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Cancer Screening via Infrared Spectral Cytopathology (SCP): Results for the Upper Respiratory and Digestive Tracts
Abstract
Instrumental advances in infrared micro-spectroscopy have made possible the observation of individual human cells and even subcellular structures. The observed spectra represent a snapshot of the biochemical composition of a cell; this composition varies subtly but reproducibly with cellular effects such as progression through the cell cycle, cell maturation and differentiation, and disease. The aim of this summary is to provide a synopsis of the progress achieved in infrared spectral cytopathology (SCP) - the combination of infrared micro-spectroscopy and multivariate methods of analysis - for the detection of abnormalities in exfoliated human cells of the upper respiratory and digestive tract, namely the oral and nasopharyngeal cavities, and the esophagus.
... The authors concluded that infrared cytopathology of exfoliated cells from the oral cavity is a method of superior sensitivity for detecting oral diseases. Notably, the findings from this group indicate that FTIR cytopathology can detect biochemical changes in morphologically normal cells from patients with pre-cancerous oral diseases, providing strong support for the use of FTIR in early oral cancer detection [97]. ...
... Part III: Cells, inoculated tissues and human tissues. [90] Infrared microspectroscopy of Oral Squamous Cell Carcinoma: Spectral signatures of cancer grading [91] Fourier transform infrared imaging analysis in discrimination studies of squamous cell carcinoma [92] Fourier-transform-infrared-spectroscopy based spectral-biomarker selection towards optimum diagnostic differentiation of oral leukoplakia and cancer [93] FTIR-ATR and FT-Raman Spectroscopy for Biochemical Changes in Oral Tissue [94] Spectral cytopathology: new aspects of data collection, manipulation and confounding effects [95] Infrared micro-spectroscopy for cyto-pathological classification of esophageal cells [96] Oral cell studies Cancer Screening via Infrared Spectral Cytopathology (SCP): Results for the Upper Respiratory and Digestive Tracts [97] Chemometric analysis of integrated FTIR and Raman spectra obtained by non-invasive exfoliative cytology for the screening of oral cancer [98] In vitro FTIR microspectroscopy analysis of primary oral squamous carcinoma cells treated with cisplatin and 5-fluorouracil: a new spectroscopic approach for studying the drug-cell interaction [99] Oral cancer diagnostics based on infrared spectral markers and wax physisorption kinetics [100] ...
Oral cancer is one of the most common cancers worldwide. Despite easy access to the oral cavity and significant advances in treatment, the morbidity and mortality rates for oral cancer patients are still very high, mainly due to late-stage diagnosis when treatment is less successful. Oral cancer has also been found to be the most expensive cancer to treat in the United States. Early diagnosis of oral cancer can significantly improve patient survival rate and reduce medical costs. There is an urgent unmet need for an accurate and sensitive molecular-based diagnostic tool for early oral cancer detection. Fourier transform infrared spectroscopy has gained increasing attention in cancer research due to its ability to elucidate qualitative and quantitative information of biochemical content and molecular-level structural changes in complex biological systems. The diagnosis of a disease is based on biochemical changes underlying the disease pathology rather than morphological changes of the tissue. It is a versatile method that can work with tissues, cells, or body fluids. In this review article, we aim to summarize the studies of infrared spectroscopy in oral cancer research and detection. It provides early evidence to support the potential application of infrared spectroscopy as a diagnostic tool for oral potentially malignant and malignant lesions. The challenges and opportunities in clinical translation are also discussed.
... While cancerous cells can be collected from the surface, this does not constitute a diagnosis, which requires histological proof of breach of basement membrane. Despite this, brush biopsy may be very useful for patients with multiple oral lesions or for monitoring OPMDs A limited number of FTIR microspectroscopy studies have been reported, demonstrating the feasibility of diagnosis of oral cancers using single exfoliated cells prepared by cytocentrifugation onto low e microscope slides [13,[51][52][53][54]. Papamarkakis et al. demonstrated that FTIR microspectroscopy could classify oral cells according to anatomical site and that the compositional changes were attributed to the expression of keratins in the cells of the tongue and to the expression of collagen in the cells of the floor of the mouth [52]. ...
... Spectral differences in oral cells infected with the herpes simplex virus were also reported. Further studies from the same group showed that spectra from exfoliated cells from healthy volunteers could be discriminated from exfoliated cells from patients with oral dysplasia and cancer [13,53,54]. ...
Vibrational spectroscopy, based on either infrared absorption or Raman scattering, has attracted increasing attention for biomedical applications. Proof of concept explorations for diagnosis of oral potentially malignant disorders and cancer are reviewed, and recent advances critically appraised. Specific examples of applications of Raman microspectroscopy for analysis of histological, cytological and saliva samples are presented for illustrative purposes, and the future prospects, ultimately for routine, chairside in vivo screening are discussed.
... The most relevant bibliography involving FTIR cell analyses can be found in recent reviews [9,10]. Regarding the discrimination of cells from the diagnostic point of view, commonly referred to as Spectral Cytopathology (SCP), the most important works using FTIR microscopy are mainly related to Diem's collaborations [11][12][13][14][15][16][17][18]. All these studies are focused on smear cells directly extracted from different parts of the patients rather than in cell cultures. ...
... All these studies are focused on smear cells directly extracted from different parts of the patients rather than in cell cultures. They have covered different types of cancer pathologies: urine [14], cervix [15] or upper respiratory and digestive tract [16][17][18]. In most of them, cells were deposited on low-e slides and measured in transflection. ...
Fourier transform infrared (FTIR) spectroscopy is a highly versatile tool for cell and tissue analysis. Modern commercial FTIR microspectroscopes allow the acquisition of good-quality hyperspectral images from cytopathological samples within relatively short times. This study aims at assessing the abilities of FTIR spectra to discriminate different types of cultured skin cell lines by different computer analysis technologies. In particular, 22700 single skin cells, belonging to two non-tumoral and two tumoral cell lines, were analysed. These cells were prepared in three different batches that included each cell type. Different spectral preprocessing and classification strategies were considered, including the current standard approaches to reduce Mie scattering artefacts. Special care was taken for the optimisation, training and evaluation of the learning models in order to avoid possible overfitting. Excellent classification performance (balanced accuracy between 0.85 and 0.95) was achieved when the algorithms were trained and tested with the cells from the same batch. When cells from different batches were used for training and testing the balanced accuracy reached values between 0.35 and 0.6, demonstrating the strong influence of sample preparation on the results and comparability of cell FTIR spectra. A deep study of the most optimistic results was performed in order to identify perturbations that influenced the final classification.
... A quality control method for the National certification Program of Breast Centers certification was to ensure that patients received this document within six months after finishing their treatment [8]. Studies revealed that while the SCP was a helpful tool, it did not have the same impact on patient-reported results. ...
... FTIR hyperspectral images were taken of slices of oesophageal tissue taken at resection from patients with diagnoses on the continuum from normal squamous to oesophageal adenocarcinoma, including pre-cancerous An image of the reference H + E stained section at the same magnification as that in the chemical image (Â40) is provided for comparison spectroscopic methods with machine learning for similar classification tasks [18,28,42], especially considering the patient biopsy-based blind independent testing procedure used in this study. Importantly, no previous study using tissue biopsies for pathological classification with chemical imaging has employed a sample set with the scale of that employed in this study. ...
Fourier Transform Infrared (FTIR) based chemical imaging is a powerful, non‐destructive, label‐free biophotonic technique which spatially acquires bio‐molecularly relevant information in histopathology. Cancer detection with objective chemical imaging techniques is relatively well established, though detection of pre‐cancer stages within a continuum from normal tissue to cancer remains challenging. Here machine learning with chemical imaging was used to provide an objective classification pipeline for oesophageal tissues pathologically classified as normal, oesophagitis, dysplasia, Barrett’s disease and cancer. Spectral images were segmented using a k‐means cluster validity indices approach and clustered spectra were classified using partial least squares discriminant analysis. Classification performances approached a receiver operator characteristic area‐under‐the‐curve (ROC‐AUC) of 0.90 for binary classification tasks (eg, normal vs Barrett’s). Isolated histopathological substructures were identified which delivered a ROC‐AUC in of ~0.69 in classifying into each of the five‐classes. This work may provide the means to assist pathologist diagnoses of intermediate pre‐cancer stages.
