Browsing by Author "Stone, Nicholas"

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    Advancing the clinical application of Raman spectroscopic diagnosis of oesophageal pre-malignancies
    (Cranfield University, 2009-01) Hutchings, J.; Stone, Nicholas; Kendall, Catherine; Barr, H.
    Raman spectroscopy is a technique that utilises inelastic scattering processes to provide a biochemical fingerprint that has been shown to successfully discriminate oesophageal pathologies. The aim of this study was to develop Raman spectroscopy as a clinical tool; both in vivo for ‘targeted biopsy’, and in ex vivo for ‘automated histopathology’. Two different Raman probes were evaluated and compared and tissue classification models generated ex vivo. A preliminary classification model of a novel single collection fibre probe demonstrated potential for the probe design. Both probes were shown to discriminate three different oesophageal pathology groups. A cross-validated tissue classification model (88 samples) discriminated normal, Barrett’s and neoplasia with an overall accuracy of 86.5% with a sensitivity of 83.3-89.5% and specificity of 89.2-97.1%. A novel rapid Raman mapping technique was evaluated. It was shown that sufficient biochemical information for pathology diagnosis could be extracted from low signal to noise ratio data using multivariate analysis providing the dataset was sufficiently large, thus demonstrating the feasibility of automated histopathology in a clinically realistic time frame. Furthermore, it was demonstrated that high spatial resolution imaging was not necessarily required for automated histopathology using novel interpretation of multivariate techniques. A tissue classification model generated from two rapid Raman maps containing separated substrate, normal, HGD, luminescence and fibrous connective tissue with an overall training performance of 97.5% Problems limiting clinical implementation of Raman techniques were investigated and methods of overcoming devised.
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    Anisotropy visualisation from X-ray diffraction of biological apatite in mixed phase calcified tissue samples
    (Springer, 2025-02-14) Scott, Robert; Lyburn, Iain D.; Cornford, Eleanor; Bouzy, Pascaline; Stone, Nicholas; Greenwood, Charlene; Gosling, Sarah; Arnold, Emily L.; Bouybayoune, Ihsanne; Pinder, Sarah E.; Rogers, Keith
    X-ray diffraction is widely used to characterise the mineral component of calcified tissue. Broadening of the diffraction peaks yields valuable information on the size of coherently diffracting domains, sometimes loosely described as crystallite size or crystallinity. These domains are markedly anisotropic, hence a single number describing their size is misleading. We present a novel variation on a method for visualising crystallographic anisotropy in X-ray diffraction data. This provides an intuitively interpretable depiction of crystalline domain size and anisotropy. The new method involves creating a polar plot of calculated domain thickness for peaks in a diffractogram versus crystallographic direction. Points with the least error are emphasised. Anisotropic domain dimensions are calculated by refining an ellipsoidal model in a whole pattern fit. These dimensions are then used to overlay an ellipse on the peak broadening plot. This is illustrated by application of the method to calcifications in breast tissue with suspected cancer, which frequently contain whitlockite as well as nanocrystalline apatite. Like most biogenic apatite, this exhibits markedly anisotropic peak broadening. The nature of this anisotropy offers potentially useful information on normal function and pathology of calcified tissue and is a frequently neglected crystallographic feature of these materials.
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    Applications of Raman spectroscopy to urology
    (Cranfield University, 2006-07) Hart Prieto, Maria Consuelo; Wright, M.; Ritchie, A. W.; Stone, Nicholas
    Raman spectroscopy is an optical technique that can interrogate biological tissues. In doing so it gives us an understanding of the changes in the molecular structure that are associated with disease development. The Kerr gating technique uses a picosecond pulsed laser and fast temporal gating of inelastically (Raman) scattered light. The tissue samples used were taken following fully informed consent and ethics approval. Bladder samples were obtained by taking a biopsy during a TURBT or TURP, prostate samples were taken during TURP and the liver and kidney (pigs) were bought at a supermarket. The bladder and prostate samples were snap frozen in liquid nitrogen and stored in an -80°C freezer until required for experimentation. The liver and kidney tissue were used fresh. The constituent samples were bought from Sigma – Aldrich. Multivariate and least squares analysis were used to ascertain the biochemical basis of the differing pathologies within the bladder and the prostate gland, as well as to test diagnostic algorithms produced by a colleague in our group. Depth profiling through the bladder and prostate gland was shown to be feasible by utilizing the Kerr gating technique as was the suppression of fluorescence from dark tissue (liver and kidney). We have shown for the first time, that we can utilise Raman spectroscopy to determine the biochemical basis of pathologies of the bladder and the prostate gland. With the help of the Kerr gating technique we also obtained spectra from different depths through them. We also suppressed fluorescence and resonantly enhanced Raman spectra from dark tissue. These have major implications in terms of understanding pathogenesis and disease progression and also the potential to accurately assess depth of tumour invasion.
