Dr. Tom Niessink (1,2), Florina Ionete (3,4), Dr. Martijn Gerritsen (5), Pr. Dr. Ea Hang Korng (6), Pr. Dr Pascal Richette (6), Pr. Dr. Tim Welting (7), Pr. Dr. Tristan Pascart (8), Dr. Matthijs Janssen (1), Pr. Dr. Cees Otto (2), Dr. Tim L Jansen (1)
Affiliation(s):
1. Department of Rheumatology, VieCuri Medical Centre, Venlo, Netherlands
2. Technical Medicine Centre, University of Twente, Enschede, Netherlands
3. Department of Rheumatology, Dr. Ion Cantacuzino Clinical Hospital, Bucharest Romania
4. Carol Davila University of Mericine and Pharmacy, Bucharest, Romania
5. Department of Rheumatology, Reade Medical Centre, Amsterdam, Netherlands
6. Department of Rheumatology, , Lariboisière hospital, AP-HP, Université Paris Cité, Paris, France
7. Experimental Orthopedics, MUMC, Maastricht, Netherlands
8. Department of rheumatology, Lille Catholic University, Lille, France
Objective: Due to their wide diversity in shape, size, and birefringence, the chemical composition of calcium pyrophosphate (CPP) crystals has intrigued rheumatologists since their first discovery in the 1960’s by Dr. Daniel McCarty. Most well-known is the distribution of CPP crystals per morphology; CPP can present as either rod-like or rhomboid-like structures. A total of four CPP crystal phases have been identified in vitro, including monoclinic CPP, triclinic CPP, monoclinic CPP tetrahydrate beta, and an amorphous variant. Whether these four phases are present in vivo, especially in the synovial fluid, remains unknown. This is of importance, as it was previously proven that CPP crystals of different chemical composition invoke different levels of inflammation (Campillo-Gimenez et al. Front. Immunol. 2018). In this study we used Raman spectroscopic analysis of CPP crystals to characterize their chemical composition.
Status: In this project 468 samples were collected and 458 (97.8%) of them analysed. Out of 5 sites, samples from 4 are fully analysed. 1 site (Lille, France) remains tbd: planned for February 2025.
Methodology: We collected 468 synovial fluid samples from patients with swollen joints in five clinical centra, three in the Netherlands (Amsterdam, Maastricht, and Venlo) and two in France (Paris and Lille). We analysed all samples with our integrated Raman polarized light microscope (Hybriscan Technology B.V.) and collected all data collected from CPP crystals. Hyperspectral data was pre-processed in MATLAB r2023b, which included baseline correction with Whittaker smoothing and min/max normalization. The final dataset was further analysed with Ramapp (Vanna et al. Anal. Chem. 2024), using k-means cluster analysis to cluster similar spectra in our dataset. K-means is highly sensitive to the subtle spectral differences expected between the different hypothetical types of CPP.
Findings: Out of 458 analysed samples, 72 were positive for CPP crystals. In total, 2118 spectra of crystals were collected. All identified CPP crystals were positive for peaks at 361, 498, 756, 1050, 1081, 1117, and 1185 cm-1 and were therefore identified as triclinic calcium pyrophosphate (tCPP). No other phases of CPP were identified, regardless of morphology. Other types of calcium- and sodium urate crystals were identified in the samples, including BCP, calcium carbonate (calcite, dolomite), and calcium oxalate (monohydrate and dihydrate).
Significance: Based on our results, only one phase of CPP crystal (tCPP) was identified in the synovial fluid. This was the conclusion of our analysis of a large dataset, from a diverse cohort of patients. This conclusion is contradicting earlier hypotheses of multiple phases of CPP crystals in ex vivo samples and is also contradicting earlier findings by Rosenthal et al. who identified only monoclinic CPP in five synovial fluids from the knee using synchrotron Fourier transform infrared spectroscopy. As we have based our analyses on optical localization of CPP, we can only detect crystals with a size > 300nm and very small crystals might have been missed. A head-to-head comparison between different spectroscopic techniques will be highly interesting and required for a definitive conclusion on the composition of CPP.