chloride spray may be used, if needed, on the overlying skin to lessen the sensation of the needle penetration. Fluid for cytologic evaluation should be placed in an EDTA tube. Note, however, that EDTA interferes with the mucin clot test. If a mucin clot test is to be run, another aliquot of synovial fluid should be placed in a tube with no anticoagulant (eg, red-top tube) or one containing heparin.

Mucin Clot Test

The mucin clot test provides a semiquantitative index of the degree of polymerization of hyaluronic acid in synovial fluid. It should not be performed on samples collected in EDTA; samples collected in heparin or with no anticoagulant should be used. The test is performed by mixing about 1 part of the supernatant from centrifuged synovial fluid with 4 parts of 2.5% glacial acetic acid. The acid causes precipitation or clumping of synovial fluid mucin. After gently mixing, the clumped mucin is observed.

A four-level subjective grading scheme is used for recording results of mucin clot tests. The clot is classified as good if a single, compact clot forms in an otherwise clear solution. It is classified as fair if a single, soft clot forms in a slightly turbid solution. A poor clot is friable and may break apart if the tube is gently agitated. The surrounding fluid in such specimens is cloudy. A very poor clot should be recorded if the tube contains only flecks of precipitated mucin in an otherwise cloudy solution.

Hyaluronic acid content has been measured directly in synovial fluid, but this test is not performed in most clinical laboratories.⁵

Total Protein Concentration

Most plasma proteins, especially those with higher molecular weight, are excluded from synovial fluid by the dialyzing properties of the synovial membrane and hyaluronic acid in the perisynovial connective tissue. Some glycoprotein is synthesized by the synovial lining cells and secreted into the fluid. Values reported in the veterinary literature for the protein concentration of normal equine synovial fluid vary widely. One author, using the biuret technique, reported a reference range of 0.92 to 3.11 g/dl.⁶ Another investigator reported a reference range of 0.5 to 1.0 g/dl using the Coomassie blue method. This latter author attributed the different results to methodologic differences.⁷ In limited comparisons, we have found that the biuret and Coomassie blue methods yield very similar values and that these values are in the range reported above for the biuret method.

Refractometric measurements have also been used to qualify protein in equine synovial fluid.⁸ While refractometry has been suggested by some to provide at least an index of synovial fluid protein concentration, others have criticized its use.^3,9

Other Tests

In addition to direct measurement of hyaluronic acid content, other chemical tests that have been performed on equine synovial fluid include measurement of creatine kinase and lactate dehydrogenase total activity and isoenzyme activity, alkaline phosphatase activity, and glucose content.^6,10 None of these has been used widely in diagnosis of joint disease in animals.

Nucleated Cell Counts

Nucleated cell counts of synovial fluid can be determined manually with a hemacytometer or automatically with an electronic cell counter. The techniques give similar results with canine synovial fluid.¹¹ Because normal equine synovial fluid often has a much lower nucleated cell count than feline or canine synovial fluid, the validity of electronically determined counts on some equine specimens is open to question. Cell counts of synovial fluid from normal equine joints may be so low that they are below the background threshold level of electronic cell counters. Manual electronic counters can provide accurate counts on specimens with at least 500 cells/μl. If the electronically determined count is near the threshold level of the instrument, a manual count should be performed.

Manual cell counts should be performed by loading the hemacytometer chamber with undiluted synovial fluid if the total cell count is low. The number of nucleated cells in the four corner primary squares should be counted; the total number of cells in the four corners is multiplied by 2.5 to obtain the number of cells per microliter. Specimens with high cell counts must first be diluted with a white blood cell pipette in the same fashion as when performing manual white blood cell counts on blood. Saline should be used as the diluent for synovial fluid rather than the dilute acetic acid that is sometimes used as a diluent for blood. Acetic acid causes precipitation of the mucin in synovial fluid. A standard white blood cell pipette may be filled to the 0.5 mark with synovial fluid and then filled to the 11 mark with saline. After the diluted synovial fluid is mixed and the first few drops discarded, a hemacytometer chamber should be charged with fluid. Nucleated cells in the four corner squares should be counted as above. The total is then multiplied by 50 to obtain the number of cells per microliter of undiluted synovial fluid.

It may be difficult to obtain counts on markedly exudative specimens because of the presence of cell clumps, fibrin, and necrotic debris. Another potential technical difficulty is that cell counts of routinely diluted synovial fluid may be falsely low because the viscous synovial fluid does not mix evenly with the diluent in the pipette. Pretreatment of such synovial fluid with hyaluronidase reportedly causes an apparent increase in the cell count of more than twofold.¹² This technique is not routinely used in most clinical laboratories.