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    Breast calcification micromorphology classification
    (British Institute of Radiology, 2022-07-25) Robert Scott, Robert Scott; Iain Lyburn, Iain Lyburn; Cornford, Eleanor; Bouzy, Pascaline; Stone, Nicholas; Greenwood, Charlene; Bouybayoune, Ihsanne; Pinder, Sarah; Rogers, Keith
    Objectives: The importance of consistent terminology in describing the appearance of breast calcifications in mammography is well recognised. Imaging of calcifications using electron microscopy is a globally growing field of research. We therefore suggest that the time is ripe to develop a lexicon of terms for classifying the micromorphology of breast calcifications. Methods: Calcifications within a wide range of histological sections of breast tissue, both benign and malignant, were imaged by Scanning Electron Microscopy (SEM). These images were examined, and the micromorphology of calcifications present was grouped to create a classification system. Results: Based on the appearance of the calcifications observed, we propose five main categories for classification of the micromorphology of breast calcifications, namely Dense Homogenous, Punctulate, Banded, Spongy, and Aggregate. Conclusions: Use of the descriptive categories outlined here will help to ensure consistency and comparability of published observations on the micromorphology of breast calcifications. Advances in knowledge: This is the first time a lexicon and classification system has been proposed for the micromorphology of breast calcifications, as observed by scanning electron microscopy of histological sections. This will facilitate comparability of observed relationships between micromorphology, mammographic appearance, chemistry, and pathology.
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    Calcification microstructure reflects breast tissue microenvironment
    (Springer, 2019-12-05) Gosling, Sarah; Scott, Robert; Greenwood, Charlene; Bouzy, Pascaline; Nallala, Jayakrupakar; Lyburn, Iain Douglas; Stone, Nicholas; Rogers, Keith
    Microcalcifications are important diagnostic indicators of disease in breast tissue. Tissue microenvironments differ in many aspects between normal and cancerous cells, notably extracellular pH and glycolytic respiration. Hydroxyapatite microcalcification microstructure is also found to differ between tissue pathologies, including differential ion substitutions and the presence of additional crystallographic phases. Distinguishing between tissue pathologies at an early stage is essential to improve patient experience and diagnostic accuracy, leading to better disease outcome. This study explores the hypothesis that microenvironment features may become immortalised within calcification crystallite characteristics thus becoming indicators of tissue pathology. In total, 55 breast calcifications incorporating 3 tissue pathologies (benign – B2, ductal carcinoma in-situ - B5a and invasive malignancy - B5b) from archive formalin-fixed paraffin-embedded core needle breast biopsies were analysed using X-ray diffraction. Crystallite size and strain were determined from 548 diffractograms using Williamson-Hall analysis. There was an increased crystallinity of hydroxyapatite with tissue malignancy compared to benign tissue. Coherence length was significantly correlated with pathology grade in all basis crystallographic directions (P < 0.01), with a greater difference between benign and in situ disease compared to in-situ disease and invasive malignancy. Crystallite size and non-uniform strain contributed to peak broadening in all three pathologies. Furthermore, crystallite size and non-uniform strain normal to the basal planes increased significantly with malignancy (P < 0.05). Our findings support the view that tissue microenvironments can influence differing formation mechanisms of hydroxyapatite through acidic precursors, leading to differential substitution of carbonate into the hydroxide and phosphate sites, causing significant changes in crystallite size and non-uniform strain.
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    Critique of fourier transform infrared microspectroscopy applications to prostate pathology diagnosis
    (Cranfield University, 2010-01) Aning, Jonathan; Stone, Nicholas
    Prostate cancer is a biologically heterogenous disease with considerable variation in clinical aggressiveness. Gleason grade, the universally accepted method for classification of prostate cancer, is subjective and gives limited predictive information regarding prostate cancer progression. There is a clinical need for an objective, reliable tool to help pathologists improve current prostate tissue analysis methods and better assess the malignant potential of prostate tumours. Fourier Transform Infrared (FTIR) microspectroscopy is a powerful bioanalytical technique that uses infrared light to interrogate biological tissue. The studies detailed in this thesis examine the ability of FTIR combined with multivariate analysis to discriminate between benign, premalignant and malignant prostate pathology in snap frozen, paraffinated and deparaffinated tissue. Prostate tissue was collected during and after urological procedures performed between 2005 and 2008. The tissue was analysed utilising a bench top FTIR system in point and image mapping modes. The histology under interrogation was identified by a uro- pathologist. Multivariate analysis was applied to the spectral dataset obtained. FTIR performance was evaluated. FTIR was able to reproducibly discriminate between benign and malignant prostate tissue in a pilot study. Cross validated diagnostic algorithms, constructed from the spectral dataset in this experiment, achieved sensitivities and specificities of 95% and 89% respectively. FTIR analysis of transverse paraffinated and deparaffinated radical prostatectomy sections achieved good differentiation of the benign, premalignant and malignant pathology groups. However the performance of diagnostic algorithms constructed from this dataset under cross validation was poor. The work in this thesis illustrates the potential of FTIR to provide an objective method to assist the pathologist in the assessment of prostate samples. The limitations of the technique and directions for future work are presented.
