weekly news updates on | june 2013 | … · 2017-07-17 · to ﬁnd out more about our range of...

Also in this issue :

Sleeping sickness elimination Pg. 22

Women and heart disease Pg. 26

New prognostic biomarker for coronary artery disease Pg. 30

ELISA & IFA automated platform Pg.33

Sampling tube for effective glycolysis inhibition Pg.35

Multiparametric immunofluorescence assay for hantaviruses Pg.36

Exciting perspectives for mass spectrometry in clinical labs Pg.6

Weekly news updates on www.cli-online.com | June 2013 | Volume 37

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Frances Bushrod, Ph.D.

Around 80% of people in Western countries experience low back pain at some point in their life; indeed during a single year up to half of the adult population will experi-ence back pain. In the majority no single clear cause can be identified, and the condition is self limiting. However for the approximately 7% of patients who develop chronic low back pain, quality of life can be significantly impaired. Chronic low back pain also has a serious financial impact in terms of health-care costs and lost working days; in most industrialized countries it is the most common reason for workplace absence.Now researchers at the Spine Centre of Southern Denmark have pub-lished the results of two very inter-esting and potentially far-reaching studies. The first involved sixty-one patients who had been suffering from low back pain for more than six months. Lumbar disc hernia-tion was confirmed by MRI and the patients underwent primary surgery for removal of nucleus material. When this material was cultured for micro-organisms, forty-six percent of patients had positive cultures, predominantly with the normally commensal anaerobic bacterium Propionibacterium acnes. A sig-nificantly higher number of patients with anaerobic infections developed new Modic changes (MC), MRI-visible bone edema associated with low back pain, in vertebrae adja-cent to the previous disc herniation compared with patients with nega-tive cultures or cultures positive for aerobic organisms.The second study was a double blind randomized controlled trial involv-ing 162 patients who had been suf-fering from low back pain for more than six months and with MC in a disc adjacent to a previous disc her-niation. Patients received either pla-cebo or the antibiotic Bioclavid for a hundred days and were followed up at the end of treatment and after one year; outcome measures included both pain and workplace absence. Improvement was highly

significant in the group treated with the antibiotic.The authors suggest that when the lumbar disc is herniated, the anaerobic bacteria penetrate it and precipitate an insidious infec-tion and chronic low back pain. Although they stress that antiseptic

techniques were rigorously followed when the nucleus material was removed, it is surely still necessary to find a method of demonstrating anaerobic infection in patients who have low back pain and relevant MC but who have not had surgery. If this could be done many desperate

chronic lower back pain sufferers might finally be able to stop taking analgesics or visiting osteopaths, chiropractors and acupunctur-ists, and get relief from a course of antibiotics instead.

Chronic low back pain: could an anaerobic infection be responsible?

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– June 20133EDITOR’S LETTER

ContentsFRONT COVER

FEATURES [6 - 20] MASS SPECTROMETRY

[6 - 8] Mass spectrometry: exciting perspectives for clinical labs

[10 - 12] The evolution of mass spectrometry for endocrine medicine

[14 - 16] LC-MS/MS in clinical diagnostic laboratories: screening for catecholamine-producing tumours [18 - 20] The use of MS for the investigation of irritable bowel syndrome and inflammatory bowel disease

[22 - 24] PARASITOLOGY

Sleeping sickness elimination: are we dreaming?

[26] INTERVIEW

Women and heart disease

[27 - 32] CORONARY ARTERY DISEASE

[27 - 29] Use of global hemostatic markers for risk stratification and personalized treatment of coronary artery disease

[30 - 32] YKL-40: a new prognostic biomarker in patients with coronary artery disease

REGULARS[3] Editor’s letter

[33] Product news

Mass 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 per-sonalized 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.

The cover image shows an AB SCIEX QTRAP®5500 system with SelexION® (for research use only).

Also in this issue :

Sleeping sickness elimination Pg. 22

Women and heart disease Pg. 26

New prognostic biomarker for coronary artery disease Pg. 32

ELISA & IFA automated platform Pg.33

Sampling tube for effective glycolysis inhibition Pg.35

Multiparametric immunofluorescence assay for hantaviruses Pg.36

Exciting perspectives for mass spectrometry in clinical labs Pg.6

Weekly news updates on www.cli-online.com | June 2013 | Volume 35

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Superior sensitivity and specificity, samples reusableMass spectrometry identifies a molecule by its unique mass-to-charge ratio, and is both highly sensitive and specific. In spite of con-cerns 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 polycys-tic ovarian syndrome or endometriosis.

Another advantage of mass spectrometry is its ability to use the same serum for mul-tiple analyte profiling. This makes it useful in large-scale clinical studies, where sam-ples have often been archived. Another NCI study, for instance, used mass spec-trometry 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 chromatographyAs a technology, mass spectrometry is not new in a lab setting. Gas chromatograph MS (GC-MS) has been used for ages in the diag-nosis of organic metabolic disorders. More recently, liquid chromatograph mass spec-trometry (LC-MS) has become a recom-mended resource for screening newborns.

The longer use of GC-MS means a bigger user base, as well as a more extensive leg-acy 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 standardizationHowever, there is still some way to go before mass spectrometry attains wider use. One key barrier is a lack of standardi-zation, above all in the preparation of sam-ples. Clinical labs have different approaches to this issue, especially in terms of purifica-tion. 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 spectrometryTypically, two steps are involved in prepar-ing a sample: the concentrating of analytes, followed by ionization. The sample itself consists of two parts: the analytes of inter-est, and other components which are collec-tively 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 spec-trum, since the kidneys have already done most of the job of concentrating analytes.

Techniques for preparing samples include solid-phase extraction (SPE), immunoex-traction (or immunoaffinity purification) and so-called ‘dilute-and- shoot’. In SPE,

analytes and other matrix compounds are separated on the basis of their physi-cal and chemical properties, among them charge and polarity. SPE systems consist of a liquid, mobile phase and a solid station-ary 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 immobi-lized antibody, unwanted components are washed away, and the enriched analyte is then eluted; another method is to concen-trate 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 gener-ally 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 essentialThe process of analysis relies wholly on successful ionization, as mass spectrom-eters can only detect charged analytes in a gaseous phase. Ionization can be either positive (cationic) or negative (anionic). The most common techniques for ioniza-tion 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 vapor-ize the sample, the plasma (also known as a corona discharge) ionizes the evapo-rated solvent. Following this, mechani-cal 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,

Mass spectrometry: exciting perspectives for clinical labsMass 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.

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proteins) desorb from the liquid droplet surface into a gas phase and then enter the mass spectrometer.

Challenges for vendorsUntil 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 sam-ples. 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 spectrom-etry 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 bun-dle 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 spec-trometry, 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 useOn 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 detec-tor 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 microfluid-ics, 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 inevitableFor both researchers and industry, the Holy Grail is to devise ade-quate 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 tes-tosterone 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 growthNo one doubts that the market for mass spectrometry is poten-tially huge. Globally, sales have been rising briskly, after falling due to the recession. A study from Los Angeles-based Strategic Direc-tions 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 pharmaceuti-cal companies (a share of 20% of sales, with mass spectrometers increasingly used for metabolomic screening and drug discov-ery). Government follows closely (with an 18.5% share), univer-sities (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.



