Currently, the diagnosis of bowel diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) relies on invasive and expensive procedures. Identification of biomarker-based tests to aid diagnosis is an important area of research. Here we review the use of mass spectrometry in this search and discuss recent findings.
by Dr B. De Lacy Costello, Professor N. M. Ratcliffe and S. Shepherd
Inflammatory bowel disease (IBD) is an inflammatory autoimmune disease caused by an inappropriate response of the immune system to commensal gut microbes [1]. There are two types of IBD, ulcerative colitis (UC) and Crohn’s disease (CD). UC affects the large bowel only, affecting variable lengths of the colon continuously from the rectum, primarily affecting the mucosa [Fig. 1]. CD can affect any part of the GI tract, and is a transmural disease [2]. Common symptoms of IBD are severe abdominal pain, defecation urgency and diarrhoea, which can contain blood.
Irritable bowel syndrome (IBS) is a functional disorder of the digestive tract. It is characterized by its symptoms, with no physiological changes in the GI tract. IBS can be diarrhoea predominant (IBS-D), constipation predominant (IBS-C) or symptoms can alternate between the two (IBS-A). Common symptoms include abdominal pain and cramps, bloating and flatulence, and unusual bowel habit. IBS has, as yet, no known cause. People with IBS show abnormal gut motility and hypersensitivity to pain in the GI tract. Stress and anxiety are known to cause changes in gut motility [3] with stress and anxiety being common symptoms of IBS. When under physical or psychological stress IBS patients showed increased gastro-intestinal sensitivity when compared to healthy controls [4]. Recently it has been thought that there may be changes in the gut microbiota in patients with IBS, the evidence being that IBS symptoms often occur after infective gastroenteritis or in patients in remission from IBD or diverticulitis. SIBO (small intestinal bowel overgrowth) has also been implicated in IBS and other function bowel disorders. One current hypothesis is that an altered microbiota activates the immune system within the mucosa, leading to an increase in epithelial permeability, causing dysregulation of the enteric nervous system [5]. Genome-wide association studies have successfully identified many genetic loci involved in susceptibility to IBD, and it is thought that genetic factors may also play a role in IBS [1].
Diagnosis of GI disease
IBS-D can present with symptoms similar to IBD and other non-functional bowel conditions. The diagnosis of IBS is often one of exclusion, where more serious bowel diseases, such as IBD or colon cancer which present with common symptoms, are ruled out. The current gold standard for diagnosis of IBD is endoscopic and histological testing; however, these investigations are both invasive and costly, and have associated risks. Of the patients referred for endoscopy few actually have organic bowel disease [6]. The costs associated with functional bowel disease are significant, with healthcare costs for IBS patients being significantly higher than non IBS controls [7].
There are currently no known biomarkers of IBS. There are various biomarkers that have potential in the differentiation of functional from inflammatory gastrointestinal disease, but there is still a need to identify biomarkers and to develop quicker, lower cost and less invasive testing for diagnosis of gastro-intestinal disease.
Biomarkers such as lactoferrin, calprotectin, c-reactive protein (CRP) and erythrocyte sedimentation rate (ECR) have all been used to help distinguish functional from inflammatory bowel disorders and to diagnose IBD. Serological markers such as antibodies to bacterial and fungal antigens that can indicate an abnormal response to commensal microbes can also be useful in identifying IBD.
Fecal calprotectin and lactoferrin are protein biomarkers of inflammation. In 2010 a meta-analysis of six studies (n=670) in adults by Van Rheenen et al. [8] found that screening patients by testing fecal calprotectin levels would have reduced the number of endoscopies performed by 67%, although its diagnosis would have been delayed in 6% of patients. When taking a weighted mean of 19 studies including 1001 patients, where IBD patients were compared with controls of IBS and other colonic diseases, fecal lactoferrin has a sensitivity and specificity of 80% and 82%, respectively [9].
Although these biomarkers can be useful as part of the screening process when establishing a diagnosis [6, 8], there is currently no biomarker or test that can replace the need for endoscopic and histological investigations. Mass spectrometry techniques are at the forefront of research for biomarker prospecting for IBS/IBD.
Mass spectrometry
Mass spectrometry (MS) has the ability to identify numerous compounds in a single sample. It is also high throughput allowing rapid analysis of many samples, which is especially useful for large studies or for the diagnosis of many samples. The ability to obtain results quickly, usually in less than 1 hour makes it attractive for clinical use.
Proteomic approach
Although MS (with associated sample vaporisation methods) was originally limited to low molecular weight volatile compounds, in the last 2 decades advances in MS technology have enabled its use with high molecular weight compounds, changing the way proteins are analysed. The soft ionization techniques electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) allow for the analysis of proteins and other macromolecules [10]. The identification of proteins through peptide mass fingerprinting, or peptide sequencing using MS is more rapid than techniques such as de novo protein sequencing and data can be analysed automatically. MS can also be used to determine the abundance of a molecule in a sample [10].
Differential protein expression can identify different diseases, and can indicate the degree of the disease state, or be used to assess the effects of treatment – for example the response of IBD patients to anti-TNF alpha antibodies (infliximab) [11]. It also has applications in the identification of protein biomarkers.
In 2011 MALDI-MS was used by M’koma et al. for tissue analysis; through profiling of the proteome of the colonic submucosa they were able to distinguish UC from CD by comparing proteomic spectra. Definitive diagnosis of either UC or CD is important as people with UC also have an increased risk of colon cancer [12].
Goo et al. have investigated protein biomarkers for IBS. ESI with LC-MS was used on protein fragments from the urine of women with IBS. They found differences in some specific components of the urinary proteome, and demonstrated that there is a possibility for future biomarker studies for IBS [13].
There are still limitations to mass spectrometric protein analysis, for example the difficulty in detecting hydrophobic membrane proteins. However, it seems promising that, with the advances in mass spectrometry technology, there will be an increase in the discovery of protein biomarkers and key pathogenic factors of gastro–intestinal disease, and improved diagnosis and therapy.
Metabolomic approach
The metabolome is the set of small molecule metabolites found in a biological sample. Unlike proteomics, metabolomics can be a direct measure of production of compounds and activity of cells or systems in an organism. This can be especially useful when looking for disease biomarkers in IBS and other bowel diseases as it can be used to understand the environment of the GI tract, as well as factors such as digestion and absorption of dietary products and gut microbial activity [14], which are implicated in IBS pathogenesis.
Researchers have explored the use of various techniques incorporating MS on breath [15], urine [16] and stool [17] samples in search of metabolic biomarkers of bowel disease for non-invasive testing and many possible candidates have been identified.
The commonly used analytical techniques in metabolomics are GC-MS (gas chromatography-mass spectrometry) or LC-MS (liquid chromatography-mass spectrometry) and NMR (nuclear magnetic resonance) spectrometry. NMR has the advantage that there is no need to have the compounds in the vapour phase, although the limit of detection using NMR is much poorer than MS.
LC-MS metabolomic studies have been recently undertaken using urine to identify putative colon inflammation biomarkers [18]. The authors note that urinary biomarkers would be preferable to sampling intestinal tissue or blood as the collection of urine samples is non-invasive and multiple samples are more
readily obtained.
The analysis of volatile organic compounds (VOCs) or metabolites (VOMs) is an emerging area of disease diagnosis. VOCs are small molecules that are readily analysed by GC-MS. Other commonly used methods of VOC detection are selected ion flow tube mass spectrometry (SIFT-MS) [Fig. 2], and the similar technique of PTR-MS (proton transfer MS).
There are already several FDA approved tests using volatiles from breath. These include testing for heart transplant rejection, hemoglobin breakdown in children and measurement of hydrogen or methane to diagnose GI lactose or fructose malabsorption. The measurement of breath hydrogen has also been used to diagnose SIBO. Recent work by Španĕl et al. using SIFT-MS quantified the breath pentane concentration of study subjects using the reaction of O2+ with pentane. It was found that patients with CD and UC had significantly elevated breath pentane levels compared to healthy controls [15].
Testing for fecal biomarkers of bowel disease is facile as samples are easily obtained and have been in contact with the gastro intestinal tract. The changes in the odour of feces and flatus reported in many bowel conditions are due to changes in the VOC profile. This altered VOC profile could lead to identification of biomarkers of disease state. A recent pilot study carried out by Ahmed et al. using GC-MS on fecal samples from IBD and IBS patients identified a key set of VOMs which were able to distinguish IBS-D from Active IBD with a sensitivity of 96% and a specificity of 80% [19].
Conclusions
MS techniques show promise for the identification of biomarkers of various GI disease states, which have the potential to reduce invasive testing, improve patient care and reduce healthcare costs.
Instrumentation is still expensive and relatively large, limiting its use in hospital settings and particularly limiting its use for near-patient testing. Also biomarker discovery is still in its infancy and much remains to be clarified in relation to the significance of markers to disease and the underlying metabolic pathways.
However, work to reduce the size and cost of mass spectrometers is well advanced and would open up the possibility of instruments being deployed for point-of-care detection and monitoring of diseases including IBS and IBD.
References
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5. Simrén M, Barbara G, Flint HJ, Spiegel BM, Spiller RC, et al. Gut 2013; 62(1): 159–176.
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15. Hrdlicka L, Dryahina K, Spanel P, Bortlik M, et al. Gastroenterology 2012; 142(5): S-784.
16. Rao AS, Camilleri M, Eckert DJ, Busciglio I, Burton DD, Ryks M, Zinsmeister AR. Am J Physiol Gastrointest Liver Physiol 2011; 301(5): G919–G928.
17. Garner CE, Smith S, de Lacy Costello B, White P, Spencer R, Probert C, Ratcliffe NM. FASEB J. 2007; 21(8): 1675–1688.
18. Otter D, Cao M, Lin H-M, Fraser F, Edmunds S, et al. J Biomed Biotechnol. 2011; 2011: 974701
19. Ahmed I, Greenwood R, de Lacy Costello B, Ratcliffe NM, Probert CS. PloS one, 2013; 8(3): e58204.
The authors
Ben De Lacy Costello PhD, Norman M. Ratcliffe*PhD and Sophie Shepherd BSc
Institute of Bio-Sensing Technology, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol, BS16 1QY
*Corresponding author
E-mail: Norman.Ratcliffe@uwe.ac.uk
Mass spectrometry: exciting perspectives for clinical labs
, /in Featured Articles /by 3wmediaMass spectrometry is poised for a new era, as clinical labs and researchers, hospital managers and industry prepare themselves for expansion in its use. Fuelling growth are trends towards personalized healthcare, the identification of novel biomarkers for translational medicine, large-scale epidemiological screenings as well as everyday clinical chemistry tests beyond just toxicology and endocrinology. There is room for such growth. At present, clinical lab applications of mass spectrometers account for only about 5% of the market.
Superior sensitivity and specificity, samples reusable
Mass spectrometry identifies a molecule by its unique mass-to-charge ratio, and is both highly sensitive and specific. In spite of concerns about cost and steep learning curves, the superiority of mass spectrometry versus immunoassays has never been disputed. Indeed, a study by the US National Cancer Institute (NCI) in 2008 focused on using mass spectrometry to distinguish between breath samples from patients with ovarian epithelial cancer versus those with polycystic ovarian syndrome or endometriosis.
Another advantage of mass spectrometry is its ability to use the same serum for multiple analyte profiling. This makes it useful in large-scale clinical studies, where samples have often been archived. Another NCI study, for instance, used mass spectrometry to identify biomarkers in blood from patients with acute myeloid leukemia; some of the samples were almost 10 years old. Dated samples have also been used for a range of other biomarkers, including malignant melanoma, soft tissue sarcomas and non-small cell lung cancer.
Gas chromatography and liquid chromatography
As a technology, mass spectrometry is not new in a lab setting. Gas chromatograph MS (GC-MS) has been used for ages in the diagnosis of organic metabolic disorders. More recently, liquid chromatograph mass spectrometry (LC-MS) has become a recommended resource for screening newborns.