... Only three specific cases are presented here, but we can see the similarity in all solid tumors. Previous studies also used HSI as a diagnostic tool in cancers of the prostate (34,(65)(66)(67), lung (43,(68)(69)(70), cervix (71), colon (39,44,67,(72)(73)(74)(75)(76), kidney (77), brain (78,79), skin (80,81), oral cavity (82), and bladder (83), etc. ...
Hyperspectral imaging (HSI) is an emerging new technology in solid tumor diagnosis and detection. It incorporates traditional imaging and spectroscopy together to obtain both spatial and spectral information from tissues simultaneously in a non-invasive manner. This imaging modality is based on the principle that different tissues inherit different spectral reflectance responses that present as unique spectral fingerprints. HSI captures those composition-specific fingerprints to identify cancerous and normal tissues. It becomes a promising tool for performing tumor diagnosis and detection from the label-free histopathological examination to real-time intraoperative assistance. This review introduces the basic principles of HSI and summarizes its methodology and recent advances in solid tumor detection. In particular, the advantages of HSI applied to solid tumors are highlighted to show its potential for clinical use.
... Max Diem used infrared microspectrocopy with the help of statistical analysis for detection of abnormalities in the exfoliate human cells in the upper respiratory and digestive tract [107]. His group [108] worked on 388 lung biopsies from 374 patients and correlates the classical with the infrared results. ...
Background:
Cancer is a major global health issue. It causes extensive individual suffering and gives a huge burden on the health care in society. Despite extensive research and different tools have been developed it still remains a challenge for early detection of this disease. FTIR imaging has been used to diagnose and differentiate the molecular differences between normal and diseased tissues.
Methods:
Fourier Transform Infrared Spectroscopy (FTIR) is able to measure biochemical changes in tissue, cell and biofluids based on the vibrational signature of their components. This technique enables to the distribution and structure of lipids, proteins, nucleic acids as well as other metabolites. These differences depended on the type and the grade of cancer.
Results:
We emphasize here, that the FTIR spectroscopy and imaging can be considered as a promising technique and will find its place on the detection of this dreadful disease because of high sensitivity, accuracy and inexpensive technique. Now the medical community started using and accepting this technique for early stage cancer detection. We discussed this technique and the several challenges in its application for the diagnosis of cancer in regards of sample preparations, data interpretation, and data analysis. The sensitivity of chemotherapy drugs on individual specific has also discussed.
Conclusion:
So far progressed has done with the FTIR imaging in understanding of cancer disease pathology. However, more research is needed in this field and it is necessary to understand the morphology and biology of the sample before using the spectroscopy and imaging because invaluable information to be figured out.
Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol takes 2 h 20 min for sample preparation, measurement and data preprocessing and up to 8 h for a complete analysis. A key feature of the protocol is that it uses the same sample preparation procedure as commonly used in diagnostic cytology laboratories (i.e., liquid-based cytology on glass slides), ensuring compatibility with clinical workflows. Our protocol also covers methods to correct for the spectral contribution of glass and sample pretreatment methods to remove contaminants (such as blood and mucus) that can obscure spectral features in the exfoliated cells and lead to variability. The protocol establishes a standardized clinical routine allowing the collection of highly reproducible data for Raman spectral cytopathology for cancer diagnostic applications for cervical and lung cancer and for monitoring suspicious lesions for oral cancer. This protocol details how to use Raman spectroscopy for cancer cytopathology diagnosis. Detailed instructions are provided for sample preparation (cervical, oral and lung exfoliated cells), data acquisition, preprocessing and analysis.
Results of studies of the use of FTIR spectroscopy in cancer diagnosis are reviewed. Materials studied include biological fluids, tissues, and model systems for studying experimental neoplasia. The methods for working with each system are described. A detailed description is given for the use of IR spectroscopy for cancer diagnosis and in the monitoring of cancer therapy for the evaluation of drug effectiveness and determination of the disease state. Statistical methods for processing the IR spectral data are presented. The main limitations to the use of IR spectroscopy for the diagnosis of oncological diseases and the potential for its introduction into clinical practice are described.
Fourier transform infrared (FTIR) spectroscopy techniques and data analyses have become widely available, easy to use, and convenient for studies of various biosamples, especially in biomedical science. Yet, cultivation of cells and purification of cell components is costly, often methodically challenging, and time and labor consuming. Therefore, reduction of sample amount is of high value. Here we propose a novel method for analysis of small quantities of biosamples by FTIR-microscopy of dry films using diamond-anvil cell (DAC). This approach allows to decrease the sample volume at least hundred times compared to a high-throughput screening device with zinc selenide microplates while still obtaining homogenous films, acquiring qualitative spectra, and using conventional 15x objective instead of ATR-objective. Both FTIR methods were applied for analyses of human colorectal cancer cell lines SW480 and SW620 cultured under hypoxic conditions to estimate the strengths and weaknesses of each approach. FTIR absorption spectra acquired by both methods were compared and no significant spectral differences were detected. It was shown that FTIR-microscopy of films on DAC can be used for evaluation, screening, discrimination and identification of biochemical markers in biosamples like cells. We conclude that the DAC can be transferred to other biosamples like tissues, biofluids, their components and extracellular matrix, and is especially valuable when the available quantities of biosamples is limited.
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.
We present a novel approach for detection of cancerous kidney tissue areas by measuring surface enhanced Raman scattering (SERS) spectra of extracellular fluid taken from kidney tissue. The method is based on spectral analysis of the cancerous and normal tissue areas in order to find the specific spectral markers. The samples were prepared by sliding the kidney tissue over a substrate—calcium fluoride optical window. For producing the SERS signal, the dried extracellular fluid film was covered by silver nanoparticle colloidal solution. In order to suppress fluorescence background, the measurements of the dried samples were performed in the NIR spectral region with the Raman excitation wavelength of 1,064 nm. The most significant spectral differences—spectral markers—were found in the wavenumber region between 400 and 1,800 cm−1, where spectral bands related to various vibrations of carbohydrates, amino acids, and nucleic acids are located. Spectral markers in the SERS spectra are different from those in IR absorption spectra. The SERS spectroscopic method has a potential to be used directly during the surgery.
Given the increased glucose uptake in cancer cells than normal cells, near-infrared (NIR) fluorescent glucose analogues have been previously synthesized and applied in cancer cell imaging. However, most NIR dyes usually have one or more charge in their structures, which may cause low cell membrane permeability and hamper their application in cell imaging. Here we report the synthesis and characterization of a series of DCPO-conjugated glucose analogues (N0-N4), which have no charge in their structures and have different lengths of the spacer arm. Experiments in different cancer cell lines showed the uptake of N0-N4 was dependent on the protein levels of GLUT-1. The distance between the dyes and glucose was adjusted by the length of PEG. Of these five glucose analogues, the length of the linker in N2 which contains a diethylene glycol was the most appropriate spacer arm, a longer or shorter linker exhibited reduced cellular uptake efficiency. Moreover, the uptake of DCPO-conjugated glucose analogues could be inhibited by phloretin, a GLUT-1 inhibitor or competitively inhibited by unlabeled d-glucose. Therefore, our study has reported a novel type of NIR-conjugated glucose analogues, whose cell permeability ensured the potential application for cancer cell bioimaging in the NIR region. We also demonstrated, for the first time, that the length of the linker between the dyes and glucose was also an important factor that will affect the delivery efficiency of the glucose analogues to cells.