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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 chromatogra-phy-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 expand-ing for the application of quantitative peptide

hormone and protein measurement. LC-MS/MS offers a number of considerable advan-tages over conventional immunoassay (IA) technology: greater analytical sensitivity and specificity, lack of susceptibility to interfer-ence 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 analyti-cal 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 syndromeThe 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 meas-urement of serum cortisol that by the vast majority of laboratories is performed by routine IA. However, metyrapone treatment causes altered steroid metabolism and there-fore serum cortisol measurement is suscepti-ble 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 com-parison with LC-MS/MS [2]. A modest correlation between plasma adrenocorti-cotropic 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 erro-neous 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.

The evolution of mass spectrometry for endocrine medicineLiquid 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


IMMUNOASSAY [9].

Process Mechanism Example

Macro-complexes Immunoglobulin-analyte complex with immunoreactivity in the IA.

Macroprolactin (most documented example). Macro-TSH. Macro-CK (macroenzyme).

Antireagent antibodies *Binding of anti-reagent antibodies to the capture and/or detection antibodies in IA.

Human anti-mouse antibodies (HAMA). Heterophilic antibodies. Rheumatoid factor.

Autoantibodies Autoantibody-analyte complex formation. Susceptible IA include: thyroglobulin, insulin and thyroid hormone assays.

Cross-reactivity Detection of structurally-related compounds in the specimen by IA.

hCG cross-reactivity in LH assay. Prednisolone cross-reactivity in cortisol assay.

High-dose hook effect

Capture and detection Ab in two-site immunometric assays become saturated at very high analyte concentration, inhibiting sandwich complex formation.

May affect compounds that circulate at high concentration in disease, including hCG, prolactin and thyroglobulin.

Hereditary binding protein abnormalities

Genetic variants of binding proteins with altered affinity.

**The familial dysalbuminaemic hyperthyroxinaemia (FDH) albumin variant has enhanced binding affinity for T4 causing elevated FT4 result.

LC-MS/MS [10].

Ionisation

Matrix effects: Presence of compounds in the column eluent that cause ionization yield attenuation or effect droplet formation during the ionisation process.

Matrix effects have the potential to confound any LC-MS/MS method; appropriate ion suppression studies should be performed during method validation.

Choice of internal standard

Naturally occurring isotopes of the analyte of interest may interfere with bi-deuterated IS.

Naturally occurring isotope of cortisol at m/z 365 acts in the same way as D2-cortisol and fragments to give a daughter ion of the same m/z [11].

Choice of solvent for ISLoss of deuterium labels due to back-exchange of label for hydrogen ions present in solvent.

Loss of 3 deuteriums from d5 5-HIAA internal standard [12].

Mass transition selection

Isobaric compounds that have the same nominal molecular mass as the target analyte, or higher molecular weight compounds that are multiply charged and share the same m:z as target analyte.

Isobaric compounds 21-deoxycortisol, corticosterone and 11-deoycortisol (all mass 346.46 g mol-1).

Ionic cross-talk

Cross talk may occur if several mass transitions are acquired. If the collision cell is not completely purged within the interscan delay period, spurious signal will manifest in the subsequent mass transition trace.

Overestimation of 3-methoxytyramine due to ionic cross-talk during the measurement of plasma metanephrines [13].

Table 1. Potential sources of analytical interference and inaccuracy in immunoassay and LC-MS/MS*More likely to affect two-site immunometric assay format.**More likely to affect one-step analogue assays.

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Improved sensitivity: estradiol measurementProgress in both LC-MS/MS and online sample preparation tech-nology (pre-analytics) has advanced the analytical sensitivity of this methodology to the extent that for the measurement of many ster-oid hormones, modern MS applications have now transcended con-ventional IA methods in this regard. An example of this is the high sensitivity measurement of serum estradiol. External quality assur-ance 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 meth-ods to address the current poor performance of assays for measur-ing 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 prospectsLC-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 pro-teins 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 meth-ods 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 spec-trometry 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.

References1. 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 chromatog-raphy-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 measure-ment 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 hyperparathy-roidism 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: Minimiz-ing 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 over-estimation of 3-methoxytyramine during quantification of metanephrines by mass spectrometry. Clin Chem 2012; 58: 1156–1158.

The authorsPhillip J. Monaghan*1 PhD, Laura J. Owen2 MSc, Peter J Trainer3 MD, and Brian G Keevil2 MSc1Department 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 authorE-mail: [email protected]


Figure 1. Mean day curve concentration of cortisol and 11DOC for a patient as metyrapone is titrated from 500mg to 1000mg t.d.s. Dashed lines represent the target cortisol range. Figure adapted from Monaghan, et al [2].

Figure 2. The on-line solid phase extraction (OSM) system (Waters, Manchester, UK) used by the authors.

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Liquid chromatography – tandem mass spectrometry (LC-MS/MS) The development of electrospray ioniza-tion (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 spectrom-etry (MS/MS) represented a further break-through 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 filter-ing 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 inter-nal standards, has facilitated the applica-tion 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 chroma-tographic (HPLC) procedures employing ultraviolet, electrochemical or fluorimet-ric detection. This, and other develop-ments 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 sys-tems minimize time spent on sample prep-aration 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 instru-ment. Recognized impediments to imple-menting 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-lab-oratory comparison programs for qual-ity assurance represent other limitations to acceptance by clinical laboratories.

LC-MS/MS in clinical diagnostics laboratoriesThe improvements in precision and accu-racy offered by LC-MS/MS are now well recognized as offering critical advances over standard HPLC and immunoassay proce-dures, which are subject to analytical inter-ferences or do not allow precise and accu-rate identification of structurally-related compounds, such as steroid hormones. Such advances are important to the fields of endocrinology and clinical laboratory med-icine where accurate quantitative analysis is crucial for diagnostic purposes [2].

LC-MS/MS in clinical diagnostic laboratories: screening for catecholamine-producing tumoursAccurate 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


Figure 1. Schematic set-up for the analysis of metanephrines, including sample preparation by solid phase extraction (A), LC-MS/MS (B) and computerized output (C).

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 thera-peutic drug monitoring and in new-born screening for assessment of inborn errors of metabolism.