The longer use of GC-MS means a bigger user base, as well as a more extensive legacy database, richer software libraries and advanced algorithms. Although GC-MS requires more complex processes for sample preparation (discussed below), it is relatively inexpensive compared to LC-MS systems, and has been considered effective enough for the bulk of applications.
The challenge of standardization
However, there is still some way to go before mass spectrometry attains wider use. One key barrier is a lack of standardization, above all in the preparation of samples. Clinical labs have different approaches to this issue, especially in terms of purification. This leads to sometimes-significant differences in results. Confounding the problem are continuing changes in the methods used for sample preparation, over time even within individual laboratories.
In the US, the Clinical and Laboratory Standards Institute has published two sets of recommendations on the use of MS. However, these leave quite a bit of room for interpretation and are considered no more than broad guidelines.
Preparation of samples for mass spectrometry
Typically, two steps are involved in preparing a sample: the concentrating of analytes, followed by ionization. The sample itself consists of two parts: the analytes of interest, and other components which are collectively known as the sample matrix. Sample preparation is considered the most difficult when whole blood or fractions are involved, given a relatively low density of analytes. Urine lies at the the other end of the spectrum, since the kidneys have already done most of the job of concentrating analytes.
Techniques for preparing samples include solid-phase extraction (SPE), immunoextraction (or immunoaffinity purification) and so-called ‘dilute-and- shoot’. In SPE, analytes and other matrix compounds are separated on the basis of their physical and chemical properties, among them charge and polarity. SPE systems consist of a liquid, mobile phase and a solid stationary phase (usually disposable cartridge-based). The liquid phase uses two different solvents, one for binding and washing, and another for elution.
Immunoextraction separates antibodies bound to the analytes from ‘free’ matrix components, by immobilizing them to a chromatographic column or polystyrene beads. After incubation with an immobilized antibody, unwanted components are washed away, and the enriched analyte is then eluted; another method is to concentrate the sample by drying, followed by re-suspension and injection into the chromatography system.
The third mechanism for preparing MS samples, dilute-and-shoot, is generally used in samples with a relatively high concentrations of analytes (e.g. urine). Here, dilution is usually effective enough to reduce matrix components to a
manageable level.
Successful ionization essential
The process of analysis relies wholly on successful ionization, as mass spectrometers can only detect charged analytes in a gaseous phase. Ionization can be either positive (cationic) or negative (anionic). The most common techniques for ionization in a clinical lab consist of chemical ionization and electrospray.
Chemical ionization generates ions by combining heat and plasma (produced by high-voltage electricity), at atmospheric pressure. While high temperatures vaporize the sample, the plasma (also known as a corona discharge) ionizes the evaporated solvent. Following this, mechanical interaction of the sample components (including analytes of interest) leads to the formation of negative or positive ions.
On its part, electrospray ionization uses electricity, heat and air to successively reduce the size of droplets that elute off the chromatographic column and sharply increase their charge. Ions (above all, proteins) desorb from the liquid droplet surface into a gas phase and then enter the mass spectrometer.
Challenges for vendors
Until recently, industry has focused on process improvements, while researchers have concentrated on improving the specificity and sensitivity of mass spectroscopy. Innovations from vendors have aimed at increasing the efficiency of ionization and of ion transfer, and accelerating discovery of biomarkers by combining size exclusion and affinity capture to enrich low molecular weight proteins, and more quickly separate diseased from clear samples. Some companies have also coupled reference databases of micro-organisms to their mass spectroscopy systems.
The greatest challenge for industry, however, has been to increase user acceptability. Research scientists rather than clinical lab technologists have been the traditional target for mass spectrometry manufacturers. The former, typically, have more interest in top-of-the-line technical specifications and performance than user-friendliness. The potential demand from clinical labs is forcing vendors to change approach. As a result, several are now beginning to package equipment sales with training and support.
Industry is also paying attention to systems integration, to bundle sample preparation instrumentation into a mass spectrometry suite and control its findings. Indeed, software has so far proved to be one of the biggest impediments to the growth of mass spectrometry, once again given the delicate balance between enabling new users to operate a system on the one side, while permitting complex adjustment of performance parameters on the other. OEMs have sought to plug this gap with bespoke add-ons but, as all IT systems designers know, this adds to system cost.
Researchers aim for more precision, ease of use
On the R&D side, a potentially promising area consists of so-called time-of-flight (TOF) mass spectrometers. TOF provides accuracy of 1 part per million by accelerating gas phase ions toward a detector via an electric field. Other initiatives are focused on robotic assistance, turbulent-flow chromatography and ion mobility – with considerable potential seen in linear ion traps. Scientists are also exploring the use of nanospray interfaces as well as microfluidics, though most successes to date have been at bench scale. In the future, such improvements will permit a reduction of detection thresholds, along with greater precision, ease of use and efficiency.
Some trade-offs inevitable
For both researchers and industry, the Holy Grail is to devise adequate user-configurability for trade-offs between high throughput on one side (required, for example, in epidemiological studies or newborn screening), and sensitivity and specificity on the other. Even now, detection of steroids such as cortisol, estradiol and testosterone remain a challenge at the lower end of their reference range, but require high precision in certain categories of patients, for example elderly female patients.
Lab use of mass spectrometry still minor, room for growth
No one doubts that the market for mass spectrometry is potentially huge. Globally, sales have been rising briskly, after falling due to the recession. A study from Los Angeles-based Strategic Directions International estimates the 2011 mass spectrometer market at USD 3.9 billion, with projections of USD 4.8 billion by 2014. The US and Canada hold the largest share of the market (38%) followed by Europe (31%) and Japan (13%), with other countries accounting for the remainder. Leaders in the mass spectrometer market include AB Sciex, Thermo Fisher Scientific, Waters and Agilent Technologies (all from the US), along with Hitachi and Shimadzu. European companies have a smaller presence, and include Germany’s GSG, Spectromat and Thermolinear.
As mentioned before, the clinical lab segment accounts for a very small share of total sales. The biggest users are pharmaceutical companies (a share of 20% of sales, with mass spectrometers increasingly used for metabolomic screening and drug discovery). Government follows closely (with an 18.5% share), universities (12.6%) and environmental/general testing services (9.4%). Electronics, the food and chemical industries also buy more mass spectrometers than clinical laboratories or hospitals.
However, the hope is that continuing growth in this entrenched base of other users will drive down unit costs of mass spectrometers, just as clinical labs get ready to increase their own requirements.
The evolution of mass spectrometry for endocrine medicine
, /in Featured Articles /by 3wmediaLiquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is rapidly emerging as the technology of choice for measuring steroid hormones. This review will focus on the utility of clinical mass spectrometry for the assessment of endocrine disorders.
by Dr P. Monaghan, L. Owen, Prof. P. Trainer and B. Keevil
Mass spectrometry or immunoassay?
The technological armamentarium of the modern day clinical laboratory has been greatly enhanced by the introduction and continued evolution of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. This technique is almost universally applicable to the measurement of small molecule compounds such as steroid hormones and is proving to be invaluable for the endocrinologist towards diagnosing and managing complex endocrine disorders [1]. Furthermore, LC-MS/MS is rapidly expanding for the application of quantitative peptide hormone and protein measurement. LC-MS/MS offers a number of considerable advantages over conventional immunoassay (IA) technology: greater analytical sensitivity and specificity, lack of susceptibility to interference from anti-reagent antibodies and cross-reacting compounds, multiplexing capability for steroid profiles, and low running costs for consumables in comparison to antibody-based reagents. However, like IA, LC-MS/MS is also vulnerable to interference that can compromise the analytical integrity of the method. The potential sources of analytical interference and inaccuracy to consider for IA and LC-MS/MS methodologies are summarized in Table 1.
Improved specificity: safer medical management of Cushing’s syndrome
The use of the 11β-hydroxylase inhibitor metyrapone generally has an adjunctive role in the medical management of Cushing’s syndrome with the aim of improving the medical status of patients prior to surgery or radiotherapy. Patients receiving adrenal-directed anti-steroidogenic drugs such as metyrapone require frequent clinical and biochemical monitoring to minimize the risk of treatment-induced hypoadrenalism.
Current clinical guidance advocates that metyrapone dose is titrated against serum cortisol concentration and some centres, including our own, assess normalization of cortisol production via the measurement a day curve with a mean serum cortisol target between 150–300 nmol/L. The monitoring of metyrapone therapy relies on the measurement of serum cortisol that by the vast majority of laboratories is performed by routine IA. However, metyrapone treatment causes altered steroid metabolism and therefore serum cortisol measurement is susceptible to positive interference when performed by IA due to cross-reactivity with precursor steroids such as 11-deoxycortisol (11DOC) that build up in the circulation as a result of the metyrapone blockade of the adrenal steroidogenic pathway.
Our group has recently quantified the level of positive interference in serum cortisol IA for patients receiving metyrapone therapy by employing a direct quantitative comparison with LC-MS/MS [2]. A modest correlation between plasma adrenocorticotropic hormone (ACTH) concentration and the extent of positive interference in the IA for serum cortisol was also observed as 90% of patients in our study had ACTH-driven Cushing’s syndrome [3]. Our study concluded that for patients receiving metyrapone therapy, cortisol analysis by LC-MS/MS mitigates the potential for erroneous clinical decisions concerning dose titration [Figure 1] and is likely to reduce the risk of unrecognized hypoadrenalism which may result in symptoms that mimic the side-effects of metyrapone treatment, or at worst be fatal.
Improved sensitivity: estradiol measurement
Progress in both LC-MS/MS and online sample preparation technology (pre-analytics) has advanced the analytical sensitivity of this methodology to the extent that for the measurement of many steroid hormones, modern MS applications have now transcended conventional IA methods in this regard. An example of this is the high sensitivity measurement of serum estradiol. External quality assurance data reveals that a wide range of concentrations can be obtained by immunoassay when measuring samples for estradiol at lower concentrations. Furthermore, a recent position statement from the Endocrine Society has stressed the need for better analytical methods to address the current poor performance of assays for measuring low concentrations of estradiol [4]. To this end, our group has developed a novel direct assay that is applicable to routine clinical use for the measurement of estradiol and estrone (therefore permitting calculation of total estrogen status) in male patients and patients on aromatase inhibitors [5]. This high sensitivity assay uses ammonium fluoride in the mobile phase to facilitate more efficient ionization and thereby increase analytical sensitivity. Additionally, an on-line solid phase extraction (OSM) system [Figure 2 (Waters, Manchester, UK)] allows a large volume of extract to be loaded and this coupled with a XEVO™TQS tandem mass spectrometer enables unprecedented analytical sensitivity to be achieved.
Conclusions and future prospects
LC-MS/MS is a very powerful tool which is enabling substantial innovations in the endocrine laboratory. Indeed, it is likely that the majority of emerging small molecules will be addressed by LC-MS/MS analysis. There are two keys areas in which future research and development for LC-MS/MS ought to be directed. Firstly, the utility of LC-MS/MS for the quantification of peptide hormones and proteins is already becoming a reality with published methods available for measurement of renin activity [6], parathyroid hormone [7] and insulin-like growth factor-1 [8] amongst others. These current methods require the skills of highly trained personnel in order to develop and run these assays, and it is hoped that continued innovation in this area will culminate in the development of rapid protein assays that are applicable to routine clinical use. Secondly, it seems feasible with existing technology to develop fully automated random-access LC-MS/MS analysers that will enable greater ease of use in non-specialist laboratory settings. However, the automation of mass spectrometry will not be achieved without a concerted effort from the in vitro diagnostics industry to fully realize the potential of LC-MS/MS across clinical medicine.
References
1. Monaghan PJ, Keevil BG, Trainer PJ. The use of mass spectrometry to improve the diagnosis and the management of the HPA axis. Rev Endocr Metab Disord 2013 Mar 15. [Epub ahead of print].
