Raman microspectroscopic mapping studies of human bronchial tissue

Characterization of the biochemical composition of normal bronchial tissue is a prerequisite for understanding the biochemical changes that accompany histological changes during lung cancer development. In this study, 12 Raman microspectroscopic mapping experiments are performed on frozen sections of normal bronchial tissue. Pseudocolor Raman images are constructed using principal component analysis and K-means cluster analysis. Subsequent comparison of Raman images with histologic evaluation of stained sections enables the identification of the morphologic origin (e.g., bronchial mucus, epithelium, fibrocollagenous stroma, smooth muscle, glandular tissue, and cartilage) of the spectral features. Raman spectra collected from the basal side of epithelium consistently show higher DNA contributions and lower lipid contributions when compared with superficial epithelium spectra. Spectra of bronchial mucus reveal a strong signal contribution of lipids, predominantly triolein. These spectra are almost identical to the spectra obtained from submucosal glands, which suggests that the bronchial mucus is mainly composed of gland secretions. Different parts of fibrocollagenous tissue are distinguished by differences in spectral contributions from collagen and actin/myosin. Cartilage is identified by spectral contributions of glycosaminoglycans and collagen. As demonstrated here, in situ analysis of the molecular composition of histologic structures by Raman microspectroscopic mapping creates powerful opportunities for increasing our fundamental understanding of tissue organization and function. Moreover, it provides a firm basis for further in vitro and in vivo investigations of the biochemical changes that accompany pathologic transformation of tissue.     

Microarray analysis of MRI-defined tissue samples in glioblastoma reveals differences in regional expression of therapeutic targets.

Microarray technologies have come into prominence for the assessment of molecular diagnostic profiles in cancer tissue biopsies. To better understand the effect of sampling bias, we paired image-guided stereotactic biopsy and microarray technology to study regional intratumoral differences in tumor periphery and core regions of untreated glioblastoma. RNA was extracted from serial frozen sections using an integral histopathologic scoring approach. Gene expression analysis was performed using high-density oligonucleotide microarrays (22,283 probe sets). A consensus list of 643 genes (784 probe sets) with greater than 2-fold difference between intratumoral periphery and core samples was obtained using Microarray Suite 5.0, model-based expression indexes, and robust multiarray analysis algorithms. Results were validated using quantitative polymerase chain reaction and Western blotting analyses. Reproducible profiles emerged, in which multiple therapeutic targets significant to glioblastoma [matrix metalloproteinases, AKT1 (v-akt murine thymoma viral oncogene homolog 1), epidermal growth factor receptor, vascular endothelial growth factor] showed significant differences in regional expression that may affect treatment response. This study suggests important intratumoral regional differences in the molecular phenotype of glioblastoma.     

Use of picosecond Kerr-gated Raman spectroscopy to suppress signals from both surface and deep layers in bladder and prostate tissue

Raman spectroscopy is an optical technique able to interrogate biological tissues, giving us an understanding of the changes in molecular structure that are associated with disease development. The Kerr-gated Raman spectroscopy technique uses a picosecond pulsed laser as well as fast temporal gating of collected Raman scattered light. Prostate samples for this study were obtained by taking a chip at the transurethral resection of the prostate (TURP), and bladder samples from a biopsy taken at transurethral resection of bladder tumor (TURBT) and TURP. Spectra obtained through the bladder and prostate gland tissue, at different time delays after the laser pulse, clearly show change in the spectra as depth profiling occurs, eventually showing signals from the uric acid cell and urea cell, respectively. We show for the first time, using this novel technique, that we are able to obtain spectra from different depths through both the prostate gland and the bladder. This has major implications in the future of Raman spectroscopy as a tool for diagnosis. With the help of Raman spectroscopy and Kerr gating, it may be possible to pick up the spectral differences from a small focus of adenocarcinoma of the prostate gland in an otherwise benign gland, and also stage the bladder cancers by assessing the base of the tumor post resection.     