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    The development of novel adjuncts to aid in the diagnosis of Epithelial Misplacement
    (Cranfield University, 2013-06) Carey, Duane Owen; Kendall, Catherine; Breckon, Toby P.; Shepherd, Neil A.; Stone, Nicholas
    Epithelial Misplacement (EM) is a benign phenomenon that occurs within polyps most commonly associated with the sigmoid colon. It is brought about because of the colons convulsive nature and this forces a polyps surface epithelium into its submucosa and also causes bleeding. This is problematic as the Bowel Cancer Screening Programme (BCSP) uses positive Faecal Occult Blood (FOB) test results to identify patients that require pathological review. As EM polyps bleed, they get selected for assessment and this results in them being sectioned and stained. In these cross sections, submucosal glandular tissue will be found that looks like it has formed due to metastatic mechanisms. This can lead to ambiguous diagnoses that will cause some patients to undergo unnecessary surgery. It is postulated that this can be prevented if the continuity of the EM samples could be measured. This is because only in the EM cases will the submucosal epithelial tissue remain in continuity with the surface. To test this, volumes representative of 9 samples of cancer and 13 cases of EM were segmented and their number of 26 three dimensional (3D) connected components were recorded. These were used with the 99% confidence limits of the two tailed Mann Whitney U Statistic and tested the null hypothesis that the cancer cases were as connected as the EM samples. In this instance, no significant differences were found and so the benefit of measuring the connectivity of these pathologies is questionable. It was because of this that Immunohistochemical (IHC) alternatives were considered. It was found that Collagen IV antibody staining correctly differentiated nine samples of EM from ten cases of cancer. The Mann Whitney U Statistic found this to be highly significant, p < 0.001, and future investigations should concentrate on automating this analysis. Although, Collagen IV provided a good classification it relied upon the subjective assessment of a pathologist. Therefore, the use of epithelial specific IR spectra was also investigated and this enabled the eleven EM and nine cancer cases that were investigated to be accurately classified 80% of the time upon cross validation. The collection of epithelial specific spectra relied upon a novel digital staining technique that has much application within future research. This study demonstrates that the intermodal registration of complementary modalities is of benefit to the disease classification problem. This technique has potential to be used in the correct identification of EM but more work is required.
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    Elemental and phase composition of breast calcifications
    (2017-06-14) Scott, Robert; Rogers, Keith; Kendall, Catherine; Stone, Nicholas
    Despite the importance of calcifications in early detection of breast cancer, and their proposed association with tumour growth, remarkably little detail is known about their chemical composition, or how this relates to pathology. One reason for this gap is the difficulty of systematically and precisely locating calcifications for analysis, particularly in sections taken from diagnostic archives. Two simple methods were developed which can achieve this in sections cut from wax embedded breast tissue. These are based on micro-CT and x-ray fluoroscopy mapping, and were used to locate calcifications for further study. The elemental composition of calcifications in histological sections was measured using energy-dispersive x-ray spectroscopy in an environmental scanning electron microscope. Variations in Ca:P ratio could in principle be detected non-invasively by dual energy absorptiometry, as demonstrated in a proof of principle experiment. However, the Ca:P ratio was found to lie in a narrow range similar to bone, with no significant difference between benign and malignant. In contrast, a substantial and significant difference in Na:Ca ratio was found between benign and malignant specimens. This has potential for revealing malignant changes in the vicinity of a core needle biopsy. The phase composition and crystallographic parameters within calcifications was measured using synchrotron x-ray diffraction. This is the first time crystallite size and lattice parameters have been measured in breast calcifications, and it was found that these both parallel closely the changes in these parameters with age observed in foetal bone. It was also discovered that these calcifications contain a small proportion of magnesium whitlockite, and that this proportion increases from benign, to carcinoma in-situ, to invasive cancer. When combined with other recent evidence on the effect of magnesium on hydroxyapatite precipitation, this suggests a mechanism explaining observations that carbonate levels within breast calcifications are lower in malignant specimens.