2. Monaghan PJ, Owen LJ, Trainer PJ, Brabant G, Keevil BG, Darby D. Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11β-hydroxylase inhibitor metyrapone. Ann Clin Biochem 2011; 48: 441–446.
3. Monaghan PJ, Owen LJ, Trainer PJ, Brabant G, Keevil BG, Darby D. Response to ‘Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11β-hydroxylase inhibitor metyrapone’ by Halsall DJ and Gurnell M. Ann Clin Biochem 2012; 49: 204–205.
4. Rosner W, et al. Challenges to the measurement of estradiol: An Endocrine Society Position Statement. J Clin Endocrinol Metab 2013; 98: 1376–1387.
5. Owen LJ, Wu FC, Labrie F, Keevil BG. A rapid direct assay for the routine measurement of oestradiol and oestrone by LC-MS/MS. Ann Clin Biochem [In press].
6. Carter S, Owen LJ, Kerstens MN, Dullaart RP, Keevil BG. A liquid chromatography tandem mass spectrometry assay for plasma renin activity using online solid-phase extraction. Ann Clin Biochem 2012; 49: 570–579.
7. Kumar V, Barnidge DR, Chen LS, Twentyman JM, Cradic KW, Grebe SK, Singh RJ. Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry. Clin Chem 2010; 56: 306–313.
8. Kay R, Halsall DJ, Annamalai AK, et al. A novel mass spectrometry-based method for determining insulin-like growth factor 1: assessment in a cohort of subjects with newly diagnosed acromegaly. Clin Endocrinol 2013; 78: 424–430.
9. Sturgeon CM, Viljoen A. Analytical error and interference in immunoassay: Minimizing risk. Ann Clin Biochem 2011; 48: 418–432.
10. Vogeser M, et al. Pitfalls associated with the use of liquid chromatography-tandem mass spectrometry in the clinical laboratory. Clin Chem 2010; 56: 1234–1244.
11. Duxbury K, Owen LJ, Gillingwater S, Keevil BG. Naturally occurring isotopes of an analyte can interfere with doubly deuterated internal standard measurement. Ann Clin Biochem 2008; 45: 210–212.
12. Davison AS, Milan AM, Dutton JJ. Potential problems with using deuterated internal standards for liquid chromatography-tandem mass spectrometry. Ann Clin Biochem 2013; 50: 274.
13. Twentyman JM, Cradic KW, Singh RJ, Grebe SK. Ionic cross talk can lead to overestimation of 3-methoxytyramine during quantification of metanephrines by mass spectrometry. Clin Chem 2012; 58: 1156–1158.
The authors
Phillip J. Monaghan*1 PhD, Laura J. Owen2 MSc, Peter J Trainer3 MD, and Brian G Keevil2 MSc
1Department of Clinical Biochemistry, 3Department of Endocrinology, The Christie NHS Foundation Trust, Wilmslow Road, Manchester M20 4BX, UK.
2Department of Clinical Biochemistry, University Hospital of South Manchester, Southmoor Road, Manchester, M23 9LT, UK.
*Corresponding author
E-mail: Phillip.Monaghan@nhs.net
LC-MS/MS in clinical diagnostic laboratories: screening for catecholamine-producing tumours
, /in Featured Articles /by 3wmediaAccurate quantitative targeted analysis of low molecular weight compounds is one of the most important needs in clinical diagnostic laboratories. The enhanced analytical specificity and sensitivity of modern liquid chromatography–tandem mass spectrometry based methods satisfy this requirement for screening of endocrine related disorders, including those affecting steroidogenic systems or overproduction of catecholamines.
by Dr M. Peitzsch and Professor G. Eisenhofer
Liquid chromatography – tandem mass spectrometry (LC-MS/MS)
The development of electrospray ionization (ESI) enabled the introduction of aqueous chromatographic eluates into mass spectrometers, an advance for which John Bennett Fenn was awarded the Nobel Prize in chemistry in 2002. Subsequent refinements in liquid chromatography coupled with ESI mass spectrometry led to analytical applications directed at a broad range of macromolecules from peptides, proteins, glycoproteins and glycolipids to lower molecular weight polar and non-polar compounds, including fatty acids, vitamins, nucleic acids, steroids, amino acids and biogenic amines.
Introduction of tandem mass spectrometry (MS/MS) represented a further breakthrough enabling analyses of relationships between ‘parent or precursor ions’ in the first stage and ‘daughter or product ions’ in the second stage of the instrument [1]. For targeted quantitative analyses, the filtering capabilities and the multiple reaction monitoring (MRM) possible through MS/MS triple quadrupole instruments provide not only high selectivity, but also improved signal-to-noise ratios. In recent years, the increasing commercial availability of stable isotope labelled substances, used as internal standards, has facilitated the application of stable isotope dilution internal standardization as the gold standard for accurate quantitative analyses. Since the physicochemical properties of the target analyte and the stable-isotope-labelled internal standard are similar, this approach compensates for all variations which occur during sample extraction, injection, chromatography, ionization, and ion detection with dow stream improvements in analytical precision and accuracy [2].
The high analytical specificity of LC-MS/MS allows less rigorous sample purification and chromatographic resolution than for standard high performance liquid chromatographic (HPLC) procedures employing ultraviolet, electrochemical or fluorimetric detection. This, and other developments in column chemistry, such as those allowing ultra-high performance liquid chromatography (UPLC), in turn enables higher sample throughput than offered by conventional HPLC procedures. Fusion of LC-MS/MS with other technologies, such as multiplexing parallel LC systems and turbo-flow technology, provides additional advantages for efficient and accurate high-throughput quantitative analyses. Further, automated online sample extraction systems minimize time spent on sample preparation and allow multiple applications to be efficiently handled by one instrument.
All the above possibilities for extending sample throughput, combined with the versatility of a single LC-MS/MS system to take over the jobs of multiple standard HPLC systems, provide advantages that justify the initial high cost of the instrument. Recognized impediments to implementing LC-MS/MS technology include the complexity of the instrumentation associated with the necessity for highly skilled personnel, especially for method development. A lack of standardization combined with a shortage of inter-laboratory comparison programs for quality assurance represent other limitations to acceptance by clinical laboratories.
LC-MS/MS in clinical diagnostics laboratories
The improvements in precision and accuracy offered by LC-MS/MS are now well recognized as offering critical advances over standard HPLC and immunoassay procedures, which are subject to analytical interferences or do not allow precise and accurate identification of structurally-related compounds, such as steroid hormones. Such advances are important to the fields of endocrinology and clinical laboratory medicine where accurate quantitative analysis is crucial for diagnostic purposes [2].
LC-MS/MS applications are now used in clinical and forensic toxicology, such as for drugs-of-abuse testing. In clinical laboratory medicine, LC-MS/MS is used for measurements of endocrine hormones such as steroids, biogenic amines and thyroid hormones, as well as for therapeutic drug monitoring and in new-born screening for assessment of inborn errors of metabolism.
In contrast to commonly used immunoassays, LC-MS/MS enables measurements of multiple analytes for each sample processed. Such determination of analyte profiles includes those for the various thyroid hormones, different vitamin D metabolites and steroid profiles, all available in single analytical runs. Profiles of steroid hormones, although mainly used in research applications, hold considerable promise for the routine clinical assessment of a wide range of
steroidogenic disorders.
LC-MS/MS based screening for catecholamine producing tumours
Pheochromocytomas and paragangliomas (PPGLs) are tumours arising respectively in adrenal and extra-adrenal chromaffin cells that are characterized by an overproduction of catecholamines. Without diagnosis and an appropriate treatment, the excessive secretion of catecholamines by PPGLs can lead to disastrous consequences.
For initial biochemical screening different tests are available, including plasma or urinary measurements of the catecholamines – norepinephrine, epinephrine and dopamine – and their respective O-methylated metabolites – normetanephrine, metanephrine and 3-methoxytyramine. Whereas the free metabolites are usually measured in plasma, analyses in urine are commonly performed after acid hydrolysis in which free metabolites are liberated from sulfate conjugates.
In 2002, Taylor and Singh presented an LC-MS/MS method for the analysis of deconjugated urinary fractionated metanephrines [3]. The outlined advantages of this method over other methods, such as immunoassay and HPLC-ECD (electrochemical detection), included relative freedom from drug interferences, high sample throughput and short chromatographic run times. Subsequently, there has been a plethora of related methods published, including many that enable detection of the much lower concentrations of plasma free metanephrines than urinary deconjugated metanephrines.
Development of new sample preparation procedures, either offline or online to the LC-MS/MS system, have been particularly useful for automated high-throughput procedures [4, 5]. More recent improvements in LC-MS/MS instrumentation have led to improved analytical sensitivity, now even enabling accurate and precise measurements of picomolar plasma concentrations of 3-methoxytyramine, the O-methylated metabolite of dopamine [5–7]. This valuable biomarker not only allows detection of dopamine producing PPGLs, but can also be used to detect malignancy [9]. Using LC-MS/MS, the diagnostic performance of 3-methoxytyramine as a marker of malignancy was characterized by an enhanced diagnostic sensitivity of 86% and specificity of 96% [8] [Fig. 1].
Problems with drug interferences in HPLC-ECD and immunoassay-based methods are largely overcome using LC-MS/MS. For example, problems of acetaminophen (paracetamol) interferences in HPLC-ECD procedures are not a problem for LC-MS/MS [8, 10]. Chromatographic disruptions associated with certain disorders, such as renal insufficiency, are also less of a problem by LC-MS/MS than by HPLC-ECD [8].
Use of plasma free normetanephrine, metanephrine and methoxytyramine for reliable diagnosis of PPGLs requires collection of blood samples after 30 minutes of supine rest and an overnight fast. These conditions pose difficulties for many clinicians, which can result in excessive false-positive results or worse, missed diagnoses when inappropriately high upper cut-offs have been derived from seated sampling. Measurements of urinary metanephrines provide a reasonable alternative test for those situations where blood samples cannot be collected appropriately.
As mentioned above, urinary metanephrines are commonly measured after an acid-hydrolysis deconjugation step. This procedure is based mainly on historical convention, where initially less sensitive instruments did not allow measurements of the much lower urinary concentrations of free rather than deconjugated metanephrines. Improvements in analytical sensitivity now, however, allow analysis of urinary free metanephrines, [11, 12]. Unlike the sulfate-conjugated derivatives, which are produced by a sulfotransferase enzyme located in the gastrointestinal tract, the free metabolites are produced within chromaffin cells. This provides a potential advantage for measurements of the free metabolites. Another advantage is that there are no suitable quality controls or calibrators for measurements of urinary deconjugated metanephrines [12, 13]. Those that are available are almost entirely in the free form so that procedures will always pass quality control even if the deconjugation step is missed and values for patient samples are grossly under-estimated. Measurements of urine free metanephrines avoid this potential pitfall in quality assurance.
Finally, with measurements of urinary free metanephrines it is possible to combine the measurements with urinary catecholamines in a single run [12;14]. This also provides an advantage over measurements of urinary deconjugated metanephrines, where the deconjugation step does not allow measurements of free catecholamines.
The difficulties in applying LC-MS/MS in the clinical chemistry laboratory, such as associated with high initial instrument costs and need for expertise, are easily overshadowed by the analytical advantages. High sample throughput and the analytical versatility offered by LC-MS/MS, which enables rapid method switching, in particular represent important advantages over standard HPLC methods. Nevertheless, such advantages are not easily realized by the small hospital-based laboratory where high sample throughput is not an important consideration. In the US the highly competitive nature of the heath care system is an incentive for centralized testing where efficiency and low operating costs associated with high sample throughput (economy of scale) are more easily realized. In the US the switch from immunoassays or HPLC-based methodology to superior LC-MS/MS technology is therefore likely to remain more advanced than in Europe.
