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 multi-disciplinary 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 international level, teams are now working together to set parameters in terms of harvesting of clinical material, storage, preparation for imaging, and preprocessing 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 biochemical 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.¹  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 μm can be achieved by bench top IR machines and 0.3 μm×0.3 μm×0.5 μm by Raman,²  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 non-invasive 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:

A sensitivity of 100% recognises all people with the condition.

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.

Cancer represents a global health challenge. In 2002 there were 10.9 million new cases of cancer globally, with 6.7 million deaths.³  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.⁴ 

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.⁵  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 inexpensive 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.

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.^6,7  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. Sankaranarayanan 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.⁸ 

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.⁹  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.¹⁰  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.¹¹ 

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%.¹²  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.¹³ 

A major potential for vibrational techniques is to screen body fluids, such as plasma, saliva and urine, or breath. If a reliable vibrational spectroscopy dataset can be developed such tests could be offered from a community setting, especially important in areas where the population is scattered, such as Canada or parts of Australia, or where economic costs make hospital based screening impossible, such as rural India. Madhuri et al. (2003) compared native fluorescence spectra derived from plasma in subjects with oral cancer or liver disease and healthy subjects, showing good discrimination.¹⁴  Harris et al. (2009) compared 20 plasma samples from patients with respiratory disease with 20 from patients with head and neck cancer, thus ensuring a similar population in terms of gender, age, smoking, alcohol consumption and the likely presence of inflammatory mediators.¹⁵  They used Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA) and a ‘trained’ evolutionary algorithm, achieving sensitivity and specificity levels of 75%.

The detection of lung cancer through breath analysis has become recently an area of high interest. Sulé-Suso et al. (2009) showed variations in the amount of volatile compounds released by different lung cells (epithelial, fibroblast, and cancer).¹⁶  Further work is required to assess whether volatiles released by ‘in vitro’ growing cells could also be detected in breath and used in clinical practice for lung cancer diagnosis.

Treatment for cancer involves chemotherapy (systemic drug treatment), radiotherapy, which can be external beam or by insertion of radioactive wires directly into the cancer (brachytherapy), and/or surgery. Treatment choice depends on the type of cancer, where it is in the body, how extensive it is and its likely natural history. The experience of a cancer patient can be very variable with some cancers, such as breast, having a very long natural history, often years from diagnosis to death even in advanced disease and others, such as head and neck cancer, having a rapid course with death in less than a year being common for those whose disease cannot be controlled. Each modality of treatment comes at a price, both in terms of individual experience and health economics. Chemotherapy targets the cancer, but also affects the parts of the body where cells divide rapidly. Extreme fatigue, lethargy, anaemia, oral mucositis, vomiting and diarrhoea are common. Hair loss used to be one of the most obvious signs that someone was having chemotherapy. Many new agents spare the patient this side effect but skin reactions are common and are often considered to correlate with the degree of response to the treatment.

The toxicity profile of radiotherapy is similar. Early side effects include those listed above. Late effects include fibrosis, scarring, stiffness and reduction in blood supply to the tissue in the treatment field which extends beyond the site(s) directly involved with the cancer. Complications can be devastating, for instance radiotherapy to the pelvis can cause ‘frozen pelvis’ with marked functional disturbance. Fistulation between, for example, bowel and major blood vessels can occur with fatal sequelae. In head and neck cancer, treatment followed by late fibrosis can impair mouth opening, speech and swallowing, with unacceptable psychosocial and functional consequences.

Progress is being made in these areas by constant searches for new and more specific cancer drugs. As noted in the introduction, IR has a good record in the pharmaceutical environment and if cancer characteristics could be matched to therapeutic agent, morbidity might be minimised and efficacy maximised. If we knew which patients would, or would not, respond to a given treatment, we could be more positive in our therapies and, one hopes, spare those patients for whom radical but ineffective therapy simply takes away their final period of reasonable health. For radiotherapy, careful imaging and volume prediction has resulted in advanced computer guided treatment plans which maximise the dose to the cancer whilst sparing as much surrounding tissue as possible. Some aspects of response relate to hypoxia. Ability to image during treatment, looking at this kind of response, or even at the residual cancer burden, might allow further refinement. Recent advances in Raman imaging might make this feasible in the future.