In contrast to commonly used immu-noassays, LC-MS/MS enables meas-urements of multiple analytes for each sample processed. Such determination of analyte profiles includes those for the various thyroid hormones, different vita-min 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 tumoursPheochromocytomas and paragangliomas (PPGLs) are tumours arising respectively in adrenal and extra-adrenal chromaffin cells that are characterized by an overproduc-

tion 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 uri-nary measurements of the catecholamines – norepinephrine, epinephrine and dopa-mine – and their respective O-methylated metabolites – normetanephrine, metane-phrine and 3-methoxytyramine. Whereas the free metabolites are usually measured in plasma, analyses in urine are com-monly 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 analy-sis of deconjugated urinary fractionated metanephrines [3]. The outlined advan-tages of this method over other methods, such as immunoassay and HPLC-ECD (electrochemical detection), included rela-tive freedom from drug interferences, high sample throughput and short chromato-graphic run times. Subsequently, there has been a plethora of related methods pub-lished, including many that enable detec-tion 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 pro-cedures [4, 5]. More recent improvements in LC-MS/MS instrumentation have led to improved analytical sensitivity, now even enabling accurate and precise measure-ments of picomolar plasma concentrations of 3-methoxytyramine, the O-methylated metabolite of dopamine [5–7]. This valu-able biomarker not only allows detection of dopamine producing PPGLs, but can also be used to detect malignancy [9]. Using LC-MS/MS, the diagnostic perfor-mance 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) interfer-ences in HPLC-ECD procedures are not a problem for LC-MS/MS [8, 10]. Chromato-graphic disruptions associated with certain

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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 col-lection of blood samples after 30 minutes of supine rest and an overnight fast. These conditions pose difficulties for many clini-cians, 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 metane-phrines 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 metane-phrines. Improvements in analytical sensi-tivity now, however, allow analysis of uri-nary 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 chromaf-fin cells. This provides a potential advan-tage for measurements of the free metabo-lites. Another advantage is that there are no suitable quality controls or calibrators for measurements of urinary deconju-gated 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 sam-ples are grossly under-estimated. Measure-ments of urine free metanephrines avoid this potential pitfall in quality assurance.

Finally, with measurements of urinary free metanephrines it is possible to com-bine the measurements with urinary cat-echolamines in a single run [12;14]. This also provides an advantage over measure-ments of urinary deconjugated metane-phrines, 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 instru-ment 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 switch-ing, in particular represent important advantages over standard HPLC methods. Nevertheless, such advantages are not eas-ily realized by the small hospital-based lab-oratory 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 operat-ing costs associated with high sample throughput (economy of scale) are more easily realized. In the US the switch from immunoassays or HPLC-based methodol-ogy to superior LC-MS/MS technology is therefore likely to remain more advanced than in Europe.

Summary and conclusionModern LC-MS/MS systems provide well-recognized accuracy for quantitative tar-geted measurements of analytes used for clinical diagnostics. The high-throughput capabilities and versatility of LC-MS/MS instrumentation enable multiple applica-tions 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.

References1. Glish GL, Vachet RW. The basics of mass spec-

trometry in the twenty-first century. Nature Reviews Drug Discovery 2003; 2: 140–150.

2. Vogeser M, Parhofer KG. Liquid chromatogra-phy tandem-mass spectrometry (LC-MS/MS) – Technique and applications in endocrinol-ogy. Exp Clin Endocrinol Diabetes 2007; 115: 559–570.

3. Taylor RL, Singh RJ. Validation of liquid chroma-tography-tandem mass spectrometry method for analysis of urinary conjugated metanephrine and normetanephrine for screening of pheochromo-cytoma. 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 spec-trometry 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-pro-ducing paragangliomas: Utility of plasma meth-oxytyramine. J Clin Endocrinol Metab 2005; 90: 2068–2075.

7. Eisenhofer G, Lenders JW, et al. Plasma meth-oxytyramine: A n ovel biomarker of metastatic pheochromocytoma and paraganglioma in rela-tion 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 chro-matography - 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 char-acteristics of an LC–MS/MS method for the determination of plasma metanephrines. Cli-nica Chimica Acta 2012; 413: 1459–1465.

11. Boyle JG, Davidson DF, et al. Comparison of diagnostic accuracy of urinary free metane-phrines, vanillyl mandelic acid, and catechola-mines and plasma catecholamines for diagno-sis 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 metane-phrines 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 spec-trometric detection. Ann Clin Biochem 2009; 46: 129–136.

The authorsMirko Peitzsch*1 PhD and Graeme Eisenhofer1,2 PhD

1 Institute for Clinical Chemistry and Labo-ratory Medicine, University Hospital Carl Gustav Carus at the Technical University Dresden, Dresden, Germany2 Department of Medicine III, University of Dresden, Dresden, Germany



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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 dis-ease (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 func-tional 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 hyper-sensitivity 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-intesti-nal sensitivity when compared to healthy controls [4]. Recently it has been thought that there may be changes in the gut micro-biota 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 func-tion bowel disorders. One current hypoth-

esis is that an altered microbiota activates the immune system within the mucosa, leading to an increase in epithelial perme-ability, causing dysregulation of the enteric nervous system [5]. Genome-wide asso-ciation studies have successfully identified many genetic loci involved in susceptibil-ity to IBD, and it is thought that genetic factors may also play a role in IBS [1].

Diagnosis of GI diseaseIBS-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 diagno-sis of IBD is endoscopic and histological testing; however, these investigations are both invasive and costly, and have asso-ciated risks. Of the patients referred for endoscopy few actually have organic bowel disease [6]. The costs associated with func-tional bowel disease are significant, with healthcare costs for IBS patients being sig-nificantly higher than non IBS controls [7].

There are currently no known biomark-ers of IBS. There are various biomarkers that have potential in the differentiation of functional from inflammatory gastro-intestinal 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, calprotec-tin, c-reactive protein (CRP) and eryth-rocyte 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 pro-tein 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 tak-ing a weighted mean of 19 studies includ-ing 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 use-ful as part of the screening process when establishing a diagnosis [6, 8], there is cur-rently no biomarker or test that can replace the need for endoscopic and histological investigations. Mass spectrometry tech-niques are at the forefront of research for biomarker prospecting for IBS/IBD.

Mass spectrometryMass 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 approachAlthough MS (with associated sample vaporisation methods) was originally lim-ited to low molecular weight volatile com-pounds, in the last 2 decades advances in MS technology have enabled its use with high molecular weight compounds, chang-ing the way proteins are analysed. The soft ionization techniques electrospray ioniza-tion (ESI) and matrix-assisted laser des-orption/ionization (MALDI) allow for the analysis of proteins and other macromol-ecules [10]. The identification of proteins

The use of MS for the investigation of irritable bowel syndrome and inflammatory bowel diseaseCurrently, 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


through peptide mass fingerprinting, or peptide sequencing using MS is more rapid than techniques such as de novo protein sequenc-ing 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 anal-ysis; through profiling of the proteome of the colonic submu-cosa 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 approachThe 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 incor-porating 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 (liq-uid chromatography-mass spectrometry) and NMR (nuclear mag-netic 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 pref-erable 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 metabo-lites (VOMs) is an emerging area of disease diagnosis. VOCs are small molecules that are readily analysed by GC-MS. Other com-monly 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, hemo-globin 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 reac-tion of O2+ with pentane. It was found that patients with CD and UC had significantly elevated breath pentane levels compared to healthy controls [15].

– June 201319

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Figure 1. Diagram of the gut showing the areas where inflammatory disease is most likely to occur.