Evaluation of linear discriminant analysis for automated Raman histological mapping of esophageal high-grade dysplasia

Rapid Raman mapping has the potential to be used for automated histopathology diagnosis, providing an adjunct technique to histology diagnosis. The aim of this work is to evaluate the feasibility of automated and objective pathology classification of Raman maps using linear discriminant analysis. Raman maps of esophageal tissue sections are acquired. Principal component (PC)-fed linear discriminant analysis (LDA) is carried out using subsets of the Raman map data (6483 spectra). An overall (validated) training classification model performance of 97.7% (sensitivity 95.0 to 100% and specificity 98.6 to 100%) is obtained. The remainder of the map spectra (131,672 spectra) are projected onto the classification model resulting in Raman images, demonstrating good correlation with contiguous hematoxylin and eosin (HE) sections. Initial results suggest that LDA has the potential to automate pathology diagnosis of esophageal Raman images, but since the classification of test spectra is forced into existing training groups, further work is required to optimize the training model. A small pixel size is advantageous for developing the training datasets using mapping data, despite lengthy mapping times, due to additional morphological information gained, and could facilitate differentiation of further tissue groups, such as the basal cells∕lamina propria, in the future, but larger pixels sizes (and faster mapping) may be more feasible for clinical application.     

立体定向活检在颅内疑难病变诊断中的临床应用

目的 探讨立体定向活检在颅内疑难病变诊断中的临床应用价值。方法 选取我院2011年3月~2017年3月20例患者全部行立体定向活检术,术前均因临床症状或体征多次接受过CT 、MRI 或MRS等影像学检查, 已明确患者颅内病变, 但未对病理性质确诊, 无法采取有效的治疗方法而接受立体定向活检手术。结果 行立体定向活检术确诊病理诊断,星形细胞瘤Ⅲ级6例,星形细胞瘤Ⅱ级2例,淋巴瘤2例,肺癌脑转移瘤2例,肿瘤放射性坏死灶2例,其它5例（胶质母细胞瘤１例,转移性小细胞恶性肿瘤1例,炎细胞１例,脱髓鞘1例,脑梗塞1例）,性质不明1例,手术活检阳性率95.00%。结论　立体定向活检术具有准确性高、创伤小、并发症少等优点,能够有效的诊断颅内疑难病变,并为下一步治疗提供病理学依据。     

Discriminating basal cell carcinoma from perilesional skin using high wave-number Raman spectroscopy

An expanding body of literature suggests Raman spectroscopy is a promising tool for skin cancer diagnosis and in-vivo tumor border demarcation. The development of an in-vivo diagnostic tool is, however, hampered by the fact that construction of fiber optic probes suitable for Raman spectroscopy in the so-called fingerprint region is complicated. In contrast, the use of the high wave-number region allows for fiber optic probes with a very simple design. We investigate whether high wave-number Raman spectroscopy (2800 to 3125 cm(-1)) is able to provide sufficient information for noninvasive discrimination between basal cell carcinoma (BCC) and noninvolved skin. Using a simple fiber optic probe, Raman spectra are obtained from 19 BCC biopsy specimens and 9 biopsy specimens of perilesional skin. A linear discriminant analysis (LDA)-based tissue classification model is developed, which discriminates between BCC and noninvolved skin with high accuracy. This is a crucial step in the development of clinical dermatological applications based on fiber optic Raman spectroscopy.     

Multiplex DNA short tandem repeat analysis. A useful method for determining the provenance of minute fragments of formalin-fixed, paraffin-embedded tissue

A tiny fragment of high-grade carcinoma was found in histologic sections and in the paraffin block of a benign cervical polyp from a patient with no clinical evidence of malignancy. Thus, it raised the suspicion of block contamination. No malignant tumor was processed the same day as the polyp; however, a similar tumor had been processed 6 days earlier. Multiplex DNA short tandem repeat analysis was applied to paraffin-extracted tissue samples obtained from the polyp, the suspected contaminant, the patient's additional cervical biopsy specimen, and the putative source of contamination. The results demonstrated that the suspected contaminant and the patient's cervical tissue could not have come from the same patient and that the suspected contaminant derived from the tumor processed earlier, without reasonable doubt. We hypothesize that this friable tumor escaped from cassettes into the processor and contaminated the polyp specimen. Multiplex DNA short tandem repeat analysis can be applied to determine the provenance of minute tissue samples in surgical pathology.     

