section epub:type=”chapter” id=”c0011″ role=”doc-chapter”> After studying this chapter, the student should be able to: In areas of the skeleton where friction could develop, such as the joints, bursae, and tendon sheaths, viscous synovial fluid is present. Within articulated diarthrodial joints (e.g., the knee), the ends of apposing bones are covered with articular cartilage, the joint space is lined by a synovial membrane (except in weight-bearing areas), and synovial fluid bathes and lubricates the joint (Fig. 11.1). The surface of the synovial membrane surrounding the joint consists of numerous microvilli with a layer, one to three cells deep, of synovial cells called synoviocytes (Fig. 11.2). Two types of synoviocytes are present in the synovial membrane. The most prevalent type is actively phagocytic and synthesizes degradative enzymes (e.g., collagenases). The second type of synoviocyte synthesizes hyaluronate, a mucopolysaccharide linked with approximately 2% protein. The synoviocytes are loosely organized in the synovial membrane and differ from cells in other lining membranes in that they have no basement membrane, and adjacent synovial cells are not joined with desmosomes. Beneath the synoviocytes is a thin layer of loose connective tissue containing a vast network of blood vessels, lymphatics, and nerves. Variable numbers of mononuclear cells are also found in this connective tissue layer. Synovial fluid is formed by the ultrafiltration of plasma across the synovial membrane and from secretions by synoviocytes. The resultant viscous fluid serves as a lubricant for the joint and is the sole nutrient source for the metabolically active articular cartilage, which lacks blood vessels, lymphatics, and nerves. The composition of synovial fluid is unique in that its glucose and uric acid concentrations are equivalent to that of the blood plasma; however its total protein and immunoglobulin concentrations vary from one-fourth to one-half that of the plasma. Table 11.1 (and Appendix C) lists reference values for various characteristics and constituents of normal synovial fluid obtained from a knee joint. Table 11.1 aValues for fluid obtained from a knee joint. bSynovial fluid values are equivalent to blood plasma values if obtained from a fasting patient. cNormal lactate values are assumed to be similar to those in blood and cerebrospinal fluid; actual reference intervals have yet to be established. Arthritis and other joint diseases are common, and laboratory analysis of synovial fluid assists in the diagnosis and classification of these conditions. When synovial fluid is removed from a joint space, laboratory examination enables classification of the disease process into one of four principal categories: noninflammatory, inflammatory, septic, or hemorrhagic. These general classifications aid in differential diagnosis of joint disease and are summarized in Table 11.2. An important note is that (1) these categories partially overlap, (2) several conditions can occur in the joint at the same time, and (3) variations in test results can occur depending on the stage of the disease process. Consequently, these classifications are used only as a guide for the clinician in the evaluation and diagnosis of joint disease. In contrast to the tentative diagnoses possible on the basis of laboratory findings, a definitive diagnosis is possible when microorganisms are identified (septic arthritis) or crystals are present (crystal synovitis) in the synovial fluid. Table 11.2 aThe plasma–synovial fluid difference in glucose concentration when specimens are obtained simultaneously. bWith chronic or subsiding conditions, crystal synovitis may present as group I. cEarly stages of rheumatoid arthritis may present as group I. dPreviously known as Reiter’s syndrome.11 Synovial fluid is removed by arthrocentesis, which is the percutaneous aspiration of fluid from a joint using aseptic technique. Disposable, sterile needles and syringes are used most often to prevent birefringent contaminants associated with the cleaning and resterilization of reusable supplies. If possible, patients should be fasting a minimum of 4 to 6 hours to allow for the equilibration of chemical constituents between plasma and synovial fluid. A blood sample is collected at approximately the same time as the arthrocentesis procedure is performed when determination of the plasma–synovial fluid difference in glucose is requested. The volume of synovial fluid in a joint varies with the size of the joint cavity and is normally small—about 0.1 to 3.5 mL.1 Consequently, arthrocentesis of a joint when an effusion (or fluid buildup) is not present can result in a “dry tap”—a small yield of synovial fluid. Sometimes synovial fluid is present only in the aspiration needle; this requires that the contents of the needle be expressed into an appropriate small-volume container or, at times, directly into culture media. Alternatively, the clinician may insert the needle into a sterile cork and transport the entire syringe to the laboratory for processing. However, this practice represents a significant potential biohazard and should be avoided. Note that specimens should not be rejected because of a low volume of fluid. Diagnostic crystals, cell counts with differentials, and microbial growth are possible from a few drops of synovial fluid. Synovial fluid handling and volume requirements are summarized in Table 11.3. It is recommended by the Clinical Laboratory Standards Institute (CLSI) that the first portion of fluid obtained (tube #1) be placed into a plain red-top tube (no