Synovial or diarthrodial joints are the most sophisticated in the body. Unlike fibrous or cartilaginous joints in which the bone ends are effectively tethered together with bands of motion-limiting fibrous tissue or cartilage, in synovial joints each bone end is covered by an independent layer of hyaline cartilage. The two cartilage-covered bone ends are separated by a narrow space containing the lubricant, synovial fluid. This arrangement allows two adjacent bones the freedom to move in multiple directions.

The downside of increased movement is decreased stability and as a consequence, all diarthrodial joints are inherently unstable. Stability comes from the joint capsule, ligaments and muscle tone. The capsule consists of dense fibrous tissue which, rather than joining the bone ends, forms a strong flexible sleeve that surrounds the joint and envelops the peripheral segments of the two bones. This creates a cavity inside the joint which, except for the cartilage, is completely lined by a specialised form of connective tissue called synovium. The synovium is covered by an incomplete layer of cells – the synoviocytes. There are two main types of synoviocyte: type A, derived from macrophages that phagocytose any debris that falls into the fluid from the joint lining, and type B, derived from synovial fibroblasts with a synthesising function.

Synovial fluid is a transudate of plasma supplemented with high-molecular-weight saccharide-rich molecules, notably hyaluronans, and a molecule called lubricin, produced by the type B synoviocytes.

Synovial fluid differs from all other body fluids in that the surfaces of synovium and cartilage (the tissues in immediate contact with the synovial fluid) are covered by an incomplete layer of cells. This means that there is no intact basement membrane, which in other tissues is a significant physical and chemical barrier to the movement of molecules and cells. It also means that the matrix of cartilage and synovium are in contact with the synovial fluid, allowing a relatively homogeneous chemical environment to develop within the joint. Because of this unusual arrangement it is perhaps better to regard the synovial fluid as a semi-liquid, avascular, hypocellular connective tissue rather than a true body fluid, such as may form in the pericardial, abdominal or pleural cavity.

Variations in the volume and composition of synovial fluid reflect pathological processing occurring within the joint. Because of the relationship between the tissues within the joint, chemically mediated events such as inflammation or enzyme-mediated degradation occurring within the synovium and cartilage are reflected in changes within the synovial fluid. These changes include the production of factors responsible for the accumulation of different cell types within the fluid, and it is this that explains why the complement of cells within the joint varies between diseases.

It is normal for samples to pass through a 4 step sequential analysis after receipt in the laboratory:

Because synovial fluids from inflamed joints have a tendency to clot, they should be received in the laboratory in anticoagulant. Choosing the most appropriate anticoagulant is problematic. Because one of the key elements of synovial fluid analysis is examination for crystals, crystalline anticoagulants have to be avoided, as do chelating anticoagulants, which destroy crystals by removing core structural metal ions such as Ca²⁺. We find lithium heparin to be the best anticoagulant.

It is not possible to fix synovial fluid and the specimen therefore represents fresh tissue, and should be treated as such in every case. Even with refrigeration the optimum cytological information can only be extracted if the sample is examined within 48 hours of aspiration, and preferably as soon as possible within the first 24 hours.

Upon arrival in the laboratory the synovial fluid should be examined macroscopically. Macroscopic analysis involves a subjective assessment of colour, clarity and viscosity.

A sample of synovial fluid, agitated to achieve the most uniform distribution of cells, is diluted to a known concentration with normal saline containing methyl violet as a supravital stain. A count of the number of nucleated cells is then performed, either ‘manually,’ using a haemocytometer chamber or by machine using an automated cell counting instrument such as a Coulter Counter.⁴ The number of cells is expressed per unit volume (usually /mm³). To convert this to cell counts per mL necessitates multiplying the cell count by 1000. Normal synovial fluid contains approximately 200 cells/mm³. In inflammatory joint disease the cell count exceeds 1000 cells/mm³ and in non-inflammatory arthropathies is usually less than 1000 cells/mm³. Cell counts in excess of 30 000 cells/mm³ are found predominantly in three clinical settings: rheumatoid arthritis, septic arthritis and reactive arthritis (a form of arthropathy associated with infection at an extra-articular site and caused by the presence of, and reaction to, epitopes of the organism, but not the whole organism, within the joint).

Synovial fluid often contains small particles that can be recognised with the naked eye. In making the ‘wet prep’ the specimen is agitated and a small aliquot containing as many of these particles as possible is aspirated into a glass pipette, then placed as a large drop onto a microscope slide. The drop is gently squeezed flat beneath a coverslip and viewed unstained with a conventional microscope. For optimal results the microscope condenser diaphragm should be nearly closed to produce diffused light in which the unstained cells and particles are more clearly seen. In addition to particulate matter including crystals and fragments of tissues from joint associated structures such as cartilage, meniscus and ligament, the preparation is examined for one type of cell, the ragocyte.

Several classes of crystalline material are found in joints.^5,6 They consist of the following:

Monosodium urate

These are typically needle-shaped, highly birefringent crystals usually 5–30 μm long (Fig. 30.1). They can be distinguished from other crystals in that they are negatively birefringent when viewed in polarised light with an interposed quarter wave plate. These crystals are diagnostic of gout. If found within the background of a high cell count fluid their presence usually signifies acute gout,⁶ but even if the cell count is low the diagnosis is beyond doubt.

Fig. 30.1 Unstained synovial fluid ‘wet prep’ viewed between crossed polarisers with an interference plate in the light path giving the magenta background. The upper picture is of highly birefringent needle-shaped urate crystals and the lower of less birefringent rhomboidal CPPD. Note that crystals with their long axis running top left to bottom right are yellow in the top image and blue in the lower. This allows crystals to be distinguished easily from one another.

Hydroxyapatite

Crystals of hydroxyapatite within synovial fluid indicate damage either to the calcified zone of cartilage or underlying subarticular bone. Loss of cartilage sufficient to expose these structures to the synovial fluid is seen in the non-inflammatory disorder osteoarthritis and in the erosive inflammatory arthropathies of which easily the most common is rheumatoid arthritis. Sometimes the crystals are too small and amorphous to be seen with the conventional light microscope but staining with Alizarin red stain produces a birefringent bright red product (calcium alizarate) which is easily visualised (Fig. 30.2).⁷ Some patients have an aggressive destructive arthropathy caused by the presence of hydroxyapatite crystal spheroids within the joint. This was first described in the shoulder⁸ (Milwaukee shoulder) in association with calcification in the tendons of the rotator cuff, but has since been described in most large joints.

Fig. 30.2 Hydroxyapatite crystalloids in a fibrin clot stained with Alizarin red.

Lipids

Lipids enter the synovial fluid from the blood in inflammatory joint disease, intra-articular haemorrhage and following trauma.⁹ Different lipids have different crystalline shapes varying from the notched plates of cholesterol to the spherical liquid crystals of cholesterol esters (Fig. 30.3).¹⁰

Fig. 30.3 Lipid crystals. Compare the large flat cholesterol plate with the smaller brighter Maltese cross of a lipid liquid crystal.

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