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

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 Color pale yellow Clarity clear Viscosity forms 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. Crystals NONE Glucose <10 mg/dL (lower than blood glucose) Lactate <250 mg/dL Total protein <3 g/dL Uric acid = blood value

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

Color:

Normal clear, pale yellow Red to brown: indicates trauma of procedure or

Viscosity:

disorder Turbidity: associated with presence of WBCs Milky: may indicate presence of crystals of syringe

Measured at bedside by ability to form a string from tip Ropes test (mucin clot test) measurement of
Normal: 4-6 cm

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

Cell Count WBCs

Method
Use Neubauer counting chamber May pretreat viscous fluids with hyaluronidase & incubate at 37oC 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

Differential Count

Normal = <200 / uL

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 nonseptic

inflammation

 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

Synovial lining cell

Neutrophils in synovial fluid

Lymphs in synovial fluid

LE cell in synovial fluid

Crystals

Crystal formation may be due to:

Fluid is examined using the wet preparation technique

Metabolic disorders Decreased renal excretion Cartilage and bone degeneration Medicinal injection (ex: corticosteroids)

Under polarizing light (Direct polarization)

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 compensated polarizing light

Birefringent substances appear as bright objects on a black background Intensity varies between substances 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)

 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

Acute gout (uric acid crystals)

Uric acid crystals

 Cholesterol Nonspecific indications

Associated with chronic inflammation

Exhibit negative birefringence (compensated polarized light) Usually seen extracellularly Polarized light strongly birefringence Rhombic plates Associated with calcific deposition conditions May produce an acute inflammatory reaction Intracellular Not birefringent Require an electron microscope to examine Small, needle shaped

Hydroxyapatite (HA) (Calcium phosphate)

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

 Calcium Oxalate
Following renal dialysis

Birefringent Artifacts:
Anticoagulant crystals (calcium oxalate, lithium heparin) Starch granules Prosthesis fragments Collagen fibers Fibrin Dust particles

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 < serum plasma glucose value

Total protein

Should be run within 1 hour of collection Draw in sodium fluoride prevents glycolysis Not routinely performed Normal = < 1/3 of serum value (~3g/dL)

Increased protein

Large molecule, not easily filtered by membrane Changes in membrane permeability Increased joint synthesis Indicates an inflammatory process

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

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

Autoantibody detection (same as found in serum)

Rheumatoid arthritis (RA) Lupus erythematosus (LE)

Antibody detection in patients serum

Borrelia burgdorferi
Causative agent of Lyme disease Cause of arthritis

Group Classification I. Noninflammatory II. Inflammatory

I. II.

Septic Hemorrhagic

Significance Degenerative joint disorders Immunologic problems (RA, LE)Gout&pseudogout (crystal induced) Microbial infection Traumatic 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