Picosecond spectral coherent anti-Stokes Raman scattering imaging with principal component analysis of meibomian glands

The lipid distribution in the mouse meibomian gland was examined with picosecond spectral anti-Stokes Raman scattering (CARS) imaging. Spectral CARS data sets were generated by imaging specific localized regions of the gland within tissue sections at consecutive Raman shifts in the CH(2) stretching vibrational range. Spectral differences between the location specific CARS spectra obtained in the lipid-rich regions of the acinus and the central duct were observed, which were confirmed with a Raman microspectroscopic analysis, and attributed to meibum lipid modifications within the gland. A principal component analysis of the spectral data set reveals changes in the CARS spectrum when transitioning from the acini to the central duct. These results demonstrate the utility of picosecond spectral CARS imaging combined with multivariate analysis for assessing differences in the distribution and composition of lipids in tissues.     

In vivo immunohistochemical identification of tumor necrosis factor/cachectin in human lymphoid tissue.

To find an alternative approach to the in vivo detection of tumor necrosis factor/cachectin (TNF alpha), an immunohistochemical method to identify TNF alpha in histologic sections was developed. This method employs the streptavidin-biotin immunoperoxidase technique, and TNF alpha-specific monoclonal and polyclonal antibodies, on cryostat sections of fresh frozen human lymphoid tissue. Staining was evident in most specimens displaying follicular hyperplasia, but was absent from histologically normal tissue. Both tingible body macrophages and follicular dendritic reticulum cells appeared from phenotype analysis in serial sections and by double staining experiments to constitute the main source of TNF alpha. This technique complements other systemically oriented assays that may fail to detect significant in vivo TNF alpha production and activity at a cellular level.     

Infrared spectral histopathology (SHP): a novel diagnostic tool for the accurate classification of lung cancer

We report results of a study utilizing a recently developed tissue diagnostic method, based on label-free spectral techniques, for the classification of lung cancer histopathological samples from a tissue microarray. The spectral diagnostic method allows reproducible and objective diagnosis of unstained tissue sections. This is accomplished by acquiring infrared hyperspectral data sets containing thousands of spectra, each collected from tissue pixels about 6?μm on edge; these pixel spectra contain an encoded snapshot of the entire biochemical composition of the pixel area. The hyperspectral data sets are subsequently decoded by methods of multivariate analysis, which reveal changes in the biochemical composition between tissue types, and between various stages and states of disease. In this study, a detailed comparison between classical and spectral histopathology (SHP) is presented, which suggests SHP can achieve levels of diagnostic accuracy that is comparable to that of multi-panel immunohistochemistry.     

Recent developments in stereotactic breast biopsy methodologies: an update for the surgical pathologist

Recently, there have been a number of new devices introduced for stereotactic biopsy of nonpalpable, mammographically detected lesions. The vacuum-assisted core biopsy (VACB) (Minimal Invasive Breast Biopsy (MIBB), U.S. Surgical, Norwalk, CT; Mammotome, Biopsys Medical, Cincinnati, OH) obtains multiple tissue cores (11-gauge) in a circumferential manner around the biopsy probe, inserted under stereotactic guidance. It provides more complete sampling of mammographic lesions than the conventional 14-gauge stereotactic core biopsy, reducing the number of unsatisfactory biopsies. The advanced breast biopsy instrumentation (ABBI) (United States Surgical Corporation, Norwalk, CT) system utilizes stereotactic technique and an oscillating blade-cutting mechanism to obtain a single large diameter (5 mm to 20 mm) tissue core, with the aim of obtaining an intact lesion in its entirety for histologic assessment. Its potential as a treatment option is still under investigation. Suggested protocols for specimen handling are presented together with a review of the recent literature. Close liaison with radiologists and surgeons performing these biopsies will allow the collection of further outcome data to evaluate the strengths and weaknesses of each technique.     

