ARTHROCENTESIS

In the verification, localization, diagnosis, and management of arthritis, synovial fluid examination is a key component of an initial medical database that includes clinical history, physical examination, radiographs, complete blood count, biochemical profile, and urinalysis. See Boxes 12-1 and 12-2 for indications and contraindications for arthrocentesis.

RESTRAINT

As temperament under physical immobilization and tolerance for discomfort of each individual is different, the clinician must judge which method of restraint is appropriate to allow for controlled manipulation and centesis of the joint. Complications of inadequate restraint can include damage to blood vessels, nerves, synovial membrane, and articular cartilage surfaces, along with blood contamination and retrieving a diagnostically insufficient volume of synovial fluid.

ASEPSIS

Routine aseptic technique should be followed. Normal joint spaces are sterile.

EQUIPMENT

Sterile disposable 3-ml syringes and 1-inch, 22-gauge or 25-gauge (small dogs and cats), hypodermic needle are recommended. In large breed dogs sampling of the elbow or shoulder joints may require a 1½-inch needle and the hip joint may necessitate a 3-inch spinal needle. Microscope glass slides with frosted ends, red-top tubes, and ethylene-tetra-acetic acid (EDTA) blood tubes should be readied and labeled with the patient’s name and the joint sampled. See Box 12-3 for a complete list of materials.

APPROACHES

In most cases arthrocentesis is performed with the patient in lateral recumbency and the joint to be sampled uppermost. Palpation of the joint during manual flexion and extension helps identify the space to be entered. In all cases the needle should be advanced gently toward and through the joint capsule to avoid damaging the articular cartilage. Once the needle is inside the joint space, the volume of fluid obtained depends on the particular joint and the disorder. Ordinarily, some synovial fluid is readily collected from the stifle joint, but it is most difficult to obtain from the carpal and tarsal joints. Obviously, when joint spaces are swollen, fluid is more easily aspirated. The plunger of the syringe should be released before the needle is removed from the joint space. This minimizes blood contamination of the sample as the needle is withdrawn.

SAMPLE HANDLING AND TEST PRIORITIES

Laboratory tests performed can be limited by volume of synovial fluid collected. While the sample is in the syringe, volume, color, and turbidity should be noted. Viscosity is then assessed as the sample is expelled onto a glass slide for direct smears. Direct smears are immediately made for subsequent cytologic examination, nucleated cell differential count, and subjective assessment of cellularity. See Tables 12-1 and 12-2 for specific volumes needed and sequence of testing. When larger volumes of fluid are collected, a total nucleated cell count, mucin clot test, and total protein estimation, in order of priority, can be added to the aforementioned procedures.

TABLE 12-1 Test Priorities for 2 ml or More Synovial Fluid

TABLE 12-2 Test Priorities for Less than 1 ml of Synovial Fluid

Normal synovial fluid does not clot. However with the possibility of incidental blood contamination, intraarticular hemorrhage, or protein exudation in various inflammatory diseases, it is best to put some portion into an EDTA anticoagulant blood tube. The smallest EDTA blood tube available should be used for storage or preservation of the synovial fluid retrieved, since gross mismatches by using large EDTA tubes can lead to erroneous test results. EDTA is preferred for cytologic examination, whereas heparin or a plain blood tube is recommended for the mucin clot test. Either anticoagulant (EDTA or heparin) is suitable for other routine tests.

When sufficient fluid is collected for cell counting, various types of preparations can be made in accordance with the sample’s cellularity. When the nucleated cell count is <5000 cells/μl, cytologic examination is enhanced by cytocentrifuge concentration. About 5 minutes at 1000-1500 rpm in a cytocentrifuge is satisfactory. Fluids with nucleated cell counts >5000 cells/μl can be smeared directly onto glass slides. Although cytocentrifuge concentration is helpful, it is not essential to a good evaluation, and practitioners can get accurate results from direct smears only.

Cells in sediment smears and direct smears of fluid with the normally high viscosity may not spread out well on slides, making cell identification and differential cell count difficult. If this problem is encountered, it can be overcome by mixing an equal volume of hyaluronidase at 150 IU/ml with the synovial fluid and incubating for at least 10 minutes.¹ The result is a fluid that facilitates better presentation of cell morphology and more complete cytologic evaluation.

A cytocentrifuge is a low-speed centrifuge that allows for concentration of poorly cellular fluids directly onto a glass slide with a minimum number of cells destroyed in the process. Samples with good to fair viscosity must be pretreated with hyaluronidase, otherwise the synovial fluid mucin clogs the cytocentrifuge filter paper and interferes with proper slide preparation. This technique is helpful and used by many commercial laboratories, but is not essential for an adequate evaluation in most cases, and the practicing veterinarian can obtain diagnostically useful information from a direct smear.

Slides can be stained with any Romanowsky-type stain for routine cytologic evaluation. It is advisable to make synovial fluid smears soon after collection. Delays of several hours, particularly at warm temperatures, can result in artificial vacuolation of macrophages along with pyknosis and karyorrhexis of nucleated cells.

