Raman molecular imaging: a novel spectroscopic technique for diagnosis of bladder cancer in urine specimens

Background

Raman molecular imaging (RMI) is an optical technology that combines the molecular chemical analysis of Raman spectroscopy with high-definition digital microscopic visualization. This approach permits visualization of the physical architecture and molecular environment of cells in the urine. The Raman spectrum of a cell is a complex product of its chemical bonds.

Objective

In this work, we studied the possibility of using the Raman spectrum of epithelial cells in voided urine for diagnosing urothelial carcinoma (UC).

Design, setting, and participants

Raman signals were obtained from UC tissue, then from UC touch preps obtained from surgical specimens and studied using the FALCON microscope (ChemImage, Pittsburgh, PA, USA), with a ×100 collection objective and green laser illumination (532 nm). Then, urine samples were obtained from 340 patients, including 116 patients without UC, 92 patients with low-grade tumors, and 132 patients with high-grade tumors. Spectra were obtained from an average of five cells per slide.

Measurements

Raman spectroscopy of cells from bladder cancer (BCa) tissues and patients.

Results and limitations

The Raman spectra from UC tissue demonstrate a distinct peak at a 1584 cm⁻¹ wave shift not present in benign tissues. The height of this peak correlated with the tumor's grade. The signal obtained from epithelial cells correctly diagnosed BCa with sensitivity of 92% (100% of the high-grade tumors), specificity of 91%, and a positive predictive value of 94% and a negative predictive value of 88%. The signal correctly assigned a tumor's grade in 73.9% of the low-grade tumors and 98.5% of the high-grade tumors. RMI for diagnosis of BCa is limited by the need for specialized equipment and training of laboratory personnel.

Conclusions

RMI has the potential to become a powerful diagnostic tool that allows noninvasive, accurate diagnosis of UC.     

兔骨关节炎软骨缺损动物模型的建立

目的探讨建立一种便捷实用的兔骨关节炎软骨缺损动物模型的方法，以适应软骨组织工程技术修复骨关节炎软骨缺损研究的要求。方法5-7月龄新西兰大白兔22只，雌雄不限，体重2．5～3．0kg。根据侧别，分为改良侧（左侧膝）及对照侧（右侧膝）。改良侧分别切除兔内侧半月板、前十字韧带并在股骨髌沟部制造一直径4mm，深3mm的软骨缺损，对照侧仅切除内侧半月板和前十字韧带。分别在术后第3周和第6周在双侧股骨髁部和髌沟部取材，比较两种骨关节炎动物模型的大体形态及病理变化，进行Mankin评分及统计学分析。结果术后6周，改良侧股骨髌沟软骨缺损仍明显存在，但缺损面直径减小，股骨髌沟墨汁染色均达Ⅳ级，光学显微镜下示股骨髌沟软骨缺损达钙化层以下；而对照侧股骨髌沟墨汁染色均未达Ⅳ级。术后3周，改良侧股骨髌沟部Mankin法OA评分（11．82±1．07）分，对照侧（2．37±0．62）分；术后6周，改良侧股骨髌沟部Mankin法OA评分（13．29±1．15）分，对照侧（5．65±1．03）分；改良侧与对照侧股骨髌沟部Mankin评分比较差异有统计学意义（P〈0．01），但股骨髁部Mankin评分两组比较，差异无统计学意义（P〉0．05）。改良侧关节软骨退变进行性加重。结论改良侧和对照侧均能获得满意的骨关节炎动物模型。改良侧在股骨髌沟处形成一个明显的陈旧性软骨缺损，为应用软骨组织工程技术研究骨关节炎软骨缺损修复提供了一种便捷实用的动物模型。     

