1. SYNOVIAL FLUID
CHAPTER 11

2. Learning Objectives
Upon completing this chapter, the reader will be able to
Describe the formation and function of synovial fluid.
Relate laboratory test results to the four common classifications of joint disorders.
State the five diagnostic tests most routinely performed on synovial fluid.
Determine the appropriate collection tubes for requested laboratory tests on synovial fluid.
Describe the appearance of synovial fluid in normal and abnormal states.
Discuss the normal and abnormal cellular composition of synovial fluid.

3. Learning Objectives (cont’d)
List and describe six crystals found in synovial fluid.
Explain the differentiation of monosodium urate and calcium pyrophosphate crystals using polarized and compensated polarized light.
State the clinical significance of glucose and lactate tests on synovial fluid.
List four genera of bacteria most frequently found in synovial fluid.
Describe the relationship of serologic serum testing to joint disorders.

4. Physiology Synovial fluid functions Formation
Lubrication for the movable joints: diarthroses
Nutrients for articular cartilage
Lessens shock of joint compression
Formation
Ultrafiltrate of plasma across synovial membrane
No high weight molecules
Synoviocytes in synovial membrane secrete hyaluronic acid that makes the fluid viscous (lubrication)

5. Anatomy

6. Disorders Four classifications of disorders (arthritis)
Noninflammatory: degenerative, osteoarthritis
Inflammatory: immunologic, lupus erythematosus (LE), rheumatoid arthritis (RA), Lyme disease
Crystal-induced, gout and pseudogout
Septic: microbial infection
Hemorrhagic: trauma, tumors, coagulation deficiencies

7. Classification and Pathologic Significance of Joint Disorders
Table 11–1 Synovial Fluid Reference Values2
Volume
<3.5 mL
Color
Colorless to pale yellow
Clarity
Clear
Viscosity
Able to form a string 4 to 6 cm long
Leukocyte count
<200 cells/µL
Neutrophils
<25% of the differential
Crystals
None present
Glucose:plasma difference
<10 mg/dL lower than the blood glucose level
Total protein
<3 g/dL

8. Specimen Collection & Handling
Needle aspiration called arthrocentesis
Normal knee fluid amount 3.5 mL
>25 mL if inflamed
Normal fluid does not clot; diseased fluid clots
Collect in
Sterile heparinized or SPS for microbiology
Liquid EDTA (no powdered) for hematology
Heparinized or nonanticoagulated for other tests
Centrifuge nonanticoagulated tube and separate
Sodium fluoride for glucose
Test ASAP to avoid cellular lysis and changes in crystals

9. Appearance Normal: clear and pale yellow (egg white)
Deeper yellow with noninflammatory and inflammatory; green tinge = infection
Hemorrhagic or traumatic tap = red
Traumatic tap: look for decreasing blood in tubes
Crystal induced: milky
Turbidity: white blood cells (WBCs) or cellular debris, fibrin

10. Viscosity Polymerization of hyaluronic acid
Essential for joint movement
Arthritis decreases polymerization
4 to 6 cm string from aspirating needle = OK
Ropes (mucin clot test)
Add fluid to 2% to 5% acetic acid to form clot
Rate, good: solid clot; clear fluid, fair: soft formed clot; low: friable clot; poor: no clot
Use acetic acid to identify synovial fluid

11. Cell Counts WBCs most common
Do not use normal WBC diluting fluid; use normal saline/methylene blue
May have to treat viscous fluid with hyaluronidase or 37°C incubation first
Perform on a Neubauer counting chamber
Automated instrument, may need more hyaluronidase
Normal: <200 WBCs/μL, septic may reach >100,000

12. Counting Procedure Line petri dish with moist filter paper.
Place hemocytometer on two small sticks above paper.
Fill both sides of the hemocytometer for compatibility.
For counts less than 200 WBCs/μL, count all nine large squares.
For counts greater than 200 WBCs/μL in the above count , count the four corner squares.
For counts greater than 200 WBCs/μL in the above count, count the five small squares used for a RBC count.2<<AU: What does superscript 2 refer to?>>

13. Differential Count Incubate with hyaluronidase, then cytocentrifuge
Normal cells: monocytes, macrophages, synovial tissue cells
Neutrophils: <25%, increase in sepsis
Lymphocytes: <15%, noninflammatory higher
All cells may appear more vacuolated