Testing for fecal biomarkers of bowel dis-ease 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 sam-ples from IBD and IBS patients identified a key set of VOMs which were able to distin-guish IBS-D from Active IBD with a sensi-tivity of 96% and a specificity of 80% [19].

ConclusionsMS techniques show promise for the iden-tification of biomarkers of various GI dis-ease 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.

References1. 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, Berge-mann 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. Inflamma-tory 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. Jour-nal 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 Gas-trointest 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 authorsBen De Lacy Costello PhD, Norman M. Ratcliffe*PhD and Sophie Shepherd BSc

Institute of Bio-Sensing Technology, Univer-sity of the West of England, Frenchay Cam-pus, Coldharbour Lane, Bristol, BS16 1QY



The terminology of mass spectrometry

GC-MS: Gas chromatography-mass spectrometry This is the gold standard method for pros-pecting for volatile biomarkers. The first part, GC, separates the volatile compounds using a flow of gas (mobile phase) through a long coated silica column. The coating (stationary phase) retains different volatiles according to their boiling point or polarity etc. The second part, the mass spectral detector, fragments the separated molecules into positive ions and their mass to charge ratio is identified. They are then matched to an extensive library of mass spectral frag-mentations to aid structural identification.

LC-MS: Liquid chromatography-mass spectrometry This is similar to GC-MS except that a solvent rather than gas is used as the mobile phase

SIFT-MS: Selective ion flow transfer-mass spectrometry This method permits real time anal-yses of known volatile compounds and can be used readily to analyse the headspace of breath, urine and stool. Unlike the GC- or LC-MS there is no chromatography step so is extremely fast and gives quantitative analyses. The reaction of the analyte with precursor ions (H3O+, NO+, O2+) forms product ions. Detection of the precursor and product ions of interest is used to quantify the absolute concentration of a particular VOC in the sample using the known reac-tion kinetics.

PTR-MS: Proton transfer reaction mass spectrometry A PTR-MS instrument consists of an ion source directly connected to a drift tube (in contrast to SIFT-MS no mass filter is interconnected) and an analyzing system (quadrupole mass analyser or time-of-flight mass spectrometer).

ESI: Electrospray ionization Liquid solvent containing the analyte is aerosolised by electro-spray, a high voltage is applied to the liquid causing it to aerosolise into charged droplets. As the solvent evaporation occurs, the droplet shrinks until it reaches the point that the surface tension can no longer sustain the charge (the Rayleigh limit) at which point a ‘Coulombic explosion’ occurs and the droplet is ripped apart. This produces smaller droplets. The process is repeated with the smaller droplets until ‘naked’ charged analyte molecules are formed. This process is useful for molecules such as proteins as it is a fairly gentle method if ionisation which doesn’t disrupt the non-covalent bonds in the molecule.

MALDI: Matrix assisted laser desorption The analyte is embedded in a matrix, a laser beam is then focussed on the matrix which absorbs light of the frequency of the laser, this causes excitation of the molecules and desorption of matrix-analyte ions from the matrix. The matrix molecules then evaporate away leaving free analyte ions.

Peptide mass fingerprinting Proteins are digested into their constituent peptide fragments (the protein fingerprint), the proteins are then identified by matching the mass obtained for the fragments to the mass of known fragments using a database.

Figure 2. Photo of a SIFT mass spectrometer analysing VOCs in the breath of a volunteer in real time. Reproduced with kind permission of Patrik Španel.

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Sleeping sickness, also called human Afri-can 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 pathologyAfter 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 biologi-cal mechanism called antigenic variation. Eventually, the parasites invade the brain causing intrathecal inflammation associ-ated 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 chil-dren, never recover completely and remain disabled for the rest of their life. Sleep-ing 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 backgroundAt the turn of the 20th century, both gambiense and rhodesiense sleeping sick-ness caused devastating epidemics kill-ing about one million people within two decades. By sustained implementation of vector control (including habitat destruc-tion and insecticide spraying), culling of wild animals, and systematic screening of the population and treatment of patients by specialized teams, the colonial govern-ments gained control over the epidem-ics 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]. Coun-tries 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 situationToday, 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 availabil-ity of performing diagnostic tests and effec-tive 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 coop-erations and Non Governmental Organi-zations 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 popu-lation at risk with a serological antibody detection test, to examine seropositive suspects by microscopy and to treat para-sitologically confirmed patients in their villages or to refer them to the nearest spe-cialized treatment centre. Since more than 20 years now, the recommended screen-ing 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 elimi-nated 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 fea-sible in some isolated foci with only one species transmitting trypanosomes, prob-ably 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 dis-ease, not only from man to man but also from the domestic and wild animal reservoir to man.

Diagnosis and treatment of infectionToday, treatment of sleeping sickness patients relies on toxic drugs and most often requires several weeks of hos-pitalization. Therefore, treatment is

Sleeping sickness elimination: are we dreaming?Recent 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

– June 2013 Parasitology22

Figure 1. Giemsa stained thin smear with Trypanosoma brucei.

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 detec-tion tests remain negative in 10% to 20% of actually infected patients, untreated patients may continue to act as a para-site 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. How-ever, HAT patients have to be diagnosed in rural environments that are not com-patible with today’s DNA- or RNA-based diagnostics and molecular test do not perform better than parasitology. There-fore, it is questionable if the individual patient will ever benefit from molecular diagnostics for sleeping sickness [4].

New control toolsShould 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 technol-

ogy allows to combine the GPS coordi-nates of all villages where HAT patients are reported with demographic and envi-ronmental data, and to precisely map the populations at risk [6].

New rapid testAlso, a new rapid diagnostic test for gam-biense HAT serodiagnosis has been devel-oped (HAT Sero-K-SeT, Coris BioConcept, Belgium). The HAT Sero-K-SeT is individ-ually 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 compat-ible for use in foci with very low prevalence and in fixed health centres with minimal infrastructure [Figure 2]. In addition, strat-egies 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 pipelineFinally, the search for new drugs has iden-tified 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.

ConclusionElimination of at least one form of sleep-ing 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 devel-oped control tools in rationally designed elimination strategies adapted to the local epidemiological situation.

References1. World Health Organization. Control and surveil-

lance of African trypanosomiasis. WHO Technical Report Series 1998; 881: 1-113.

2. World Health Organization. Report of a WHO meeting on elimination of African trypanoso-miasis (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. Improv-ing 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 authorsPhilippe Büscher1* PhD, Quentin Gilleman2 MSc, and Pascal Mertens2 PhD

1 Institute of Tropical Medicine, Department of Biomedical Sciences, Nationalestraat 155, B-2000 Antwerp, Belgium2 Coris BioConcept, Crealys Park, Rue Jean Sonet 4a, B-5032 Gembloux, Belgium *Corresponding author E-mail: [email protected] Tel. +32 3247 6371

– June 2013 Parasitology24

Figure 2. The HAT Sero-K-SeT is individually packed, thermostable, equipment-free , robust and has shown excellent diagnostic performance in a phase I evaluation.

