Synovial Fluid, Atlas of Synovial Fluid, Graff's Textbook of Urinalysis and Body Fluids
Joint fluid is called synovial fluid because of its resemblance to egg white. It is a viscous, mucinous substance that lubricates most joints. Analysis of synovial fluid is important in the diagnosis of joint disease. Aspiration of joint fluid is indicated for any patient with a joint effusion or inflamed joints. Aspiration of asymptomatic joints is beneficial for patients with gout and pseudogout as these fluids may still contain crystals. Evaluation of physical, chemical, and microscopic characteristics of synovial fluid comprise routine analysis. This chapter includes an overview of the composition and function of synovial fluid and laboratory procedures and their interpretations
PHYSIOLOGY AND COMPOSITION
All human joints, except those that are weight bearing, are lined with a tissue called synovium. Synovium produces synovia, also called synovial fluid.1 This fluid capsule cushions diarthrotic joints allowing the bones to freely articu- late. A dense connective tissue layer of collagen surrounds the synovial capsule. Figure 11-1 illustrates an articulated joint. Figure 11-2 shows the synovial lining of the synovial capsule.
Synovial fluid is an ultrafiltrate or dialysate of plasma and contains levels of glucose and uric acid that are equivalent to plasma. Synovial fluid protein, however, is at a lower level (about one third) than that of plasma. Plasma constituents
Figure 11-1. Articulated joint.
Figure 11-2. Synovial membrane from a normal knee joint shows joint space,
synovial membrane composed of synovial cells embedded in a loose
connective tissue stroma overlying dense collagen (hematoxylin and eosin).
that enter joint fluid must cross a double-barrier membrane. First, the endothelial lining of the capillaries is traversed followed by movement through a matrix that surrounds synovial cells. This ultrafiltrate is combined with a mucopolysaccharide (hyaluronate) synthesized by the synovium.
SPECIMEN COLLECTION
After finding positive results with a “bulge test” (Fig. 11-3), the physician will perform an arthrocentesis and aspirate the effected joint. An appropriate gauge needle is attached to a syringe and the entry site is cleansed. A two-step process is employed for arthrocentesis in which the first puncture is made through the skin followed by a second thrust into the synovial capsule. Figure 11-4 illustrates needle placement in arthrocentesis of elbow and knee joints.
After fluid is aspirated and the needle withdrawn from the joint, the needle is removed and an end cap placed on the tip of the syringe. The syringe is properly labeled and sent to the laboratory for testing.1 Some laboratories
Figure 11-3. Bulge test of joint for the detection of synovial effusion
Figure 11-4. Placement of needle in arthrocentesis of (A) elbow and (B) knee joints
require that synovial fluid specimens be placed in specimen containers appropriate for the tests ordered. A heparinized tube is preferable to ethylenediaminetetraacetic acid (EDTA) or other anticoagulants for cells counts; sterile containers for microbiology testing; and plain tubes are normally used for chemistry and immunology testing of synovial fluid.
Synovial fluid specimens should be handled like STAT spec- imens and delivered immediately to the laboratory for testing to avoid alteration of chemical constituents, cell lysis, and problems in microorganism detection and identifica- tion. If a glucose test is to be performed, the patient should be fasting for at least 6 hours prior to collection of joint fluid. A 6-hour fast is necessary to establish an equilibrium between plasma and joint glucose levels.
LABORATORY TESTING
■ Volume. The amount of fluid contained in joints is usually small. The knee joint normally contains up to 4 mL of fluid. The volume of the aspirate is usually recorded at bedside, but some laboratories may include volume in their reports as well.
■ Color and clarity. Normal synovial fluid is colorless and clear. Other appearances may indicate various disease states. Yellow/clear synovial fluids are typical in noninflammatory effusions, whereas yellow/cloudy fluids usually involve an inflammatory processes. A white/ cloudy synovial fluid may contain crystals; and synovial fluid that is red, brown, or xanthochromic indicates hemorrhage into the joint. In addition, synovial fluid may contain various types of inclusions. Free- floating aggregates of tissue appear as rice bodies. Rice bodies are seen in rheumatoid arthritis (RA) and result from degenerated synovium enriched with fibrin.