Summary and conclusion
Modern LC-MS/MS systems provide well-recognized accuracy for quantitative targeted measurements of analytes used for clinical diagnostics. The high-throughput capabilities and versatility of LC-MS/MS instrumentation enable multiple applications for rare diseases to be handled by a single instrument. Furthermore, single analyte assays can be extended to accurate profiling by LC-MS/MS assays, providing deeper insight into endocrine metabolic disorders. This, however, remains largely a research-based application and for LC-MS/MS to be readily adapted for routine use in the clinical laboratories, other advantages such as those associated with economy of scale must be appreciated and realized.
References
1. Glish GL, Vachet RW. The basics of mass spectrometry in the twenty-first century. Nature Reviews Drug Discovery 2003; 2: 140–150.
2. Vogeser M, Parhofer KG. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) – Technique and applications in endocrinology. Exp Clin Endocrinol Diabetes 2007; 115: 559–570.
3. Taylor RL, Singh RJ. Validation of liquid chromatography-tandem mass spectrometry method for analysis of urinary conjugated metanephrine and normetanephrine for screening of pheochromocytoma. Clinical Chemistry 2002; 48: 533–539.
4. Lagerstedt SA, O’Kane DJ, et al. Measurement of plasma free metanephrine and normetanephrine by liquid chromatographym-tandem mass spectrometry for diagnosis of pheochromocytoma. Clinical Chemistry 2004; 50: 603–611.
5. Peaston RT, Graham KS, et al. Performance of plasma free metanephrines measured by liquid chromatography-tandem mass spectrometry in the diagnosis of pheochromocytoma. Clinica Chimica Acta 2010; 411: 546–552.
6. Eisenhofer G, Goldstein DS, et al. Biochemical and clinical manifestations of dopamine-producing paragangliomas: Utility of plasma methoxytyramine. J Clin Endocrinol Metab 2005; 90: 2068–2075.
7. Eisenhofer G, Lenders JW, et al. Plasma methoxytyramine: A n ovel biomarker of metastatic pheochromocytoma and paraganglioma in relation to established risk factors of tumour size, location and SDHB mutation status. European Journal of Cancer 2012; 48(11): 1739–1749.
8. Peitzsch M, Prejbisz A, et al. Analysis of plasma 3-methoxytyramine, normetanephrine and metanephrine by ultra performance liquid chromatography – tandem mass spectrometry: utility for diagnosis of dopamine-producing metastatic phaeochromocytoma. Ann Clin Biochem 2013; 50: 147–155.
9. Eisenhofer G, Tischler AS, et al. Diagnostic Tests and Biomarkers for Pheochromocytoma and Extra-adrenal Paraganglioma: From Routine Laboratory Methods to Disease Stratification. Endocrine Pathology 2012; 23: 4–14.
10. Petteys JB, Graham KS, et al. Performance characteristics of an LC–MS/MS method for the determination of plasma metanephrines. Clinica Chimica Acta 2012; 413: 1459–1465.
11. Boyle JG, Davidson DF, et al. Comparison of diagnostic accuracy of urinary free metanephrines, vanillyl mandelic acid, and catecholamines and plasma catecholamines for diagnosis of pheochromocytoma. J Clin Endocrinol Metab 2007; 92: 4602–4608.
12. Peitzsch M, Pelzel D, et al. Simultaneous liquid chromatography tandem mass spectrometric determination of urinary free metanephrines and catecholamines, with comparisons of free and deconjugated metabolites. Clinica Chimica Acta 2013; 418: 50–58.
13. Simonin J, Gerber-Lemaire S, et al. Synthetic calibrators for the analysis of total metanephrines in urine: Revisiting the conditions of hydrolysis. Clinica Chimica Acta 2012; 413: 998–1003.
14. Whiting MJ. Simultaneous measurement of urinary metanephrines and catecholamines by liquid chromatography with tandem mass spectrometric detection. Ann Clin Biochem 2009; 46: 129–136.
The authors
Mirko Peitzsch*1 PhD and
Graeme Eisenhofer1,2 PhD
1 Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus at the Technical University Dresden, Dresden, Germany
2 Department of Medicine III, University of Dresden, Dresden, Germany
*Corresponding author
E-mail: Mirko.Peitzsch@uniklinikum-dresden.de
The use of MS for the investigation of irritable bowel syndrome and inflammatory bowel disease
, /in Featured Articles /by 3wmediaCurrently, the diagnosis of bowel diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) relies on invasive and expensive procedures. Identification of biomarker-based tests to aid diagnosis is an important area of research. Here we review the use of mass spectrometry in this search and discuss recent findings.
by Dr B. De Lacy Costello, Professor N. M. Ratcliffe and S. Shepherd
Inflammatory bowel disease (IBD) is an inflammatory autoimmune disease caused by an inappropriate response of the immune system to commensal gut microbes [1]. There are two types of IBD, ulcerative colitis (UC) and Crohn’s disease (CD). UC affects the large bowel only, affecting variable lengths of the colon continuously from the rectum, primarily affecting the mucosa [Fig. 1]. CD can affect any part of the GI tract, and is a transmural disease [2]. Common symptoms of IBD are severe abdominal pain, defecation urgency and diarrhoea, which can contain blood.
Irritable bowel syndrome (IBS) is a functional disorder of the digestive tract. It is characterized by its symptoms, with no physiological changes in the GI tract. IBS can be diarrhoea predominant (IBS-D), constipation predominant (IBS-C) or symptoms can alternate between the two (IBS-A). Common symptoms include abdominal pain and cramps, bloating and flatulence, and unusual bowel habit. IBS has, as yet, no known cause. People with IBS show abnormal gut motility and hypersensitivity to pain in the GI tract. Stress and anxiety are known to cause changes in gut motility [3] with stress and anxiety being common symptoms of IBS. When under physical or psychological stress IBS patients showed increased gastro-intestinal sensitivity when compared to healthy controls [4]. Recently it has been thought that there may be changes in the gut microbiota in patients with IBS, the evidence being that IBS symptoms often occur after infective gastroenteritis or in patients in remission from IBD or diverticulitis. SIBO (small intestinal bowel overgrowth) has also been implicated in IBS and other function bowel disorders. One current hypothesis is that an altered microbiota activates the immune system within the mucosa, leading to an increase in epithelial permeability, causing dysregulation of the enteric nervous system [5]. Genome-wide association studies have successfully identified many genetic loci involved in susceptibility to IBD, and it is thought that genetic factors may also play a role in IBS [1].
Diagnosis of GI disease
IBS-D can present with symptoms similar to IBD and other non-functional bowel conditions. The diagnosis of IBS is often one of exclusion, where more serious bowel diseases, such as IBD or colon cancer which present with common symptoms, are ruled out. The current gold standard for diagnosis of IBD is endoscopic and histological testing; however, these investigations are both invasive and costly, and have associated risks. Of the patients referred for endoscopy few actually have organic bowel disease [6]. The costs associated with functional bowel disease are significant, with healthcare costs for IBS patients being significantly higher than non IBS controls [7].
There are currently no known biomarkers of IBS. There are various biomarkers that have potential in the differentiation of functional from inflammatory gastrointestinal disease, but there is still a need to identify biomarkers and to develop quicker, lower cost and less invasive testing for diagnosis of gastro-intestinal disease.
Biomarkers such as lactoferrin, calprotectin, c-reactive protein (CRP) and erythrocyte sedimentation rate (ECR) have all been used to help distinguish functional from inflammatory bowel disorders and to diagnose IBD. Serological markers such as antibodies to bacterial and fungal antigens that can indicate an abnormal response to commensal microbes can also be useful in identifying IBD.
Fecal calprotectin and lactoferrin are protein biomarkers of inflammation. In 2010 a meta-analysis of six studies (n=670) in adults by Van Rheenen et al. [8] found that screening patients by testing fecal calprotectin levels would have reduced the number of endoscopies performed by 67%, although its diagnosis would have been delayed in 6% of patients. When taking a weighted mean of 19 studies including 1001 patients, where IBD patients were compared with controls of IBS and other colonic diseases, fecal lactoferrin has a sensitivity and specificity of 80% and 82%, respectively [9].
Although these biomarkers can be useful as part of the screening process when establishing a diagnosis [6, 8], there is currently no biomarker or test that can replace the need for endoscopic and histological investigations. Mass spectrometry techniques are at the forefront of research for biomarker prospecting for IBS/IBD.
Mass spectrometry
Mass spectrometry (MS) has the ability to identify numerous compounds in a single sample. It is also high throughput allowing rapid analysis of many samples, which is especially useful for large studies or for the diagnosis of many samples. The ability to obtain results quickly, usually in less than 1 hour makes it attractive for clinical use.
Proteomic approach
Although MS (with associated sample vaporisation methods) was originally limited to low molecular weight volatile compounds, in the last 2 decades advances in MS technology have enabled its use with high molecular weight compounds, changing the way proteins are analysed. The soft ionization techniques electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) allow for the analysis of proteins and other macromolecules [10]. The identification of proteins through peptide mass fingerprinting, or peptide sequencing using MS is more rapid than techniques such as de novo protein sequencing and data can be analysed automatically. MS can also be used to determine the abundance of a molecule in a sample [10].
Differential protein expression can identify different diseases, and can indicate the degree of the disease state, or be used to assess the effects of treatment – for example the response of IBD patients to anti-TNF alpha antibodies (infliximab) [11]. It also has applications in the identification of protein biomarkers.
In 2011 MALDI-MS was used by M’koma et al. for tissue analysis; through profiling of the proteome of the colonic submucosa they were able to distinguish UC from CD by comparing proteomic spectra. Definitive diagnosis of either UC or CD is important as people with UC also have an increased risk of colon cancer [12].
Goo et al. have investigated protein biomarkers for IBS. ESI with LC-MS was used on protein fragments from the urine of women with IBS. They found differences in some specific components of the urinary proteome, and demonstrated that there is a possibility for future biomarker studies for IBS [13].
There are still limitations to mass spectrometric protein analysis, for example the difficulty in detecting hydrophobic membrane proteins. However, it seems promising that, with the advances in mass spectrometry technology, there will be an increase in the discovery of protein biomarkers and key pathogenic factors of gastro–intestinal disease, and improved diagnosis and therapy.
Metabolomic approach
The metabolome is the set of small molecule metabolites found in a biological sample. Unlike proteomics, metabolomics can be a direct measure of production of compounds and activity of cells or systems in an organism. This can be especially useful when looking for disease biomarkers in IBS and other bowel diseases as it can be used to understand the environment of the GI tract, as well as factors such as digestion and absorption of dietary products and gut microbial activity [14], which are implicated in IBS pathogenesis.
Researchers have explored the use of various techniques incorporating MS on breath [15], urine [16] and stool [17] samples in search of metabolic biomarkers of bowel disease for non-invasive testing and many possible candidates have been identified.
The commonly used analytical techniques in metabolomics are GC-MS (gas chromatography-mass spectrometry) or LC-MS (liquid chromatography-mass spectrometry) and NMR (nuclear magnetic resonance) spectrometry. NMR has the advantage that there is no need to have the compounds in the vapour phase, although the limit of detection using NMR is much poorer than MS.
LC-MS metabolomic studies have been recently undertaken using urine to identify putative colon inflammation biomarkers [18]. The authors note that urinary biomarkers would be preferable to sampling intestinal tissue or blood as the collection of urine samples is non-invasive and multiple samples are more
readily obtained.
The analysis of volatile organic compounds (VOCs) or metabolites (VOMs) is an emerging area of disease diagnosis. VOCs are small molecules that are readily analysed by GC-MS. Other commonly used methods of VOC detection are selected ion flow tube mass spectrometry (SIFT-MS) [Fig. 2], and the similar technique of PTR-MS (proton transfer MS).
