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Vol. 31 No.4 • 6-7/2014ISSN 1068-1760

W O R L D ’ S C L I N I C A L L A B O R A T O R Y N E W S L E A D E RDAILY CLINICAL LAB NEWS

V I S I T

Blood Test Predicts Obesity in Children

AA simple blood test, which can ana-lyze DNA, could be used to pre-

dict obesity levels in children and thetest can assess the levels of epigeneticswitches in a gene that regulates fatstorage in the body. Epigenetic switch-es take place through a chemicalchange called DNA methylation,

Cont’d on page 8

DNA Biomarkers ImproveColon Cancer Diagnosis

AA large-scale clinical study showedthat a noninvasive test that de-

tects several DNA biomarkers andblood in stool samples was significant-ly more sensitive than the currentlyused fecal immunochemical test (FIT)for diagnosis of colorectal cancer. TheExact Sciences (Madison, WI, USA;

Cont’d on page 10

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AA pproximately one out of threechildren screened for high cho-

lesterol between the ages of 9 and 11had borderline or high cholesterol,potentially placing them at greaterrisk for future cardiovascular disease.

While cardiovascular disease inchildren is rare, the presence of cer-tain risk factors in childhood can in-

crease the chances of developingheart disease as an adult and previousstudies have demonstrated that ather-osclerosis can begin in childhood.

Scientists at Texas Children’s Hos-pital examined the medical recordsof 12,712 children who had beenscreened for cholesterol levels as partof the largest studies of outpatient

Study Calls for Universal Pediatric Cholesterol Screening

Blood Test Offers EarlierAlzheimer’s Diagnosis

AA team of researchers has discov-ered biomarkers and validated

the first blood test that accuratelypredicted whether an asymptomaticperson will develop amnestic mildcognitive impairment or Alzheimer’sdisease within 3 years before onsetof clinical symptoms, heralding thepotential for developing treatment

Microarray GlycoprofileTest for Ovarian Cancer

CCancer researchers have devel-oped a microarray assay that

identifies the abnormally glycosylat-ed surface residues associated withovarian tumor biomarker proteinssuch as CA125. The CA125 bio-marker assay plays an important rolein the diagnosis and management ofinvasive ovarian cancer. However, a

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Nano-Based Strip Test Could Transform Cancer Detection

Image: Low-cost urine test developed by MIT engineersSee article on page 6

A noninvasive paper-stripurine test using innova-

tive nanoparticle technologycould offer widespread andinexpensive point-of-carescreening for cancer andother diseases. Developedby MIT scientists, the newtest could reveal resultswithin minutes.

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Gene Test PredictsMelanoma Metastases

AA gene expression profile (GEP)test can identify primary cuta-

neous melanoma tumors that arelikely to metastasize in patients whohad a negative sentinel lymph nodebiopsy. The noninvasive 31-geneGEP test that is widely used to de-termine metastatic risk in Stage Iand II melanoma patients has been

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An international multicenterstudy has been carried out to

assess the performance of a newgeneration of slide-staining de-vices. The aim of the study wasto evaluate the capability of theinstrument to achieve good sen-sitivity and to adjust the staining

Slide-Staining System for Tuberculosis Offers Flexible Programming

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4LabMedica InternationalJune-July/2014

LabMedicaLabMedica

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to individual circumstances and preferences through-out the world by providing a wide range of staining set-tings via an intuitive user interface.

According to the World Health Organization(WHO), microscopy of stained sputum samples is rec-ognized as an accurate and economical approach forearly detection of pulmonary Tuberculosis (TB) infec-tions. Staining Mycobacterium tuberculosis and otheracid-fast bacilli (AFB) require Carbol Fuchsin or Fluo-rescent staining techniques, both approaches comingwith variations. Carbol Fuchsin staining usually uti-lizes Ziehl-Neelsen or Kinyoun primary stains with ei-ther Brilliant Green or Methylene Blue counterstains.Fluorescent staining techniques usually utilize Au-ramine O or Auramine Rhodamine primary stains andPotassium Permanganate or Thiazine Red as the coun-terstain. These staining methods are well recognizedand it is generally accepted that Fluorescence mi-croscopy provides a more rapid smear examination ofstained smears and is more suitable for use in region-al laboratories with large workloads. Given the in-creasing importance of fluorescence staining, the mul-ticenter evaluation of the new system focused partic-ularly on this approach.

ELITechGroup Biomedical Systems (Logan, UT,USA; www.elitechgroup.com/north-america/home)has developed a new Aerospray TB Stainer/Cytocen-trifuge (Model 7722). Like previous Aerospray TB mod-els, this stainer automates the staining process of micro-scope slides without any risk of cross-contamination.The stainer utilizes significantly less stain than manualstaining techniques and stains slides in only a quarter ofthe time usually required by manual staining. The op-

tional Cytocentrifuge function of the instrument allowsusers to prepare samples evenly and uniformly on themicroscope slide prior to staining, which is a significantadvantage to increase the overall sensitivity of TBscreening in patients with extra- pulmonary TB or withHIV co-infection.

The new Aerospray TB “series 2” utilizes a state-of-the-art touchscreen user interface and provides trace-ability for users, samples, and reagents. Above all, it iscustomizable to each laboratory’s needs, yet simple tooperate. Various reagent combinations and stain routineadjustments are available in order to achieve the needsof AFB staining in the laboratory.

The stainer has been used in an international studycarried out in Belgium, Namibia, South Africa, andUSA, which included over 500 samples. These samplesincluded direct and digested specimens (N-Acetylcys-tein – Phosphate buffer method), stained with differentreagent combinations. Each sample was made in dupli-cate. At least one slide from each sample was stained bymanual staining techniques and at least one slide wasstained on the new Aerospray TB stainer. The slideswere classified as AFB negative or AFB positive. Theslide stained on the stainer was classified the same asthe manually stained slide on >99.6% of the samples,thus demonstrating the high correlation between theautomated and manual approaches.

Dennis Briscoe, General Manager of ELITechGroupBiomedical Systems said, “With the series 2 AerosprayTB (Model 7722), we’ve maintained the unique advan-tages of the Aerospray stainers and introduced a newslide-staining system for tuberculosis with highly flexi-ble programming, good sensitivity and a user interfacethat is intuitive enough for routine clinical use.”

Slide-Staining System for Tuberculosis Offers Flexible Programming

SSoon it may be possible to make a rapid, early di-agnosis of prostate cancer using a noninvasive

electronic nose that sniffs a urine sample.Prostate cancer is the second most common malig-

nancy in men and one of the leading causes of deathfrom cancer. It is difficult to diagnose and make reliableprognoses about the disease because it does not ap-pear consistently in prostate tissue.

Scientists at the University of Tampere (Finland;www.uta.fi) tested 50 patients with confirmedprostate cancer and 24 samples from 15 patientswith benign prostatic hyperplasia. Fifteen patientsprovided urine preoperatively and nine patients pro-vided samples three months postoperatively and allpatients were scheduled to undergo robotic assistedlaparoscopic radical prostatectomy or transurethralresection of the prostate.

The electronic nose or eNose used in the studywas a commercially available model ChemPro100(Environics Inc.; Mikkeli, Finland; www.environics.fi)based on the ion mobility spectrometry principle. Thedevice contains an ion mobility cell that consists ofeight electrode strips producing two-channel outputand a metal oxide based semiconductor cell. Togeth-er these sensors produce 18-channel measurementdata. The sensors do not specify molecules, but pro-duce a characteristic smell print of the sample.

