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Biomedical Applications of Synchrotron Infrared Microspectroscopy
By David Moss
The Royal Society of ChemistryCopyright © 2011 Royal Society of Chemistry
All rights reserved.
Vibrational Spectroscopy: What Does the Clinician Need?
SHEILA E. FISHER, ANDREW T HARRIS, NITISH KHANNA AND JOSEP SULE-SUSO
Modern medicine demands rapid, consistent and reliable techniques for population screening, clinical and laboratory diagnosis, prediction of treatment outcomes and to guide the use of ever more expensive therapeutic agents. This chapter will explore clinical need using examples of major diseases and highlight the potential of vibrational spectroscopy to play a part in future clinical management.
To achieve success it is vitally important that basic scientists, spectroscopists, biologists and clinicians are able to communicate effectively and understand each other's requirements and challenges. The advancement of multidisciplinary working has been a key feature of the Diagnostic Applications of Synchrotron Infrared Microspectroscopy (DASIM) initiative, a Specific Support Action funded by the European Union to bring these groups together. During this period, the work of the group has encompassed, in addition to synchrotron based work, the wider aspects of diagnostic vibrational technology and the range of diseases and disorders to which it can be applied. In 2005, when the network began its work, although existing networks between biologists, clinicians and synchrotron scientists were making substantial progress in IR microspectroscopy of cells and tissues leading to the identification of spectroscopic biomarkers of potential diagnostic relevance, the European dimension was missing in these efforts because very few countries had synchrotron IR microspectroscopy facilities. Global networking was established between scientists but the technology had made limited impact on clinical practice and was poorly understood by doctors and other health professionals. During the life of the network, collaborations have been established which cross disciplines and new technology and techniques are constantly being developed and improved, potentially bringing the power and resolution previously offered only by synchrotron sources to the hospital laboratory, the clinic and to the patient. Understanding of the strengths and weaknesses of different spectral modalities, e.g. infrared (IR), Raman and fluorescence, and exploration as to the place of each in biomedical work is an emerging theme which continues to advance apace. At inter national level, teams are now working together to set parameters in terms of harvesting of clinical material, storage, preparation for imaging, and pre processing of data, all of which are essential given the changes which happen in biological samples as soon as they are separated from their host tissue and blood supply and also the tremendous complexity of the systems and bio chemical processes to be imaged. Since disease may change the biochemical composition, not only of cells and tissues but also of blood and other body fluids, the potential to use these as 'biomarkers' of disease processes is an important area for clinically based research. To map disease related changes it is necessary to carry out spectroscopic measurements at a microscopic level, matched to the size of cells or subcellular structures such as the nuclei and major organelles.
Clinically based research on vibrational techniques has focused on IR and Raman spectroscopy. The scientific basis of this approach relies on measurement of the natural vibrational frequencies of the atomic bonds in molecules. These frequencies depend on the masses of the atoms involved in the vibrational motion (i.e. on their elemental and isotopic identity), on the strengths of the bonds, and on the resting bond lengths and angles – in other words, on all the parameters that constitute the structure of the molecule. For this reason, IR spectroscopy is a powerful technique for the identification, quantification and structural analysis of small molecules, and has been established for many decades as an indispensible tool in organic chemistry, polymer chemistry, pharmaceuticals, forensic science, and many other areas. Thus very high resolution material can be imaged at subcellular level and beyond to allow detailed understanding of biological processes.
Resolutions as small as 7 µm×7 µm×2 mm can be achieved by bench top IR machines and 0.3 µm×0.3 µm×0.5 mm by Raman, allowing the level of exploration required for understanding of clinically important spectral changes. Images can be acquired by transmission or reflectance modes and increasing use is being made of confocal techniques. The resolution offered by Raman and its freedom from difficulties imaging aqueous based preparations or environments makes it a promising modality for biological imaging, both 'in vitro' in the laboratory setting and 'in vivo' for noninvasive diagnosis in the clinic. Its spatial resolution permits detection of subcellular components (mitochondria, nucleoli, condensed chromatin) in cells, and opens new avenues of monitoring cellular processes without the use of stains or marker molecules, using the inherent spectral properties of molecular constituents. Evolving techniques are increasing the depth of imaging possible 'in vivo', which is critically important for clinical utility.
