TOPIC: SYNOVIAL FLUID

Topic Outline:
1. Physiology and Composition of Synovial Fluid
2. Classification of Joint Disorders
3. Specimen Collection
4. Laboratory Testing
a. Macroscopic Evaluation
b. Microscopic Examination
c. Chemical Examination
d. Microbiology Examination
e. Serological Examination

PHYSIOLOGY

 Synovial fluid
 Synovium refers to the tissue lining synovial tendon sheaths, bursae, and
arthrodial joints, except for the articular surface.
 It is composed of one to three cell layers that form a discontinuous surface
overlying fatty, fibrous, or periosteal joint tissue
 Synovial = syn (like) + ovia (egg)
 It is referred to as "joint fluid" is a viscous liquid found in the joint cavities of the
movable joints (diarthroses).
 Refers to the tissue lining synovial tendon sheaths, bursae and diarthrodial joints
except articular surface.
 Functions
 Lubrication for the movable joints: diarthroses
 It is a lubricant and adhesive and provides nutrients for the avascular articular
cartilage.
 Lessens shock of joint compression
 Formation
 Synovial fluid is formed as an ultrafiltrate of plasma across the synovial
membrane
 The synovial membrane contains specialized cells called synoviocytes.
i. Synoviocytes secrete a mucopolysaccharide containing hyaluronic acid
and a small amount of protein (approximately one fourth of the plasma
concentration) into the fluid.
ii. The large hyaluronate molecules contribute the noticeable viscosity to the
synovial fluid.
Disorders
 Damage to the articular membranes produces pain and stiffness in the joints, collectively
referred to as arthritis
 Four classifications of disorders
I. Noninflammatory
 Degenerative joint disorders, osteoarthritis
 II. Inflammatory
 Immunologic disorders, rheumatoid arthritis, systemic lupus
erythematosus, scleroderma, polymyositis, ankylosing spondylitis,
rheumatic fever, Lyme arthritis
 Crystal-induced gout, pseudogout
III. Septic
 Microbial infection
IV. Hemorrhagic
 Traumatic injury, tumors, hemophilia, other coagulation disorders
 Anticoagulant overdose
Clinical Significance
 .Laboratory results of synovial fluid analysis can be used to determine the pathologic
origin of arthritis.
 The beneficial tests most frequently performed on synovial fluid are the white
blood cell (WBC) count, differential, Gram stain, culture, and crystal examination.

 Laboratory Findings in Joint Disorders

Group Classification Laboratory Findings

I. Noninflammatory Clear, yellow fluid
Good viscosity
WBCs <1000 L
Neutrophils <30%
Similar to blood glucose
II. Inflammatory
Cloudy, yellow fluid
Poor viscosity
WBCs 2,000 to 75,000 L
Immunologic Origin
Neutrophils >50%
Decreased glucose level
Possible autoantibodies present
 Cloudy or milky fluid
Low viscosity
WBCs up to 100,000 L
Crystal-induced origin
Neutrophils <70%
Decreased glucose level
Crystals present
III. Septic Cloudy, yellow-green fluid
Variable viscosity
WBCs 50,000 to 100,000 L
Neutrophils >75%
Decreased glucose level
Positive culture and Gram stain
IV. Hemorrhagic Cloudy, red fluid
Low viscosity
WBCs equal to blood
Neutrophils equal to blood
Normal glucose level

SPECIMEN COLLECTION AND HANDLING

 Collected by need aspiration called Arthrocentesis

 Normal amount of fluid in the adult knee cavity is less than 3.5 mL, but can increase to
greater than 25 mL with inflammation.

 Collect in sterile, disposable needles and plastic syringes 

 Tubes

 Sterile heparinized or sodium polyanethol sulfonate for gram stain and culture
 Liquid EDTA for hematology (Powdered anticoagulants should not be used)

 Heparinizes or nonanticoagulated tube for other tests

i. It must be centrifuged and separated to prevent cellular elements from

interfering with chemical and serologic analyses.