The team found that the eNose, which analyzedmolecules in the urine headspace, was able to dis-criminate prostate cancer from benign prostatic hy-

perplasia with a sensitivity of 78% and a specificity of67%, on a par with the prostate specific antigen(PSA) tests. Niku Oksala, MD, PhD, DSc, the seniorauthor of the study said, “PSA is known to correlatepositively with prostate volume, which is a potentialsource of diagnostic error when comparing prostatecancer with benign disease. According to our currentanalysis, prostate volume did not affect the eNose re-sults, potentially indicating high specificity of our sen-sor array to cancer.” The study was published onlineon February 25, 2014, in the Journal of Urology.

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Electronic Nose Sniffs Out Prostate Cancer

Image: The ChemPro100 handheld chemical

detector (Photo cour-tesy of Environics).

AA simple, noninvasive paper strip-urine test us-ing nanoparticle technology could help trans-

form worldwide screening for cancer and otherdiseases. Developed by Massachusetts Institute ofTechnology (MIT; Cambridge, MA, USA; www.mit.edu) scientists, the urine sample based assay,which works like a pregnancy test, could revealwithin minutes whether a person has cancer. Thisapproach has helped detect infectious diseases,and the new technology would enable some non-communicable diseases to be detected using thesame strategy.

The technology, developed by a team led byMIT professor Sangeeta Bhatia, relies on nanopar-ticles designed to interact with abnormally upreg-

ulated proteases from growing tumors, whereeach protease can trigger release of hundreds ofsynthetic molecular biomarkers easily detectablein a patient’s natural, unprocessed urine. Prof.Bhatia and colleagues had developed the technol-ogy to thus amplify “signals” from tumor matrixmetalloproteinases (MMPs), which facilitate thespread of cancer cells from their original site bycutting through proteins of the extracellular ma-trix, which holds the cells in place. The MITnanoparticles are coated with peptides targetedby different MMPs. The nanoparticles congregateat tumor sites, where tumor MMPs cleave hun-dreds of the coating peptides, which are excretedin the urine.

“When we invented this new class of synthet-ic biomarker, we used [mass spectrometry] to dothe analysis,” says Prof. Bhatia; “For the develop-ing world, we thought it would be exciting toadapt it instead to a paper test that could be per-formed on unprocessed samples in a rural set-ting, without the need for any specialized equip-ment. The simple readout could even be trans-mitted to a remote caregiver by a picture on amobile phone.” The team adapted the particlesso they could be analyzed using the simple later-al flow assay (LFA), as in pregnancy tests. Detec-tion is by sandwich immunoassay – nitrocellu-lose paper strips are coated with antibodies tothe peptides, as the antibody-captured peptidesflow along the strip they are exposed to severalinvisible test lines made of other antibodies spe-cific to different tags preattached to the peptides.Also, the capture antibody specificity allows theprobes to be multiplexed in vivo and quantifiedsimultaneously by the paper lateral flow assay orby ELISA.

This technology can easily be modified to de-tect multiple types of peptides released by othertypes or stages of disease. “This is a clever and in-spired technology to develop new exogenouscompounds that can detect clinical conditionswith aberrantly high protease concentrations,”said Samuel Sia, associate professor of biologicalengineering at Columbia University (not involvedin the research); “Extending this technology todetection by strip tests is a big leap forward inbringing its use to outpatient clinics and decen-tralized health settings.”

“This is a new idea – to create an excreted bio-marker instead of relying on what the body givesyou,” said Prof. Bhatia. In mouse models, thismethod accurately identified colorectal solid tu-mors, as well as thrombosis blood clots. A grantfrom MIT’s Deshpande Center for TechnologicalInnovation will help fund clinical trials and com-mercializing the technology. It would likely firstbe applied to high-risk populations, such as peoplewho have had cancer previously or a family mem-ber with the disease.

Prof. Bhatia said she would like to see iteventually used throughout developing areas,but such technology could in many cases also beused in more developed areas as a simple and in-expensive alternative or complement to moreexpensive advanced diagnostics. Such “point-of-care, image-free cancer detection [...] could real-ly be transformative,” said Prof. Bhatia. Withthe current version, patients would first receivean injection of the nanoparticles, then urinateonto the paper test strip. The team is now work-ing on a nanoparticle formulation that could beimplanted under the skin for more convenienceand for longer-term monitoring. The team is al-so working to identify signatures of MMPs thatcould be exploited as biomarkers for other typesof cancer, as well as for tumors that have metas-tasized.

The assay is described by Warren et al. in thejournal Proceedings of the National Academy ofSciences of the United States of America (PNAS),ahead of print Feb. 24, 2014.


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Nano-Based Strip Test Could Transform Cancer Detection

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AA new test utilizes fluorescence immunoassaytechnology to detect and measure two bio-

markers that reflect the risk of develop-ing acute kidney injury (AKI). The NephroCheckTest measures tissue inhibitor of metallopro-teinase 2 (TIMP-2) and insulin-like growth factorbinding protein 7(IGFBP-7) in human urine. Bothare involved in G1 cell cycle arrest during thevery early phases of cellular stress, and are robustmeasures of risk for AKI manifesting within thefollowing 12-24 hours. TIMP-2 is an inhibitor ofthe matrix metalloproteinases (MMPs) that direct-ly suppress the proliferation of endothelial cells.IGFBP-7 is involved in the regulation of tissueavailability of insulin and IGFs, andstimulates cell adhesion implicated inepithelial cell-cycle arrest.

For the test, a sample of a patient’surine is mixed with reagents onto a sin-gle-use cartridge inserted into the propri-

etary Astute140 Meter. The meter employs a sand-wich immunoassay technique along with fluores-cence detection technology to quantitatively meas-ure the biomarker concentrations, combining theminto a single numerical test result. Multiple levels ofcontrols (internal, external, and electronic) are usedto ensure reliable and accurate results in about 20minutes. The NephroCheck Test and Astute140 Me-ter are products of Astute Medical (San Diego, CA,USA; www.astutemedical.com), and have receivedthe European Community CE marking of approval.

AKI is a significant global health hazard thatstrikes up to seven percent of hospitalized pa-tients. AKI often occurs in patients that are al-

ready suffering from myriad issues such as sepsis,trauma, surgery, or being treated with nephrotox-ic drugs, further complicating diagnosis, while al-so increasing mortality rates.

Rapid Urine Test Detects Early Kidney Injury

LabMedicaLabMedicafor daily laboratory medicine news click to www.labmedica.comfor daily laboratory medicine news click to www.labmedica.com

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Study Calls for Universal Pediatric Cholesterol Screeningcont’d from cover

clinic visits. Of these, 4,709, or 30% had bor-derline or elevated total cholesterol. In thestudy, the investigators found that boys weremore likely than girls to have elevated totalcholesterol, low-density lipoprotein (LDL),and triglycerides, while girls had lower high-density lipoprotein (HDL). Obese childrenwere more likely to have elevated total cho-lesterol, LDL and triglycerides, with lowerHDL in comparison to non-obese children.Mean total cholesterol, LDL, non-HDL, andHDL were all within the normal range, 162mg/dL, 92 mg/dL, 113 mg/dL and 52mg/dL, respectively and mean triglycerideswere borderline or abnormal at 103 mg/dL.