Five in One Blood Test Technology

– June 201325Blood gas analysis

It has been over 50 years since the first blood gas analyser was-invented. Blood gas analysis technology has always played a very important role in acute respiratory failure diagnosis, surgery, patient monitoring as well as rescue procedures. With the progress of science and technology, and the spe-cial demands set by critical patient care, medical profes-sionals have a number of new requirements for improving traditional blood gas analysers, including rapid and accurate results, minimal maintenance, integration of a variety of tests, portable design, simple opera-tion procedure etc.

In order to meet these require-ments, the POCT blood gas and chemistry analyser has been developed in recent years. However, ensuring the precision and accuracy of the analyser while incorporat-ing instrument miniaturiza-tion and easy, maintenance-free operation puts higher demands on the whole sys-tem - the analyser itself, the testing procedure and the reagents. The technical char-acteristics of the test cassettes are the most important item: what is the most desirable design for a test cassette?

1. MLC: Microchip liquid control technology ensures the tightness of the circuit as well as the reaction speed of the liquid flow to improve precision and accuracy, while achieving the required minia-turization and maintenance-free operationof the analyser.

2. MMM: Microsensor multi-function membrane technolo-gyis the core technology that enables the high sensitivity and increases the variety of tests.

3. SAM: The innovative sen-sor array microfluidics design

makes it possible to accu-rately measure the volume of sample and reagent, thereby improving the preci-sion and accuracy and making point-of-care testing a reality.

4. AC: Reliable analyser autocalibration features a multipoint calibration curve

set in the factory and only requires onepoint checking by the end user; this helps elimi-nate errors between different analysers and tests.

5. TR: Adopting interna-tionally accepted traceabil-ity of calibrator and quality control allows the analyser

to produce results that are highly comparable to those of the central lab. Critical care needs critical technol-ogy innovation of the ana-lyser, exemplified in the five-in-one testing technol-ogy solution that brings in a new era for POCT blood gas analysis.


Q. What impact does cardiovascular disease have on women?

Cardiovascular disease, or CVD, is a sig-nificant 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 pres-

sure, 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 obe-sity and depression, are more prevalent in women. There are also some risk factors unique to women, including pregnancy complications, oral contraceptive use, hor-mone 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 Asso-ciation (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 com-monly present with symptoms other than chest pain, which makes diagnosis challenging. Rather, women often expe-rience 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 con-sequences. 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 treat-ment. Consider that women receive only about 34 percent of interventional treatments, with and witout the place-ments 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 provid-ing 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, clini-cians can help enhance the assessment, diagnosis and follow-up care for women with CVD.

Reference1 . h t t p : / / g a m a p s e r v e r. w h o . i n t / g h o /

i n t e r a c t i v e _ c h a r t s / w o m e n _ a n d _h e a l t h / c a u s e s _ d e a t h / c h a r t . h t m l ; a c c e s s e d 1 1 / 2 7 / 1 2

Women and heart diseaseCardiovascular 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.

– June 2013 Interview26

Jean Onofrio, Senior Director, Global Assay Marketing, Siemens Healthcare Diagnostics

Global hemostatic markers in coronary artery diseaseHemostasis is the process by which bleed-ing is stopped, involving blood coagula-tion and platelet aggregation. This process depends on the delicate balance of many pro- and anti-coagulant factors, and when hemostatic balance is disrupted, patho-logical clot formation may occur leading to potentially fatal venous or arterial throm-bosis. Appropriate fibrinolysis, the break-down 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 infarc-tion. Additionally, the presence of coro-nary 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 unnecessar-ily 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 individ-ual coagulation and fibrinolytic factors, or by global coagulation assays. Such imbal-ances 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 fibrinoly-sis, including prothrombin fragment 1+2, fibrinopeptide A, thrombin–antithrom-bin and plasmin–antiplasmin complexes, D-dimer, plasminogen activator inhibi-tor-1, thrombin activatable fibrinolysis

inhibitor and tissue plasminogen activator [2, 3]. However, measuring single factors does not reflect the overall hemostatic bal-ance as other pro- or anti-coagulant, and pro- and anti-fibrinolytic factors may com-pensate for the deficient or elevated fac-tor. 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 dis-orders, but do not reliably detect hyper-coagulability in this context. Recently, there has been interest in global assays of coagulation and fibrinolysis as meth-ods 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.

ThromboelastographyThromboelastography is a method meas-uring 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 interfer-ence is detected by the pin. Thromboelas-tography (TEG, Haemonetics, Braintree,

Use of global hemostatic markers for risk stratification and personalized treatment of coronary artery diseaseCoronary 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

– June 201327Coronary artery disease

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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, hyperfibrinoly-sis 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 addi-tionally 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 assayThe thrombin generation assay was first described in 1953, but has more recently been simplified, standardized and commercial-ized, including in the form of the Calibrated Automated Throm-bogram (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 assess-ment has suggested the assay can predict bleeding and ischemic events in patients with coronary artery disease [9].

Overall Hemostatic Potential (OHP) assayThe 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 iden-tified 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 throm-boelastography 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 hypercoagula-bility and hypofibrinolysis in patients with acute and stable coronary artery disease [Fig. 2] [7, 11]. The observations in this latter popu-lation 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 requirementsThere is a growing body of evidence that ex vivo hypercoagula-bility of patients’ blood or plasma has prognostic value in arte-rial or venous thrombotic events. Global markers of hemostasis,

– June 2013 Coronary artery disease28

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Figure 1. Thrombin generation curves showing the increase in thrombin generation in stable angina patients (data published in Reddel CJ, et al. [7]).

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 thrombo-prophylaxis. Further large-scale prospective trials are needed to directly address this.

References1. Anandasundaram B, Lane DA, Apostolakis S, Lip GY. The

impact of atherosclerotic vascular disease in predicting a stroke, thromboembolism and mortality in atrial fibrilla-tion 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? Patho-physiol Haemost Thromb. 2003; 33: 302–308.

3. Gorog DA. Prognostic value of plasma fibrinolysis activa-tion 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 appli-cations 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 thrombo-gram (CAT): a universal routine test for hyper- and hypo-coagulability. 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 Hae-most. 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 asso-ciate with myocardial infarction. Thromb Haemost. 2012; 107: 1092–1099.

The authorsCaroline Reddel* PhD; Jennifer Curnow MBBS, PhD, FRACP, FRCPA; David Brieger MBBS, PhD, FRACP, FACCANZAC Research Institute, Concord Repa-triation General Hospital, Concord NSW, 2139, Australia


– June 201329

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Figure 2. Overall haemostatic potential curves (fibrin generation and fibrinolysis) showing the increase in fibrin generation and reduction in fibrinolysis in stable angina patients (data published in Reddel CJ, et al. [7]).

Clinical backgroundThe number of patients with coronary artery disease (CAD) is increasing world-wide, 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 inva-sive 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 indi-rect costs of CAD in the US in 2010 were $503.2 billion [1].