For those cancers where surgery is the modality of choice, the most important aspects of efficacy are achieving full excision of the cancer, together with any related premalignant tissue. In oral cancer, close or involved margins have an adverse impact on both locoregional recurrence (return or persistence of the cancer at the primary site and/or the lymphatics which drain the area) and survival.¹⁷  For some cancer sites a radical resection is possible without marked morbidity. For all sites, there is eventually an anatomical structure which limits further surgery. For sites such as the head and neck, the more radical the surgery, the greater is the postoperative morbidity and this has an impact on key functions like eating, speech and swallowing. Cancer infiltrates tissue at microscopic level, so the boundaries of the cancer are not visible to the treating surgeon. It is usual practice to remove the cancer with a ‘safety margin’ of normal tissue. Figure 1.1 shows the invasive front of a cancer. This can be ‘pushing’ in character, and the true boundary relates closely to palpable disease (cancer feels firm). Where the disease spreads as islands of cells, as in the example shown, palpation will not be a reliable guide to surgical margins. There will also be problems with the method many surgeons use to try to check margins, frozen sections. In this approach, small pieces of tissue from the planned margins are snap frozen, stained and examined by a pathologist. If islands of cancer remain beyond the margins sampled, recurrence is likely. In head and neck cancer, even where margins are reported, after full and careful examination, as free of cancer, 10% of patients will suffer a locoregional relapse of their disease. Salvage after recurrence or residual disease is unlikely. A tool to immediately assess margins would be invaluable in surgical practice. Challenges are the ability to image to the necessary depth, to pick up microscopic deposits of a few cells only and to function in an environment contaminated with blood and saliva.

In addition to the primary cancer, spread can take place, either through the lymphatic system, which in health drains tissue fluids and filters pathogens in structures called lymph nodes, or through the blood stream. Spread to lymph nodes which drain the primary cancer site is often the first sign of systemic disease and removal of the lymph nodes often forms part of radical surgical treatment with curative intent. As with the primary site, this operation comes at a price in terms of morbidity but leaving involved nodes in situ will result in locoregional treatment failure. Attempts have been made to minimise the need for surgery using techniques which image involved nodes. One which has undergone clinical trials is sentinel node biopsy where a dye and/or radioactive tracer is injected into the primary cancer and the nodes become apparent by simple inspection and/or radioimaging.¹⁸  This assessment is time sensitive if the correct nodes are to be identified and has yet to gain widespread clinical acceptance. As noted above, imaging by a vibrational spectroscopy probe may perhaps become feasible in the future.

Once the cancer has been removed, it is the task of the pathologist to determine if the cancer has been fully removed. If one imagines the volume and surface area of the specimen, the time required to do a systematic margin check at microscopic level can be appreciated. The time to similarly fully examine a specimen containing lymph nodes is a similar challenge. If ‘sampling’ is practised, looking only at selected areas, the risk of understaging (underestimating the extent of spread of) the cancer exists and the patient is denied the option of adjuvant radiotherapy or chemoradiotherapy which would increase their chance of locoregional disease control and overall survival. The need to examine the whole specimen carefully is illustrated by the presence of very small deposits of cancer outside the capsule which forms the boundary of lymph nodes (Figure 1.2). Such disease, which can be very small in volume and which can occur in lymph nodes less than 3 mm in diameter, has a marked adverse effect on disease control and survival.^19,20  This is an area where clinically important advances are certainly feasible. One advantage of vibrational techniques is that the threshold for identification of a meaningful difference is a function of the processing of data. A screening tool through which sections were passed could hypothetically be programmed for high sensitivity, selecting out those sections which the pathologist should carefully examine. Some authorities have suggested that automated diagnosis might be feasible in the future but the analysis of cancer and treatment recommendations rely on a complex interpretation of the disease and its patterns of spread. A tool to help pathologists focus their skills most effectively is feasible; this should be a primary aim of research towards the clinical setting.