A potential study limitation is that it is un-known if testing was ordered in a universalmanner or selectively based upon individualrisk factors or a family history of prematurecardiac disease. Thomas Seery, MD, pediatriccardiologist at Texas Children’s Hospital(Houston, TX, USA; www.texaschildrens.org), assistant professor of Pediatrics at BaylorCollege of Medicine, said, “Children need tohave their cholesterol panel checked at somepoint during this timeframe of 9 to 11 yearsold. In doing so, it presents the perfect oppor-tunity for clinicians and parents to discuss theimportance of healthy lifestyle choices on car-diovascular health. Our findings give a com-pelling reason to screen all children’s bloodcholesterol.”

The American Academy of Pediatrics(Elk Grove Village, IL, USA; www.aap.org)calls for universal cholesterol screening ofchildren between the ages of 9 and 11years and, again between 17 and 21 yearsof age. The study was presented the Amer-ican College of Cardiology’s 63rd AnnualScientific Session held March 29–31, 2014, in Washington DC (USA; http://accscientificsession.cardiosource.org).

Image: The NephroCheck test cartridge and Astute140 meter

(Photo courtesy of Astute Medical).

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compared to sentinel lymph node biopsy (SLNB)which has traditionally been the best prognostictest available for melanoma patients.

Scientists at Castle Biosciences Inc. (Friends-wood, TX, USA; www.castlebiosciences.com) com-pared their test called DecisionDx-Melanoma to re-sults from134 patients with Stage I, II, or III cuta-neous melanoma. These 134 patients representedall patients in the initial clinical validation studies

who had a documented sentinel lymph node proce-dure. The DecisionDx-Melanoma is a proprietarytest carried out in the company’s laboratories.

In patients with a negative SLNB, a result inter-preted as a lower risk for metastasis, the GEP testidentified the vast majority of melanomas that ulti-mately progressed over the subsequent five year pe-riod. The rate of five year metastasis-free survival(MFS) was 55% for SLNB negative patients com-pared to 37% for SLNB positive patients The GEP

test results showed improved prognostic accuracyin these same patients with an MFS of 87% for thelow-risk (Class 1) patients and 31% for the high risk(Class 2) patients.

The GEP test showed improved prognostic accu-racy, with an overall survival (OS) for GEP Class 1patients of 92% compared to 49% for Class 2 pa-tients. The GEP test was also analyzed in combina-tion with SLNB status. The 20% of patients whohad high-risk results for both tests (GEP Class 2 andSLNB positive) had lower survival rates(MFS=34%; OS=53%). Similarly, the 31% of pa-tients who had low risk results for both tests (GEPClass 1 and SLNB negative) had higher survivalrates (MFS=82%; OS=92%). Importantly, in the49% of patients who had results that were discor-dant, high-risk outcome for one test, low risk forthe other, the GEP test result correctly predictedthe patients’ clinical outcomes. Net reclassificationimprovement of GEP class over SLNB status wasgreater than 50%.

Pedram Gerami, MD, the study author and an as-sociate professor of Dermatology at NorthwesternUniversity (Evanston, IL, USA; www.northwestern.edu), said, “The results from this study show theGEP test is an independent predictor of metastasisand death, and significantly improves upon sentinellymph node biopsy for staging melanoma patients.Based upon this data, optimal use of the GEP testmay be to identify high risk patients among thosewith a negative SLNB result, or for patients who areineligible for or who decline a SLNB procedure.” Thestudy was presented at the 72nd Annual Meeting ofthe American Academy of Dermatology held March21-25, 2014, in Denver (CO, USA).

Gene Test Predicts Melanoma Metastases



cont’d from cover

which controls how genes work and is set duringearly life and can be used to differentiate betweenchildren with a high body fat and those with a lowbody fat when they were older.

Scientists at the University of Southampton(UK; www.soton.ac.uk) and their colleagues ana-lyzed DNA methylation in the peroxisomal prolif-erator-γ co-activator-1α promoter gene (PGC1a)in blood from 40 children (20 of each sex) collect-ed annually between 5 and 14 years by pyrose-quencing. PCG1a is central to energy homeostasisthrough regulation of mitochondrial function,pancreatic β-cell function and adipogenesis and istherefore of potential relevance to obesity and car-dio-metabolic disease (CMD) risk.

Pyrosequencing was carried out using Pyro-Mark Gold Q96 Reagents on a PyroMark Q96MD Pyrosequencer (Qiagen; Manchester, UK;www.qiagen.com) and the percentage methyla-tion was calculated. Electrophoretic mobility shiftassays (EMSA) were performed using LightShiftChemiluminescent EMSA Kit (Thermo Scientific,Loughborough, UK; www.thermoscientific.com).The team found that the tests, when carried outon children at five years old, can distinguish be-tween children with adiposity and those with alow body fat when they were older. Resultsshowed that a rise in DNA methylation levels of

10% at five years was as-sociated with up to 12%more body fat at 14 years.

All the results were in-dependent of the child’sgender, their amount ofphysical activity and theirtiming of puberty. SevenCpG loci were identifiedthat showed no signifi-cant temporal change orassociation with leuko-cyte populations. TheCpG sites are regions ofDNA where a cytosinenucleotide occurs next toa guanine nucleotide in the linear sequence ofbases along its length.

The authors concluded that their findings sup-port the view that epigenetic marks measured inchildhood which exhibit temporal stability mayhave utility in predicting future disease risk. Thesefindings suggest that temporally stable CpG locimeasured in childhood may have utility in predict-ing CMD risk. Identification of such marks in bloodmay increase the number of individuals in whomsuch associations can be tested beyond those forwhich fetal tissue is available and provide opportu-nities for further investigation of longitudinal asso-

ciations and the impact of therapeutic intervention.Graham C. Burdge, PhD, the lead author of

the study said, “It can be difficult to predict whenchildren are very young, which children will puton weight or become obese. It is important toknow which children are at risk because help,such as suggestions about their diet, can be of-fered early and before they start to gain weight.”The study was published on March 12, 2014, inthe journal Diabetes.

Image: The PyroMark CpG assays are predesignedfor quantification of CpG methylation by Pyrose-quencing (Photo courtesy of Qiagen).

Blood Test Predicts Obesity in Children


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AAnew blood test is being devel-oped that one day could rapid-

ly confirm whether someone is hav-ing a stroke and what kind, will leadto faster diagnosis and treatment,which could mean the differencebetween life and death.

A device has been constructedthat can process whole blood andisolate genetic material for two po-tential stroke biomarkers withinminutes, and by identifying morebiomarkers could aid in diagnosis asthe device can analyze a total of fourbiomarkers at the same time.

Scientists at the Louisiana StateUniversity (Baton Rouge, LA, USA;www.lsu.edu) designed and testedthe performance of a polymer mi-crofluidic device that can affinity se-lect multiple types of biological cellssimultaneously with sufficient re-

covery and purity to allow for theexpression profiling of messenger ri-bonucleic acid (mRNA) isolatedfrom these cells.

