Diagnosis of Crystalopathies via Raman Spectroscopy

描述(由申请人提供)：关节间隙中晶体种类的存在和身份的误诊往往会因对关节病变的错误治疗而受到惩罚。现有的偏振成像诊断标准缺乏特异性和敏感性，而且很大程度上局限于显微镜下可见的双折射晶体。在目前的研究中，我们建议将拉曼光谱作为一种诊断技术的替代方案，以改进对关节空间中晶体物种的识别。在拉曼光谱中，分子振动是通过激光激发来触发的，反射的光包含了关于观察区域中物种的身份和数量的信息。该技术目前被用于关节镜下对其他疾病的活体诊断；因此，基于这一临床潜力，有必要探索其在评估关节疾病中所涉及的晶体种类方面的分析和临床可行性。拟议的探索性研究的第一阶段将揭示拉曼光谱可以检测到的各种晶体的识别阈值。尿酸一钠、二水焦磷酸钙、羟基磷灰石、磷酸八钙、磷酸三钙、草酸钙、胆固醇、半胱氨酸和黄嘌呤的合成类似物将以不同浓度与健康滑液样本混合，这些混合物将在宏观水平上进行分析，以检测晶体发出的累积信号。我们将尝试通过使用表面增强拉曼光谱和对光谱数据进行主成分分析来降低识别阈值。在第二阶段，将从患者身上采集滑液样本，并通过补偿偏振显微镜、X射线衍射和茜素染色来确认晶体的存在或不存在。这些样品的一等份将进行拉曼分析，以确定拉曼分析是否可以检测到临床样本中晶体的存在和身份。如果这项探索性研究表明，拉曼分析的分析极限足以涵盖晶体物种的临床浓度，则可以进行全面的临床研究，以评估该技术的特异性和敏感性，最终导致关节镜拉曼设备的开发。

DESCRIPTION (provided by applicant): Misdiagnosis of the presence and the identity of crystal species in joint space are often penalized with the incorrect treatment of the arthropathic condition. The existing diagnostic standard of polarized imaging lacks specificity and sensitivity and it is largely limited to microscopically visible birefringent crystals. In the current study we propose Raman spectroscopy as an alternative for diagnostic technology for improved identification of crystal species in joint spaces. In Raman spectroscopy molecular vibrations are triggered through laser excitation and the reflected light holds information on the identity as well as on the amount of species in the observed region. The technique is currently utilized for arthroscopic in vivo diagnosis of other diseases; thus, based on this clinical potential it would be worthwhile to explore its analytical and clinical feasibility towards the assessment of crystal species involved in arthropathic conditions. The first phase of the proposed exploratory study will reveal the identification thresholds of various crystals that can be detected by Raman spectroscopy. Synthetic analogs of monosodium urate, calcium pyrophosphate dihydrate, hydroxyapatite, octacalcium phosphate, tricalcium phosphate, calcium oxalate, cholesterol, cystine and xanthine will be mixed with healthy synovial fluid samples at various concentrations and these mixtures will be analyzed at the macro level to detect the cumulative signal emanating from crystals. We will attempt to lower the identification thresholds by employing surf ace-enhanced Raman spectroscopy and by principal component analysis of the spectral data. During the second phase synovial fluid samples will be taken from patients and the presence or absence of crystals will be confirmed via compensated polarized microscopy, X-ray diffraction and alizarin staining. Aliquots of these samples will be subjected to Raman analysis to determine whether Raman analysis can detect the presence and identity of crystals in clinical samples. If this exploratory study demonstrates that the analytical limits of Raman analysis suffice to cover clinical concentrations of crystal species than comprehensive clinical studies can be conducted to assess the specificity and the sensitivity of the technique finally leading to the development of an arthroscopic Raman device.