Urolithiasis is one of the most common urologic diseases; it has a high recurrence rate and results in substantial pain and an increased risk of renal failure.[1–3] The prevalence of urolithiasis is approximately 4–15% in North America, Asia, Europe, and Australia.[4] Urinary stones mainly contain of calcium oxalate dehydrate (COD), calcium oxalate monohydrate (COM), uric acid (UA), hydroxyapatite (HAP), dicalcium phosphate dehydrate (DCPD), struvite, and cystine. After identifying the urine stone type of their patients, clinicians could provide adequate medical treatment and dietary advice to prevent the recurrence of kidney stones.[5]
The formation of urine stones is a multistep process that includes nucleation, growth, aggregation, and retention.[4, 6] The urine stones are composed of urine crystals and other metabolites. The types of crystals in the urine have a high correlation (90.4%) with the composition of urine stones in urolithiasis patients.[7] In a previous study, we found the existence of urine crystals in the urine of over 80% of the urolithiasis patients.[8] Knowing the types of urine crystals maybe helpful in understanding the cause of urolithiasis and further preventing its recurrence.
Manual microscopic examination of urine sediment is the gold standard in clinics for analyzing crystal types based on crystal morphology.[9] Automatic microscopic instruments have been widely used for examining urine sediment in clinical practice, with high-throughput and high concordance rate.[10] All abnormal results identified through the automatic procedure should be further confirmed by manual microscopic examination.[11]
Spectroscopic and imaging techniques have been used for urine crystal analysis. Raman technique has many advantages for analyzing amorphous irregularly shaped urine crystals. Raman spectroscopy is not affected by water in the surrounding environment, yielding a high signal-to-noise ratio peak, high specificity, and selective qualitative information in biological samples.[12, 13] Micro-Raman spectroscopy (MRS) provide a non-destructive identification of specific vibration peaks in the spectra of urine crystals; the types of the crystals can be characterized accurately and rapidly.[14] Overall, analyzing urine crystals by Raman spectroscopy has the following benefits: (i) simple sample treatment, (ii) independence of water in the environment, (iii) no additional reagents required, and (iv) real-time monitoring.
Chiu et al. developed a nanoplatform, based on Fe3O4 nanoparticles, to collect crystals from urine and identify the types of crystals through Raman spectroscopy.[7] Since urine crystals are small, amorphous, irregularly shaped, transparent, and colorless, they are difficult to observe under a microscope. Lo et al. further modified the Fe3O4 nanomaterial by labeling dyes (crystal violet) to show the position of crystals for faster automatic crystal analysis.[8] Chen et al. analyzed the urine crystals by micro-Raman system and confocal microscopy for studying the correlation between urine crystals formation and gout patients.[15]
In our recent studies, we discovered that some urine crystals have autofluorescence. To the best of our knowledge, these findings have never been presented in the literature. Furthermore, we found that the autofluorescent urine crystals appeared in most urolithiasis patients. We think it is interesting to identify the type of fluorophores and investigate the role of the existence of the fluorophores in urine crystals. In this study, we aim to characterize the urine crystals through confocal laser scanning microscopy (CLSM) and Fourier-transform infrared spectroscopy (FTIR) and further identify the type of fluorophores present in the autofluorescent urine crystals through mass spectroscopy (MS).