The microfluidic device consist-ed of four independent selectionbeds with curvilinear channels thatwere 25 μm wide and 80 μm deepand were modified with antibodiestargeting antigens specifically ex-pressed by two different cell types.Bifurcated and Z-configured devicegeometries were evaluated for cellselection. Whole blood sampleswere obtained from anonymoushealthy donors. To analyze the cellsthe scientists used flow cytometricanalysis and evaluated carboxylicacid surface densities.

Cell characterization was per-formed by acquiring fluorescent im-ages on an IX71-DSU Spinning Disk

Confocal inverted microscope(Olympus America; Center Valley,PA, USA; www.olympusamerica.com). Reverse transcription andpolymerase chain reaction (PCR) ofCD4+ T-cells and neutrophils wasperformed after they were isolatedon a chip and lysed by infusing withlysis buffer. Reverse transcription(RT) was accomplished using a Pro-toScript II First Strand cDNA Syn-thesis Kit (New England BioLabs; Ip-swich, MA, USA; www.neb.com).

The expression of possible strokebiomarker genes from isolated T-cells and neutrophils, such as thecalcium binding protein A9(S100A9), the T cell receptor beta(TCRB), and the formyl peptide re-ceptor 1 (FPR1), was evaluated us-ing RT-PCR. The modification andisolation procedures can also be

used to analyze other cell types aswell where multiple subsets need tobe investigated. The study was pub-lished online on March 20, 2014, inthe journal Analytical Chemistry.

Image: The IX71-DSU spinning diskconfocal inverted microscope (Photocourtesy of Olympus).

Blood Biomarkers Accurately Diagnose Stroke

cont’d from cover

www.exactsciences.com) Cologuardtest includes quantitative molecularassays for KRAS mutations, aberrantNDRG4 and BMP3 methylation, andbeta-actin, plus a hemoglobin im-munoassay. It was compared to acommercially available FIT assay andto the colonoscopy “gold standard” ina screen of nearly 10,000 patients.

Colonoscopy examinations of the9,989 participants who could be eval-uated detected 65 (0.7%) with col-orectal cancer and 757 (7.6%) withadvanced precancerous lesions. Com-paring these results to the DNA assayand FIT revealed that the sensitivityfor detecting colorectal cancer was

92.3% with DNA testing and 73.8%with FIT. The sensitivity for detectingadvanced precancerous lesions was42.4% with DNA testing and 23.8%with FIT. The rate of detection ofpolyps with high-grade dysplasia was69.2% with DNA testing and 46.2%with FIT; the rates of detection of ser-rated sessile polyps measuring onecentimeter or more were 42.4% and5.1%, respectively. Specificities withDNA testing and FIT were 86.6% and94.9%, respectively. Among partici-pants with nonadvanced or negativefindings on colonoscopy the resultswere 89.8% and 96.4%, respectively.Thus, while the DNA test was moresensitive than the FIT test, it did pro-

duce more false positive results,which would have led to unneces-sary colonoscopies.

“Cologuard detection rates of earlystage cancer and high-risk precancer-ous polyps validated in this large studywere outstanding and have not beenachieved by other noninvasive ap-proaches,” said contributing authorDr. David Ahlquist, a gastroenterolo-gist at the Mayo Clinic (Rochester,

MN, USA; www.mayo.edu) and co-in-ventor of the Cologuard test. “It is ourhope that this accurate and user-friendly test will expand screening ef-fectiveness and help curb colorectalcancer rates in much the same way asregular Pap smear screening has donefor cervical cancer.”

The study was published in March19, 2014, online edition of the NewEngland Journal of Medicine.

DNA Biomarkers Improve Colon Cancer Diagnosis

AA lthough there have been ad-vancements in instrumentation

within hematology laboratories, thereis still a need for review of a peripher-al blood films (PBF). Morphologicalassessment is extremely subjectiveand dependent on the acceptablestandard of the film being assessed,and it is important to determinewhether or not automated slidemak-er-stainers are able to consistently andreproducibly prepare and stain bloodfilms of exemplary quality, withoutcarryover between specimens.

Hematologists at the LondonHealth Sciences Center (London, ON,Canada; www.lhsc.on.ca) selected atotal of 131 specimens, 46 morpholog-ically normal and 85 hematologicallyabnormal, from their Health Center,which specializes in cancer care, pedi-atrics, and obstetrics. A broad speci-men pool of potential disease stateswas recruited from the day-to-dayworkload over an eight-day period.Four blood films were prepared manu-ally and stained with the laboratory’s

routine method of Wright-Giemsa.The scientists evaluated the Uni-

Cel DxH slidemaker stainer (DxHSMS, Beckman Coulter; Brea, CA,USA; www.beckmancoulter.com),which is a fully automated, integrat-ed slidemaker, and stainer intendedfor the hematology laboratory. TheDxH SMS is available either as astand-alone instrument or part of theDxH workcell configuration. Aunique patented device, the hemas-phere, is incorporated into the systemto measure “residual clinging” of theblood sample to an internal surface asan analogue for the way in whichblood would behave as it is spread ona glass slide. Two samples were se-lected to perform this study evaluat-ing the degree of cellular carryoverthat has the potential to occur in cir-cumstances when a sample with ex-treme leukocytosis is followed by onewith extreme leukopenia.

The study was published in theApril 2014 issue of InternationalJournal of Laboratory Hematology.

110LMI-07-14LINKXPRESS COM 10LabMedica InternationalJune-July/2014


Automated Slidemaker-Stainer Evaluated for Performance

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SSevere and sometimes fatal lung disease causedby a group of bacteria in the same family as

those that cause tuberculosis is much more com-mon than previously thought.

Nontuberculous mycobacteria (NTM) includemore than 150 types of bacteria found in water andsoil that can infect the lungs when inhaled, but un-like tuberculosis, NTM is not contagious and can-not spread from person to person.

Scientists at the University of Illinois (Chicago,IL, USA; www.uic.edu) analyzed mortality relatedto nontuberculous mycobacterial infections from1999 through 2010 by examining multiple-cause-of-death data from the National Center for HealthStatistics (Atlanta, GA, USA; www.cdc.gov). Theyanalyzed age-adjusted mortality rates, trends, asso-ciations with demographic variables, and comorbidconditions and correlated this information withsimilar data for tuberculosis-related mortality dur-ing the same time.

The investigators found that NTM was listed asimmediate cause of death for 2,990 people, ofwhom 87% were 55 or older, 85% were white, and52% were female. NTM-related deaths also corre-

lated with smoking, cancer, human immunodefi-ciency virus (HIV) infection, and chronic obstruc-tive pulmonary disease. They also found that theage-adjusted NTM mortality rates were unevenacross the USA. Tuberculosis-related deathsamong ethnic groups were also disproportional asamong those with tuberculosis, 4,847 (59%) werewhite.

The team suggested that both warm and dryclimates may contribute to NTM disease and influ-ence on mortality by different environmental fac-tors, such as temperature, soil and water condi-tions. NTM demonstrated a strong relationshipwith some comorbid conditions such as chronic ob-structive pulmonary disease, chronic respiratoryfailure, bronchiectasis, HIV, interstitial lung dis-eases, rheumatoid arthritis, hematopoietic malig-nancies and tobacco use. The infection is treatable,but antibiotic therapy is expensive and can take upto two years. Examples of nontuberculous my-cobacteria that should be distinguished from tuber-cle bacilli are Mycobacterium chelonae, M. absces-sus, M. fortuitum, and M. cosmeticum.

