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Fiber based infrared spectroscopy of cancer tissues
Abstract
We present the infrared spectroscopy study of three types of human healthy and cancerous tissues using standard ATR and fiber optics based ATR techniques. The use of fiber based ATR probes eliminates the need to bring the sample under the study to the spectroscopic instrument and opens the path for the application of infrared spectroscopy for the studies in situ and in vivo. The study revealed that conventional portable FTIR spectrometers with room temperature DTGS detectors cannot be readily used when coupled with fiber probes due to the high loses of the optical signal, but this problem can be overcome by combining an additional more sensitive liquid nitrogen cooled MCT detector to the system. The potential of the system is demonstrated in the cases of the studies of three types of tumorous tissues: kidney, bladder and brain. The studies of the tissue smears and untreated (wet) tissues indicated that “water free” fingerprint region has clear spectral signatures for the tumor identification. Depending on the tissue, the signatures are based on the glycogen infrared absorption bands for kidney tissue and the collagen absorption bands for bladder tissue. Brain tissue examination also shows some promising spectral markers for which precise establishment the higher data sets are still needed.
... Details of the system are described elsewhere. 17,18 The cone-shaped Ge ATR crystal tip was pressed onto the sample tissue during the measurement covering a surface of ∼5 mm 2 . Before each measurement, tip cleaning was performed by wiping the tip surface by a soft cotton swab dampened with distilled water followed by ethanol. ...
Significance:
Pancreatic surgery is a highly demanding and routinely applied procedure for the treatment of several pancreatic lesions. The outcome of patients with malignant entities crucially depends on the margin resection status of the tumor. Frozen section analysis for intraoperative evaluation of tissue is still time consuming and laborious.
Aim:
We describe the application of fiber-based attenuated total reflection infrared (ATR IR) spectroscopy for label-free discrimination of normal pancreatic, tumorous, and pancreatitis tissue. A pilot study for the intraoperative application was performed.
Approach:
The method was applied for unprocessed freshly resected tissue samples of 58 patients, and a classification model for differentiating between the distinct tissue classes was established.
Results:
The developed three-class classification model for tissue spectra allows for the delineation of tumors from normal and pancreatitis tissues using a probability score for class assignment. Subsequently, the method was translated into intraoperative application. Fiber optic ATR IR spectra were obtained from freshly resected pancreatic tissue directly in the operating room.
Conclusion:
Our study shows the possibility of applying fiber-based ATR IR spectroscopy in combination with a supervised classification model for rapid pancreatic tissue identification with a high potential for transfer into intraoperative surgical diagnostics.
... Some authors report results of application of IR spectroscopy for human BC investigation, however, most of them cover applications for specially prepared tissue samples such as formalin-fixed and paraffin-embedded tissues [20,21] or cultured cell lines fixed in glutaraldehyde in Phosphate-Buffered Saline (PBS) [22]. In our previous studies we showed that fiber-based IR spectroscopy can be successfully applied for examination of various freshly resected human tissues [23,24]. Here we represent the study of 54 patients by using fiber-based IR spectroscopy to delineate normal and tumorous human bladder tissues under ex vivo conditions. ...
Surgical treatment is widely applied curative approach for bladder cancer. White light cystoscopy (WLC) is currently used for intraoperative diagnostics of malignant lesions but has relatively high false-negative rate. Here we represent an application of label free fiber-based attenuated total reflection infrared spectroscopy (ATR IR) for freshly resected human bladder tissue examination for 54 patients. Defined molecular spectral markers allow to identify normal and urothelial carcinoma tissues. While methods of statistical analysis (Hierarchical cluster analysis (HCA) and Principal component analysis (PCA)) used for spectral data treatment allow to discriminate tissue types with 91% sensitivity and 96–98% specificity. In the present study the described method was applied for tissue examination under ex vivo conditions. However, after method validation the equipment could be translated from laboratory studies to in situ or even in vivo studies in operating room.
... From a technical point of view, this can be easily implemented in the clinical workflow, as acquisition times are short (seconds). ATR IR spectroscopy [35]and Raman spectroscopy [36] can be performed using fiber-based systems that were already demonstrated to provide high quality spectra of human brain tumors. Moreover, we have already shown that prognostic relevant molecular markers like IDH1 mutation and tumor stem cells can be addressed by both techniques, with large potential for intraoperative tumor characterization [17,25,37]. ...
Purpose
Infrared (IR) spectroscopy has the potential for tumor delineation in neurosurgery. Previous research showed that IR spectra of brain tumors are generally characterized by reduced lipid-related and increased protein-related bands. Therefore, we propose the exploitation of these common spectral changes for brain tumor recognition.