There is a considerable literature on the differentiation of cancer and non-cancer so we have limited our description to examples of work in this area. Tobin et al. (2004) used synchrotron IR to differentiate oral cancer tissue, using air dried tissue sections, from the surrounding normal tissue using PCA and LDA.²¹  Sections were subsequently stained and good correlation was achieved; indeed, in one section where visually the tissue appeared to be ‘non-cancer’ but for which the spectra fitted the ‘cancer’ profile, after staining it became clear that there were cancer cells in the area from which the spectra had been obtained. The possibility of obtaining IR spectra from cells which have already been stained opens up a new avenue in the study of pathology samples with Fourier Transform Infra-Red (FTIR) spectroscopy.²² 

For some cancers the prognosis (outcome) is defined by tissue characteristics. This is true in prostate cancer, one of the most common cancers in men, and the aggressiveness is decided using a rating system known as Gleason grading. The higher the Gleason grade, the poorer the prognosis will be. Baker et al. (2008, 2009) have reported high correlation between FTIR and Gleason grades in a series of prostate cancer patients.^23,24  Other groups have concentrated on attempting to understand the biological basis for the spectral changes seen, as typified by the studies of Anastassopoulou.²⁵ 

In addition to controlling the locoregional disease, cancer causes mortality by distant spread, a process known as metastasis. Metastasis occurs primarily at sites with a high blood flow, typically lung, bone, brain, liver and kidneys. Once spread has occurred, it is highly likely that metastases will prove to be multiple so a systemic therapy is needed. Chemotherapy is given either through a catheter inserted into a vein or, increasingly in modern practice, orally. Much effort has been placed into development of new and more specific agents to target cancers and reduce effects on normal tissue. Despite that aim, reduction in cancer size comes at a price, usually fatigue, anaemia, nausea, and vomiting and diarrhoea. There remains a small but significant mortality. If it were to prove possible to predict which patients will respond to which agent, this would be a significant health gain.

In summary there is good and increasing evidence for the efficacy of vibrational spectroscopy techniques in cancer screening and diagnosis. The next phase will be validation of these techniques in carefully designed studies of sufficient statistical power to prove efficacy in clinical practice.

This part of the chapter reviews the present stage of optical spectroscopy in the diagnosis of vascular pathology. Ischaemic heart disease (IHD) and peripheral vascular disease constitute a massive burden to the western world.^26–29  Approximately 250 000 people per year die in Britain of cardiac disease.³⁰  Of those the preponderance are males, usually over 50 years of age. However, with genetic and increased lifestyle risk factors many younger males, and females, are also suffering from this disease.

Both these disease patterns originate from the same source, i.e. atheromatous plaques within arterial vessel walls. In order to understand this pathology it is necessary to be familiar with the anatomical structure of arteries. The arteries are composed of three layers: an intima, which lines the interior of the blood vessel, the media, a structural support for the vessel, and then the adventitia, surrounding the outer surface of the vessel (Figure 1.3). The constituents of these layers are endothelial cells, smooth muscle cells, and collagen and proteoglycan elements which make up the extracellular matrix.³¹  There are three types of artery: the large or elastic artery, of which the aorta is an example, medium sized vessels, which include branches of the aorta such as the coronary arteries, and then the small arteries (usually less than 2 mm in diameter). Atherosclerosis affects only the large and medium sized vessels.³¹ 

Atherosclerosis literally means hardening of the arteries. This is caused by smooth muscle proliferation within the intimal layer. These smooth muscle cells originate in the media layer, but migrate into the intima in response to damage to the intima from hypertension, smoking and other toxins.^32,33  Lipid accumulation occurs at the site, with macrophage infiltration. The macrophages engulf the accumulating lipids and form foam cells, which give the macroscopic appearance of fatty streaks, forming a plaque.³¹  The plaque grows and reduces the size of the vessel lumen; as this plaque enlarges it will decrease lumen size. The plaque is covered by a fibrotic cap, which if it ruptures may cause acute thrombus formation which will result in vessel occlusion and ischaemia distally.

At present assessment of patients with atheromatous lesions within the cardiac or peripheral arterial system is by X-ray angiography (injection of a radio-opaque dye and imaging the flow by sequential X-ray films); this can quantify the severity of the vessel stenosis.³⁴  Patients may also undergo other forms of assessment such as external ultrasound scanning, with spiral computer tomography or magnetic resonance angiography as options. These investigative modalities can all provide assessment of vessel stenosis and whether calcification is present, but they do not possess the ability to detect the chemical composition of the atherosclerotic plaque.