Evaluation of the tissue microarray technique for immunohistochemical analysis in rectal cancer

BACKGROUND: Immunohistochemical staining for tumor-associated proteins is widely used for the identification of novel prognostic markers. Recently, a tissue-conserving, high-throughput technique, tissue microarray, has been introduced. This technique uses 0.6-mm tissue core biopsy specimens, 500 to 1000 of which are brought into a new paraffin array block, which can be sectioned up to 100 times. METHODS: We evaluated the tissue microarray technique for immunohistochemical analysis in 20 rectal cancers. Immunohistochemical staining was performed for the proliferation marker Ki-67 and the tumor suppressor protein p53 in whole tissue sections and in tissue core biopsy specimens. RESULTS: The whole tissue sections were assessed by counting all cells in 10 high-power fields (x40), which resulted in a mean fraction of Ki-67-expressing tumor cells of 0.81 (range, 0.54-1.0). p53 expression assessed in whole tissue sections showed nuclear staining in 15 (75%) of 20 rectal carcinomas. For the tissue microarray technique, a median of 3 (range, 3-5) 0.6-mm tissue core biopsy specimens were studied from each of the 20 tumor specimens. The tissue microarray method gave a mean Ki-67 expression of 0.85 (range, 0.50-1.0) in tumor cell nuclei and showed p53 protein expression in the same 15 of 20 tumors as in the whole tissue sections. CONCLUSION: We conclude that the tissue microarray technique for immunohistochemical staining in rectal cancer yields staining of good quality and expression data for Ki-67 and p53 comparable to those obtained with whole tissue staining. The feasibility of tissue microarray thus enables time- and tissue-preserving studies of multiple markers in large tumor series.     

Raman spectroscopy,a potential tool for the objective identification and classification of neoplasia in Barrett's oesophagus

Histopathology remains the gold standard technique for the diagnosis of intraepithelial neoplasia (dysplasia) in Barrett's oesophagus, but it is highly subjective and relies on blind biopsy targeting. The aim of this study was to evaluate Raman spectroscopy, a rapid, non-invasive, molecular, specific analytical technique, for the objective identification and classification of Barrett's neoplasia in vitro. A secondary objective was to demonstrate the need for a rigorous gold standard in the development of new diagnostic techniques. Forty-four patients with a mean age of 69 years (range 34-89 years) undergoing surveillance for Barrett's oesophagus were included in the study. Three consultant pathologists independently assessed snap-frozen oesophageal biopsy specimens. Raman spectra were measured on 87 histopathologically homogeneous samples. Spectral classification models were developed using multivariate analysis for the prediction of pathology. Histopathology and Raman classification results were compared. Raman spectral prediction with a consensus pathology classification model gave sensitivities between 73% and 100% and specificities of 90-100%. A high level of agreement (kappa = 0.89) was demonstrated between the three-subset biopsy targeting model and consensus pathology opinion. This compares favourably with the agreement measured between an independent pathologist and the consensus pathology opinion for the same spectra (kappa = 0.76). Raman spectroscopy appears to provide a highly sensitive and specific technique for the identification and classification of neoplasia in Barrett's oesophagus.     

Characterization of Malignant Brain Tumor Using Elastic Light Scattering Spectroscopy

We report a pilot study designed to test elastic light-scattering (ELS) spectroscopy for characterizing normal, tumor, and tumor-infiltrated brain tissues. ELS spectra were measured from 393 sites on 36 ex vivo tissue specimen obtained from 29 patients. We employed and compared the performances of three methods of spectral classification for tissue characterization, including spectral slope analysis, principle component analysis (PCA), and artificial neural network (ANN) classification. The ANN classifier yielded the best correlation between spectral pattern and histopathological diagnosis, with a typical sensitivity of 80% and specificity of 93% for differentiating tumor from normal brain tissues. We also demonstrate that all three classification methods discriminate between tumor and normal tissue and have the potential to identify and quantitatively characterize tumor-infiltrated brain tissues.     