Methods
Attenuated total reflection IR spectroscopy was performed on fresh specimens of 790 patients within minutes after resection. Using principal component analysis and linear discriminant analysis, a classification model was developed on a subset of glioblastoma (n = 135) and non-neoplastic brain (n = 27) specimens, and then applied to classify the IR spectra of several types of brain tumors.
Results
The model correctly classified 82% (517/628) of specimens as “tumor” or “non-tumor”, respectively. While the sensitivity was limited for infiltrative glioma, this approach recognized GBM (86%), other types of primary brain tumors (92%) and brain metastases (92%) with high accuracy and all non-tumor samples were correctly identified.
Conclusion
The concept of differentiation of brain tumors from non-tumor brain based on a common spectroscopic tumor signature will accelerate clinical translation of infrared spectroscopy and related technologies. The surgeon could use a single instrument to detect a variety of brain tumor types intraoperatively in future clinical settings. Our data suggests that this would be associated with some risk of missing infiltrative regions or tumors, but not with the risk of removing non-tumor brain.
... From a technical point of view, this can be easily implemented in the clinical work ow, as acquisition times are short (seconds). ATR IR spectroscopy [31]-and Raman spectroscopy [32] can be performed using berbased systems that were already demonstrated to provide high quality spectra of human brain tumors. ...
Purpose
Infrared (IR) spectroscopy has the potential for tumor delineation in neurosurgery. Previous research showed that IR spectra of brain tumors are generally characterized by reduced lipid-related and increased protein-related bands. Therefore, we propose the exploitation of these common spectral changes for brain tumor recognition.
Methods
Attenuated total reflection IR spectroscopy was performed on fresh specimens of 790 patients within minutes after resection. Using principal component analysis and linear discriminant analysis, a classification model was developed on a subset of glioblastoma (n = 135) and non-neoplastic brain (n = 27) specimens, and then applied to classify the IR spectra of several types of brain tumors.
Results
The model correctly classified 82% (517/628) of specimens as “tumor” or “non-tumor”, respectively. While the sensitivity was limited for infiltrative glioma, this approach recognized GBM (86%), other types of primary brain tumors (92%) and brain metastases (92%) with high accuracy and all non-tumor samples were correctly identified.
Conclusion
The concept of differentiation of brain tumors from non-tumor brain based on a common spectroscopic tumor signature will accelerate clinical translation of infrared spectroscopy and related technologies. The surgeon could use a single instrument to detect a variety of brain tumor types intraoperatively in future clinical settings. Our data suggests that this would be associated with some risk of missing infiltrative regions or tumors, but not with the risk of removing non-tumor brain.
Urothelial bladder carcinoma (BC) is primarily diagnosed with a subjective examination of biopsies by histopathologists, but accurate diagnosis remains time-consuming and of low diagnostic accuracy, especially for low grade non-invasive BC. We propose a novel approach for high-throughput BC evaluation by combining infrared (IR) microscopy of bladder sections with machine learning (partial least squares-discriminant analysis) to provide an automated prediction of the presence of cancer, invasiveness and grade. Cystoscopic biopsies from 50 patients with clinical suspicion of BC were histologically examined to assign grades and stages. Adjacent tissue cross-sections were IR imaged to provide hyperspectral datasets and cluster analysis segregated IR images to extract the average spectra of epithelial and subepithelial tissues. Discriminant models, which were validated using repeated random sampling double cross-validation, showed sensitivities (AUROC) ca. 85% (0.85) for the identification of cancer in epithelium and subepithelium. The diagnosis of non-invasive and invasive cases showed sensitivity values around 80% (0.84-0.85) and 76% (0.73-0.80), respectively, while the identification of low and high grade BC showed higher sensitivity values 87-88% (0.91-0.92). Finally, models for the discrimination between cancers with different invasiveness and grades showed more modest AUROC values (0.67-0.72). This proves the high potential of IR imaging in the development of ancillary platforms to screen bladder biopsies.
Background: Intraoperative frozen section analysis is frequently used to obtain a histological diagnosis at the time of resection and to assess resection margins. Although many surgeons perceive a clinical benefit, particularly with respect to the transected resection margins, the limitations and pitfalls of frozen section analysis have not been well documented.
Case: Here, we report a case of serous cystadenoma with background pancreatitis masquerading on frozen section as an invasive pancreatic ductal adenocarcinoma. This interpretation was a surprise in light of preoperative imaging that was highly suggestive of a benign cystic tumor, but nevertheless prompted intraoperative consideration of a more radical operation to ensure a complete resection was achieved.
Conclusions: Frozen section analysis is an imperfect test, and misdiagnoses can potentially impact patient outcomes adversely. Intraoperative decisions must carefully integrate the preliminary pathological interpretation with the overall clinical context. Further studies are warranted to more fully characterize the accuracy, utility, and cost-effectiveness of intraoperative frozen section analysis for pancreatic surgery.