Ochronotic shards are debris from metal and plastic joint prosthesis. These shards look like ground pepper. Figure 11-5 compares normal and blood synovial fluids, whereas Figure 11-6 demonstrates the appearance of synovial fluid inclusions.
■ Viscosity. Synovial fluid is very viscous due to its high concentration of polymerized hyaluronate. A string test can be used to evaluate the level of synovial fluid viscosity. After removing the needle or cap from the syringe, synovial fluid is expressed into a test tube one drop at a time. Normal synovial fluid will form a “string” approximately 5 cm long before breaking. In addition, the fluid may cling to the side of the test tube rather than running down to the bottom. Synovial fluids with poor viscosity will form shorter stings
Figure 11-5. Synovial fluid. A. Normal. B. Bloody
Figure 11-6. Synovial fluid inclusions. A. “Ground pepper” ochronotic
shards. B. “Rice bodies” fibrin-enriched synovium fragments.
(<3 cm) or run out of the syringe and down the side of the test tube like water. Low viscosity of synovial indicates the presence of an inflammatory process. Figure 11-7 illustrates the performance of the string test for synovial fluid viscosity.
■ Clotting. Clotting of synovial fluid can result when fibrinogen is present. Fibrinogen may have entered into the synovial capsule during damage to the synovial membrane or as a result of a traumatic tap. Clots in specimens interfere with performance of cell counts. Depositing part of the specimen into a tube containing heparin may help avoid clotting of synovial fluid.
■ Mucin clot. The mucin clot test, also known as Rope’s test, is an estimation of the integrity of the hyaluronic acid–protein complex (mucin). Normal synovial fluid forms a tight ropy clot upon the addition of acetic acid.
Figure 11-7. String test showing normal synovial fluid viscosity
The procedure for mucin clot varies among laboratories as evidenced by differing fluid to acid ratios appearing in various texts. Clinical laboratory professionals should use the procedure adopted by their laboratories. Table 11-1 demonstrates this variability. In all cases, the interpretation of clot formation is the same. A good mucin clot indicates good integrity of the hyaluronate. A poor mucin clot, one that breaks up easily, is associated with destruction or dilution of hyaluronate.Figure 11-8 illustrates the tight clot of normal synovial fluid.
CHEMICAL EXAMINATION
■ Protein. Synovial fluid contains all proteins found in plasma, except various high–molecular weight proteins. These high–molecular-weight proteins include fibrinogen, beta 2 macroglobulin, and alpha 2 macroglobulin, and can be absent or present in very low amounts. Most commonly used serum protein proce- dures can be used to measure synovial fluid protein. The normal range for synovial fluid protein is 1–3 g/dL. Increased synovial fluid protein levels are seen in ankylosing spondylitis, arthritis, arthropathies that
Figure 11-8. Mucin clot test of normal synovial fluid.
accompany Crohn disease, gout, psoriasis, Reiter syn- drome, and ulcerative colitis.
■ Glucose. Synovial fluid glucose levels should be inter- preted using serum glucose levels. A fasting specimen should be used or at least one 6–8 hours postprandi- ally. Normally, synovial fluid glucose levels are less than 10 mg/dL lower than serum levels. Joint disor- ders that are classified as infectious demonstrate large decreases in synovial fluid glucose and can be as much as 20–100 mg/dL less than serum levels. Other groups of joint disorders demonstrate a less of a decrease in synovial fluid glucose, 0–20 mg/dL.
■ Uric acid. Synovial fluid uric acid normally ranges from 6 to 8 mg/dL. The presence of uric acid in synovial fluid is helpful in diagnosis gout. Usually, crystal identification is used for this determination, but synovial fluid uric acid levels may be performed in laboratories that do not a have light polarizing microscope.
■ Lactic acid. Lactic acid is rarely measured in synovial fluid but can be helpful in diagnosing septic arthritis. Normally, synovial fluid lactate is less than 25 mg/dL but can be as high as 1000 mg/dL in septic arthritis.
