Development of Raman spectroscopy-integrated calcium oxalate crystallization index (rCOCI) test and its clinical validation for diagnosing calcium oxalate urolithiasis

Abstract

Urinary stone disease is mostly formed in the kidney and is highly recurrent. The most common type of urinary stones is calcium oxalate (CaOx). The development of urinary screening tests for estimating risk of CaOx stone formation is still challenging. Recently, we developed a urinary test, called indole-reacted calcium oxalate crystallization index (iCOCI) test, that provided a high diagnostic accuracy for CaOx urolithiasis. However, the iCOCI test is time-consuming and highly acidic chemicals are required for quantifying amount of CaOx. In this study, we proposed to develop a new method, called Raman spectroscopy-integrated calcium oxalate crystallization index (rCOCI) test, that used Raman spectroscopy to quantify amount of CaOx produced in the supersaturated urine instead of the indole reaction. Urinary rCOCI value calculated from the Raman peak intensity was the best value for CaOx quantitation. Method validation of the rCOCI test was conducted, and analytical validation parameters including specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), repeatability, and reproducibility were reported. The rCOCI method was validated within the linear range of 50-100 mM. The LOD and LOQ of the test were 24 mM and 72 mM, respectively. The repeatability and reproducibility of the test were acceptable. For clinical validation, the rCOCI levels were determined in 24-h urine samples to evaluate the performance for distinguishing CaOx stone patients from non-stone forming (NSF) subjects. Urinary rCOCI levels in CaOx stone patients (n = 44) were significantly higher than NSF subjects (n = 98). Receiver operating characteristic (ROC) analysis of urinary rCOCI test for separating CaOx stone patients from NSF subjects revealed an area under the ROC curve of 0.8244 (95%CI: 0.7438-0.9051). At the cutoff of 1.40, the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and accuracy of urinary rCOCI test for diagnosing CaOx urolithiasis were 80.00%, 57.00%, 45.45%, 86.15%, 1.86, 0.36 and 64.00%, respectively. In conclusion, the urinary rCOCI test was successfully developed. Method validation parameters suggested that urinary rCOCI test was suitable for quantifying CaOx produced in supersaturated urine. Clinical accuracy testing demonstrated that urinary rCOCI test had a good performance for separating CaOx urolithiasis from NSF individuals. We believed that this innovative urinary rCOCI test could be used for screening CaOx urolithiasis, and it could provide a significant commercial impact in the future.