Detection of calcium phosphate crystals in the joint fluid of patients with osteoarthritis - analytical approaches and challenges

Abstract

Clinically, osteoarthritis (OA) is characterised by joint pain, stiffness after immobility, limitation of movement and, in many cases, the presence of basic calcium phosphate (BCP) crystals in the joint fluid. The detection of BCP crystals in the synovial fluid of patients with OA is fraught with challenges due to the submicroscopic size of BCP, the complex nature of the matrix in which they are found and the fact that other crystals can co-exist with them in cases of mixed pathology. Routine analysis of joint crystals still relies almost exclusively on the use of optical microscopy, which has limited applicability for BCP crystal identification due to limited resolution and the inherent subjectivity of the technique. The purpose of this Critical Review is to present an overview of some of the main analytical tools employed in the detection of BCP to date and the potential of emerging technologies such as atomic force microscopy (AFM) and Raman microspectroscopy for this purpose.

Fig. 1. A joint with severe osteoarthritis. In osteoarthritis, the cartilage becomes worn away. Spurs grow out from the edge of the bone, and synovial fluid increases. Altogether, the joint feels stiff and sore.

Fig. 2. A group of negatively birefringent monosodium urate (MSU) crystals aspirated from a tophus (magnification × 1400, original magnification × 400). Reproduced with permission from ref. . (Copyright 1992, Ciba-Geigy Corp.)

Fig. 3. Calcium pyrophosphate dihydrate (CPPD) crystals extracted from the synovial fluid of a patient with pseudogout viewed under polarised light microscopy. Note the fact that some of the particles do not appear to exhibit birefringence. Reproduced with permission from ref. . (Copyright 1999, BMJ Publishing Group Ltd.)

Fig. 4. Typical calcium oxalate (CO) crystals obtained from a synovial fluid effusion in a patient on long-term dialysis (magnification × 1800, original magnification × 400). Reproduced with permission from ref. . (Copyright 1992, Ciba-Geigy Corp.)

Fig. 5. A synovial fluid wet smear stained with Alizarin red S. The darker red deposits have the appearance of hydroxyapatitc accumulations. The specimen is from a patient with end-stage renal disease (magnification × 720, original magnification × 200). Reproduced with permission from ref. . (Copyright 1992, Ciba-Geigy Corp.)

Fig. 6. Micrographs of solid particles in the synovial fluids of normal (a), osteoarthritis (b), and traumatic synovitis (c) subjects. Reproduced with permission from ref. . (Copyright 2007, Elsevier Ltd.)

Fig. 7. AFM images of HA and CPPD crystals from a patient with HA-induced arthritis. (A) HA crystals clump to form pseudospherical microaggregates (100–150 nm in diameter). (B) CPPD crystals shown here were an unexpected finding in this specimen. Reproduced with permission from ref. . (Copyright 1995, Elsevier Ltd.)

Fig. 8. Infrared spectrum of hydroxyapatite. Reproduced with permission from ref. . (Copyright 2007, Elsevier Ltd.)

Fig. 9. Caption typical infrared spectra of synovial joint fluid. Reproduced with permission from ref. . (Copyright 2004, IEEE.)

Fig. 10. Infrared difference spectra generated by the subtraction of the class-average spectrum of non-arthritic synovial fluid films from the class-average spectra of synovial fluid films for (1) osteoarthritis (2) rheumatoid arthritis and (3) spondyloarthropathy. Reproduced with permission from ref. . (Copyright 1997, John Wiley & Sons, Inc.)

Fig. 11. IR spectra of osteoarthritis and rheumatoid arthritis synovial fluid samples. Both are transmission spectra in the range of 2500 to 2000 cm^–1. A reference spectrum of water was subtracted from each sample. Bands at 2362 and 2340 cm^–1 result from CO₂ vibrations. Reproduced with permission from ref. . (Copyright 2002, Elsevier Ltd.)

Fig. 12. Raman reference spectra of synthetic BCP, HA, CPPD and MSU crystals.

Fig. 13. Pamidronate-IRDye78.

Fig. 14. Capillary electropherograms of human synovial fluid: (a) normal sample; (b) osteoarthritis; (c) rheumatoid arthritis. A and B: unidentified substances; C: presumably hyaluronan, D: uric acid. Reproduced with permission from ref. . (Copyright 1994, Royal Society of Chemistry.)

Alexander Yavorskyy

Gillian McMahon