It is well recognised that lesion composition is a very important aspect of this disease.^35–37  Plaques can be divided into stable or unstable, based on their chemical composition not their area or volume. Unstable plaques have lipid pools which can rupture creating a thrombus cascade, causing occlusion of the vessel.³⁸  This is thought to be the predominant pathophysiology for acute myocardial infarctions, and these patients are thought to have a poorer prognosis. However, densely calcified plaques are slow growing in nature, and are inherently stable, thus producing progressive clinical symptoms giving a prior warning of future events.^39,40 

Cardiac risk factors cannot predict how disease will progress, and as these important chemical changes occur at an asymptomatic phase, there is a need to delineate underlying chemical structure at this early stage, thus allowing optimal planning of treatment. At present the majority of patients present after a clinical vascular event, such as a myocardial infarction (MI, ‘heart attack’) or cerebrovascular accident (CVA, ‘stroke’). At this stage, permanent morbidity may well be present and reversal of disability is unlikely to be achieved. Intravascular ultrasound can provide more detailed anatomy of plaques, as can magnetic resonance imaging, but they cannot detail chemical composition.^41,42  To be able to enumerate the compounds present in the atherosclerotic plaque, with quantities present, would provide the ability to intervene early in those patients at high risk of plaque rupture and prevent clinical vascular events and their attendant morbidity and mortality.

Spectroscopy, more specifically Raman spectroscopy, has the ability to give ‘in vivo’ recordings of chemical compositions of atheromatous plaques.

The initial work in this field consisted of ‘in vitro’ work carried out on human vessels. Early work centred on fluorescence spectroscopy. Bartorelli and co-workers (1991) and Richards-Kortum and colleagues (1989) studied the use of spectra in the ultraviolet (UV) light region on diseased arterial tissue.^43,44  They independently demonstrated the ability of this technique to distinguish between atheromatous and normal tissue. However, recent work has centred on the use of Raman as this has an ability to distinguish different chemical compositions.^45–56  Raman spectra are unique to compounds (which produce ‘fingerprints’ as stated above) whereas fluorescence spectra are limited in their differences.

Buschman and colleagues (2001) utilised Raman spectroscopy to differentiate normal versus diseased arterial walls.⁵⁷  They described a technique for Raman spectroscopy which could potentially be performed in situ. From 16 patients, hearts were obtained from explantation during transplant procedures; 200 samples of human coronary artery tissue were gathered. Of these 16 patients, 7 were awaiting transplant due to dilated cardiomyopathy, and the rest were on the organ waiting list for severe ischaemic heart disease. The methodology included the arterial samples being washed then immediately frozen in liquid nitrogen. The samples were placed into two categories. The first consisted of 113 samples which were used in the original data collection. They were thawed and underwent spectroscopic evaluation, formalin (10%) fixed and underwent histological evaluation, in which the two pathologists were blinded to the Raman results. The remaining 87 samples were evaluated against the first set to give comparable data.

Their results indicated that non-atherosclerotic tissue, non-calcified atherosclerotic plaque and calcified atherosclerotic plaque all give different Raman spectra, and hence this technique can differentiate these three states of a vessel wall. Microscopic Raman spectra were obtained to study the chief constituents of the vessels under examination: elastic laminae, collagen fibres, smooth muscle cells, fat cells, foam cells, necrotic cores, cholesterol crystals, β-carotene containing crystals, and calcium mineralisations. The results indicate different levels of each depending on which arterial sample was being observed. For example, calcified atherosclerotic plaques contained a lot more foci of calcium mineralisation than normal tissue but fewer fat cells than normal tissue, whereas atherosclerotic tissue contained much higher concentrations of foam cells than normal tissue, as would be expected. This paper gives a thorough, detailed methodology and indicates the potential for the clinical use of Raman spectroscopy in atheromatous disease. All techniques have limitations which must be understood and addressed, such as the difficulty in obtaining clear distinctions between cellular constituents such as foam cells and cholesterol crystals, which gave similar spectra. These spectra also are very close to those of smooth muscle cells, collagen fibres and elastic laminae.

