ML S MTA P1

L E S SO N 9 : SY NOV IAL F L U ID

SUMMARY • Damage to the articular membranes produces

I Introduction pain and stiffness in the joints, collectively
II Specimen Collection and Handling referred to as arthritis
A Color and Clarity • Laboratory results of synovial fluid analysis can be
B Viscosity used to determine the pathologic origin of
C Cell Counts arthritis
i Differential Count
• The beneficial tests most frequently performed on
ii Other Cell Abnormalities
G Neutrophils, LE, and Crystals in Synovial Fluid synovial fluid are:
H Crystals o White blood cell (WBC) count
I Chemistry Tests o Differential
J Microbiology Tests o Gram stain
K Serologic Tests o Culture
o Crystal examination

Table 1. Characteristics of Synovial Fluid

Volume <3.5mL
Color Pale yellow
Clarity Clear
Able to form a string 4-6 cm
Viscosity
long
Erythrocyte count <2000 cells/ul
Leukocyte count <200 cells/ul
Neutrophils <20% of the differential
Lymphocytes <15% of the differential
Monocytes and 65% of the differential
macrophages count
Crystals None present
<10% mg/dL lower than
Glucose
blood glucose
Lactate <250 mg/dL
Total protein <3 g/dL
Uric acid Equal to blood volume
Figure 1. Synovial joint
Table 2. Classification and Pathologic Significance of
Joint Disorders
Group Classification Pathologic Significance
INTRODUCTION Non-inflammatory Degenerative joint
disorders, osteoarthritis
• Synovial fluid - joint fluid (lubricant) Inflammatory Immunologic disorders,
• Viscous liquid found in the cavities of the movable rheumatoid arthritis,
joints (diarthroses) or synovial joints lupus erythematosus,
scleroderma,
• The bones in the synovial joints are lined with polymyositis,
smooth articular cartilage and separated by a anklylosing spondylitis,
cavity containing the synovial fluid rheumatic fever, and
• The synovial membrane contains specialized cells Lyme arthritis Crystal-
induced gout and
called synoviocytes pseudogout
• Importance: Septic Microbial infection
o Reduces friction between the bones during Hemorrhagic Traumatic injury, tumors,
joint movement hemophilia, other
coagulation disorders
o Provides lubrication in the joints
anticoagulant overdose
o Provides nutrients to the articular cartilage
o Lessens the shock of joint compression that
occurs during activities such as walking and
SPECIMEN COLLECTION AND HANDLING
jogging
• Synovial fluid is formed as an ultrafiltrate of • Synovial fluid is collected by needle aspiration
plasma across the synovial membrane called arthrocentesis
• The synoviocytes secrete a mucopolysaccharide • The amount of fluid present varies with the size of
containing hyaluronic acid and a small amount of the joint and the extent of fluid buildup in the joint
protein into the fluid
• The large hyaluronate molecules contribute the
noticeable viscosity to the synovial fluid
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Figure 2. Viscosity of synovial fluid
• Normal synovial fluid does not clot; however, fluid
from a diseased joint may contain fibrinogen and
will clot
• Therefore, fluid is often collected in a syringe that
has been moistened with heparin
• When sufficient fluid is collected, it should be
distributed into the following tubes based on the
required tests:
o A sterile heparinized tube for gram stain and
culture (microbiology)
o A heparin or ethylenediaminetetraacetic acid
(EDTA) tube for cell counts (hematology)
o A nonanticoagulated tube for other tests
(chemistry)
o A sodium fluoride tube (antiglycolytic agent)
for glucose analysis
• Powdered anticoagulants should NOT be used
because they may produce artifacts that interfere
with crystal analysis
• The non-anticoagulated tube for other tests must
be centrifuged and separated to prevent cellular
elements from interfering with chemical and
serologic analyses
• Ideally, all testing should be done as soon as
possible to prevent cellular lysis and possible
changes in crystals
COLOR AND CLARITY
• Normal synovial fluid appears colorless to pale
yellow
• The word “synovial” comes from the Latin word for
egg; normal viscous synovial fluid resembles egg
white
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• The color becomes a deeper yellow in the presence
of non-inflammatory and inflammatory effusions
and may have a greenish tinge with bacterial
infection (septic)
• As with cerebrospinal fluid, in synovial fluid the
presence of blood from a hemorrhagic arthritis
must be distinguished from blood from a
traumatic aspiration
• Differentiation between hemorrhagic arthritis and
traumatic aspiration by observing the uneven
distribution of blood in the specimens obtained
from a traumatic aspiration
• Turbidity is frequently associated with the
presence of WBCs; however, synovial cell debris
and fibrin also produce turbidity
• The fluid may appear milky when crystals are
present
VISCOSITY
• Viscosity of the synovial fluid comes from the
polymerization of the hyaluronic acid and is
essential for the proper lubrication of the joints
• Arthritis affects both the production of
hyaluronate and its ability to polymerize, thus
decreasing the viscosity of the fluid
• Several methods are available to measure the
viscosity of the fluid, the simplest being to
observe the ability of the fluid to form a string
(string test) from the tip of a syringe, and can be
done at the bedside; string that measures 4 to 6
cm is considered normal
• Measurement of the amount of hyaluronate
polymerization can be performed using ropes, or
mucin clot test
2
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o When added to a solution of 2% to 5% acetic OTHER CELL ABNORMALITIES