anticoagulant) for chemical and immunologic evaluation. The next portion (tube #2) is collected in an anticoagulant tube for microscopic examinations, and the last portion (tube #3) is placed in a sterile anticoagulant tube for microbiological studies. Because the volume of synovial fluid present can vary significantly, the amount collected and distributed into each collection tube will also vary. Table 11.3 EDTA, Ethylenediaminetetraacetic acid. aSodium heparin concentration at 25 U/mL synovial fluid. bNo upper limit to the amount of fluid that can be submitted; large volumes of fluid increase the recovery of cellular elements. cLarge fluid volumes may increase the recovery of viable microbial organisms. Note that using larger volumes of synovial fluid for cultures can enhance the recovery of microbial organisms; similarly, greater volumes of fluid will increase the numbers of cells recovered for cytologic evaluation. For cell counts and crystal identification, the best anticoagulant for synovial fluid is “liquid” ethylenediaminetetraacetic acid (EDTA) or sodium heparin at approximately 25 units (U) per milliliter of synovial fluid. These anticoagulants prevent clotting when fibrinogen is present but do not form crystals. Oxalate, lithium heparin, and powdered EDTA must be avoided because they can produce crystalline structures that resemble monosodium urate crystals, thereby interfering with the microscopic examination for in vivo crystals. For microbial studies, synovial fluid placed in a sterile tube without anticoagulant or with sodium polyanetholsulfonate (SPS, yellow-top tube) is acceptable.2 Note that the concentration of SPS must not exceed 0.025% (wt/vol) because some Neisseria spp. and certain anaerobes are inhibited by higher concentrations.3 For anaerobic culture, fluid can be placed into an anaerobic transport vial or tube. Because synovial fluid specimens are often sent to different laboratories for testing, the total volume of fluid removed should be recorded in the patient’s chart and on specimen test request forms at the time of fluid collection. Synovial fluid should be transported and analyzed at room temperature. As with other body fluids, it should be processed and tested as soon as possible after collection. If processing is delayed, several adverse changes can occur: (1) Cells in the synovial fluid can alter the chemical composition, (2) detection of microbial organisms can be jeopardized, and (3) blood cells (white blood cells [WBCs], red blood cells [RBCs]) can lyse. Note that refrigeration adversely affects the viability of microorganisms and could erroneously induce in vitro crystalline precipitation. The hematology laboratory often performs the physical and microscopic examinations of synovial fluid. Physical examination includes visual assessment for color, clarity, and viscosity. Normally, synovial fluid appears pale yellow or colorless and is clear. Color variations of red and brown are associated with trauma during the arthrocentesis and with disorders that disrupt the synovial membrane, allowing blood to enter the joint cavity, such as joint fracture, tumor, and traumatic arthritis. A traumatic procedure is indicated when the amount of blood in the fluid decreases as collection continues, or when a streak of blood is noticed in the fluid. With some joint disorders, particularly infections, synovial fluid can appear greenish or purulent; with other conditions (e.g., tuberculous arthritis, systemic lupus erythematosus), synovial fluid can appear milky. Numerous substances can modify the clarity of synovial fluid; these substances include WBCs, RBCs, synoviocytes, crystals, fat droplets, fibrin, cellular debris, rice bodies, and ochronotic shards. The specific entity or entities causing the observed turbidity are usually identified by microscopic examination. Some substances are evident upon gross visual examination of synovial fluid. Rice bodies are white, free-floating particles made up of collagen covered by fibrinous tissue.4 They resemble polished, shiny grains of rice and can vary greatly in size. Although they may be seen in many arthritic conditions, they are observed most commonly with rheumatoid arthritis. Dark, pepper-like particles called ochronotic shards can be present in synovial fluid from individuals with ochronotic arthropathy, a consequence of alkaptonuria and ochronosis.5 These pepper-like particles are pieces of pigmented cartilage that has eroded and broken loose into the fluid. Synovial fluid has high viscosity compared to water because of its high concentration of the mucoprotein hyaluronate. This high-molecular-weight polymer of repeating disaccharide units is secreted by synoviocytes and serves as a joint lubricant. During inflammatory conditions, hyaluronate can be depolymerized by the action of the enzyme hyaluronidase, which is present in neutrophils, as well as by some bacteria (e.g., Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens). In addition, some disease processes inhibit the production and secretion of hyaluronate by synoviocytes. Synovial fluid viscosity can be assessed at the bedside by observing the fluid as it is expelled from the collection syringe. A drop of normal synovial fluid forms a string at least 4 cm long before breaking. The viscosity of the fluid is considered abnormally low when the string breaks earlier or forms discrete water-like droplets. Because viscosity measurements have little diagnostic or clinical value and are not reliable, they are rarely performed. Spontaneous clot formation in synovial fluid indicates the abnormal