Discriminating model for diagnosis of basal cell carcinoma and melanoma in vitro based on the Raman spectra of selected biochemicals

Raman spectroscopy has been employed to identify differences in the biochemical constitution of malignant [basal cell carcinoma (BCC) and melanoma (MEL)] cells compared to normal skin tissues, with the goal of skin cancer diagnosis. We collected Raman spectra from compounds such as proteins, lipids, and nucleic acids, which are expected to be represented in human skin spectra, and developed a linear least-squares fitting model to estimate the contributions of these compounds to the tissue spectra. We used a set of 145 spectra from biopsy fragments of normal (30 spectra), BCC (96 spectra), and MEL (19 spectra) skin tissues, collected using a near-infrared Raman spectrometer (830 nm, 50 to 200 mW, and 20 s exposure time) coupled to a Raman probe. We applied the best-fitting model to the spectra of biochemicals and tissues, hypothesizing that the relative spectral contribution of each compound to the tissue Raman spectrum changes according to the disease. We verified that actin, collagen, elastin, and triolein were the most important biochemicals representing the spectral features of skin tissues. A classification model applied to the relative contribution of collagen III, elastin, and melanin using Euclidean distance as a discriminator could differentiate normal from BCC and MEL.     

Raman microspectroscopy of human coronary atherosclerosis: biochemical assessment of cellular and extracellular morphologic structures in situ.

BACKGROUND: We have previously shown that Raman spectroscopy can be used for chemical analysis of intact human coronary artery atherosclerotic lesions ex vivo without tissue homogenization or extraction. Here, we report the chemical analysis of individual cellular and extracellular components of atherosclerotic lesions in different stages of disease progression in situ using Raman microspectroscopy. METHODS: Thirty-five coronary artery samples were taken from 16 explanted transplant recipient hearts, and thin sections were prepared. Using a high-resolution confocal Raman microspectrometer system with an 830-nm laser light, high signal-to-noise Raman spectra were obtained from the following morphologic structures: internal and external elastic lamina, collagen fibers, fat, foam cells, smooth muscle cells, necrotic core, beta-carotene, cholesterol crystals, and calcium mineralizations. Their Raman spectra were modeled by using a linear combination of basis Raman spectra from the major biochemicals present in arterial tissue, including collagen, elastin, actin, myosin, tropomyosin, cholesterol monohydrate, cholesterol linoleate, phosphatidyl choline, triolein, calcium hydroxyapatite, calcium carbonate, and beta-carotene. RESULTS: The results show that the various morphologic structures have characteristic Raman spectra, which vary little from structure to structure and from artery to artery. The biochemical model described the spectrum of each morphologic structure quite well, indicating that the most essential biochemical components were included in the model. Furthermore, the biochemical composition of each structure, indicated by the fit contributions of the biochemical basis spectra of the morphologic structure spectrum, was very consistent. CONCLUSIONS: The Raman spectra of various morphologic structures in normal and atherosclerotic coronary artery may be used as basis spectra in a linear combination model to analyze the morphologic composition of atherosclerotic coronary artery lesions.     

Infrared spectroscopic imaging of renal tumor tissue

Fourier transform infrared (FTIR) spectroscopic imaging has been used to probe the biochemical composition of human renal tumor tissue and adjacent normal tissue. Freshly resected renal tumor tissue from surgery was prepared as a thin cryosection and examined by FTIR spectroscopic imaging. Tissue types could be discriminated by utilizing a combination of fuzzy k-means cluster analysis and a supervised classification algorithm based on a linear discriminant analysis. The spectral classification is compared and contrasted with the histological stained image. It is further shown that renal tumor cells have spread in adjacent normal tissue. This study demonstrates that FTIR spectroscopic imaging can potentially serve as a fast and objective approach for discrimination of renal tumor tissue from normal tissue and even in the detection of tumor infiltration in adjacent tissue.     