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    Elemental vs. phase composition of breast calcifications
    (Nature Publications, 2017-03-09) Scott, Robert; Kendall, Catherine; Stone, Nicholas; Rogers, Keith
    Despite the importance of calcifications in early detection of breast cancer, and their suggested role in modulating breast cancer cell behaviour, very little detail is known about their chemical composition or how this relates to pathology. We measured the elemental composition of calcifications contained within histological sections of breast tissue biopsies, and related this to both crystallographic parameters measured previously in the same specimens, and to the histopathology report. The Ca:P ratio is of particular interest since this theoretically has potential as a non-invasive aid to diagnosis; this was found to lie in a narrow range similar to bone, with no significant difference between benign and malignant. The Mg:Ca ratio is also of interest due to the observed association of magnesium whitlockite with malignancy. The initially surprising inverse correlation found between whitlockite fraction and magnesium concentration can be explained by the location of the magnesium in calcified tissue. Sodium was also measured, and we discovered a substantial and significant difference in Na:Ca ratio in the apatite phase between benign and malignant specimens. This has potential for revealing malignant changes in the vicinity of a core needle biopsy.
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    FT-infrared spectroscopic studies of lymphoma, lymphoid and myeloid leukaemia cell lines
    (2007-12-31T00:00:00Z) Babrah, Jaspreet; McCarthy, Keith P.; Lush, Richard; Rye, Adam D.; Bessant, Conrad M.; Stone, Nicholas; Dietrich, Schweitzer; Maryann, Fitzmaurice
    This paper presents a novel method to characterise spectral differences that distinguish leukaemia and lymphoma cell lines. This is based on objective spectral measurements of major cellular biochemical constituents and multivariate spectral processing. Fourier transform infrared (FT-IR) maps of the lymphoma, lymphoid and myeloid leukaemia cell samples were obtained using a Perkin-Elmer Spotlight 300 FT-IR imaging spectrometer. Multivariate statistical techniques incorporating principal component analysis (PCA) and linear discriminant analysis (LDA) were used to construct a mathematical model. This model was validated for reproducibility. Multivariate statistical analysis of FTIR spectra collected for each cell sample permit a combination of unsupervised and supervised methods of distinguishing cell line types. This resulted in the clustering of cell line populations, indicating distinct bio-molecular differences. Major spectral differences were observed in the 4000 to 800 cm- 1 spectral region. Bands in the averaged spectra for the cell line were assigned to the major biochemical constituents including; proteins, fatty acids, carbohydrates and nucleic acids. The combination of FT-IR spectroscopy and multivariate statistical analysis provides an important insight into the fundamental spectral differences between the cell lines, which differ according to the cellular biochemical composition. These spectral differences can serve as potential biomarkers for the differentiation of leukaemia and lymphoma cells. Consequently these differences could be used as the basis for developing a spectral method for the detection and identification of haematological malignancies.
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    The further assessment of a handheld Raman spectroscopy probe for the intraoperative diagnosis of axillary lymph nodes in breast cancer
    (Cranfield University, 2012-09) Tydeman, C.; Stone, Nicholas; Bristol, J.; Chan, C.
    Axillary sentinel lymph node biopsy plays an important role in breast cancer management in determining further surgical and medical treatment options. Intraoperative assessment of the sentinel lymph node might allow immediate axillary surgery, which would incur benefits to both the patient and healthcare trusts. A handheld Raman spectroscopy probe has already been shown to be a comparable option for intraoperative assessment through previous published and unpublished studies, delivering a sensitivity of up to 92% and specificity of up to 99%. This research aims to define further the role of the hand-held Raman spectroscopy probe as an accurate, rapid and non-destructive technique for intra-operative axillary node assessment, making it a strong competitor in the clinical market. It also looks to improve the sensitivity of the probe by altering the methodology used in previous studies. 122 lymph node halves were collected intraoperatively from 37 patients diagnosed with breast cancer and spectra measured using a commercially available handheld Raman spectroscopy probe. Spectra were then fed into a specialist software programme and analysed using principal component fed linear discriminant analysis trained by histopathology results. A “2 group” training model defining the probe‟s ability to distinguish between benign and malignant tissue produced an overall performance of 86.4%, with a sensitivity of 71% and specificity of 91%. The results were not as impressive as previous studies. This was possibly due to a broken probe, leading to four different phases of measurements (original probe/failing probe/temporary replacement/mended probe). Secondly a smaller, less balanced data set, in terms of spectra per pathology group, was collected and there appeared to be more fluorescence in some of the data which may have originated from varying blue dye injection protocols. However, that said further research using a robust, high specification system may help establish its role as a reliable assessment tool intraoperatively as well as a non-invasive means of assessing lymph nodes in the initial assessment clinic.
