SYNOVIAL FLUIDSynovial fluid referred as to “joint fluid” is a viscous liquid in the cavities of diarthroses (synovial joints lines with smooth articulate cartilage and separated be a cavity. It an ultrafiltrate of plasma which lubricates the joint, provide nutrients to the articular cartilage and lessen the shocks of joint compression. It

The joint is enclosed in a fibrous joint capsule lined by synovial membrane containing synoviocytes. Synoviocytes secretes a mucopolysaccharide substance, hyaluronic acid and small amount of proteins.

Arthritis occurs when articular membrane is damage causing pain and joint stiffness. Condition as infection, inflammation, metabolic disorder, trauma, physical stress and advanced aged may be associated.

There are four classifications of joint disorders: noninflammatory, inflammatory, septic, and hemorrhagic and its corresponding pathologic disorder. Arthrocentesis is the collection of synovial fluid by needle aspiration. Tests most frequently used for synovial fluid are WBC count, differential count, Gram stain, culture, and crystal examination. Normally, there is less than 3.5 ml in an adult knee cavity, but it increases up to greater than 25 ml with inflammation. A normal synovial fluid does not clot but a fluid from a disease joint may contain fibrinogen and will clot. Thus, fluid is collected through a syringe moistened with heparin. When sufficient fluid is collected, it should be distributed into the following tubes based on the required tests:

A sterile heparinized tube for Gram stain and culture A heparin or EDTA tube for cell count A nonanticoagulated tube for other tests A sodium fluoride tube for glucose analysis

Powdered anticoagulants should not be used because it may produce artifacts. The nonanticoagulant tube for other tests must be centrifuged and separated to prevent cellular elements from interfering with chemical and serologic analyses. Ideally, test should be performed as soon as possible.

Figure 1 Synovial joint

Table 1. Classification of Synovial Fluid based on Laboratory ExaminationTest Normal Noninflamatory Inflammato

rySeptic Hemorrha

gicVolume

(ml)<3.5 >3.5 >3.5 >3.5 >3.5

Color Pale yellow

Yellow Yellow-white Yellow-green

Red-brown

Viscosity High High Low Low DecreasedLeukocyte

count (cells/μL)

<200 <3000 3000 to 50,000

>50,000 <10,000

Neutrophils

<25% <25% >50% >75% >25%

Glucose concentrat

ion

Approx. equal to that of plasma

Approx. equal to that of plasma

< that of plasma

< that of plasma

Approx. equal that of plasma

Glucose: P-SF*

difference

<10 mg/dL

< 25 mg/dL > 25 mg/dL > 40 mg/dL

< 25 mg/dL

Culture Negative Negative Negative Positive NegativePathologic

al significanc

e

Degenerative joint disorder

OsteoarthritisOsteochondritisOsteochondrmat

osisTraumatic

arthritisNeuroarthropath

y

Immunologic disorder

Rheumatoid arthritis

Crystal synovitis (Gout, Psuedogout)

Lupus erythromatosus

SclerodermaPolymyositisAnkylosing

sponylitisRheumatic

feverLyme

arthritisReiter’s

disease

Microbial infection

Traumatic injury

TumorsHemophiliaJoint prosthesis

Anticoagulant

overdose

Normal synovial fluid appears colorless to pale yellow. It becomes deeper yellow in the presence of noninflammatory and inflammatory effusions, and may have tinge of green due to microbial infection. Hemorrhagic arthritis must be distinguished from blood from a traumatic aspiration. Uneven distribution of blood specimen is

seen from a traumatic aspiration. Presence of turbidity is associated in the presence of WBCs. Milky fluid is caused by crystals.

Viscosity comes from polymerization of the hyaluronic acid and is essential for proper join lubrication. It is measured though the Ropes , or mucin clot test. Acetic acid (2-5%) is added in the fluid. A normal synovial fluid I forms a solid clot surrounded by clear fluid. As polymerization decreases, clot becomes less firm. Mucin clot test is reported in terms of good (solid clot), fair (soft clot), low (friable clot) and poor (no clot). However, this test is not routinely performed.

Total leukocyte count is the most frequently performed test. RBC count is seldom requested. To prevent cellular degradation, counts must be performed as soon as possible. Very viscoud fluid may be pretreated by adding a pinch of hyalurodinase to 0.5 ml of fluid or 0.05% hyalurodinase in phosphate buffer per milliliter and incubate at 37°C for 5 minutes. Manual counting is done using Neubauer counting chamber. Clear fluid can be usually counted undiluted, but dilution is necessary if turbid or bloody. Traditional WBC diluting fluid cannot be used because it contains acetic acid. Normal saline can be used as diluents. If RBC is necessary to lyse, hypotonic saline (0.3%) or saline containing saponin is suitable. Methylene blue is added to stain WBC nuclei. WBC counts less than 200 cells/μL are considered normal. Severe infection may reach 100,000 cells/μL or higher.