Pilot study for detection of early changes in tissue associated with heterotopic ossification: moving toward clinical use of Raman spectroscopy

Abstract

Over 60% of combat-wounded patients develop heterotopic ossification (HO). Nearly 33% of them require surgical excision for symptomatic lesions, a procedure that is both fraught with complications and can delay or regress functional rehabilitation. Relative medical contraindications limit widespread use of conventional means of primary prophylaxis, such as nonspecific nonsteroidal anti-inflammatory medications and radiotherapy. Better methods for risk stratification are needed to both mitigate the risk of current means of primary prophylaxis as well as to evaluate novel preventive strategies currently in development. We asked whether Raman spectral changes, measured ex vivo, could be associated with histologic evidence of the earliest signs of HO formation and substance P (SP) expression in tissue biopsies from the wounds of combat casualties. In this pilot study, we compared normal muscle tissue, injured muscle tissue, very early HO lesions (?<?16 d post-injury), early HO lesions (?>?16 d post-injury) and mature HO lesions. The Raman spectra of these tissues demonstrate clear differences in the Amide I and III spectral regions of HO lesions compared to normal tissue, denoted by changes in the Amide I band center (p?<?0.01) and the 1340/1270?cm^?1 (p?<?0.05) band area and band height ratios. SP expression in the HO lesions appears to peak between 16 and 30 d post-injury, as determined by SP immunohistochemistry of corresponding tissue sections, potentially indicating optimal timing for administration of therapeutics. Raman spectroscopy may therefore prove a useful, non-invasive and early diagnostic modality to detect HO formation before it becomes evident either clinically or radiographically.     

Thymoma with extensive necrosis: a case report and review of literature

Thymoma exhibiting extensive necrosis is extremely rare and remains a diagnostic challenge for both radiologists and pathologists. We describe such a thymoma in an 18-year-old African-American female. Core needle biopsy contained only necrotic tumor tissue. A well-encapsulated 13-cm anterior mediastinal thymoma with extensive necrosis and areas of hemorrhage was resected. Microscopically, the thymus was almost completely replaced by tumor composed of plump epithelial cells with vesicular nuclei and prominent nucleoli in a background of lymphocytes and extensive necrosis. The clinical, immunohistochemical and diagnostic pitfalls of this tumor in core needle biopsies is discussed.     

Giant cell tumor of bone. A cytologic study of 24 cases

Twenty-three patients with radiologic diagnoses of giant cell tumor of bone underwent fine needle aspiration cytology and needle biopsy for tissue diagnosis before curettage or resection. One patient had two tumors, making a total of 24 cases. The accuracy of the cytologic diagnosis was compared with that of tissue biopsy. Cytologically there were mononucleated and multinucleated cells. The former often occurred in clusters or, less often, were dispersed. They had spindle or plump cell bodies with moderate amounts of cytoplasm and well-defined cytoplasmic membranes. The oval nuclei demonstrated fine, evenly distributed chromatin and small nucleoli. The multinucleated cells were osteoclastlike and were associated with the clusters of mononucleated cells or lying freely. They had a well-demarcated cytoplasm and contained from a few to several dozen monomorphic nuclei. Cytologic diagnosis was made in 20 of 24 cases, and histologic diagnosis was made in 21 of 24. Insufficient diagnostic material for cytology was the reason for failure in 4 cases. This was attributed to faulty technique (2 cases), cystic change (1 case), and massive necrosis (1 case). As other benign and malignant bone tumors may contain benign giant cells, cytologic or histologic findings alone are not diagnostic of giant cell tumor of bone, but should be complemented with the clinicoradiologic findings. Aspiration cytology is as accurate as tissue needle biopsy, may be of high diagnostic value in deeply located lesions not amenable to cutting needle biopsy, and should be done with full knowledge of the clinicoradiographic information.