Determination of cancerous and normal kidney tissues during partial, simple or radical nephrectomy surgery was performed by using differences in the IR absorption spectra of extracellular fluid taken from the corresponding tissue areas. The samples were prepared by stamping of the kidney tissue on ATR diamond crystal. The spectral measurements were performed directly in the OR during surgery for 58 patients. It was found that intensities of characteristic spectral bands of glycogen (880-1200 cm⁻¹) in extracellular fluid are sensitive to the type of the tissue and can be used as spectral markers of tumours. Characteristic spectral band of lactic acid (1730 cm⁻¹) - product of the anaerobic glycolysis, taking place in the cancer cells is not suitable for use as a spectral marker of cancerous tissue, since it overlaps with the band of carbonyl stretch in phospholipids and fatty acids. Results of hierarchical cluster analysis of the spectra show that the spectra of healthy and tumour tissue films can be reliably separated into two groups. On the other hand, possibility to differentiate between tumours of different types and grades remains in question. While the fluid from highly malignant G3 tumour tissue contains highly pronounced glycogen spectral bands and can be well separated from benign and G1 tumours by principal component analysis, the variations between spectra from sample to sample prevent from obtaining conclusive results about the grouping between different tumour types and grades. The proposed method is instant and can be used in situ and even in vivo.
This article summarizes the methods employed, and the progress achieved over the past two decades in applying vibrational (Raman and IR) micro-spectroscopy to problems of medical diagnostics and cellular biology. During this time, several research groups have verified the enormous information content of vibrational spectra; in fact, information on protein, lipid and metabolic composition of cells and tissues can be deduced by decoding the observed vibrational spectra. This decoding process is aided by the availability of computer workstations and advanced algorithms for data analysis. Furthermore, commercial instrumentation for the fast collection of both Raman and infrared micro-spectral data has enabled the collection of images of cells and tissues based solely on vibrational spectroscopic data. The progress in the field has been manifested by a steady increase in the number and quality of publications submitted by established and new research groups in vibrational spectroscopy in the biological and biomedical arenas.
We report results from a study utilizing infrared spectral cytopathology (SCP) to detect abnormalities in exfoliated esophageal cells. SCP has been developed over the past decade as an ancillary tool to classical cytopathology. In SCP, the biochemical composition of individual cells is probed by collecting infrared absorption spectra from each individual, unstained cell, and correlating the observed spectral patterns, and the variations therein, with against classical diagnostic methods to obtain an objective, machine-based classification of cells. In the past, SCP has been applied to the analysis and classification of cells exfoliated from the cervix and the oral cavity. In these studies, it was established that SCP can distinguish normal and abnormal cell types. Furthermore, SCP can differentiate between truly normal cells, and cells with normal morphology from the vicinity of abnormalities. Thus, SCP may be a valuable tool for the screening of early stages of dysplasia and pre-cancer.
The use of vibrational spectroscopy, FTIR and Raman, for cytology and cellular research has the potential to revolutionise the approach to cellular analysis. Vibrational spectroscopy is non-destructive, simple to operate and provides direct information. Importantly it does not require expensive exogenous labels that may affect the chemistry of the cell under analysis. In addition, the advent of spectroscopic microscopes provides the ability to image cells and acquire spectra with a subcellular resolution. This introductory review focuses on recent developments within this fast paced field and highlights potential for the future use of FTIR and Raman spectroscopy. We particularly focus on the development of live cell research and the new technologies and methodologies that have enabled this.
Transflection-mode FTIR spectroscopy has become a popular method of measuring spectra from biomedical and other samples due to the relative low cost of substrates compared to transmission windows, and a higher absorbance due to a double pass through the same sample approximately doubling the effective path length. In this publication we state an optical description of samples on multilayer low-e reflective substrates. Using this model we are able to explain in detail the so-called electric-field standing wave effect and rationalise the non-linear change in absorbance with sample thickness. The ramifications of this non-linear change, for imaging and classification systems, where a model is built from tissue sectioned at a particular thickness and compared with tissue of a different thickness are discussed. We show that spectra can be distorted such that classification fails leading to inaccurate tissue segmentation which may have subsequent implications for disease diagnostics applications.
This chapter describes a methodology to diagnose transmissible spongiform encephalopathy (TSE) of terminally ill or preclinically infected animals from serum by a combination of Fourier transform infrared (FT-IR) spectroscopy and chemometrics. Within the scope of experimental and field studies, it could be shown that scrapie infections in hamsters and BSE infections in cattle are associated with reproducible disease-related structural and compositional alterations in the sera of infected donors. Fingerprint analyses of the IR serum pattern by artificial neural networks allowed the identification of infected individuals with an accuracy of prediction of larger than 90%.
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.
To determine the accuracy and acceptability to patients of non-endoscopic screening for Barrett's oesophagus, using an ingestible oesophageal sampling device (Cytosponge) coupled with immunocytochemisty for trefoil factor 3.
Prospective cohort study.
12 UK general practices, with gastroscopies carried out in one hospital endoscopy unit.
504 of 2696 eligible patients (18.7%) aged 50 to 70 years with a previous prescription for an acid suppressant (H(2) receptor antagonist or proton pump inhibitor) for more than three months in the past five years.
Sensitivity and specificity estimates for detecting Barrett's oesophagus compared with gastroscopy as the ideal method, and patient anxiety (short form Spielberger state trait anxiety inventory, impact of events scale) and acceptability (visual analogue scale) of the test.
501 of 504 (99%) participants (median age 62, male to female ratio 1:1.2) successfully swallowed the Cytosponge. No serious adverse events occurred. In total, 3.0% (15/501) had an endoscopic diagnosis of Barrett's oesophagus (≥1 cm circumferential length, median circumferential and maximal length of 2 cm and 5 cm, respectively) with intestinal metaplasia. Compared with gastroscopy the sensitivity and specificity of the test was 73.3% (95% confidence interval 44.9% to 92.2%) and 93.8% (91.3% to 95.8%) for 1 cm or more circumferential length and 90.0% (55.5% to 99.7%) and 93.5% (90.9% to 95.5%) for clinically relevant segments of 2 cm or more. Most participants (355/496, 82%, 95% confidence interval 78.9% to 85.1%) reported low levels of anxiety before the test, and scores remained within normal limits at follow-up. Less than 4.5% (2.8% to 6.1%) of participants reported psychological distress a week after the procedure.
The performance of the Cytosponge test was promising and the procedure was well tolerated. These data bring screening for Barrett's oesophagus into the realm of possibility. Further evaluation is recommended.
Infrared spectroscopic cytology is potentially a powerful clinical tool. However, in order for it to be successful, practitioners must be able to extract reliably a pure absorption spectrum from a measured spectrum that often contains many confounding factors. The most intractable problem to date is the, so called, dispersion artefact which most prominently manifests itself as a sharp decrease in absorbance on the high wavenumber side of the amide I band in the measured spectrum, exhibiting a derivative-like line shape. In this paper we use synchrotron radiation FTIR micro-spectroscopy to record spectra of mono-dispersed poly(methyl methacrylate) (PMMA) spheres of systematically varying size and demonstrate that the spectral distortions in the data can be understood in terms of resonant Mie scattering. A full understanding of this effect will enable us to develop strategies for deconvolving the scattering contribution and recovering the pure absorption spectrum, thus removing one of the last technological barriers to the development of clinical spectroscopic cytology.
Aim
Spectral Cytopathology (SCP) is a novel spectroscopic method for objective and unsupervised classification of individual exfoliated cells. The limitations of conventional cytopathology are well-recognized within the pathology community. In SCP, cellular differentiation is made by observing molecular changes in the nucleus and the cytoplasm, which may or may not produce morphological changes detectable by conventional cytopathology. This proof of concept study demonstrates SCP’s potential as an enhancing tool for cytopathologists by aiding in the accurate and reproducible diagnosis of cells in all states of disease.
