ANALYSIS OF URINE AND OTHER BODY FLUIDS SYNOVIAL FLUID ANALYSIS OUTLINE • Introduction • Synovial Fluid o Physiology o Specimen Collection and Handling o Classification and Pathologic Significance of Joint Disorders o Appearance o Viscosity o Cell Counts o Differential Count o Synovial Fluid Crystals o Chemistry Tests o Microbiology Tests o Serological Tests INTRODUCTION • Synovial fluid is the viscous fluid present in areas of the skeleton that are prone to friction, such as joints, bursae, and tendon sheaths. This fluid acts as a lubricant and provides nourishment for the developing adjacent cartilage, which lacks blood vessels, lymphs, and nerves. Analysis of the synovial fluid gives information regarding inflammatory and non-inflammatory conditions of the area of collection as well as septic or hemorrhagic conditions. SYNOVIAL FLUID • Synovial Fluid is often referred to as “joint fluid”, is a viscous liquid found in the cavities of the movable joints or synovial joints. • Function o The synovial fluid reduces friction between the bones during joint movement. o It provides lubrication in the joints. o It also provides nutrients to the articular cartilage. o It lessens the shock of joint compression occurring during activities such as walking and jogging. PHYSIOLOGY • It is formed as an ultrafiltrate of plasma across the synovial membrane. • Majority of the chemical constituents have concentrations similar to plasma values. • Cells lining the synovial membrane (synoviocytes) secrete a mucopolysaccharide containing hyaluronic acid and a small amount of protein into the fluid. • Damage to the articular membranes produces pain and stiffness in the joints, collectively referred to as arthritis . • A variety of conditions including infection, inflammation, metabolic disorders, trauma, physical stress, and advanced age are associated with arthritis. • Normal Synovial Fluid Values Volume <3.5 mL Color Pale yellow Clarity Clear Viscosity Able to form a string 4-6 cm long Erythrocyte count <2000 cells/µL Leukocyte count <200 cells/µL Neutrophils <20% of the differential Lymphocytes <15% of the differential Monocytes and Macrophages 65% of the differential Crystals None present Glucose <10mg/dL lower than blood glucose Lactate <250 mg/dL Total protein <3 g/dL Uric acid Equal to blood value SPECIMEN COLLECTION AND HANDLING • Synovial fluid is collected by needle aspiration called arthrocenthesis. • Amount of fluid present will vary with the size of the joint and the degree of fluid buildup in the joint. • The volume of fluid collected should be recorded. • Normal synovial fluid does not clot; however, fluid from a diseased joint may contain fibrinogen and may clot. • To avoid clotting, the fluid is usually collected in a syringe that has been moistened with heparin. • Specimen should be distributed into 3 tubes. o sterile heparinized tube for the microbiology section o a liquid EDTA tube for hematology section o nonaticogulated tube for other tests • Powdered anticoagulants should not be used because it may produce artifacts that will interfere with crystal analysis. CLASSIFICATION AND PATHOLOGIC SIGNIFICANCE OF JOINT DISORDERS Group Classification Pathologic Significance I. Non-inflammatory Degenerative joint disorders II. Inflammatory Immunologic problems, including rheumatoid arthritis and lupus erythematosus Crystal-induced gout and pseudogout III. Septic Microbial infection IV. Hemorrhagic Traumatic injury Coagulation deficiencies LABORATORY FINDINGS IN JOINT DISORDERS • I. Non-inflammatory o Clear, yellow fluid o Good viscosity o WBCs • II. Inflammatory o Immunologic origin ▪ Poor viscosity ▪ WBCs 2000 – 5000 µL ▪ Neutrophils >50% ▪ Decreased glucose level ▪ Possible autoantibodies present o Crystal-induced origin ▪ Cloudy or Milky fluid ▪ Poor viscosity ▪ WBCs up to 50 000 µL ▪ Neutrophils >90% ▪ Decreased glucose level ▪ Elevated uric acid level ▪ Crystals present ▪ Cloudy, yellow-green fluid
• III. Septic o Poor viscosity o WBCs 10 000 – 200 000 µL o Neutrophils >90% o Decreased glucose level o Positive culture and Gram stain • IV. Hemorrhagic o Cloudy, red fluid o Poor viscosity o WBCs <5000 µL o Neutrophils <50% o Normal glucose level o RBCs present APPEARANCE • Normal synovial fluid appears clear and pale yellow. • The color becomes deeper yellow in the presence of inflammation and may have a greenish tinge with bacterial infection. • Presence of blood from a hemorrhagic arthritis may be distinguished from blood from traumatic aspiration by observing the uneven distribution of blood in a traumatic aspiration. • Turbidity is frequently associated with the presence of WBCs (synovial cell debris and fibrin also produce turbidity). The fluid may appear milky when crystals are present. VISCOSITY • Viscosity comes from the polymerization of hyaluronic acid and is essential for the proper lubrication of the joints. • Arthritis affects both the production and ability to polymerize of hyaluronate thus decreasing viscosity of the fluid. • Simplest way to measure viscosity is to observe the ability of the fluid to form a string from the tip of the syringe. 