of adrenocortical carcinoma - Clinlabint.com...adrenocortical carcinoma. Also in this issue :...

Also in this issue :

Immunohistochemistry for determining mismatch repair status of colorectal carcinoma Pg. 16

Improved method to assess BRCA status in hereditary breast and ovarian cancer Pg. 22

Measurement uncertainty report inQC data management software

Pg.15

Assay for monitoring 6 antibioticsin serum/plasma Pg.33

LC-MS/MS method improves diagnosis of adrenocortical carcinoma Pg.10

News updates on www.clinlabint.com | September 2018 | Volume 42

by Bio-Rad

by Jenoptik

by Chromsystems

Augmented microscopy platformfor pathology applications Pg.32

StatStrip is a registered tradmark of Nova Biomedical.Accu-Chek is a registered trademark of Roche Diagnostics.www.novabiomedical.com

1. StatStrip Glucose Hospital Meter System. MAUDE (Manufacturer and User Facility Device Experience) database. Accessed 17 Jan 2018.www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfMaude/search.CFM2. ACCU-CHEK Inform II Blood Glucose Monitoring Test Strips. MAUDE database. Accessed 17 Jan 2018. www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfMaude/search.CFM3. StatStrip market share of 57% and Inform II market share of 30% from 2017 market share survey. Data available on request.

The FDA requires manufacturers and users to report all hospital adverse events, including patient deaths that are caused by their glucose meters. These reports are then summarized on the FDA’s MAUDE database. The most recent 2015-2017 MAUDE data shows a dramatic improvement in patient care when advanced technology Nova StatStrip glucose meters are used in place of other meters. This publically available data shows that hospitals using Nova’s technology have an adverse event rate 40x lower than others. This remarkable reduction in Nova adverse events, including no patient deaths, comes from using the more accurate (no known clinical interferences) Nova StatStrip meter. These dramatic improvements in patient outcomes are obtained despite the fact StatStrip is the only meter cleared by the FDA for use on critically ill patients who have the most analytically challenging samples.

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Alison SleighPh.D.

– September 20183Editor’S lEttEr

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Lyme disease is caused by Borrelia spirochaetes: predominantly Borrelia burgdorferi in North America (but also present in Europe), and pre-dominantly B. afzelii and B. garinii in Europe and Asia and is spread to people via infected deer ticks. Infec-tion occurs after only a minority of tick bites, but is typified by three stages. Stage 1, early localized lyme disease is characterized by the bull’s eye rash (erythema migrans (EM)). Stage 2, early disseminated infection occurs within days to weeks after the local infection as the bacteria begin to spread through the bloodstream. Stage 3, late disseminated infection, where the infection has spread throughout the body, can occur several months later in untreated or inadequately treated patients involving chronic symptoms that can be severe and disabling. Treatment by antibiotics is effective in the early localized stage of the disease but this is often hampered by late diagnosis. Diagnosis can be delayed for a number of reasons: there is a lack of awareness in the general public (as well as GPs outside of what are thought to be the high-risk areas); approximately 25% of people do not get the typical bull’s eye rash; and symptoms can be so varied and vague that, when occurring weeks or months later, are difficult to relate back to the time of the tick bite. Knowledge of a tick bite and an associated EM rash is sufficient for diagnosis. However, in cases where there is a clinical suspi-cion of Lyme disease but no EM rash, laboratory testing is advised. Testing for antibodies is done via a two-tiered approach, starting with a sensitive ELISA, which, if positive or equivocal, is followed by a more specific immu-noblot. However, the overall sensitiv-ity of the two-tiered tests is only 64% when done in the early stages of infec-tion, which is when accurate diagno-sis is most needed. Because of these diagnostic limitations, the prevalence of Lyme disease is likely to be far higher than is currently thought. With increasing incidence and geographic spread of the disease, better testing for diagnosis, particularly in the early

stages of infection, is perhaps required. Research is ongoing into PCR meth-ods as well as and for the detection of OspA antigens that are shed into urine. An LLT-MELISA (lympho-cyte transformation test-memory

lymphocyte immunostimulation assay) has been developed and is sug-gested to be a useful supportive diag-nostic tool, particularly in infections acquired in Europe. In the USA, next-generation sequencing (NGS) has

been used for specific pathogen iden-tification and to guide treatment deci-sions. With technological advances making NGS quicker and cheaper, could this eventually become the next gold standard test for Lyme disease?

Lyme disease diagnosis: waiting for the next gold standard

ContentsFRONT COVER

FEATURES[6 - 15] MASS SPECTROMETRY[6 - 8] Urine ethyl glucuronide and ethyl sulphate measurement using liquid chromatography-tandem mass spectrometry

[10 - 12] Use of an lC-MS/MS 13-steroid serum panel in the diagnosis of adrenocortical carcinoma

[14 - 15] technology Watch: Automating lC-MS/MS analysis for streamlined clinical testing workflows

[16 - 21] PATHOLOGY[16 - 18] Use of immunohistochemistry in the determination of mismatch repair status of colorectal carcinoma

[20 - 21] Scientific literature review

[22 - 24] MOLECULAR DIAGNOSTICS Competitive PCr-high resolution melting analysis: an improved approach to assess BrCA status in hereditary breast and ovarian cancer patients

[28] AWARD Winner of first global CArES HiV/AidS award announced at iAS 2018

[31] PRODUCT PROfILE Gonotec: a continued success story since 1979

REGULARS[3] Editor’s letter

[25 - 27] News in brief

[29 - 30] industry news

[32 - 34] Product news

[30] Calendar

liquid chromatography-tandem mass spectrometry (lC-MS/MS) is increasingly being used in clinical biochemistry laboratories to measure steroid hor-mones in order to overcome the issue of cross-reac-tivity that traditional immunoassays can be subject to. We have developed an lC-MS/MS method for the measurement of 13 steroids from a single blood sample, in order to improve the diagnosis of adrenocortical carcinoma.

Also in this issue :

Immunohistochemistry for determining mismatch repair status of colorectal carcinoma Pg. 16

Improved method to assess BRCA status in hereditary breast and ovarian cancer Pg. 22

Measurement uncertainty report inQC data management software

Pg.15

Assay for monitoring 6 antibioticsin serum/plasma Pg.33

LC-MS/MS method improves diagnosis of adrenocortical carcinoma Pg.10

News updates on www.clinlabint.com | September 2018 | Volume 42

by Bio-Rad

by Jenoptik

by Chromsystems

Augmented microscopy platformfor pathology applications Pg.32

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BackgroundEthyl glucuronide (EtG) and ethyl sul-phate (EtS) are minor ethanol metabo-lites that can be used to detect recent alcohol consumption [1, 2]. Following the ingestion of alcohol, over 95% is metabolized by alcohol dehydrogenase to acetaldehyde. Up to 5% of ethanol is excreted unchanged in breath, sweat and urine. A small amount of ethanol (<0.1%) is metabolized in the liver by conjugation of glucuronic acid or sul-phate to form EtG and EtS (Fig. 1). Fol-lowing alcohol consumption, ethanol itself can only be detected in breath or urine for up to 6 or 12 hours, respec-tively (depending on the amount of alcohol consumed) [3]. In comparison, it has been demonstrated that EtG and EtS can be detected in urine for at least

24 hours and over 48 hours with heavy alcohol consumption [4].

The ability of these markers to detect alcohol intake over a longer time period means that they can be useful to identify alcohol relapses in alcohol-dependent individuals in treatment programmes [5]. In the UK, alcohol treatment programmes rely on breath ethanol and self-reporting to detect recent alcohol intake. However, this will only detect a proportion of indi-viduals who are continuing to drink alco-hol; this has been a low as 7% in one study comparing breathalyser/self-reported alcohol intake to urine EtG measurement [6]. Therefore, EtG and EtS can be help-ful to detect those in alcohol treatment who are continuing to drink alcohol but deny it and have a negative breath ethanol

test [7]. This allows additional interven-tions in individuals who are continuing to drink, which may ultimately improve out-comes. During 2016–17, 80 454 individu-als entered alcohol treatment in England; of those 61% were free of alcohol depend-ence following the standard 12-week pro-gramme [8]. Therefore, improved detec-tion of continuing alcohol consumption could lead to initiation of earlier inter-vention and altered strategies to increase the numbers successfully completing treatment.

Measurement of ethyl glucuronide and ethyl sulphateLiquid chromatography (LC) to separate analytes with detection using mass spec-trometry (MS) is now routinely used in clinical laboratories for an increasing number of tests. It is routine practice in urine toxicology testing for results to be confirmed by either LC or gas chromatog-raphy with detection using MS and it has been recommended by the United States Substance Abuse and Mental Health Ser-vices Administration (SAMHSA) that MS confirmation should be used for the measurement of EtG and EtS [9].

In tandem MS, two mass spectrometers are arranged sequentially with a ‘collision cell’ placed between the two instruments (Fig. 2). Using selective reaction monitor-ing, the first mass spectrometer (MS1) selects the ion with the mass/charge (m/z) ratio of interest. The selected ion (par-ent ion) is fragmented into small ions that enter the second mass spectrometer (MS2) where an ion with a specific m/z ratio is selected (daughter). Detection of analytes using an m/z ratio is very specific and sensitive allowing detection of very small amounts of EtG and EtS.

A number of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for EtG and EtS have been published and a reference method has been proposed for EtG using solid phase extraction followed by LC-MS/MS [10]. Deuterated standards (EtG-d5 and EtS-d5) are readily available to purchase

urine ethyl glucuronide and ethyl sulphate measurement using liquid chromatography-tandem mass spectrometryEthyl glucuronide and ethyl sulphate are ethanol metabolites that increase the time window for detecting recent alcohol intake in comparison to measuring breath or urine ethanol. These markers are a useful additional tool for monitoring individuals in alcohol dependence treatment programmes. To measure these analytes, liquid chromatography-tandem mass spectrometry methods have been developed that are suitable for use in the routine clinical laboratory.

by Jane Armer and Rebecca Allcock

– September 2018 Mass spectrometry6

Figure 1. The metabolism and excretion of ethanol. The size of the arrow demonstrates the proportion of the ethanol consumed that is excreted via each mechanism. Over 95% is metabolized to acetaldehyde and acetic acid. Less than 0.1% is metabolized to ethyl glucuronide and ethyl sulphate.

for use as internal standards ensuring reproducibility and reliability; an inter-nal standard must mimic the analyte of interest but have a different mass to allow the MS detector to differentiate between the analyte of interest and the internal standard.

Sample preparation in published meth-ods ranges from solid phase extraction to protein precipitation to dilution of urine in mobile phase. Solid phase extraction or protein precipitation of urine samples prior to LC-MS/MS can reduce the pres-ence of potentially interfering substances

which may cause ion suppression. It may also help to increase the lifespan of the column. For chromatographic separa-tion of EtG and EtS, the mobile phases are usually formic acid in HPLC grade water and acetonitrile. Published methods have used both isocratic and gradients of mobile phase A and B to achieve separa-tion of EtG and EtS; this is dependent on the sample preparation, the exact compo-sition of the mobile phases and the col-umn chosen. A rapid sample preparation of diluting urine samples in mobile phase A and then adding internal standard has been shown to be effective with no ion

suppression or enhancement at or near the retention times for EtG and EtS [11]. Our experience has been to use an increasing gradient of mobile phase B (acetonitrile) from 1% to 10% over the first 2 minutes and then 10% to 100% from 2 minutes to 2.5 minutes. The increase from 1% to 10% acetonitrile elutes EtS/EtS-d5 at 1.27 min-utes and the increase from 10% to 100% elutes EtG/EtG-d5 at 2.03 minutes. Figure 3 shows an example chromatogram for a urine sample collected from an individual attending the community based alcohol treatment programme; the high EtG and EtS results demonstrate that this person was continuing to drink alcohol.

Using MS to measure EtG and EtS requires the availability of LC-MS/MS equipment within the laboratory, the technical exper-tise required to set up an LC-MS/MS method and a dedicated member of staff to perform the analysis. In laboratories already using LC-MS/MS for other assays, there should be no difficulty in setting up a method to measure urine EtG and EtS.

An enzyme immunoassay method is also available to measure EtG and may be adapted for use on many automated laboratory analysers. This method has been shown to compare well to an LC-MS method [12]. For routine use, an immunoassay for EtG on an auto-mated analyser has a number of advan-tages including rapid turnaround times, availability of EtG analysis out of rou-tine working hours and the same staff members performing the analyses of multiple tests at the same time. How-ever, there is no requirement for urine EtG and EtS analysis to be performed 24/7 as they would not be required in an acute setting. Generally, clients in a community treatment programme attend weekly, so once or twice weekly analysis using LC-MS/MS should be adequate for feedback of results to cli-ents at their next visit. Not requiring a dedicated member of staff (as would be required for LC-MS/MS) is advanta-geous but according to SAMHSA guide-lines, immunoassay results will require confirmation using a MS method. In addition, there is currently no immuno-assay method available to measure EtS. This is important as there are a number of scenarios that can cause a false posi-tive EtG result with a negative EtS result. For example, ‘positive’ EtG results (but not EtS results) have been demonstrated after the consumption of non-alcoholic beers (alcohol content 0.5%) [13]. EtG

– September 20187

Figure 2. Selective reaction monitoring using a triple quadrupole tandem mass spectrometer. Following ionization, MS1 selects the ion with the m/z ratio of interest. In the collision cell, the selected ion (parent ion) is fragmented into smaller ions. All of these smaller ions enter MS2 where an ion with a specific m/z ratio is selected (daughter ion). For example, for EtG the parent ion has an m/z ratio of 221 and the two daughter ions have an m/z ratio of 75 and 85. note that the optimal transitions need to be determined for each MS/MS.

Figure 3. Chromatogram of a urine sample from an individual attending a community alcohol treatment programme. EtG = 1.14 mg/L (cut-off 0.26 mg/L) and EtS = 0.78 mg/L (cut-off 0.22 mg/L) (Data originally published in Armer & Allcock 2017 [11]).

could also be formed in subjects with glycosuria and E.coli infection. If etha-nol was formed due to the fermentation of sugars in the urine, this could be con-verted to EtG by bacteria present in the urine [14]. EtS would not be produced so again EtS can verify whether the EtG result is a true positive. Both EtG and EtS have been detected in individuals who used ethanol-based mouthwash or hand gel; however, the mouthwash was gar-gled 4 times/day which is much higher than the recommended frequency of use [15]. Owing to these factors, it is advisable to measure both EtG and EtS, which is currently only possible if using LC-MS/MS.