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    A multi-modal exploration of heterogeneous physico–chemical properties of DCIS breast microcalcifications
    (Royal Society of Chemistry, 2022-03-21) Gosling, Sarah; Calabrese, Doriana; Nallala, Jayakrupakar; Greenwood, Charlene; Pinder, Sarah; King, Lorraine; Marks, Jeffrey; Pinto, Donna; Lynch, Thomas; Lyburn, Iain Douglas; Hwang, Shelley; Grand Challenge PRECISION Consortium; Rogers, Keith; Stone, Nicholas
    Ductal carcinoma in situ (DCIS) is frequently associated with breast calcification. This study combines multiple analytical techniques to investigate the heterogeneity of these calcifications at the micrometre scale. X-ray diffraction, scanning electron microscopy and Raman and Fourier-transform infrared spectroscopy were used to determine the physicochemical and crystallographic properties of type II breast calcifications located in formalin fixed paraffin embedded DCIS breast tissue samples. Multiple calcium phosphate phases were identified across the calcifications, distributed in different patterns. Hydroxyapatite was the dominant mineral, with magnesium whitlockite found at the calcification edge. Amorphous calcium phosphate and octacalcium phosphate were also identified close to the calcification edge at the apparent mineral/matrix barrier. Crystallographic features of hydroxyapatite also varied across the calcifications, with higher crystallinity centrally, and highest carbonate substitution at the calcification edge. Protein was also differentially distributed across the calcification and the surrounding soft tissue, with collagen and β-pleated protein features present to differing extents. Combination of analytical techniques in this study was essential to understand the heterogeneity of breast calcifications and how this may link crystallographic and physicochemical properties of calcifications to the surrounding tissue microenvironment.
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    Optimisation of machine learning methods for cancer detection using vibrational spectroscopy
    (Cranfield University, 2011-01) Sattlecker, Martine; Bessant, Conrad; Stone, Nicholas
    Early cancer detection drastically improves the chances of cure and therefore methods are required, which allow early detection and screening in a fast, reliable and inexpensive manner. A prospective method, featuring all these characteristics, is vibrational spectroscopy. In order to take the next step towards the development of this technology into a clinical diagnostic tool, classification and imaging methods for an automated diagnosis based on spectral data are required. For this study, Raman spectra, derived from axillary lymph node tissue from breast cancer patients, were used to develop a diagnostic model. For this purpose different classification methods were investigated. A support vector machine (SVM) proved to be the best choice of classification method since it classified 100% of the unseen test set correctly. The resulting diagnostic models were thoroughly tested for their robustness to the spectral corruptions that would be expected to occur during routine clinical analysis. It showed that sufficient robustness is provided for a future diagnostic routine application. SVMs demonstrated to be a powerful classifier for Raman data and due to that they were also investigated for infrared spectroscopic data. Since it was found that a single SVM was not capable of reliably predicting breast cancer pathology based on tissue calcifications measured by infrared micro-spectroscopy a SVM ensemble system was implemented. The resulting multi-class SVM ensemble predicted the pathology of the unseen test set with an accuracy of 88.9%, in comparison a single SVM assessed with the same unseen test set achieved 66.7% accuracy. In addition, the ensemble system was extended for analysing complete infrared maps obtained from breast tissue specimens. The resulting imaging method successfully detected and staged calcification in infrared maps. Furthermore, this imaging approach revealed new insights into the calcification process in malignant development, which was not previously well understood.
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    Raman Spectroscopy of Biological Tissue for Application in Optical Diagnosis of Malignancy
    (Cranfield University, 2010-03) Orr, L. E.; Stone, Nicholas; Tothill, Ibtisam E.; Thomas, M.