Mehdi Mirsaeidi, MD, MPH, the lead author

said, “People and physicians need to be aware ofNTM, especially as we see more of it, and becauseit can look like a lot of other pulmonary diseases.Patients usually present with a cough, and correctdiagnosis can take years. Having a better under-standing of the risk factors associated with NTMwill give us a better chance of diagnosing it faster.This is important, because it’s largely a curable in-fection.” The study was published on March 14,2014, in the journal Public Library of ScienceONE.

Image: Researchers have found that Nontuberculousmycobacteria (NTM), a severe and sometimes fatallung disease, is much more common than previouslythought (Photo courtesy of the University of Illinois).

Risk Factors Identified for Little-Known Lung Infections

TThe heart muscle protein cMyBP-C (cardiacmyosin binding protein-C) is rapidly released into

the bloodstream after cardiac damage and may be asensitive biomarker for diagnosing the onset of my-ocardial infarction (MI).

Myosin-binding protein C is a myosin-associatedprotein found in the cross-bridge-bearing zone (C re-gion) of A bands in striated muscle. It is found at reg-ularly spaced intervals and is hypothesized to act likea “barrel hoop” and hold the thick filament together.cMyBP-C, the cardiac isoforms of the protein, is ex-pressed exclusively in heart muscle.

Detection of elevated plasma cardiac troponin(cTn) levels is the “gold standard” for early discoveryof MI. However, troponin levels peak only four to sixhours after heart attack and lack the sensitivity re-

quired to detect the onset of MI at its earliest stages.Investigators at the Loyola University School of

Medicine (Chicago, IL, USA; www.lumc.edu) soughtto evaluate the usefulness of cMYBP-C as an ultra-ear-ly biomarker of MI. To this end, they studied the re-lease kinetics of cMyBP-C in a porcine model of MIand in two human cohorts.

They measured cMyBP-C levels in serum and plas-ma samples from MI pigs and patients serially from30 minutes to 14 days after coronary damage using acustom immunoassay based on electrochemilumines-cence. The anti-cMyBP-C antibodies used in thisstudy were specific to cMyBP-C and were generatedagainst the C0 domain of cMyBP-C, which is exclu-sively present in the cardiac isoform and does notcross react with skeletal MyBP-C isoforms. The assay

was comparable to a previously described ELISA andused the same capture and detection antibodies. Thechemiluminescence immunoassay had improved sen-sitivity, compared to ELISA.

Results revealed that in the pig model systemcMyBP-C plasma levels were increased from baseline(around 76 nanograms per liter) to about 767nanograms per liter three hours after onset of cardiacdamage and then peaked at about 2,418 nanogramsper liter after six hours. Plasma troponin and myosinlight chain levels were all increased after six hours. Ina cohort of 12 patients with hypertrophic obstructivecardiomyopathy, cMyBP-C was significantly in-creased from baseline about 49 nanograms per liter ina time-dependent manner, peaking at about 560nanograms per liter after four hours. In a second co-hort of 176 patients with non-ST segment elevation,cMyBP-C serum levels were significantly higher(about 7,615 nanograms per liter) than those in a con-trol cohort of 153 normal individuals (about 416nanograms per liter).

“This is a potential ultra-early biomarker thatcould confirm whether a patient has had a heart at-tack, leading to faster and more effective treatment,”said senior author Dr. Sakthivel Sadayappan, assistantprofessor of cell and molecular physiology at LoyolaUniversity School of Medicine. “These findings sug-gest that cMyBP-C has potential as an ultra-early bio-marker for the diagnosis of [heart attack], but this stillneeds to be validated using a large cohort study. AcMyBP-C blood test might lead to an earlier diagnosisin patients who present at the emergency departmentshortly after coronary artery blockage. However, a sys-temic prospective investigation is required to establishsuch data for clinical use.”

The study was published in the February 15,2014, issue of the American Journal of Physiology –Heart and Circulatory Physiology.

Rise in Blood Levels of Heart Muscle Protein an Early Diagnostic Marker for Cardiac Injury




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AAnew cryogenic storage solution providesmore reliable biobanking of samples while re-

ducing space utilization.Greiner Bio-One (Kremsmuenster, Austria;

www.gbo.com), a technology partner for the diag-nostic, pharmaceutical, and biotechnology indus-tries, is expanding its product portfolio to includea solution for high-throughput sample storage inautomated storage systems. At Analytica 2014(April 1-4, Munich, Germany), Greiner Bio-OneGmbH (Frickenhausen, Germany), division ofGreiner Bio-One, will be presenting this solutionfor valuable sample storage in biorepositories us-ing the new “Cryo.s” biobanking tubes. Cryo.stubes feature a 30% reduction in height as com-pared to standard cryo tubes, allowing for a moreoptimized utilization of available space in deepfreezers and liquid nitrogen tanks. Thus, Cryo.stubes also contribute to substantial savings on en-

ergy consumption and costs for equipment andmaintenance.

Made of medical-grade polymer with provenlow content of leachables and an optimizedscrew-cap design with silicon gasket, Cryo.s tubesare ideal for storage over long periods at extreme-ly low temperatures, such as in gas phase over liq-uid nitrogen. Both features minimize risk of sam-ple contamination or sample loss over time.

The new tubes are available in volumes of 300 μL, 600 μL, and 1,000 μL, and are suppliedin automation-friendly cryo racks. Equipped withdecapper-friendly screw caps, they can be openedand closed with automated equipment. Further-more, a unique datamatrix code on the tubesguarantees for machine-assisted, error-proof iden-tification of individual samples, making Cryo.stubes well suited also for automated processing oflarge sample quantities.

Image: Cryos with 2D Datamatrixcode - with the op-tion for Datamatrix coding, a beneficial feature isadded to the well-tried range of Cryo.s (Photo cour-tesy of Greiner Bio-One).

Innovative Cryo Tubes Feature Advantages for High-Throughput Storage

Routine Blood Glucose Measurements Can

Accurately Estimate HbA1c

GG lycated hemoglobin or HbA1c is the standardmeasurement for assessing glycemic control

over time in people with diabetes and blood levels ofHbA1c are typically measured every few months in alaboratory. The relationship between HbA1c and av-erage glucose levels could determine whetherHbA1c could be expressed and reported as averageglucose in the same units as used in self-monitoring,which could increase individuals’ motivation to im-prove diabetes control.

Scientists at the University of Virginia (Char-lottesville, VA, USA; www.virginia.edu) workingwith those at Sanofi-Aventis Deutschland GmbH(Frankfurt, Germany; www.sanofi.de) developed adata-based model that accurately estimates HbA1cusing self-monitored blood glucose (SMBG) readings.The computer algorithm they developed was basedon a training data set drawn from 379 subjects andthen evaluated for accuracy on an independent testdata set. The average HbA1c level was 7.6% (±1.1%), with minimum and maximum values of 5.2%and 12.2%, respectively.

A conceptually new, clinically viable procedurewas developed for real-time tracking of average gly-caemia from self-monitoring data. The average glu-cose tracing can then be converted into running es-timates of HbA1c, which can be presented to the pa-tient daily. The computational demands of the proce-dure are low and therefore readily implementable in-to devices with limited processing power, such ashome SMBG meter.