Intraoperative frozen section (FS) diagnostics is an important diagnostic tool in neurosurgery, but agreement with final histopathology diagnoses may vary. In the present study we assess the diagnostic properties of intraoperative FSs in suspected intracranial tumors.
Retrospective single-center review of consecutive patients with suspected intracranial brain tumors from January 2008 to December 2012. We included all cases were both an intraoperative FS and a formalin-fixed paraffin-embedded (FFPE) section had been acquired. Agreement with final diagnosis, sensitivity, specificity, and predictive values were explored. Time between date of surgery and first final diagnosis based on FFPE section, whether the patients had undergone previous brain surgery and/or prior cerebral radiotherapy were also registered.
Agreement between FS diagnoses and final FFPE section diagnoses was seen in 504/558 (90.3%), while there was lack of agreement in 54/558 (9.7%). In 20 cases, agreement was not classifiable. Agreement was lower in low-grade gliomas (82.5%) than in high-grade gliomas (93.2%). Agreement between FS and FFPE was significantly higher in primary operations (92.1%) than in re-do operations (81.5%) (P = 0.001). Sensitivity of FS ranged from 30.8% in lymphomas to 94.6% in meningiomas.
Intraoperative FS diagnoses demonstrate high diagnostic accuracy. However, agreement varies among histopathological entities and is lower in low-grade tumors than in high-grade tumors. Sensitivity for diagnosing CNS lymphomas is low. A variable degree of reservation is always necessary when interpreting and communicating FS diagnoses.
Herein, a technique to analyze air-dried kidney tissue impression smears by means of Attenuated Total Reflection Infrared (ATR-IR) spectroscopy is presented. Spectral tumor markers - absorption bands of glycogen - are identified in the ATR-IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo-sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze-thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92 % for normal tissue and 94 % for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real-time intra-operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.
Objective:
To assess the accuracy of intraoperative frozen section of ovarian tumours at our institution and to identify the possible reasons for misdiagnosis.
Study design:
Between January 2002 and August 2013, a total of 684 patients were included in the study. Frozen section diagnosis was compared with the final paraffin section diagnosis as the gold standard. The sensitivity, specificity, and positive and negative predictive values of frozen-section diagnosis were calculated for benign, borderline and malignant tumours. Clinicopathological parameters influenced by misdiagnosis were evaluated performing multivariate logistic regression analysis.
Results:
The overall accuracy was detected as 96.1%. Frozen-section diagnoses of 26 patients (3.8%) showed discordance. The specificity (99.7%) and PPV (99.4%) of frozen-section diagnosis was highest in the malignant category. In BOTs, diagnostic agreement was observed in 57 of 70 (81.4%) cases. The PPV (81.4%) was lowest for these patients. Tumour diameter of ≥10 cm (OR [95% CI]= 3.0 [1.1 to 8.2]; P=0.030) and mucinous histology (OR [95% CI]= 2.5 [1.0 to 6.2]; P=0.042) were significant predictors of misdiagnosis. With the increase in the number of sections, the accuracy rate of frozen section diagnosis was decreased. While not statistically significant (p=0.361).
Conclusion:
The number of sections is increased parallel to increase in tumor diameters. On the contrary, the diagnostic accuracy was no significantly increased with an increase in the number of sections. This discrepancy may be associated with falling tumor size per frozen section. A prospective study based on a certain tumour diameter per frozen section may better demonstrate the positive effect of the number of sections.
A Fourier transform infrared/attenuated total reflectance technique has been developed to study protein adsorption onto surfaces. The application of this technique to an ex vivo model using a beagle dog as the source of whole, flowing blood is described (currently, high-quality infrared spectra are being collected at 5-s intervals of protein adsorption). This approach has enabled the authors to identify albumin and glycoproteins as the initially adsorbing species, with the subsequent competitive replacement of part of this protein layer with fibrinogen and other proteins. The exact relationship between the pattern of protein adsorption from whole blood and the generation of a thrombus (clot) is not yet clear, but it is hoped that this type of experimental approach will help clarify the relationship.
