A Quantitative Spectrophotometric Assay Matched With Environmental Scanning Electron Microscopy to Measure Calcium...

In the field of osteoarthritis (OA), the identification of reliable diagnostic and prognostic biomarkers in patients with hip lesions such as femoroacetabular impingement (FAI) could have an immeasurable value. Calcium crystal detection in synovial fluids (SFs) is one tool currently available to diagnose patients with rheumatologic disorders. Crystals, such as monosodium urate (MSU) and calcium pyrophosphate (CPP), are identified qualitatively by compensated polarized light, whereas basic calcium phosphate (BCP) crystals are visualized under conventional light microscopy by Alizarin red S (ARS) staining. Here, we present an efficient and straightforward protocol to quantify calcium crystals by spectrophotometric analysis in human osteoarthritic SFs after staining with ARS. The type and size of the different crystal species are confirmed by environmental scanning electron microscopy (ESEM). Key features • This protocol provides a quantitative assay to measure calcium crystals in human synovial fluids. • ARS is specific for hydroxyapatite, calcium phosphate, tetrasodium pyrophosphate, sodium phosphate, calcium chloride/pyrophosphate dihydrate, and oxalate crystals; does not show any combination with SFs components. • The standard curve for crystal quantification was prepared with a synthetic hydroxyapatite, which allows to prepare a series of stable and reproducible standards. • The analysis with ESEM determines the elemental composition of all visible particles without any pretreatment of the biological sample prior to observation.