POS0375 characterization of  calcium pyrophosphate crystals in synovial fluid with Raman spectroscopy reveals presence of triclinic CPP only

Background:
Calcium pyrophosphate deposition disease (CPPD) is a prevalent form of arthritis primarily affecting older adults, characterized by the deposition of calcium pyrophosphate (CPP) crystals in joint tissues. To date, CPP crystals identified in synovial fluid have been classified into two main forms: monoclinic rods (m-CPP) and triclinic rhomboids (t-CPP). Additionally, two other phases of CPP crystals have been described—tetrahydrate forms (m-CPPTβ) and amorphous CPP (a-CPP)—which are believed to act as precursors to m- and t-CPP crystals under in vitro conditions [1]. Traditionally, polarized light microscopy (PLM) has been the primary diagnostic tool for CPPD. However, PLM is limited in its ability to differentiate crystal types based solely on morphology. Integrated Raman spectroscopy with Polarized Light Microscopy (iRPolM) has demonstrated enhanced diagnostic accuracy over traditional methods, successfully identifying CPP crystals in cases where PLM and other techniques failed [2].

Objectives:
We aimed to determine whether different phases of CPP crystals exist in clinical samples, based on their morphology observed under PLM and their chemical composition analyzed via Raman spectroscopy. Specifically, we sought to identify if variations in crystal morphology correlate with distinct CPP phases in vivo based on their Raman spectra.

Methods:
In this project 468 synovial fluid samples from different joints were collected (458 of them were analyzed, 97.8%) from patients across five specialized European centers: three in the Netherlands and two in France. The samples were examined using integrated Raman spectroscopy with polarized light microscopy (iRPolM), combining PLM for morphological analysis and Raman spectroscopy for spectra collection and cluster analysis. Each patient's sample allowed for the evaluation of up to 10–20 different objects of varying sizes and shapes analyzed per sample to capture a diverse range of crystal morphologies and structures. The resulting Raman spectra were compared to reference spectra previously reported for calcium pyrophosphate crystals (CPP). A patient was classified as positive for CPP crystals if at least one matching spectrum was identified in the sample. All measurements were conducted following a standardized protocol to ensure reproducibility and accuracy.

Results:
Among the 458 analyzed synovial fluid samples, 2118 crystal spectra were collected and 72 of the samples were positive for CPP crystals after matching the Raman spectra. All CPP-positive samples exhibited Raman spectra consistent with the triclinic calcium pyrophosphate (t-CPP) phase, identified by peaks at 361, 498, 756, 1050, 1081, 1117, and 1185 cm−1. No monoclinic (m-CPP), tetrahydrate (m-CPPTβ), or amorphous CPP (a-CPP) phases were detected, regardless of their shapes or sizes. Although crystals exhibited varying morphology (rod-like, cuboid, rhomboid), all spectra indicated a single crystal type, t-CPP. Other calcium-based crystals, such as basic calcium phosphate (BCP), calcium carbonate, and calcium oxalate, were identified in a subset of samples.

Conclusion:
Our study analyzed a large dataset, from a diverse cohort of patients, using a Raman spectroscopic analysis of CPP crystals to characterize their chemical composition. The results showed that triclinic calcium pyrophosphate (t-CPP) is the sole CPP phase identified with Raman spectroscopy in clinical samples from CPPD patients. Despite shape and size, all crystals shared the same Raman spectral signature, suggesting that crystal morphology does not always correspond to different chemical compositions. This raises important questions about the environmental and biological factors that favor the formation of t-CPP over other phases in vivo. While our results challenge previous assumptions about CPP crystal diversity and highlight Raman spectroscopy's diagnostic potential, confirmation with additional analytical methods is necessary before drawing definitive conclusions.