1. Synovial Fluid I. Physiology & Composition Movable joints (diarthroses) composed of: Bones lined with articular cartilage Separated by a cavity containing synovial fluid enclosed in a synovial membrane Synovial membrane synoviocytes: Phagocytic – synthesizes degradative enzymes Synthesizes hyaluronate Connective tissue Blood vessels, lymphatics & nerves Fluid formation Ultrafiltrate of plasma across synovial membrane Non selective Excludes proteins of high molecular weight Synoviocytes Secrete mucopolysaccharite which contains: Hyaluronic acid protein

2. Cartilage & fluid function: Reduce friction between bones Lubricates joints Fluid provides nutrients to cartilage Lessens shock of walking and jogging impact Synovial Fluid – Normal Values Volume<3.5 mL Colorpale yellow Clarityclear Viscosityforms string 4-6 cm long Erythrocytes<2000 cells/uL Leukocytes<200 cells/uL Neutrophils<20% of diff. Lymphocytes<15 % of diff. Monocytes & macrophages65% of diff. CrystalsNONE Glucose<10 mg/dL (lower than blood glucose) Lactate<250 mg/dL Total protein<3 g/dL Uric acid= blood value

3. Collection: arthrocentesis – needle aspiration of synovial fluid Volume: Normal= 3.5 mL Diseased / inflamed = up to 25 mL Collect 2 tubes Heparin tube : microbiology Plain top: chemistry and immunology EDTA (liquid) : hematology *Avoid all powdered anticoagulants – interfere with crystal analysis Fluid verification Mucin clot test- Add fluid to dilute acetic acid  turbidity (clot formation) due to hyaluronate Metachromatic staining Place fluid on filter paper + few drops of toluidine blue  metachromatic staining

4. III. Physical Examination Color: Normal – clear, pale yellow Red to brown: indicates trauma of procedure or disorder Turbidity: associated with presence of WBCs Milky: may indicate presence of crystals Viscosity: Measured at bedside by ability to form a string from tip of syringe Normal: 4-6 cm Ropes test (mucin clot test)– measurement of hyaluronate polymerization Fluid forms a clot surrounded by clear fluid when added to acetic acid Clot quality is reported: Good = solid clot Fair = soft clot Poor = friable clot Very poor = no clot Test is of questionable precision and seldom used

6. IV. Microscopic Examination Cell Count – WBCs Method Use Neubauer counting chamber May pretreat viscous fluids with hyaluronidase & incubate at 37 o C for 5 min. Dilution with hypotonic saline is used to lyse any RBCs OR Dilute with normal saline/methylene blue mixture to differentiate WBCs from RBCs Normal = <200 / uL Differential Count Cytocentrifuge specimen and prepare typical blood smear Normal: 60% monocytes, macrophages neutrophils: <20% lymphocytes: <15% (* values vary between texts) Increased neutrophils – possible septic condition Increased lymphocytes – indicate nonspetic inflammation

7. Other cell abnormalities: Increased eosinophils – rheumatic fever, parasitic infections, metastatic carcinoma, post radiation therapy or arthrography LE cells – patients with lupus erythematosus Reiter cells – macrophages with ingested neutrophils RA cells (ragocytes) – precipitated rheumatoid factor appearing as cytoplasmic granules in neutrophils Hemosiderin granules – due to hemorrhagic process or cases of pigmented villonodular synovitis Cartilaginous cells – observed in cases of osteoarthritis Rice bodies – found in septic and rheumatoid arthritis and Tuberculosis Fat droplets – indicate traumatic injury

8. Synovial lining cell

9. Neutrophils in synovial fluid

10. Lymphs in synovial fluid

11. LE cell in synovial fluid

12. Crystals Crystal formation may be due to: Metabolic disorders Decreased renal excretion Cartilage and bone degeneration Medicinal injection (ex: corticosteroids) Fluid is examined using the wet preparation technique ASAP examination as pH and temperature affect observation Ideally examined prior to WBC disintegration Examine under both direct and compensated polarizing light *may also be observed in Wright stain preparations Under polarizing light (Direct polarization) Birefringent substances appear as bright objects on a black background Intensity varies between substances Under compensated polarizing light A red compensator plate is placed between the crystal and slide Crystals aligned parallel to the compensator appear yellow (negative birefringence) Crystals aligned perpendicular to the compensator appear blue (positive birefringence)