Method
Infrared spectra are collected from cervical cells deposited onto reflectively coated glass slides. Each cell has a corresponding infrared spectrum that describes its unique biochemical composition. Spectral data are processed and analyzed by an unsupervised chemometric algorithm, Principal Component Analysis (PCA).
Results
In this blind study, cervical samples are classified by analyzing the spectra of morphologically normal looking squamous cells from normal samples and samples diagnosed by conventional cytopathology with low grade squamous intraepithelial lesions (LSIL). SCP discriminated cytopathological diagnoses amongst twelve different cervical samples with a high degree of specificity and sensitivity. SCP also correlated two samples with abnormal spectral changes: these samples had a normal cytopathological diagnosis but had a history of abnormal cervical cytology. The spectral changes observed in the morphologically normal looking cells are most likely due to an infection with human papillomavirus, HPV. HPV DNA testing was conducted on five additional samples, and SCP accurately differentiated these samples by their HPV status.
Conclusions
SCP tracks biochemical variations in cells that are consistent with the onset of disease. HPV has been implicated as the cause of these changes detected spectroscopically. SCP does not depend on identifying the sparse number of morphologically abnormal cells within a large sample in order to make an accurate classification, as does conventional cytopathology. These findings suggest that the detection of cellular biochemical variations by SCP can serve as a new enhancing screening method that can identify earlier stages of disease.
Spectral cytopathology (SCP) is a novel approach for diagnostic differentiation of disease in individual exfoliated cells. SCP is carried out by collecting information on each cell's biochemical composition through an infrared micro-spectral measurement, followed by multivariate data analysis. Deviations from a cell's natural composition produce specific spectral patterns that are exclusive to the cause of the deviation or disease. These unique spectral patterns are reproducible and can be identified and used through multivariate statistical methods to detect cells compromised at the molecular level by dysplasia, neoplasia, or viral infection. In this proof of concept study, a benchmark for the sensitivity of SCP is established by classifying healthy oral squamous cells according to their anatomical origin in the oral cavity. Classification is achieved by spectrally detecting cells with unique protein expressions: for example, the squamous cells of the tongue are the only cell type in the oral cavity that have significant amounts of intracytoplasmic keratin, which allows them to be spectrally differentiated from other oral mucosa cells. Furthermore, thousands of cells from a number of clinical specimens were examined, among them were squamous cell carcinoma, malignancy-associated changes including reactive atypia, and infection by the herpes simplex virus. Owing to its sensitivity to molecular changes, SCP often can detect the onset of disease earlier than is currently possible by cytopathology visualization. As SCP is based on automated instrumentation and unsupervised software, it constitutes a diagnostic workup of medical samples devoid of bias and inconsistency. Therefore, SCP shows potential as a complementary tool in medical cytopathology.
Infrared spectra of single biological cells often exhibit the 'dispersion artefact' observed as a sharp decrease in intensity on the high wavenumber side of absorption bands, in particular the Amide I band at approximately 1655 cm(-1), causing a downward shift of the true peak position. The presence of this effect makes any biochemical interpretation of the spectra unreliable. Recent theory has shed light on the origins of the 'dispersion artefact' which has been attributed to resonant Mie scattering (RMieS). In this paper a preliminary algorithm for correcting RMieS is presented and evaluated using simulated data. Results show that the 'dispersion artefact' appears to be removed; however, the correction is not perfect. An iterative approach was subsequently implemented whereby the reference spectrum is improved after each iteration, resulting in a more accurate correction. Consequently the corrected spectra become increasingly more representative of the pure absorbance spectra. Using this correction method reliable peak positions can be obtained.
In this report the applicability of an improved method of image segmentation of infrared microspectroscopic data from histological specimens is demonstrated. Fourier transform infrared (FT-IR) microspectroscopy was used to record hyperspectral data sets from human colorectal adenocarcinomas and to build up a database of spatially resolved tissue spectra. This database of colon microspectra comprised 4120 high-quality FT-IR point spectra from 28 patient samples and 12 different histological structures. The spectral information contained in the database was employed to teach and validate multilayer perceptron artificial neural network (MLP-ANN) models. These classification models were then employed for database analysis and utilised to produce false colour images from complete tissue maps of FT-IR microspectra. An important aspect of this study was also to demonstrate how the diagnostic sensitivity and specificity can be specifically optimised. An example is given which shows that changes of the number of teaching patterns per class can be used to modify these two interrelated test parameters. The definition of ANN topology turned out to be crucial to achieve a high degree of correspondence between the gold standard of histopathology and IR spectroscopy. Particularly, a hierarchical scheme of ANN classification proved to be superior for the reliable classification of tissue spectra. It was found that unsupervised methods of clustering, specifically agglomerative hierarchical clustering (AHC), were helpful in the initial phases of model generation. Optimal classification results could be achieved if the class definitions for the ANNs were carried out by considering the classification information provided by cluster analysis.
We report microscopically collected infrared spectra of cells found in human urine in an effort to develop automatic methods for bladder cancer screening. Unsupervised multivariate analysis of the observed spectral patterns reveals distinct spectral classes, which correlated very well with visual cytology. Therefore, we believe that spectral analysis of individual cells can aid cytology in rendering reliable diagnoses based on objective measurements and discriminant algorithms.
This chapter presents novel microscopic methods to monitor cell biological processes of live or fixed cells without the use of any dye, stains, or other contrast agent. These methods are based on spectral techniques that detect inherent spectroscopic properties of biochemical constituents of cells, or parts thereof. Two different modalities have been developed for this task. One of them is infrared micro-spectroscopy, in which an average snapshot of a cell's biochemical composition is collected at a spatial resolution of typically 25 mum. This technique, which is extremely sensitive and can collect such a snapshot in fractions of a second, is particularly suited for studying gross biochemical changes. The other technique, Raman microscopy (also known as Raman micro-spectroscopy), is ideally suited to study variations of cellular composition on the scale of subcellular organelles, since its spatial resolution is as good as that of fluorescence microscopy. Both techniques exhibit the fingerprint sensitivity of vibrational spectroscopy toward biochemical composition, and can be used to follow a variety of cellular processes.
Fourier-transform IR (FT-IR) spectra of pelleted exfoliated cervical cells from patients with cervical cancer or dysplasia differ from those from normal women. To study the origin of these spectral changes, we obtained the FT-IR spectra of individual cervical cells from normal, dysplastic, and malignant cervical samples. Ninety five percent of normal superficial and intermediate cells displayed two distinct spectral patterns designated A and B, and 5% displayed an intermediate pattern, suggesting extensive structural heterogeneity among these cells. Parabasal and endocervical cells showed pattern B spectra. The spectra of malignant, dysplastic, and other abnormal cells also were characterized. Analysis of FT-IR spectra of over 2, 000 individual cells from 10 normal females, 7 females with dysplasia, and 5 females with squamous cell carcinoma revealed that the spectra of normal-appearing intermediate and superficial cells of the cervix from women with either dysplasia or cancer differed from those of normal women. Chemometric and classical spectroscopic analysis showed a continuum of changes paralleling the transition from normalcy to malignancy. These findings suggest that (i) the structural changes underlying the spectroscopic changes are involved in or are a product of cervical carcinogenesis and (ii) the neoplastic process may be more extensive than currently recognized with morphological criteria. This approach may be useful for the structural study of neoplasia and also may be of help in the diagnosis or classification of cervical disorders.