Microbiologic evaluation of samples collected aseptically can be done if cytologic and clinical findings suggest an infectious agent is present. If possible, synovial fluid should be placed into a culture system immediately after collection. Use of an EDTA tube is undesirable because EDTA interferes with growth of some bacteria; a red-top tube is undesirable because it may not be sterile.

LABORATORY ANALYSIS AND REFERENCE VALUES

Viscosity

Normal synovial fluid is very viscous because of its high concentration of hyaluronic acid. Viscosity can be measured using a viscometer; however, this is rarely done in small animal practice, but instead, is assessed subjectively. When slowly expressed from a needle attached to a syringe held horizontal, normal synovial fluid forms a long strand that is at least 2.5 cm before separating from the needle. When a drop of fluid is placed between the thumb and forefinger, a similar strand bridges the two digits as they are moved apart. Viscosity is usually recorded as normal, decreased, or markedly decreased.

Viscosity is easily assessed at the time of collection. However, if it must be evaluated after the sample is added to an anticoagulant, heparin is probably preferable to EDTA for sample preservation. EDTA tends to degrade hyaluronic acid and may decrease the sample’s viscosity.⁴

Viscosity can also be subjectively assessed when cytologically evaluating direct or sediment smears such that smears of fluids with normally high viscosity tend to have cells aligned in a linear pattern that is sometimes referred to as windrowing (Figure 12-1). In contrast, synovial fluid samples with decreased viscosity have cells more randomly arranged on the smear (Figure 12-2).

Figure 12-1 Direct smear of synovial fluid from a dog with acute suppurative arthritis. Note the markedly increased cell count and linear arrangement of cells. The latter, referred to as windrowing, suggests normal viscosity. (Wright’s stain, original magnification 160×.)

(From Parry: In Pratt PW: Laboratory Procedures for Veterinary Technicians, ed 2. Goleta, Calif, American Veterinary Publications, 1992.)

Figure 12-2 Sediment smear of synovial fluid from a dog with degenerative arthropathy. Note the normal (low) cell count and random distribution of cells. The latter suggests decreased viscosity. (May-Grünwald-Giemsa stain, original magnification 200×.)

Total Cell Counts

Nucleated cell counts in normal synovial fluid vary from joint to joint within an individual animal.¹ However, surveys have not shown these differences to be either statistically significant or clinically relevant. Various canine reference intervals have been reported (Table 12-3). As a generalization from these studies, most normal joints have nucleated cell counts <3000 cells/μl.

TABLE 12-3 Reference Intervals for Synovial Fluid Total Nucleated Cell Counts in Healthy Dogs

Range (cells/μl)	Joints sampled
33-24951	12 joints: stifle, shoulder, carpus
0-29002	55 joints: hip, stifle, hock, elbow, shoulder, carpus
700-440010	20 stifles
327-145011	14 stifles
50-272512	58 stifles
209-20705	19 stifles

A recent study of synovial fluid samples from clinically normal cats, showed white blood cell (WBC) counts of 161 ± 209 cells/μl (mean ± SD) and median WBC of 91 cells/μl with a range of 2 to 1134 cells/μl.³ Samples were excluded from this study when there was gross evidence of blood contamination, radiographic evidence of osteoarthritis, or histologic evidence of synovitis or if postmortem physical examination revealed abnormalities. As a generalization from this study, most normal joints have nucleated cell counts <1000 cells/μl (Table 12-4).

TABLE 12-4 Reference Intervals for Synovial Fluid in Healthy Dogs and Cats

A comparison of manual hemacytometer and electronic, automatic, particle counting of nucleated cells in canine synovial fluid revealed that the mean electronic total nucleated cell count was statistically higher than the mean manual count.⁵ Manual counting methods can demonstrate within-day and between-day analytical imprecision that is statistically higher than that of automated particle counting instruments.^6,7 In general, differences in mean cell counts and precision have not proven to be clinically relevant; therefore, the efficiency and speed of automatic particle counters offer an advantage over manual methods.

EDTA is preferred as an anticoagulant and preservative for cytologic examination and nucleated cell counts. In comparison of EDTA versus heparin anticoagulants as a preservative for synovial fluid, samples stored in heparin showed a 4-fold and 9-fold greater decrease in total nucleated cell counts over 24 hours and 48 hours at 4°C, respectively.⁶ On the other hand, EDTA reportedly decreases synovial fluid mucin quality; therefore, total nucleated cell counts on fluid samples collected into EDTA may not be increased by hyaluronidase. Regardless of the anticoagulant/preservative used, synovial fluid should be pretreated with hyaluronidase when automated hematology analyzers are used for cell counts.^8,9

Nucleated cell counts may also be performed using a hemocytometer. In clear or nonturbid specimens (i.e., specimens that appear to have a low total nucleated cell count) the sample may be counted undiluted. However, if the sample is turbid and the anticipated nucleated cell count is high, the specimen should be diluted. A WBC-diluting pipette and physiologic saline are suitable for this purpose. The BD Unopette brand test for manual WBC/platelets counts can also be used; however, the use of an acetic acid diluent should be avoided because it causes mucin to clot and invalidates the results.