4 to 6 cm is considered normal. • Measurement of the degree of hyaluronate polymerization can be performed using Ropes, or mucin clot test. • In 2 – 5% acetic, normal synovial fluid will form a solid clot surrounded by clear fluid. As the ability of the hyaluronate to polymerize decreases, the clot becomes less firm and the surrounding fluid increases in turbidity. • The mucin clot test is reported in terms of: o good – solid clot o fair – soft clot o poor – friable clot o very poor – no clot CELL COUNTS • The total leukocyte count is the most frequently performed cell count on synovial fluid. • Red blood cell counts may be requested unless evidence of a traumatic tap exists. • Very viscous fluid should be pretreated by adding a pinch of hyaluronidase to 0.5 mL of fluid or one drop of 0.05 percent hyaluronidase in phosphate buffer per mL of fluid and incubating at 37°C for 5 minutes. • Manual counts are done using the Neubauer counting chamber in the same manner as CSF counts. • WBC diluting cannot be used because it contains acetic acid, which will cause the formation of mucin clots. • Normal saline can be used as diluent. • Methylene blue added to normal saline will stain the WBC nuclei, permitting separation of the RBCs and WBCs during counts. • WBC counts less than 200 cells/µL are considered normal and may reach 100 000 cells/µL or higher in severe infections. DIFFERENTIAL COUNT • Neutrophil o Description: Polymorphonuclear leukocyte o Significance: Bacterial Sepsis, crystal induced inflammation • Lymphocyte o Description: Mononuclear leukocyte o Significance: Nonseptic inflammation • Synovial lining cell o Description: Similar to macrophage, but may be multinucleated, resembling a mesothelial cell o Significance: Normal • LE cell o Description: Neutrophil containing characteristic ingested: “round body” o Significance: Lupus erythematosus • Reiter ce ll o Description: Vacuolated macrophage with ingested neutrophils o Significance: Reiter’s syndrome, nonspecific inflammation • RA cell (ragocyte) o Description: Neutrophil with dark cytoplasmic granules containing immune complexes o Significance: Immunologic inflammation • Cartilage cells o Description: Large, multinucleated cells o Significance: Osteoarthritis • Rice bodies o Description: Macroscopically resemble polished rice, microscopically show collagen and fibrin o Significance: Tuberculosis, septic and rheumatoid arthritis • Fat droplets o Description: Refractile intracellular and extracellular globules, stain with Sudan dyes o Significance: Traumatic injury • Hemosiderin o Description: Inclusions within clusters of synovial cells o Significance: Pigmented villonodular synovitis SYNOVIAL FLUID CRYSTALS • Monosodium urate o Needle-shape o Negative bifringence, run parallel to the long axis of the crystal, with slow vibration and produces a yellow color in Compensated Polarized Light o Found in Intracellular and Extracellular o Found in cases of gout
• Calcium pyrophosphate o Rod, Needles and Rhombic shape o Positive Bifringence, Run perpendicular to the long axis, fast vibration and produces a blue color in Compensated Polarized Light o Found in Intracellular and Extracellular o Found in cases of pseudogout • Cholesterol o Notched rhombic plates shape o Negative bifringence in Compensated Polarized light o Found in Extracellular • Corticosteroid o Flat, variable-shaped plates in shape o Positive and negative birefringence in Compensated Polarized Light o Found primarily in Intracellular CHEMISTRY TESTS • Because synovial fluid is chemically an ultrafiltrate of plasma, chemistry test values are approximately the same as serum values. • Glucose Test o Glucose is the most requested test because markedly decreased values are indicative of inflammatory (group II) or septic (group III) disorders. o Normal synovial fluid glucose should not be more than 10mg/dL. o Glycolysis upon long standing of the specimen causes falsely decreased values. • Lactate Test o Synovial fluid lactate provides rapid differentiation between inflammatory and septic arthritis. o Synovial fluid levels greater than 250 mg/dL are found consistently with septic arthritis, but may also be seen in rheumatoid arthritis. • Other Chemistry Test o Total protein and Uric acid test may be requested because the large protein molecules are not filtered through the synovial membranes, normal synovial fluid contains less than 3 g/dL of protein. o Increased levels are found in inflammatory and hemorrhagic disorders. MICROBIOLOGY TESTS • Gram stains and cultures are two of the most important tests performed on the synovial fluid. • Bacterial infections are frequently seen; however, fungal, tubercular, and viral infections can also occur. • The most frequently seen bacterial species are Staphylococcus and Streptococcus , but other fastidious organisms such as Hemophilus and Neisseria gonorrhea may also be found. SEROLOGIC TESTS • The autoimmune diseases rheumatoid arthritis and lupus erythematosus cause very serious inflammation of the joints and are diagnosed in the serology laboratory by demonstrating the presence of their particular autoantibodies in the patient’s serum. • Arthritis is a frequent complication of Lyme disease. • Demonstration of antibodies to the causative agent Borrelia burgdorfri in the patient’s serum can confirm the cause of the arthritis.