Cut-off values for EtG and EtSThere has been a lot of debate in the litera-ture about suitable cut-off values to use for EtG and EtS. Some authors have suggested using the lower limit of detection (LLOD) or lower limit of quantitation (LLOQ) for the method so that any detectable EtG and EtS is a ‘positive’ result. However, the LLOD and LLOQ in LC-MS/MS meth-ods will be variable between laboratories depending on a number of factors includ-ing sample preparation, column choice, chromatography and the tandem MS optimization. For EtG and EtS, the pub-lished LLOQs range from 0.05–0.20 mg/L and 0.04–0.10 mg/L respectively. New Clinical & Laboratory Standards Institute (CLSI) guidelines were published in 2016 and these should help to improve stand-ardization between LC-MS/MS methods [16]. Alternatively, cut-off values could be defined by measuring EtG and EtS in a non-drinking population and incorporat-ing measurement uncertainty (0.26 mg/L and 0.22 mg/L for EtG and EtS respec-tively) [11]. For EtG, a cut-off of 0.50 mg/L has been proposed to reduce the risk of false positive results. The disadvantage of a higher EtG cut-off is a reduction in sensi-tivity. Jatlow et al. demonstrated that using a 0.50 mg/L cut-off would only detect the intake of a low dose of alcohol 12 hours earlier (estimated blood alcohol 20 mg/dL) in 50% of participants. However, all participants had results above 0.10 mg/L and 0.20 mg/L after the same low alco-hol dose 12 hours earlier [4]. SAMHSA have suggested separating EtG results into ‘high’ positive (>1.00 mg/L), ‘low’ posi-tive (0.50–1.00 mg/L) and ‘very low’ posi-tive (0.10–0.50 mg/L). They suggest that a ‘very low’ positive result may indicate previous heavy drinking (1–3 days ago), previous light drinking (12–36 hours ago) or ‘extraneous’ exposure [9].

Another consideration for urine EtG and EtS analysis is the dilution of urine samples; in urine toxicology testing, it is standard practice to measure creatinine to check the validity of a urine sample. There is limited data on the utility of EtG and EtS creatinine ratios. However, it is good practice to measure creatinine and ques-tion the validity of the EtG and EtS results if the creatinine is ≤2.0 mmol/L [17].

ConclusionUrine EtG and EtS are valuable additional tools to detect recent alcohol intake in individuals undergoing treatment for alcohol dependence to ensure continued abstinence. Owing to the risk of false pos-itive EtG results from unintentional expo-sure (e.g. non-alcoholic beer, urine infec-tion with glycosuria, ethanol-based hand gel/mouthwash), the measurement of EtS in addition to EtG is recommended. An immunoassay is available for EtG but only MS allows the detection of both EtG and EtS to confidently confirm recent alcohol intake. There are a number of published methods for LC-MS/MS for EtG and EtS which are applicable for routine use in a clinical laboratory.

references1. Dahl H, Stephanson N, Beck O, Helander A. Comparison of urinary excretion characteristics of ethanol and ethyl glucuronide. J Anal Toxicol 2002; 26: 201–204.2. Helander A, Beck O. Ethyl Sulphate – a metabo-lite of ethanol in humans and a potential biomarker of acute alcohol intake. J Anal Toxicol 2005; 29: 270–274.3. Helander A, Beck O, Jones W. Laboratory test-ing for recent alcohol consumption: comparison of ethanol, methanol and 5-hydroxytryptophol. Clin Chem 1996; 42: 618–624.4. Jatlow P, Agro A, Wu R, Nadim H, Toll BA, Ralevski E, Nogueira C, Shi J, Dziura JD, et al. Eth-ylglucuronide and ethyl sulfate assays in clinical tri-als, interpretation and limitations: results of a dose ranging alcohol challenge study and two clinical trials. Alcohol Clin Exp Res. 2014; 38: 2056–2065.5. Dahl H, Voltaire Carlsson A, Hillgren K, Helander A. Urinary ethyl glucuronide and ethyl sulphate for detection of recent drinking in an outpatient treat-ment program for alcohol and drug dependence. Alcohol Alcohol 2011; 46: 278–282.6. Wetterling T, Dibbelt L, Wetterling G, Göder R, Wurst F, Margraf M, Junghanns K. Ethyl glucuro-nide (EtG): better than breathalyser or self-reports to detect covert short-term relapses into drinking. Alcohol Alcohol 2014; 49: 51–54.7. Armer J, Gunawardana L, Allcock R. The perfor-mance of alcohol markers including ethyl glucuro-nide and ethyl sulphate to detect alcohol use in cli-ents in a community alcohol treatment programme.

Alcohol Alcohol 2017; 52: 29–34.8. Knight J, Brand P, Willey P, van der Merwe J. Adult substance misuse statistics from the National Drug Treatment Monitoring System (NDTMS): 01 April 2016 – 31 March 2017. Public Health England 2017(https://assets.publishing.service.gov.uk/govern-ment/uploads/system/uploads/attachment_data/file/658056/Adult-statistics-from-the-national-drug-treatment-monitoring-system-2016-2017.pdf).9. The role of biomarkers in the treatment of alco-hol use disorders. Substance Abuse and Mental Health Services Administration (SAMHSA) Advi-sory 2012; 11(2) (https://store.samhsa.gov/shin/content/SMA12-4686/SMA12-4686.pdf).10. Helander A, Kenan N, Beck O. Comparison of analytical approaches for liquid chromatography/mass spectrometric determination of the alcohol biomarker ethyl glucuronide in urine. Rapid Com-mun Mass Spectrom 2010: 24: 1737–1743.11. Armer J, Allcock R. Urine ethyl glucuronide and ethyl sulphate using liquid chromatography-tan-dem mass spectrometry in a routine clinical labora-tory. Ann Clin Biochem 2017; 54: 60–68.12. Bottcher M, Beck O, Helander A. Evaluation of a new immunoassay for urine ethyl glucuronide test-ing. Alcohol Alcohol 2008; 43: 46–48.13. Thierauf A, Gnann H, Wohlfarth A, Auwärter V, Perdekamp MG, Buttler KJ, Wurst FM, Weinmann W. Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of “non-alco-holic” beer. Forensic Sci Int 2010; 202: 82–85.14. Helander A, Ollson I, Dahl H. Postcollection synthesis of ethyl glucuronide by bacteria in urine may cause false identification of alcohol consump-tion. Clin Chem 2007; 53: 1855–1857.15. Reisfield G, Goldberger B, Pesce A, Crews BO, Wilson GR, Teitelbaum SA, Bertholf RL. Ethyl glucuronide, ethyl sulfate, and ethanol in urine after intensive exposure to high ethanol content mouthwash. J Anal Toxicol 2011; 35: 264–268.16. Lynch K. CLSI C62-A: a new standard for clini-cal mass spectrometry. Clin Chem 2016; 62(1): 24–29.17. European guidelines for workplace drug testing in urine. European Workplace Drug Testing Society 2015 (http://www.ewdts.org/data/uploads/documents/ewdts-urine-guideline-2015-11-01-v2.0.pdf).

the authorsJane Armer*1 BA MSc FRCPath and Rebecca Allcock2 BSc MSc FRCPath

1Department of Blood Sciences, East Lan-cashire Hospitals NHS Trust, Blackburn, UK2Department of Clinical Biochemistry, Lancashire Teaching Hospitals NHS Foun-dation Trust, Preston, UK

*Corresponding authorE-mail: [email protected]


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BackgroundLiquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly becoming the method of choice in the clinical laboratory for the measurement of low molecular weight analytes. The major advantage that LC-MS/MS pos-sesses relative to conventional laboratory

techniques such as immunoassay is its higher specificity (and often sensitivity, although this is compound specific) and its ability to measure multiple compounds in a single run (multiplexing). LC-MS/MS thus provides the opportunity for more accurate and precise biochemical diag-nosis and monitoring of human disease.

One example of the increasing adoption of LC-MS/MS by clinical laboratories is the measurement of steroid hormones in various matrices (serum, saliva, urine).

Steroid metabolismAll steroids share a cyclopentanoperhy-drophenanthrene nucleus, with individual species varying according to the presence of different functional groups attached to this four-ring structure, as well as by the oxidation state of the rings. Cortisol struc-ture is given as an example in Figure 1. In humans, the major sites of steroid hor-mone production are the adrenal gland and the gonads. Steroids are synthesized from cholesterol via a series of enzyme-catalysed steps (Fig. 2), which are under tight regulation in healthy individuals by feedback mechanisms involving the hypo-thalamus and anterior pituitary. Steroids have a wide range of physiological func-tions which are summarized in Table 1.

Adrenocortical carcinoma – a diag-nostic challengeThere are many endocrine disorders that result in the improper synthesis of steroids, and one of the rarest and most severe is adrenocortical carci-noma (ACC). ACC is a malignancy of the adrenal cortex with an annual inci-dence of 1 or 2 cases per million [1]. The majority of ACC cases are sporadic and occur in the fifth or sixth decade of life and more commonly in women; although ACC can be associated with several familial syndromes including Li-Fraumeni, Beckwith-Wiedemann, Lynch syndrome and multiple endo-crine neoplasia type 1 [2]. Functional steroid hormone-producing tumours occur in around two-thirds of cases [3], presenting with varied signs and symptoms of steroid overproduction, most commonly Cushing’s syndrome (cortisol excess) and hyperandrogen-ism. ACC can progress rapidly in some patients, therefore it is vital that it is dis-tinguished from benign adrenal adeno-mas, as ACC has a 5-year survival rate of <50% [2]. A surgical cure is only pos-sible if the carcinoma is detected in its

use of an LC-MS/MS 13-steroid serum panel in the diagnosis of adrenocortical carcinomaLiquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly being used in clinical biochemistry laboratories to measure steroid hormones in order to overcome the issue of cross-reactivity that traditional immunoassays can be subject to. We have developed an LC-MS/MS method for the measurement of 13 steroids from a single blood sample, in order to improve the diagnosis of adrenocortical carcinoma.

by Victoria Treasure and Dr David Taylor


Figure 1. Cortisol structure. The four-ringed cyclopentaperhydrophenanthrene nucleus (labelled A-D) is common to all steroids, carbons are numbered. Steroids differ according to the presence (glucocorticoids/minerolcorticoids) or absence (androgens) of a sidechain at C19, the reduction status of ring A, and the presence of hydroxyl group or carbonyl groups at carbons 3, 11, 17 and 21.

localized stage, otherwise the median survival period is <15 months [4].

The diagnosis of ACC is challenging as there is no single diagnostic tool that is able to distinguish ACC from other adre-nal masses, including benign adenomas with glucocorticoid or mineralocorticoid excess, phaeochromocytoma and non-functioning adenomas. Imaging alone is insufficient for diagnosis, as although patients with ACC almost always present with tumours ≥4 cm, the presence of a large mass only has a clinical specificity of 61% [5]. Additionally, whereas up to two-thirds of tumours are functional, less than half of ACC cases present with clinical signs of steroid overproduction [3], with a further proportion presenting with other symptoms including abdominal pain. However, a significant proportion are dis-covered incidentally [2].

The European Network for the Study of Adrenal Tumours (ENSAT) currently recommends that the initial biochemi-cal work-up for suspected ACC includes measurement of serum cortisol (both basal and assessment of suppression after dexa-methasone), dehydroepiandrostenedione sulphate (DHEAS), androstenedione, testosterone, 17-hydroxyprogesterone, estradiol and aldosterone (if the patient is hypokalemic or hypertensive). An alterna-tive approach is to measure steroid metab-olites in urine using gas chromatography-mass spectrometry (GC-MS); increases in the excretion of metabolites of the steroid precursors 11-deoxycortisol, 17-hydroxy-pregnenolone and pregnenolone have been shown to provide particularly high diagnostic utility in ACC. Unfortunately, urine steroid profiling is not commonly available in clinical laboratories owing

to lengthy sample preparation and com-plex result interpretation. Further, serum 11-deoxycortisol, 17-hydroxypregne-nolone or pregnenolone measurements are rarely performed either because of lack of demand, or specificity of the avail-able immunoassays which may be subject to significant levels of cross-reactivity.

As a result of these limitations, the use of LC-MS/MS is increasingly being adopted to provide more specific steroid hormone measurements. An approach we have taken in our laboratory is to develop and fully evaluate a multiplexed LC-MS/MS method panelling 13 steroids in serum [6] to include many of the steroid synthetic pathway intermediates currently not available for ACC work-up.

Use of a serum steroid panelThe steroids included in our serum panel are highlighted in Figure 2 and are as follows:• androstenedione• corticosterone• cortisol• cortisone• 11-deoxycorticosterone• 11-deoxycortisol• 21-deoxycortisol• DHEAS• 17-hydroxypregnenolone• 17-hydroxyprogesterone• pregnenolone• progesterone• testosterone.

Samples are prepared for analysis by an initial protein precipitation step to remove steroids from their binding pro-teins, followed by liquid-liquid extrac-tion in order to cleanly extract the ster-oids from remaining matrix components.

Prepared extracts are then analysed by LC-MS/MS in which steroids are first resolved on a reverse phase C18 column by gradient elution followed by MS/MS detection using positive atmospheric pressure chemical ionization (APCI) operated in multiple reaction monitor-ing mode. Chromatographic separation of several isobaric (same mass to charge ratio) steroids is essential, as is the use of deuterated internal standards for all ster-oids in the method.

When we applied our method to adrenal tumour samples [6], we were able to show that between 4 and 7 steroids were elevated in all ACC cases in comparison to non-ACC adrenal tumours where a maximum of 1–2 steroids were abnormal. The corti-sol precursor 11-deoxycortisol was most useful in the discrimination between ACC and non-ACC adrenal lesions, whereas other steroids markedly elevated in ACC included 17-hydroxypregnenolone and pregnenolone. Indeed, all steroids except testosterone in males and corticosterone and cortisone in both sexes were of use in discriminating ACC. This validates the use of a panelling approach when investi-gating adrenal masses.

Our findings compare well with urine steroid profiling studies. Although urine steroid profiling using 24-hour collections may offer greater clinical sensitivity compared to a single blood measurement owing to diurnal rhythms of steroid production, urine measure-ments rely on accurately timed collec-tions that are often performed incor-rectly and are inconvenient to the patient. Advantages of our LC-MS/MS serum panel compared to urine ster-oid profiling by GC-MS include a less

– September 201811

Figure 2. Steroid biosynthetic pathway in the adrenal cortex. Steroids highlighted in red are measured in our LC-MS/MS serum steroid panel.

labour intensive sample preparation, as well as less expertise required for the interpretation of complex profiles, as the serum method only targets selected steroids rather than the large number of their metabolites in urine.

Use of our LC-MS/MS serum steroid panel in ACC patients has further dem-onstrated the limitations of assessing serum steroids by immunoassay. We observed evidence of notable interfer-ence in ACC patients in the cortisol, progesterone, 17-hydroxyprogesterone and androstenedione immunoassays, inferred to be due to elevated concen-trations of structurally related steroid precursors.

Future workCurrently, our 13-steroid serum panel has been used to study a rela-tively small number of ACC patients (because of the rarity of the disease), and clearly larger prospective studies are required to more fully determine the diagnostic utility of our panel in ACC. Further work is also required to clarify the effects of age, sex and diurnal variation on serum steroid panelling; nonetheless the most useful markers of ACC are markedly elevated above variation attributable to these biological factors. In addition to the complexity of interpreting biomarker panels, it is not only important to con-sider specific reference ranges, but to

also consider the patterns in results which require an omics-based analy-sis approach to interpretation. The challenge surrounding this, as well as the requirement for clear presentation and reporting of results to clinicians requires close involvement of clinical colleagues for the development and introduction of such testing strategies.

The analysis of steroid panels by LC-MS/MS can also undoubtedly be used in other conditions including inborn errors of steroid metabolism such as congenital adrenal hyperplasia (CAH) and polycystic ovarian syndrome (PCOS).

