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Purpose:The detection of basic calcium phosphate (BCP) crystals in the synovial fluid (SF) of patients with osteoarthritis (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 inherent subjectivity of the technique. In this respect, we have been developing semi-quantitative, colorimetric assays for detecting calcium phosphate crystals in SF samples
Methods:Spiked samples were prepared by suspending synthetic calcium phosphate crystals (HA/CPPD) in simulated SF (3% hyaluronan in human serum) at concentrations normally found in vivo. SF samples were aspirated from the affected joints (mainly knees and shoulders) of 20 patients with OA and/or other arthropathies. SF samples (100 µl) were first treated with sodium hypochlorite and ultrasonication to digest the organic matrix. Samples were then centrifuged to pellet the crystals followed by a number of washing steps with water. The pellet was dissolved in 1N nitric acid to break down the crystals into their constituent ions. Calcium content was measured using an o-cresolpthalein complexone assay (1-10 mg/l range) and phosphate measured using a modified molybdate blue method (1-50 mg/l) on a Biotek Powerwave XS 96-well reader. Scanning electron microscopy (SEM) and energy dispersive x-ray (EDX) spectroscopy (Hitachi SN-3000) was used to confirm the presence/absence of crystals isolated within the pellet.
Results:Sample pre-treatment is necessary in order to reduce interference from other components in SF. Sodium hypochlorite was found suitable for this purpose since it degrades the organic material but does not break down HA and CPPD crystals due to their basic properties. SEM of spiked SF showed that HA/CPPD crystals were successfully isolated from the organic matrix with no signs of degradation. Calcium levels found in patient SF samples ranged from 40 - 200 (mg/l). Typically, high levels of calcium were consistent with the presence of crystals in the sample as found in SEM. Furthermore, we envisage that the relative ratio of calcium to phosphate may be indicative of the type of crystal present.
Conclusions:We present a simple, semi-quantitative method for detecting calcium phosphate crystals in SF. The main challenge is to correlate the high levels of calcium/phosphate with the presence of crystals and the proposed diagnosis. This aspect will require the analysis of a larger pool of samples and may provide a tool for improved diagnosis and characterisation of OA and other crystal related pathologies.
Methods:Spiked samples were prepared by suspending synthetic calcium phosphate crystals (HA/CPPD) in simulated SF (3% hyaluronan in human serum) at concentrations normally found in vivo. SF samples were aspirated from the affected joints (mainly knees and shoulders) of 20 patients with OA and/or other arthropathies. SF samples (100 µl) were first treated with sodium hypochlorite and ultrasonication to digest the organic matrix. Samples were then centrifuged to pellet the crystals followed by a number of washing steps with water. The pellet was dissolved in 1N nitric acid to break down the crystals into their constituent ions. Calcium content was measured using an o-cresolpthalein complexone assay (1-10 mg/l range) and phosphate measured using a modified molybdate blue method (1-50 mg/l) on a Biotek Powerwave XS 96-well reader. Scanning electron microscopy (SEM) and energy dispersive x-ray (EDX) spectroscopy (Hitachi SN-3000) was used to confirm the presence/absence of crystals isolated within the pellet.
Results:Sample pre-treatment is necessary in order to reduce interference from other components in SF. Sodium hypochlorite was found suitable for this purpose since it degrades the organic material but does not break down HA and CPPD crystals due to their basic properties. SEM of spiked SF showed that HA/CPPD crystals were successfully isolated from the organic matrix with no signs of degradation. Calcium levels found in patient SF samples ranged from 40 - 200 (mg/l). Typically, high levels of calcium were consistent with the presence of crystals in the sample as found in SEM. Furthermore, we envisage that the relative ratio of calcium to phosphate may be indicative of the type of crystal present.
Conclusions:We present a simple, semi-quantitative method for detecting calcium phosphate crystals in SF. The main challenge is to correlate the high levels of calcium/phosphate with the presence of crystals and the proposed diagnosis. This aspect will require the analysis of a larger pool of samples and may provide a tool for improved diagnosis and characterisation of OA and other crystal related pathologies.
G. McMahon, None; A. Hernandez-Santana, None; S. Donnelly, None; C.J. McCarthy, None; A. Yavorskyy, None; G.M. McCarthy, Bristol Myers Squibb, 5; Wyeth, 5; Abbott, 5.