 Sodium fluoride for glucose

 All testing should be done as soon as possible to prevent cellular lysis and possible
changes in crystal

COLOR AND CLARITY

Appearance Indications
Clear and pale yellow (egg white) Normal
Deeper yellow Noninflammatory and inflammatory
Greenish tinge Bacterial infection
Red Hemorrhagic or traumatic tao
Turbidity White blood cells, synovial cell debris, fibrin
Milky Crystal induced

VISCOSITY

 Synovial fluid viscosity comes from polymerization of the hyaluronic acid

o Essential for the proper joints lubrication

 Arthritis decreases polymerization

 Methods to measure the synovial fluid viscosity

o Observing fluid’s ability to form a string from the tip of a syringe

 a test that easily can be done at the bedside

 A string measuring 4 to 6 cm is considered normal

o Ropes or Mucin clot Test

 a test that measured hyaluronate polymerization

 Add fluid to 2% to 5% acetic acid to form clot

 As the ability of the hyaluronate to polymerize decreases, the clot

becomes less firm, and the surrounding fluid increases in turbidity.

 Reported in terms:

 good (solid clot)

 fair (soft clot)

 low (friable clot)

 poor (no clot)

 Formation of a mucin clot after adding acetic acid can be used to identify
a questionable fluid as synovial fluid.
CELL COUNTS

 Total leukocyte count is the most frequently performed cell count on synovial fluid
  Counts should be performed as soon as possible, or the specimen should be
refrigerated

 Very viscous fluid may need to be pretreated by adding one drop of 0.05%
hyaluronidase in phosphate buffer per milliliter of fluid and incubating at 37°C for 5
minutes

 Do not use normal WBC diluting fluid; use saline/methylene blue

 Perform on a Neubauer counting chamber

 Counting Procedure

o Line a petri dish with moist paper and place the hemocytometer on two small
sticks to elevate it above the moist paper.

o Fill and count both sides of the hemocytometer for compatibility.

o Acceptable ranges are determined by the laboratory

 For counts less than 200 WBCs/uL, count all 9 large squares.

 For counts greater than 200 WBCs /uL in the above count, count the 4
corner squares.

 For counts greater than 200 WBCs /uL in the above count, count the 5
small squares used for a RBC count

 Automated cell counters can be used for synovial fluid counts

o Highly viscous fluid may block the apertures, and the presence of debris and
tissue cells may falsely elevate counts

 WBC counts less than 200 cells/uL are considered normal and may reach 100,000
cells/uL or higher in severe infections
DIFFERENTIAL COUNT

 Performed on cytocentrifuged preparations or on thinly smeared slides

 Fluid should be incubated with hyaluronidase prior to slide preparation

 Mononuclear cells, including monocytes, macrophages, and synovial tissue cells, are the
primary cells seen in normal synovial fluid.

 Neutrophils: <25%, increase in sepsis

 Lymphocytes: <15%, noninflammatory higher

  In both normal and abnormal specimens, cells may appear more vacuolated than they
do on a blood smear.

 Other cell abnormalities

o eosinophils

o LE cells

o Reiter cells (or neutrophages, vacuolated macrophages with ingested

neutrophils)

o RA cells (or ragocytes, neutrophils with small, dark cytoplasmic granules

consisting precipitated rheumatoid factor)

 Lipid droplets may be present after crush injuries

 Hemosiderin granules are seen in cases of pigmented villonodular synovitis

CRYSTAL IDENTIFICATION

 important diagnostic test in evaluating arthritis

 Crystal formation in a joint frequently results in an acute, painful inflammation or chronic

 Causes of crystal formation

o metabolic disorders

o decreased renal excretion that produce elevated blood levels of crystallizing

chemicals

o degeneration of cartilage and bone, and injection of medications, such as

corticosteroids, into a joint

 Types of crystals

o Primary crystal seen in synovial fluid

 Monosodium urate (uric acid) (MSU) – gout

 Increased serum uric acid resulting from impaired metabolism of

purine
  increased consumption of high-purine-content foods, alcohol, and
fructose

 chemotherapy treatment of leukemias

 decreased renal excretion of uric acid

 Calcium pyrophosphate dihydrate (CPPD) – Pseudogout

 degenerative arthritis, producing cartilage calcification and

endocrine disorders that produce elevated serum calcium levels

o Hydroxyapatite (basic calcium phosphate)

 calcified cartilage degeneration

 needle shape

o Cholesterol crystals

 chronic effusions from patients with osteoarthritis or RA

 Notched, rhomboid plates shape

o Calcium oxalate crystals

 seen in renal dialysis patients

 envelopes shaped

o Corticosteroid

 after injection

 Flat, variable-shaped plates

o Apatite crystals

 calcific periarthritis

 osteoarthritis

 inflammatory arthritis

 Artifacts present may include talcum powder and starch from gloves, precipitated
anticoagulants, dust, and scratches on slides and cover slips.