Chemometrics in Analytical Spectroscopy provides students and practising analysts with a tutorial guide to the use and application of the more commonly encountered techniques used in processing and interpreting analytical spectroscopic data. In detail the book covers the basic elements of univariate and multivariate data analysis, the acquisition of digital data and signal enhancement by filtering and smoothing, feature selection and extraction, pattern recognition, exploratory data analysis by clustering, and common algorithms in use for multivariate calibration techniques. An appendix is included which serves as an introduction or refresher in matrix algebra. The extensive use of worked examples throughout gives Chemometrics in Analytical Spectroscopy special relevance in teaching and introducing chemometrics to undergraduates and post-graduates undertaking analytical science courses. It assumes only a very moderate level of mathematics, making the material far more accessible than other publications on chemometrics. The book is also ideal for analysts with little specialist background in statistics or mathematical methods, who wish to appreciate the wealth of material published in chemometrics.
Modern Vibrational Spectroscopy and Micro-Spectroscopy: Theory, Instrumentation and Biomedical Applications unites the theory and background of conventional vibrational spectroscopy with the principles of microspectroscopy. It starts with basic theory as it applies to small molecules and then expands it to include the large biomolecules which are the main topic of the book with an emphasis on practical experiments, results analysis and medical and diagnostic applications. This book is unique in that it addresses both the parent spectroscopy and the microspectroscopic aspects in one volume. Part I covers the basic theory, principles and instrumentation of classical vibrational, infrared and Raman spectroscopy. It is aimed at researchers with a background in chemistry and physics, and is presented at the level suitable for first year graduate students. The latter half of Part I is devoted to more novel subjects in vibrational spectroscopy, such as resonance and non-linear Raman effects, vibrational optical activity, time resolved spectroscopy and computational methods. Thus, Part 1 represents a short course into modern vibrational spectroscopy. Part II is devoted in its entirety to applications of vibrational spectroscopic techniques to biophysical and bio-structural research, and the more recent extension of vibrational spectroscopy to microscopic data acquisition. Vibrational microscopy (or microspectroscopy) has opened entirely new avenues toward applications in the biomedical sciences, and has created new research fields collectively referred to as Spectral Cytopathology (SCP) and Spectral Histopathology (SHP). In order to fully exploit the information contained in the micro-spectral datasets, methods of multivariate analysis need to be employed. These methods, along with representative results of both SCP and SHP are presented and discussed in detail in Part II.
Spectral cytopathology (SCP) is a robust and reproducible diagnostic technique that employs infrared spectroscopy and multivariate statistical methods, such as principal component analysis to interrogate unstained cellular samples and discriminate changes on the biochemical level. In the past decade, SCP has taken considerable strides in its application for disease diagnosis. Cultured cell lines have proven to be useful model systems to provide detailed biological information to this field; however, the effects of sample fixation and storage of cultured cells are still not entirely understood in SCP. Conventional cytopathology utilizes fixation and staining methods that have been established and widely accepted for nearly a century and are focused on maintaining the morphology of a cell. Conversely, SCP practices must implement fixation protocols that preserve the sample's biochemical composition and maintain its spectral integrity so not to introduce spectral changes that may mask variance significant to disease. It is not only necessary to evaluate the effects on fixed exfoliated cells but also fixed cultured cells because although they are similar systems, they exhibit distinct differences. We report efforts to study the effects of fixation methodologies commonly used in traditional cytopathology and SCP including both fixed and unfixed routines applied to cultured HeLa cells, an adherent cervical cancer cell line. Data suggest parallel results to findings in Part 1 of this series for exfoliated cells, where the exposure time in fixative and duration of sample storage via desiccation contribute to minor spectral changes only. The results presented here reinforce observations from Part 1 indicating that changes induced by disease are much greater than changes observed as a result of alternate fixation methodologies. Principal component analysis of HeLa cells fixed via the same conditions and protocols as exfoliated cells (Part 1) yield nearly identical results. More importantly, the overall conclusion is that it is necessary that all samples subjected to comparative analysis should be prepared identically because although changes are minute, they are present. F or the past decade, infrared (IR) microspectroscopy has climbed its way to being considered a competitive alternative to conventional cytopathology practices. Traditional cytopathology includes the inspection of stained cells, visually measuring predetermined parameters, such as nucleus-to-cytoplasm (N/C) ratio, staining patterns, morphology of nuclear membrane, etc., and assigning a diagnosis based on these parameters. 1,2 IR microspectroscopy is at the forefront of new methods being developed because it is a label free and reproducible method that evaluates a physical measurement, the biochemical composition, of each unstained cell; the term " spectral cytopathology (SCP) " has been coined to describe the combination of microscopic infrared data acquisition and analysis of the spectral data via multivariate methods. 3−5 After IR acquisition, samples can then be subjected to traditional staining protocols and evaluated via conventional cytopathol-ogy means to compare results from both techniques. Since the early successes of SCP, many groups have begun investigating cultured cell lines to provide additional information regarding disease diagnosis and biological information. 6−8 Cultured cells serve several purposes ranging from distinguishing between different cell lines to their behavior in understanding disease and evaluation of drug effects and uptake. 8−10 Cultured cell lines offer a microscopic model system to explore and probe mechanisms, pathways, drug interactions, etc. Most importantly, diseased cells can be potentially biopsied from a patient's organ and propagated in cell culture conditions to be investigated thoroughly. 8,11 Often fixation procedures are applied to preserve cells for extended periods; however, the spectral effects of fixation on cultured cells are not entirely understood. 12 Previous reports claim fixation protocols introduce large spectral changes and obstruct proper analyses, speculating fixation methods as obstacles to be avoided. 13−15 This is the second paper in a series aimed at addressing the effects of fixation and storage conditions on spectral data of cellular samples. In the first paper, we described the influence of these factors to exfoliated oral (buccal) mucosa cells and demonstrated that exceedingly small variances occurred upon various fixation methods that were negligible in comparison to
Background & Aims: Endoscopic screening and periodic surveillance for patients with Barrett's esophagus has been shown to be cost-effective in patients with esophageal dysplasia, with treatment for esophageal cancer limited to esophagectomy. Most gastroenterologists refer patients with high-grade dysplasia for esophagectomy, and effective endoscopic therapies are available for nonoperative patients with esophageal cancer. The cost-effectiveness of screening strategies that incorporate these nonsurgical treatment modalities has not been determined. Methods: We designed a Markov model to compare lifetime costs and life expectancy for a cohort of 50-year-old men with chronic reflux symptoms. We compared 10 clinical strategies incorporating combinations of screening and surveillance protocols (no screening, screening with periodic surveillance for both dysplastic and nondysplastic Barrett's esophagus, or periodic surveillance for dysplasia only), treatment for high-grade dysplasia (esophagectomy or intensive surveillance), and treatment for cancer (esophagectomy or surgical and endoscopic treatment options). Results: Screening and surveillance of patients with both dysplastic and nondysplastic Barrett's esophagus followed by esophagectomy for surgical candidates with high-grade dysplasia or esophageal cancer and endoscopic therapy for cancer patients who were not operative candidates cost $12,140 per life-year gained compared to no screening. Other screening strategies, including strategies that had no endoscopic treatment options, were either less effective at the same cost, or equally effective at a higher cost. Conclusions: The cost-effectiveness of screening and subsequent surveillance of patients with dysplastic as well as nondysplastic Barrett's esophagus followed by endoscopic or surgical therapy in patients who develop cancer compares favorably to many widely accepted screening strategies for cancer.
Due to chronic duodeno-gastro-esophageal reflux, the epithelial lining of the esophagus can undergo a change to a more intestinal like type of epithelium. This condition, referred to as Barrett’s esophagus, markedly increases the risk of developing esophageal adenocarcinoma [1]. This type of cancer is rapidly increasing in western society and shows a very high mortality, primarily due to late detection. Early detection of malignancy can significantly increase survival rates. Most detection methods depend on histological examination from endoscopically collected biopsies. However, there are no endoscopically clearly observable differences between healthy tissue, metaplasia and severe displasia. Therefore it is necessary to randomly collect biopsies, with a significant risk of missing malignant degenerations.
