Cheng Xingguo, Haggins Donard G, York Russel H, Yeni Yener N, Akkus Ozan
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907, USA.
Appl Spectrosc. 2009 Apr;63(4):381-6. doi: 10.1366/000370209787944280.
The current study assessed the feasibility of the application of Raman spectroscopy toward the diagnosis of gout and pseudogout. First, the lowest concentrations of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) crystals detectable by Raman spectroscopy were investigated by mixing known amounts of synthetic crystals with synovial fluid in the concentration range of 1 to 100 microg/mL. Second, a digestion protocol was developed for clinical samples to improve crystal extraction. The ensuing centrifugation of the digest congregated crystals at a well-defined point and allowed for point-and-shoot Raman analysis without having to conduct an extensive search for individual crystals. Finally, synovial fluid samples obtained from patients (n = 35) were cross-analyzed by polarized light microscopy (PLM) and the Raman method to compare and contrast the diagnoses of the two methods. It was found that Raman spectroscopy can detect MSUM and CPPD crystals with good sensitivity and specificity at concentrations as low as 5 microg/mL and 2.5 microg/mL, respectively, using the current method. This detection limit of Raman analysis is lower than that reported for PLM. Raman and PLM diagnoses of clinical samples agreed in 32 out of 35 samples in the entire sample pool. However, the rate of disagreement between PLM-based and Raman-based diagnoses was noteworthy within the subset of diseased samples (3 out of 10), indicating that PLM has limitations and that the confirmation by a secondary method is essential for a reliable outcome. The proposed protocol of sample preparation and Raman analysis ascribes baseline feasibility to the diagnosis of gout and pseudogout by Raman spectroscopy, thus justifying further studies using a larger clinical sample set for obtaining sensitivity and specificity.
本研究评估了拉曼光谱法在痛风和假性痛风诊断中的应用可行性。首先，通过将已知量的合成晶体与浓度范围为1至100微克/毫升的滑液混合，研究了拉曼光谱法可检测到的单水尿酸钠（MSUM）和二水焦磷酸钙（CPPD）晶体的最低浓度。其次，开发了一种针对临床样本的消化方案以改善晶体提取。随后对消化液进行离心，使晶体在一个明确的点聚集，从而可以进行定点拉曼分析，而无需对单个晶体进行广泛搜索。最后，对从患者（n = 35）获得的滑液样本采用偏振光显微镜（PLM）和拉曼方法进行交叉分析，以比较和对比两种方法的诊断结果。结果发现，使用当前方法，拉曼光谱法分别在低至5微克/毫升和2.5微克/毫升的浓度下，能够以良好的灵敏度和特异性检测到MSUM和CPPD晶体。拉曼分析的这一检测限低于报道的PLM检测限。在整个样本库中，35个样本中有32个样本的拉曼和PLM临床样本诊断结果一致。然而，在患病样本子集中，基于PLM和基于拉曼的诊断之间的不一致率值得注意（10个中有3个），这表明PLM存在局限性，并且通过第二种方法进行确认对于获得可靠结果至关重要。所提出的样本制备和拉曼分析方案为拉曼光谱法诊断痛风和假性痛风赋予了基线可行性，因此有理由使用更大的临床样本集进行进一步研究以获得灵敏度和特异性。