 Slides and cover slips should be examined and if necessary cleaned again before us
SLIDE PREPARATION

 Examine ASAP

o Crystal changes, MSU and CPPD are seen intracellularly and cells disintegrate

 Initial examination is wet preparation unstained under low and high power

 Crystal may be seen differential

 Crystals may be observed in Wright’s-stained smears

 Use of polarized and red-compensated polarized light for identification

 MSU crystals

o needle-shaped crystals.

o They may be extracellular or located within the cytoplasm of neutrophils.

o They are frequently seen sticking through the cytoplasm of the cell.

 CPPD crystals

o rhomboid-shaped or square but may appear as short rods.

o usually located within vacuoles of the neutrophil

CRYSTAL POLARIZATION

 Both MSU and CPPD crystals polarize light

 MSU is highlu birefringent and appears brighter than CPPD

 Confirm identification using compensated polarized light

 Compensated Polarized Light

o a red compensator is placed in the microscope between the crystal and the
analyzer

o The compensator separates the light ray into slow moving and fast-moving
vibrations and produces a red background

o MSU crystals
  run parallel to the long axis of the crystal

 when aligned with the slow vibration, the velocity of the slow light passing
through the crystal is not impeded as much as the fast light, which runs
against the grain

 produces a yellow color.

 NEGATIVE BIREFRINGENCE

o CPPD crystals

 run perpendicular to the long axis of the crystal

 when aligned with the slow axis of the compensator, the velocity of the
fast light passing through the crystal is much quicker

 producing a blue color

 POSITIVE BIREFRINGENCE

CHEMISTRY TEST

 Synovial fluid is chemically an ultrafiltrate of plasma, test values are approximately the
same as serum values

 Glucose determination

o most frequently requested test

o markedly decreased glucose values indicate inflammatory (group II) or septic

(group III) disorders

o normal synovial fluid glucose values are based on the blood glucose level,
simultaneous blood

o synovial fluid samples should be obtained, preferably after the patient has fasted
for 8 hours to allow equilibration between the two fluids

o Normal: not less than 10 mg/dL of plasma glucose

 Total protein determination

o large protein molecules are not filtered through the synovial membranes

o Normal synovial fluid contains less than 3 g/dL protein (approximately one third
of the serum value).
 o Increased levels are found in inflammatory and hemorrhagic disorders; however,
synovial fluid protein measurement does not contribute greatly to the
classification of these disorders

 Uric acid determination

o elevation of serum uric acid in cases of gout is well known

o elevated synovial fluid uric acid level may be used to confirm the diagnosis when
the presence of crystals cannot be demonstrated in the fluid.

o Serum uric acid is often measured as a first evaluation in suspected cases of

gout

 Fluid lactate or acid phosphatase levels maybe requested to monitor the severity and
prognosis of rheumatoid arthritis (RA)
MICROBIOLOGIC TEST
 Gram stains and cultures
o most important tests performed on synovial fluid
o Both tests must be performed on all specimens, as organisms are often missed
on Gram stain
 Infectious organisms
o Bacteria
o Fungi
o Mycobacteria
o Viruses
 Route of entry
o Bloodstream
o Penetrating wounds
o Osteomyelitis rupture
o Arthroscopy
o intra-articular steroid injections
o prosthetic joint surgery

SEROLOGIC TEST
 important in the diagnosis of joint disorders
 most of these tests are performed on serum
 Synovial fluid analysis serves as a confirmatory measure in cases that are difficult to
diagnose
 Demonstrating antibodies to the causative agent Borrelia burgdorferi in the patient’s
serum can confirm the cause of the arthritis.

Sources
Stransinger, S. & Di Lorenzo, M., Urinalysis and Body Fluids, Sixth Edition
Mundt, L. & Shanahan K., Graff’s Textbook of Urinalysis and Body Fluids, Second Edition