    The use of Raman spectroscopy in the detection and classification of malignancy within the human larynx and lymph nodes of the head and neck has been evaluated. Currently histopathology is considered the diagnostic gold standard. The potential for Raman spectroscopy to be used as an in vivo diagnostic tool in the detection of dysplasia and malignancy has been demonstrated. A consensus opinion from three expert histopathologists has been obtained and spectral diagnostic models developed by correlation with these results. The ability of Raman spectroscopy to differentiate between disease entities and normal tissue within the larynx has been shown. Raman spectroscopy was able to identify non-neoplastic vocal cord mucosa (sensitivity 85 %, specificity 95%) from laryngeal mucosa showing neoplastic change (sensitivity 95 %, specificity 85%) with an increase in sensitivity to 89% for the non-neoplastic tissue and a reduction to73% in tissues showing neoplastic changes after cross-validation. For the first time benign changes in the structure of vocal cords such as those exhibiting hyperkeratosis and hyperplasia, where also identified with sensitivity of 97.9% for tissue exhibiting hyperplasia/hyperkeratosis and 100% for normal squamous cell epithelium. Research into the ability of Raman spectroscopy to interrogate lymphoid tissue in order to differentiate reactive nodes (sensitivity 90 %, specificity 88%) from those containing cancer (sensitivity 88 %, specificity 90%) was successful and fully independently validated. This work was further developed and the efficacy of Raman spectroscopy in differentiating between squamous cell carcinoma (sensitivity 76%, specificity 95%), adenocarcinoma (sensitivity 93 %, specificity 99%), Hodgkin‘s lymphoma (sensitivity 80%, specificity 90%) and reactive lymph nodes (sensitivity 81%, specificity 88%) was shown. This model was also independently cross-validated by node producing further improvements to give a spectral performance of sensitivity/specificity for SCC of 75/97%, adenocarcinoma 100/99%, Hodgkin‘s lymphoma 83/92% and reactive lymph nodes 85/86%.
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    Raman spectroscopy of biological tissue for application in optical diagnosis of malignancy
    (2009-11-25T17:41:59Z) Stone, Nicholas; Ahmad, S. R.; Barr, Hugh
    The utilisation of near-infraredR aman spectroscopyfo r the discrimination of cancersa nd pre-cancers from normal tissue in the acro-digestive tract has been evaluated. A commercially available Raman microspectrometehr as been modified to provide optimum throughput, sensitivity and fluorescence suppression for epithelial tissue measurements. Laser excitation at 830nmw as demonstratedto be optimum. High quality (SN ratio 15-20) NIR-Raman spectrah ave been acquired from oesophageaal nd laryngeal tissues in time scales under 30 seconds. Pathological groupings covering the full range of normal and neoplastic tissues in the organs of interest have been studied. Both fresh (snap frozen) and formalin fixed tissue samples were investigated,f irstly to indicate whether tissue-typesc an be distinguishedi n vivo and secondlyt o demonstrateth e use of Raman spectroscopya s a tool for classificationi n the pathology lab. Results using multivariate statistical techniques to distinguish between spectra from specimens exhibiting different tissue pathologies have been extremely promising. Crossvalidation of the spectral predictive models has shown that three groups of larynx tissue can be separated with sensitivities and specificities of between 86 and 90% and 87 and 95% respectively. Oesophageal prediction models have demonstrated sensitivities and specificities of 84 to 97% and 93 to 98% respectively for a three-group consensus model and 73 to 100% and 92 to 100% for an eight-groupc onsensusm odel. Epithelial tissues including stomach, tonsil, endometrium, bladder and prostate have been studiedt o identify further tissuesw hereR amans pectroscopym ay be employedf or detection of disease.S pectraw ere similar to those obtainedf rom oesophagusa nd larynx, although sufficiently different for distinct discriminant models to be required. This work has demonstratedth e genericn atureo f Ramans pectroscopyfo r the detectiona nd classification of cancersa nd pre-cancerousle sionsi n many tissues.T he evidencep rovided by this study indicatest hat utilisation of Ramans pectroscopyfo r non-invasived etectiona nd classification of diseaseis a distinct possibility. Potentiald ifficulties in the transferabilityf rom in vitro to in vivo have been evaluated and no significant barriers have been observed. However, further in vivo probe development and optimisation will be required before 'optical biopsy' with Ramans pectroscopyc anb ecomea reality.
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    Relationships between pathology and crystal structure in breast calcifications: an in situ X-ray diffraction study in histological sections
    (2016-09-28) Scott, Robert; Stone, Nicholas; Kendall, Catherine; Geraki, Kalotina; Rogers, Keith
    Calcifications are not only one of the most important early diagnostic markers of breast cancer, but are also increasingly believed to aggravate the proliferation of cancer cells and invasion of surrounding tissue. Moreover, this influence appears to vary with calcification composition. Despite this, remarkably little is known about the composition and crystal structure of the most common type of breast calcifications, and how this differs between benign and malignant lesions. We sought to determine how the phase composition and crystallographic parameters within calcifications varies with pathology, using synchrotron X-ray diffraction. This is the first time crystallite size and lattice parameters have been measured in breast calcifications, and we found that these both parallel closely the changes in these parameters with age observed in fetal bone. We also discovered that these calcifications contain a small proportion of magnesium whitlockite, and that this proportion increases from benign to in situ to invasive cancer. When combined with other recent evidence on the effect of magnesium on hydroxyapatite precipitation, this suggests a mechanism explaining observations that carbonate levels within breast calcifications are lower in malignant specimens.
