Synovial Fluid Examination Lecture PDF

SYNOVIAL FLUID EXAMINATION LECTURER: MS.YEAMA SINGH PHYSIOLOGY ◼ It is often referred to as “joint fluid,” is a viscous liquid found in the cavities of the movable joints (diarthroses) or synovial joints. ◼ The bones in the synovial joints are lined with smooth articular cartilage and separa...

SYNOVIAL FLUID EXAMINATION LECTURER: MS.YEAMA SINGH PHYSIOLOGY ◼ It is often referred to as “joint fluid,” is a viscous liquid found in the cavities of the movable joints (diarthroses) or synovial joints. ◼ The bones in the synovial joints are lined with smooth articular cartilage and separated by a cavity containing the synovial fluid. ◼ The joint is enclosed in a fibrous joint capsule lined by the synovial membrane that is lubricated by synovial fluid.  PHYSIOLOGY ◼ Apart from providing lubrication in the joints, synovial fluid provides nutrients to the articular cartilage and lessens the shock of joint compression that occurs during activities such as walking and jogging. ◼ Laboratory results of synovial fluid analysis can be used to determine the pathological origin of arthritis. ◼ The beneficial tests performed most frequently on synovial fluid are the white blood cell (WBC) count, differential, Gram stain, culture, and crystal examination. ◼ A variety of conditions, including infection, inflammation, metabolic disorders, trauma, physical stress, and advanced age, are associated with arthritis. SYNOVIAL JOINT PHYSIOLOGY ◼ The joint is enclosed in a fibrous joint capsule lined by the synovial membrane that is lubricated by synovial fluid. The synovial membrane contains specialized cells called synoviocytes. Two types of synoviocytes are present in the synovial membrane. ◼ Type A cells are macrophage-like cells located in the superficial layer of the synovial membrane and play an important role in phagocytosis. ◼ Type B cells are fibroblast-like cells with prominent endoplasmic reticulum located in a deeper layer of the synovial membrane and produce hyaluronic acid, fibronectin, and collagen to produce synovial fluid INTRODUCTION Importance of synovial fluid analysis: ◼ Diagnose joint disorders (e.g., arthritis, gout, infections). Components of routine examination: ◼ Gross examination ◼ Cell counts ◼ Morphologic examination ◼ Common chemical tests SPECIMEN COLLECTION AND HANDLING ◼ Synovial fluid is collected from a joint by needle aspiration called arthrocentesis. ◼ The amount of fluid present varies with the size of the joint and the extent of fluid buildup in the joint. ◼ In some instances, only a few drops of fluid are obtained, but these still can be used for microscopic analysis or culturing. The volume of fluid collected should be recorded. ARTHROCENTESIS SPECIMEN COLLECTION AND HANDLING ◼ Normal synovial fluid does not clot; however, fluid from a diseased joint may contain fibrinogen and will clot. ◼ Therefore, fluid is collected in a syringe that has been moistened with heparin. ◼ When sufficient fluid is collected, it should be distributed into specific tubes based on the required tests. SPECIMEN COLLECTION AND HANDLING The Clinical Laboratory Standards Institute recommends that synovial fluid should be collected as follows: ◼ Tube 1: The first 4 to 5 mL of the synovial fluid obtained should be placed into a plain, non-anticoagulated red stopper tube and observed for clotting. The tube is centrifuged to remove cellular and other components. The supernatant is used for chemical or immunologic analysis. ◼ Tube 2: The next 4 to 5 mL is collected into a tube to which 25 units (U) of sodium heparin per mL (green stopper) is added or to a ethylenediaminetetraacetic acid (EDTA) tube (lavender stopper) for cell count, differential count, and crystal identification.. SPECIMEN COLLECTION AND HANDLING ◼ Tube 3: The last 4 to 5 mL is placed into a sterile tube to which 25 U per mL heparin is added (green stopper) or to a sodium polyanethol sulfonate (yellow stopper) tube for microbiological studies. ◼ The non-anticoagulated tube for other tests must be centrifuged and separated to prevent cellular elements from interfering with chemical and serological analyses. Ideally, all testing should be done as soon as possible to prevent cellular lysis and possible changes in crystals. GROSS