Due to the lack of clinical application, RBC counts are not typically reported. Samples from normal patients contain very few RBCs and are a result of incidental blood contamination at the time of collection.

CYTOLOGIC EXAMINATION

When only a few drops of fluid are collected, cytologic reports should include subjective assessments of the amount of blood present, total nucleated cell count, and sample viscosity. When a cell count and mucin clot test can be performed, incongruities with the subjective assessments should be reported.

Normal synovial fluid contains very few RBCs. Increased RBC numbers may result from hemorrhage associated with collection and trauma or inflammation involving the joint capsule. Generalizations regarding the cellularity of synovial fluid can be consistently made via freshly prepared direct smears. The body of the smear of normal specimens contains about 2 cells/field at 400× magnification (40× objective). Cellularity of direct smears are categorized as normal (see Figure 12-2), slightly increased, moderately increased (Figure 12-3), or markedly increased (see Figure 12-1). Due to unpredictable variation among processing techniques and instrumentation, such assessments are impractical with concentrated specimens (sediment and cytocentrifuge smears).

Figure 12-3 Synovial fluid from a cat with suppurative (neutrophilic) arthritis. Fluid protein is observed as pink granular or stippled material and crescents. (Wright’s stain, original magnification 500×.)

Because of the high viscosity of normal synovial fluid, cells of direct and centrifuged sediment smears tend to line up in rows (i.e., windrowing, see Figure 12-1). This characteristic arrangement can be used to comment on sample viscosity, when volume is not sufficient for viscosity and mucin clot tests. However, in smears from synovial fluid with low cell counts, windrowing may not be apparent, even though viscosity is normal.

Smears of normal, and sometimes abnormal, synovial fluid can have a pink granular proteinaceous background (see Figures 12-3 and 12-13) that must not be confused with bacteria.

Nucleated cells should be classified as neutrophils, large mononuclear cells, lymphocytes, or eosinophils. Classification as mononuclear cells encompasses those that are phagocytically active. These cells could be derived from blood monocytes, tissues macrophages, or synovial lining cells. The origin of these cells has little practical importance regarding clinical diagnosis and therapy. The proportion of large mononuclear cells that have phagocytized debris, cells, or microorganisms should be recorded. On smears that are freshly made or from fluid not exposed to EDTA, the degree of vacuolated large mononuclear cells should be noted and reported as mild, moderate, or marked. Overall assessment of nucleated cell morphology ought to include comments on the degree of karyolysis, pyknosis, and karyorrhexis. Delayed processing can lead to nuclear degeneration and increased numbers of markedly vacuolated large mononuclear cells.¹ Synovial fluid nucleated cell differentials are reported as percentage values and are incorporated into the interpretation of a total nucleated cell count or subjective assessment of cellularity.

Reports of normal canine synovial fluid nucleated cell differentials indicate neutrophils can make up as much as 12% of nucleated cells, but frequently compose < 5% of all nucleated cells.^1,2,10,11 Eosinophils are absent.^1,2,10–12 Lymphocyte values can be quite variable, with studies reporting 0% to 100% (mean 44%) and 3% to 28% (mean 11%).^1,2 The balance of nucleated cells in normal joints are large mononuclear cells, ranging from 64% to 97% in one study, to 60% to 92% in another study. In samples of normal joints processed immediately by direct smear, the percentage of large mononuclear cells that were markedly vacuolated was about 9%.¹ As with delayed processing, pre-treatment of fluid with hyaluronidase can increased the percentage of large mononuclear cells that are markedly vacuolated to about 14% to 18%.

A study of normal feline synovial fluids reports that mononuclear cells predominate, ranging from 61% to 100% of all nucleated cells (mean 96.4%), with smaller proportions of neutrophils that vary from 0% to 39% of all nucleated cells (mean 3.6%).³ Within the mononuclear cell component, lymphocytes or small mononuclear cells make up 0% to 45% of all mononuclear cells (mean 9.1%) and large mononuclear cells include 0% to 100% of all mononuclear cells (mean 81%). Freshly prepared smears from these cats reveal that among mononuclear cells, 0% to 100% are vacuolated (mean 9.9%).

BACTERIOLOGIC CULTURE

Conventional agar-based or broth-type bacteriologic culture methods have been shown to lack sensitivity when compared with blood culture methods.^13–15 Liquid blood culture media offers several advantages, including culture of larger volumes compared with culture plates, resins that decrease the inhibitory affects of antibiotics and substances intrinsic to synovial fluid, and lytic agents that release microorganisms phagocytized by inflammatory cells. Because relatively small volumes are retrieved with arthrocentesis of cats and dogs, pediatric blood culture bottles or tubes are most appropriate. Blood culture tubes or bottles should be inoculated at the time of fluid collection and incubated for 24 hours at 37°C, at which time the result is transferred to appropriate growth media.

It has been suggested that culturing a synovial membrane biopsy specimen may be superior to culture of synovial fluid, but this was not shown to be the case when stifle joints were experimentally infected with Staphylococcus intermedius.¹⁵