Although we have demonstrated the advantages of our LC-MS/MS steroid panel compared to routine immunoas-says, there are undoubtedly disadvan-tages of using LC-MS/MS. These include the initial cost of instrument purchase, the increased expertise required and often a more laborious sample prepa-ration. Additionally, the specificity of mass spectrometry should not be read-ily assumed; careful selection of multi-ple reaction monitoring (MRM) transi-tions and chromatography conditions are essential to separate isobaric ster-oids and other interfering compounds. However, in the context of improving the biochemical tools available to us to aid the diagnosis of ACC, the advan-tages of LC-MS/MS far outweigh these limitations.

SummaryIn summary, LC-MS/MS serum steroid panelling offers an additional tool for the challenge that is the diagnosis of ACC. Our method combines measurement of both common and rarely measured ster-oids in a single sample, which we have shown provides useful data to aid the dis-crimination of ACC from benign adrenal tumours. Use of LC-MS/MS gives several advantages over the immunoassay and GC-MS-based methods currently used to assess steroid overproduction, but fur-ther work is required to demonstrate the full potential of its use in the diagnosis of ACC.

references1. Fassnacht M, Kroiss M, Allolio B. Update in adrenocortical carcinoma. J Clin Endo-crinol Metab 2013; 98: 4551–4564.2. Else T, Kim AC, Sabolch A, Ramond VM, Kandathil A, Caoili EM, Jolly S, Miller BS, Giordano TJ, Hammer GD. Adrenocortical carcinoma. Endocr Rev 2014; 35: 282–326.3. Arlt W, Biehl M, Taylor AE, Hahner S, Libé R, Hughes BA, Schneider P, Smith DJ, Stiekema H, et al. Urine steroid metabo-lomics as a biomarker tool for detecting malignancy in adrenal tumours. J Clin Endocrinol Metab 2011; 96: 3775–3784.4. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade-Brittinger C, Lacroix A, et al. Combination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med 2012;366:2189–2197.5. Hamrahian AH, Ioachimescu AG, Remer EM, Motta-Ramirez G, Bogabathina H, Levin HS, Reddy S, Gill IS, Siperstein A, Bravo EL. Clinical utility of noncontrast computed tomography attenuation value (Hounsfield units) to differentiate adrenal adenomas/hyperplasias from nonadeno-mas: Cleveland Clinical experience. J Clin Endocrinol Metab 2005; 90: 871–877.6. Taylor DR, Ghataore L, Couchman L, Vincent RP, Whitelaw B, Lewis D, Diaz-Cano S, Galata G, Schulte KM, et al. A 13-steroid serum panel based on LC-MS/MS: use in detection of adrenocortical car-cinoma. Clin Chem 2017; 63: 1836–1846.

the authorsVictoria Treasure* MSc and Dr David Taylor PhDDepartment of Clinical Biochemistry (Viapath), King’s College Hospital NHS Foundation Trust, London, UK



Steroid group Major source Principle steroids Functions

Mineralocorticoids Adrenal cortex Aldosterone Minor: 11-Deoxycorticosterone (DOC)

Regulate blood pressure through sodium and water retention

Glucocorticoids Adrenal cortex CortisolMinor: Corticosterone

Increase blood glucose concentration, decrease lipolysis, modulate immune system

Sex steroids Gonads (minor contribution: adrenal cortex)

Testosterone Dihydrotestosterone OestradiolProgesteroneMinor activity as weak androgens: AndrostenedioneDHEA

Secondary sexual characteristics, auxiliary hair growth, fertility

Table 1. Major steroids and their functions.

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LC-MS/MS methods have been widely adopted by a broad range of research labo-ratories, due in large part to their ability to accurately measure multiple analytes in various matrices with high specificity and resolution.

Within clinical laboratories, however, LC-MS/MS methods are used across a relatively limited number of disciplines, most notably endocrinology, immunosup-pressant and therapeutic drug monitoring, toxicology, new born screening, microbiol-ogy, as well as small molecule, peptide and protein marker analysis. This powerful tech-nique brings many advantages to clinical workflows, enabling laboratory scientists to analyse multiple analytes with greater speci-ficity and sensitivity than alternative meth-ods, such as some immunoassays.

Despite its numerous benefits for patient care, LC-MS/MS technology has not been adopted across the wider clinical setting. One of the biggest barriers preventing its broader use has been the lack of commer-cially available automated systems that address the specific needs of the clinical laboratory.

the importance of fast turnaround timesConventional LC-MS/MS workflows typi-cally involve a large number of manual and time-consuming processes. Indeed, while advances in the performance of LC separation and MS analysis techniques mean that meas-urement acquisition steps now take a matter of minutes to complete, batching of samples, sample preparation, data analysis and equip-ment maintenance can significantly extend

the length of time that must be invested in each sample run. Moreover, the burden asso-ciated with manual data entry can signifi-cantly lengthen timelines. To ensure quality, further data validation steps are required before final reporting, resulting in significant amount of time devoted to processes that do not add real value to operations.

Traditional LC-MS/MS methods also require users to ensure careful batch-ing and multiple runs need scheduling at appropriate times, which may prove chal-lenging when faced with shift patterns or a weekend testing service. Overall, the labor intensive LC-MS/MS workflows limit sam-ple throughput, while requiring a high level of human input and incurring a significant operating expense. As such, these methods do not fit well with the working practices of the clinical laboratory.

the need for accurate analysisManual methods also leave measurements vul-nerable to human error. Even when analyses are performed by the most experienced labo-ratory scientists, these multi-step workflows are susceptible to mistakes, omissions or even small variations in the way protocols are con-ducted. If errors are identified, repeat experi-ments are required to correct them. This can significantly add to the time taken to obtain clinically useful insights, prevent timely patient treatment decisions and even undermine con-fidence in the accuracy of findings.

Given the complexity of conventional LC-MS/MS workflows, and to reduce the potential of human error, the operation of these systems has traditionally been assigned to highly skilled scientists with specialist kno-whow. High levels of expertise are also essen-tial for sample preparation and data analysis. As a consequence, many clinical laboratories have been facing the need to train their per-sonnel, which can place an additional burden on budgets and bandwidth.

Automated lC-MS/MS driving process optimizationAnalytical methods within the clinical lab must be automated, reliable and provide walk-away capabilities to meet clinicians’ need for rapid turnaround of accurate results. By eliminating many of the error-prone and time-consuming manual steps

Automating LC-MS/MS analysis for streamlined clinical testing workflowsLiquid chromatography-tandem mass spectrometry (LC-MS/MS) technology has emerged as an important tool for a wide range of analytical applications. However, the manual, multi-step processes involved in LC-MS/MS workflows have limited its adoption in clinical laboratories. In this article, we look at how the latest automated LC-MS/MS technology designed specifically for the clinical laboratory is simplifying these workflows, allowing laboratory scientists to harness the full potential of this approach.

– September 2018 technology watch14

Cascadion SM Clinical Analyser

involved in traditional workflows, fully automated, random access LC-MS/MS sys-tems are well placed to simplify and accel-erate the collection of high quality data. This ability to assess samples quickly, while maintaining a high level of accuracy, would be especially beneficial to those assays that involve more complex processes, since they could be streamlined and automated to sim-plify workflow.

Undoubtedly, the future of clinical analysis is trending towards the broader adoption of fully automated systems. Automation will greatly benefit LC-MS/MS workflows, making this powerful technique accessible for a wide range of clinical applications, without the need to create a new team of highly trained experts. Laboratories that are already performing clinical LC-MS/MS testing will also be able to better manage their highly trained experts and apply their talents to the development and early imple-mentation of newer, more esoteric, high value analytes – expanding the laboratory’s overall service capabilities as a result. Fur-thermore, while the capital cost of currently available LC-MS/MS systems is relatively high, operational costs related to materials are actually low. If the volume of samples is high enough, then the economy of scale will make cost of ownership comparable to alternative clinical testing methods.

Labs already performing laboratory devel-oped tests (LDTs) using LC-MS/MS may be more resistant to automation. The develop-ment and validation of MS assays takes a significant amount of time and expertise, so there may be concern over the impact that automation will have on their existing LDT protocols. However, automation will not be a limiting factor in a laboratory’s ability to develop and implement LDTs. Automation has the potential to reduce the need for highly trained staff to apply themselves to the repetitive tasks, allowing them to focus on the development of emerging, clinically needed LDTs.

Meeting the needs of clinical lC-MS/MS analysisThe need for an automated LC-MS/MS system that addresses the unique require-ments of the clinical laboratory has led to the development of the new Thermo Sci-entific™ Cascadion™ SM Clinical Analyser*. Designed to eliminate, automate and sim-plify many of the manual processes involved in traditional LC-MS/MS workflows, the system gives users all of the power of this important technology in an easy to imple-ment tool.

Owing to its random access capability, the Cascadion SM Clinical Analyser removes the need for long periods of batch loading, and instead facilitates continuous, uninter-rupted operation for rapid turnaround of results. This is particularly important for the out-of-hours service and processing of STAT samples. Moreover, by minimizing the potential for human error, the technology is enabling the collection of accurate measure-ments, the first time around. When imple-mented in the clinical setting, this level of dependability is helping to accelerate clini-cal outcomes and deliver real value for clini-cal laboratories.

Furthermore, because the system can be operated by non-LC-MS/MS experts, experienced scientists have more time to work in other capacities. With more time back in their daily routine, this gives clinical researchers, for example, the opportunity to develop new tests to meet an urgent unmet need or to support bet-ter patient care. This level of ease-of-use and simplicity not only relates to run-to-run performance, it also extends to system maintenance too. A recent study from Argent Global Services has found that monthly maintenance takes approxi-mately 18 minutes, meaning that signifi-cant amounts of time can be saved and put to better use.

ConclusionLC-MS/MS systems offer clear benefits for clinical applications. However, the lack of auto-mated systems has posed a barrier to their broader uptake in the clinical setting. Requir-ing expert operation and the investment of sig-nificant time and resources to ensure compat-ibility with sample preparation processes and data review and reporting systems, traditional technologies have, until now, not adequately addressed the needs of clinical laboratories.

Fully automated, random access LC-MS/MS technology, designed specifically for clinical use, is alleviating these pain points and ena-bling clinicians to benefit from quality results at high throughputs, while reducing the need to perform repetitive manual tasks. The impact of these systems is benefitting clinical laboratories, helping to improve operational efficiencies and ensure clinicians receive the results they need to make informed treat-ment decisions in a timely fashion.

*This product is IVD/CE-marked. Product is not 510(k) cleared and not yet available for sale in the U.S.

referenceZhang V & Rockwood A. “Impact of Automation on Mass Spectrometry”. Clinica Chimica Acta 450 (2015): 298-303.

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Bio-Rad Laboratories recently announced the availability of a new Measurement Uncertainty Report in their Quality Control Data Management Software Unity Real Time 2 Service Pack 5 upgrade. Under ISO 15189, Measurement Uncertainty is a man-datory requirement and allows labs to calculate their analytical test dispersion. This new report allows laboratories to calculate their Measurement Uncertainty according to one of three dif-ferent calculations. The three different calculations include the standard expanded Uncertainty based on the lab’s imprecision

(consistent with RCPA and NABL), and two combined expanded uncertainty calcula-tions which contain either the bias or the calibrator uncertainty (consistent with the SH GTA 14 Guideline from France).Unity Real Time 2 is Bio-Rad’s expert QC Data Management solution for desktop users which facilitates compliance under CLIA and ISO 15189. It provides run validation with real-time bench and supervisor QC data review with comprehensive audit trails. It allows labs to participate in Bio-Rad’s Unity Interlaboratory Program with over 50,000 participating instruments. All QC data can be uploaded from any LIS, middleware or instrument via the Unity Connect software. Users can also reduce non-essential retests with Analytical Goal options and implement the best QC rules with the Westgard Advi-sor or use Bio-Rad’s latest QC design tool Mission: Control which provides a risk based approach to Quality Control.http://www.qcnet.com/datamanagement/

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introductionOwing to recent remarkable advances in our understanding of the molecular and genetic basis of disease, it is now known that colorectal carcinoma (CRC) is a het-erogenous clinical entity characterized by multiple molecular subtypes [1]. One such molecular pathway involved in CRC pathogenesis is the microsatellite insta-bility (MSI) pathway, where a deficient mismatch repair (dMMR) system leads to unchecked errors in DNA replication [2]. These errors result in a propensity for abnormal insertion or deletion of short, repetitive sequences of DNA (microsat-ellites), resulting in mutations in cancer-related genes and ultimately neoplasia. Up to 15–20% of colorectal carcinomas are of MSI phenotype. An inherited predisposi-tion to dMMR cancers, particularly CRC, is present in Lynch syndrome, the most common heritable cancer syndrome. It is due to autosomal dominant mutations in four mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) or more rarely by mutations in EPCAM, a gene upstream of MSH2. Patients present at an earlier age and have an increased inci-dence of synchronous and metachronous CRCs. Histologically, tumours are poorly differentiated, frequently exhibiting a mucinous or signet ring cell morphology. Tumour infiltrating lymphocytes are often prominent and a Crohn’s-like inflam-matory response may be present at the tumour periphery. However, the majority of dMMR CRCs arise sporadically and are a result of MLH1 promoter hypermeth-ylation. Unlike in Lynch syndrome, these

tumours affect the right side of the colon, are diagnosed at advanced age and have a female preponderance. They are, however, histologically similar to Lynch syndrome CRCs. Mutation of the BRAF V600E gene is present in 60–70% of sporadic dMMR tumours and is almost never seen in Lynch syndrome. As such, incorporating BRAF and/or MLH1 methylation status into MMR diagnostic algorithms offers poten-tial exclusion criteria for genetic testing [3–5].

Why is it important to identify dMMr in colorectal carcinoma?Diagnosing a patient with a dMMR cancer has a number of advantages:

1. Identification of patients with Lynch syndromeOnce diagnosed, these patients benefit from increased surveillance, prophylactic aspirin therapy and more radical surgery in order to facilitate the prevention and/or early detection of potential tumours (both colonic and extracolonic) [5].

2. It provides prognostic informationSeveral studies have shown that dMMR CRC has a better prognosis than MMR proficient (pMMR) CRC. dMMR tumours are less likely to develop lymph node and liver metastases. However, in advanced disease (stage IV) dMMR status can por-tend a poorer prognosis [6–8].

3. It provides predictive informationdMMR tumours likely have a reduced response to 5-flurouracil based

chemotherapy. In addition, advanced dMMR tumours have been shown to have a better response rate and progression free survival to the anti PD-1 drug pem-brolizumab when compared to pMMR tumours [7–9].

Reliance by clinicians on clinical criteria such as the revised Bethesda guidelines to determine which patients should undergo screening for Lynch syndrome results in inaccurate determination of eligibility for screening in up to 28% of cases [10]. Con-sequently, a number of organizations have recently published guidelines endorsing reflex MMR testing of all diagnosed CRCs, including the National Institute for Health and Care Excellence (NICE), the Ameri-can Society for Clinical Pathology (ASCP) and the American Society for Clinical Oncology (ASCP), among others [11–12]. The cost effectiveness of such a screening approach has been proven by several stud-ies [13].

diagnosisDiagnosis of dMMR tumours is either via PCR amplification of specific microsatel-lite repeats in formalin-fixed, paraffin-embedded tumour tissue or by immuno-histochemistry (IHC) which confirms the absence or presence of MMR proteins. Both MSI testing and IHC have virtually equivalent informative value in predicting germline mutation [3, 14]. Given that IHC is more widely available in general pathol-ogy laboratories and is a rapid, efficient and cost-effective method of testing, it is the more frequently used test. It also has the added benefit of directing germline testing to the particular mutated gene.