However, the complexity and variation in these processes are a challenge. Alterations in cell function may be a product of changes in biochemical pathways but are likely to represent changes in magnitude or amplification of cell pathways, requiring quantitative measurements of molecules. Identification of specific 'biomarkers' of disease is likely to be possible only in a limited range of conditions. Ranges of quantitative change with cross over between disease state and normality are likely to prove a challenge when making careful assessment of any proposed clinical instruments in a well conducted clinical trial to establish sensitivity and specificity. These are defined as follows:
Sensitivity = number of true positives/[number of true positives + number of false negatives] (1:1)
A sensitivity of 100% recognises all people with the condition.
Specificity = number of true negatives/[number of true negatives + number of false positives] (1:2)
A specificity of 100% means that the test recognises all healthy people as healthy.
This kind of data is imperative for quality assurance and to give confidence to doctors who may be considering use of vibrational technology based devices. Taking the current promise into the clinical environment will require robust scientific and clinical collaborations.
This chapter will consider clinical applications which might be met by vibrational spectroscopy, using cancer, infective diseases and vascular surgery as examples and gives a brief overview. This is by no means exhaustive but suffices to illustrate the potential applications of the technology, which are addressed in greater detail in the appropriate following chapters.
1.2 Vibrational Spectroscopy in Cancer
Cancer represents a global health challenge. In 2002 there were 10.9 million new cases of cancer globally, with 6.7 million deaths. It was estimated at this time that 24.6 million people were living with cancer, within 3 years of their diagnosis. These figures will undoubtedly be underestimates as remote populations will be under recorded. Breast cancer is the most prevalent cancer with lung cancer having the highest mortality. The World Health Organization states that by 2020 the rates of cancer will have doubled.
Therapeutics research has embraced the concept of 'designer' drugs, based on exploration of biochemical pathways, giving considerable impetus to the biomedical disciplines of genomics, epigenetics, proteomics and metabolomics, all of which look in complementary ways to understand disease processes at molecular level. The result is that by 2006, approximately 2000 new chemotherapeutic agents were in the pathway towards clinical use. This explosion in science and therapeutics provides challenges both in management of individual patients and health economics. There is considerable pressure through the media to offer new therapies, often with little consideration of side effects and lack of efficacy. In the UK, the management of funding decisions has become the work of the National Institute for Health and Clinical Excellence (NICE). Their remit is to take decisions on risk versus benefit and cost per quality adjusted life year (QALY), a health economics based measure, allowing comparison between patient groups and treatments, in a system which will always have cost pressures.
The areas to which vibrational spectroscopy could contribute to clinical practice include:
Screening – Who is likely to develop the disease? How reliable is the prediction? Can this be achieved without invasive methods? Can inex pensive devices be produced for use in population screening, ideally at venues close to home?
Diagnosis – Is the disease present? How advanced is it? Can vibrational methods be combined with other technologies?
Intraoperative monitoring – Has the cancer been fully removed?
Prediction of response to therapy – Will the proposed treatment work? Is there any evidence of residual cancer?
Follow-up – Has the disease returned?
These clinical challenges require some common approaches in terms of recognition of abnormal cell pathways, cell morphology and tissue characteristics but also important individual features. In the next section, we will briefly examine each of these needs in turn.
