E.

MANAHAN
 Synovial Fluid Physiology
- Often referred as “joint fluid”

- A viscous fluid found in the cavities of the movable

joints (diarthroses) or synovial joints

- Contained in a cavity that separates the articular

cartilage which lines the bones in the synovial joints

- Smooth articular cartilage & synovial fluid reduce

friction between the bones during joint movement

E. MANAHAN
- Provides nutrients to the articular cartilage &
lessens the shock of compression occurring during
activities such as walking & jogging

- Formed as an ultrafiltrate of plasma across the

synovial membrane (nonselective filtration except
for high molecular weight proteins)

- Viscosity is caused by the secreted

mucopolysaccharide containing hyaluronic acid of
the synoviocytes

E. MANAHAN
 Normal Synovial Fluid Values
 Volume <3.5mL
 Color pale yellow
 Clarity clear
 Viscosity able to form a string 4-6cm long
 Erythrocyte count <2000 cells/µL
 Leukocyte count <200 cells/µL
 Neutrophils <20% of the differential
 Lymphocytes <15% of the differential
 Monocytes & macrophages 65% of the differential
 Crystals none present
 Glucose <10mg/dL lower than the blood glucose
 Lactate equal to the plasma level
 Total protein <3.0 g/dL
 Uric acid equal to blood volume

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- Lab results of synovial fluid analysis can be used to
determine pathologic origin of arthritis

 Arthritis – damage to the articular membranes that

produces pain & stiffness in the joints

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Disorders are classified into 4 groups:

I. Non inflammatory - Degenerative joint disorder

II. Inflammatory - Immunologic problems, including
rheumatoid arthritis & lupus
erythematosus
- Crystal induced gout & pseudogout
III. Septic - Microbial infection
IV. Hemorrhagic - Traumatic injury
-Coagulation deficiencies

 Overlapping of test results among the group may occur,

& the patient’s clinical history must also be considered
when assigning a category.
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Most frequently performed tests :
 WBC count
 Differential
 Gram stain
 Culture
 Polarized microscopy examination for crystals

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Specimen Collection & Handling

- Collected by needle aspiration (arthrocentesis)

- Amount of fluid present varies with joint size &
degree of fluid build up in the joint
- Volume of liquid collected should be recorded
- Normal synovial fluid does not clot; fluid from a
diseased joint may contain fibrinogen & will clot
- Fluid is usually collected in a syringe moistened with
heparin

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Specimen Collection & Handling
- Sufficient fluid is distributed into 3 tubes:
#1- sterile heparinized: microbiology lab
#2 – liquid EDTA tube: hematology lab
#3 – nonanticoagulated tube: other tests
- must be centrifuged & separated from to prevent
cellular elements from interfering with chemical &
serologic analyses

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Specimen Collection & Handling
Powdered coagulants should not be used because they
may produce artifacts that will interfere with crystal
analyses.
All testing should be done as soon as possible to
prevent cellular lysis & possible changes in crystals.

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Appearance & Viscosity
 Normal - clear & pale yellow
 Deeper yellow - presence of inflammation
 Greenish - bacterial infection
 Turbidity - presence of WBCs
-May also be due to synovial cell debris & fibrin
 Milky – presence of crystals

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Appearance & Viscosity
 Viscosity of the Synovial Fluid
- comes from the polymerization of the hyaluronic
acid & is essential for the proper lubrication of joints
- arthritis decreases viscosity
- simplest method to measure viscosity is the ability
of the fluid to form a string from the tip of a syringe.
Normal string measures 4 to 6cm.

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Appearance & Viscosity
 Ropes, or Mucin Clot Tests
- used to measure the degree of hyaluronate
polymerization

Mucin Clot Test:

Normal synovial fluid + 2-5% HOAc solid clot
surrounded by clear fluid

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Appearance & Viscosity
 Mucin clot test is reported as:
Good – solid clot was formed
Fair – soft clot was formed
Poor – friable clot was formed
Very poor – no clot was formed

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Cell Counts
 Total leukocyte count – most frequently performed cell
count on synovial fluid
 RBC count may also be requested unless evidence of
traumatic tap exists
 Should be performed ASAP to prevent cellular
disintegration or the specimen should be refrigerated

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Cell Counts
 Very viscous fluid may be pretreated by adding a pinch
amount of hyaluronidase to 0.5mL of fluid or one drop
of 0.05% hyaluronidase in PO4- buffer per mL of fluid
& incubate at 37°C for 5mins
 Manual count on thoroughly mixed specimens –
Neubauer counting chamber

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Cell Counts
 Clear fluids can be counted undiluted.
 Dilutions for bloody or turbid fluids.
 Traditional WBC diluting fluid cannot be used – acetic
acid will cause formation of mucin clot
 Normal saline can be used as diluent.
 To lyse RBCs use hypotonic saline (0.3%) or saline that
contains saponin as diluent.