EXAMINATION Appearance: ◼ Normal: Clear, pale yellow, viscous. ◼ Abnormal: Cloudy (infection), bloody (trauma), greenish (infection). Viscosity: ◼ Normal: Stringy due to hyaluronic acid. ◼ Decreased: Indicates inflammation or infection. Volume: ◼ Increased in joint effusion. Odor: Odorless, but foul smelling in pyogenic infections COLOR & CLARITY ◼ Normal synovial fluid appears colorless to pale yellow. ◼ The word “synovial” comes from the Latin word for egg, ovum. Normal viscous synovial fluid resembles egg white. ◼ The color becomes a deeper yellow in the presence of non-inflammatory and inflammatory effusions and may have a greenish tinge with bacterial infection. ◼ In synovial fluid, the presence of blood from a hemorrhagic arthritis must be distinguished from blood from a traumatic aspiration. COLOR & CLARITY ◼ This is accomplished primarily by observing the uneven distribution of blood or even a single blood streak in the specimens obtained from a traumatic aspiration. ◼ Clarity is determined by the presence of WBCs, red blood cells (RBCs), synoviocytes, crystals, fat droplets, fibrin, and cellular debris in the synovial fluid. ◼ Turbidity is associated frequently with the presence of WBCs; however, synovial cell debris and fibrin also produce turbidity. ◼ The fluid may appear milky when crystals are present SYNOVIAL FLUID VISCOSITY ◼ Synovial fluid viscosity comes from polymerization of the hyaluronic acid and is essential for the proper lubrication of joints. ◼ Arthritis affects both the production of hyaluronate and its ability to polymerize, thus decreasing the fluid viscosity. ◼ Several methods are available to measure the synovial fluid viscosity, the simplest being to observe the fluid’s ability to form a string from the tip of a syringe a test that can be done easily at the bedside. ◼ A string measuring 4 to 6 cm is considered normal. VISCOSITY ◼ Hyaluronate polymerization is measured using a Ropes, or mucin clot, test. ◼ When it is added to a solution of 2% to 5% acetic acid, normal synovial fluid forms 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. VISCOSITY ◼ The mucin clot test is reported in terms of good (solid clot), fair (soft clot), low (friable clot), and poor (no clot). ◼ The mucin clot test is not performed routinely because all forms of arthritis decrease viscosity and little diagnostic information is obtained. ◼ Formation of a mucin clot after adding acetic acid can be used to identify a questionable fluid as synovial fluid.  MORPHOLOGIC EXAMINATION Polarized Microscopy: ◼ Detects crystals: Urate (gout), calcium pyrophosphate (pseudogout). Crystal Types: ◼ Monosodium urate: long, thin, and pointed, and appear yellow and needle-shaped under polarized light microscopy. ◼ Calcium pyrophosphate: shorter and less sharp than MSU crystals, and appear blue under polarized light microscopy Differential Cell Count: ◼ Neutrophils: Infection. ◼ Lymphocytes: Autoimmune conditions. CELL COUNTS ◼ The total leukocyte count is the cell count performed most frequently on synovial fluid. ◼ RBC counts are seldom requested. ◼ To prevent cellular disintegration, counts should be performed as soon as possible, or the specimen should be refrigerated. ◼ Very viscous fluid may need to be pretreated by adding one drop of 0.05% hyaluronidase in phosphate buffer per milliliter of fluid and incubating at 37°C for 5 minutes CELL COUNTS ◼ Manual counts on specimens that have been mixed thoroughly are done using the Neubauer counting chamber. ◼ Usually, clear fluids can be counted undiluted, but dilutions are necessary when fluids are turbid or bloody. ◼ Traditional WBC diluting fluid cannot be used because it contains acetic acid that causes the formation of mucin clots. ◼ Normal saline can be used as a diluent.. ◼ Methylene blue added to the normal saline stains the WBC nuclei, permitting separation of the RBCs and WBCs during counts performed on mixed specimens. CELL COUNTS ◼ The recommended technique is to line a petri dish with moist paper and place the hemocytometer on two small sticks to elevate it above the moist paper. ◼ Fill and count both sides of the hemocytometer for compatibility. Acceptable ranges are determined by the laboratory. Counting procedure: 1. For counts less than 200 WBCs/µL, count all nine large squares. 