软骨细胞外基质与壳聚糖复合制备组织工程软骨支架及其性能研究

 目的 探讨采用壳聚糖与脱细胞软骨基质复合制备组织工程软骨支架的可行性,检测其理化性能和细胞相容性。方法 取天然人软骨粉碎.取 100 nm~5μm 软骨微丝,脱细胞处理后制备为质量浓度 1%悬液.与质量浓度 2%壳聚糖醋酸溶液按 1颐1(重量比)充分搅拌混合,冷冻干燥制备复合支架。对支架交联,并进行组织学、扫描电镜、孔隙率及吸水性测定、生物力学评估, MTT法分析支架浸提液毒性。分离培养犬软骨细胞.种植到支架上.倒置显微镜、电镜观察细胞在支架的生长、分化情况。结果 组织学显示支架中无细胞碎片残留.II型胶原免疫组化染色阳性。扫描电镜显示支架内孔洞相互连通似海绵状.孔径为(136.2±34.9)μm.孔隙率为 81.4%±3.5%.吸水性约为 1525.7±129.3%。支架纵向弹性模量为(1.940±0.335)MPa。 MTT法显示不同浓度支架浸提液与对照培养液吸光度值比较.差异无统计学意义(P>0.05)。倒置显微镜观察.细胞在支架上粘附良好.扫描电镜下细胞在支架上均匀分布.呈圆形或椭圆形.有基质分泌。结论 软骨细胞外基质和壳聚糖复合制备的仿生三维多孔双相支架.具有较高的孔隙率和吸水性.良好的生物力学特性.无毒.生物相容性良好.是组织工程软骨的良好支架载体。     

Analysis of near-infrared Raman spectroscopy as a new technique for a transcutaneous non-invasive diagnosis of blood components

. Near-infrared Raman spectroscopy can be a new technique for physical evaluations, allowing the measurement of lactic acid concentrations, in blood or muscles, during the physical activity in a transcutaneous non-invasive way. Lactic acid accumulation in the human body is one of the factors that leads to fatigue and therefore it should be continually monitored during physical training. Our proposal is to use Raman spectroscopy to monitor the lactic acid present in an athlete without interrupting his exercise for sample collection. The experimental set-up for Raman spectroscopy comprised a near infrared laser at 830 nm, a Kaiser f/1.8 spectrometer and a liquid nitrogen cooled CCD detector. The radiation from the exciting laser is blocked in the collecting system by Kaiser holographic filters. A personal computer controls the entire system, saving and processing the Raman spectra. Experiments were undertaken to verify the presence of lactic acid in the Raman spectra of solutions of lactic acid in human serum and in blood from a Wistar rat. After these two experiments, another was developed in vivo in a Wistar rat, injecting intraperitoneally 1 ml of a 0.12 mol/l lactic acid aqueous solution. An optical fibre catheter touching the skin of the rat groin, over the ileac vein collected the Raman signal. The presence of lactic acid was detected inside a live organism, in a transcutaneous non-invasive way. The minimum lactic acid concentration that the equipment can detect was also studied. An experiment was undertaken for that purpose, in which the laser illuminated directly a quartz cuvette containing solutions with decreasing lactic acid concentrations up to values near to the physiological level in the human body. The results indicated that the technique can be suitable for the physical evaluation of athletes. Paper received 12 May 2000; accepted after revision 29 June 2000.     

Ex vivo characterization of articular cartilage and bone lesions in a rabbit ACL transection model of osteoarthritis using MRI and micro-CT

OBJECTIVE: To characterize the rabbit anterior cruciate ligament transection (ACLT) model of osteoarthritis (OA) at various stages of disease using high-resolution 3-D medical imaging systems, which, in turn, will facilitate future longitudinal studies evaluating disease progression and response to therapy in live animals. METHODS: Degenerative changes in femorotibial cartilage, volumetric bone mineral density (vBMD), bone volume fraction (BV/TV), and osteophyte volume were characterized ex vivo using 4-T magnetic resonance imaging (MRI) and micro-computed tomography (micro-CT) at 4, 8, and 12 weeks post-ACLT. These changes were subsequently correlated to macroscopic joint evaluation. RESULTS: Macroscopic assessment demonstrated progressive cartilage degeneration post-surgery, which was significantly correlated to MRI evaluation (r=0.82, P<0.0001). Linear regression analysis indicated that vBMD and BV/TV are linearly related such that as vBMD increases, BV/TV increases (P<0.0001). Micro-CT revealed bone loss at 4 and 8 weeks post-ACLT, but recovery to control values at 12 weeks post-ACLT. Volumetric BMD was not strongly correlated with macroscopic assessment of articular cartilage degeneration (r=-0.35, P<0.0001). Quantitative measurement of osteophyte volume demonstrated a statistically significant difference (with respect to control groups) at both 8 and 12 weeks post-ACLT, but not at 4 weeks post-ACLT. CONCLUSIONS: The rabbit ACLT model of OA demonstrates progressive cartilage degeneration and intermediate bone changes at 4, 8, and 12 weeks post-surgery. Cartilage and bone lesions were characterized ex vivo using 4-T MRI and micro-CT, and MRI assessment of cartilage degeneration was correlated to macroscopic grading.     

