SynovianalysisKS Godkhindi, GH Tilve, KG Nair
Department of Medicine, Seth G. S. Medical College and K.E.M. Hospital, Parel, Bombay-400 012., India
Synovial fluid reflects the pathology of joint diseases. The presence of synovial fluid, was known from the 16th century. Examination of joint fluid did not attract attention, until 1953 when Ropes and Bauer, published their monograph on the subject, in which the diagnostic value of synovial fluid examination was stressed. , Examination of synovial fluid can be the only way in diagnosing conditions like septic arthritis, villonodular synovitis and crystal synovitis, and in other arthritides adds to the accuracy of diagnosis. With advances in immunological and biochemical techniques synovial fluid has become a ground for research in understanding the pathogenesis of various joint diseases.
Synovial fluid is dialysed from plasma and enriched in hyaluronate by the synovial membrane. Hyaluronate, a high molecular weight polymer, by the virtue of its long coiled structure, imparts the unique quality of viscosity, to the joint fluid. , Highly viscous synovial fluid film, subserves the function of lubrication of joints and nourishes the joint structures. During the inflammation, there is depolymerisation of hyaluronate and viscosity is reduced, exposing the joint surfaces to more friction and damage. The polymerisation can be increased by salicylate and indomethacin therapy.  The normal protein content of synovial fluid is one third that of plasma, with 6070%0 as albumin, and 6-7% as alpha 2 , globulin. This distribution prevails as long as synovial fluid is normal. During inflammation gamma globulins are raised and fibrinogen enters the fluid making it coagulable.
Pathogenetic mechanisms of joint disease
Inflammatory response to a disease may be an attempt to eliminate or destroy the causative agent which may be an organism, antigen antibody complex, or deposited crystal. Extension of such a response may damage the delicate structure of joints causing disability. Immune mechanisms causing damage to the joint are considered in two ways, one in which the antibodies are directed to the specific component of the body tissues and the other in which the antigen antibody complexes are trapped at the synovial membrane, initiating the inflammatory reaction. The former concept, that of autoimmunity, is based on the description of systemic lupus erythymatosus (SLE) phenomenon. It is known, that a factor in serum of patients with SLE reacts with the nucleoprotein (DNA) of the damaged leukocyte. The resulting complexes undergo phagocytosis resulting in L.E cells. Various antinuclear factors arc seen by immunofluorescent techniques it SLE, scleroderma and rheumatoid arthritis.  It was shown, that the tissue specific antibodies were found in the organ affected as well as in circulation. Cross reacting antibodies are found in rheumatic fever. Hepten induced sensitivity was described with an example of Sidormid purpura, where a drug altered the platelets, to induce anti-platelet antibodies causing purpura. Similar cause may b: present in hydralazine or procainamid induced SLE like picture, though these drugs may just be uncovering pre-existing SLE
Second mechanism in which a wide spread tissue damage occurs, is antigen antibody complex as a causative agent This is evidenced by such complexes seer at the site of damage as in glomerulonephritis and SLE. Rheumatoid factor: (RF) are found in high titres in sera of patients suffering from rheumatoid arthritis with severe vascular involvement and in neuropathy. The leukocyte inclusions, the low complement activity in synovial fluid and the rheumatic nodule which begins as vasculitis, suggest the combination of rheumatoid factor with immunoglobulin complexes as pathogenic. Such a combination is shown to generate inflammatory mediator kinins.
When antigen engages antibody, reaction with complement occurs, followed by sequential addition of other components resulting in potentiation of antibody activity. Polymorphs are attracted tc the site under the influence of chemotactic factors. This process leads to a release of proteolytic enzymes from lysosomes which are present in synovial cells, causing additional damage. Cartilagenous cells and macrophages contain lysosomal enzymes which disrupt the mast cell granules to aggravate the inflammation.  Kinins liberated during the process are potent vasodilators, leucotactic, and cause increased permeability; these are released when kallekrein enzymes act on kniniogen. 
Collection of the synovial fluid
The synovial fluid can be aspirated easily from the knee joint in presence of an effusion. Aspiration from other joints is usually difficult, and more so from normal joints. When carried out under strict aseptic precautions, the aspiration of the joints is safe. The knee joint aspiration is done as follows:
A small area of the skin between lateral condyle and patella is infiltrated with Xylocaine. Using wide bore needle, the anaesthetised skin is punctured and the needle is directed inwards under the patella gently into the cavity. Extreme care is taken to avoid the scoring of the joint surfaces. The fluid aspirated, is collected in three different test tubes. The first sample is collected in EDTA, second sample is used as such for microscopy, third one is used for bacteriological culture.