13. Monosodium Urate Crystals (MSU) Indicate gouty arthritis due to: Increased serum uric acid Decreased renal excretion of uric acid Impaired metabolism of nucleic acid Exhibit negative birefringence Intracellular (acute stages) & extracellular location Polarized light – strongly birefringent Compensated polarized light – yellow when parallel blue when perpendicular Needle shaped Calcium pyrophosphate (CCPD) Indicates pseudogout due to: Degenerative arthritis Endocrine disorders with increased serum calcium Calcification of cartilage Exhibit positive birefringence Seen intracellular- and extracellularly Polarized light – weakly birefringent Compensated polarized light – blue when parallel (yellow when perpendicular) Blunt rods or rhombic shapes

14. Acute gout (uric acid crystals)

15. Uric acid crystals

16. Cholesterol Nonspecific indications Associated with chronic inflammation Exhibit negative birefringence (compensated polarized light) Usually seen extracellularly Polarized light – strongly birefringence Rhombic plates Hydroxyapatite (HA) (Calcium phosphate) Associated with calcific deposition conditions May produce an acute inflammatory reaction Intracellular Not birefringent Require an electron microscope to examine Small, needle shaped Corticosteroid Associated with intra-articular injections; NO clinical significance Primarily intracellular Exhibit positive and negative birefringence Can closely resemble MSU and CCPD Flat, variable shaped plates

17. Calcium Oxalate Following renal dialysis Birefringent Artifacts: Anticoagulant crystals (calcium oxalate, lithium heparin) Starch granules Prosthesis fragments Collagen fibers Fibrin Dust particles

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19. V. Chemistry Tests Glucose Done simultaneously with blood sample (prefer 8 hour fast) Difference between blood and synovial glucose values is evaluated Normal = < 10 mg/dL Inflammatory conditions = > 25mg/dL Sepsis = >40 mg/dL Considered low if < ½ serum plasma glucose value Should be run within 1 hour of collection Draw in sodium fluoride – prevents glycolysis Total protein Not routinely performed Normal = < 1/3 of serum value (~3g/dL) Large molecule, not easily filtered by membrane Increased protein Changes in membrane permeability Increased joint synthesis Indicates an inflammatory process

20. Uric Acid Alone, not diagnostic May determine gout in conjunction with plasma uric acid, esp. when crystals are undetectable Normal = serum level Lactate May differentiate between inflammatory and septic arthritis Septic arthritis = >250 mg/dL Gonococcal arthritis = normal to low levels Production results from : Increased demand for energy Tissue hypoxia Severe inflammatory conditions

21. VI. Microbiology Tests Gram stain Performed on all specimens Most infections are bacterial: Staphylococcus Streptococcus S. pyogenes S. pneumoniae Hemophilus Neisseria gonorrhea Fungal, viral and tubercular agents may also be observed Culture Routine culture Enrichment medium (chocolate agar Specialty media depending on clinician orders and indications

22. VII. Serologic Tests Autoantibody detection (same as found in serum) Rheumatoid arthritis (RA) Lupus erythematosus (LE) Antibody detection in patient’s serum Borrelia burgdorferi Causative agent of Lyme disease Cause of arthritis

23. VIII. Joint disorder classification Group ClassificationSignificance I. NoninflammatoryDegenerative joint disorders II. InflammatoryImmunologic problems (RA, LE)Gout & pseudogout (crystal induced) III. SepticMicrobial infection IV. HemorrhagicTraumatic injury Coagulation deficiency Note: * categories overlap * multiple conditions can occur simultaneously * disease stage can vary laboratory results *see text for details of associated abnormal laboratory findings