Diagnostic accuracy of Raman spectroscopy for prostate cancer: a systematic review and meta-analysis

BackgroundAlthough various studies have been conducted to demonstrate the possibility of Raman spectroscopy (RS) as a diagnostic tool for prostate cancer (PC), it is difficult to use it in the real clinical area because of imitations in various research processes. Therefore, we did a systematic review and meta-analysis about the accuracy in diagnostic use of RS for PC.MethodsA literature search was done using PubMed, Embase, and Cochrane library databases in March 2019 to analyze the accuracy of RS for diagnosis of PC. The accuracy of RS for diagnosis of PC was evaluated by means of pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and summary receiver operating characteristic (SROC).ResultsFive studies were included for qualitative analysis by screening the remaining articles according to the inclusion and exclusion criteria by means of a systematic review. The pooled sensitivity and specificity of RS were 0.89 (95% CI: 0.87–0.91) and 0.91 (95% CI: 0.89–0.93), respectively. The overall PLR and NLR were 9.12 (95% CI: 4.15–20.08) and 0.14 (95% CI: 0.07–0.29), respectively. The DOR of RS demonstrated high accuracy (73.32; 95% CI: 18.43–291.73). The area under the curves (AUCs) of SROC curves was 0.93.ConclusionsRS is an optical diagnostic method with high potential for diagnosis and grading of PC and has advantages of real-time and convenient use. In order to consider real-time use of RS in an actual clinical setting, more studies for standardization and generalization of RS performance and analytical method must be conducted.     

Use of Raman spectroscopy to identify active spermatogenesis and Sertoli‐cell‐only tubules in mice

Microdissection testicular sperm extraction (micro‐TESE) has become the first line therapy to harvest spermatozoa for men with nonobstructive azoospermia. However, the pitfall is that the selection of seminiferous tubules depends on subjective assessment of the colour and size of tubules, which cannot guarantee successful retrieval of spermatozoa. The aim of this study was to determine whether Raman spectroscopy (RS) could distinguish tubules with spermatogenesis from Sertoli‐cell‐only (SCO) tubules, and potentially serve as a useful tool to improve sperm retrieval rates. Fourteen male adult mice were divided into two groups: SCO group received a single intraperitoneal injection of busulfan (40 mg per kg body weight), and the control group received a placebo dose of 0.9% saline solution. Mice were sacrificed after 4 weeks, and the testicular tissue was assessed by RS and then confirmed with histopathology. The results indicated that tubules with spermatogenesis had intensified Raman peaks at 748, 1124, 1309, 1446 and 1658 cm^?1 compared to SCO tubules, except a decreased peak at 1582 cm^?1. RS was able to distinguish the two groups with a sensitivity of 91.2% and specificity of 82.9%. In conclusion, RS may serve as a useful diagnostic tool prior to sperm retrieval.     

Raman spectroscopy for optical diagnosis in normal and cancerous tissue of the nasopharynx-preliminary findings

BACKGROUND AND OBJECTIVES: Raman spectroscopy (RS), which can detect molecular changes associated with cancer, was explored as a means of distinguishing normal and cancerous nasopharyngeal tissue. STUDY DESIGN/PATIENTS AND METHODS: Tissue from six patients with normal and cancerous biopsies was studied using a rapid acquisition Raman spectrometer. RESULTS: Spectra were obtainable within 5 seconds. Consistent differences were noted between normal and cancer tissue in three bands 1,290-1,320 cm(-1) (P = 0.005), 1,420-1,470 cm(-1) (P = 0.006), and 1,530-1,580 cm(-1) (P = 0.002). CONCLUSIONS: Spectral differences appear to exist between normal and cancerous nasopharyngeal tissue. The ability to obtain spectra rapidly supports the potential for future in vivo application.     

