InfoLink Issue 9


*Issue 9 2000*

SYNOVIAL FLUID ANALYSIS IN THE INVESTIGATION OF ARTHRITIS

Dr Rodger Laurent
Immuno-Rheumatology, PaLMS
tel: +61 2 9926 7507 or 9926 7319
e-mail: rlaurent@med.usyd.edu.au

Rosemary Gleeson
Immuno-Rheumatology, PaLMS
tel: +61 2 9926 7732
e-mail: rgleeson@doh.health.nsw.gov.au

Introduction

Synovial fluid analysis is an important test in the evaluation of a patient with arthritis. It can help determine whether the fluid is inflammatory or non-inflammatory and the type of arthritis. It is particularly of value in a patient who presents with arthritis in one joint which is often due to gout, pseudogout or septic arthritis.

1. Normal synovial fluid

Normal synovial fluid is a clear pale yellow or straw-coloured, viscous liquid which does not clot. It is a plasma dialysate which contains 0.2 - 0.5% of hyaluronan. The high viscosity is due to the hyaluronan which polymerises to form large molecular weight complexes. Hyaluronan is synthesised by the type B synoviocyte, a cell with similar properties to fibroblasts. Hyaluronan holds water and provides a liquid cushion for the cartilage, acts as a transport system for nutrients to the chondrocytes and is a stable film of lubricant. There are proteins within the synovial fluid but the large molecular weight proteins are excluded from the synovial fluid. Synovial fluid is relatively acellular and has a white cell count of usually less than 100 x 106/L. These cells are about 70% monocytes and 30% lymphocytes.

2. Synovial Fluid Analysis

There are several components to synovial fluid analysis; these include viscosity, clarity, white cell count, crystals, rheumatoid factor and culture. Changes in these are summarised in Table 2 (page 3). The volume of synovial fluid aspirated from the joint is not of any diagnostic value.

3. Viscosity

Viscosity is helpful in distinguishing between inflammatory and non-inflammatory synovial fluid. Synovial fluid in osteoarthritis is non-inflammatory and the viscosity is usually normal or slightly reduced. Rheumatoid arthritis or similar inflammatory arthritides have a low viscosity synovial fluid. The low viscosity is due to reduced production of hyaluronan as well as a reduction in its polymerisation, with the resulting hyaluronan being of low molecular weight. The viscosity can be determined at the bedside. Normal synovial fluid has a positive string test where the fluid when dripped from the syringe forms a string of greater than 10 - 15 cm. Inflammatory synovial fluid drips like water, forming small drops.

4. Colour and Clarity

Inflammatory synovial fluid develops a yellow colour. In active rheumatoid arthritis, the synovial fluid may have a green tinge. The clarity relates to the number of cells and particles in the synovial fluid.

5. White Cell Count

The total white cell count and differential provide important information. It is used to determine whether fluid is inflammatory or non-inflammatory. The accepted level to differentiate between inflammatory and non-inflammatory is 2,000 x 106/L. Within each disease group there is a wide range of values. Specific cells seen in synovial fluid are detailed in Table 1. One of the problems with measuring white cell counts in synovial fluid is that its viscosity causes clumping of the cells producing a falsely low total white cell count. This effect can be reduced by collecting the synovial fluid in EDTA which depolymerises hyaluronan. It does not modify the cells for routine staining for cell morphology but does affect them for functional studies.

Table 1. Specific Cells in Synovial Fluids

Ragocytes	Neutrophils or macrophages which contain distrinctive granules The granules are thought to consist of immune complexes and contain immunoglobulins, including anti-nuclear antibody, rheumatoid factor and fibrin Most commonly found in septic arthritis but can occur in rheumatoid arthritis, gout and psuedogout
Apoptotic neutrophils	Dead or dying cells which lack a nucleus Have several small, dense, dark purple nuclear fragments scattered within their cytoplasm
Cytophagocytic Mononuclear Cells	Large mononuclear cells which contain phagocytosed neutrophils The neutrophils are apoptotic and there is a relationship between the number of cytophagocytic mononuclear cells and the number of apoptotic neutrophils These are most commonly seen in reactive arthritis. They are also found in rheumatoid arthritis
Eosinophils	These are occasionally seen in synovial fluid Levels are increased if there has been: intra-articular haemorrhage arthrography with radio contrast medium parasitic infection or Lyme arthitis
Miscellaneous	Very rarely, malignant cells or lupus erythematosus cells may be seen in synovial fluid