This paper presents a short review on the improvements in data processing for spectral cytopathology, the diagnostic method developed for large scale diagnostic analysis of spectral data of individual dried and fixed cells. This review is followed by the analysis of the confounding effects introduced by utilizing reflecting "low-emissivity" (low-e) slides as sample substrates in infrared micro-spectroscopy of biological samples such as individual dried cells or tissue sections. The artifact introduced by these substrates, referred to as the "standing electromagnetic wave" artifact, indeed, distorts the spectra noticeably, as postulated recently by several research groups. An analysis of the standing wave effect reveals that careful data pre-processing can reduce the spurious effects to a level where they are not creating a major problem for spectral cytopathology and spectral histopathology.
The use of near-infrared Raman spectroscopy to interrogate epithelial tissue biochemistry and hence distinguish between normal and abnormal tissues was investigated. Six different epithelial tissues from the larynx, tonsil, oesophagus, stomach, bladder and prostate were measured. Spectral diagnostic models were constructed using multivariate statistical analysis of the spectra to classify samples of epithelial cancers and pre-cancers. Tissues were selected for clinical significance and to include those which develop into carcinoma from squamous, transitional or columnar epithelial cells. Rigorous histopathological protocols were followed and mixed pathology tissue samples were discarded from the study. Principal component fed linear discriminant models demonstrated excellent group separation, when tested by cross-validation. Larynx samples, with squamous epithelial tissue, were separated into three distinct groups with sensitivities ranging from 86 to 90% and specificities from 87 to 95%. Bladder specimens, containing transitional epithelial tissue, were separated into five distinct groups with sensitivities of between 78 and 98% and specificities between 96 and 99%. Oesophagus tissue can contain both squamous and columnar cell carcinomas. A three group model discriminated the columnar cell pathological groups with sensitivities of 84–97% and specificities of 93–99%, and an eight group model combining both columnar and squamous tissues in the oesophagus was able to discriminate pathologies with sensitivities of 73–100% and specificities of 92–100%. It is likely that any overlap between pathology group predictions will have been due to a combination of the difficulty in histologically distinguishing between pre-cancerous states and the fact that there is no biochemical boundary from one pathological group to the next, i.e. there is believed to be a continuum of progression from the normal to the diseased state. Copyright © 2002 John Wiley & Sons, Ltd.
Spectral cytology, the diagnosis of disease based on objective physical measurements on individual cells and subsequent computer‐based algorithmic interpretation, promises to provide faster and more reliable results than classical cytology. The measurements described in this review are based on well‐established vibrational microspectroscopic techniques, which provide a snapshot of the biochemical composition of a cell, or parts thereof. The spectral data are subsequently diagnosed by unsupervised and supervised methods of multivariate analysis.
More than any other recent attempts to improve on cytology, these spectral methods exhibit exquisite sensitivity toward small changes in cellular conditions. In fact, these cellular conditions, for example, fixation or proliferation state, have to be controlled very carefully to eliminate spurious effects. This chapter provides the ground work for later applications of this methodology in medical diagnostics. Furthermore, the application of novel research tools, such as confocal Raman microscopy, followed by cluster analysis of the hyperspectral data sets, and their relevance to research in cellular biology are described.
This chapter outlines the use of Raman spectroscopy for discrimination of pathologies in the bladder and esophagus based upon the biochemical signature accompanying the disease process. This has great strength in that objective molecular-specific analysis becomes possible for diagnosis and understanding of carcinogenesis processes. The importance of the gold-standard histopathology, sample handling procedures and spectrometer standardization are outlined. Methods of sampling in vivo and in vitro are discussed and discrimination methodologies are demonstrated. Finally exploitation of the inherent biochemical signature found within the tissues is explored and an attempt is made to provide relative concentrations of significant biochemical components.
Keywords:
esophagus;
bladder;
Raman;
biochemistry;
standardization;
histopathology;
discrimination;
cancer;
dysplasia
This paper explores different phenomena that cause distortions of infrared absorption spectra by mixing of reflective and absorptive band shape components of infrared spectra, and the resulting distortion of observed band shapes. In the context of this paper, we refer to the line shape of the variations of the refractive index in spectral regions of an absorption maximum (i.e., in regions of "anomalous dispersion") as "dispersive" or "reflective" line shape contributions, in analogy to previous spectroscopic literature. These distortions usually result in asymmetric bands with a negative intensity contribution at the high wavenumber of the band, accompanied by a shift toward lower wavenumber, and confounded band intensities. In extreme cases of band distortions caused by the "resonance Mie" (RMie) mechanism, spectral peaks may be split into doublets of peaks, change from positive to negative peaks, or appear as derivative-shaped features.
We investigated the hormonal influences on cervical cells using infrared microspectroscopy and found that there were observable spectral changes occurring throughout the cycle. The main differences were seen in the glycogen region (1200–1000 cm−1) and the greatest cyclical variation was observed in spectra of ectocervical cells of women not taking any form of oral contraception. Ectocervical cells from women taking monophasic contraception and endocervical cells from both groups did not display the same degree of variation. Principal component analysis revealed that, although there is cyclical variation, these cells are normal and discrimination between histologically normal and abnormal (high-grade dysplasia) cells was maintained.
In this paper we describe the advantages of collecting infrared microspectral data in imaging mode opposed to point mode. Imaging data are processed using the PapMap algorithm, which co-adds pixel spectra that have been scrutinized for R-Mie scattering effects as well as other constraints. The signal-to-noise quality of PapMap spectra will be compared to point spectra for oral mucosa cells deposited onto low-e slides. Also the effects of software atmospheric correction will be discussed. Combined with the PapMap algorithm, data collection in imaging mode proves to be a superior method for spectral cytopathology. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
IntroductionMethods
Results and DiscussionConclusions
AcknowledgmentsReferences
We report a computational method to remove or reduce dispersion artifacts from infrared microspectral data collected in transflection (reflection/absorption) mode. This artifact occurs along the edges of tissue samples, in particular if the tissue does not adhere well to the substrate. The method proposed for the removal of the artifact is similar to the phase correction used in standard Fourier transform infrared spectroscopy.
We have optimized an imaging methodology capable of monitoring individual live HeLa cells using non-synchrotron FTIR in an aqueous environment. This methodology, in combination with MATLAB based pre-processing techniques, allows fast and efficient collection of data with high signal-to-noise ratio in comparison with previous methods using point mode data collection, which required manual operation and more collection time. Also, presented are early results that illustrate interpretable spectral differences from live cells treated with chemotherapeutic drugs, demonstrating the potential of this methodology to develop more desirable modes of treatment for patients in their diagnoses and treatments for disease.
Spectral cytopathology (SCP) is a novel approach for disease diagnosis that utilizes infrared spectroscopy to interrogate the biochemical components of cellular samples and multivariate statistical methods, such as principal component analysis, to analyze and diagnose spectra. SCP has taken vast strides in its application for disease diagnosis over the past decade; however, fixation-induced changes and sample handling methods are still not systematically understood. Conversely, fixation and staining methods in conventional cytopathology, typically involving protocols to maintain the morphology of cells, have been documented and widely accepted for nearly a century. For SCP, fixation procedures must preserve the biochemical composition of samples so that spectral changes significant to disease diagnosis are not masked. We report efforts to study the effects of fixation protocols commonly used in traditional cytopathology and SCP, including fixed and unfixed methods applied to exfoliated oral (buccal) mucosa cells. Data suggest that the length of time in fixative and duration of sample storage via desiccation contribute to minor spectral changes where spectra are nearly superimposable. These findings illustrate that changes influenced by fixation are negligible in comparison to changes induced by disease.
Reprint requests Address requests for reprints to: Chair, Clinical Practice and Quality Management Committee, AGA National Office, 4930 Del Ray Avenue, Bethesda, Maryland 20814. e-mail: drobin@gastro.org; telephone: (301) 272-1189.