A number of commercially available MMR IHC antibodies are available for laboratory use. A protocol using a panel of four immunohistochemical antibodies to the four mismatch repair gene proteins (MLH1, MSH2, MSH6, PMS2) is recom-mended (Fig. 1). Complete loss of expres-sion of one or more MMR protein is sug-gestive of dMMR. Loss of MLH1 often occurs in conjunction with loss of PMS2. This is due to the fact that MLH1 protein

use of immunohistochemistry in the determination of mismatch repair status of colorectal carcinomaMicrosatellite instability, reflective of a defective mismatch repair system, has been implicated as one of the main pathways involved in the pathogenesis of colorectal carcinoma. Herein, we describe the role of the mismatch repair system in the development of colorectal carcinoma, the advantages and disadvantages of using immunohistochemistry as the primary method of determining mismatch repair status, and compare the suitability of colorectal endoscopic biopsy versus resection specimens as the testing material of choice.

by Dr Odharnaith O’Brien, Dr Éanna Ryan and Prof. Kieran Sheahan

– September 2018 Pathology16

forms a heterodimer complex with PMS2. Isolated loss of PMS2 Is indicative of a defect in the PMS2 gene. However, com-bined loss of PMS2 and MLH1 indicates the defect lies in MLH1, as MLH1 con-fers stability to PMS2. A similar situation is seen with MSH2 and MSH6; isolated loss of MSH6 indicating defective MSH6, whereas loss of expression of both proteins indicates the defect involves MSH2. Back-ground positive IHC staining in intratu-moural lymphocytes or of adjacent nor-mal colonic epithelium, if present, serve as reliable internal positive controls [5].

Once loss of expression of any IHC MMRP is confirmed, further testing is required. In cases where there is loss of MLH1, testing for the presence of BRAF V600E mutation and MLH1 hypermeth-ylation, as mentioned previously, can further stratify those patients who likely have sporadic dMMR tumours. Patients demonstrating loss of MSH2, MSH6 or PMS2, and patients demonstrating loss of MLH1 who are BRAF V600E negative and MLH hypermethylation negative, should undergo germline testing to confirm Lynch syndrome (Fig. 2).

MMR IHC testing is typically performed on CRC resection specimens. Data has recently begun to accumulate that the yield of IHC testing performed on endo-scopic biopsy material may be as good as that performed on surgical resections. We recently published a study evaluat-ing the reliability of MMR IHC in CRC

from preoperative endoscopic biopsy tis-sue when compared to matched surgical resection specimens and demonstrated 100% concordance in 53 cases of dMMR (n=10) and pMMR (n=43) tumours [14]. Our results corroborate the results of other studies that indicate endoscopic biopsies are a suitable source of tissue for MMR IHC analysis [15–17].

Preferential testing of MMR status on endoscopic biopsy samples over resection specimens carries a number of advantages. Immunostaining is highly sensitive to the degree of tissue fixation; given the small size of biopsy samples, faster and more thorough fixation may result in supe-rior quality staining. Additionally, neo-adjuvant chemoradiotherapy used in the standard treatment of locally advanced rectal tumours may result in a complete pathologic response, with no residual tumour available for testing. Neoadjuvant treatment can also occasionally alter the MMRP status of the tumour. In these two scenarios, the pretreatment biopsy could provide reliable testing material.

Endoscopic biopsies could also be used to initiate earlier and indeed preopera-tive genetic testing, allowing informed clinical decisions regarding the extent of resection to be made before surgery in those patients confirmed as having Lynch syndrome. The option of total colectomy as an alternative to segmental colectomy could be discussed, particu-larly with younger patients, to reduce the


Figure 1. (a) H&E section of colorectal carcinoma exhibiting mismatch repair protein loss. There is loss of expression of MLH1 (b) and PMS2 (c), with retention of MSH2 (d) and MSH6 (e) staining. Note the positive staining of intratumoural lymphocytes in (b) and (c), acting as a positive internal control.

Figure 2. Recommended algorithmic approach in the molecular work-up of all patients with newly-diagnosed colorectal carcinoma. [Adapted from O’Brien O, et al. Correlation of immunohistochemical mismatch repair protein status between colorectal carcinoma endoscopic biopsy and resection specimens Journal of Clinical Pathology 2018;71:631-636 (15)].

(a)

(b)

(c)

(d)

(e)

risk of metachronous CRC and the need for intense postoperative surveillance. In addition, females identified as having Lynch syndrome, who have completed their families, could be considered for concurrent hysterectomy, with/without bilateral salpingo-oophorectomy, in order to prevent the development of a gyneco-logical tract malignancy and spare them a potential additional future procedure.

Recent studies suggest that dMMR tumours may respond well to immuno-therapy in patients with advanced dis-ease [9]. In the instance that an advanced tumour is inoperable at diagnosis, meta-static or endoscopic biopsy tissue could be used to screen for dMMR and Lynch syn-drome, and direct immunotherapy.

Despite these advantages, some limita-tions exist in the use of IHC to determine MMR status which are not just specific to biopsy tissue. Rare missense mutations have been reported in MLH1 and MSH6 genes that affect MMR protein function but not translation and antigenicity – in this scenario the tumour harbours a defec-tive protein, but one which demonstrates retention of IHC staining, giving a false result [19].

Intratumoural heterogeneity, where there is heterogeneity of MMR protein expres-sion within a single tumour, also repre-sents a potential pitfall [20]. This may be of particular concern in biopsy samples as they represent only a small proportion of a tumour and could erroneously misclassify the MMR status by virtue of inadequate sampling. Another issue is the small size of endoscopic biopsies; adequate material may not be available for IHC. Encouraging generous tumour sampling at the time of biopsy could reduce the risk of such limi-tations. Heterogeneity in the MMR status of CRC is rare and is thought in many instances to be a result of suboptimal tis-sue fixation. Given biopsies are usually of small size, adequate fixation of tissue can be assured.

ConclusionUp to 15–20% of CRCs are of MSI phenotype, secondary to either spo-radic methylation-induced silencing or inherited mutations in MMR-related genes. IHC is an effective and reliable testing modality for determining MMR status in CRC. Colorectal endoscopic biopsy and resection specimens are both suitable sources of testing material, with resection specimens currently the

preferred specimen type. Endoscopic biopsy samples may become increas-ingly important as a testing material as the potential of tailored approaches to surgery, chemotherapy and immuno-therapy becomes a standard of care in this era of personalized medicine.

references1. Guinney J, Dienstmann R, Wang X, de Reyniès A, Schlicker A, Soneson C, Marisa L, Roepman P, Nyamundanda G, et al. The consensus molecu-lar subtypes of colorectal cancer. Nat Med 2015; 21(11): 1350–1356.2. Poulogiannis G, Frayling IM, Arends MJ. 2010. DNA mismatch repair deficiency in sporadic colo-rectal cancer and Lynch syndrome. Histopathol-ogy 2010; 56(2): 167–179.3. Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ, Walsh-Vockley C, Petersen GM, Walsh MD, et al. Immunohisto-chemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol 2002; 20(4): 1043–1048.4. Bouzourene H, Hutter P, Losi L, Martin P, Ben-hattar J. Selection of patients with germline MLH1 methylation and BRAF mutation. Fam Cancer 2010; 9: 167–172.5. Richman S. Deficient mismatch repair: read all about it (Review). Int J Oncol 2015; 47: 1189–1202.6. Saridaki Z, Souglakos J, Georgoulias V. Prog-nostic and predictive significance of MSI in stages II/III colon cancer. World J. Gastroenterol 2014; 20(22): 6809–6814.7. Guastadisegni C, Colafranceschi M, Ottini L, Dogliotti E. Microsatellite instability as a marker of prognosis and response to therapy: a meta-analysis of colorectal cancer survival data. Eur J Cancer 2010; 46(15): 2788–2798.8. Mohan HM, Ryan E, Balasubramanian I, Ken-nelly R, Geraghty R, Sclafani F, Fennelly D, McDer-mott R, Ryan EJ, et al. Microsatellite instability is associated with reduced disease specific survival in stage III colon cancer. Eur J Surg Oncol 2016; 42(11); 1680–1686.9. Le DT, Uram JN, Wang H, Bartlett BR, Kember-ling H, Eyring AD, Skora AD, Luber BS, Azad NS, et al. PD-1 blockade in tumors with mismatch-repair deficiency. N Eng J Med 2015; 372(26): 2509–2520.10. Mukherjee A, McGarrity TJ, Ruggiero F, Kol-tun W, McKenna K, Poritz L, Baker MJ. The revised Bethesda guidelines: extent of utilization in a university hospital medical center with a cancer genetics program. Hered Cancer Clin Pract 2010; 8: 9.11. Diagnostics guidance 27 (DG27). Molecular testing strategies for Lynch syndrome in people with colorectal cancer. NICE 2017 (https: //www.nice.org.uk/guidance/dg27).12. Sepulveda AR, Hamilton SR, Allegra CJ, Grody W, Cushman-Vokoun AM, Funkhouser WK, Kopetz SE, Lieu C, Lindor NM, et al. ASCO, A.

C. A. Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the Ameri-can Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology. J Clin Oncol 2017; 35: 1453–1486.13. Snowsill T, Huxley N, Hoyle M, Jones-Hughes T, Coelho H, Cooper C, Frayling I, Hyde C. A systematic review and economic evaluation of diagnostic strategies for Lynch syndrome. Health Technol Assess 2014; 18(56): 1–406.14. Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P, Clendenning M, Sotamaa K, Prior T, et al. Feasibility of screening for Lynch syndrome among patients with colorectal cancer. J Clin Oncol 2008; 26: 5783–5788.15. O’Brien O, Ryan É, Creavin B, Kelly ME, Mohan HM, Geraghty R, Winter DC, Sheahan K. Correlation of immunohistochemical mismatch repair protein status between colorectal carci-noma endoscopic biopsy and resection specimens. J Clin Pathol 2018; 71(7): 631–636.16. Kumarasinghe AP, de Boer B, Bateman AC, Kumarasinghe MP. DNA mismatch repair enzyme immunohistochemistry in colorectal cancer: a comparison of biopsy and resection material. Pathology 2010; 42(5): 414–420.17. Warrier SK, Trainer AH, Lynch AC, Mitchell C, Hiscock R, Sawyer S, Boussioutas A, Heriot AG. Preoperative diagnosis of Lynch syndrome with DNA mismatch repair immunohistochemistry on a diagnostic biopsy. Dis Colon Rectum 2011; 54(12): 1480–1487.18. Vilkin A, Leibovici-Weissman Y, Halpern M, Morgenstern S, Brazovski E, Gingold-Belfer R, Wasserberg N, Brenner B, Niv Y, et al. Immuno-histochemistry staining for mismatch repair pro-teins: the endoscopic biopsy material provides useful and coherent results. Hum Pathol 2015; 46(11): 1705–1711.19. Klarskov L, Holck S, Bernstein I, Okkels H, Rambech E, Baldetorp B, Nilbert M. Challenges in the identification of MSH6-associated colorectal cancer: rectal location, less typical histology, and a subset with retained mismatch repair function. Am J Surg Pathol 2011; 35(9): 1391–1399.20. Watson N, Grieu F, Morris M, Harvey J, Stewart C, Schofield L, Goldblatt J, Iacopetta B. Heteroge-neous staining for mismatch repair proteins dur-ing population-based prescreening for hereditary nonpolyposis colorectal cancer. J Mol Diagn 2007; 9: 472–478.

the authorsDr Odharnaith O’Brien* MB BCh BAO, Dr Éanna Ryan MB BCh BAO, and Prof. Kieran Sheahan MB BCh BAODepartment of Pathology, St. Vincent’s University Hospital, Dublin, Ireland

*Corresponding authorE-mail: odharnaithobrien@ gmail.com

– September 2018 Pathology18

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An assessment of the effect of haemoglobin variants on detection by faecal immunochemical testsCarroll MR, John C, Mantio D, Djedovic NK, Benton SC. Ann Clin Biochem 2018; doi: 10.1177/0004563218778716 [Epub ahead of print]

BACKGROUND: Faecal immunochemical tests (FIT) for hae-moglobin (Hb) are being used in the investigation of colorectal cancer. These tests use antibodies raised to the globin moiety of human Hb. Where the globin structure is abnormal or reduced, it is possible that antibody binding, and thus Hb-detection may be affected.

METHODS: Lysates prepared from whole blood samples of patients with known variants were diluted in manufacturer-spe-cific buffer to 10, 100 and 500 μg Hb/g feces. These samples were analysed on four FIT analysers and the results compared with samples with no known variant present (normal samples).

RESULTS: The results from this study show that of 20 variants tested, three showed a decrease in detection by all four analys-ers. These were β-thalassemia major and two fetal cord blood samples.

CONCLUSIONS: Of 20 common Hb variants studied, 17 did not affect detection of Hb by the FIT systems tested. Hb variants leading to a reduction in the presence of a globin chain caused a reduction in Hb detection; in such cases, cancers could be missed.

thirty-three-day storage of dithiothreitol-treated red blood cells used to eliminate daratumumab interfer-ence in serological testingLorenzen H, Lone Akhtar N, Nielsen M, Svendsen L, Andersen P. Vox Sang 2018; doi: 10.1111/vox.12699 [Epub ahead of print]

BACKGROUND AND OBJECTIVES: Daratumumab binds CD38 on red blood cells causing interference with indirect anti-globulin tests. Dithiothreitol is used to eliminate interference allowing detection of alloantibodies. Hemolysis is observed during storage of dithiothreitol-treated antibody identification panel cells. The objective of this study was to develop a modified method for dithiothreitol treatment to reduce the hemolysis dur-ing 33 days of storage and still be able to eliminate daratumumab interference.

MATERIALS AND METHODS: Panel cells were treated with various volumes of 0·2 m dithiothreitol supplied by various manu-facturers. Hemolysis Index of dithiothreitol-treated and untreated panel cells was measured and compared on days 1, 15 and 33. Antibody screening tests with dithiothreitol-treated screening cells were performed on samples from 15 daratumumab-treated patients (dose 16 mg/kg) and 34 patients with known alloantibod-ies. Antibody identifications with dithiothreitol-treated panel cells were performed on seven additional known alloantibodies.

RESULTS: Dithiothreitol treatment with a ratio of 30:25 (red blood cells:dithiothreitol) showed the same degree of hemolysis as with untreated panel cells. Daratumumab interference was eliminated in all 15 samples from daratumumab-treated patients. Twenty-six of 34 alloantibodies were detected, and all seven addi-tional alloantibodies were identified using the modified dithi-othreitol treatment. Eight alloantibodies within the Kell system were negative. No decrease in the reaction strength was observed during the 33-day storage period.

– September 2018 SCiENtiFiC litErAtUrE rEViEW:20

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CONCLUSION: The modified dithiothreitol method was able to reduce hemolysis during storage and to detect and identify alloantibodies in the presence of daratumumab.

the assessment of iodine status – populations, individuals and limitationsWainwright P, Cook P. Ann Clin Biochem 2018; doi: 10.1177/0004563218774816 [Epub ahead of print]

Iodine deficiency is a significant global health concern, and the single greatest cause of preventable cognitive impairment. It is also a growing public health concern in the UK particularly among pregnant women. Biomarkers such as urinary iodine concentra-tion have clear utility in epidemiological studies to investigate population-level iodine status, but determination of iodine status in individuals is much more problematic with current assays. This article reviews the available biomarkers of iodine status and their relative utility at the level of both populations and individuals for the investigation of iodine deficiency and iodine excess.