There are already several FDA approved tests using volatiles from breath. These include testing for heart transplant rejection, hemoglobin breakdown in children and measurement of hydrogen or methane to diagnose GI lactose or fructose malabsorption. The measurement of breath hydrogen has also been used to diagnose SIBO. Recent work by Španĕl et al. using SIFT-MS quantified the breath pentane concentration of study subjects using the reaction of O2+ with pentane. It was found that patients with CD and UC had significantly elevated breath pentane levels compared to healthy controls [15].
Testing for fecal biomarkers of bowel disease is facile as samples are easily obtained and have been in contact with the gastro intestinal tract. The changes in the odour of feces and flatus reported in many bowel conditions are due to changes in the VOC profile. This altered VOC profile could lead to identification of biomarkers of disease state. A recent pilot study carried out by Ahmed et al. using GC-MS on fecal samples from IBD and IBS patients identified a key set of VOMs which were able to distinguish IBS-D from Active IBD with a sensitivity of 96% and a specificity of 80% [19].
Conclusions
MS techniques show promise for the identification of biomarkers of various GI disease states, which have the potential to reduce invasive testing, improve patient care and reduce healthcare costs.
Instrumentation is still expensive and relatively large, limiting its use in hospital settings and particularly limiting its use for near-patient testing. Also biomarker discovery is still in its infancy and much remains to be clarified in relation to the significance of markers to disease and the underlying metabolic pathways.
However, work to reduce the size and cost of mass spectrometers is well advanced and would open up the possibility of instruments being deployed for point-of-care detection and monitoring of diseases including IBS and IBD.
References
1. Khor B, Gardet A, Xavier RJ. Nature 2011; 474(7351): 307–317.
2. Geboes K. Churchill Livingstone Elsevier 2003; 255–276.
3. Drossman DA, Camilleri M, Mayer EA, Whitehead WE. Gastroenterology 2002; 123(6): 2108–2131.
4. Murray CD, Flynn J, Ratcliffe L, Jacyna MR, et al. Gastroenterology, 2004; 127(6): 1695–1703.
5. Simrén M, Barbara G, Flint HJ, Spiegel BM, Spiller RC, et al. Gut 2013; 62(1): 159–176.
6. Kok L, Elias SG, Witteman BJ, Goedhard JG, Muris JW, et al. Clinical chemistry 2012; 58(6): 989–998.
7. Maxion-Bergemann S, Thielecke F, Abel F, Bergemann R. Pharmacoeconomics 2006; 24: 21–37.
8. Van Rheenen PF, Van de Vijver E, Fidler V. BMJ 2010; 341: doi 10.1136/bmj.c3369.
9. Gisbert JP, McNicholl AG, Gomollon F. Inflammatory bowel diseases 2009; 15(11): 746–1754.
10. Alberici RM, Simas RC, Sanvido GB, Romão W, Lalli PM, Benassi M, Eberlin MN. Analytical and bioanalytical chemistry 2010; 398(1): 265–294.
11. Han NY, Kim EH, Choi J, Lee H, Hahm KB. Journal of Digestive Diseases 2012; 13(10): 497–503.
12. M’Koma AE, Seeleyv EH, Washington MK, Schwartz DA, Muldoon RL, Herline A, Caprioli RM. Inflammatory bowel diseases 2011; 17(4): 875–883.
13. Goo YA, Cain K, Jarrett M, Smith L, et al. Journal of Proteome Research 2012; 11(12): 5650–5662.
14. Collino S, Martin FPJ, Rezzi S. British journal of clinical pharmacology 2013; 75(3): 619–629.
15. Hrdlicka L, Dryahina K, Spanel P, Bortlik M, et al. Gastroenterology 2012; 142(5): S-784.
16. Rao AS, Camilleri M, Eckert DJ, Busciglio I, Burton DD, Ryks M, Zinsmeister AR. Am J Physiol Gastrointest Liver Physiol 2011; 301(5): G919–G928.
17. Garner CE, Smith S, de Lacy Costello B, White P, Spencer R, Probert C, Ratcliffe NM. FASEB J. 2007; 21(8): 1675–1688.
18. Otter D, Cao M, Lin H-M, Fraser F, Edmunds S, et al. J Biomed Biotechnol. 2011; 2011: 974701
19. Ahmed I, Greenwood R, de Lacy Costello B, Ratcliffe NM, Probert CS. PloS one, 2013; 8(3): e58204.
The authors
Ben De Lacy Costello PhD, Norman M. Ratcliffe*PhD and Sophie Shepherd BSc
Institute of Bio-Sensing Technology, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol, BS16 1QY
*Corresponding author
E-mail: Norman.Ratcliffe@uwe.ac.uk
Sleeping sickness elimination: are we dreaming?
, /in Featured Articles /by 3wmediaRecent sleeping sickness epidemics killed over 400,000 people in less than 20 sub-Saharan African countries. Serological screening of populations at risk and treatment of confirmed patients have drastically reduced the annually reported cases. Elimination seems feasible but only with new control tools and strategies adapted to the new epidemiological situation.
by Dr Philippe Büscher, Quentin Gilleman and Dr Pascal Mertens
Sleeping sickness, also called human African trypanosomiasis (HAT), is caused by two subspecies of the protozoan parasite Trypanosoma brucei (T.b.). The disease is transmitted by blood sucking tsetse flies that only occur in sub-Saharan Africa. T.b. gambiense causes a rather chronic disease and is found in West and Central Africa. T.b. rhodesiense causes a more fulminant form of the disease in Eastern Africa. Other Trypanosoma species cause diseases in animals, including cattle and small ruminants [Figure 1].
Infection and pathology
After inoculation with the saliva of an infective tsetse fly, the parasites invade lymph, blood and all peripheral organs where they multiply and survive the immune response of the host by a biological mechanism called antigenic variation. Eventually, the parasites invade the brain causing intrathecal inflammation associated with neurological disorders such as altered sleep-wake rhythm, behavioural changes, motor disabilities etc.
Except for some very rare cases, the disease is always lethal and even after successful treatment, many patients, especially children, never recover completely and remain disabled for the rest of their life. Sleeping sickness is a rural disease affecting poor populations living in the forests and wooded savannah where tsetse flies breed. Today, T.b. rhodesiense is mainly found in wild animals in game parks and natural reserves where it is often transmitted to rangers and visiting tourists.
Epidemiological background
At the turn of the 20th century, both gambiense and rhodesiense sleeping sickness caused devastating epidemics killing about one million people within two decades. By sustained implementation of vector control (including habitat destruction and insecticide spraying), culling of wild animals, and systematic screening of the population and treatment of patients by specialized teams, the colonial governments gained control over the epidemics and reduced the annual number of cases to less than 5000 cases around 1960. However, around 1990, a new epidemic of gambiense HAT was rampant in many countries with several tens of thousands of annually reported patients [1]. Countries most affected were typically poor and socio-politically unstable such as Angola, Central African Republic, D.R. of the Congo, Rep. of Congo, Sudan and Uganda to name a few.
Current situation
Today, about 20 years later, the number of reported cases has fallen again to about 7000 in 2012 of which 85% were diagnosed and treated in one single country, the D.R. of the Congo [2]. This achievement was made possible by a combination of different factors among which the availability of performing diagnostic tests and effective treatment, the recognition of sleeping sickness as Neglected Tropical Disease (NTD), thus attracting attention by donor agencies, humanitarian organizations and the private sector, and the combined effort of the World Health Organization, national HAT control programmes, bilateral cooperations and Non Governmental Organizations to organize large scale active case finding in the affected regions.
Active case finding is typically done by mobile teams that consist of up to seven persons trained in diagnosis and treatment of HAT. They go out in the field for several weeks, carrying all necessary equipment, diagnostics and drugs to screen the population at risk with a serological antibody detection test, to examine seropositive suspects by microscopy and to treat parasitologically confirmed patients in their villages or to refer them to the nearest specialized treatment centre. Since more than 20 years now, the recommended screening test is the Card Agglutination Test for Trypanosomiasis (CATT), a rapid test that detects gambiense specific antibodies [3].
Neglected Tropical Disease (NTD)
The recent success in HAT control has led to the inclusion of gambiense HAT in the WHO’s list of NTD’s that could be eliminated as a public health problem in Africa by 2020 with zero transmission in 2030 [2]. However, with the currently available tools for HAT control, elimination may remain an elusive target. Indeed, eradication of the tsetse flies, although proven to be feasible in some isolated foci with only one species transmitting trypanosomes, probably will never be achieved in endemic countries with dense forests and with large protected zones. As a consequence, tsetse flies will continue to transmit the disease, not only from man to man but also from the domestic and wild animal reservoir to man.
Diagnosis and treatment of infection
Today, treatment of sleeping sickness patients relies on toxic drugs and most often requires several weeks of hospitalization. Therefore, treatment is
administered only to patients in which the parasites have been detected in the blood, lymph or cerebrospinal fluid. Given that even the most sensitive parasite detection tests remain negative in 10% to 20% of actually infected patients, untreated patients may continue to act as a parasite reservoir, sometimes for years before they are treated or die. With the venue of molecular diagnostics, it was believed that such tests would sooner or later replace microscopic parasite detection. However, HAT patients have to be diagnosed in rural environments that are not compatible with today’s DNA- or RNA-based diagnostics and molecular test do not perform better than parasitology. Therefore, it is questionable if the individual patient will ever benefit from molecular diagnostics for sleeping sickness [4].
New control tools
Should we then despair about sleeping sickness elimination? Not at all, at least not for gambiense HAT. History shows that in countries that are socio-politically stable, where the rural population has access to functional primary healthcare facilities and where changing land use has suppressed the tsetse fly population, sleeping sickness has disappeared as is the case in Benin, Burkina Faso, Ghana and Togo [5]. For countries where these conditions cannot be met in the near future, newly developed HAT control tools may play a major role in disease elimination. For example, GIS technology allows to combine the GPS coordinates of all villages where HAT patients are reported with demographic and environmental data, and to precisely map the populations at risk [6].
New rapid test
Also, a new rapid diagnostic test for gambiense HAT serodiagnosis has been developed (HAT Sero-K-SeT, Coris BioConcept, Belgium). The HAT Sero-K-SeT is individually packed, thermostable, equipment-free , robust and has shown excellent diagnostic performance in a phase I evaluation [7]. Its target product profile, and especially its very high specificity, makes it fully compatible for use in foci with very low prevalence and in fixed health centres with minimal infrastructure [Figure 2]. In addition, strategies involving newly developed small size (0,25 x 0,25 m) insecticide-treated targets to kill the riverine tsetse fly are more cost effective than former models [8].
New drugs in the pipeline
Finally, the search for new drugs has identified a new class of compounds of which one, the SCYX-7158 has been selected for the development of a safe, one-dose oral treatment of both stages of sleeping sickness [9]. Once such a drug becomes available, parasite detection and stage determination that can only be accurately performed by expert medical staff, may become dispensable and decision to treat might be taken on the serodiagnostic evidence of infection.
Conclusion
Elimination of at least one form of sleeping sickness seems possible but only with the long-term commitment of donor agencies and ministries of health in endemic countries and with the cost efficient deployment of the newly developed control tools in rationally designed elimination strategies adapted to the local epidemiological situation.
References
1. World Health Organization. Control and surveillance of African trypanosomiasis. WHO Technical Report Series 1998; 881: 1-113.
2. World Health Organization. Report of a WHO meeting on elimination of African trypanosomiasis (Trypanosoma brucei gambiense), 3-5 December 2012, Geneva, Switzerland. WHO/HTM/NTD/IDM 2013.4 http://apps.who.int/iris/bitstream/10665/79689/1/WHO_HTM_NTD_IDM_2013.4_eng.pdf (accessed 27 May 2013)
3. Chappuis F, Loutan L, Simarro P, Lejon V and Büscher P. Options for the field diagnosis of human African trypanosomiasis. Clinical Microbiology Reviews 2005; 18: 133-146.