Septic arthritis usually has a white cell count greater than 50,000 x 10^6 /L with more than 90% of the cells being neutrophils. There is no relationship between the degree of leukocytosis and the infecting organism. However, a septic arthritis in immunosupressed patients, may have a low synovial fluid white cell count.

Gout has a white cell count usually between a 2,500 - 25,000 x 10^6 /L. Greater than 90% of the cells are also neutrophils. High white cell counts usually occur in acute gout, but in gout that has been present for at least 3-4 days, the white cell counts are usually lower. About one quarter of people with gout have a synovial fluid white cell count within the non-inflammatory range. The white cell count is usually proportional to the concentration of crystals within the synovial fluid.

Pseudogout is similar to gout, but the average white cell count is usually lower, and once again, the cells are predominantly neutrophils. Acute pseudogout synovial fluid has higher white cell counts than chronic effusions. One third of patients with pseudogout, have white cell counts within the non-inflammatory range.

Rheumatoid arthritis has white cell counts between 2,500 and 25,000 x 10^6 /L . It is the opposite to gout and pseudogout where the chronic effusions are more likely to have higher white cell counts than acute effusions. The predominant cell is also a neutrophil. About 5% of patients with rheumatoid arthritis have a white cell count greater than 50,000 x 10^6 /L which causes concern about septic arthritis.

Viral arthritis synovial fluid white cell count and differential is variable. It can be in the inflammatory or non-inflammatory range. The predominant cell is usually a mononuclear cell either lymphocyte or monocyte. The cells in the more inflammatory fluids may be predominantly neutrophils.

Traumatic arthritis, the arthritis that occurs after injury or overuse is non-inflammatory with a lymphocyte predominance.

Collection
Synovial fluid should be collected into:

an EDTA tube for white cell count and differential (0.5 to 1 mL required)
a plain tube for crystal analysis and rheumatoid factor **
a sterile plain tube for Gram stain and culture **
maximum volume available should be sent for analysis
**The specimens should be sent to PaLMS as soon as possible.

6. Crystals

Numerous crystals are present in the synovial fluid. Pictures of crystals found in synovial fluid can be viewed in this article on the PaLMS intranet or internet sites. However only monosodium urate and calcium pyrophosphate dihydrate crystals are of diagnostic relevance. The crystals are identified by their shape and characterised using polarised light microscopy. Synovial fluid also contains other birefringent material that needs to be distinguished from crystals. The absence of crystals does not exclude them as the cause of the arthritis. They are more difficult to find very early or late in the arthritis.

6.1. Monosodium Urate Crystals:
These crystals are needle shaped, negatively birefringent and soluble in water. They occasionally arrange themselves radially to form spherules. To produce gout the crystals must have been phagocytosed by neutrophils. The presence of intracellular urate crystals in the synovial fluid is diagnostic of gout. Monosodium urate crystal may be present in synovial fluid of people who are hyperuricaemic who do not have gout. In this situation they are always extracellular. People who are hyperuricaemic with an arthritis may still have synovial fluid crystals within the joint but they will not be the cause of the arthritis if there are no intracellular crystals.

Fig. 1. Monosodium Urate Crystals
(Click on image for high resolution version)

6.2. Calcium Pyrophosphate Dihydrate Crystals (CPPD)
These are associated with pseudogout, which most frequently involves the wrists and knees. These crystals are various shapes but are usually rhomboid in shape and are positively birefringent. They must also be intracellular to be considered as the cause for the inflammatory arthritis. They may also be present in osteoarthritis without any associated acute inflammation.