How low can you go? Analytical performance of five automated testosterone immunoassaysLa’ulu SL, Kalp KJ, Straseski JA. Clin Biochem 2018; 58: 64–71

BACKGROUND: Testosterone is commonly measured using immunoassays, yet concerns with the accuracy and quality of testing by these methods exist, particularly for low testosterone concentrations. Study objectives were to evaluate selective per-formance characteristics, including functional sensitivity (FS), of five automated immunoassays for total testosterone.

METHODS: FS, imprecision, assay interference, limit of blank, linearity, and accuracy were assessed using the Abbott ARCHITECT i2000SR, SIEMENS ADVIA Cen-taur and IMMULITE 2000, Beckman Coulter DxI 800, and Roche MODULAR E170. Comparisons to an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were performed using patient samples from men, women, boys, and girls.

RESULTS: FS at 20% coefficient of variation (CV) for the ARCHI-TECT, Centaur, DxI, E170 and IMMULITE assays were 0.14, 1.23, 0.36, 0.77, 3.49 nmol/L, respectively. Total CVs for the 5-day imprecision study were ≤9.0% for all methods. All assays met manufacturer’s claims for hemolysis, icterus, and lipemia interfer-ence and limit of blank. Dilution linearity studies had deviations from the target recoveries ranging from 3.4% (ARCHITECT) to 14.3% (DxI). Using National Institute of Standards and Tech-nology Standard Reference Material 971, recoveries ranged from 79.2–149.2% (DxI, male and female, respectively). When compared to LC-MS/MS, more immunoassays under-recovered in men and women and over-recovered in boys and girls. Slopes ranged from 0.71 (IMMULITE, women) to 1.35 (DxI, boys). The combined aver-age for percent bias was higher in boys (28.0%) than men (11.6%), women (22.8%), and girls (25.7%).

CONCLUSIONS: Challenges with accurately measuring testos-terone appear to remain for some immunoassays, but not all. While most immunoassays remain optimized for concentrations observed in healthy men, some showed acceptable performance when challenged at lower concentrations.

– September 201821PAtHoloGY

www.cli-online.com & search 27682

the relevance of BrCA analysisThe identification of BRCA pathogenic variants (PVs) is the major concern for the genetic counselling in families with a high risk of breast (BC) and ovarian cancer (OC). BRCA1 (breast cancer early onset 1) and BRCA2 (breast cancer early onset 2) are the two major susceptibility genes in BC/OC, conferring a lifetime risk up to 87% for BC and up to 44% for OC. BRCA muta-tions have been found in 4–14% of all OC, with a higher occurrence of about 22% in the high-grade serous OC [1]. The clinical relevance of the identification of BRCA PV carriers concerns many aspects of a patient’s evaluation. The first relevant implication is the assessment of the lifetime cancer risk. Additionally, BRCA testing has a relevant impact on the therapeutic approach and on the treatment outcomes owing to the pos-sibility of selecting patients for biomarker-directed therapy based on the mutational status [2]. BRCA-positive patients with OC, particularly, respond well to platinum-based chemotherapy, especially in the high-grade serous OC subtype, and tend to retain plati-num-sensitivity for longer than those with-out BRCA PVs. Additionally, the treatment

with poly (ADP-ribose) polymerase (PARP) inhibitor (e.g. olaparib) was approved as a target therapy in patients with both germline and somatic BRCA PVs. PARP inhibitor therapy is able to improve progression-free survival in response to a recent platinum-based chemotherapy [3]. To date, licensed PARP inhibitor is part of the standard care and, consequently, BRCA evaluation is con-sidered a routine investigation tool, useful before treatment management. With respect to these benefits, BRCA testing should be offered to all patients with OC on the basis of histological subtype, regardless of age, or family and personal history of malignancy. This issue causes an increase of the demand for BRCA testing with a strong challenge into the diagnostic laboratories commit-ted in fulfilling the need of an efficient and rapid molecular evaluation [4].

the challenge of BrCA testingTo date 1700 PVs in BRCA1 and 1900 PVs in BRCA2 have been reported. Most of them are single nucleotide polymorphisms (SNPs) or small insertion-deletion mutations (indels), with a significant impact on the structure and function of the protein. Also

large genomic rearrangements (LGRs), con-sisting mainly in large deletions or duplica-tions, represent an important part of BRCA molecular lesions. To date, a total of 98 dif-ferent BRCA LGRs have been reported, 81 in BRCA1 and 17 in BRCA2 [5] with a prev-alence that varies considerably. Interestingly, deletion of BRCA1 exon 1a-2 is reported in several populations worldwide and is con-sidered a recurrent BRCA LGRs in BC/OC patients [6]. Owing to the broad complexity in the mutational landscape of BRCA genes, comprehensive screening including the effi-cient assessment of both qualitative (SNPs, indels) and quantitative (LGRs) alterations is mandatory (Fig. 1). Diagnostic laborato-ries are adopting next-generation sequenc-ing (NGS) technology for BRCA testing, which offers the potential of fast, cost-effi-cient and comprehensive sequencing. By choosing NGS technology, many considera-tions should be made, such as the selection of an NGS platform, including the enrich-ment methods, the sequencing chemistries, the analytical procedures and the variant calling for both germline and somatic PVs [2]. NGS is highly recommended as the ref-erence sequencing method for BRCA test-ing because of the size of coding region and the method’s sensitivity in tumour sample evaluation. In fact, Sanger sequencing is not suitable for the analysis of somatic muta-tions, especially in samples where the per-centage of tumour cells is under 50%, and it requires also a large amount of starting DNA [4]. Several methods are commonly used for LGR analysis, including multiplex ligation-dependent probe amplification (MLPA) and multiplex amplicon quantification (MAQ). However, these approaches are expensive

Competitive PCR-high resolution melting analysis: an improved approach to assess BRCA status in hereditary breast and ovarian cancer patientsWidespread use of BRCA molecular testing has been observed in recent decades relating to the approval of PARP inhibitor as a target therapy for breast and ovarian cancer in BRCA-positive patients. This article provides an overview of the crucial issues of the BRCA test, focusing on our innovative cPCR-HRMA technology.

by Elisa De Paolis, Dr Angelo Minucci, Dr Giovanni Luca Scaglione, Maria De Bonis and Prof. Ettore Capoluongo

– September 2018 Molecular diagnostics22

Figure 1. Integrated BRCA molecular workflow.

and time-consuming, and consequently these are not always suit-able for all laboratories. In this case, LGR evaluation of BRCA genes represents a bottleneck in terms of time and costs. In this con-text, the great benefit of the NGS approach is the opportunity to obtain both qualitative and quantitative information from the same sequencing data by using tailored bioinformatics algorithms [7]. Only a positive bioinformatics result needs to be confirmed using an alternative conventional method. In order to optimize our rou-tine diagnostic procedures for BRCA testing, we recently developed a new molecular approach called competitive PCR-high resolution melting analysis (cPCR-HRMA) [8], as an alternative method for LGR identification in BRCA genes. HRMA is a simple and robust closed-tube method commonly used for diagnostics, forensic and research purposes. This method consists of a PCR amplification performed in the presence of saturating binding dyes followed by a melting reaction. Specifically, the incremental increase of the reac-tion temperature causes the denaturation of double-stranded DNA with the concomitant release of intercalated dye and a decrease of fluorescence signal. The specific sequence of the analysed amplicon, primarily relating to the GC content and the length, determines the melting behaviour observed in a fluorescence signal versus tem-perature plot. Additionally, the melting temperature (Tm) may be calculated as the derivative of the melting curve. The shape of the curves and the specific Tm value obtained in the output plots is used for the genotyping. The advantages of this technique include rapid turn-around times and a closed-system environment that decrease the risk of laboratory contamination [9].

cPCr-HrMA for lGr evaluationHRMA technology is typically applied to detect a single substitu-tion, as well as small indels variants [6, 10]. The new cPCR-HRMA represents an optimized HRMA method that allows an efficient evaluation of BRCA1 copy number variation (CNV) by relying on the melting behaviour of target BRCA amplicon compared to a reference amplicon in the same HRMA reaction. In particular, specific albumin sequences were chosen as unchanged CNV refer-ences and analysed by coupling them with specific BRCA1 exons in a duplex PCR assay preceding the melting analyses. The landmarks of this new HRMA rely on the primers and the amplification pro-tocol design. First of all, primer pairs for the simultaneous ampli-fication of target and reference sequences are selected in order to produce paired amplicons with comparable lengths (similar ampli-fication efficiencies) and different melting temperatures (no overlap between amplicons melting peaks). Furthermore, the primer con-centration used for both target and reference amplification was set in order to produce comparable PCR performance between the two amplicon types and to obtain melting profiles more suggestive of CNV. In addition, the PCR thermal cycling was carried on until the exponential phase, in which the amplification performance reflects the CNV status of the target region. These optimized features lead to melting profiles specifically tailored for CNV investigations allowing a rapid detection of samples affected by a change in copy number. Genotype association was assessed by direct interpretation of melt-ing profiles, as shown in Figure 2: samples with similar profiles were clustered into the same genotype group and CNV positive samples showed a typical melting profile with a detectable shape compar-ing to the wild type. In addition to the qualitative evaluation, we provide also a semi-quantitative analysis of melting behaviour with

the calculation of the fluorescence peak height ratio (R) of target the amplicon (BRCA1) to the reference amplicon (albumin), according to the formula:

The mean and the standard deviation of the R values calculated in a consistent number of control CNV samples allowed the identification of the reference range for the R parameter: WT sample (mean±2SD; 2 copies), deletion (≤mean−2SD; n copies) and duplication (≥mean+2SD; 3n copies). The R value calculated in each analysed sample is normalized with the average of the ratios calculated in the WT sample group, obtaining the normal-ized fluorescence peak height ratio (Rn). The latter is compared to the reference range in order to obtain the copy number inter-pretation. Taken together, the qualitative and the semi-quanti-tative evaluations of the cPCR-HRMA assay allow the correct identification of copy number status in BRCA gene, resulting as a rapid and alternative method for the analysis of LGRs. Advan-tages of cPCR-HRMA are the ease and fast handling of samples. Furthermore, this application needs the same reagents and equip-ment for standard HRMA protocols commonly used in many


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laboratories routines. By introducing this efficient alternative method, our first aim was the optimization of the BRCA work-flow, promoting a more rational use of confirmatory testing, such as MLPA and MAQ. Finally, we are confident that a gen-eral implementation of BRCA testing is

now necessary as an emerging challenge. After a complete genetic counselling and a multidisciplinary activity that involves geneticists, oncologist and all other pro-fessionals, the patient should be directed to specialized laboratories. The complex-ity of the potential BRCA mutations,

coupled with their clinical relevance, leads to the mandatory adoption of a compre-hensive molecular workflow for BRCA analysis that must be characterized by a low wait-time and efficient clinical report-ing in order to guarantee a useful medical application.

references1. Antoniou A, Pharoah PD, Narod S, et al. Aver-age risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet 2003; 72(5): 1117–1130.2. Capoluongo E, Ellison G, López-Guerreroc JA, et al. Guidance statement on BRCA1/2 tumor testing in ovarian cancer patients. Semin Oncol 2017; 44(3): 187–197.3. George A, Kaye S, Banerjee S. Delivering wide-spread BRCA testing and PARP inhibition to patients with ovarian cancer. Nat Rev Clin Oncol 2017; 14(5): 284–296.4. Capoluongo E, Scambia G, Nabholtz JM. Main implications related to the switch to BRCA1/2 tumor testing in ovarian cancer patients: a proposal of a consensus. Oncotarget 2018; 9(28): 19463–19468.5. Sluiter MD, van Rensburg EJ. Large genomic rear-rangements of the BRCA1 and BRCA2 genes: review of the literature and report of a novel BRCA1 muta-tion. Breast Cancer Res Treat 2011; 125: 325–349.6. Mazoyer S. Genomic rearrangements in the BRCA1 and BRCA2 genes. Hum Mutat 2005; 25(5): 415–422.7. Scaglione GL, Concolino P, De Bonis M, et al. A whole germline BRCA2 gene deletion: how to learn from CNV in silico analysis. Int J Mol Sci 2018; 19(4): pii: E961.8. Minucci A, De Paolis E, Concolino P, et al. Com-petitive PCR-high resolution melting analysis (C-PCR-HRMA) for large genomic rearrangements (LGRs) detection: a new approach to assess quanti-tative status of BRCA1 gene in a reference labora-tory. Clin Chim Acta 2017; 470: 83–92.9. Erali M, Voelkerding KV, Wittwer CT. High reso-lution melting applications for clinical laboratory medicine. Exp Mol Pathol 2008; 85(1): 50–58.10. De Paolis E, Minucci A, De Bonis M, et al. A rapid screening of a recurrent CYP24A1 pathogenic variant opens the way to molecular testing for idi-opathic infantile hypercalcemia (IIH). Clin Chim Acta. (2018) Mar 21; 482: 8–13.

the authorsElisa De Paolis MSc, Angelo Minucci PhD, Giovanni Luca Scaglione PhD, Maria De Bonis MSc, Ettore Capoluongo* PhDCatholic University of The Sacred Heart, Rome, Italy


– September 2018 Molecular diagnostics24

Figure 2. Examples of germline cPCR-HRMA profiles of BRCA1 exon 2 performed on LighCycler 480 Real-time PCR system (Roche Diagnostics). The panels show: normalized and temperature-shifted plot (a), difference plot (b) and derivative plot (c). Data are reported in duplicate for WT (blue) and deleted (red) sample. In the derivative plot: BRCA1 exon 2 peak at Tm: 77.3 °C and albumin peak at Tm: 82.6 °C.