1.2.2 Screening, Early Diagnosis and Surveillance
Screening is the process by which those who have the disease or, in some conditions, are likely to suffer it in the future, can be identified with the aim of maximising cure and reducing morbidity to the lowest possible level. Well known examples include mammographic screening for breast cancer and the cervical smear programme. The expenditure involved in bringing patients into the service and analysing their results can only be justified economically (on cost per patient diagnosed) and ethically (in terms of the anxiety and morbidity of invasive investigation when screening suggests disease but subsequently this is not confirmed by more intense investigation [false negative result]), when the disease is sufficiently prevalent and the test sufficiently accurate. No screening test achieves 100% sensitivity and specificity and some patients will be falsely reassured by screening tests. The requirement is for a test which will be reliable, with high sensitivity and specificity, easy to administer and which is acceptable to patients, and is ideally achieved by a non-invasive modality.
Some screening tools are aimed at visible areas, mainly skin where differentiation between melanoma and non-melanoma is topical. Moncrieff et al. (2002) and Claridge et al. (2003) have used colour of lesions and matched this to spectral characteristics. The oral mucous membrane is another area of interest as up to one third of patients who subsequently develop cancer go through a phase where visibly identifiable changes are present. However clinical prediction as to which changes are likely to transform to cancer is difficult. Histological assessment, where a piece of tissue is surgically removed from the patient, usually under a local anaesthetic, processed, stained and examined under a microscope, can help but there remains a lack of concordance amongst even specialist pathologists as to which lesions are of concern. Sankaranar ayanan et al. (2007) in a large population study in India, where oral cancer is most prevalent, showed that a screening programme can result in early diagnosis, with a reduction in morbidity and mortality.
There are two advances which could make a major impact on this area of medicine. One is a non-invasive technique which could accurately predict the potential for malignant transformation by 'in vivo' mucosal surveillance. Work has been done in a number of premalignant entities; for example, Kendall et al. (2003) have studied Barrett's oesophagus and cervical dysplasia with promising results. The cervical cancer screening programme has been in operation for many years, and depends on smears of exfoliated cells, examined using staining patterns and morphological parameters. Suspect cases then require a formal operation, known as cone biopsy, to allow definitive staging. A reliable imaging tool could eliminate this procedure with less delay and less discomfort to the patient. Utzinger et al. (2001) suggest that a simple algorithm based on two specific intensity ratios, taken from 13 patients 'in vivo', can discriminate between high-grade squamous dysplasia and other pathologies, misclassifying only one sample. Teh et al. (2009) have achieved a sensitivity of 90.5% and specificity of 90.9% in the diagnosis of gastric dysplasia using Raman spectroscopy.
Given the prevalence of cancer and the improvements in survival achieved at some anatomical sites, it is not surprising that more and more people face this diagnosis more than once in their lives. Whether there is a general predisposition to cancer as a generic behavioural change in cells or whether susceptibility is site specific is something which will only be determined by extensive epidemiological work. Certainly for some sites, of which head and neck cancer is a good example, the chance of a second primary cancer is of the order of 15%. The justification for longer term follow up of these patients is to identify and intervene early if a second cancer is found. The site can be anywhere in the upper aerodigestive tract as the entire mucosal surface is subject to incremental cellular changes which ultimately lead to cancer. Multifocal disease is also observed. The challenges here are those described for screening, to determine which anatomical area is at immediate risk. Current practice involves regular examination, either in the clinic or under sedation or full anaesthesia. Where there is suspicion of new disease, patients are scanned using computed tomography (CT) or magnetic resonance imaging (MRI). The personal cost of this surveillance is anxiety and a requirement for regular hospital visits. The cost to healthcare budgets is considerable, especially where the cancer is common and surveillance requires formal admission. Lung and colorectal cancer both require formal endoscopy and biopsy at intervals during the surveillance period. To test by a non-invasive probe or to develop a 'surrogate' test using a body fluid would result in a considerable health gain. Candidate systems include breath for lung, faeces for colorectal, and urine for kidney and bladder surveillance. Of wider impact is the possibility of markers in serum or saliva.
Excerpted from Biomedical Applications of Synchrotron Infrared Microspectroscopy by David Moss. Copyright © 2011 Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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