Examination of synovial fluid
1. Appearance of synovial fluid: Colour and clarity of the fluid are noted.
2. Fibrin clot test: Plain synovial fluid is observed for formation of clot, and the clotting may be graded 1 to 4 plus depending on the firmness.
3. Rope's test: The synovial fluid is centrifuged and supernatant (1 ml) is added to 4 ml. of distilled water, and 0.13 nil of 7 N acetic acid. A precipitate is formed at the junction of the acid and the fluid.
The clot floats normally. Firmness and thickness of clot are recorded.
4. Viscosity: Fluid from the syringe can be allowed to drip in the form of a string. Longer the string formed more is the viscosity. Viscosimeter is used for more accurate readings.
5. Microscopy: A drop of fluid mixed with EDTA, is put on a slide and a cover slip is laid on it. Doubly refractile crystals may be seen under light microscope, but polarised light microscopy will enhance the identification of crystals. When there is a difficulty in distinguishing crystals of urate and calcium pyrophosphate the pH of the fluid may be raised to 9 or the fluid may be incubated in uricase to dissolve the urate crystals leaving the calcium pyrophosphate crystals unaffected.
6. Cytology: Cellular structures are counted using white blood cell counting areas on a haemocytometer. The fluid is diluted to 1: 20, by using 0.85 per cent saline and 0.1 per cent methylene blue in distilled water. The differential count is done on a smear prepared of the sediment from synovial fluid. A relative percentage of polymorphonuclear cells to mononuclear cells is noted as an indication of synovial reaction.
Immunological, biochemical and other studies may be carried out on the same fluid in advanced laboratories.
Pathological changes in synovial fluid
An easy aspiration from a joint indicates some synovial reaction in that joint. A decrease in the contents of hyaluronate and albumin together can be noted on a poor flocculation formed on addition of acetic acid whereas normally a thick ropy clot is formed. The glucose levels are decreased in synovial fluid during inflammation in proportion to the number of leucocytes. Many enzymes like beta glucoronidase, pepsin, trypsin, amylase, lipase, peroxidase, transaminases, alkaline and acid phosphatases are found in the fluid. The levels of enzymes are in proportion to the number of white cells in the fluid 
Complement activity is decreased and kinins are increased in rheumatoid synovial fluid. Rheumatoid factors may be found in synovial fluid in the absence of the same in serum. The leucocytes, macrophages, and synovial cells are seen in the fluid during inflammation. Relative percentage of polymorphs to mononuclear cells is a reliable index of the type of synovial reaction. Doubly refractile crystals of sodium urate in gout and of sodium pyrophosphate in chondrocalcinosis are diagnostic. , Cholesterol crystal plates with notched corners are found in rheumatoid arthritis.  In degenerative joint diseases desquamated cartilagenous fragments are seen. Rheumatoid arthritis cell, which is a leukocyte with intracytoplasmic inclusions carrying gamma globulin determinants, is found in rheumatoid synovial fluids. 
Depending on the analysis of synovial fluid the joint diseases are grouped into non-inflammatory, inflammatory, septic and haemorrhagic types.  [Table 1].
A clear straw coloured, viscous fluid with a good clot on addition of acetic acid, and minimal increase in cell count upto 2000, indicates mild synovial reaction. Such a type of fluid is found in traumatic arthritis, osteoarthritis, rheumatic fever, and systemic lupus erythematosus.
A turbid, yellow coloured fluid with low viscosity, showing fibrin clot but poor clot on addition of acetic acid, and having a high cell count with predominent polymorphonuclear cells, indicates definite inflammation of the joints, as found in cases of rheumatoid arthritis, gout and pseudogout.
In septic arthritis, the synovial fluid is inflammatory with a very high cell count, most of the cells being polymorphs and the culture of such a fluid is usually positive.
The last group, where the synovial fluid is haemorrhagic, is found in cases of trauma, haemorrhagic diathesis, pigmented villonodular synovitis and neoplasms.
However the groups are overlapping e.g. an inflammatory type of fluid may be found in osteoarthritis where one has to consider the possibility of erosive forms of osteo-arthritis which produces inflammation, or the presence of pre-existing rheumatoid arthritis. , An increase in cell count may follow an intra-articular administration of drugs which may mimic sepsis or rheumatoid process. The bacteriological studies in such cases are mandatory, which may yield a growth of streptococci, staphylococci, pneumococci, or coliform bacteria.
Synovial fluid aspiration is a simple and safe procedure. Synovianalysis aids in diagnosis and prognosis as well. With the advanced laboratory techniques, synovial fluid examination has been a great help in understanding the complex pathogenesis of joint disorders.