SYNOVIAL FLUID CHAPTER 11

Learning Objectives Upon completing this chapter, the reader will be able to Describe the formation and function of synovial fluid. Relate laboratory test results to the four common classifications of joint disorders. State the five diagnostic tests most routinely performed on synovial fluid. Determine the appropriate collection tubes for requested laboratory tests on synovial fluid. Describe the appearance of synovial fluid in normal and abnormal states. Discuss the normal and abnormal cellular composition of synovial fluid.

Learning Objectives (cont’d) List and describe six crystals found in synovial fluid. Explain the differentiation of monosodium urate and calcium pyrophosphate crystals using polarized and compensated polarized light. State the clinical significance of glucose and lactate tests on synovial fluid. List four genera of bacteria most frequently found in synovial fluid. Describe the relationship of serologic serum testing to joint disorders.

Physiology Synovial fluid functions Formation Lubrication for the movable joints: diarthroses Nutrients for articular cartilage Lessens shock of joint compression Formation Ultrafiltrate of plasma across synovial membrane No high weight molecules Synoviocytes in synovial membrane secrete hyaluronic acid that makes the fluid viscous (lubrication)

Anatomy

Disorders Four classifications of disorders (arthritis) Noninflammatory: degenerative, osteoarthritis Inflammatory: immunologic, lupus erythematosus (LE), rheumatoid arthritis (RA), Lyme disease Crystal-induced, gout and pseudogout Septic: microbial infection Hemorrhagic: trauma, tumors, coagulation deficiencies

Classification and Pathologic Significance of Joint Disorders Table 11–1 Synovial Fluid Reference Values2 Volume <3.5 mL Color Colorless to pale yellow Clarity Clear Viscosity Able to form a string 4 to 6 cm long Leukocyte count <200 cells/µL Neutrophils <25% of the differential Crystals None present Glucose:plasma difference <10 mg/dL lower than the blood glucose level Total protein <3 g/dL

Specimen Collection & Handling Needle aspiration called arthrocentesis Normal knee fluid amount 3.5 mL >25 mL if inflamed Normal fluid does not clot; diseased fluid clots Collect in Sterile heparinized or SPS for microbiology Liquid EDTA (no powdered) for hematology Heparinized or nonanticoagulated for other tests Centrifuge nonanticoagulated tube and separate Sodium fluoride for glucose Test ASAP to avoid cellular lysis and changes in crystals

Appearance Normal: clear and pale yellow (egg white) Deeper yellow with noninflammatory and inflammatory; green tinge = infection Hemorrhagic or traumatic tap = red Traumatic tap: look for decreasing blood in tubes Crystal induced: milky Turbidity: white blood cells (WBCs) or cellular debris, fibrin

Viscosity Polymerization of hyaluronic acid Essential for joint movement Arthritis decreases polymerization 4 to 6 cm string from aspirating needle = OK Ropes (mucin clot test) Add fluid to 2% to 5% acetic acid to form clot Rate, good: solid clot; clear fluid, fair: soft formed clot; low: friable clot; poor: no clot Use acetic acid to identify synovial fluid

Cell Counts WBCs most common Do not use normal WBC diluting fluid; use normal saline/methylene blue May have to treat viscous fluid with hyaluronidase or 37°C incubation first Perform on a Neubauer counting chamber Automated instrument, may need more hyaluronidase Normal: <200 WBCs/μL, septic may reach >100,000

Counting Procedure Line petri dish with moist filter paper. Place hemocytometer on two small sticks above paper. Fill both sides of the hemocytometer for compatibility. For counts less than 200 WBCs/μL, count all nine large squares. For counts greater than 200 WBCs/μL in the above count , count the four corner squares. For counts greater than 200 WBCs/μL in the above count, count the five small squares used for a RBC count.2<<AU: What does superscript 2 refer to?>>

Differential Count Incubate with hyaluronidase, then cytocentrifuge Normal cells: monocytes, macrophages, synovial tissue cells Neutrophils: <25%, increase in sepsis Lymphocytes: <15%, noninflammatory higher All cells may appear more vacuolated