4. Deborggraeve S and Büscher P. Molecular diagnostics for sleeping sickness: where’s the benefit for the patient? The Lancet Infectious Diseases 2010; 10: 433-439.
5. Simarro PP, Diarra A, Ruiz Postigo JA, Franco JR, and Jannin JG. The human african trypanosomiasis control and surveillance programme of the world health organization 2000-2009: the way forward. PLoS Neglected Tropical Diseases 2011; 5: e1007.
6. Simarro PP, Cecchi G, Franco JR et al. Estimating and mapping the population at risk of sleeping sickness. PLoS Neglected Tropical Diseases 2012; 6: e1859.
7. Büscher P, Gilleman Q and Lejon V. Novel rapid diagnostic tests for sleeping sickness. New England Journal of Medicine 2013; 368: 1069-1070.
8. Esterhuizen J, Rayaisse JB, Tirados I et al. Improving the cost-effectiveness of visual devices for the control of riverine tsetse flies, the major vectors of human African trypanosomiasis 3. PLoS Neglected Tropical Diseases 2011; 5: e1257.
9. Jacobs RT, Nare B, Wring SA et al. SCYX-7158, an Orally-Active Benzoxaborole for the Treatment of Stage 2 Human African Trypanosomiasis. PLoS Neglected Tropical Diseases 2011; 5: e1151.
The authors
Philippe Büscher1* PhD, Quentin Gilleman2 MSc, and Pascal Mertens2 PhD
1 Institute of Tropical Medicine, Department of Biomedical Sciences, Nationalestraat 155, B-2000 Antwerp, Belgium
2 Coris BioConcept, Crealys Park, Rue Jean Sonet 4a, B-5032 Gembloux, Belgium
*Corresponding author
E-mail: pbuscher@itg.be
Tel. +32 3247 6371
Women and heart disease
, /in Featured Articles /by 3wmediaCardiovascular disease (CVD) is still widely considered as a middle-aged man’s disease and this is clearly a misconception. In actual fact, CVD is the number one cause of death for women worldwide. Also, compared with men, women have a number of additional risk factors that are specific to them and should not be ignored by medical professionals. Laboratory testing has a key role to play in the diagnosis and follow up of women with CVD. CLI talked to Jean Onofrio, Senior Director, Global Assay Marketing, Siemens Healthcare Diagnostics, about this important health issue for women.
Q.What impact does cardiovascular disease have on women?
Cardiovascular disease, or CVD, is a significant health concern for women. In fact, it’s the number one killer of women globally, [1] and according to the World Health Organization (WHO), accounts for one-third of deaths in women.
CVD also is the main cause of death for older women. Women generally develop CVD about 10 years later in life than men, likely due to the protective, anti-oxidant effects of estrogen prior to menopause.
Unfortunately, the misperception that CVD is a middle-aged man’s disease still persists. Understanding CVD’s global impact on women is one positive step toward battling the disease.
Q. What are the risk factors for CVD in women? How do these compare to risk factors in men?
While many CVD risk factors, such as age, family history and high blood pressure, are similar in both genders, there are some, including diabetes, tobacco use and high triglyceride levels, that put women at higher risk. Other risk factors, like obesity and depression, are more prevalent in women. There are also some risk factors unique to women, including pregnancy complications, oral contraceptive use, hormone replacement therapy and polycystic ovary syndrome. It’s important for women to understand their CVD risk factors and discuss their concerns with their physician.
Q. How does the mortality rate of women with CVD compare to the mortality rate of CVD in men?
While the mortality rate is high for older women, a heart attack can occur at any age. For younger women, heart attacks are actually more deadly than for men. According to the American Heart Association (AHA), among adults aged 45-62, women are twice as likely as men to die within the first year after a heart attack.
Also, more than twice as many women will develop heart failure within five years of surviving a heart attack compared to men, and three times more women than men will suffer a stroke after surviving a heart attack.
Q. What are some of the challenges associated with diagnosing CVD in women?
Women having a heart attack commonly present with symptoms other than chest pain, which makes diagnosis challenging. Rather, women often experience such less common symptoms as fatigue, indigestion, appetite loss and
“heart flutters.”
Even though these symptoms may not be severe, they may still lead to deadly consequences. Unfortunately, many women, and often clinicians, disregard their symptoms, attributing them to other non-life-threatening conditions.
Adding to this challenge, women with CVD aren’t as likely as men to receive aggressive diagnosis and treatment. Consider that women receive only about 34 percent of interventional treatments, with and witout the placements of stents.
Q. What role does laboratory testing play in the diagnosis and management of women with CVD? What about biomarkers?
CVD is largely preventable, and simple laboratory tests can help assess a person’s risk.
Laboratory professionals play an increasingly important role in providing access to both traditional and novel cardiac biomarkers that are available throughout the disease continuum. Also, whether conducted in the central lab or at the point-of-care, cardiac tests, such as high-sensitivity troponin, are key diagnosis tools.
By leveraging the appropriate use of laboratory diagnostic testing, clinicians can help enhance the assessment, diagnosis and follow-up care for women with CVD.
Reference
1.http://gamapserver.who.int/gho/interactive_charts/women_and_health/causes_death/ chart.html; accessed 11/27/12
Use of global hemostatic markers for risk stratification and personalized treatment of coronary artery disease
, /in Featured Articles /by 3wmediaCoronary artery disease has been linked to a hypercoagulable state of the blood, and the use of global hemostatic assays such as thromboelastography, thrombin generation or the overall hemostatic assay may allow for prediction of adverse events in these patients as well as targeted, individualized treatment.
by Dr C. Reddel, Dr J. Curnow and Professor D. Brieger
Global hemostatic markers in coronary artery disease
Hemostasis is the process by which bleeding is stopped, involving blood coagulation and platelet aggregation. This process depends on the delicate balance of many pro- and anti-coagulant factors, and when hemostatic balance is disrupted, pathological clot formation may occur leading to potentially fatal venous or arterial thrombosis. Appropriate fibrinolysis, the breakdown of blood clots, is also essential to the process of hemostasis.
Coronary artery disease is considered an inflammatory disease in which patients are predisposed to arterial thrombosis, which can lead to myocardial infarction. Additionally, the presence of coronary artery disease can increase the risk of venous thrombosis [1]. This points to an overall hypercoagulable state of the blood in this disease. Although the use of antiplatelet and anticoagulant therapies is a common and necessary method of reducing this risk, this may unnecessarily expose patients to a risk of bleeding. There is a need to risk stratify patients and individually tailor thromboprophylaxis.
Imbalances in the hemostatic system can be assessed in citrated plasma samples from patients either by measuring individual coagulation and fibrinolytic factors, or by global coagulation assays. Such imbalances have been found to be associated with various pro-thrombotic states, such as cancer, pregnancy or trauma. In stable and acute coronary artery disease, there is evidence for links between prognosis and markers of coagulation and fibrinolysis, including prothrombin fragment 1+2, fibrinopeptide A, thrombin–antithrombin and plasmin–antiplasmin complexes, D-dimer, plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor and tissue plasminogen activator [2, 3]. However, measuring single factors does not reflect the overall hemostatic balance as other pro- or anti-coagulant, and pro- and anti-fibrinolytic factors may compensate for the deficient or elevated factor. Therefore measurement of the overall coagulable state of the blood may provide a more relevant picture.
Standard laboratory coagulation tests, such as prothrombin time (PT) or activated partial thromboplastin time (APTT), can be useful for patients with bleeding disorders, but do not reliably detect hypercoagulability in this context. Recently, there has been interest in global assays of coagulation and fibrinolysis as methods of assessing the overall potential of a patient’s blood to form or lyse a clot. These include assays of thrombin generation, thromboelastography and the overall hemostatic potential assay.
Thromboelastography
Thromboelastography is a method measuring clot formation and lysis in whole blood. A pin is suspended into a cuvette of whole blood heated to 37°C, and the cup and pin move relative to each other, so that when the clot forms the interference is detected by the pin. Thromboelastography (TEG, Haemonetics, Braintree, Massachusetts, USA) and Thromboelastometry (ROTEM, Tem International GmbH, Munich, Germany) are two commercial variants of the assay. The assay measures not only time to clot, but speed of clot formation, clot strength and elasticity, and can be modified to assess platelet function, fibrinogen, hyperfibrinolysis and effect of anticoagulant treatment. The use of whole blood means the role of the cell is incorporated into the assay, although this necessitates immediate use of the sample.
Thromboelastography is a point-of-care assay which is used to measure and characterize peri-operative bleeding. It may additionally be useful in monitoring antiplatelet therapy such as aspirin or clopidogrel. Recently, it has also been used to detect hypercoagulability in patients with coronary artery disease, and further, has been demonstrated to predict thrombotic events in patients who have undergone coronary stenting or coronary artery bypass grafting [4, 5].
Thrombin generation assay
The thrombin generation assay was first described in 1953, but has more recently been simplified, standardized and commercialized, including in the form of the Calibrated Automated Thrombogram (Thrombinoscope BV, Maastricht, The Netherlands) and Technothrombin (TGA, Technoclone, Vienna, Austria) [6]. In this assay, ex vivo potential for thrombin generation is measured in platelet-rich or platelet-poor plasma. In a 96-well plate, thrombin generation is triggered by addition of tissue factor, phospholipids and calcium at 37°C, and conversion of a substrate for thrombin measured over an hour by fluorescence.
Thrombin is central to the process of hemostasis, and various pro-thrombotic states have been associated with variations in plasma potential to generate thrombin. Patients with stable coronary artery disease have elevated thrombin generation [Fig. 1] [7], and patients with acute coronary syndrome have still higher thrombin potential [8]. Antiplatelet therapies most likely do not affect the thrombin generation assay in platelet-poor plasma, but it may be possible to monitor the effect of anticoagulant drugs (including novel oral anticoagulants) using the assay, and preliminary assessment has suggested the assay can predict bleeding and ischemic events in patients with coronary artery disease [9].
Overall Hemostatic Potential (OHP) assay
The Overall Hemostatic Potential (OHP) assay is a test of fibrin generation and fibrinolysis first described in 1999 [10]. Similar to the thrombin generation assay, it is performed in citrated plasma in 96-well plates and triggered by tissue factor or thrombin and calcium at 37°C. It is a turbidometric assay, measuring the change in absorbance over an hour at 405nm, which allows for a kinetic analysis of fibrin clot formation. Tissue plasminogen activator is also added to half the wells, which triggers fibrinolysis. The assay measures coagulation potential and fibrinolytic potential, and is carried out on stored plasma samples.
A limitation of the plasma-based thrombin generation and OHP assays is the absence of cells. These assays have nonetheless identified differences between patients with pro-thrombotic states and healthy controls, and the use of plasma allows for samples to be stored and batch-tested, which is an advantage for screening large numbers of patients. The OHP assay additionally requires no specialized equipment, apart from a standard plate reader, and although not standardized, it is inexpensive. Unlike thromboelastography which is relatively insensitive to hypofibrinolysis, the OHP assay can detect and quantify hypofibrinolysis as well as hyperfibrinolysis.
Very recently the OHP assay has been used to show hypercoagulability and hypofibrinolysis in patients with acute and stable coronary artery disease [Fig. 2] [7, 11]. The observations in this latter population suggest the potential for this assay to predict future events, and prospective studies are required to determine its utility in this context.
Future trends and requirements
There is a growing body of evidence that ex vivo hypercoagulability of patients’ blood or plasma has prognostic value in arterial or venous thrombotic events. Global markers of hemostasis, including results of thromboelastography, the thrombin generation and OHP assays, may prove clinically relevant in identifying individual patients at risk of adverse event, and thus allow the tailoring of thromboprophylaxis. Further large-scale prospective trials are needed to directly address this.
References
1. Anandasundaram B, Lane DA, Apostolakis S, Lip GY. The impact of atherosclerotic vascular disease in predicting a stroke, thromboembolism and mortality in atrial fibrillation patients: a systematic review. J Thromb Haemost. 2013; 11: 975–987.