The ability of Raman spectroscopy to distinguish diseased from normal vessel wall is also supported by Nogueira et al. (2005),⁵⁸  utilising FTIR spectroscopy, and studying carotid artery samples from post-mortem examinations. Seventy-five vessel samples were obtained from 22 subjects. Once spectral results had been obtained the sample was marked with India ink and then formalin treated and underwent histopathological study. The results indicate that calcified plaques have very different spectra, but those of atherosclerotic and non-atherosclerotic tissues are very similar. Their study highlights another problem faced; the initial diseased state is in between the intima and media layers, possibly being just too deep for spectral analysis when an endovascular approach is used, acquiring the spectra through the vessel wall. Hence atheromatous vessels produce spectra similar to ‘normal’ tissue.

Römer and co-workers (1998) sought to evaluate the ability of Raman spectroscopy to correlate with histopathological assessment of human coronary arteries.⁵⁹  One hundred and sixty-five coronary artery samples were obtained from explanted recipients’ hearts. Their report aimed not only to show an ability to distinguish between ‘normal’ and diseased tissue but to quantify the chemical composition of diseased vessels. Again calcium rich lesions produce different spectra, with diseased and normal tissue having slight differences which when analysed through mathematical models show a clear distinction. Their data suggest cell constituents differ in diseased states and can be quantified by spectroscopy, although subject to errors as previously mentioned.

In order to progress from the laboratory to a clinical setting work was undertaken on a probe which could be utilized for in situ vessel analysis. Motz et al. (2004) undertook work on a Raman probe.⁶⁰  There are many difficulties when designing such an instrument; for example, it must be long enough and flexible to access remote organs; have a diameter tolerable to the vessel; contain optical filters strong enough to reduce the background ‘noise’ from the fibres themselves. Background ‘noise’ can often mask the signal of the tissue. The laser must have a low enough exposure to be unable to cause any damage to the vessel or organs in the beam. The authors produced a probe which underwent evaluation in simulation tests with experimental models and ‘in vitro’ analysis. Other authors have also described optical probes for Raman spectroscopy ‘in vivo’ work, and outlined the design problems to overcome.^61–66 

Motz and colleagues (2006) used their Raman probe ‘in vivo’ approximately two years later.⁶⁷  Spectra were taken from 14 femoral bypass and 6 carotid endarterectomy operations. The sites from which spectra were taken were marked with a suture and a small biopsy was taken for histopathological analysis. However, sutures could not be used for the carotid endarterectomy samples, and so estimates were made of where these samples were taken. The field was bloodless due to saline flushes being used prior to sampling. The authors state that the results obtained correlate well with the pathology report, indicating that spectra could distinguish the presence of markers of atheromatous disease.

The other practical issue is that all lights have to be turned off in theatre to reduce background ‘noise’; this may not be appropriate or even safe in all circumstances.

The promise centres on the ability of Raman probes to image the chemical composition of plaques and thus these techniques may be able to predict which patients are at risk of acute arterial occlusion, an aim which would have considerable benefit to patients.

Infective disease remains one of the main healthcare burdens faced by both the developing and the developed world. Control of infective outbreaks proves challenging in terms of:

• Identification of the causal organism

• Determination of the correct therapy

• Recognition of biological change, e.g. a mutation which renders the organism resistant to a therapy which has been reliably used to contain it.

Pathogenic (capable of causing disease) organisms include bacteria, viruses, and yeasts.