This article reviews some of the recent advances on FTIR spectroscopy in areas related to natural tissues and cell biology. It is the second review publication resulting from a detailed study on the applications of spectroscopic methods in biological studies and summarizes some of the most widely used peak frequencies and their assignments. The aim of these studies is to prepare a database of molecular fingerprints, which will help researchers in defining the chemical structure of the biological tissues introducing most of the important peaks present in the natural tissues. In spite of applying different methods, there seems to be a considerable similarity in defining the peaks of identical areas of the FTIR spectra. As a result, it is believed that preparing a unique collection of the frequencies encountered in FTIR spectroscopic studies can lead to significant improvements both in the quantity and quality of research and their outcomes. This article is the first review of its kind that provides a precise database on the most important FTIR characteristic peak frequencies for researchers aiming to analyze natural tissues by FTIR spectroscopy and will be of considerable assistance to those who are focusing on the analysis of cancerous tissues by FTIR spectroscopy.
Frozen section diagnosis provides critical information for immediate surgical management decision making. Over the last several years, there have been some significant advances in treatment of genitourinary cancer, particularly with regard to surgical techniques. These changes in turn impact the type and frequency of intraoperative frozen section requests. In this review, we describe the main indications and diagnostic challenges of frozen section diagnosis during surgeries of each genitourinary organ system including prostate, kidney, bladder, testis, and penis. The pitfalls and approaches to different diagnostic situations are discussed. It is also stressed that pathologists must not only be familiar with the histological diagnosis, but also understand the limitations of frozen section diagnosis and communicate with urologists during the intraoperative treatment decision making process.
As a molecular probe of tissue composition, infrared spectroscopic imaging can potentially serve as an adjunct to histopathology in detecting and diagnosing disease. This study demonstrates that human gliomas are distinguishable from control tissue on the basis of IR image used in combination with chemometric imaging processing. Using an iterative two-step algorithm – comprised of a linear discriminant analysis guided genetic optimal spectral region selection – tissue types can be discriminated from one another thus providing insight into the malignancy grade of the tissue. A series of classification models were built using a k-fold cross validation scheme and the classification predictions from the various models were combined to provide an aggregated prediction. The validation of the aggregated model reveals an improvement in the classification success rate to 64%.
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.
The discrimination of cell types is a crucial task in cell biology. Available techniques, based on an irreversible treatment of the cells, do not allow a sensitive label-free characterization under in situ conditions. Infrared spectroscopic imaging is a new and useful tool for studying individual cells. It has established itself as a powerful method to probe the molecular composition and to indicate the biochemistry of cells. Monolayers of cultivated U343, T1115 and T508 human glioma cells were characterized using infrared spectroscopic imaging. A classification algorithm based on linear discriminant analysis was developed to distinguish different cells without labeling. The classification is based upon spectral features which mainly arise from proteins, nucleic acids, and cholesterol. An accuracy of 91% and 84% was obtained for cells of U343 and T1115, respectively. Cells of the T508 cell line exhibit some misclassifications resulting in a lower accuracy rate of 73%. As the results demonstrate, the potential of infrared spectroscopic imaging method to assess the overall molecular composition of cells in a non-destructive manner opens the possibility to characterize cells on a molecular level without labels or an irreversible treatment.
The applications of infrared spectroscopy to pharmaceutical sciences is small compared to the applications of infrared spectroscopy to the fields of chemistry, biology, and biochemistry. This is unfortunate because modern routine infrared spectrometers are excellent research tools that provide very high signal-to-noise, high resolution, and extensive data-manipulation computer software packages. This review summarizes basic principles of infrared spectrometers and the use of Fourier self-deconvolution.
Collagen fibers impart tensile strength and transfer tension from bladder smooth muscle cells. We have previously shown that fibrotic bladders are characterized by an increased type III:type I collagen ratio. To determine the effect of decreased type III collagen on bladder function, type III collagen-deficient mice (COL3A1) were studied physiologically.
Bladders from wild-type (+/+) and heterozygous (+/-) COL3A1 mice were biochemically characterized to determine total collagen (hydroxyproline analysis) and collagen subtype concentration (cyanogen bromide digestion and ELISA). Alterations in collagen fiber diameter were assessed by electron microscopy. Bladder muscle strips were used to assess physiologic function.
Hydroxyproline content decreased in heterozygous bladders, which had 50% less type III collagen. Wild-type bladders had a biphasic distribution of collagen fiber sizes, whereas heterozygous bladder collagen fibers spanned a broad range. Physiologically, there were no differences in contractile responses between wild-type and heterozygotes when stimulated with ATP, carbachol or KCl, indicating normal contraction via purinergic and muscarinic receptors, and in response to direct membrane depolarization. In contrast, tension generation in heterozygotes was decreased after field stimulation (FS), indicating decreased synaptic transmission. Length-tension studies showed that the heterozygote muscle strips generated less tension per unit length, indicating that they were more compliant than wild-type controls.
Critical levels of type III collagen appear to be a requirement for normal bladder tension development and contraction. Our data show that a decrease in the type III:type I collagen ratio, and altered fiber size, results in a more compliant bladder with altered neurotransmitter function.