presence of fibrinogen. Because of its high molecular weight (340,000), fibrinogen cannot pass through a normal or healthy synovial membrane. Pathologic processes that damage the synovial membrane or a traumatic arthrocentesis with blood contamination can cause fibrinogen to be present in synovial fluid, which will result in clot formation. Therefore a portion of a synovial fluid collection should always be anticoagulated using liquid EDTA or sodium heparin to prevent potential fibrin clots that interfere with the microscopic examination. To eliminate the potential for clot formation and reduce the viscosity of synovial fluid, before analysis a well-mixed portion is transferred to another tube and a few grains of hyaluronidase are added. This enzyme will eliminate hyaluronate, thereby preventing clot formation, reducing viscosity, and enhancing an even distribution of cells in the counting chambers of the hemacytometer. Alternatively when the fluid requires dilution because it is cloudy or turbid, a hyaluronidase buffer solution can be used as the diluent.6 Note that if the synovial fluid has not been “pretreated” with hyaluronidase, an acetic acid diluent cannot be used because it will cause hyaluronate to form a mucin clot and cells to clump, which interfere with the microscopic examination. A manual microscopic examination is performed using a hemacytometer and well-mixed synovial fluid, either undiluted or diluted. See Chapter 17 for procedural details for performing manual cell counts and Appendix D for optional diluents when needed. Analysis should occur as soon as possible to avoid potential changes in cell counts (lysis) and crystal formation. Normally, the number of RBCs in synovial fluid is fewer than 2000 RBCs/μL. Some RBCs originate from the procedure itself; those resulting from hemorrhagic effusions are usually obvious from their large numbers and the initial red-brown appearance of the fluid. When the number of RBCs present is excessive and they must be eliminated to allow performance of an accurate WBC and differential count, hypotonic saline (0.3%) is used as the diluent because it will selectively lyse the RBCs while retaining the WBCs. WBCs are normally present in synovial fluid at cell counts lower than 200 WBCs/μL. Although WBC counts greater than 2000 cells/μL are typically associated with bacterial arthritis, leukocytosis can occur with other conditions, such as acute gouty arthritis and rheumatoid arthritis. Although a total WBC count has limited value in identifying a specific disease process, when abnormal, it is providing clinically valuable information. Synovial fluid can be concentrated by several techniques; however, cytocentrifugation preserves cellular morphology better than routine centrifugation procedures.7 Normally, about 60% of synovial fluid’s nucleated cells are monocytes or macrophages, approximately 30% are lymphocytes, and approximately 10% are neutrophils.8 Nucleated cell differentials have limited clinical value because they can vary in a particular disease as well as with the stage of the disease. A nucleated cell differential with more than 80% neutrophils is associated with bacterial arthritis and urate gout, regardless of the total cell count. An increase in the lymphocyte percentage often occurs in the early stages of rheumatoid arthritis, whereas neutrophils predominate in later stages. An increased eosinophil count (greater than 2%) has been associated with a variety of disorders, including rheumatic fever, parasitic infestations, and metastatic carcinoma, and often follows procedures such as arthrography and radiation therapy. The nucleated cell types that can be present in synovial fluid include granulocytes (i.e., neutrophils, eosinophils, basophils/mast cells), lymphocytes, plasma cells, mononuclear phagocytes (i.e., monocytes, macrophages), synoviocytes (lining cells of synovial membrane), and, although rare, malignant cells. Note that all nucleated cells are counted in the differential, including synoviocytes, the lining cells of the synovial membrane. Synoviocytes can be difficult to differentiate from monocytes and they closely resemble mesothelial cells that are found in pleural and peritoneal fluids (compare Fig. 11.2 with Fig 10.6). As with mesothelial cells in serous fluid, the presence of synoviocytes in synovial fluid is expected and does not have any diagnostic value. Many institutions group synoviocytes, monocytes, and macrophages in a single category without differentiating between them. Some nucleated cells are associated with a specific disease process, such as LE cells with lupus erythematosus, cells with hemosiderin inclusions following a hemorrhagic process, multinucleated cartilaginous cells in patients with osteoarthritis, and malignant cells in patients with metastatic tumors. One of the most important laboratory tests routinely performed on synovial fluid is microscopic examination for crystals. Identification of some crystals is pathognomonic of a specific joint disease, thereby enabling a rapid definitive diagnosis (Table 11.4). Because temperature and pH changes affect crystal formation and solubility, synovial fluid specimens should be maintained at room temperature and examined as soon as possible after collection. Time delays before microscopic examination can result in a decrease in WBCs (lysis) and in phagocytosis of crystals by WBCs during storage. Note that pretreatment with hyaluronidase has no effect on crystals if present. Table 11.4 aCharacteristics of typical crystalline forms; however, other crystalline forms can also be present.
