A proof of principle experiment into the added value of raman spectroscopy for synovial crystal research author(s)
Tom Niessink, Charline Kuipers, Brighton De Jong, Aufried Lenferink, Matthijs Janssen, Tim Jansen, Cees Otto Presenting Author Tom Niessink
University Of Twente, Enschede
Objective: Compensated polarized light microscopy (CPLM) is the standard method of identification in crystal induced arthritis, but this technique is known to be subjective and lacks the possibility to accurately identify novel synovial crystals. CPLM is highly sensitive in the detection of monosodium urate crystals, but lacks sensitivity/specificity for CPP associated arthritis and other more complex and rare forms of crystalline disease. Raman spectroscopy is a non-destructive chemical analytical tool which is approaching the field of rheumatology with high specificity and objectivity to identify crystals. In this study, we apply raman spectroscopy on a microscopic scale to study individual synovial crystals.
Method: We retrieved synovial fluids from 28 patients with suspected gout or CPPD and two oxalate kidney stones from viecuri medical center, venlo. From each patient, we located 5-10 crystals with polarized light microscopy which we then scanned with an integrated raman spectroscope. To be able to identify the spectra of measured crystals we also scanned synthetic crystals and retrieved spectra from mineralogical databases.
Results: We identified a total of 10 different types of crystals. These include the classical known crystals MSU, TCPP, hydroxyapatite, and com but also include titanium dioxide polymorphs anatase and rutile, calcium carbonate polymorphs calcite and aragonite, sodium sulphate crystal thenardite and calcium magnesium carbonate crystal dolomite. Additionally, we also measured spectra of deposited carotenoids and lipid spherules also known as maltese crosses. The raman spectra of these compounds are shown in figure 1, the distribution of all found crystals over the different patients is shown in table 1. We can see that multiple samples show a range of identified crystals, including samples having multiple ‘pathologically relevant’ crystals such as patient 12, wo presented MSU and hydroxyapatite and patient 13, who presented MSU, TCPP and hydroxyapatite.
Discussion: Raman spectroscopy shows to be a method which can accurately identify singular objects in synovial fluid and can add to our knowledge of synovial crystals. It was able to identify well-known crystals with a pivotal role in auto-inflammation, such as MSU, TCPP, plus additional hydroxyapatite (BCP) and com but also crystals where such a relation is not yet established, such as calcium carbonate and titanium dioxide. The question rises as to how these particles are accumulated in synovial fluids and what role they play in enhancing arthritis/synovitis. At least one patient who presented titanium particles also had a titanium alloy hip replacement five years prior to the swollen ankle which the studied synovial fluid was extracted from. We could not establish such a relation for other patients in this study. In the future we want to further explore our findings, increase the sample size and systematically analyze synovial fluids. We aim to establish a highly accurate and fully objective method to diagnose all forms of crystal-associated arthritis based on raman spectroscopy.