骨髓基质干细胞与软骨脱细胞基质多孔支架异位构建组织工程化软骨的实验研究

目的 探讨骨髓基质干细胞(bone marrow mesenchymal stem cells,BMSCs)与软骨脱细胞基质多孔支架(cartilage ECM-derived porous scaffold,CEDPS)在裸鼠体内异位构建软骨的可行性,并建立利用PKH26荧光和分子荧光活体成像系统无创评估组织工程化细胞-支架复合体在体内生长情况的新方法.方法 PKH26荧光标记成软骨诱导的BMSCs,接种入CEDPS支架,体外进行电镜、Desd/Live荧光染色观察,然后植入裸鼠背部,4周后利用分子荧光活体成像系统无创伤性评估组织工程化组织在裸鼠体内生长情况,取材进行组织学以及Ⅱ型胶原免疫荧光检测,与荧光图像比较.结果 体外培养的BMSCs-CEDPS复合体电镜检查结果表明随着培养时间的增加,细胞在支架中增殖显著,细胞基质分泌增加,Dead/Live染色表明BMSCs在支架内部活性良好.4周后活体荧光示踪显示BMSCs在支架内生长良好,无扩散趋势,BMSCs-CEDPS复合体在裸鼠体内生成软骨样组织,番红"O"、甲苯胺蓝染色、Ⅱ型胶原免疫组化染色阳性,免疫荧光检查表明构建的软骨样组织内的细胞来源为接种的PKH26标记的BM-SCs.结论 利用BMSCs和CEDPS支架能够在裸鼠皮下异位构建类软骨样组织.PKH26标记与分子荧光活体成像系统结合,能够无创伤性评估组织工程化组织的种子细胞在动物体内生长情况与转归.     

The Protective Effects of Parathyroid Hormone (1‐34) on Cartilage and Subchondral Bone Through Down‐Regulating JAK2/STAT3 and WNT5A/ROR2 in a Collagenase‐Induced Osteoarthritis Mouse Model

ObjectiveTo assess the effects of PTH (1‐34) on bone and cartilage metabolism in a collagenase‐induced mouse model of osteoarthritis (OA) and examine whether PTH (1‐34) affects the expression of JAK2/STAT3 and WNT5A/ROR2 in this process.MethodsEighteen 12‐week‐old male C57Bl/6 mice were randomly assigned into three groups as follows: sham group (Group A), the collagenase + saline injection group (Group B), and the collagenase + PTH (1‐34) treatment group (Group C). Collagenase was injected (intra‐articular) into the knee joint of Group B and C. The PTH (1–34)‐treatment was started at 6 weeks after the operation and lasted for 6 weeks. Cartilage pathology was evaluated by gross visual, histological, and immunohistochemical assessments. Subchondral bone was evaluated by microcomputed tomography (micro‐CT) and immunohistochemical analyses.ResultsThe OARSI macroscopic and microscopic scores of Group B were higher than those of Group A (P = 0.026; P = 0.002, respectively). Group C showed statistically significant differences in macroscopic and microscopic scores from Group B (P = 0.041; P = 0.008, respectively). The results showed that the Col‐II and AGG expression levels in the cartilage tissue were significantly lower in Group B than Group A (P < 0.001; P = 0.008, respectively). The Col‐II and AGG expression levels were significantly higher in Group C than Group B (P = 0.009; P = 0.014, respectively). MMP‐13, ADAMTS‐4, Caspase‐3, P53, and Bax expression levels were significantly higher in Group B than the Group A (P < 0.001; P < 0.001; P = 0.04; P < 0.001; P = 0.005, respectively); however, the cartilage tissue in Group C showed significantly less ADAMTS‐4, MMP‐13, Caspase‐3, P53, and Bax expression than Group B (P < 0.001, P < 0.001, P = 0.044; P = 0.002; P = 0.005, respectively). Over‐expressed JAK2/STAT3 and WNT5A/ROR2 were observed in both cartilage and subchondral bone in this model; however, these changes were prevented by PTH (1‐34) treatment. These parameters (bone mineral density, bone volume ratio, trabecular bone pattern factor, and structure model index) of micro‐CT indicated subchondral bone loss and architecture changes in Group B, but improvements in these parameters in Group C.ConclusionsPTH (1‐34) exhibits protective effects on both cartilage and subchondral bone in a collagenase‐induced OA mouse model, and it may be involved in down‐regulating the expression of JAK2/STAT3 and WNT5A/ROR2.