Very often it is the most vulnerable, the very young, infirm, or those individuals suffering other conditions, who are at greatest risk of infections. The EPIC study (European Prevalence of Infection in Intensive Care) of 1417 intensive care units (ICUs) in 17 different countries found that almost 45% of patients on these units had a microbiologically confirmed infection.⁶⁸  At present, septic patients (broadly defined as patients with either a confirmed infection, or highly suspected infection with clinical signs of a systemic inflammatory response syndrome), whether on ICUs or other hospital wards, are initially treated with empirical broad spectrum (essentially, best guess) antimicrobial therapy until microbiology data on culture results and sensitivities are available, usually one to two days later.⁶⁹ 

Despite the use of broad spectrum antimicrobial therapy, 10–30% of patients still receive inappropriate antibiotics, which may impact on mortality.^70,71  It is in the ICU patients where appropriate antimicrobial therapy can lead to a 30–60% reduction in mortality rates.⁷²  Furthermore, despite adequate antimicrobial therapy, mortality rates amongst patients with microbiologically confirmed infections are statistically greater when compared to patients with no infection.⁷³  These infections can be community or hospital acquired (nosocomial); however it is estimated that nosocomial infections are 5 to 10 times greater in ICUs than on general medical wards.^74–77  Patients in ICUs are at the highest risk, due to immunosuppression, inserted lines, catheters, mechanical ventilators, and multiple illnesses.

The most clinically important disadvantage of using broad spectrum antibiotics empirically is the emergence of drug resistance.⁷⁸  Concerns have focused on methicillin-resistant Staphylococcus aureus (MRSA),^79,80  vancomycin-resistant enterococci (VRE),^81–83  extended spectrum β-lactamase producing Gram-negative bacilli,⁸⁴  multi-drug resistant Mycobacterium tuberculosis,⁸⁵  and resistant Candida species.⁸⁶  In the USA and Japan there have also been reports of isolation of S. aureus with a reduced susceptibility to vancomycin.⁸⁷ 

Any drug has the potential for adverse reactions, ranging from simple diarrhoea to pseudomembraneous colitis, or possible anaphylactic shock. It is apparent that the use of unnecessary medication can be detrimental to a patient. However, due to the inability to quickly determine the nature of an organism, an empirical approach is utilised to prevent a patient clinically deteriorating prior to the availability of microbiological data. Furthermore, there is a large body of evidence suggesting that the initial use of antimicrobial agents to which the identified pathogens are resistant increases the risk of hospital mortality.^88–96 

At present, after harvesting, the biological material (usually a blood culture, aspirate or swab) is taken to the laboratory where it is manually ‘plated out’ on dishes of nutrient material until colonies of the organism can be grown. These are then characterised, using simple morphological characteristics, e.g. Gram staining. Colonies are treated with candidate antibiotics and the zone around the antibiotic (zone of inhibition) is measured. From this, a therapy which is likely to work in clinical practice can be derived. All of this is labour intensive and takes time. It is often as long as 48 hours before a definitive recommendation can be made to the treating physician.

In order to attain a faster diagnosis and begin targeted antibiotic therapy, studies have focused on the use of the polymerase chain reaction (PCR).^97,98  Belgrader and colleagues (1999) investigated a high speed PCR technique to detect bacteria in seven minutes.⁹⁹  PCR requires a degree of sample preparation prior to analysis. In addition, due to the extremely high sensitivity of PCR, contamination from non-template DNA present in the laboratory environment is problematic. Although automated microbial detection systems frequently identify common organisms, a number of organisms have proven to be difficult to identify consistently. Evidence exists that enterococci,¹⁰⁰  pneumococci,¹⁰¹  and certain β-lactam resistant Enterobacteriaceae¹⁰²  pose problems for certain automated systems. Furthermore, these systems can misidentify rare organisms, leading to inadequate or inappropriate antimicrobial therapy.^103,104  Bacterial detection systems requiring minimal specimen processing and with the ability to detect small numbers of organisms accurately and consistently would be highly advantageous.

Maquelin et al. (2003) reported a clinical trial of vibrational spectroscopy.¹⁰⁵  Firstly, a reference library was created of bacterial and fungal pathogens. The reference strains were seeded in blood samples from healthy volunteers. These blood samples were then inoculated into standard blood culture bottles appropriate for the organism. Once the blood cultures were flagged as positive via a BACTEC blood culture system, the material was incubated on a culture medium for six hours and then underwent Raman spectroscopy, from the culture medium. For the IR detection, the biomass from the blood culture bottle was incubated overnight and then transferred to detection plates. References were then obtained for the 106 different strains of bacterial species used and for 34 yeast strains. Recordings were taken until the system was calibrated through a mathematical model to identify the various pathogens. Next, the prospective data collection began from blood culture bottles from two separate hospitals over three- to four-month periods. These were incubated for six to eight hours and then underwent spectroscopic evaluation. Raman correctly identified 92% of the samples as compared to conventional phenotypic identification. The IR method correctly identified 98% of the organisms. These are very promising results, as the phenotypic mechanism system currently used in practice cannot guarantee 100% accuracy.