Figure 2 Comparison between (a) a healthy normal synovial membrane and it l components from (b) a rheumatic arthritis becoming hyperplatic and the presence of inflammatory cell.

Differential count should be performed on cytocentrifuge preparations or on thinly smeared slides. Fluid should be incubated with hyaluronidase prior to slide

preparation. Mononuclear cells are the primary cells seen in the normal synovial fluid. In both normal and abnormal specimens, cells appear more vacuolated than they do on blood smear. Inceased number of normal cells, other than ablnormalities includes the presence of eosinophils, LE cells, Reiter cells, and RA cells (ragocytes). Lipid droplet may be present following crush injuries, hemosiderin granules are seen in pigmented villonodular synovitis.

Table 2. Cells and Inclusion in Seen in the Synovial FluidCell/Inclusion Description Significance

Neutrophil Polymorphonuclear leukocyte

Bacterial sepsisCrystal-induced inflammation

Lymphocyte Mononuclear leukocyte

Nenseptic inflammation

Macrophage (Monocyte)

Large mononuclear

leaukocyte (may be vacuolated)

NormalViral infection

Synovial lining cell

Similar to macrophage, but

may be multinucleated,

resembling a mesothelial cell

Normal

LE cell Neutrophil containing

characteristics ingested: “round

body”

Lupus erthromatosus

Reiter cell Vacuolated macrophage with

ingested neutrophils

Reiter syndrome

Nonspecific inflammation

Ragocyte (RA cell)

Neutrophil with dark cytoplasmic

granules containing immune

complexes

Rheumatoid arthritis

Immunologic inflammation

Cartillage cells

Large, multinucleated

cells

Osteoarthritis

Rice bodies Macroscopic: polished riceMicroscopic:

collagen & fibrin

Tuberculosis,Septic and

Rheumatoid arthritis

Microscopic

Macroscopic

Fat droplets Refractile intracellular & extracellular

globules

Traumatic injury

Chronic inflammation

(Sudan dye stain)

(Micrograph)

Hemosiderin Inclusions with clusters of

synovial cells

Pigmented villonodular synovitits

Crystals are important diagnostic test in microscopic examination for arthritis. This may result to an acute, painful inflammation and may become chronic. Metabolic disorder and decreased renal excretion cause crystal formation producing elevated blood levels of crystal chemicals, degeneration of cartilage and bone and injection of medication. Primarily, monosodium urate (uric acid) (MSU) is found in cases of gout and calcium pyrophosphate (CPPD) in pseudogout. Increased serum uric acid resulting from metabolim of purines, increased consumption of high purine content foods, alcohol, and fructose, chemotheraphy treatment of leukemias, and decreased renal excretion of uric acid are the most frequent causes of gout. Pseudogout is most often associated with degenerative arthritis, producing cartilage calcification and endocrine disorders that produce elevated serum calcium levels.

Other crystals that may be present are as follows: Hydroxyl-apatite (basic calcium phosphate) associated with calcified cartilage

degeneration; Cholesterol crystals associated with chronic inflammation; Corticosteroids following injections; and Calcium oxalate crystals in renal dialysis patients.

Table 3. Characteristics of Crystals

Crystal Shape

Compensated

Polarized Light

Significance

Monosodium urate

NeedlesNegative

birefringence

Gout

Calcium pryophosp

hate

Rhombic square,

rods

Positive birefringen

ce

Pseudogout

CholesterolNotched, rhombic plates

Negative birefringen

ce

Extracellular

Corticosterois

Plat, variable-shaped plates

Positive and

Negative birefringen

ce

Injection

Calcium oxalate

Envelopes

Negative birefringen

ce

Renal dialysis

Apatite (Ca phosphate)

Small particlesRequire electon

microscopy

No birefringen

ce

Osteoarthritis

Examination under slide preparation must be performed as soon as possible and examined unstained wet preparation (LPO and HPO). Wright stain is used for observing crystals, but this should not replace the wet preparation and the use of polarized and compensated polarized light for identification.

Chemical tests are clinically important in synovial fluid, an ultrafiltrate of plasma. Glucose concentration which is based on blood glucose level is preferably measured to a patient who fasted for 8 hours to allow equilibration. Glucose should not be more than 10 mg/dl lower than the blood value. To prevent falsely decreased values (glycolysis), specimen should be analyzed within 1 hour or preserved with sodium fluoride. Other tests that may be requested are total protein and uric acid determination.

Gram stain and culture are two of the most important test performed in synovial fluid in case of infection. Fastidious Haemophilus species and N. gonorrhoeae are the common microbes that infect the synovial fluid. On the other hand, serologic tests are performed to diagnose autoimmune disease rheumatoid arthritis and lupus erythromatosus. Arthritis is also a frequent complication of Lyme disease of Borrelia burdorferi in patient’s serum. The extent of inflammation can be

determined through measurement of the concentration of acute phase reactant (fibrinogen and CRP).