Currently, there is a lack of new bio-markers 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 inflammationIt has been well established that inflammation plays an important role in development and progression of

atherosclerosis in the coronary arter-ies [2]. Moreover, inflammation is also involved in the inflammatory pathways inducing extracellular matrix remodel-ling 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 atheroscle-rosis remains controversial. Therefore, it is not very useful for continuous monitor-ing of treatment effects and progression of the disease.

The inflammatory biomarker YKL-40YKL-40 is a glycoprotein mainly pro-duced 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 character-ized 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 adhe-sion, angiogenesis, remodelling of the extracellular matrix, cell proliferation and differentiation [5]. Macrophages in athero-sclerotic 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 subjectsThe normal YKL-40 value in a healthy subject from the general population has recently been published [7]. In 3130 sub-jects the median YKL-40 value was 40 μg/L and increased exponentially with age.

YKL-40 in coronary artery diseaseSerum 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 infarc-tion and thereafter consistently decreased from a maximum value just after the myo-cardial infarction and during a 360 day follow-up period towards its normal levels. Plasma YKL-40 levels were found to cor-relate inversely with left ventricular ejec-tion 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, revasculariza-tion with balloon angioplasty of signifi-cant 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 meas-urement 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.

YKL-40: a new prognostic biomarker in patients with coronary artery diseaseInflammation 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


Figure 1. The 6 year cumulative frequency of all-cause mortality in patients with stable coronary artery disease and serum YKL-40 cuts at 110, 129, 153, 191, and 256 μg/L.

Therefore, it is very interesting that serum YKL-40 was a very strong prognostic bio-marker 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 failureThe 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 hos-pital 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 activityStatin treatment is used in CAD for lower-ing 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 sta-ble CAD on statin treatment compared to patients without [14] [Fig. 2].

This difference seems to be independent of the effect that statins have on lower-

ing 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 corre-late to cholesterol levels in statin-treated CAD patients [14].

– June 201331

EUROIMMUNM e d i z i n i s c h eL a b o r d i a g n o s t i k aA G

EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Telephone +49 451 58550 · Fax 5855591 · E-mail [email protected]

For further information contact our product manager Dr. J. Aldag ([email protected]; +49 451 5855 25453)

Autoantibodies against Phospholipase A2 Receptor A milestone in the diagnosis of primary membranous glomerulonephritis (MGN)

PLA2R-transfected cells Control transfection

Gold standard for serological diagnosis: cells transfected with

PLA2R as IF substrate or purifi ed PLA2R as ELISA substrate

Specifi city > 99 %, sensitivity up to 80 % in idiopathic MGN

Quantitative detection of anti-PLA2R IgG for determination of

disease activity and for therapy monitoringPodocyte foot processes(with PLA2R on the surface)

Capillaryendothelium

BloodPrimary urine

Filtration

Filtration barrier of the kidney

Anti-PLA2R

Basement membrane

Renal corpuscule

Capillary loopswith podocytes


Figure 2. Median YKL-40 levels (μg/L) healthy controls and CAD patients with and without statin treatment.

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 perspectiveYKL-40 is a new inflammatory biomarker in ischemic heart dis-ease. It is increased in both acute and chronic coronary artery dis-ease and is a very strong diagnostic biomarker for death. It is sug-gested to be a mirror of the active inflammatory atherosclerotic processes in CAD, more than a measurement of degree of myo-cardial 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 interestA patent application (WO 2009/092382) is published and pending.

References1. 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 Ath-

eroscler 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, Kas-trup 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 authorsJens 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



www.jib-sdbio.fr

58e EDITION

13-14-15 NOV. 2013CNIT PARIS LA DÉFENSEF R A N C E

INTERNATIONAL DAYS OF BIOLOGY

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In-office diabetes testingThe DCA® HbA1c test kit, used for more than two decades to monitor patients, can now be used as an aid to diagnose diabetes and identify patients at risk for developing the dis-ease. The dual use kit, for both monitoring and diagnosing, is available for sale in EU coun-tries and countries not requir-ing any local regulatory clear-ances under part #10698915. Early detection and tight gly-cemic control help mitigate the serious conditions that accompany diabetes: heart and kidney disease, limb

neuropathy, retinopathy, and stroke. More importantly, patients identified as being at risk can stop or reverse disease progression through lifestyle adjustments and/or pharmacological treatment. Availability of results at the time of visit provides healthcare pro-viders and educators the opportunity to have meaningful, face-to-face discussions regarding treatment options. From a con-venience and scheduling standpoint, it eliminates multiple visits for pre-visit blood draws or post-testing follow-up consultation. The DCA Vantage® analyser is just one of two point-of-care HbA1c analysers that meet NGSP performance criteria.

SIEMENS HEALTHCARE DIAGNOSTICSAACC Booth 3449 www.cli-online.com & search 26348

Celiac disease quick test Celiac quick test is the latest addi-tion to Biohit’s GI tract quick test portfolio and makes it easy to test for celiac disease right at the point of care. Results are obtained by testing antitissue transglutaminase (tTG) antibodies from a blood sample. The blood sample is taken from finger tip and incubated with

buffer on the test plate. Already after 10 minutes the result is ready for visual reading. The test is useful for general practitioners, endoscopists, outpatient departments and dietetics. (The product is not available in the United States or Canada).

BIOHIT OYJAACC Booth 2709 www.cli-online.com & search 26347

– June 201333PRODUCT NEWS

LabUMat 2 & UriSed 2

Complete Urine Laboratory System

77 Elektronika Kft. Fehérvári út 98. H-1116 Budapest, Hungary Phone: +36-1-206-1480 Fax: +36-1-206-1481 E-mail: [email protected] Web: www.e77.hu

est Hungary

Whole field of view images automatically: the professional choice

Automated Urine Chemistry- and Sediment AnalyzerOutstanding performance in test strip evalua on and par cle recogni onUnique measurement technique automates tradi onal microscopyCost-e ec ve opera on; No need for any liquid reagents


ELISA & IFA automated platformSKYLAB 752 is a fully automated analyser able to process simulta-neously up to 7 EIA microplates and 28 IFA slides. The flexibility of the working area makes SKYLAB 752 suitable for any laboratory requirement. It is equipped with an innovative pipetting system:

two pipettors working simultaneously and independently. This important feature makes it possible to dispense a whole micro-plate in half the time it takes with other similar analysers on the market, hence minimizing the drift effect. Standard high-quality disposable tips ensure zero carry over. The innovative and exclu-sive dispensing technology guarantees precision and accuracy of the results. Disposable high-quality tips are ideal for infectious disease tests (Hepatitis, ToRCH etc). The built-in barcode reader allows sample positive identification and reagents detection. SKY-LAB 752’s unique features (such as the possibility to have 5 differ-ent buffers on board, as well as being able to carry up to 4 orbital shakers) make it suitable for any kind of ELISA test. Addition-ally, SKYLAB 752 is equipped with an innovative optical reading system: LEDs, instead of the traditional halogen lamp, guarantee stable reliability of the results.

