Determination of calcium in synovial fluid samples as an aid to diagnosing osteoarthritis

Microscopic inorganic crystals are commonly observed in the synovial fluid of patients suffering from arthritic diseases. Basic calcium phosphate (BCP) crystals are known to occur quite commonly in the joint fluid of osteoarthritis (OA) patients and are insoluble at physiological pH. Current analysis of patient synovial fluid depends on light microscopy and staining with Alizarin Red-S. Both methods cannot identify crystals < 1µm in size and are highly subjective. This article investigates the use of o-cresolphthalein complexone (OCP), a colorimetric reagent, to quantify calcium from crystals isolated from synovial fluid samples as a means of identifying the presence of BCP and, hence, improving the diagnosis of OA.

Inorganic crystals were isolated following degradation of the biological sample matrix with hyaluronidase. 1-M HNO(3) was used for crystal dissociation into ions and the colorimetric response of OCP to calcium was measured in a basic environment of 2-amino-2-methyl-1-propanol. The average calcium content in OA patient samples was up to 40% higher than in rheumatoid arthritis (RA) patient samples. RA samples were used as a comparison, because they are generally accepted to be crystal free. Within the OA group, higher levels of calcium were detected in three out of 12 synovial fluid samples, which correlated with a significantly greater number of BCP crystals detected during microscopic examination.

A simple method based on colorimetry for measurement of calcium content and semiquantification of BCP crystals in synovial fluid samples has been described. Sample pretreatment following addition of hyaluronidase proved to be effective in reducing viscosity and aiding the dissociation of BCP crystals in synovial fluid samples.