acid, normal synovial fluid forms a solid clot
surrounded by clear fluid; as the ability of the • Increased eosinophils – rheumatic fever, parasitic
hyaluronate to polymerize decreases, the clot infections, metastatic carcinoma, post radiation
becomes less firm and the surrounding fluid therapy or arthrography (infla)
increases in turbidity • LE cells – patients with lupus erythematosus
o The mucin clot test is reported in terms of (infla)
good (solid clot), fair (soft clot), low (friable • Reiter cells – macrophages with ingested
clot), and poor (no clot) neutrophils (septic)
• RA cells (ragocytes) – precipitated rheumatoid
CELL COUNTS factor appearing as cytoplasmic granules in
neutrophils (infla)
• The total leukocyte count is the most frequently
• Hemosiderin granules – due to hemorrhagic
performed cell count on synovial fluid
process or cases of pigmented villonodular
• Red blood cell (RBC) counts are seldom requested
synovitis (hemorrhagic)
• To prevent cellular disintegration, counts should
• Cartilaginous cells – observed in cases of
be performed as soon as possible or the specimen
osteoarthritis
should be refrigerated
• Rice bodies – found in septic and rheumatoid
• Very viscous fluid may need to be pretreated by
arthritis and Tuberculosis (septic & infla)
adding a pinch of hyaluronidase to 0.5 ml of fluid
• Fat droplets – indicate traumatic injury (hemo)
or one drop of 0.05% hyaluronidase in phosphate
buffer per milliliter of fluid and incubating at 37°c
for 5 minutes
• Manual counts on thoroughly mixed specimens
are done using the Neubauer counting chamber
• Clear fluids can usually be counted undiluted, but
dilutions are necessary when fluids are turbid or
bloody
• WBC diluting fluid cannot be used because it
contains acetic acid that causes the formation of
mucin clots
• Normal saline can be used as a diluent; If it is
necessary to lyse the RBCs, hypotonic saline
(0.3%) or saline that contains saponin is a suitable
diluent Figure 3. Neutrophils in synovial fluid
• Methylene blue is added to the normal saline
stains the WBC nuclei, permitting separation of
the RBCs and WBCs during counts performed on
mixed specimens
• WBC counts < 200 cells/uL are considered normal
and may reach 100,000 cells/uL or higher in severe
infections
• There is, however, considerable overlap of elevated
leukocyte counts between septic and
inflammatory forms of arthritis
• Pathogenicity of the infecting organisms also
produces varying results in septic arthritis, as
does antibiotic administration
Figure 4. LE cells in synovial fluid
DIFFERENTIAL COUNT
CRYSTALS
• Cytocentrifuge specimen and prepare typical
blood smear • Crystal formation may be due to:
o Metabolic disorders
• Normal:
o Decreased renal excretion
o 60% monocytes, macrophages
o Cartilage and bone degeneration
o Neutrophils: <20%
o Medicinal injection (ex: corticosteroids)
o Lymphocytes: <15%
• Fluid is examined using the wet preparation
• Increased neutrophils – possible septic condition
technique
• Increased lymphocytes – indicate nonspetic
o ASAP examination as pH and temperature
inflammation (inflammatory, noninflammatory,
affect observation
or hemorrhagic)
o Ideally examined prior to WBC disintegration