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Normalized and shifted melting curves

Normalized and temperature-shifted difference plot

Melting peaks

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tracing the footprints of a tumour: genomic “scars” allow cancer profiling

DNA mutations driving cancer development are caused by dif-ferent mecha-nisms, each of

them leaving behind specific patterns, or “scars” in the genome. Using CRISPR-Cas9 technology, researchers at CeMM and the Wellcome Trust Sanger Institute at Cambridge, UK were able to show for the first time in cell culture that specific genetic alterations indeed lead to the predicted pattern of mutational signa-tures observed in human cancers. When a cell develops into a tumour, something has gone terribly wrong: the uncontrolled growth, invasion of nearby tissues and finally metastasis are the result of many consecutive DNA muta-tions. Such an accumulation of demol-ished genetic material often derives from initial environmental exposures, enzymatic activities or defects in DNA replication or DNA repair mechanisms. Each of those initial mutagenic condi-tions creates their own pattern of DNA damage called mutational signature. Deciphering them could theoretically allow us to trace back the initial cause of a tumour, profile its properties and help find a therapeutic strategy.However, reading those mutational sig-natures in tumour samples is a difficult task, as the large amount of mutations that a patient acquires during its lifetime creates a noisy and uncontrolled system – even the best clinical data will, at most, provide only associations. Therefore, the group of Joanna Loizou, Principal Inves-tigator at CeMM in collaboration with researchers from the Wellcome Trust Sanger Institute, developed an experi-mental setup to validate the concept of mutational signatures in cell culture.The findings of this study not only confirm an analytical principle that describes mutational processes and can-cer development, they show that muta-tional signatures are a direct mechanis-tic read-out of specific dysfunctions of a cell. Thus, even if the underlying gene defect is unknown, mutational signa-tures could be used as biomarkers for the molecular characterization of tumu-ors – a new diagnostic tool to improve the precise and personalized treatment of cancer.CeMMcemm.at/news/

New biomarker identified for early diagnosis of lung cancer

High levels of c ytoskeleton-associated pro-tein 4 (CKAP4) have been

identified in the blood of patients with lung cancer. In a novel study investiga-tors found that CKAP4 levels were sig-nificantly higher in patients with lung cancer than in healthy individuals. They further determined that CKAP4 levels are already elevated in the blood of patients with stage I disease, making it a poten-tial non-invasive diagnostic marker that could change current practices in the diagnosis and treatment of some types of lung cancer, including non-small-cell lung cancer and squamous cell carcinoma, and improve patient outcomes.Lung cancer is the leading cause of can-cer deaths in both men and women in the United States and worldwide. The dis-ease is associated with a poor prognosis because most lung cancers are only diag-nosed at an advanced stage.“The identification of patients at an early stage of cancer when it can be treated sur-gically is extremely important to improve prognosis,” explained Yuichi Sato, PhD, Department of Molecular Diagnostics, Kitasato University School of Allied Health Sciences, Sagamihara, Kanagawa, Japan, who led the study. “We need better biomarkers for early diagnosis.”Current biomarkers for lung cancer include carcinoma embryonic antigen (CEA), sialyl Lewis X antigen (SLX), squa-mous cell carcinoma (SCC) antigen, and cytokeratin fragment (CYFRA) 21-1, but these are not sensitive enough to detect tumours early, according to co-investiga-tor Ryo Nagashio, PhD, from the Kitasato University School of Allied Health Sci-ences. “The results of our study provide evidence that the CKAP4 protein may be a novel early sero-diagnostic marker for lung cancer.”Researchers performed reverse-phase protein array analysis using a monoclonal antibody designated as KU-Lu-1 antibody on the blood of 271 lung cancer patients and 100 healthy individuals. KU-Lu-1 reacted only with tumour cells and tumour stromal fibroblasts in lung cancer tissues and not with normal lung tissues. Using immunoprecipitation and mass spectrometry, they confirmed that the KU-Lu-1 antibody recognized CKAP4 in lung cancer cells and tissues, and its secre-tion into the culture supernatant was also confirmed. In addition, a validation set

consisting of samples from 100 patients with lung cancer and 38 healthy controls was also studied.CKAP4 was recently identified as a recep-tor of Dickkopf1 (DKK1). Expressions of DKK1 and CKAP4 were frequently observed in tumour lesions of human pancreatic and lung cancers, and the simultaneous expression of both proteins in tumour tissues was inversely correlated with prognosis and relapse-free survival.Across disease stages I-IV, the sensitivities of serum CEA, CYFRA, and SCCA are reported with 30 to 52, 17 to 82, and 24 to 39 percent, respectively. In this study, the sensitivity of serum CKAP4 was 81 per-cent in the training set and 69 percent in the validation set. These rates are higher than those of the current sero-diagnostic markers. Furthermore, the sensitivity of serum CKAP4 was also high even in stage I non-small-cell lung cancer and squa-mous cell carcinoma.“The use of CKAP4 as a biomarker could change current practices regarding the treatment of lung cancer patients, and the diagnostic accuracies may be markedly improved by the combination of CKAP4 and conventional markers,” concluded Dr. Sato.EurekAlerthttps://tinyurl.com/y8tfjm86

insight into blood signatures of inflammation

A new study from BUSM and BUSPH identifies a pattern of i n f l a m m a -tion associ-

ated with cardio-metabolic risks among participants in the Black Women’s Health Study, as well as two independent groups of vulnerable women. These findings could help underserved patients benefit from precision medicine and personal-ized profiles of disease risk.According to the researchers, body mass index alone is an imperfect measure of obesity-associated disease risks, such as for Type 2 diabetes, because there are some individuals with chronic obesity who are apparently protected from cardio-meta-bolic complications and lean individuals with high cardiovascular and diabetes risks. Abnormal, unresolved inflamma-tion in blood and adipose (fat) tissue, rather than obesity per se, is thought to be important for development of dis-ease. Certain biomarkers show promise

– September 201825NEWS iN BriEF

in predicting obesity-associated diabetes risk; however, the clinical utility of single biomarkers is limited for complex disease phenotypes such as these.The research team took a data-driven, systems biology approach to discover six cytokine signatures associated with Type 2 diabetes risk in a vulnerable population: African American women with obesity and varying degrees of metabolic health. These six distinct signatures are patterns of sixteen cytokines/chemokines that pro-mote or reduce inflammation.Analyses of plasma samples from partici-pants in the Black Women’s Health Study, formed the basis for the discovery dataset, which was then validated in two separate groups, African American women vol-unteers with obesity who had donated plasma to the Komen Tissue Bank, and African American women with obe-sity who were breast reduction surgical patients at a safety net hospital in Greater Boston. The patterns or signatures in the validation cohorts closely resembled the distributions in the discovery cohort.“These findings are highly relevant to an understudied and underserved popula-tion that experiences elevated risks for co-morbidities of obesity. The overall impact of this report is high because of the potential utility of the new signatures just discovered and validated, which could assist clinical decision making with more personalized information,” explained cor-responding author Gerald V. Denis, PhD, Associate Professor of Pharmacology and Medicine at BUSM.Boston University School of Medicinehttps://tinyurl.com/y7hkgpou

Gene study spots clues to heart risk for statin patients

A Vanderbilt-led research team has dis-covered genetic variations that increase the

risk of heart attack even when patients are receiving a statin drug like Lipitor or Crestor to lower their blood cholesterol.The finding helps explain why some patients experience a heart attack or the need for coronary revascularization to open blocked heart arteries while taking statins. It suggests that drugs targeting the genetic variations could lower the heart risk in these patients.The study demonstrates the power of genome-wide association studies and lon-gitudinal electronic health records (EHRs)

to find links between genetic variation and disease, said the paper’s first author, Wei-Qi Wei, MD, PhD, assistant professor of Biomedical Informatics in the Vander-bilt University School of Medicine.Some of the patients were followed for heart disease for up to a decade after start-ing on their statin drug. The study found that the effect of the genetic variations or variants was independent of how much their cholesterol improved while taking statins.“People with these genetic variants were at a higher risk for heart disease, even con-sidering those who have ideal cholesterol levels on their statin,” said Joshua Denny, MD, MS, Vice President of Personalized Medicine at Vanderbilt University Medi-cal Center (VUMC) and the paper’s cor-responding author.The researchers searched four sites in the Electronic Medical Records and Genom-ics (eMERGE) network, a nationwide consortium of experts, biorepositories and electronic medical record systems supported by the National Institutes of Health (NIH), including BioVU, VUMC’s DNA databank.They found 3,099 people who had expe-rienced a heart attack or the need for revascularization while on statins, and compared them to 7,681 “control” patients on statins who did not experience heart events.From this comparison, the research-ers were able to identify seven genetic variations, called single nucleotide poly-morphisms or SNPs, in the LPA locus of genes that were associated with these heart events in patients receiving statin treatment.The LPA gene encodes apolipoprotein (a), a fatty protein that binds to low-density lipoprotein (LDL), the form of blood cho-lesterol that is the target of statin drugs. High levels of bound LDL, called Lp(a) for short, is well known to be an independent risk factor for heart disease.One of the SNPs was highly associated with an increased risk of heart events. When the researchers examined the full EHRs of 11,566 individuals who carried the SNP for more than 1,000 physical con-ditions, they found significantly higher rates of coronary heart disease and heart attack but not of other diseases.The approach, called a phenome-wide association study, was pioneered by Denny and his colleagues at Vanderbilt.“The study highlights the need to consider targeting Lp(a) levels as an important independent factor to reduce cardiovas-cular risk in patients on statin therapy,”

Wei concluded.Efforts to reduce Lp(a) levels using exist-ing or new drugs could reduce heart events in the proportion of patients on statins who carry LPA variations, he added, although clinical trials would be needed to detect potential side effects and confirm the safety of any such treatment.Vanderbilt University Medical Centrehttps://tinyurl.com/y8rrjhbu

Cause of resistance to breakthrough breast and ovarian cancer drug

Scientists have identified a mutation that gives cancer cells resistance to the break-

through cancer treatment olaparib and other PARP poly ADP Ribose polymer-ase) inhibitors.The study findings could help predict which patients will develop resistance to PARP inhibitors and allow doctors to alter treatment at the earliest possible opportunity.A team at The Institute of Cancer Research, London, used gene editing to identify a specific mutation in the PARP1 protein that prevents PARP inhibitors from working.Testing for this mutation could add another level of personalization to an already targeted treatment – helping guide decisions about whether to use PARP inhibitors in the first place, and when to switch to other drugs, such as platinum-based therapies.PARP1 is crucial for the repair of dam-aged DNA and is an important target for olaparib and other PARP inhibitors. These drugs are especially effective in patients who already have weaknesses in DNA repair because of inherited errors in the BRCA (BReast CAncer) genes – a discov-ery that was made at the ICR.The scientists used new ‘CRIPSR-Cas9’ gene-editing technology to generate mutations in small, targeted sections of the PARP1 gene, and tagged the mutant protein with a fluorescent protein so their effects could be tracked.This approach allowed the researchers to observe the effect of specific mutations on PARP1 and on the sensitivity of cancer cells to PARP inhibitors, such as olaparib and talazoparib.Olaparib is available on the NHS for women with ovarian cancer who have inherited BRCA mutations, and is

NEWS iN BriEF – September 2018 26

currently being evaluated for breast can-cer. It was the first ever cancer drug to be approved that is targeted against an inher-ited genetic fault.The study identified specific PARP1 muta-tions which disrupt the ability of the pro-tein to bind to DNA, which means PARP inhibitors can no longer trap the protein molecules at the site of DNA damage.The researchers found that, contrary to their original predictions, cancer cells with certain mutations in the BRCA1 gene could survive this loss of PARP1’s DNA repair function – making them resistant to PARP inhibitors.It is thought that in these cases the BRCA1 gene retains some function, provid-ing some residual ability to repair DNA despite the loss of PARP1.The scientists emphasized that further research needs to be carried out to exam-ine more PARP1 mutations in patients as only one example in humans was found in this study.The team is looking to apply this same gene-editing approach to study how resist-ance arises to other drugs, and whether it is possible to predict how quickly this resistance will progress.institute of Cancer researchhttps://tinyurl.com/y7kc5y8u

rNA molecules predict adverse heart growth and function that can lead to atrial fibrillation and death

Researchers have iden-tified that enlargement of the left atrium of the heart is linked to abnor-mal activity of mole-cules that are associated

with adverse changes in the heart’s size, shape, structure, and function — condi-tions that can lead to atrial fibrillation and death.The new study, conducted by research-ers at the Intermountain Medical Center Heart Institute in Salt Lake City, is the first time this association has been con-nected to the human heart in a clinical setting.In conducting the study, researchers noted that under stress conditions, car-diac fibroblasts, which play a role in normal cardiac function and changes in the heart, release greater quantities of exosomes, which are small pieces of cells circulating in the blood that contain cel-lular components and convey informa-tion to distant tissues.The Intermountain Medical Center

Heart Institute researchers found that in patients with atrial fibrillation, exosomes and plasma are enriched with Micro-RNA (miR)-21-3p — which is associated to abnormal enlargement of the heart muscle.Scientists are interested in exosomes because initially they were thought to be a waste by-product as cells shed. But now researchers are learning that not only are exosomes communicators between cells, but they influence the spread of proteins, lipids, mRNA, miRNA, and DNA and are contributing factors in the development of several diseases.“Our study gives us a better understand-ing of the process of how atrial fibrillation begins and advances,” says Victoria Jacobs, NP, PhD, a member of the Intermountain Medical Center Heart Institute research team. “Once atrial fibrillation happens, we have some ‘band-aids’ to fix its symptoms, but we want to learn how to keep atrial fibrillation and atrial enlargement from happening in the first place.”While an enlarged atria may have sev-eral causes, recent studies have linked enlargement to an increased risk of atrial fibrillation. Researchers are interested in learning more about atrial fibrillation because it, along with coronary artery disease, is the number one killer of people in America. Atrial fibrillation affects more than 3.4 million Americans, primarily older adults.An enlarged left atrium has been linked to atrial fibrillation, as it can prevent the heart from pumping blood properly and may increase risk of an irregular heartbeat.Researchers at the Intermountain Medi-cal Center Heart Institute examined bio-markers, which are biological molecules used to see how well the body responds to a treatment for a disease or condition, that could specifically predict the occur-rence and severity of adverse growth in the left atrium of the heart. A basic study previously done in Germany that focused on cell cultures and small lab rodents sug-gested that miR-21-3p played a role in that growth. But no one has connected it to the human heart in a clinical setting until now.“We know patients with atrial fibrilla-tion develop thickening of heart tissue, or fibrosis,” said Dr. Jacobs. “As atrial fibrillation progresses, we know there’s more fibrosis in the left atrium. But this is the first time we’ve shown miR-21-3p is associated with left atrial fibrillation in patients.”the intermountain Medical Centerhttps://tinyurl.com/y7u6uymb

depleted metabolic enzymes promote tumour growth in kidney cancer

Kidney cancer, one of the ten most prevalent malignancies in the world, has increased in incidence over the last decade, likely due to

rising obesity rates. The most common subtype of this cancer is “clear cell” renal cell carcinoma (ccRCC), which exhibits multiple metabolic abnormalities, such as highly elevated stored sugar and fat deposition.By integrating data on the function of essential metabolic enzymes with genetic, protein, and metabolic abnormalities asso-ciated with ccRCC, researchers at the Perel-man School of Medicine at the University of Pennsylvania determined that enzymes important in multiple pathways are univer-sally depleted in ccRCC tumours. “Kidney cancer develops from an extremely complex set of cellular malfunctions,” said senior author Celeste Simon, PhD, the sci-entific director of the Abramson Family Cancer Research Institute and a professor of Cell and Developmental Biology. “That’s why we approached studying its cause from many perspectives.”Using human tissue provided by the National Cancer Institute’s Cooperative Human Tissue Network and Penn Medi-cine physicians Naomi Haas, MD, an asso-ciate professor of Hematology/Oncology, and Priti Lal, MD, an associate professor of Pathology and Laboratory Medicine, the team found that the expression of certain enzymes is strongly repressed in ccRCC tumours. For example, reduced activity of one enzyme, arginase, promotes ccRCC tumour growth through at least two dis-tinct biochemical pathways. One is by con-serving a critical molecular cofactor and the second is by avoiding toxic accumula-tion of organic compounds. The enzymes whose activities are depressed are involved in the breakdown of urea, a by-product of protein being used in the human body. In addition, loss of these enzymes results in decreased ability of the immune system to eradicate these tumours.“Pharmacological approaches to restore the expression of urea cycle enzymes would greatly expand treatment options for ccRCC patients, whose current thera-pies only benefit a small subset,” Simon said. Penn Medicinehttps://tinyurl.com/yc223sgc