The procedure, tracking average glycaemia andHbA1c in real time, could provide valuable assis-tance to the daily optimization of diabetes control.The procedure is not intended as a substitute for lab-oratory assessments of HbA1c and it should beviewed as a surrogate measure that allows conven-ient tracing of average glucose, readily imple-mentable in a point-of-care SMBG device.

The study was published on April 23, 2014, inthe journal Diabetes Technology & Therapeutics.


To receive prompt and free information on products, log on towww.LinkXpress.com or fill out reader service form located on last pagePRODUCT NEWS

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CLINICAL SYSTEMAdvanced Instruments

The Anoxomat system creates anaerobic, mi-croaerophilic, hypoxic, and capnophilic environ-ments in jars in minutes, with superior precisionand reproducibility. Anoxomat's simple operationmakes user work process more flexible, and built-in QA guarantees reliable results.

URINALYSIS SYSTEMAVE Science & Technology

The AVE-766 and AVE-752 are designed tostreamline urine chemistry and sediment analysisin the busiest laboratories. Key features includefully automated analysis, and a fast throughput of200 tests per hour, ensuring maximum lab pro-ductivity and workflow.

MOLECULAR DIAGNOSTICS SYSTEMBeckman Coulter

The VERIS MDx fully automated, random accesssystem is designed for the quantitative and quali-tative analysis of molecular targets from patientspecimens. Features include continuous access,one-step loading, and individual test reporting thatstreamline workflow and system management.

AA tool has been developed that allows physi-cians to quickly and accurately predict the

risk of death in children with septic shock, a sys-temic infection that damages vital organs.

The prognostic tool has been validated andhelps doctors decide much faster which severelysick children need to receive aggressive life-savingtherapy for septic shock, which is one of the lead-ing causes of death among hospitalized children.

A multi-institutional study led by scientists atthe Cincinnati Children’s Hospital Medical Center(OH, USA; www.cincinnatichildrens.org) testedthe tool on a group of 182 pediatric patients fromthe intensive care units of 17 pediatric institu-tions. The diverse group of study participants fromage 1 to 13 years covered a wide range of differ-ent conditions and illness severity. All the serumsamples from the children were tested within 24hours of admission to intensive care.

The tool, a pediatric sepsis biomarker risk modelis called Persevere. Persevere includes the biomark-ers C-C chemokine ligand 3 (CCL3), interleukin 8(IL8), heat shock protein 70-kDa 1B (HSPA1B),

granzyme B (GZMB), and matrix metallopeptidase8 (MMP8). Serum concentrations of these biomark-ers were measured using a multiplex magnetic beadplatform (MILLIPLEX MAP) designed for the proj-ect by the EMD Millipore Corporation (Billerica,MA, USA; www.emdmillipore.com). Biomarkerconcentrations were measured in a Lminex 100/200 System (Luminex Corporation, Austin, TX,USA; www.luminex.com).

The tool showed that children who tested pos-itive for high-risk sepsis had a 34% chance of notsurviving, but those children who tested negativehad only a 3% chance of dying. The scientists alsoshowed that children who tested positive for high-risk sepsis, but survived the infection, 21% ofstudy patients, had greater degrees of organ fail-ure and longer stays in the intensive care unit.The investigators noted that the treatments re-ceived for their sepsis was probably reason thesepatients survived even though the test showedthey were at very high risk.

The authors concluded that they had prospec-tively tested the prognostic accuracy of the updat-

ed version of Persevere and found that it can beused to assign a reliable mortality probability inchildren with septic shock. This tool has variouspotential applications in the field of pediatric sep-tic shock. The development of a rapid assay plat-form to generate biomarker data in a timely man-ner is still required and although no assay plat-form currently exists, the technology to developsuch an assay is readily available. The study waspublished on January 29, 2014, in the journalPublic Library of Science One.

Image: The model 100/200, designed for multiplex-ing (Photo courtesy of Luminex).

IIndividuals with various forms of heart disease havebeen found to be a risk for a near term, potentially

fatal cardiac event if they display high levels of urinaryglobotriaosylceramide (Gb3).

Elevated urinary Gb3, which reflects the buildupof the lipid in various organs due to defects in the en-zyme alpha-galactosidase (GLA), has been considereda hallmark of Fabry disease, an X-linked lysosomal dis-order with risk for most types of heart disease. Elevat-ed Gb3 had not been connected previously to non-Fabry heart conditions.

Investigators at Baylor Research Institute (Dallas,TX, USA; www.baylorhealth.edu) were screeningheart disease patients for Fabry disease when theyfound that even in the absence of Fabry many of thepatients had elevated Gb3 in their urine. They hadscreened 1,421 consecutive patients with commonforms of heart disease for Fabry disease by measuring

urinary Gb3 in whole urine using tandem mass spec-trometry. They also determined GLA activity in driedblood spots, and they looked for GLA mutations byparallel sequencing of the whole gene (exons and in-trons) in pooled genomic DNA samples followed bySanger sequencing verification.

They reported finding GLA variants in 13 patients.In the 1,408 patients without GLA mutations, urinaryGb3 levels were significantly higher in heart diseasepatients compared to 116 apparently healthy controls.

Urinary lipid profiling showed that levels of fiveother lipids could distinguish between urine of sevenpatients with Fabry disease and six other heart diseasepatients with elevated urinary Gb3. Sphingomyelinand Gb3 levels were abnormal in the left ventricularwall of patients with ischemic heart failure. Elevatedlevels of urinary Gb3 were independently associatedwith increased risk of death in the average follow-up

period of 17 months.“To our surprise, we noticed after a few months

that some heart disease patients who did not have Fab-ry disease did have elevated Gb3 in the urine,” saidsenior author Dr. Raphael Schiffmann, medical directorof the institute of metabolic disease at the Baylor Re-search Institute. “Simultaneously, we also found thatsome of those patients had died in the short intervalthat had passed since we had last seen them for thisscreening study. This was a very surprising, yet encour-aging, discovery, given the fact that Gb3 elevation was,until now, thought to be the exclusive hallmark of Fab-ry disease. Remarkably, this biomarker is significantlydifferent from existing ones and could be of great sig-nificance for the future study of heart disease.”

The study was published in the February 4, 2014,online edition of the Journal of the American HeartAssociation.

Elevated Urinary Globotriaosylceramide Suggested as Heart Disease Biomarker

Prognostic Tool Predicts Pediatric Septic Shock Mortality Risk

AApowerful set of software tools is now avail-able to simplify the use of automated PCR

workstations for both the clinical and researchlaboratory. The Swiss instrument manufacturerTecan (Männedorf, Switzerland; www.tecan.com) has launched the TouchTools PCR Wizard,an easy-to-use add-on for the Freedom EVO fami-ly of workstations, which offers straightforwardautomation of PCR reaction set-ups.

The Tecan Freedom EVO series offers workta-bles with building-block modularity that ensuresprecision, reliable liquid handling, and easy-to-userobotics. Each platform can be combined with awide choice of robotic arms, liquid handling tools,and application options powered by straightfor-ward software that can be programmed to meetthe needs of each individual laboratory. The EVOplatform allows a choice of pipetting technologieson the same platform, including the possibility ofcombining both air and liquid displacement on a

single workstation.The PCR Wizard simplifies reaction

set-up for a wide range of applications,from end-point, real-time and multi-plex PCR protocols, to sequencing,genotyping and gene expression meth-ods, as well as pathogen and mutagen-esis detection. The PCR Wizard’s intu-itive TouchTools interface helps to re-duce training time and costs for re-search and diagnostic laboratories.