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Table 3. Cells and Inclusions Seen in Synovial Fluid

Cell/Inclusion Description Significance
Neutrophil Polymorphonuclear Bacterial sepsis
leukocyte Crystal induced inflammation
Lymphocyte Mononuclear Nonseptic inflammation
leukocyte
Macrophage (monocyte) Large mononuclear leukocyte, may Normal
be vacuolated Viral infections
Synovial lining cell Similar to macrophage, but may be Normal
multinucleated,
resembling a mesothelial cell
LE cell Neutrophil containing Lupus erythematous
characteristic ingested “round body”
Reiter cell Vacuolated macrophage with Reiter’s syndrome
ingested neutrophils Nonspecific inflammation
RA cell (ragocyte) Neutrophil with dark cytoplasmic Rheumatoid arthritis
granules containing immune Immunologic inflammation
complexes
Cartilage cells Large, multinucleated cells Osteoarthritis
Rice bodies Macroscopically resemble polished Tuberculosis, septic and
rice rheumatoid arthritis
Microscopically show collagen and
fibrin
Fat droplets Refractile intracellular and Traumatic injury
extracellular globules
Stain with Sudan dyes
Hemosiderin Inclusions within clusters of Pigmented villonodular synovitis
synovial cells

o Examine under both direct and compensated o Compensated polarized light – blue when
polarizing light parallel (yellow when perpendicular)
o May also be observed in Wright stain o Blunt rods or rhombic shapes
preparations • Cholesterol
• Under polarizing light (direct polarization) o Nonspecific indications
o Birefringent substances appear as bright ▪ Associated with chronic inflammation
objects on a black background o Exhibit negative birefringence (compensated
o Intensity varies between substances polarized light)
• Under compensated polarizing light o Usually seen extracellularly
o A red compensator plate is placed between o Polarized light – strongly birefringence
the crystal and slide
o Crystals aligned parallel to the compensator
appear yellow (negative birefringence)
o Crystals aligned perpendicular to the
compensator appear blue (positive
birefringence)
• Monosodium Urate Crystals (MSU)
o Indicate gouty arthritis due to:
▪ Increased serum uric acid
▪ Decreased renal excretion of uric acid
o Impaired metabolism of nucleic acid
o Exhibit negative birefringence
o Intracellular (acute stages) & extracellular
location
o Polarized light – strongly birefringent Figure 5. Acute gout (uric acid crystals)
o Compensated polarized light
▪ Yellow when parallel
▪ Blue when perpendicular
o Needle shaped
• Calcium Pyrophosphate (CCPD)
o Indicates pseudogout due to:
▪ Degenerative arthritis
▪ Endocrine disorders with increased
serum calcium
▪ Calcification of cartilage
o Exhibit positive birefringence
o Seen intracellularly and extracellularly
o Polarized light – weakly birefringent
Figure 6. Uric acid crystals)

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Table 4. Patholog Significance of Laboratory Findings in Synovial Fluid