Synovial Fluid Analysis
Key Terms1⁎ *
Physiology and Composition
Physical Examination
Total volume
0.1–3.5 mL
Color
Pale yellow
Clarity
Clear
Viscosity
High; forms “strings” 4–6 cm long
Spontaneous clot formation
No
Microscopic Examination
Erythrocyte count
<2000 cells/μL
Leukocyte count
<200 cells/μL
Differential cell count
Monocytes and macrophages
≈60%
Lymphocytes
≈30%
Neutrophils
≈10%
Crystals
None present
Chemical Examination
Glucose
Equivalent to plasma valuesb
Glucose: P-SF difference
<10 mg/dLb
Uric acid
Equivalent to plasma valuesb
Total protein
1–3 g/dL
Lactate
9–33 mg/dLc
Hyaluronate
0.3–0.4 g/dL
Classification of Joint Disorders
Test
Normal
Group I Noninflammatory
Group II Inflammatory
Group III Septic
Group IV Hemorrhagic
Volume, mL
<3.5
>3.5
>3.5
>3.5
>3.5
Color
Pale yellow
Yellow
Yellow-white
Yellow-green
Red-brown
Viscosity
High
High
Low
Low
Decreased
WBC count, cells/μL
<200
<3000
2000–100,00012
10,000– >100,00012
>500012
Neutrophils
<25%
<25%
>50%
>75%
>25%
Glucose concentration
Approximately equal to plasma level
Approximately equal to plasma level
Less than plasma level
Less than plasma level
Approximately equal to plasma level
Glucose: P – SFa difference
≤10 mg/dL12 (≤ 0.55 mmol/L)
<20 mg/dL (< 1.11 mmol/L)
>20 mg/dL (range, 0–80)6 (> 1.11 mmol/L)
>40 mg/dL (range, 20–100)6 (> 2.22 mmol/L)
<20 mg/dL (< 1.11 mmol/L)
Culture
Negative
Negative
Negative
Positive
Negative
Associated diseases
—
Specimen Collection
Collection Tube Order
Test
Volume
Tube Type
All tubes
Physical examination
≈1 mL
Color, clarity, viscosity
#1
Chemical examination
Lactate, lipids (cholesterol, triglycerides), protein, uric acid
1–3 mL
No anticoagulant (red top)
Glucose
1–3 mL
No anticoagulant (red top) or sodium fluoride (gray top)
#2
Microscopic examination
Total cell count
2–5 mL
Liquid EDTA or sodium heparina
Differential cell count
Crystal identification
Cytologic studies (e.g., malignant cells)
5–50 mLb
Sodium heparina
#3
Microbiological studies
Culture
3–10 mLc
Sterile tube; no anticoagulant (red top), sodium heparin,a or sodium polyanethole sulfonate (yellow top)
Physical Examination
Color
Clarity
Viscosity
Clot Formation
Microscopic Examination
Total Cell Count
Differential Cell Count
Crystal Identification
Crystal
Microscopic Characteristicsa
Clinical Conditions
Monosodium urate monohydrate
Fine, needle-like, with pointed ends; strong negative birefringence
Urate arthritis (gouty arthritis)
Calcium pyrophosphate dihydrate
Rod-like or rhombic; weak positive birefringence
Pseudogout (i.e., chondrocalcinosis)
Cholesterol
Flat, parallelogram-shaped plates, with notched corners; negative birefringence; intensity varies with crystal thickness
Chronic arthritic conditions (e.g., rheumatoid arthritis), monoarthritis
Hydroxyapatite
Requires electron microscopy for visualization; not birefringent
Apatite-associated arthropathies
Corticosteroid
Irregular with jagged or serrated edges; broken pieces; varies with corticosteroid used
Indicates previous intraarticular injection
Calcium oxalate
Calcium oxalate dihydrate (Weddellite) and monohydrate (Whewellite)
Oxalate gout in long-term renal dialysis patients or those with primary hyperoxaluria (rare, inherited disorder)
Hematin
Yellow-brown (golden) rhomboid crystals under brightfield microscopy
Indicates a previous hemorrhage in the joint
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Synovial Fluid Analysis
Learning Objectives