– September 201827NEWS iN BriEF

A 28-year-old African community youth worker, Evah Namakula, has won the first, global CARES HIV/AIDS award, designed to recognize ordinary people who have shown ‘care, dedication and commitment’ in their communities as part of the fight against the dis-ease. Ms Namakula was also part of the 2018 award launch at the International Aids Society meeting in Amsterdam (July 23 – 27).In its first year, the CARES award focused on the dedication of ordinary people in Africa, one of the areas in the world most affected by HIV/AIDS. The award has two categories of winners – an individual, Ms Namakula, and an organization, the Hillcrest AIDS Centre Trust (HACT). This is a South African charity that cares for some of the poorest and most disad-vantaged people in Africa. Each year the winning organization will receive a grant of 5500 USD provided by Beckman Coulter Life Sciences through the Beckman Coulter Foundation. The grant is made in the name of the individual winner, but their work cannot be linked. An independent judging panel described as ‘remarkable’ Ms Namakula’s achievements in her local Ugandan community to dispel the stigma of HIV/AIDs. She is also global youth ambassador for Reach Out Integrity (ROI) Africa, where she helps to promote health and sexual responsibility to young people. Evah has recently founded her own charity, IGNITE, to carry her work forward.Ms Namakula is part of the Young African Leaders Initiative (YALI) set up by President Barak Obama to empower leadership skills in African youth. As a YALI volunteer, she has been working as a leadership mentor in local communities and schools, helping to develop public speaking skills.Inspired as a child by the determination of her mother and siblings, Evah said: “l had already become a campaigner, but it was while l was working in my local hospital laboratory that I realized how I could use my medical knowl-edge to reduce the myth young people in my community had about HIV/AIDs.”“Evah is an inspirational young woman and will be a hard act to follow,” said Samuel Boova, Beckman Coulter’s Director Alliance Devel-opment, High Burden HIV Markets. “She is exactly the kind of youth leader that President Obama wanted to encourage to develop the Africa of the future and we are honoured not only to have her as our first winner, but to have

her support in launching the global initiative.“The award gives a platform to the work and stories of those we see as the unsung heroes of individual communities. These are people who have shown individual dedication, commit-ment and courage or who have made a differ-ence in the battle against HIV/AIDS. “However, it is not just the final winner we want to publically recognize. We hope the award will encourage communities to learn about and honour the work of every nominee, so that more people will come forward to help and support those living with HIV/AIDS.”

Potential candidates for the CARES award can be nurses, healthcare workers, national coor-dinators, lab scientists and even clinicians. It could include lay people who are active in com-munity outreach work or a social worker pro-viding AIDS counselling.CARES supports the UNAIDS 90-90-90 target to ensure that by the year 2020, 90% of people living with HIV will know their status, 90% of those with diagnosed HIV infection will receive sustained antiretroviral therapy, and 90% of all people receiving antiretroviral therapy will have viral suppression.It focuses on encouraging innovative solutions for the monitoring of HIV and AIDS treatment. It was inspired by the work of Professor Debbie Glencross, a leading South African laboratory

pathologist, who found an inexpensive way to measure a patient’s CD4 count, a special type of white blood cell that can indicate how compro-mised a person’s immune system might be. Prof Glencross is Director and Principle Pathologist in the Flow Cytometry unit of the Department of Hematology at the Charlotte Maxeke Johan-nesburg Academic Hospital. Monitoring a patient’s immune system by counting the CD4 cells has to be carried out by laser technology in a special blood ana-lyser, the flow cytometer. However, in many parts of rural Africa, the equipment and infra-structure simply hasn’t been available to test patients, get their blood samples to a labora-tory, and then report the results. As Prof Glen-cross explained: “We are working to empower smaller community laboratories so that they can extend the availability of the test to meet demand while still meeting the requirements of the National Health Laboratory Service. This will enable best clinical and laboratory practice while reducing the time it takes to deliver the result.”When counting CD4 cells, large global hospi-tal labs first differentiate between the types of white blood cells, count them and then work out the number of CD4 cells in each mil-lionth of a liter of blood. While accurate, this method can be laborious. In contrast, rather than going through the time-consuming and costly process of isolating individual anti-bodies, Glencross’s ingenious approach uses a mathematical equation. She realized that using the white cell count as a stable reference point would eliminate the need for additional quality control steps, while still maintaining standards.

http://info.beckmancoulter.com/CArES-Award-intro

AWArd – September 2018 28

Winner of first global CArES HiV/AidS award announced at iAS 2018

The award recognizes the ‘care, dedication and commitment’ of ordinary people in the battle against HIV/AIDS.

In late July, Svar Life Science signed a strategic agreement to transfer the port-folio of radioimmunoassays (RIA) and the Chromogranin A Neolisa™ (ELISA) product to DIAsource ImmunoAssays, a BioVendor group company. Svar Life Science AB (formerly Euro Diagnostica AB), a Swedish life science company that has been working across the clinical diagnostic value chain for over 30 years, and DIAsource Immuno-Assays, a leading diagnostics company delivering manual RIA and ELISA kits and open automation solutions to inter-national markets, today announced a strategic agreement, under which Svar Life Science will transfer its portfolio of RIA products and the Neolisa™ CgA ELISA product to DIAsource, securing the continued production and sales of these products. Svar Life Science will continue to invent, develop and apply the best analytical technologies, with a focus on helping deliver the answers needed in drug dis-covery and clinical diagnostics, and the impact this has on human lives. This transaction strengthens DIAsource and BioVendor’s position as one of the

top RIA and larger ELISA manufactur-ers, committed to servicing customers worldwide that use manual assays and open automation to complement their portfolio on closed automated systems. The portfolio to be transferred includes the complete line of radioimmunoassays for the quantification of a number of peptide hormones involved in critical physiological processes. These endo-crinological parameters are mainly used as tumour markers and for diabetology and salt balance analysis. The portfolio transfer also includes the ELISA Chro-mogranin A Neolisa™ product which complements the RIA Chromogranin A product. In order to support a smooth transi-tion with minimal disruption for cus-tomers, the companies have agreed to a transition period. Starting 1st of Sep-tember 2018 DIAsource ImmunoAs-says assumes sales of the RIA product portfolio. During the remainder of 2018 the RIA production will be transferred. Sales of the Neolisa™ CgA ELISA prod-uct will be assumed by DIAsource as of January 2019, with production transfer to follow during 2019.

Ron Long, CEO, Svar Life Science, said: “The agreement with DIAsource ImmunoAssays forms part of our strat-egy of focusing our product portfolio and core technologies to help deliver the answers needed in drug discovery and clinical diagnostics today. It also enables us to strengthen our support for life science customers providing high quality solutions, the best analyti-cal technologies for drug development and clinical research. DIAsource ImmunoAssays are com-mitted to being a complete diagnostic provider and we’re confident that this agreement enables them to increase their support for customers in the field of radioimmunoassays.” Jef Vangenechten, CEO of DIAsource ImmunoAssays, said: “This acquisition is another step in our strategy to position DIAsource as a consolidator of manual specialty assays, after previous acquisi-tions of the Intertech RIA product line in 2012, Viro-Immun ELISA and IFA product lines in 2017, and more recently the RIA product line from ZenTech.”

www.diasource-diagnostics.com

– September 201829iNdUStrY NEWS

18-19 OCTOBRE 2018LA BIOLOGIE AU SERVICE DU PROGRÈS MÉDICAL

ESPACE GRANDE ARCHE PARIS - LA DÉFENSE WWW.JIB-INNOVATION.COM

61e ÉDITION

JOURNÉES DE L’INNOVATION EN BIOLOGIE

SDB-JIB 2018-Flyer.indd 1 08/03/2018 18:04



61e ÉDITION





61e ÉDITION





61e ÉDITION





61e ÉDITION



61ST EDITION

DAYS OF INNOVATION IN BIOLOGY 18-19 OCTOBER 2018BIOLOGY AT THE SERVICE OF MEDICAL PROGRESS

diasource immunoAssays and Svar life Science (formerly Euro diagnostica) sign milestone agreement

FdA grants Breakthrough device designation for roche’s Elecsys cer-ebrospinal fluid (CSF) assays to sup-port diagnosis of Alzheimer’s disease

Roche announced in July that the U.S. Food and Drug Administration (FDA) has granted Breakthrough Device Designation to Elecsys® ß-Amyloid (1-42) CSF and Elecsys® Phos-pho-Tau (181P) CSF. These in vitro diagnos-tic immunoassays are for the measurement of the ß-Amyloid (1-42) and Phospho-Tau concentrations in cerebrospinal fluid (CSF) in adult patients with cognitive impairment who are being evaluated for Alzheimer’s dis-ease (AD) or other causes of dementia. Currently, the diagnosis of AD is largely based on clinical symptoms, including cog-nitive testing, with a significant number of patients diagnosed when their disease has already advanced. A diagnosis of AD based on cognitive measures alone is only correct in 70 – 80 percent of cases. Measuring bio-markers with CSF immunoassays, associated with AD pathology, increases certainty of a diagnosis of AD and can help to evaluate the progression of the disease. The Breakthrough Device Designations are for indication of use with Elecsys β-Amyloid (1-42) CSF and Elec-sys Phospho-Tau (181P) CSF in concordance with amyloid PET visual read result and risk of cognitive or functional decline. The Break-through Devices Program is a voluntary pro-gram for certain medical devices that provide for more effective treatment or diagnosis of a life-threatening or irreversibly debilitat-ing disease or condition. This program is designed to expedite the development and review of these medical devices.

“We are excited about FDA’s recognition of the potential clinical benefit the Elecsys CSF assays can bring to clinicians, laboratories and their patients in diagnosing AD at an early stage,” said Roland Diggelmann, CEO of Roche Diagnostics. “Roche was one of the first companies to use biomarkers in clinical trials and we will continue to explore high-performing diagnostic and disease-moni-toring solutions.”www.roche.com

UCi, Beckman Coulter diagnostics announce strategic collaboration Beckman Coulter Diagnostics has embarked on a strategic collaboration with the Univer-sity of California, Irvine – one of the first targeted academic partnerships the company envisions around the globe. Beckman Coulter Diagnostics will access UCI research in innovative diagnostic plat-forms, life sciences applications, devices, and data analytics to further its mission of advancing healthcare. UCI faculty, stu-dents and entrepreneurs will benefit from Beckman Coulter Diagnostics’ expertise in accelerating the commercialization of technology and other world-class UCI breakthroughs. “We are honoured that Beckman Coulter Diagnostics has selected UCI as a strategic innovation partner,” said Richard Sudek, Ph.D., chief innovation officer and executive director at UCI Applied Innovation. “This is a new type of industry collaboration which aims to significantly change how industry and universities partner together. We look forward to teaming up with Beckman Coul-ter to increase the speed and quality of how UCI discoveries make it to market.”“We are excited to tap into the broad exper-tise of UCI researchers as we focus on identifying innovative solutions to clini-cal unmet needs“ said Fiona Adair, Ph.D., vice president of strategy and innovation at

Beckman Coulter Diagnostics. “We believe this type of academic-industry partner-ship can lead to development of innova-tive diagnostic technologies to improve healthcare. UCI Applied Innovation is a place where valuable new ideas are incu-bated. In turn, we can provide promising students, researchers, and entrepreneurs industry-specific feedback and mentor-ship opportunities. We will have a Beckman Coulter office at the Cove @ UCI Applied Innovation for seamless collaboration with academic units as well as to integrate into the innovation ecosystem.” This level of collaboration is an industry model of synthesizing research, commercial expertise and clinical needs to produce ben-eficial results. As a first step, Beckman Coul-ter will fund Proof of Product grants to help UCI innovations bridge the gap between the lab and early commercialization. Through these grants, Beckman Coulter will deter-mine a specific focus area for university entrepreneurial teams. Additionally, Beckman Coulter will also seek eligible UCI graduate students to enter its competitive talent onboarding program, in which they’ll get the opportunity to work across multiple divisions of the company. “The partnership with UCI represents a land-mark in Beckman Coulter’s strategic initiatives to drive translational innovation and extend the company’s leadership in clinical diagnos-tics.” said John Blackwood, senior vice presi-dent and general manager of products and ser-vices at Beckman Coulter. “Beckman Coulter is engaging with academic partners that excel in applying the latest technology to develop supe-rior solutions for better patient outcomes. UCI maintains an ecosystem of innovation that facilitates academic-industry partnerships and we are excited about the opportunity to lever-age UCI’s research expertise for the benefit of patients around the world.”

www.uci.edu www.beckman.com

iNdUStrY NEWS – September 2018 30

CAlENdAr oF EVENtS

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.clinlabint.com/events/

Sept 9-12, 201830th European Congress of Pathology Bilbao, Spainwww.esp-congress.org

Sept 9-13, 2018MSACL 2018 EUSalzburg, Austriawww.msacl.org

Oct 2-3, 2018Genomic Medicine 2018 Nordic Odense, Denmarkhttps://biotexcel.com/event/genomic-medicine-2018-nordic/

Oct 2-4, 2018Medlab EuropeBarcelona, Spainwww.medlabeurope.com/en/home.html

Oct 9, 2018Genomic Medicine 2018 Nijmegen The Netherlands https://biotexcel.com/event/genomic-medicine-2018-nijmegen/

Oct 18-19, 2018JIB 2018Paris, Francewww.jib-innovation.com

Oct 29-Nov 1, 2018CMEF AutumnShanghai, Chinawww.cmef.com.cn

Nov 12-15, 2018MedicaDüsseldorf, Germanywww.medica.de

Gonotec: a continued success story since 1979The following products by Gonotec, the “osmometer people”, are especially designed for use in clinical laboratories. They feature ease of handling, quick operational readiness and provide for rapid measurement. In addition, accurate data can be obtained from small samples.

“We are the osmometer people.”

osmomat 3000 Freezing point osmometer The gonotec® Single-Sample Freezing Point osmometer is especially designed for routine measurements in the medical field and is also very suitable for measurements in research. The osmomat 3000 determines the total osmolality of aqueous solutions. The instrument requires very small sample volumes and can thus be applied for extreme measuring tasks. Its rapidity allows serial measurements in a very short time.

osmomat 3000 basic Freezing point osmometer Specially designed for the operator who is looking for simple handling and precise measurements rather than automatic documentation, the Osmomat 3000basic is the basic version of the oSMoMAT 3000 for which measurements have to be recorded by the operator, as it is not equipped with a printer nor with data output.

CM20 Chloridemeter The Chloridemeter CM20 provides quick and exact determination of the chloride ion concentration in liquid micro samples with a minimum volume of 10 µl. This cutting-edge digital instrument has an easy, intuitive user interface with a modern touch screen and multi-language prompts which lead the operator through the entire measurement process. The instrument is factory-calibrated and equipped with an integrated printer. Results are sent in document-ready format to a PC that can be connected via uSB or RS232 for data transfer.

osmomat auto Freezing point osmometer The design of the osmomat auto is based on ten years of experience with the measuring principle of the Osmomat 030, the first cryoscopic osmometer that was launched by gonotec for clinical application. The company was.able to automate the draining of the measuring system so that it is possible to reliably obtain a high sample throughput. The simple menu-driven operation requires a minimum of user interaction and allows automated operation of the osmomat.

osmomat 050 Colloid osmometer The osmomat 050 has been especially developed for use in intensive care units.It has been designed to be used on a 24 h/day 7 days/week basis and the system is always ready to collect data. The system performs all cleaning and control operations during the stand-by period so that the user can perform a measurement at any time. After performing a measurement, the Osmomat 050 is automatically cleaned so the next measurement can be performed as required.