SCLAVO DIAGNOSTICSAACC Booth 1849 www.cli-online.com & search 26345

Flow cytometry software for hematology

Part of the com-pany’s Hemato-Flow solution c o m b i n i n g diagnostic rea-gent, hematol-ogy and flow

cytometry hardware and IT expertise, Ver-sion 2 CytoDiff CXP Autogating software is designed to give laboratories added con-fidence in the use of flow cytometry in the routine hematology lab for the auto-vali-dation of abnormal samples. Easy-to-use, the advanced Version 2 software further improves sub-population classifications. It adds new features, such as greater precision in the removal of potential interference and metrics that provide a new ‘confidence level’ on population classification. The new software simplifies the review process making it easier to validate results with greater certainty. HematoFlow with the

CytoDiff five-colour antibody cocktail and CXP Autogating Software make it possible to use the precision of flow cytometry to deliver extended white blood cell (WBC) differential results with far greater con-sistency than manual microscopic assess-ment. The five-colour antibody cocktail, the CytoDiff, uses six monoclonal antibod-ies to establish the differential. The rou-tine use of flow cytometry for validating abnormal samples can improve workflow and turnaround time as well as providing access to additional diagnostic informa-tion for patients. European laboratories are already working with Beckman Coulter to increase confidence in the routine use of flow cytometry in the hematology process for validating abnormal samples. Hospitals including University Hospital, Rennes and Bordeaux in France, Erasme University Hospital, Brussels, Belgium and several private labs are successfully adopting this approach. CytoDiff is not available for in vitro diagnostic use in the United States and other geographies.

BECKMAN COULTERAACC Booth 4751 www.cli-online.com & search 26354

Small benchtop centrifugeIn a compact footprint, the new Thermo Scientific small benchtop cen-trifuge delivers the flexibility to adapt to evolv-ing clinical and research needs alike, including clinical chemis-

try, cell culture, microbiology and hema-tology. This small benchtop centrifuge features: application flexibility, cleaning convenience, and ease-of-use with Thermo Scientific Auto-Lock rotor exchange; ver-satility to spin blood tubes, conical tubes, microtubes and microplate compatibility with sealing options – all in one versatile unit; one-handed sample protection with Thermo Scientific ClickSeal biocontain-ment lids; intuitive glove- and detergent-friendly interface with a bright backlit display, featuring one-touch operation with pre-set protocol options and optional password protection.

THERMO FISHER SCIENTIFIC AACC Booth 5140 www.cli-online.com & search 61415

– June 201335PRODUCT NEWS

www.sclavodiagnostics.com

www.axadiagnostics.com

DISTRIBUTORS WANTED

Flexibility and Reliability in Nephelometry

Automation in ELISA and IFA testing

Visit us at booth 1849!


Sampling tube for effective glycolysis inhibition

The reliable stabilization of blood sugar lev-els directly after sampling is an important pre-condition for the accuracy of the analysis results.

VACUETTE® GLUCOMEDICS was spe-cially developed for this application. The additive mixture in the tube stabilizes the blood sample immediately after sampling and thus impedes the breakdown of glucose over time (glycolysis). The combination of sodium EDTA, sodium fluoride, citric acid and sodium citrate acidifies the whole blood sample, resulting in the immediate and complete inhibition of glycolysis, thus setting it apart from conventional sodium fluoride tubes. The glucose concentration of whole blood and/or plasma samples is kept constant at room temperature for up to 48 hours at the “in-vivo value” (almost 100% of the original value). The dosed liq-uid additives ensure the easy mixing of the additive with the whole blood sample. To make up for the dilution effect of the additive, the analysis result must be multi-plied by 1.16. This is required to obtain the “in-vivo glucose concentration” in the plasma.

GREINER BIOONEAACC Booth 4511 www.cli-online.com & search 26356

Free 25 OH Vitamin D ELISA

A new and unique research use only (RUO) assay launched in partnership with Future Diagnostics will help to normalize free vitamin D (FVD) levels despite the effects of variations in Vitamin D Bind-ing proteins (VDBP) that occur in certain pathologies , e.g. pregnancy, liver disease, end stage renal disease and dialysis . Con-ventional total 25 OH Vitamin D assays by LC/MS-MS or immunoassay can give misleading results in these physiological conditions because these assays utilize a sample pre-treatment step that destroys the VDBP. The DIAsource Free 25 OH Vitamin D ELISA provides a more accurate assess-ment of the bioactive FVD in the patient sample. The levels of FVD measured in this assay are in the range of 1.1 pg/mL to 40 pg/mL with a 10 uL serum sample.

DIASOURCE IMMUNOASSAYS S.AAACC Booth 3050 (Belgium AWEX)


Strip-based test for hemoglobin A new strip-based test for quick and easy hemoglobin analysis for ane-mia determination is now available. STAT-Site M Hgb is a pocket-sized device that reliably delivers accurate hemoglobin analy-sis results within

30 seconds from 15 μL of finger-prick whole blood. This hemoglobin photometer is maintenance-free, and features intui-tive, step by step on-screen instructions minimize training and ensure ease of use. Due to its durability, portability and user-friendliness, STAT-Site M Hgb is especially suited to blood screening programmes in developing world markets, but it also has a niche in settings where operators are mobile. Using just one 3V Li type battery for every 1,000 tests and with an oper-ating temperature range of 16° to 35°C, the analyser is ideal for field use.

EKF DIAGNOSTICSAACC Booth 3614 www.cli-online.com & search 26344

RSV/Adenovirus differential test

The OSOM® RSV/Adeno Test provides differential diagnosis for respiratory syn-cytial virus (RSV) and/or Adenovirus within 10 minutes with good sensitiv-ity versus PCR (>90% RSV; 85% ver-sus Adenovirus). Sample types include nasal swabs, nasal suction fluid or throat swabs (for Adenovirus). Features include objective, two colour results and room temperature storage.

SEKISUI DIAGNOSTICSAACC Booth 1627 www.cli-online.com & search 26357

Biotinidase neonatal screening

The Neonatal Biotinidase Screening Assay is an enzymatic assay for the quantitative determination of biotinidase activity in neonates using blood spot samples dried on Whatman S&S 903 filter collection paper. A non-invasive sampling method is used where specimens are collected by applying a few drops of blood freshly drawn from the infant’s heel with a lan-cet onto specially manufactured absor-bent specimen collection (filter) paper. This method has many advantages: short collection time, low invasiveness, reduc-tion of sample volume and ease of sample storage. It allows quantitative results to minimize the needs of a confirmatory test. Convenient transport and good stability of the samples and ready to use reagents are assured.