2. For counts greater than 200 WBCs/µL in the previous count, count the four corner squares. 3. For counts greater than 200 WBCs/µL in the previous count, count the five small squares used for a RBC count. CELL COUNTS ◼ Automated cell counters can be used for synovial fluid counts; however, highly viscous fluid may block the apertures, and the presence of debris and tissue cells may elevate counts falsely. ◼ WBC counts less than 200 cells/µL are considered normal and may reach 100,000 cells/µL or higher in severe infections. ◼ There is, however, considerable overlap of elevated leukocyte counts between septic and inflammatory forms of arthritis. Pathogenicity of the infecting organisms also produces varying results in septic arthritis, as does antibiotic administration. DIFFERENTIAL COUNT ◼ Differential counts should be performed on cytocentrifuged preparations or on thinly smeared slides. ◼ Fluid should be incubated with hyaluronidase before slide preparation. Mononuclear cells, including monocytes, macrophages, and synovial tissue cells, are the primary cells seen in normal synovial fluid. ◼ Neutrophils should account for less than 25% of the differential count and lymphocytes less than 15%. ◼ Increased neutrophils indicate a septic condition, whereas an elevated cell count with a predominance of lymphocytes suggests a nonseptic inflammation. DIFFERENTIAL COUNT ◼ In both normal and abnormal specimens, cells may appear more vacuolated than they do on a blood smear. ◼ Besides increased numbers of these usually normal cells, other cell abnormalities include the presence of eosinophils, lupus erythematous (LE) cells, Reiter cells and rheumatoid arthritis (RA) cells. ◼ Lipid droplets may be present after crush injuries. CRYSTAL IDENTIFICATION ◼ Microscopic examination of synovial fluid for the presence of crystals is an important diagnostic test in evaluating arthritis. ◼ Crystal formation in a joint frequently results in an acute, painful inflammation. Also, it can become a chronic condition. ◼ Causes of crystal formation include metabolic disorders and decreased renal excretion that produce elevated blood levels of crystallizing chemicals, degeneration of cartilage and bone, and injection of medications, such as corticosteroids, into a joint. TYPES OF CRYSTALS MSU CRYSTALS MICROBIOLOGICAL TESTS ◼ Gram stains and cultures are two of the most important tests performed on synovial fluid. ◼ Both tests must be performed on all specimens, as organisms often are missed on Gram stain. When they are suspected, special culturing procedures should be used. ◼ Molecular methods using the polymerase chain reaction (PCR) are available for the detection of microorganisms. This testing is particularly beneficial for the organisms that are hard to detect and culture. ◼ https://www.youtube.com/watch?v=b8PZj8ab6Wo ◼ https://www.youtube.com/watch?v=McINCWMbseI GRAM STAINS & CULTURE MOLECULAR METHODS SEROLOGICAL TESTS ◼ Since there is an association of the immune system with the inflammation process, serological testing plays an important role in the diagnosis of joint disorders. ◼ However, most of these tests are performed on serum, and synovial fluid analysis actually serves as a confirmatory measure in cases that are difficult to diagnose. ◼ The autoimmune diseases Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE) cause very serious joint inflammation and are diagnosed in the serology laboratory by demonstrating the presence of their particular autoantibodies in the patient’s serum. COMMON CHEMICAL TESTS Glucose: ◼ Normal: Similar to blood glucose. ◼ Decreased: Infection or inflammation. Protein: ◼ Normal: ~1–3 g/dL. ◼ Increased: Inflammation or infection. Uric Acid and Lactate: ◼ Uric acid: Elevated in gout. ◼ Lactate: Elevated in septic arthritis. SUMMARY ◼ Gross appearance provides initial diagnostic clues. ◼ Cell counts and morphology identify inflammatory and infectious conditions. ◼ Crystal identification diagnoses gout and pseudogout. ◼ Chemical tests confirm metabolic or infectious diseases. SUMMARY WBCs: ◼ Normal: 100,000/µL (septic arthritis). RBCs: ◼ Presence indicates trauma or bleeding disorder. Counting Method: ◼ Manual count using a hemocytometer.  The End Questions & Clarifications