– September 201831ProdUCt ProFilE

Expanded range of safety prod-ucts for blood collection

The VACU-ETTE SAFETY Winged Set is a new addition to Greiner Bio-One’s range of

safety products and it is equipped with a safety mechanism. For greater flexibil-ity, there are three different options for activating the safety mechanism imme-diately after blood collection. The clearly audible click confirms that the needle is safely and irreversibly locked in the safety shield. To give the user more choice, the safety blood collection set is available both with and without a pre-assembled blood culture holder. The transparent product design provides optimal blood flow vis-ibility. Around a third of all occupational accidents in healthcare involve needlestick injuries. Nurses are at particular risk of being infected by blood-borne diseases such as hepatitis B, hepatitis C and HIV. It is therefore advisable to use safety prod-ucts for taking blood samples in order to lower the risk of needlestick injuries. The first VACUETTE Safety Products were brought onto the market in 2003. The products have been developed and enhanced on an ongoing basis since then. In addition to the established VACUETTE Safety Blood Collection Sets, VACUETTE Safety Needles are also available, enabling users to select from a wide range to meet their specific needs.

GREINER BIO-ONE www.clinlabint.com & search 27744

Continuous workforce education system enables addition of own learning content

Advances in t e c h n o l o g y, and the con-stant competi-tive necessity to upgrade w o r k f o r c e skills mean

that employees need to be trained con-tinuously. For that reason, Siemens Health-ineers expanded its premium subscription for workforce education management, PEPconnections, with a Virtual Library, which provides customers with the ability to add their own learning content to their group. With PEPconnections institutions can easily manage their workforce educa-tion, design learning plans, manage, assign and administer online training and profes-sional development activities for employee groups in hospitals and laboratories. Importantly, it does not matter whether the learning activities were developed by Sie-mens Healthineers, the institution itself, or any other source. Customers now can easily upload their own content as PDFs, videos, documents or links to the Virtual Library. Following upload, items in the Virtual Library are available for general usage or can be assigned to group members through a new or existing learning plan. PEPcon-nections allows subscribers to manage staff competency and skills – supporting them in transforming care delivery for enhanced patient outcomes. Institutions can ben-efit from Siemens Healthineers trainings but also have the ability to individualize

their learning experience and upload their own content. PEPconnections provides a connection to knowledge in digitalizing healthcare that is designed to increase staff competency, efficiency, and productivity. The basis is PEPconnect – the industry’s first personalized education and perfor-mance experience for healthcare profes-sionals worldwide. It provides medical professionals with customized training and professional development opportu-nities, with learning activities in topics on laboratory diagnostics, medical imag-ing and minimally-invasive therapy. The activities are designed at Siemens Health-ineers by experts in key areas, such as application design, instructional technol-ogy and information security. Learners can also construct a learning experience personalized to their needs, and share their learning experience with others via a range of social media channels or groups they are members of within PEPconnect. Almost 300,000 professionals are already using PEPconnect for their training and professional development needs in up to eight different languages.

SIEMENS HEALTHINEERS


Combined detection of anti-Zika virus igAM in acute infections

A new ELISA provides paral-lel detection of anti-Zika virus antibodies of classes IgA and IgM against

the highly specific viral non-structural pro-tein (NS-1). Anti-Zika virus NS-1 antibod-ies of class IgA are a novel alternative indi-cator of acute infection, and their detection is particularly useful in cases where IgM is not detectable. This can occur, for example, in patients who have had previous contact with a flavivirus through infection or vac-cination (e.g. dengue, yellow fever, tick-borne encephalitis, Japanese encephalitis or West Nile viruses). When these individuals become infected with another virus of the genus Flavivirus, in this case Zika virus, the specific IgM response may be absent (sec-ondary immune response). In these cases, determination of specific IgA can facilitate diagnosis of the acute Zika virus infection. Studies have shown that although not all patients with an acute Zika virus infec-tion exhibit specific IgM and specific IgA antibodies, all tested samples were reac-tive for at least one of the two parameters.

ProdUCt NEWS – September 2018 32

J e n n o p -tik Opti-cal Sys-tems have developed an inno-

vative technology to bring artificial intelligence (AI) to conventional microscopy through overlay of digi-tal content directly into a commer-cial-off-the-shelf microscope’s field of view, in collaboration with Google researchers. Google has demonstrated modeling for tumour detection in histopathology slides for both pros-tate cancer and lymph nodes from breast cancer using the platform and presented their results at the Annual

meeting for the American Associa-tion for Cancer Research (AACR) in April of 2018. Jenoptik designed and integrated the opto-mechanical tech-nology at its Jupiter, Florida location, which was made possible by leverag-ing their Systems Engineering expe-rience, optical R&D capabilities, and their portfolio of IP across a breadth of innovative applications and indus-tries. The augmented microscope solution benefitted from their optical design expertise and a digital imaging platform Jenoptik developed over the past decade.

JENOpTIK www.clinlabint.com & search 27747

Augmented microscopy platform for pathology applications

Therefore, combined testing for IgM and IgA is beneficial for reliable diagnosis of acute infections.In the study, serum samples of 31 patients with confirmed acute Zika virus infections from the Dominican Republic, where Zika and dengue viruses are endemic, and 40 patients from Vietnam with acute dengue virus infection were tested with the new ELISA. The Anti-Zika Virus IgAM yielded a sensitivity of 100% (31/31) and a speci-ficity of 95% (38/40). In comparison, the Anti-Zika Virus IgM demonstrated a sen-sitivity of 29% (9/31) and a specificity of 100% (40/40). Thus, combined detection of both antibody classes instead of just IgM leads to a threefold increase in the sensitiv-ity for acute Zika virus infections without a significant loss of specificity.Early diagnosis of acute infections with Zika viruses is particularly important for pregnant women since the virus may interfere with the neurological develop-ment of the fetus and can cause micro-cephaly. However, also in adults the viral infection may have long-term neuro-logical consequences. Therefore, timely recognition of this infectious disease is required.

EUROIMMUN www.clinlabint.com & search 27745

MrSA assay now CE marked in Europe

The Pan-ther Fusion MRSA assay has received CE mark in

Europe. This assay, the latest in a grow-ing menu of Panther Fusion and Aptima assays, brings full automation, efficiency and excellent assay performance to MRSA screening. The Panther Fusion system retains all the key benefits of the Panther platform, including full sample-to-result automation, and is suitable to any size laboratory. With rising antibi-otic resistance in Europe and the threat posed by healthcare-associated infec-tions to public health, the new assay will help customers provide a rapid service to hospitals, so cases of resistance, such as MRSA, are identified and managed quickly and effectively. The Panther Fusion MRSA assay accurately detects and differentiates Staphylococ-cus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) DNA from ESwab nasal samples. It has broad strain inclusivity including the Bengal Bay clone,

and is able to correctly identify empty cas-sette variants. The Panther Fusion MRSA assay ena-bles laboratory customers to leverage the benefits of the fully automated Panther Fusion system, which provides random and continuous access to improve labo-ratory efficiency. Laboratories can com-bine women’s health, virology, respiratory, hospital-acquired infections, and Open Access tests on the Panther Fusion sys-tem, running up to 32 different assays at the same time. In addition, MRSA samples can be loaded directly into the Panther Fusion, saving customers vital time and labour costs and delivering first results in 2.4 hours, considerably faster than com-monly-used culture and lab developed tests. In addition, Panther Fusion offers labs the potential to process up to 500 Panther Fusion and Aptima assays in an 8-hour period. Panther Fusion is available as a full sys-tem, or the Panther Fusion module can be attached to existing Panther systems in the field to extend testing capabilities. The Panther Fusion module adds the capac-ity to run PCR (polymerase chain reac-tion) assays in addition to tests based on TMA (transcription-mediated amplifica-tion), the proprietary Hologic chemistry used in the company’s Aptima brand. Key benefits include the ability to run mul-tiple tests from a single sample, random and continuous access, sample processing with rapid turnaround time, continuous loading, and STAT capabilities. Its ready to use, unit-dose lyophilized reagents, which have 60-day on-board stability, help reduce waste and the need for manual reagent preparation.

HOLOGIC www.clinlabint.com & search 27740

remote analyser monitoring via connected network

Digitization has revolutionized connectivity by increasing pro-ductivity and improving lab efficiency like never before.

Thanks to connected devices, techni-cal teams no longer need to remain in front of their workstations, or even in the laboratory. The secure Connect.One network archi-tecture allows laboratories to benefit from remote monitoring of their automated analysers, meaning Stago can check they are operating correctly and diagnose and analyse any malfunctions as part of a pro-active approach. Designed to meet international data pri-vacy and security guidelines, Connect.One will become a cornerstone for inte-grating new applications. Systems connected to Connect.One can also send QC results to My Expert QC (Stago’s externalized IQC platform), as well as activity statistics to simplify the management of “Cost Per Reportable Results” contracts. Connect.One can track analyser per-formance and send notifications in case of problems while downloading software improvements from a secure infrastructure. Using their connected devices, laborato-ries can increase productivity, efficiency and operational flexibility by receiving a higher level of support and expertise from Stago.

STAGO www.clinlabint.com & search 27746

– September 201833ProdUCt NEWS

Chromsys-tems has l a u n c h e d the first C E - I V D v a l i d a t e d

HPLC assay for antibiotics. It allows the quantitative determination of ampicillin, cefepime, ceftazidime, lin-ezolid, meropenem and piperacillin in human serum and plasma. The assay is used to monitor the blood levels of these antibiotics and also provides qualitative data for sulbactam and

tazobactam. The exceptional high sta-bility of calibrators and controls as well as two internal standards ensure a precise quantification. The assay also includes a Priming Solution that increases the stability of the patient samples. The HPLC determination with subsequent UV detection is divided into two groups and requires only one column and one sample prep for all parameters.

CHROMSySTEMS www.clinlabint.com & search 27736

Assay for monitoring of 6 antibiotics in serum/plasma

Emerging antimicrobial resistance detection system

The DxM Micro-Scan WalkAway system is a diag-nostic system for bacterial identi-fication and anti-biotic suscepti-bility testing for

microbiology laboratories. The DxM MicroScan WalkAway system uses direct minimum inhibitory concen-trations (MIC) for detection of anti-microbial resistance, offering greater confidence in results through gold-standard accuracy and the broadest breadth of first-time reporting. Emerg-ing antimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, para-sites, viruses and fungi, according to the World Health Organization. Up to 50,000 people die because of AMR in the U.S. and Europe alone, and one study claims that 10 million people a year could die from this global cri-sis by the year 2050. Timely, accurate detection of emerging AMR is of criti-cal importance in helping to ensure patients are protected against the risk of worsening infection or its progres-sion to life-threatening conditions

such as sepsis or septic shock. The DxM MicroScan WalkAway sys-tem allows detection of emerging resistance to the toughest pathogens. As new issues have arisen, such as carbapenemases or other resistance mechanisms, MicroScan has adapted. The panels have been updated to assist in identifying these new resist-ance mechanisms. At the same time, the system’s delivery of first-time gold-standard MIC accuracy helps laboratories achieve their operational goals by reducing costs associated with confirmatory—or offline—test-ing. Added workflow enhancements include a new fluid-level sensing technology for added assurance, easy-to-view external LED indicators that show status at a glance, quick bottle release that simplifies reagent main-tenance, and integrated reliability improvements that maximize uptime. The DxM MicroScan WalkAway sys-tem is part of a full line of microbiol-ogy solutions from Beckman Coulter. Automation and standardization of all core aspects of specimen testing creates workflow efficiencies for the laboratory.

BECKMAN COULTER DIAGNOSTICS www.clinlabint.com & search 27748

Glycated albumin assay kit Now available is a new Stanbio Chem-istry Glycated Albu-min (GA) assay kit. GA is a sensitive marker for inter-mediate glycemic control in diabetes

and can fill the gap between long-term and short-term measurements of blood glucose status. This is particularly useful just after the start or during an alteration in diabetes treatment, where measure-ment of GA can confirm blood glucose changes over just 2 to 3 weeks. It is also useful for prognosis in hemodialysis patients and during pregnancy when HbA1c may provide an insufficient indi-cation of glycemic levels. The Stanbio Chemistry GA assay uses an enzymatic method with liquid reagents and requires no preparation. It can be used on com-patible open channel clinical chemis-try analysers and expresses results as a ratio of Glycated Albumin to Albumin in mmol/mol from a small amount of blood serum. GA accurately reflects the latest blood glucose status as it is found throughout the body and is a type of pro-tein that can readily be glycated.

EKF DIAGNOSTICS www.clinlabint.com & search 27735

ProdUCt NEWS – September 2018 34

The 2018 Biotexcel conference ‘Genomic Medicine’ will take place at Murray Edwards College, Cambridge, UK, on 5–6 December 2018.

The running scientific theme for this meeting, like other Biotex-cel genomic meetings, will cover the areas where Next Generation Sequencing is used in the analysis of human disease. These topics will

include different disease areas where particularly promising genomic studies have been performed; large population studies; whole genome & whole exome studies; epigenomics and many other topics. In this meeting we will also hear from commercial genomic companies, whether those that currently have genomic solutions on the market as well as those that are spin-outs or are in the research/earlier phases.

Confirmed speakers include (among others):• Prof. Lucy Raymond, University of Cambridge• Dr Caroline Wright, University of Exeter Medical School• Dr Anna Middleton, Wellcome Sanger Institute• Dr Nitzan Rosenfeld, Cancer Research UK Cambridge Institute• Prof Ferenc Mueller, University of Birmingham• Prof Yanick Crow, MRC IGMM, University of Edinburgh• Prof Michael Parker, University of Oxford• Dr Athena Matakidou, Astra Zeneca.

Topics to be covered include:• Whole genome sequence analysis of critically ill children• Transforming drug discovery and development with an integrated

genomics approach• Making new genetic diagnoses with old data• How is society responding to genomics?• Genomic analysis of cell-free DNA in plasma for non-invasive can-

cer diagnostics• Genomic imprinting: lessons on the 4D genome.

The meeting will also have presentations on a variety of the latest tech-nology developments.

In addition to presentations, the meeting will also include a number of networking opportunities, such as an introductory networking session on Day 1, a panel debate on topical issues and bottlenecks, a poster ses-sion and a networking dinner at The Punter Pub.

This meeting is intended to be suitable for NGS users, researchers and students, bioinformaticians and those in the NHS and private labs, bio-tech companies, CROs and service providers.

Discounts are available for students, academics and hospital staff. https://biotexcel.com/event/genomic-medicine-2018-cambridge

MEEtiNG PrEViEW: Genomic Medicine, Cambridge, 2018

1. Percentage of samples with numerical results that do not require additional intervention or handling, such as manual smear review, spun hematocrit, dilution, or other repeat/reflex testing. DxH series side-by-side results documentation.© 2018 Beckman Coulter, Inc. All rights reserved. Beckman Coulter, the stylized logo and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries.

For more information, visit www.beckmancoulter.com/contact

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