2. Stegnar M, Vene N, Bozic M. Do haemostasis activation markers that predict cardiovascular disease exist? Pathophysiol Haemost Thromb. 2003; 33: 302–308.
3. Gorog DA. Prognostic value of plasma fibrinolysis activation markers in cardiovascular disease. J Am Coll Cardiol. 2010; 55:2 701–709.
4. Hobson AR, Agarwala RA, Swallow RA, Dawkins KD, Curzen NP. Thrombelastography: current clinical applications and its potential role in interventional cardiology. Platelets 2006; 17: 509–518.
5. McCrath DJ, Cerboni E, Frumento RJ, Hirsh AL, Bennett-Guerrero E. Thromboelastography maximum amplitude predicts postoperative thrombotic complications including myocardial infarction. Anesth Analg. 2005; 100: 1576–1583.
6. Hemker HC, Giesen P, AlDieri R, Regnault V, de Smed E, Wagenvoord R, et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb. 2002; 32: 249–253.
7. Reddel CJ, Curnow JL, Voitl J, Rosenov A, Pennings GJ, Morel-Kopp MC, et al. Detection of hypofibrinolysis in stable coronary artery disease using the overall haemostatic potential assay. Thromb Res. 2013; 131: 457–462.
8. Orbe J, Zudaire M, Serrano R, Coma-Canella I, Martinez de Sizarrondo S, Rodriguez JA, et al. Increased thrombin generation after acute versus chronic coronary disease as assessed by the thrombin generation test. Thromb Haemost. 2008; 99: 382–327.
9. Campo G, Pavasini R, Pollina A, Fileti L, Marchesini J, Tebaldi M, et al. Thrombin generation assay: a new tool to predict and optimize clinical outcome in cardiovascular patients? Blood Coag Fibrinolysis 2012; 23: 680-687.
10. He S, Bremme K, Blomback M. A laboratory method for determination of overall haemostatic potential in plasma. I. Method design and preliminary results. Thromb Res. 1999; 96: 145–156.
11. Leander K, Blomback M, Wallen H, He S. Impaired fibrinolytic capacity and increased fibrin formation associate with myocardial infarction. Thromb Haemost. 2012; 107: 1092–1099.
The authors
Caroline Reddel* PhD; Jennifer Curnow MBBS, PhD, FRACP, FRCPA; David Brieger MBBS, PhD, FRACP, FACC
ANZAC Research Institute, Concord Repatriation General Hospital, Concord NSW, 2139, Australia
*Corresponding author
E-mail: creddel@anzac.edu.au
YKL-40: a new prognostic biomarker in patients with coronary artery disease
, /in Featured Articles /by 3wmediaInflammation is of importance for the progression of coronary artery disease. Until now, there has been no biomarker to monitor the effect of treatment regimes. YKL-40 is a new biomarker of inflammation, which if highly elevated in the disease, is a strong prognostic predictor of death and potentially can be used to monitor disease activity.
by Prof. J. Kastrup, Dr M. Harutyunyan-Bønsager and Dr N. D. Mygind
Clinical background
The number of patients with coronary artery disease (CAD) is increasing worldwide, and CAD is the most common cause of death in western countries. Although the prognosis and quality of life for patients has improved due to more aggressive and invasive treatment regimes, in the US someone will have a coronary event approximately every 25 seconds, and someone will die of one approximately every minute. Therefore CAD is an increasing economic burden and the total estimated direct and indirect costs of CAD in the US in 2010 were $503.2 billion [1].
Currently, there is a lack of new biomarkers for monitoring the effect of the patients’ treatment and for predicting their risk of a heart attack, heart failure and cardiac death.
Coronary artery disease and inflammation
It has been well established that inflammation plays an important role in development and progression of atherosclerosis in the coronary arteries [2]. Moreover, inflammation is also involved in the inflammatory pathways inducing extracellular matrix remodelling and heart failure progression [3]. The inflammatory biomarker high-sensitivity C-reactive protein (hs-CRP) is associated with atherosclerosis and the incidence of coronary events [4], but its association with the extent and severity of atherosclerosis remains controversial. Therefore, it is not very useful for continuous monitoring of treatment effects and progression of the disease.
The inflammatory biomarker YKL-40
YKL-40 is a glycoprotein mainly produced by macrophages and neutrophils, which are important for the development of atherosclerosis, and is stimulated by hypoxia [5]. Serum YKL-40 is suggested to be a biomarker of diseases characterized by inflammation [5] and its plasma concentration has been shown to increase reversibly in patients by more than 25% following an inflammatory stimulus.
YKL-40 is not a disease specific biomarker, but plays a role in cell migration and adhesion, angiogenesis, remodelling of the extracellular matrix, cell proliferation and differentiation [5]. Macrophages in atherosclerotic plaques, especially those located more deeply in the atherosclerotic lesion, express YKL-40 [6], and macrophages in early atherosclerotic lesions express the highest amount of YKL-40 mRNA. As Hs-CRP is mainly produced in the liver, it is likely that biomarkers such as YKL-40 (secreted from inflammatory cells within the atherosclerotic plaque) could be superior for monitoring CAD.
YKL-40 in healthy subjects
The normal YKL-40 value in a healthy subject from the general population has recently been published [7]. In 3130 subjects the median YKL-40 value was 40 µg/L and increased exponentially with age.
YKL-40 in coronary artery disease
Serum YKL-40 has been found to be increased in both acute and coronary artery disease [8]. Serum YKL-40 levels were also significantly increased in patients with acute ST-elevation myocardial infarction and thereafter consistently decreased from a maximum value just after the myocardial infarction and during a 360 day follow-up period towards its normal levels. Plasma YKL-40 levels were found to correlate inversely with left ventricular ejection fraction (LVEF) recovery, but not with infarct size in patients with STEMI [9, 10].
Although highly increased in patients with stable CAD, it has not been possible to detect any relationship between serum YKL-40 level and the degree of CAD as evaluated by the number of vessels involved or the degree of artery stenosis [11]. In patients with stable CAD, revascularization with balloon angioplasty of significant stable coronary artery lesions has no effect on YKL-40 levels within a 6 month follow-up period (unpublished data).
This indicates that YKL-40 not is a measurement of the amount of ischemia within the myocardium. Serum YKL-40 seems to be more a measurement of ongoing inflammatory activity rather than the presence of stabilized chronic lesions.
Therefore, it is very interesting that serum YKL-40 was a very strong prognostic biomarker for death within a 2.6 and 6 year follow-up period in patients with stable CAD [12, 13] [Fig. 1].
YKL-40 and heart failure
The consequence of CAD is often the development of severe heart failure. It has recently been demonstrated that serum YKL-40 is increased in heart failure and that YKL-40 is an independent significant prognostic biomarker for death [15]. It is interesting that serum YKL-40 measured in all-comers at acute hospital admission is a very strong predictor of death, especially within the first year, in patients with heart disease [16]. Of patients admitted with disease of the heart, those with elevated YKL-40 had a hazard ratio of death within the first year after discharge from the hospital at 2.5 compared to heart patients with normal serum YKL-40 levels. YKL-40 remained an independent biomarker of mortality, even after adjusting for other known risk factors such as age, hs-CRP and NT-proBNP [16].
YKL-40 for monitoring CAD activity
Statin treatment is used in CAD for lowering cholesterol levels. However, it also has an anti-inflammatory action. Therefore, it is very interesting that serum YKL-40 is significantly lower in patients with stable CAD on statin treatment compared to patients without [14] [Fig. 2].
This difference seems to be independent of the effect that statins have on lowering cholesterol levels, indicating that the YKL-40 level can be regulated by the direct anti-inflammatory action of statins [14]. This is unlike the situation with the inflammatory biomarker hs-CRP, which has been shown to correlate to cholesterol levels in statin-treated CAD patients [14].
Moreover, the mortality is also lower in stable CAD on statins compared to non-statins [12, 13]. This indicates that YKL-40 could be used to monitor the anti-inflammatory effect of statin treatment. Whether YKL-40 is also useful for
monitoring the effects of other anti-angina medications remains to be investigated.
Conclusion and future perspective
YKL-40 is a new inflammatory biomarker in ischemic heart disease. It is increased in both acute and chronic coronary artery disease and is a very strong diagnostic biomarker for death. It is suggested to be a mirror of the active inflammatory atherosclerotic processes in CAD, more than a measurement of degree of myocardial ischemia induced by stable coronary lesions. Since YKL-40 is lower in patients on statin treatment, it can potentially be used to monitor disease activity and the effect of anti-inflammatory or stabilizing treatment regimes.
Conflict of interest
A patent application (WO 2009/092382) is published and pending.
References
1. Roger VL, Go AS, Lloyd-Jones DM, Benjamin EJ, Berry JD, Borden WB, et al. Circulation 2012; 125(1): e2–e220.
2. Hansson GK. J Thromb Haemost 2009; 7 Suppl 1: 328–331.
3. Radauceanu A, Ducki C, Virion JM, Rossignol P, Mallat Z, McMurray J, et al. J Card Fail 2008; 14(6): 467–474.
4. Corrado E, Rizzo M, Coppola G, Fattouch K, Novo G, Marturana I, et al. J Atheroscler Thromb 2010; 17(1): 1–11.
5. Kastrup J. Immunobiology 2012; 217(5): 483–491.
6. Boot RG, van Achterberg TA, van Aken BE, Renkema GH, Jacobs MJ, Aerts JM, et al. Arterioscler Thromb Vasc Biol 1999; 19(3): 687–694.
7. Bojesen SE, Johansen JS, Nordestgaard BG. Clin Chim Acta 2011; 412: 709–712.
8. Wang Y, Ripa RS, Johansen JS, Gabrielsen A, Steinbruchel DA, Friis T, et al. Scand Cardiovasc J 2008; 42(5): 295–302.
9. Nojgaard C, Host NB, Christensen IJ, Poulsen SH, Egstrup K, Price PA, et al. Coron Artery Dis 2008; 19(4): 257–263.
10. Hedegaard A, Ripa RS, Johansen JS, Jorgensen E, Kastrup J. Scand J Clin Lab Invest 2010; 70(2): 80–86.
11. Mathiasen AB, Harutyunyan MJ, Jorgensen E, Helqvist S, Ripa R, Gotze JP, et al. Scand J Clin Lab Invest 2011; 71(5): 439–447.
12. Kastrup J, Johansen JS, Winkel P, Hansen JF, Hildebrandt P, Jensen GB, et al. Eur Heart J 2009; 30(9): 1066–1072.
13. Harutyunyan M, Gotze JP, Winkel P, Johansen JS, Hansen JF, Jensen GB, Hilden J, Kjøller E, Kolmos HJ, Gluud C, Kastrup J. Immunobiology 2013; 218(7): 945–951.
14. Mygind ND, Harutyunyan MJ, Mathiasen AB, Ripa RS, Thune JJ, Gotze JP, et al. Inflamm Res 2011; 60(3): 281–287.
15. Harutyunyan M, Christiansen M, Johansen JS, Køber L, Torp-Petersen C, Kastrup J. Immunobiology. 2012; 217(6): 652–656.
16. Mygind ND, Iversen K, Køber L, Goetze JP, Nielsen H, Boesgaard S, Bay M, Johansen JS, Nielsen OW, Kirk V, Kastrup J. J Intern Med 2013; 273(2): 205–216.
The authors
Jens Kastrup* MD, DMSc; Marina Harutyunyan-Bønsager MD; and Naja Dam Mygind MD
Department of Cardiology B, The Heart Centre, Rigshospitalet Copenhagen University Hospital, Copenhagen, Denmark
*Corresponding author
E-mail: jens.kastrup@regionh.dk
A breakthrough in timely ovarian cancer diagnosis?
