Feasibility of a tetracycline-binding method for detecting synovial fluid basic calcium phosphate crystals

Abstract

Objective: Basic calcium phosphate (BCP) crystals are common components of osteoarthritis (OA) synovial fluid. Progress in understanding the role of these bioactive particles in clinical OA has been hampered by difficulties in their identification. Tetracyclines stain calcium phosphate mineral in bone. The aim of this study was to investigate whether tetracycline staining might be an additional or alternative method for identifying BCP crystals in synovial fluid.

Methods: A drop of oxytetracycline was mixed with a drop of fluid containing synthetic or native BCP, calcium pyrophosphate dihydrate (CPPD), or monosodium urate (MSU) crystals and placed on a microscope slide. Stained and unstained crystals were examined by light microscopy, with and without a portable broad-spectrum ultraviolet (UV) pen light. A small set of characterized synovial fluid samples were compared by staining with alizarin red S and oxytetracycline. Synthetic BCP crystals in synovial fluid were quantified fluorimetrically using oxytetracycline.

Results: After oxytetracycline staining, synthetic and native BCP crystals appeared as fluorescent amorphous aggregates under UV light. Oxytetracycline did not stain CPPD or MSU crystals or other particulates. Oxytetracycline staining had fewer false-positive test results than did alizarin red S staining and could provide estimates of the quantities of synthetic BCP crystals in synovial fluid.

Conclusion: With further validation, oxytetracycline staining may prove to be a useful adjunct or alternative to currently available methods for identifying BCP crystals in synovial fluid.

Figure 1. Apparatus for focusing the UV pen light on the microscope field

This is a photograph of the apparatus used to stabilize the UV pen light under the microscope stage.

Figure 2. BCP crystals stained with oxytetracycline under UV light

A Synthetic BCP crystals in water were incubated for 15 minutes at room temperature with a drop of oxytetracycline, and then placed on a microscope slide. B. Synthetic BCP crystals in porcine synovial fluid were incubated for 15 minutes at room temperature with a drop of oxytetracycline, and then placed on a microscope slide. C. One drop of unprocessed synovial fluid from a patient with Milwaukee Shoulder Syndrome was incubated with oxytetracycline for 15 minutes at room temperature, and then placed on a microscope slide. All samples were examined with a microscope under broad spectrum UV light and photographed. (Magnification 200 x)

Figure 3. Standard curve of synthetic BCP crystals in human synovial fluid stained with oxytetracycline

Crystal-free synovial fluid from a patient with osteoarthritis was spiked with 10, 20 or 50 μg/ml synthetic BCP crystals. One hundred μl of sample were added to a well of a 96 well black fluorimeter plate. Twenty-five μl of 5 mg/ml oxytetracycline were added to each sample. Samples were read in a Biotek® Synergy ™ HT plate reader with excitation at 450/50 nm and emission at 540/35 nm. Background fluorescence in the un-spiked fluid was subtracted from each value.