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Based on Clinical Microscopy of StrasingerMary Christelle G. AquitaniaDisclaimer: Images are not owned by the creator.
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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).