Synovial fluid analysis procedure

INTENDED USE:  used for diagnosis of acute painful joint, Gout, pseudogout, and traumatic conditions like meniscal /ligament injury.: Synovial fluid aspiration analysis is a low-cost diagnostic test useful in the diagnosis and prognosis of potentially serious joint pathology like differentiating inflammatory arthritis from septic arthritis, or malignancy.

Sample collection:

synovial fluid is collected by arthrocentesis, an aspiration of the joint using a syringe, moistened with an anticoagulant usually sodium heparin. ideally, the sample should be separated into 3 parts sterile tube for microbial analysis, an anticoagulant tube with sodium heparin or liquid EDTA for microscopic examination, and in a plain tube for chemical analysis

kindly note: oxalate, powdered EDTA, and lithium heparin should not be used as they produce crystalline structures similar to monosodium urates (MSU).

synovial fluid should be tested within one hour of collection to maintain the integrity of laboratory results. Glycolysis will falsely decrease glucose levels if not tested within that amount of time or preserved with sodium fluoride

Physical examination:

The normal appearance of a sample of synovial fluid is usually straw-colored, clear & moderately viscous – drops of it from a syringe needle will form a “string” a few inches long.

color: Normal synovial fluid is colorless to pale yellow .colour should be evaluated against a white background in a clear tube. septic arthritis may be yellow, brown, or green depending upon the offending organism. Noninflammatory and inflammatory produce xanthochromic  fluid with a cloudy appearance, milky colored synovial fluid can be seen in Gout

Changes in the physical characteristics may provide clues to the disease

present, such as:

1) Less viscous fluid may be seen with inflammation.

2) Cloudy synovial fluid may indicate the presence of microorganisms,

white blood cells, or crystals.

3) Reddish synovial fluid may indicate the presence of blood, but an

increased number of red blood cells may also be present in cloudy

synovial fluid.

clarity: Normal SF is transparent. leucocytes are most commonly responsible for changes in clarity. Shiny appearance can be due to cholesterol crystals.

Free-floating Rice bodies: rice bodies are fragments of degenerating proliferative synovial cells or microinfarcts synovium Rice bodies are mainly formed of fibrin and are seen about joints, bursa, or tendon sheaths in patients with rheumatoid arthritis, tuberculosis, or tenosynovitis.

Chemical examination:

Tests that may be performed on synovial fluid samples may include:

1) Glucose-typically a bit lower than blood glucose levels; maybe

significantly lower with joint inflammation and infection.

2) Protein increased with a bacterial infection. viscous fluid can be pretreated  with the enzyme hyaluronidase

3) Lactate dehydrogenase-increased LD (LDH) level may be seen in rheumatoid arthritis, infectious arthritis, or gout.

4) Uric acid increased with gout.

 Microscopic examination:

Includes the leukocyte count and red blood cell count of the whole (uncentrifuged) specimen. In addition; the sediment of the centrifuged specimen is taken for the study of the stained smear, for the direct examination of abnormal cells and differential count.

  1. Total Cell Count: Increased WBCs may be seen with infections and with conditions such as gout and rheumatoid arthritis.

Procedure for Estimation of Total Leukocyte Count:

  1. RBC: Red blood cell counts on the synovial fluid are of little value. A visual exam and a hematocrit (if necessary) are sufficient ways to identify a hemorrhagic fluid.
  2. WBCs: Increased WBCs may be seen with infections, autoimmune conditions, and malignancy

Procedure for Estimation of Total Leukocyte Count:

1) Gently mix the specimen. Clear fluids are usually loaded on the hemocytometer undiluted. for undiluted specimens, if u see fewer than 10 cells count all 9 squares .for 10-100 cells Count 4 squares. 1 square is equal to 1 millimeter in Neubauers hemocytometer.

2)cloudy fluid should be assessed by taking a drop of fluid on the slide covering it with a coverslip and observing the cellularity to determine the dilution required.

3)for example for 1:20 dilution  Add 20 µl of diluting fluid in the labeled tubes. To the tubes add 380 µl of well-mixed specimen.

3) Mix well and charge both sides of two Neubauer chambers.

4) Let it stand for 5 minutes and then observe under high power objective.

5)Count the number of WBCs in four  corner squares (take the mean of both sides)

WBC/µL (microlitre)= number of cell counted x dilution/ number of  square counted  x0.1

WBC/µL= (Number of cells counted/4) x 10

6) Each chamber is observed by two different pathologists/technician

7)Acetic acid should not be used for lysing RBC in synovial fluid because of proteins such as hyaluronic acid will precipitate.

Procedure for Estimation of Differential Leukocyte Count:

1)Using cytocentrifuge is preferred for both concentrating cells in body fluid samples and maintaining cellular morphology.

2)If cytocentrifuge is not available – manually Centrifuge the specimen at 2000 rpm for 10 minutes. Tap the sediment to mix the last drop of fluid in the centrifuge tube.

3) Make a smear on a clean slide and leave to dry (make a thin smear in case of adequate sediment and a thick smear spread in the centre of the slide in case of scanty deposit).

5) Stain the slide with Wright or Leishman stain and observe under oil immersion

6)Adding a drop of 11%albumin  to  synovial  fluid sample being cytocentrifuged or manually centrifuged  aids in preserving the cellular morphology.

Synovial lining cells(synoviocytes)
LE cell present in patients with lupus arthritis,also in Rheumatoid arthritis
Measure Normal Non-inflammatory Inflammatory Septic arthritis hemorrhagic
Volume, ml <3.5 Often>3.5 Often>3.5 Often>3.5 Usually >3.5
clarity Transparent Transparent Translucent to opaque opaque opaque
colour Clear to pale yellow Yellow Yellow to white bloody white Red brown or xanthochromic
viscosity High High low variable variable
WBC /mm3 0-150 <3000 3000-75000 50,000-200000 5010,000
Polymorphs percentage <25% <30% >50% >90% <50%
culture Negative Negative Negative Often positive Negative

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