Infrared absorption spectra of formalin-fixed, paraffin-embedded human cervical tissue are reported for normal, dysplastic and neoplastic samples. The spectral differences found in this study between these states of the tissues are far less than those observed for single cells by us and others. Nevertheless, we find a direct correspondence between spectral data from tissue sections, obtained from biopsies, and individual exfoliated cells, typically obtained during a pap procedure. We also find that spectra due to dysplastic samples fall about halfway between the spectral features of normal and cancerous samples.
Early detection of (pre-)cancerous changes improves prognosis, therefore in the UK patients at high risk of developing gastrointestinal cancers are enrolled on endoscopic surveillance programmes or the Bowel Cancer Screening Programme. The current gold standard technique for the detection of pre-cancerous changes in the gastrointestinal tract is histopathological analysis of biopsy tissue collected at endoscopy. This relies upon subjective assessment of morphological changes within the excised tissue samples and poor targeting of pre-malignant lesions. Raman spectroscopy offers a number of potential advantages for in vivo assessment of tissue at endoscopy. The performance of a custom built Raman probe as a biopsy targeting tool has been evaluated using excised biopsy material. Multivariate classification models have been used to demonstrate the likely ability of a miniature, confocal, fibre optic Raman probe to be used as an optical biopsy tool at endoscopy to provide spectral information in clinically practicable timescales. This technique could facilitate improved targeting of excisional biopsy with associated clinical benefits.
Fourier Transform Infrared (FT-IR) spectroscopic imaging is emerging as an automated alternative to human examination in studying development and disease in tissue. The technology's speed and accuracy, however, are limited by the trade-off with signal-to-noise ratio (SNR). Signal processing approaches to reduce noise have been suggested but often involve manual decisions, compromising the automation benefits of using spectroscopic imaging for tissue analysis. In this manuscript, we describe an approach that utilizes the spatial information in the data set to select parameters for noise reduction without human input. Specifically, we expand on the Minimum Noise Fraction (MNF) approach in which data are forward transformed, eigenimages that correspond mostly to signal selected and used in inverse transformation. Our unsupervised eigenimage selection method consists of matching spatial features in eigenimages with a low-noise gold standard derived from the data. An order of magnitude reduction in noise is demonstrated using this approach. We apply the approach to automating breast tissue histology, in which accuracy in classification of tissue into different cell types is shown to strongly depend on the SNR of data. A high classification accuracy was recovered with acquired data that was ∼10-fold lower SNR. The results imply that a reduction of almost two orders of magnitude in acquisition time is routinely possible for automated tissue classifications by using post-acquisition noise reduction.
This study evaluates the potential of near-infrared Raman spectroscopy for in vivo detection of squamous dysplasia, a precursor to cervical cancer. A pilot clinical trial was carried out at three
clinical sites. Raman spectra were measured from one colposcopically normal and one abnormal area of the cervix. These sites were then biopsied and submitted for routine histologic analysis. Twenty-four
evaluable measurements were made in vivo in 13 patients. Cervical tissue Raman spectra contain peaks in the vicinity of 1070, 1180, 1195, 1210, 1245, 1330, 1400, 1454, 1505, 1555, 1656, and 1760
cm-1. The ratio of intensities at 1454 to 1656 cm-1 is greater for squamous dysplasia than all other tissue types, while the ratio of intensities at 1330 to 1454 cm-1 is
lower for samples with squamous dysplasia than all other tissue types. A simple algorithm based on these two intensity ratios separates high-grade squamous dysplasia from all others, misclassifying only
one sample. Spectra measured in vivo resemble those measured in vitro. Cervical epithelial cells may contribute to tissue spectra at 1330 cm-1, a region associated with DNA. In
contrast, epithelial cells probably do not contribute to tissue spectra at 1454 cm-1, a region associated with collagen and phospholipids.
To assess the expression of estrogen receptor (ER) in oral mucosa and salivary glands, buccal mucosal biopsies from ten postmenopausal women (taken before and during the hormone replacement therapy), as well as, single biopsies from 20 healthy 19-year-old women were analyzed for ER expression. Salivary gland biopsies were taken from the minor labial salivary glands (n=6), submandibular glands (n=5) and parotid gland (n=1) from women at different ages.
total RNA extracted from the tissue samples was reverse-transcripted (RT) to single-stranded cDNA, and the RT-polymerase chain reaction (RT-PCR) product was subjected to nucleotide sequencing to confirm the match with ER cDNA. Immunohistochemistry (IHC) with a monoclonal ER antibody (ER-ICA, Abbott) and Western blot analysis with monoclonal antibody against ER-related antigen (ER-D5, Amersham) were performed on the biopsies taken from the postmenopausal women.
ER mRNA was expressed in 18/20 (90%) and 20/20 (100%) of the mucosal biopsies in the postmenopausal and 19-year-old women, respectively. The expression of mRNA was detected in all the submandibular gland samples, in the single parotid gland, as well as, in 4/6 (67%) of the labial glands. ER expression could not be detected by IHC, indicating either a very low level of expressed protein or difficulties in recognizing the epitopes by IHC. However, Western blot demonstrated 8/8 (100%) of the mucosal biopsies of postmenopausal women positive for ER-related antigen.
the presence of ER mRNA and immunoreactive ER protein suggests that estrogens have a biological role in oral mucosa and salivary glands.
Despite a critical presumption of reliability, standards of interpathologist agreement have not been well defined for interpretation of cervical pathology specimens.
To determine the reproducibility of cytologic, colposcopic histologic, and loop electrosurgical excision procedure (LEEP) histologic cervical specimen interpretations among multiple well-trained observers.
The Atypical Squamous Cells of Undetermined Significance-Low-grade Squamous Intraepithelial Lesion (ASCUS-LSIL) Triage Study (ALTS), an ongoing US multicenter clinical trial.
From women enrolled in ALTS during 1996-1998, 4948 monolayer cytologic slides, 2237 colposcopic biopsies, and 535 LEEP specimens were interpreted by 7 clinical center and 4 Pathology Quality Control Group (QC) pathologists.
kappa Values calculated for comparison of the original clinical center interpretation and the first QC reviewer's masked interpretation of specimens.
For all 3 specimen types, the clinical center pathologists rendered significantly more severe interpretations than did reviewing QC pathologists. The reproducibility of monolayer cytologic interpretations was moderate (kappa = 0.46; 95% confidence interval [CI], 0.44-0.48) and equivalent to the reproducibility of punch biopsy histopathologic interpretations (kappa = 0.46; 95% CI, 0.43-0.49) and LEEP histopathologic interpretations (kappa = 0.49; 95% CI, 0.44-0.55). The lack of reproducibility of histopathology was most evident for less severe interpretations.
Interpretive variability is substantial for all types of cervical specimens. Histopathology of cervical biopsies is not more reproducible than monolayer cytology, and even the interpretation of LEEP results is variable. Given the degree of irreproducibility that exists among well-trained pathologists, realistic performance expectations should guide use of their interpretations.
Fourier transform IR (FTIR) microspectroscopy at a spatial resolution of 18 microm was used to study skin fibroblasts and giant sarcoma cells. Both cell lines were derived from the same patient; they were metabolically active and in the exponentially growing phase. The IR spectra were acquired for the nuclei and cytosol of untreated cells, cells washed with ethanol, and cells treated with RNase or DNase. A comparison of the spectra of the two cell lines yielded only insignificant spectral differences, indicating that IR spectroscopy monitors the overall cell activity rather than specific signs of cancer.