, /in Featured Articles /by 3wmediaWhile globally ovarian cancer is the eighth most common cancer in women, in the developed countries (with the exception of Japan) the disease is much more prevalent. In Europe it is the fifth most frequently diagnosed cancer in women, with an average lifetime risk of 1 in 70, and in both Europe and North America the disease accounts for over 5% of all female cancer deaths. In addition, unlike with most other cancers, the five year survival rate of only 45% has barely improved in the last 30 years. This poor prognosis is largely due to the non-specific symptoms, resulting in diagnosis at Stage III or IV when the tumour has already metastasized. But if ovarian cancer is diagnosed early, the five year survival rate exceeds 90%.
Much work in recent decades has concentrated on finding a simple screening method that would allow more timely diagnosis; so far none has had a significant effect on mortality. An assay for the most frequently used biomarker, CA125, was developed around 30 years ago. Normally elevated in the serum of patients diagnosed with symptomatic ovarian cancer, CA125 is ideal in disease management, but its use to enable early disease detection has remained controversial. Specificity is very limited as the serum level is raised in several benign conditions (such as endometriosis) as well as in other cancers. In addition sensitivity is only about 50% in patients with Stage I or II disease. More recently human epididymis protein 4 (HE4) has been advocated as a useful marker for ovarian cancer detection. Its level is not elevated as a result of benign pelvic disease so its specificity is higher than CA125, but levels of HE4 are also raised in some other cancers. Recent work on ovarian cancer screening has suggested that screening utilizing a combination of these two biomarkers may be the best approach for early disease detection.
Now exciting preliminary data from the Anderson Cancer Center have just been published. Over 4,000 women, healthy at the start of the study, were classified into three risk groups based on a mathematical model- the ROCA- incorporating their age and CA125 serum level. Follow-up over eleven years was dependent on the evolving perceived risk. The US researchers were ‘cautiously optimistic’ about this approach, but await results from a similar trial in the UK, involving more than 200,000 women, which will be available within two years. Hopefully, though, screening using the ROCA will lead to more timely diagnosis and thus a better survival rate for ovarian cancer patients.
Phage-displayed peptides as novel reagents for norovirus detection
, /in Featured Articles /by 3wmediaCurrent methods for detecting noroviruses (NoVs) have significant limitations in sensitivity and feasibility for use in remote locations. Our group recently identified phage-displayed peptides with specific binding to NoVs and sensitivity comparable to that of existing antibodies. These reagents can be easily optimized by mutagenesis and represent promising diagnostic tools.
by Amy M. Hurwitz, Prof. Robert L. Atmar and Prof. Timothy G. Palzkill
Norovirus infection and diagnosis
Each year, norovirus (NoV) infections cause approximately 267 million new cases of gastroenteritis and 200,000 deaths worldwide [1]. Infection spreads rapidly in areas of close human contact, such as cruise ships and hospitals, and is treated only by rehydration, as no antiviral therapy currently exists. An infectious dose estimated to be as low as 18 virions and high environmental stability contributed to classification of NoVs as a category B biodefense agent in the U.S. Therefore, rapid, accurate and highly sensitive diagnosis is important for outbreak recognition and control, and also to guide physicians in patient management. The potential health and economic consequences that may be ameliorated by early NoV detection have led to a high demand for optimized detection reagents that can be used to develop reliable diagnostic assays with minimal requirements for expensive, bulky equipment or technical training.
NoVs are divided into six different genogroups (GI–GVI) based on the amino acid sequence of the major capsid protein (VP1). These are organized further into more than 30 genotypes, and finally into numerous strains or variants [2]. The VP1 protein assembles to form an icosahedral shell with an inner shell (S) domain and outer protruding (P) domain. The P domain is on the virus surface and is the most accessible, while the S domain has the highest sequence conservation across different strains. Given the ability of NoVs to evolve rapidly to result in novel or recombinant strains, continual optimization of detection reagents may be necessary in order to recognize the majority of human-infecting strains. Strains classified into GI and GII are most relevant for human infections, and thus the focus for diagnostic assay development efforts.
Current diagnostic methods and their limitations
Methods used currently for the diagnosis of norovirus infection are far from ideal as they exhibit several limitations that hinder their use for individual patient diagnoses or in rural and developing locations. The gold standard for diagnosis is reverse transcriptase (RT)-PCR, which requires multiple sets of primers to detect about 90% of human-infecting strains [3]. This method has significant equipment and expertise requirements, which are often not available outside of large institutions. Further, the expense of running multiple samples and the need for timely instrument accessibility limit the feasibility of applying RT-PCR as point-of-care applications or for preventing the rapid spread of an outbreak.
Other existing methods include immune electron microscopy (IEM) and enzyme immunoassays. IEM was the first method described for identifying NoVs and was used originally to classify viruses based on structural appearance. This method has limited sensitivity, and also requires expensive equipment and skilled expertise. Enzyme immunoassays, developed after the discovery of type-specific antibody epitopes on the NoV capsid, detect viral particles in human stool samples [4]. This method offers increased specificity and has led to the development of commercially available ELISA and lateral flow assays.
Currently, the only FDA-approved antigen detection assay is an ELISA called RIDASCREEN® (3rd Generation) produced by R-Biopharm, which uses an antibody cocktail with specificity for GI and GII NoVs [Fig. 1]. Due to limitations in sensitivity, this assay is only approved for use during outbreaks and takes several hours to produce results. Several companies, including R-Biopharm, have developed rapid diagnostic assays that use lateral flow technology and have also demonstrated strong specificity for NoV GI and GII strains. However, these have similar limitations with sensitivity and thus are only recommended for preliminary screening to be confirmed by RT-PCR, and are distributed primarily outside of the United States [5]. Overall, there is a clear need for improved diagnostic methods to detect norovirus rapidly with strong specificity, high sensitivity, and with minimal equipment and expertise requirements.
Novel diagnostic phage reagents
Recent studies in our laboratory have identified short, 12-mer peptide reagents with specific binding to the GI.1 NoV genotype [6]. The small size of these peptides displayed on phages offers the ability to access epitopes that may be buried in the capsid protein and not accessible to antibodies, and the potential for increased avidity through multiple linked peptide molecules. To identify peptides with specific binding to NoV, we used phage display technology to screen commercially available, large-scale libraries of randomized peptides that are fused to the gene III protein and expressed in five copies on one end of the phage. Rounds of biopanning were performed in which filamentous phage libraries were screened for phages displaying peptides that bind immobilized Norwalk (NV) GI.1 virus-like particles (VLPs). The phage libraries were added to VLPs and, after washing away non-binding phages, the phages displaying VLP-binding peptides were eluted with low pH [Fig. 2A]. Two to four subsequent rounds of biopanning using the resulting phage populations enriched for phages displaying peptides with the highest binding affinity for NV. DNA sequencing of individual phage clones recovered after multiple rounds of biopanning revealed three peptides, named NV-O-R5-3, NV-O-R5-6, and NV-N-R5-1, that occurred most commonly, and the phage clones displaying the peptides were further characterized for their NoV binding properties [6].
Phage-based ELISAs confirmed the binding specificity of phage-displayed peptides to NV VLPs. These affinity-binding assays used NV VLP captured by immobilized rabbit polyclonal anti-NV antibody in order to maintain the structural integrity of VLPs. Single phage clones were added to the captured VLPs and binding was detected using anti-M13 phage antibody that was conjugated to horseradish peroxidase to provide a signal for bound antibody [Fig. 2B]. Of the three peptide-displaying phage clones analysed, NV-N-R5-1 exhibited a dose-dependent response with decreasing NV VLP concentration and the highest sensitivity with a limit of detection at 1.56 ng NV VLP. Additional phage ELISAs indicated that NV-N-R5-1 binds to the P domain of the capsid protein, which extends the furthest out from the virus, and has comparable sensitivity for NV as existing antibodies used for diagnostics [6]. These results provide proof-of-concept and a strong lead reagent for developing novel phages displaying peptides as effective detection reagents for NoV. Further, the methods described establish a platform methodology for using phage display to identify antigen-specific binding reagents that may be applied to any pathogen with distinct surface epitopes.
Current status
To develop our lead phage-displayed peptide into a commercially viable tool, we are currently optimizing its binding affinity for other genogroups of NoV in order to broaden its diagnostic applications. Phage display technology provides a simple platform for constructing collections of new mutations in a lead peptide that can be used for additional rounds of biopanning to screen for variants with optimal affinity properties [Fig. 2C]. The three phage-displayed peptides discussed above share conserved amino acid sequence motifs that likely confer binding specificity for particular epitopes on the NV capsid protein. Directed evolution through mutagenesis of amino acids surrounding these consensus sequences can enable us to improve binding affinity to NV and alter binding specificities starting with the lead phage peptide, NV-N-R5-1. In particular, developing phage-displayed peptides with optimized binding affinity for the NoV GII.4 genotype, which accounts for >80% of NoV infections worldwide [1], and other GI and GII NoV genotypes will have the greatest relevance for diagnostic applications.
Future development of bacteriophage reagents
For decades, phages have been used to identify their target bacterial strains and species in order to diagnose the cause of infections by phage typing. More recent applications have begun to leverage synthetic biology and genomic engineering strategies to customize phage specificity and reporter signals to enable ‘near-real-time’ detection of a broader range of human pathogens [7]. Our recent work has established a methodology for the identification, characterization, and development of phage-based affinity reagents that may be applied to different pathogens and translated into diagnostic applications. The process outlined in Figure 2 demonstrates the progression from (A) identifying lead reagents against a target of interest, (B) characterizing binding affinity for the antigenic target, (C) optimizing leads through directed evolution or genomic engineering strategies, and finally (D) producing scalable quantities of reagent for commercial diagnostic applications. Zou and colleagues, for example, used a similar method to identify a phage-displayed peptide reagent with specific binding to transmittable gastroenteritis virus (TGEV) that also showed potential antiviral activity [8]. Several groups have also developed phage-based reagents to detect bacterial pathogens, such as Salmonella enterica and Escherichia coli [9, 10].
In summary, the use of phage-based reagents for microbial diagnostics offers many advantages in comparison to more commonly used detection reagents, such as antibodies. Phage display technology enables rapid identification and validation of candidate phage reagents with specificity for new or evolved pathogens through biopanning of commercial or custom made phage libraries (Fig. 2A, B). Phage manipulation through directed evolution facilitates development of reagents with optimized binding affinity and specificity to a target of interest (Fig. 2B). Finally, production of large quantities of phages is accomplished rapidly and inexpensively, as simple preparation methods can produce sufficient phage for hundreds of assays (Fig. 2D). As viral pathogens such as NoV continually evolve, the flexibility provided by phage-based reagents will be essential for developing next generation diagnostics for effective containment of outbreaks. A cocktail of phages, each of which binds to a specific target NoV genotype, may ultimately be the ideal strategy for producing an assay to detect the broadest possible range of NoVs without sacrificing specificity. Overall, phages have an enormous potential for use as detection reagents in clinical, agricultural, food, and environmental settings, and represent an underutilized resource for diagnostic development.
References
1. Donaldson EF, Lindesmith LC, Lobue AD, Baric RS. Norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations. Immunological Reviews 2008; 225(1): 190–211.
2. Kroneman A, Vega E, Vennema H, Vinjé J, White P, Hansman G, Green K, Martella V, Katayama K, Koopmans M. Proposal for a unified norovirus nomenclature and genotyping. Archives of Virology 2013; doi:10.1007/s00705-013-1708-5.
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The authors
Amy M. Hurwitz1 BS, Robert L. Atmar2,3 MD, Timothy G. Palzkill*2,4 PhD
1 Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA
2 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA
3 Department of Medicine, Baylor College of Medicine, Houston, Texas, USA
4 Department of Pharmacology, Baylor College of Medicine, Houston, Texas, USA
*Corresponding author
E-mail: timothyp@bcm.edu