■ Lactate dehydrogenase. Lactate dehydrogenase (LD) can be elevated in synovial fluid, while serum levels remain normal. Synovial fluid LD levels are usually increased in RA, infectious arthritis, and gout. The neutrophils that are increased during the acute phase of these disorders contribute to this increased LD level.
■ Rheumatoid factor. Rheumatoid factor (RF) is an antibody to immunoglobulins.4 RF is present in the serum of most patients with RA, whereas just more than half of these patients will demonstrate RF in synovial fluid. However, if RF is only being produced by joint tissue, synovial fluid RF may be positive while the serum RF is negative.4 False-positive RF can result from other chronic inflammatory diseases.
MICROSCOPIC EXAMINATION OF SYNOVIAL FLUID
Cell Counts
Synovial fluid cell counts, as all body fluid cell counts, should be performed within 1 hour of collection. Hemocytometer counts and manual differentials are normally performed on synovial fluid. Saline may be used as a diluent for synovial fluids with a high number of cells. Hypotonic saline, a weak acid, or commercially available white blood cell (WBC) diluent reservoirs may be used when many RBCs are present. Instruments are available to automate these counts. Cytocentrifugation of the specimen provides good smears for Wright staining and observation.
Differential
Normal synovial fluid contains small numbers of lympho- cytes and only a few neutrophils (Fig. 11-9). The WBC count on normal synovial fluid ranges from 0 to 150 cells per microliter. The mean distribution of these nucle- ated cells is neutrophils 7%, lymphocytes 24%, monocytes 48%, macrophages 10%, and synovial lining cells 4%. The
Figure 11-9. Normal cellular elements found in synovial fluid include
(A) neutrophils, (B) lymphocytes, (C) monocytes/histiocytes, and
(D) synovial lining cells. A few red blood cells are almost always present
in joint effusions (Wright–Giemsa).
Figure 11-10. Synovial fluid with acute inflammation demonstrating
neutrophilic pleocytosis (Wright–Giemsa).
presence of synovial lining cells is of no significant diagnostic concern. Neutrophils may be vacuolated or contain bacteria or crystals. In addition, cells may exhibit pyknotic nuclei or karyorrhexis. Other cells that may be seen in synovial fluid include plasma cells, eosinophils, and lupus erythematosus (LE) cells. The presence of these cells or abnormal numbers of cells normally seen in synovial fluid indicate various disease processes occurring in joints. An eosinophil count of greater than 2% has been associated with allergic disease with arthritis, hemorrhagic joint effusions, Lyme disease, parasitic arthritis, rheumatoid diseases, and tubercular arthritis.
Septic arthritis exhibits a high number of neutrophils (Fig. 11-10). A predominance of lymphocytes may be seen in the early stages of RA. Neutrophils present in later stages of RA may exhibit inclusions that contain immune complexes such as IgG, IgM, complement and RF. These neutrophils will appear to have dark cytoplasmic granules and are sometimes called RA cells or ragocytes. A high number of mono- cytes may be found in arthritis associated with serum sick- ness, viral infections, and crystal-induced arthritis. LE cells
Figure 11-11. LE cell (arrow) is a neutrophil containing
a phagocytized homogeneous nucleus (Wright–Giemsa).
Figure 11-12. Tart cell: a macrophage containing a phagocytized
nucleus that retains some nuclear detail (Wright–Giemsa).
are seen in synovial fluid in about 10% of patients with sys- temic lupus erythematosus and in some patients with RA (Fig. 11-11). LE cells are neutrophils that have engulfed a nucleus of a lymphocyte that has been altered by antinuclear antibody. Tart cells, monocytes that have engulfed nuclear material (Fig. 11-12), may be confused with LE cells. Although not specific for Reiter syndrome, Reiter cells may be present in synovial fluid. Figure 11-13 shows a Reiter cell (neutrophil-laded macrophage).
Lipids may be released from bone marrow after injury to the bone. As a result, lipophages as seen in Figure 11-14 may be present in synovial fluid.
