CHAPTER 12 Synovial Fluid Analysis
Synovium is essentially a living ultrafiltration membrane with fenestrated capillaries just below an intimal surface containing no epithelial cells, no basement membrane, no cell junctions, and wide intercellular gaps. Fenestrated synovial capillaries, up to 50 times more permeable to water than continuous capillaries, allow water and small solutes into the subintima, but exclude varied proportions of albumin and most larger proteins, such as fibrinogen and clotting factors.
As fluid enters and leaves the joint cavity, its diffusion and composition is regulated by connective tissue of the subintima and cells of the intima or synovial lining. The intima has two major cell populations: macrophages (type A cell) and secretory fibroblast-related synoviocytes (type B cell). Type A are derived from blood-borne mononuclear cells and considered resident tissue macrophages, much like hepatic Kupffer cells. The type B cells secrete components for tissue interstitium and synovial fluid that include collagens, fibronectin, hyaluronan, and lubricin.
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.
BOX 12-1 Indications for Arthrocentesis
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.
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.
BOX 12-3 Materials for Arthrocentesis
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.
Entry is obtained via the antibrachiocarpal joint or the middle carpal joint. In either case, the carpus is flexed to increase access to the joint’s spaces. The needle is introduced from the dorsal aspect, just medial of center, then inserted perpendicular to the joint. Landmarks for the antibrachiocarpal joint are the distal radius and the proximal radial carpal bone. The middle carpal joint is between the distal portion of the radial carpal bone and the second and third carpal bones.
The elbow is flexed to a 90-degree angle and the needle introduced just proximal to the olecranon and medial to the lateral epicondylar crest. The needle will be inserted parallel to the olecranon and the long axis of the ulna.
Access is gained from the lateral aspect with the needle introduced distal to the acromion of the scapula and caudal to the greater tubercle of the humerus. The needle is directed medial to the greater tubercle and distal to the supraglenoid tubercle of the scapula.
Access is gained via a cranial or lateral approach. In the cranial approach, the tarsus is slightly flexed and the needle is introduced at the space palpated between the tibia and talus (tibiotarsal) bones, just lateral to the tendon bundle. For the lateral approach, the needle can be inserted just distal to the lateral malleolus of the fibula with either a slightly cranial or plantar path.
The stifle is flexed and the needle introduced just lateral to the patellar ligament and distal to the patella. The needle is advanced in a medial and proximal direction pointing toward the medial condyle of the femur.
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.
|1 drop||Cytology and WBC differential with viscosity estimate||Glass slide|
|0.5 to 1.0 ml||Total nucleated cell count||Lavender top or plain|
|1 to 3 ml||Bacterial culture and sensitivity||20-ml (pediatric) BD BBL Septi-Chek blood culture tube|
|0.5 to 1.0 ml||Bacterial culture and sensitivity||Sterile plain blood tube|
|1 drop||Cytology and WBC differential with total nucleated cell estimate and viscosity||Glass slide|
|2 or 3 drops||Bacterial culture and sensitivity||Culturette|
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.
Synovial fluid volumes depend on patient size and which joint is being collected (within an individual there is variation from joint to joint). In normal animals, fluid volume can range from 1 drop to 1.0 ml in dogs and 1 drop to 0.25 ml in cats.1–3 Clinical experience is an extremely valuable guide to detecting an articular effusion. This judgment is based on the degree of joint capsule distension, ease of fluid collection, and volume readily obtained. The aim of arthrocentesis for synovial fluid analysis is to collect some synovial fluid, but not to drain the joint space.
Normal synovial fluid is transparent and colorless to very light yellow or straw-colored. Samples with increased cellularity exhibit variable discoloration and increased turbidity. When a fluid is blood tinged, hemarthrosis should be distinguished from iatrogenic contamination. In cases of hemarthrosis, the fluid is uniformly bloody throughout the time of collection. If the fluid was initially free of blood, but there is a subsequent admixture during the sampling procedure, contamination should be suspected. As an alternative and when the volume is sufficiently large for centrifugation, recent hemorrhage is associated with a sediment of red blood cells (RBCs) and a clear to straw-colored supernatant. The supernatant of fluids with chronic hemorrhage have a yellow to yellow-orange discoloration due to hemoglobin breakdown products.
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.)
If sufficient sample remains following slide preparation and nucleated cell count, synovial fluid mucin quality or hyaluronic acid may be assessed using a mucin clot test. When there is the potential for clotting of joint fluid, heparin is recommended as an anticoagulant because EDTA interferes with the mucin clot test by degrading hyaluronic acid.4
One part synovial fluid is added to four parts 2.5% glacial acetic acid, which causes mucin to precipitate and sometimes agglutinate or clot. The test can be performed in test tubes when sufficient fluid is collected or on glass slides when only a drop is available for this test. The mixture is gently agitated and the nature of the clot observed. Assessment is enhanced by reading the test against a dark background. In inflammatory arthropathies, hyaluronic acid is degraded by proteases from neutrophils. This results in a decreased hyaluronic acid or hyaluronate concentration and decreased viscosity.
The following subjective classifications are commonly used: good (normal), when there is a compact, ropey clot in a clear solution; fair (slightly decreased), when there is a soft clot in a slightly turbid solution; poor, when there is a friable clot in a cloudy solution; and very poor, when there is no actual clot, just some large flecks in a very turbid solution. If clot quality is initially debatable, it can be reassessed after about 1 hour at room temperature. When the solutions are gently shaken during assessment, good clots remain ropey, and poor clots fragment.
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.
|Range (cells/μl)||Joints sampled|
|33-24951||12 joints: stifle, shoulder, carpus|
|0-29002||55 joints: hip, stifle, hock, elbow, shoulder, carpus|
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).
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.
Comparatively few studies have reported baseline values for total protein concentration, which probably reflects the relatively low priority given to this value. Other tests are preferred because sample volume is usually insufficient to allow for protein measurement. Synovial fluid protein concentration is best measured by a quantitative biochemical assay because refractometry measures other solutes and protein. A study of normal stifle, shoulder, and carpal joints reported a total protein concentration reference interval of 1.8 to 4.8 g/dl, as measured by refractometer.1 Normal synovial fluid does not clot in vitro because it is essentially free of fibrinogen and other clotting factors. Joint fluid can form a thixolabile gel if left undisturbed for several hours. Because clots are not thixotropic, normal fluid is distinguishable from clotting by gently shaking the sample to restore fluidity. If a specimen forms a clot after collection, this indicates intra-articular hemorrhage or inflammation with increased vascular permeability and protein exudation into the joint space.
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%).
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