The combination of synchrotron IR microspectroscopy and fluorescence microscopy has led to the identification of specific IR signatures of apoptosis and necrosis at a single cell level. Apoptosis was induced by treatment of Fas+ tumor cell lines with anti-Fas monoclonal antibodies. Detection of the early and late stages of apoptosis was performed using conjugated annexin V-fluorescein isothiocyanate (AV-FITC) and propidium iodide. Very early cellular changes were detected by IR before externalization of phosphatidylserine and AV-FITC labeling, and they were probably linked to DNA unwinding. The IR signals at 1044, 1177, and 1222 cm(-1), as well as an intensity variation in the CHx stretching region, are the main signature changes of early and late apoptosis, in line with the hypothesis of DNA fragmentation. The increased intensity of the CHx stretching bands of the lipids was observed only at an early stage of apoptosis. Changes in the relative intensity of CH3 and CH2 stretching accompany this increased intensity, suggesting changes in the relative amount and/or type of lipids concomitant with an increased lipid content. Finally, necrotic cells were characterized by marked changes in their chemical composition because several new vibrational features were observed.
Endoscopic screening and periodic surveillance for patients with Barrett's esophagus has been shown to be cost-effective in patients with esophageal dysplasia, with treatment for esophageal cancer limited to esophagectomy. Most gastroenterologists refer patients with high-grade dysplasia for esophagectomy, and effective endoscopic therapies are available for nonoperative patients with esophageal cancer. The cost-effectiveness of screening strategies that incorporate these nonsurgical treatment modalities has not been determined.
We designed a Markov model to compare lifetime costs and life expectancy for a cohort of 50-year-old men with chronic reflux symptoms. We compared 10 clinical strategies incorporating combinations of screening and surveillance protocols (no screening, screening with periodic surveillance for both dysplastic and nondysplastic Barrett's esophagus, or periodic surveillance for dysplasia only), treatment for high-grade dysplasia (esophagectomy or intensive surveillance), and treatment for cancer (esophagectomy or surgical and endoscopic treatment options).
Screening and surveillance of patients with both dysplastic and nondysplastic Barrett's esophagus followed by esophagectomy for surgical candidates with high-grade dysplasia or esophageal cancer and endoscopic therapy for cancer patients who were not operative candidates cost $12,140 per life-year gained compared to no screening. Other screening strategies, including strategies that had no endoscopic treatment options, were either less effective at the same cost, or equally effective at a higher cost.
The cost-effectiveness of screening and subsequent surveillance of patients with dysplastic as well as nondysplastic Barrett's esophagus followed by endoscopic or surgical therapy in patients who develop cancer compares favorably to many widely accepted screening strategies for cancer.
Incidence of nasopharyngeal carcinoma has remained high in endemic regions. Diagnosing the disease in the early stages requires a high index of clinical acumen and, although most cross-sectional imaging investigations show the tumour with precision, confirmation is dependent on histology. Epstein-Barr virus (EBV)-encoded RNA signal is present in all nasopharyngeal carcinoma cells, and early diagnosis of the disease is possible through the detection of raised antibodies against EBV. The quantity of EBV DNA detected in blood indicates the stage and prognosis of the disease. Radiotherapy with concomitant chemotherapy has increased survival, and improved techniques (such as intensity-modulated radiotherapy), early detection of recurrence, and application of appropriate surgical salvage procedures have contributed to improved therapeutic results. Screening of high-risk individuals in endemic regions together with developments in gene therapy and immunotherapy might further improve outcome.
High-resolution endoscopy (HRE) may improve the detection of early neoplasia in Barrett's esophagus. Indigo carmine chromoendoscopy (ICC) and narrow-band imaging (NBI) may be useful techniques to complement HRE. The aim of this study was to compare HRE-ICC with HRE-NBI for the detection of high-grade dysplasia or early cancer (HGD/EC) in patients with Barrett's esophagus.
Twenty-eight patients with Barrett's esophagus underwent HRE-ICC and HRE-NBI (separated by 6 - 8 weeks) in a randomized sequence. The two procedures were performed by two different endoscopists, who were blinded to the findings of the other examination. Targeted biopsies were taken from all detected lesions, followed by four-quadrant biopsies at 2-cm intervals. Biopsy evaluation was supervised by a single expert pathologist, who was blinded to the imaging technique used.
Fourteen patients were diagnosed with HGD/EC. The sensitivity for HGD/EC was 93 % and 86 % for HRE-ICC and HRE-NBI, respectively. Targeted biopsies had a sensitivity of 79 % with HRE alone. HGD was diagnosed from random biopsies alone in only one patient. ICC and NBI detected a limited number of additional lesions occult to HRE, but these lesions did not alter the sensitivity for identifying patients with HGD/EC.
In most patients with high-grade dysplasia or early cancer in Barrett's esophagus, subtle lesions can be identified with high-resolution endoscopy. Indigo carmine chromoendoscopy and narrow-band imaging are comparable as adjuncts to high-resolution endoscopy.
For decades, the incidence rates for squamous cell carcinoma of the esophagus and adenocarcinoma of the distal stomach have been declining while the rates for adenocarcinomas of the esophagus and gastric cardia have increased profoundly. Recent studies have shown that the gastroesophageal junction (GEJ) is regularly exposed to concentrated gastric acid and to a variety of nitrosating species, noxious agents that may contribute to carcinogenesis in this region. For adenocarcinomas that straddle the GEJ, it can be difficult to determine whether the tumor originated in the esophagus or in the gastric cardia. This classification problem hampers studies on the epidemiology and pathogenesis of GEJ tumors. Current concepts in the prevention of cancers of the distal esophagus and proximal stomach have emerged from advances in our understanding of the specific molecular events that occur during the evolution of these tumors. This report reviews those events and focuses on current concepts in the prevention of adenocarcinomas at the GEJ. The similarities and differences in risk factors, molecular pathogenesis, and in preventive strategies for adenocarcinomas of the esophagus and gastric cardia are highlighted.
We have previously reported spectral differences for cells at different stages of the eukaryotic cell division cycle. These differences are due to the drastic biochemical and morphological changes that occur as a consequence of cell proliferation. We correlate these changes in FTIR absorption and Raman spectra of individual cells with their biochemical age (or phase in the cell cycle), determined by immunohistochemical staining to detect the appearance (and subsequent disappearance) of cell-cycle-specific cyclins, and/or the occurrence of DNA synthesis. Once spectra were correlated with their cells' staining patterns, we used methods of multivariate statistics to analyze the changes in cellular spectra as a function of cell cycle phase.
We report results from a study of human and canine mucosal cells, investigated by infrared micro-spectroscopy, and analyzed by methods of multivariate statistics. We demonstrate that the infrared spectra of individual cells are sensitive to the stage of maturation, and that a distinction between healthy and diseased cells will be possible. Since this report is written for an audience not familiar with infrared micro-spectroscopy, a short introduction into this field is presented along with a summary of principal component analysis.
There is now a clear causal relationship between symptomatic gastroesophageal reflux and esophageal adenocarcinoma (Lagergren et al, 1999). The risk factor is now identified as Barrett's metaplasia (Solaymani et al, 2004). Chronic reflux results in Barrett's metaplastic change, and the route to carcinoma is a stepwise progression, through dysplasia to invasive carcinoma (Jankowski et al, 2000). Earlier-stage disease is found in patients undergoing surveillance and is the major predictor of survival following surgery (Fountoulakis et al, 2004). Screening and surveillance by endoscopic biopsy regimen has profound implications for the allocation of healthcare resources and the provision of clinical services. Screening a high-risk group such as men with gastroesophageal reflux disease (GERD) will result in the detection of more patients with Barrett's esophagus, many of whom are asymptomatic. Once detected, questions remain as to surveillance intervals and the current methodology for surveillance. There are profound challenges with the accurate endoscopic and pathologic detection and categorization of Barrett's metaplasia, dysplasia , and, indeed, cancer. New endoscopic detection methods are being investigated to improve the diagnosis and definition of the premalignant phenotype. The detection of dysplasia requires increased surveillance and usually intervention either endoscopically or with surgery.