14. Differential Count (cont’d)
Other cells
LE cells
Eosinophils
Reiter cells/neutrophages: vacuolated macrophages with ingested neutrophils
Ragocytes (RA cells): neutrophils with small, dark granules containing RA factor (IgM)
Lipid droplets: crush injuries
Hemosiderin granules: pigmented villonodular synovitis

15. Crystal Identification
Important diagnostic test; frequently performed
Acute and chronic cases
Metabolic disorders and decreased renal function
Other causes: increased blood levels, degeneration of bone and cartilage, injection of corticosteroids

16. Types of Crystals
Primary: monosodium urate (MSU) in gout; calcium pyrophosphate dihydrate (CPPD) in pseudogout
MSU: impaired purine metabolism, high purine foods, leukemia chemotherapy, decreased renal excretion of uric acid
CPPD: degenerative arthritis, disorders causing elevated calcium levels

17. Types of Crystals (cont’d)
Hydroxyapatite: cartilage degeneration, only seen with electron microscopy
Cholesterol: systemic autoimmune diseases (LE, RA) appear similar to urine cholesterol crystals: notched corners
Corticosteroids: injections, flat, variable plates
Calcium oxalate: renal dialysis patients
Artifacts: starch, powdered anticoagulants, dust, scratches (clean slides, cover slips)

18. Slide Preparation Examine ASAP
Crystal changes, MSU and CPPD are seen intracellularly and cells disintegrate
Initial examination is wet preparation unstained under low and high power
Crystals may be seen on differential

19. Slide Examination
Continued examination is done under polarized and compensated polarized light of wet preparation
MSU crystals: needle shaped; seen intra- and extracellularly; may be seen sticking through cytoplasm
CPPD crystals: rhombic, square shaped, or short rods; often seen in vacuoles of neutrophils; MSU crystals lyse vacuole membranes

20. Crystal Polarization Both MSU and CPPD crystals polarize light
MSU is highly birefringent and appears brighter than CPPD
Confirm identification using compensated polarized light

21. Compensated Polarized Light
Red compensated polarized light: red compensator is placed between crystal and analyzer, producing a red background
Separates light into slow- and fast-moving vibrations
Align crystals with slow vibration
Linear structure of molecules causes different colors to be produced

22. MSU and CCPD Crystals Under Compensated Polarized Light

23. MSU and CPPD Under Compensated Light

24. MSU Under Compensated Light
MSU molecules run parallel to the long axis, aligned with slow vibration; fast light is impeded, producing a yellow color (negative birefringence)
CPPD molecules run perpendicular to long axis and impede the slow light producing a blue color (positive birefringence)

25. Chemistry Tests
As an ultrafiltrate of plasma, normal values are similar to those of plasma
Few are clinically important
Most frequent is glucose
Normal: not less than 10 mg/dL of plasma glucose
Draw sample same time as fluid is collected
Markedly decreased levels seen in inflammatory and septic categories

26. Chemistry Tests (cont’d)
Synovial fluid protein
Normal is less than 3 g/dL
Increased in inflammatory and hemorrhagic categories
Synovial fluid lactate and acid phosphatase
Severity and prognosis of rheumatoid arthritis (RA)

27. Reference Synovial Fluid Values
Volume: <3.5 mL
Color: colorless to pale yellow
Clarity: clear
Viscosity: able to form a string 4 to 6 cm long
Leukocyte count: <200 cells/μL
Neutrophils: <25% of the differential
Crystals: none present
Glucose: <10 mg/dL lower than the blood glucose plasma difference
Total protein: <3 g/dL

28. Microbiology Tests
Infections caused by inflammation, trauma, and systemic infections
Gram stain and cultures are routinely performed
Culture must include chocolate agar
Primary organisms are Staphylococcus, Streptococcus, Haemophilus influenzae, and Neisseria gonorrhoeae
Patient history determines fungal and Tuberculosis (TB) cultures

29. Serologic Tests
Majority of related tests are performed on serum and fluid may only serve as a confirmation
RA and LE are the most common autoimmune causes of arthritis
Lyme disease: arthritis is frequent complication; test serum for Borrelia burgdorferi antibodies
Extent of inflammation: test for C-reactive protein and fibrinogen