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    Study of calcification formation and disease diagnostics utilising advanced vibrational spectroscopy
    (Cranfield University, 2012-10) Kerssens, Marleen Maartje; Stone, Nicholas; Matousek, Pavel; Rogers, Keith
    The accurate and safe diagnosis of breast cancer is a significant societal issue, with annual disease incidence of 48,000 women and around 370 men in the UK. Early diagnosis of the disease allows more conservative treatments and better patient outcomes. Microcalcifications in breast tissue are an important indicator for breast cancers, and often the only sign of their presence. Several studies have suggested that the type of calcification formed may act as a marker for malignancy and its presence may be of biological significance. In this work, breast calcifications are studied with FTIR, synchrotron FTIR, ATR FTIR, and Raman mapping to explore their disease specific composition. From a comparison between vibrational spectroscopy and routine staining procedures it becomes clear that calcium builds up prior to calcification formation. Raman and FTIR indicate the same size for calcifications and are in agreement with routine staining techniques. From the synchrotron FTIR measurements it can be proven that amide is present in the centre of the calcifications and the intensity of the bands depends on the pathology. Special attention is paid to the type of carbonate substitution in the calcifications relating to different pathology grades. In contrast to mammography, Raman spectroscopy has the capability to distinguish calcifications based on their chemical composition. The ultimate goal is to turn the acquired knowledge from the mapping studies into a clinical tool based on deep Raman spectroscopy. Deep Raman techniques have a considerable potential to reduce large numbers of normal biopsies, reduce the time delay between screening and diagnosis and therefore diminish patient anxiety. In order to achieve this, a deep Raman system is designed and after evaluation of its performance tested on buried calcification standards in porcine soft tissue and human mammary tissue. It is shown that, when the calcification is probed through tissue, the strong 960 cm-1 phosphate band can be used as a pseudo marker for carbonate substitution which is related to the pathology of the surrounding tissue. Furthermore, the first study in which human breast calcifications are measured in bulk tissue with a thickness of several millimetres to centimetres is presented. To date, measurements have been performed at 41 specimens with a thickness up to 25 mm. Measurements could be performed through skin and blue dye. The proposed deep Raman technique is promising for probing of calcifications through tissue but will need refinement before being adopted in hospitals.
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    A study of FT-IR spectroscopy for the identification and classifcation of haematological malignancies
    (Cranfield University, 2009-06) Babrah, Jaspreet; Stone, Nicholas; Bessant, Conrad
    The aim of the work presented in this thesis was to explore the use of FT-IR spectroscopy, as a complementary clinical tool for haematological laboratory analysis. FT-IR spectra were measured from air-dried and frozen cell lines derived from lymphoma, lymphoid, myeloid leukaemia and normal and chronic lymphocytic leukaemia blood samples. Multivariate statistical analysis was used to extract important spectral information with the greatest discriminative power. Principal component fed linear discriminant spectral models have been tested with leave one out cross validation procedures. A preliminary unfiltered classification model using 50 frozen and air-dried samples correctly classified 54% of 18556 spectra. The performance improved with the three cell line group datasets, with 71% of 19903 spectra correctly classified. Furthermore, the use of the frozen spectra improved the performance of the three cell line group classification model considerably. Findings showed that 73.3% of 9920 spectra were correctly classified in the frozen datasets, whereas in the air-dried only 41.5% of 9983 spectra are correctly classified. Optimisation of the spectral models by selection of principal components, application of Savitsky-Golay filters and selecting spectra using standard deviation and absorption filter tool was investigated. Using the first 25 significant PCs, a 0 th derivative Savitsky-Golay filter and the absorbance filter tool on the frozen five cell line spectral dataset were shown to be the optimal parameters for constructing a classification model. When tested with leave one batch out cross validation 90% of the spectra were correctly classified for the five cell line model. Blood component classification models tested with leave one batch out cross validation performed well. The whole blood model correctly classified 70% of 1736 spectra, measured on 22 samples. The plasma model correctly classified 80.6% of 331 spectra and the buffy coat model correctly classified 99.5% of 1438 spectra. This demonstrated that the buffy coat (containing white blood cells) holds the key biochemical information for discrimination between the pathology of the blood samples. Partial least squares analysis has been demonstrated as a method to support whole blood count tests for real time prediction of cellular constituents. These findings demonstrate the potential of FT- IR spectroscopy as a clinical tool although more work is needed if it is to be applied in clinical practice.