Data can be exported in formatscompatible with the most popular real-time PCRcyclers from Bio-Rad (Hercules, CA, USA;www.bio-rad.com) and Life Technologies (Carls-bad, CA, USA; www.lifetechnologies.com) andoptional integrated sample tracking further in-creases process security. For maximum flexibility,

the PCR Wizard can be used in conjunction withthe Freedom EVOware Normalization Wizard,providing simple, user-oriented solutions for theentire genomics workflow.

Image: The Freedom EVO workstation (Photo cour-tesy of Tecan).

New Software Suite Simplifies Automated PCR Setup

Combined Screening Improves Detection of ALK Rearrangements

In Non-Small-Cell Lung Cancer

AAnaplastic lymphoma kinase (ALK) rearrange-ments occur in 1% to 7% of non-small-cell lung

cancers (NSCLCs), but nearly 25% of samples fromNSCLC patients with ALK rearrangements were notidentified when analyzed by either fluorescent in situhybridization (FISH) or immunohistochemistry (IHC).

Investigators at the Université de Rennes(France; www.univ-rennes1.fr) reported resultsfrom a large series of parallel FISH and IHC ALKtesting in 3244 consecutive NSCLC cases analyzedat two independent French centers. The results re-vealed FISH-positive and/or IHC-positive results in150 of the 3244 cases (4.6%). An imbalanced sexratio was detected, with women exhibiting a 2.2-fold relative risk for an alteration. Strikingly, only 80of 150 specimens were classified as ALK positive byboth techniques. The specimens with discordantFISH/IHC analyses were FISH-positive/IHC-nega-tive (36), FISH-negative/IHC-positive (19), or FISH-noncontributive/IHC-positive (15). Thus, a singleFISH or IHC analysis performed alone would havefailed to detect approximately one-fourth of theALK-positive cases.

The drug crizotinib, an ALK inhibitor, has beendemonstrated to provide dramatic clinical benefitsin ALK-positive advanced-stage NSCLC. “Data oncrizotinib response in patients who have been diag-nosed differently by FISH and IHC are still prelimi-nary,” said first author Dr. Florian Cabillic, a re-searcher at the Université de Rennes. “Thus, untillarge-scale studies in patients under therapy withcrizotinib determine which testing is the most rele-vant to predict responses to ALK inhibition, our da-ta support the need to routinely perform both analy-ses because of the difficulty in detecting thechimeric ALK protein in NSCLC and the presence offalse-negative cases for each method.”

The study was published in the March 2014 is-sue of the Journal of Thoracic Oncology.



AAteam of researchers has discov-ered and verified a set of new

blood-based biomarkers that distin-guished between healthy woman andpatients with ovarian cancer.

Prior studies have suggested thatglycans are differentially expressed in

patients with ovarian cancer versuscontrols. A team based at the Universi-ty of California Davis ComprehensiveCancer Center (Sacramento, CA, USA;www.ucdmc.ucdavis.edu/cancer) hasdiscovered a blood-based glycan bio-marker panel that may identify women

as risk for epithelial ovarian cancer(EOC), and have verified that glycanscan be used to detect this disease. Intheir report, published in the journalCancer Epidemiology, Biomarkers &Prevention, March 7, 2014 (onlineahead of print), Kim et al. describetheir glycomics approach and results.

“This is one of many papers we’vedone to see if glycans can distinguishbetween women who have ovariancancer and those who don’t,” saidstudy leader Gary Leiserowitz, chief ofthe Division of Gynecologic Oncology;“So far, the results have been consis-tent and promising.” Since glycans areoften altered when patients have can-cer, by measuring these changes theteam hopes to develop a simple bloodtest that will detects OC very early.

The team conducted a series of ex-periments to ensure their glycanmeasurements were indeed detectingcancer. “You have to do these incred-ibly rigorous validation studies, be-cause the vast majority of markersthat look favorable turn out not to bereproducible,” said Prof. Leiserowitz;“We don’t want to raise people’shopes only to find we don’t have avalid marker.”

Using mass spectrometry the re-searchers studied glycans in healthywomen (n=100) and women with ei-ther early (n=52) or late (n=147)stage EOC. The first test, the “train-ing set,” measured glycan expressionin the serum samples and helped de-termine which glycans would helpthem differentiate between thegroups. The candidate glycan-basedbiomarker panel developed with thetraining set distinguished women

with EOC from healthy controls with86% sensitivity, 95.8% specificity.

Then an independent “test set”was conducted, testing entirely newpatient samples. These results showeddetection of EOC with 70% sensitivi-ty, 86% specificity, including both ear-ly and late stage EOC. The glycancandidate markers distinguished be-tween healthy and early-stage EOC asaccurately as the current standard di-agnostic blood test CA-125.

Because sample selection can biasresults, they then swapped samples,creating a new training set with thepatients from the previous testing set,and vice versa. The results showedthat the method works well, and thatthe developed markers are robustenough to be not overly influencedby patient selection. So althoughsuch tests tend to vary in their resultsdepending on which patients are test-ed, in testing on different patientsample sets, these glycan markerscontinued to show promise as a diag-nostic test for EOC.

“We take all these rigorous step-wise approaches to eliminate the pos-sibility of bias,” said Prof. Leis-erowitz. “This paper establishes thatthese are consistent and reproduciblefindings. This is a real phenomenon.”Nevertheless, the mechanisms be-hind the glycan changes are not un-derstood – while the changes couldbe caused by cancer, they might alsorepresent the body’s reaction to can-cer – an inflammatory response, forexample. The researchers cautionthat additional study and validation isneeded before the markers are readyfor clinical use.

Blood-Based Biomarkers for Risk of Ovarian Cancer

AAnew method has been developed for rapid di-agnostic detection and antibiotic susceptibility

determination of the pathogenic Bacillus anthracisusing a bioluminescent reporter phage.

Although anthrax is a treatable disease, positivepatient prognosis is dependent on rapid diagnosisand therapy. A team at the University of Missouri(MU; Columbia, MO, USA; www.missouri.edu) as-sessed a bioluminescent reporter phage, developedby David Schofield at Guild BioSciences (Charles-ton, SC, USA; www.guildbiosciences.com), for itsvalue as a clinical diagnostic tool for Bacillus an-thracis. The reporter phage based method, pub-lished in the Journal of Microbiological Methods(November 2013), detects live B. anthracis strainsby transducing a bioluminescent phenotype. Itwas found to rule out false positives – displayingspecies specificity by its inability, or significantlyreduced ability, to detect members of the closely

related Bacillus cereus group and other commonbacterial pathogens.

The method detects low levels of B. anthracis,at clinically relevant bacterial concentrations,within 5 hours. “Normally to identify whether anorganism is present, you have to extract the mate-rial, culture it, and then pick colonies to examinethat might turn out to be anthrax bacteria,” saidProf. George Stewart, PhD, medical bacteriolo-gist. “Then you conduct chemical testing whichtakes some time – a minimum of 24 to 48 hours.Using this newly-identified method, we can re-duce that time to about 5 hours.” The method al-so provides antibiotic susceptibility informationthat mirrors the CLSI method, except that dataare obtained at least 5-fold faster.