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Cell Counts
 Methylene blue + normal saline: stain WBC nuclei
permitting separation of RBCs & WBCs on mixed
specimens
 Automated cell counters can be used
 Highly viscous fluid may block the apertures
 Presence of debris & tissue cells may falsely elevate
counts

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Cell Counts
 WBC count <200 cells/µL = normal
 WBC count may reach about 100,000 cells/µL or higher
in severe infections.

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Differential Count
 Cytocentrifuged preparations or thinly smeared slides
 Fluid should be incubated with hyaluronidase prior to
slide preparation
 Primary cells seen in normal synovial fluid:
 Monocytes
 Macrophages
 Synovial tissue cells

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Differential Count
 Neutrophils = <20% of the differential count
 Lymphocytes = <15%
 Increased neutrophils = septic condition
 Increases lymphocytes = nonseptic inflammation
 Normal & abnormal specimen – cells appear more
vacuolated than on a blood smear
 Lipid droplets may be present following crash injuries
 Hemosiderin granules in cases of pigmented
villonodular synovitis

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Differential Count
 Other Cell Abnormalities:
- eosinophils
- LE cells
- Reiter cells (vacuolated macrophages with ingested
neutrophils)
- RA cells or ragocytes (neutrophils with small, dark,
cytoplasmic granules that consist of precipitated
rheumatoid factor)

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Crystal Identification
- Important diagnostic test in evaluation of arthritis
- Crystal formation results in an acute, painful
inflammation
- Primary crystals seen:
- Monosodium urate (uric acid) (MSU) – gout
- Calcium pyrophosphate (CPPD) - pseudogout

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Crystal Identification
 Gout – often caused by increase serum uric acid
resulting from impaired metabolism of nucleic acid
associated with myeloproliferative disorders &
decreased renal secretion of uric acid
 Pseudogout – often associated with degenerative
arthritis, resulting in cartilage calcification &
endocrine disorders producing elevated serum
calcium levels

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Crystal Identification
 Additional crystals present:
- hydroxyapatite (basic calcium phosphate)
= calcified cartilage degeneration
- cholesterol crystals
- corticosteroids = following injections
- calcium oxalate crystals = renal dialysis patients

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Crystal Identification
 Artifacts may be:
 Talc & starch from gloves
 Precipitated anticoagulants
 Dust
 Scratches from slides & cover slips
 Crystal examination should be performed soon after
fluid collection to ensure crystals are not affected by
changes in temperature & pH.

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Crystal Identification
 Report MSU & CPPD crystals as being located
extracellularly & intracellularly (within neutrophils)
 Fluid is examined unstained under polarized &
compensated polarized light for detection of MSU &
CPPD crystals.
 Examine under LPO with direct polarized light, then
HPO, and finally under compensated polarized light.

E. MANAHAN
Crystal Identification
 MSU crystals – needle-shaped, appears extracellularly
& within the cytoplasm of neutrophils
- more highly birefringent & brighter against a dark
background
 CPPD crystals – rhombic-shaped, intracellular
inclusions

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Chemistry Test
 Glucose determination – most frequently requested
- blood & synovial fluid samples should be obtained
after 8hours of fasting to allow equilibration between
the two
- N.V. = not more than 10mg/dL lower than the blood
value
- specimens should be analyzed within 1hr or
preserved with sodium fluoride

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Chemistry Test
 Synovial fluid lactate
- provide rapid differentiation between
inflammatory & septic arthritis
- >250 mg/dL consistent with septic arthritis, but
may also be seen in rheumatoid arthritis
 Other Chemistry Tests:
- total protein determination: NV= <3mg/dL
- uric acid determination

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Microbiologic Test
 Gram stain & culture
- most important tests performed on synovial fluid
- Gram stain is performed on all specimens
- most frequent is bacterial infection but fungal,
tubercular & viral infections may also occur; if
suspected, special culture should be used.

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Microbiologic Test
 Most common organisms infecting synovial fluid:
 Hemophilus species
 Neisseria gonorrhoeae

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Serologic Test
- Diagnosis of joint disorders
- Rheumatoid arthritis & lupus erythromatosus are
diagnosed by demonstrating the presence of their
particular antibodies in the patient’s serum
- Demostration of antibodies to the causative agent
Borrelia burgdorgeri in the patient’s serum can confirm
the cause of the arthritis

E. MANAHAN