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    Study of Raman spectroscopy for the early detection and classification of malignancy in oesophageal tissue
    (Cranfield University, 2002-10) Kendall, Catherine; Greenhalgh, Douglas A.; Barr, H.; Stone, Nicholas
    Raman Spectroscopy for the identification and classification of malignancy in the oesophagus has been demonstrated in this thesis. The potential of Raman spectroscopy in this field is twofold; as a adjunct for the pathologist and as a biopsy targeting tool at endoscopy. This study has demonstrated the feasibility of these potential applications in vitro. Spectral diagnostic models have been developed by correlating spectral information with histopathology. This is the current 'gold standard' diagnostic method for the identification of dysplasia, the established risk factor for the development of oesophageal cancer. Histopathology is a subjective assessment and widely acknowledged to have limitations. A more rigorous gold standard was therefore developed, as part of this study, using the consensus opinion of three independent expert pathologists to train the diagnostic models. Raman spectra have been measured from oesophageal tissue covering the full spectrum of malignant disease in the oesophagus, using a near infrared Raman spectrometer customised for tissue spectral measurements. Two spectral datasets were measured with different volumes of tissue probed using twenty and eighty times magnification ultra long working distance objectives. Multivariate statistical analysis has been used to extract the required spectral information with the greatest discriminative power. Principal component fed linear discriminant spectral models have been tested with leave one out cross validation procedures. Three pathology group models have correctly classified up to 91% of spectra, and eight group models have correctly classified up to 82% of spectra. Optimisation of the spectral models by selection of significant principal components, filtering the data and using staggered models was investigated. Effort has been made to understand the findings in their clinical context, with review of patient history and clinical progress, long term follow up is required. Preliminary work projecting independent data on to the models has been encouraging with 76% of the spectra in the three group model correctly classified, approaching classification levels of the training dataset. Formalin fixed tissue models were demonstrated to perform well, with 80% of the spectra were correctly classified in the seven group model. This further demonstrates the potential of Raman spectroscopy as a pathology tool. If Raman spectroscopy is to be implemented in a clinical setting it must be transferable between different measurement systems. This has been evaluated with oesophageal tissue spectra measured on two systems using three objectives. Simple calibration has demonstrated the use of multiple systems and measurement parameters in the development and application of spectral classification models. Testing of a new design of fibre probe has provided encouraging preliminary results. There is potential for the application of Raman spectroscopy in vivo, however the technology remains immature. Spectral maps of samples taken from across the spectrum of disease have shown clear delineation of the morphological features seen on the H&E images. Furthermore the biochemical information elicited has been analysed. Initial measurements of oesophageal tissue using multiphoton imaging have demonstrated the potential of collagen autofuorescence in the diagnosis of malignant change.
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    A time-course Raman spectroscopic analysis of spontaneous in vitro microcalcifications in a breast cancer cell line
    (Nature Publishing Group, 2021-06-11) Bouzy, Pascaline; O’Grady, Shane; Madupalli, Honey; Tecklenburg, Mary; Rogers, Keith; Palombo, Francesca; Morgan, Maria P.; Stone, Nicholas
    Microcalcifications are early markers of breast cancer and can provide valuable prognostic information to support clinical decision-making. Current detection of calcifications in breast tissue is based on X-ray mammography, which involves the use of ionizing radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. Additionally, these techniques are not capable of discriminating between microcalcifications from benign and malignant lesions. Several studies show that vibrational spectroscopic techniques are capable of discriminating and classifying breast lesions, with a pathology grade based on the chemical composition of the microcalcifications. However, the occurrence of microcalcifications in the breast and the underlying mineralization process are still not fully understood. Using a previously established model of in vitro mineralization, the MDA-MB-231 human breast cancer cell line was induced using two osteogenic agents, inorganic phosphate (Pi) and β-glycerophosphate (βG), and direct monitoring of the mineralization process was conducted using Raman micro-spectroscopy. MDA-MB-231 cells cultured in a medium supplemented with Pi presented more rapid mineralization (by day 3) than cells exposed to βG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with βG revealed the presence of different precursor phases (octacalcium phosphate) during apatite crystal formation. These results demonstrate that Raman micro-spectroscopy is a powerful tool for nondestructive analysis of mineral species and can provide valuable information for evaluating mineralization dynamics and any associated breast cancer progression, if utilized in pathological samples.