Group Classification Pathologic Significance Laboratory Findings
Noninflammatory Degenerative joint disorders Clear, yellow fluid
Good viscosity
WBCs < 2000 ul
Neutrophils < 30%
Normal glucose (similar to blood
glucose)
Inflammatory Immunologic problems, including Immunologic origin:
rheumatoid arthritis and lupus Cloudy, yellow fluid
erythematosus Poor viscosity
WBCs 2000-5000 ul
Neutrophils > 50%
Decreased glucose level
Possible autoantibodies present

Crystal-induced origin:
Cloudy and milky fluid
Poor viscosity
WBCs up to 50,000 ul
Neutrophils < 90%
Decreased glucose level
Elevated uric acid level
Crystal present
Septic Microbial infection Cloudy, yellow-green fluid
Poor viscosity
WBCs 10,000-20,000 ul
Neutrophils > 90%
Decreased glucose level
Positive culture and Gram stain
Hemorrhagic Traumatic injury Cloudy, red fluid
Coagulation deficiencies Poor viscosity
WBCs < 5000 ul
Neutrophils > 50%
Normal glucose level
RBCs present

o Rhombic plates o Difference between blood and synovial

• Hydroxyapatite (HA) (Calcium phosphate) glucose values is evaluated
o Associated with calcific deposition ▪ Normal = < 10 mg/dl
conditions ▪ Inflammatory conditions = > 25mg/dl
o May produce an acute inflammatory reaction ▪ Sepsis = >40 mg/dl
o Intracellular ▪ Considered low if < ½ serum plasma
o Not birefringent glucose value
o Require an electron microscope to examine o Should be run within 1 hour of collection
o Small, needle-shaped o Draw in sodium fluoride – prevents glycolysis
• Corticosteroid • Total Protein
o Associated with intra-articular injections; NO o Not routinely performed
clinical significance ▪ Normal = < 1/3 of serum value (~3g/dl)
o Primarily intracellular o Large molecule, not easily filtered by
o Exhibit positive and negative birefringence membrane
o Can closely resemble MSU and CCPD o Increased protein
o Fat, variable shaped plates ▪ Changes in membrane permeability
• Calcium Oxalate ▪ Increased joint synthesis
o Following renal dialysis ▪ Indicates an inflammatory process
• Birefringent Artifacts: • Uric Acid
o Anticoagulant crystals (calcium oxalate, o Alone, not diagnostic
lithium heparin) ▪ May determine gout in conjunction with
o Starch granules plasma uric acid, esp. When crystals are
o Prosthesis fragments undetectable
o Collagen fibers o Normal = serum level
o Fibrin • Lactate
o Dust particle o May differentiate between inflammatory and
septic arthritis
CHEMISTRY TESTS o Septic arthritis = >250 mg/dl
o Gonococcal arthritis = normal to low levels
• Glucose o Production results from:
o Done simultaneously with blood sample ▪ Increased demand for energy
(prefer 8 hours fast) ▪ Tissue hypoxia
▪ Severe inflammatory conditions

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MICROBIOLOGY TESTS
• Gram Stain
o Performed on all specimens
o Most infections are bacterial:
▪ Staphylococcus
▪ Streptococcus
• S. pyogenes
• S. pneumoniae
▪ Hemophilus
▪ Neisseria gonorrhea
• Fungal, viral and tubercular agents may also be
observed
• Culture
o Routine culture
o Enrichment medium (chocolate agar)
o Specialty media depending on clinician
orders and indications

SEROLOGIC TESTS
• Autoantibody detection (same as found in serum)
o Rheumatoid arthritis (RA)
o Lupus erythematosus (LE)
• Antibody detection in patient’s serum
o Borrelia burgdorferi (bacteria)
▪ Causative agent of Lyme disease
▪ Cause of arthritis

REFERENCES

Urinalysis and Body Fluids ,6th Edition. Susan King

Strasinger and Marjorie Schaub Di Lorenzo

Fundamental of Urine and Body Fluid Analysis, 3rd

Edition. Nancy A. Brunzel

Notes from synchronous session by Mr. Edison D. Ramos,

RMT, MPH, MSMT

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