More recently Ibelings et al. (2005) studied the use of Raman alone on clinical samples.¹⁰⁶  They initially constructed a Raman reference library for 93 strains of 10 Candida species. Once Raman recordings had been taken the differences in spectra were detected using mathematical models. They tested this reference data set using a collection of peritoneal fluid samples, obtained from patients suffering from peritonitis. In total, 88 fluid samples were collected from 45 patients. The samples were cultured for 48 hours and underwent routine microbiological testing and Raman spectral readings: 29 samples were positive for Candida, and, 26 were correctly identified by Raman. This study does show promise for Raman in clinical practice but does not prove that it is any quicker than conventional methods, one of the main reasons for its consideration in the clinical setting.

Low resolution Raman spectroscopy has been shown to be a cost-effective model of spectroscopy and benefits from being a portable device that can easily couple with optical fibres. This raises the possibility of direct point of care testing, where the system of microbial detection can be available in the ICU itself, therefore allowing for rapid sample analysis and microbial identification using a reference library. This would ease the burden of samples processed in microbiology laboratories and reduce costs involved in specimen transport. Mello et al. (2005) managed to correctly identify a variety of pathogens associated with gastroenteritis indicating promise for future progress in this area.¹⁰⁷  Harz et al. (2009) recently reported direct analysis of clinically relevant single bacterial cells (Neisseria meningitidis) from cerebrospinal fluid using micro-Raman spectroscopy.¹⁰⁸  Bacterial meningitis is a disease for which urgent and effective therapy must be given and, if the promise of this initial communication is supported by larger studies, the use of Raman would represent a significant clinical advance.

In addition to basic organism identification, Raman spectrometry has been shown to be able to identify certain resistant strains, therefore providing vital information on treatment. Sockalingum et al. (1997) managed to identify Pseudomonas aeruginosa isolates with varying degrees of resistance to imipenem.¹⁰⁹  Although the technique does not elucidate mechanisms of antibiotic resistance, Raman spectroscopy identifies the indirect biochemical changes that occur when an organism exhibits resistant traits towards a particular antibiotic. In addition, Hosseini and colleagues (2003) developed a non-invasive and non-contact method of measuring antimicrobial concentrations in the eye using Raman spectroscopy, therefore raising the possibility of using this method to assess the pharmacokinetics of antimicrobials in normally inaccessible areas.¹¹⁰  In a significant breakthrough Amiali et al. (2007) explored the use of IR spectroscopy in MRSA detection.¹¹¹  Using mathematical modelling of their spectral results, they report 97% accuracy for MRSA within one hour. This compares to current techniques giving results in approximately three days.

The current literature purports that vibrational spectroscopy will hold a place in the clinical setting. Accuracy rates are reported to be greater than 90% with Raman or IR, and a combination of these could confer higher certainty. However, there are concerns regarding the capability of spectroscopy to detect polymicrobial infections, where the ability to identify the organisms involved is of paramount importance. Abscesses frequently contain multiple pathogenic organisms that need to be targeted if adjunctive antimicrobial chemotherapy is to be of any use. One method that has been developed to allow manipulation of a sample without direct physical contact is through the use of optical tweezers. Raman tweezers focus a near-IR laser on a sample. The tweezers fix the cell within an optical trap from which it may be manipulated, therefore allowing analysis of an individual cell.¹¹²  The continued interesting work on bacterial taxonomy through spectroscopy and its clinical application as discussed by Beekes, Naumann and co-workers (2007)¹¹³  reinforces the potential for future applications in microbiology and the classification of pathogenic organisms.

In this chapter we have placed studies using vibrational spectroscopy in a clinical context, indicating the potential for the future and for strong basic science/clinical alliances to be made to ensure that the promise seen in this area is translated through to clinical practice.