Fig. 2.Calcium Pyrophosphate Dihydrate Crystals (CPPD)
(Click on image for high resolution version)

6.3. Hydroxyapatite Crystals
Hydroxyapatite crystals are found in osteoarthritis synovial fluid. They have been associated with a destructive arthropathy of the shoulders and knees called hydroxyapatite induced arthritis or Milwaukee arthritis. They cannot be detected using polarised light microscopy. Transmission electron microscopy is required to identify these crystals. Calcium hydroxyapatite crystals will stain with Alizarin red. Unfortunately, this occurs with all calcium salts including calcium pyrophosphate crystals, so it cannot distinguish between types of calcium crystals.

6.4. Other Crystals
Crystals of corticosteroids used to treat arthritis including Beclomethasone and Methyl-prednisolone can also be detected in the synovial fluid if the patient has had a recent intraarticular corticosteroid injection. Beclomethasone crystals are needle shaped, and can be confused with monosodium urate crystals. However, there is more variation in crystal shape and size. Calcium oxalate crystals can be present in synovial fluid, particularly in renal failure. Cholesterol plates (see Fig 3 to the left) can all be seen in synovial fluid, although they do not have any diagnostic or clinical relevance.

Fig. 3. Cholesterol Crystals
(Click on image for high resolution version)

Table 2. Synovial Fluid Changes in Arthritis

Types of Synovial Fluid
Synovial Fluid Characteristics	Non-inflammatory	Inflammatory, non-infective	Severe Inflammatory
Examples	Osteoarthritis	Rheumatoid arthritis, crystal arthritis	Septic arthritis
Viscosity	High	Low	Low
Colour	Light straw	Yellow-greenish	Cream, yellow-greenish
Clarity	Clear	Mild opaque, turbid	Opaque, turbid
Leucocyte count (x10^6 /L)	50-500	1,500 - 30,000	> 50,000
Polymorphs	Occasional	30 - 70%	> 95%
Stained smear features	Unremarkable	ragocytes, macrophages with ingested PMNs, crystals	Bacteria
Plain smear from spun deposit	Cartilage and fibrin debris	Fibrin debris, crystals	Fibrin debris
Rheumatoid factor	Negative	Positive in rheumatoid arthritis	Negative

7. Rheumatoid Factor

Rheumatoid factor can be detected in synovial fluid in rheumatoid arthritis patients who are seropositive for rheumatoid factor. It can be derived from the serum or produced by the synovium. It can be present in the synovial fluid before it is present in the blood and is useful if the diagnosis is uncertain.

8. Glucose

Normal synovial fluid glucose level is similar to serum glucose levels. Low levels of synovial fluid glucose may be found in septic arthritis or rheumatoid arthritis. However, the diagnosis can usually be made by other methods and this tests is now rarely required.

9. Microbiology

Culture and Gram stain of the synovial fluid is important if infection is suspected.

10. Bacterial Antigens

Detection of bacterial antigens in synovial fluid is a potentially useful test in reactive arthritis. Reactive arthritis is an arthritis that occurs about 10 to 14 days following an enteric or genital infection. The common infecting bacteria are Salmonella, Shigella, Yersinia or Chlamydia. It is not a septic arthritis because viable organisms cannot be cultured from the synovial fluid. However, bacterial antigens can be detected in synovial fluid cells using polyclonal antibacterial antibodies. Studies have shown that there is a group who develop arthritis in one or two joints without clinical evidence of an infection. They are considered to have a reactive arthritis with a sub-clinical infection. Some of these patients have bacterial antigens in the synovial fluid. Staining of synovial fluid cells for bacterial antigens will be a useful diagnostic test in this group.

Further Reading.

Freemont AJ and Denton J. Atlas of Synovial Fluid Cytopathology (Current Histopathology Vol 18). Publ: Kluwer Academic Publishers, 1991.

Shmerling RH. Synovial Fluid Analysis: A critical reappraisal. Rheumatic Disease Clinics of North America. 1994;20:503-512.

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