Differential Count (cont’d) Other cells LE cells Eosinophils Reiter cells/neutrophages: vacuolated macrophages with ingested neutrophils Ragocytes (RA cells): neutrophils with small, dark granules containing RA factor (IgM) Lipid droplets: crush injuries Hemosiderin granules: pigmented villonodular synovitis

Crystal Identification Important diagnostic test; frequently performed Acute and chronic cases Metabolic disorders and decreased renal function Other causes: increased blood levels, degeneration of bone and cartilage, injection of corticosteroids

Types of Crystals Primary: monosodium urate (MSU) in gout; calcium pyrophosphate dihydrate (CPPD) in pseudogout MSU: impaired purine metabolism, high purine foods, leukemia chemotherapy, decreased renal excretion of uric acid CPPD: degenerative arthritis, disorders causing elevated calcium levels

Types of Crystals (cont’d) Hydroxyapatite: cartilage degeneration, only seen with electron microscopy Cholesterol: systemic autoimmune diseases (LE, RA) appear similar to urine cholesterol crystals: notched corners Corticosteroids: injections, flat, variable plates Calcium oxalate: renal dialysis patients Artifacts: starch, powdered anticoagulants, dust, scratches (clean slides, cover slips)

Slide Preparation Examine ASAP Crystal changes, MSU and CPPD are seen intracellularly and cells disintegrate Initial examination is wet preparation unstained under low and high power Crystals may be seen on differential

Slide Examination Continued examination is done under polarized and compensated polarized light of wet preparation MSU crystals: needle shaped; seen intra- and extracellularly; may be seen sticking through cytoplasm CPPD crystals: rhombic, square shaped, or short rods; often seen in vacuoles of neutrophils; MSU crystals lyse vacuole membranes

Crystal Polarization Both MSU and CPPD crystals polarize light MSU is highly birefringent and appears brighter than CPPD Confirm identification using compensated polarized light

Compensated Polarized Light Red compensated polarized light: red compensator is placed between crystal and analyzer, producing a red background Separates light into slow- and fast-moving vibrations Align crystals with slow vibration Linear structure of molecules causes different colors to be produced

MSU and CCPD Crystals Under Compensated Polarized Light

MSU and CPPD Under Compensated Light

MSU Under Compensated Light MSU molecules run parallel to the long axis, aligned with slow vibration; fast light is impeded, producing a yellow color (negative birefringence) CPPD molecules run perpendicular to long axis and impede the slow light producing a blue color (positive birefringence)

Chemistry Tests As an ultrafiltrate of plasma, normal values are similar to those of plasma Few are clinically important Most frequent is glucose Normal: not less than 10 mg/dL of plasma glucose Draw sample same time as fluid is collected Markedly decreased levels seen in inflammatory and septic categories

Chemistry Tests (cont’d) Synovial fluid protein Normal is less than 3 g/dL Increased in inflammatory and hemorrhagic categories Synovial fluid lactate and acid phosphatase Severity and prognosis of rheumatoid arthritis (RA)

Reference Synovial Fluid Values Volume: <3.5 mL Color: colorless to pale yellow Clarity: clear Viscosity: able to form a string 4 to 6 cm long Leukocyte count: <200 cells/μL Neutrophils: <25% of the differential Crystals: none present Glucose: <10 mg/dL lower than the blood glucose plasma difference Total protein: <3 g/dL

Microbiology Tests Infections caused by inflammation, trauma, and systemic infections Gram stain and cultures are routinely performed Culture must include chocolate agar Primary organisms are Staphylococcus, Streptococcus, Haemophilus influenzae, and Neisseria gonorrhoeae Patient history determines fungal and Tuberculosis (TB) cultures

Serologic Tests Majority of related tests are performed on serum and fluid may only serve as a confirmation RA and LE are the most common autoimmune causes of arthritis Lyme disease: arthritis is frequent complication; test serum for Borrelia burgdorferi antibodies Extent of inflammation: test for C-reactive protein and fibrinogen