Crystals
Examination of synovial fluid for crystals is a routine test in most laboratories. Crystal analysis is most commonly used to diagnose gout by the presence of monosodium urate (MSU) crystals. Chapter 8 contained an explanation of polarization and compensation of light in the analysis of crystals. MSU crystals that appear in synovial fluid are usually thin, needle-like crystals. MSU crystals polarize light and are negatively birefringent (crystals aligned with the compensator filter are
Figure 11-13. Reiter cell (center) is a macrophage that has phagocytosed
one or more neutrophils. This finding is not specific for Reiter syndrome.
Figure 11-14. Lipid-laden macrophage in synovial fluid
Figure 11-15. Synovial fluid with acute inflammation and
monosodium urate crystals. (Wright–Giemsa stain and polarized light).
Figure 11-16. Synovial fluid with acute inflammation and
monosodium urate crystals. The needle-shaped crystals
demonstrate negative birefringence, because they are yellow
when aligned with the compensator filter and blue when
perpendicular to the filter (Wright–Giemsa stain and polarized/compensated light)
yellow, whereas those lying perpendicular are blue). Figure 11-15, shows MSU crystals under underpolarized light, whereas Figure 11-16 demonstrates these crystals under compensated, polarized light. MSU crystals are yellow when aligned with the compensator filter and blue when lying per- pendicular to the compensator filter.
Other crystals that may be present in synovial fluid include calcium pyrophosphate dehydrate (CPPD) crystals. CPPD crystals may be present in pseudogout. Though CPPD crystals may be confused with MSU crystals, they are typically smaller and rodlike or rhomboid. CPPD crystals also polarize light but are positively birefringent (crystals aligned with the compensator filter are blue, whereas those lying perpendicular are yellow.1 Figure 11-17 shows a CPPD crystal under underpolarized light, whereas Figure 11-18 demonstrates a CPPD crystal under compensated, polarized light. Corticosteroid crystals are needle-shaped and may be seen in synovial fluid following intra-articular injec- tions. Cholesterol crystals may be present in chronic effu- sions from patients with osteoarthritis or RA. See Figure 9 in Chapter 10 for examples of cholesterol crystals in polarized and compensated, polarized light. Apatite crystals (small chunky rods) are seen in calcific periarthritis, osteoarthritis, and inflammatory arthritis)
Figure 11-18. Synovial fluid with acute inflammation and calcium
pyrophosphate dihydrate crystals. The rhomboidal intracellular
crystal (center) demonstrates positive birefringence, because
it is blue when aligned with the compensator filter
(Wright–Giemsa stain and polarized/compensated light)
Microbiologic Examination
Infectious agents that can enter the synovial fluid include bacteria, fungi, Mycobacteria, and viruses, with bacteria being the most common. Bacteria and other microorganisms enter the synovial capsule through the bloodstream, deep penetrating wounds, and rupture of osteomyelitis into the joint. In addition, bacteria may be introduced during procedures such as arthroscopy, intra-articular steroid injections, and prosthetic joint surgery.
Gram stain is performed on synovial fluid smears prepared by centrifugation or cytocentrifugation. Diluting synovial fluid with saline helps separate cells that tend to cluster. Even if Gram staining does not suggest the presence of infectious agents, both aerobic and anaerobic cultures should be performed. Synovial fluid Gram stains are positive in only 50% of cases with joint sepsis.
CLASSIFICATION OF JOINT DISORDERS
Joint disorders are classified into five groups. These groups, numbered I through V, include processes that are nonin- flammatory, inflammatory, septic, crystal induced, and hemorrhagic. Changes to normal joint chemistry and cell counts can occur as a result of bacterial, chemical, or mechanical damage to the joint. Varying degrees of inflam- matory response occur because of alterations of membrane and capillary permeability. Table 11-2 summaries labora- tory findings for groups of joint disorders.
REFERENCES
Lillian A. Mundt and Kristy Shanahan, Graff's Textbook of Urinalysis and Body Fluids, Second Edition 2011
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