ZENTECH AACC Booth 2951 www.cli-online.com & search 26355

PRODUCT NEWS – June 2013 36

Multiparametric immunofluorescence assay for hantavirusesA multiparametric indirect immunofluorescence assay (IFA) for the simultaneous detection of infections with different old and new world hantaviruses has been developed and evaluated in an international study published in PLOS Neglected Tropi-cal Diseases in April 2013. The IFA utilizes BIOCHIP Mosa-ics, with each substrate consisting of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre

(SNV) or Andes (ANDV). In the evaluation study, 184 confirmed seropositive sera from six diagnostic centres worldwide and 89 control sera were analysed for specific antibodies of immunoglobulin classes IgG and IgM against hantaviruses using the IFA. The assay dem-onstrated an overall sensitivity of 100% for all serotypes with the exception of SNV (96%) and an overall specificity of at least 98% for each serotype. 99% of the patient sera exhibited IgG antibodies and 71% IgM antibodies. Notably, the IFA was able to distinguish PUUV and ANDV infections from those with murinae-borne hantaviruses such as DOBV and SEOV, thus providing useful additional information for therapeutic decision making. The authors note that many hantavirus patients are still clinically misdiagnosed as having influenza-like infections, renal failure or idiopathic acute respiratory distress syndrome. Accurate diagnosis is, however, essential given the mortality rates of up to 35% from han-tavirus syndromes. The misdiagnosis rate combined with the increasing number of hanta-virus infections worldwide demonstrates the need for simple and reliable tests which allow detection of all clinically relevant hantavirus serotypes. Analysis of specific antibodies is the method of choice for diagnosing hantavirus infections. By combining the most important pathogenic hantaviruses in one assay, this IFA system is suited to detect any hantaviral infec-tion worldwide. The test is particularly suitable for serodiagnostic screening in regions where multiple serotypes are endemic.

EUROIMMUN AG AACC Booth 2738 www.cli-online.com & search 26346

Assays for the detection of sedative drugs

Three new ELISA testing kits for the detection of sedative drugs Zopiclone, Zale-plon and Zolpi-dem are now available. These

drugs behave in a similar ways to benzodi-azepines. The Z drugs are found in prescrip-tion medications, however, they should only be used for a short time period as extended use can lead to tolerance. The misuse of Z drugs is widespread as a result of both dependence and recreational use. Incidents of unusual or inappropriate behaviour have been linked to misuse of Z drugs, where the patients had no memory of their actions. Some users have reported sleepwalking, sleep driving, binge eating, and perform-ing other daily tasks while asleep. Randox Toxicology has developed three ELISA kits for the detection of Z drugs in urine and blood specimens. The Zolpidem ELISA offers an unbeatable Limit of Detection (LOD) of 0.4ng/ml in urine and 0.52ng/ml in blood. The Zopiclone and Zaleplon ELISA kits are the first immunoassays avail-able for the rapid detection of these drugs. Prior to this, the only way to detect Zopi-clone and Zaleplon was with timely chro-matographic analysis. The company also provides an automated ELISA plate reader, capable of reading 96 wells in 30 seconds. In addition to the Z drug ELISA kits, Randox

Toxicology provides a multiplex Biochip Array which targets Zolpidem, Zopiclone, Zaleplon and their metabolites simultane-ously from a single, undivided specimen. The advanced multiplex method con-solidates ELISA tests onto a small 9x9mm biochip, enhancing the ability for toxicolo-gists to quickly detect these sedatives.

RANDOX TOXICOLOGYAACC Booth 3245 www.cli-online.com & search 26319

Automated hematology analyser

The newly launched DS-500 series 5-part differential automated hematology ana-lyser uses only 10μl sample volume to obtain 27 parameters, 2 histograms and 2 scatter grams. The main technologies uti-lized by the instrument include laser scat-ter, flow cytometry analysis with chemical staining, impedance method and colorim-etry. Only three types of reagents are nec-essary, which is a great advantage in terms of running cost.

EDAN INSTRUMENTSAACC Booth 1318 www.cli-online.com & search 26343

PRODUCT NEWS – June 2013 38


Active-B12 now available in USA

The Active-B12 assay from Axis-Shield provides an innovative and highly accurate method for assessing Vitamin B12 status and is now available in the USA. The Axis-Shield assay measures Holotranscobalamin, the bio-active form of the vitamin. Holotranscobala-min represents only 10-30% of circu-lating B12 but is the only form taken up and utilised by the cells of the body – the Active-B12 assay therefore gives a more accurate assessment of the body’s B12 status. The use of a specific monoclonal antibody for Holotransco-balamin means that the assay does not suffer from the interference from anti-Intrinsic Factor antibodies which can be a medically serious failing of some traditional Vitamin B12 testing methods. The Axis-Shield Active-B12 assay is a standard 96-well ELISA with break-apart strips and is supplied with all required reagents, calibrators and controls. The kit is compatible with liquid-handling automates for higher throughput.

Axis-Shield Diagnostics(an Alere company)

The Technology ParkDundee DD2 1XA

Scotland, UKT: +44 (0) 1382 422000F: +44 (0) 1382 422088

E: [email protected]

www.active-b12.com

CALENDAR OF EVENTS

July 28 – Aug 1, 20132013 AACC Annual MeetingHouston, TX, USA hwww.aacc.org

July 28-31, 2013Laboratory Medicine Congress (LMC) 2013Cape Town, South Africa/www.lmcongress.com/

July 30 – Aug 1, 20133rd Annual World Congress of Microbes-2013 (WCM -2013) Wuhan, Chinawww.bitlifesciences.com/wcm2013

August 22-27, 201315th Int’l Congress of Immunology (ICI)Milan, Italywww.ici2013.org

Sept 12-14, 2013Medical Fair ThailandBangkok, Thailandwww.medicalfair-thailand.com

Nov 3-6, 2013CMEF Autumn 2013Xiamen, Chinawww.cmef.com.cn/en/

Nov 13-15, 2013JIB 2013 – Journées Internationales de BiologieParis, FranceTel. +33 1 4756 5079 e-mail: [email protected] www.jib-sdbio.fr

Jan 27-30, 2014MEDLAB at Arab Health 2014Dubai, UAEwww.arabhealthonline.com/en/Medlab/

Feb 18-24, 2014MEDLAB Asia Pacific 2013Marina Bay Sands, Singaporewww.medlabasia.com

Dates and descriptions of future events have been obtained from official industrial sources. CLi cannot be held responsible for errors, changes or cancellations.

For more events see: www.cli-online.com/events/

Pregnancy testing when every moment counts. Siemens answers give emergency departments more time for patient care.

www.siemens.com/clinitest-hcg

Answers for life.

Timing is everything in the emergency department. But for women of childbearing age, many medical procedures must wait until these patients have been tested for pregnancy.

The Siemens CLINITEST® hCG pregnancy test helps ED clinicians quickly clear women for further procedures. Paired with a CLINITEK Status® Connect analyzer and RAPIDComm® Data Management system, the CLINITEST hCG test provides one of the only instrument-read and truly connected solutions. Since no manual documentation or visual interpretation is required, the chance for error is greatly reduced.

Test results take just two to five minutes and can be transferred to electronic health records, enabling hospitals to quickly move ahead with X-rays or other routine procedures. Less time spent on pregnancy tests leaves more time for critical patient care.

Want to streamline your emergency department with more efficient pregnancy testing? Take our online product tour at www.siemens.com/clinitest-hcg to learn how Siemens can help.

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Visit us at AACC/ASCLS Booth #3449


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