In addition to saving lives, the new methodcould also save on high clean up and decontamina-tion costs of bioterrorism attacks. These costs for

the post-9/11 2001 anthrax letters attack totaledUSD 3.2 million, according to a 2012 report. “Inthe years since the post-9/11 postal attacks, wehaven’t had any bona fide anthrax attacks,” saidProf. Stewart; “That doesn’t mean that it’s not go-ing to happen, we just have to be prepared.”

Image: The most common forms of transmission foranthrax bacteria, a rod-shaped culture, are throughabrasions in the skin and inhalation (Photo courtesy ofthe University of Missouri).

Method Identifies Anthrax Bacteria Faster Than Current Approaches





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MMany patients treated for earlyLyme disease incur another

infection in subsequent years, sug-gesting that previous exposure toBorrelia burgdorferi may not elicit aprotective immune response.

At least 16 different strains of theLyme disease bacterium have beenshown to infect humans in the Unit-ed States of America, so being bittenby a tick carrying a different strain ofthe disease is entirely possible.

Scientists at the University ofPennsylvania (Philadelphia, PA,USA; www.upenn.edu) and theircollaborators analyzed the occur-rence of identical strains of B.burgdorferi in a cohort of 17 pa-tients with multiple episodes of cul-ture-confirmed erythema migrans.They used both multinomial proba-bility analyses and a stochastic simu-lation model to evaluate whetheronly one or fewer of 17 patientscould have identical strains of B.burgdorferi in recurrent infectionsdue to chance alone. All analyses es-timated the probability of recovering

a particular strain of B. burgdorferifrom a patient due to chance alone,based on empirical patient data.

The model allowed the re-searchers to vary assumptions suchas the presence or absence of type-specific immunity, the duration of im-munity, and the length of time a pa-tient was accessible to having beenbitten by a tick, or in other words,the time from the first visit to theclinic to the last visit, or from the firstvisit to the completion of the study.

The results of all of the simula-tions indicated that strain-specificimmunity would need to last a min-imum of four years in order to resultin the suite of infections that the 17patients acquired. When the modelparameters were used with actualdata from 200 patients who hadbeen infected at least once with aknown strain of B. burgdorferi, thesimulation indicated that immunitylasts in the range of six to nineyears. The only patient infected bythe same strain twice actually hadLyme disease four times in six years,

contracting K strain twice, five yearsapart, with an infection by a differ-ent strain in between.

The authors concluded that it ishighly unlikely that only one of 17patients would have been infectedwith an identical strain of B.burgdorferi in a recurrent episode ofLyme disease in the absence ofstrain-specific immunity. Further-more, the duration of strain-specificimmunity needed to be at least fouryears to explain the data actually ob-served. The presence and long dura-

tion of strain-specific immunity thatthe models suggest imply that hu-mans, once infected, are highly un-likely to acquire a subsequent infec-tion caused by the same strain of B.burgdorferi.

The study was published in theApril 2014 issue of the journal In-fection and Immunity.

Image: Research shows that humansappear to develop strain-specific im-munity to Borrelia burgdorferi, thebacteria that causes Lyme disease(Photo courtesy of the CDC).

Strain-Specific Immunity Discovered for Lyme Disease

Assay Detects Tumor-Specific Gene Amplifications in ctDNA

AA n assay for clinical use detectsamplifications in DNA shed

from tumors into the circulation.Personal Genome Diagnostics

Inc. (PGDx; Baltimore, MD, USA;www.personalgenome.com) intro-duced its METDetect Assay for thedetection of MET gene amplifica-tions in the circulation of cancer pa-tients. The assay utilizes the compa-ny’s PARE technology to uniquelyidentify structural alterations in theMET gene in circulating tumor DNA(ctDNA) directly from the patient’splasma, thereby eliminating theneed for invasive and costly tumorbiopsies.

The METDetect test uses nextgeneration sequencing (NGS) andPGDx’s proprietary high sensitivityPARE and other technologies toidentify focal amplifications of theMET gene that can help predictionof therapeutic response, overallprognosis and cancer recurrence,and for ongoing monitoring. Analy-ses take two to three weeks. Theyare performed in PGDx’s clinical lab-oratory improvement amendments(CLIA) laboratory certified for highcomplexity clinical testing. A com-prehensive report includes identifi-cation and schematic representationof tumor-specific MET amplifica-tions, annotation of predicted muta-tion consequences, data summarystatistics and integrated analysis re-

porting. The assay is immediatelyavailable for clinical and investiga-tive use.

PGDx’s proprietary PARE tech-nology enables whole genome iden-tification of changes in tumor-specif-ic ctDNA. Unlike other approaches,which can primarily detect pointmutations in ctDNA, PARE can alsodetect structural changes, includingthe genomic amplifications and re-arrangements that are critical forguiding cancer treatment. PARE wasinvented in the laboratories of PGDxco-founders Dr. Victor Velculescuand Dr. Luis Diaz at Johns HopkinsUniversity (Baltimore, MD, USA,www.jhu.edu) and PGDx has li-censed exclusive rights to the PAREtechnology from Johns Hopkins.

PGDx reported that its propri-etary PARE and other related ge-nomic technologies were used in astudy published in the February2014 issue of the journal ScienceTranslational Medicine. The studyassessed the utility of plasma-basedcell-free circulating tumor DNA forcancer detection and monitoring,and compared it to DNA analysesbased on tumor biopsies. The au-thors concluded that ctDNA is abroadly applicable, sensitive andspecific biomarker that can be usedfor a variety of clinical and researchpurposes in patients with differenttypes of cancer.

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INOCULATION SYSTEMBioMérieux

The PREVI Isola automated specimen inoculationsystem maximizes bacteria colony isolation, andstandardizes plate inoculation and results. Thesystem offers a throughput of 180 plates per hour,and it is designed specifically for no cross contam-ination and streamlined workflow.

CLINICAL CHEMISTRY ANALYZERTokyo Boeki Machinery

The Biolis 50i Superior is designed to measure480 tests per hour, or 580 tests per hour with ISE.Key features of the open system include HbA1cautomatic preparation, clot detection, user-friend-ly interface, multilingual software, and a dedicatedISE probe.

HbA1c SYSTEMCaretium Instruments

The KH-101 walkaway system offers reliable re-sults by HPLC method, and one-step operation(with automated addition of lyse and lyse at hightemperature). Feature highlights include a color,touch screen interface, autosampler, built-in print-er, and results available in five minutes.

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strategies to prevent, reverse, or slow disease pro-gression. In a collaborative study between severalinstitutions, mainly Georgetown University Med-ical Center (GUMC; Washington DC, USA;http://gumc.georgetown.edu) and University ofRochester School of Medicine (URSM; Rochester,NY, USA; www.urmc.rochester.edu/smd), a blood-based biomarker panel has been discovered thatmay identify preclinical Alzheimer’s disease. In thereport, published in the journal Nature Medicine,March 9, 2014, online ahead of print, Mapstone etal. describe their lipidomic approach examiningcognitively normal senior adults. They discovered

and validated a set of lipids from peripheral bloodplasma that predicted with over 90% accuracy thephenoconversion within 2-3 years from cognitivelynormal to either amnestic mild cognitive impair-ment (aMCI) or on to Alzheimer’s disease (AD).

The study has yielded the first blood test that ac-curately identified individuals at risk for developingAD. The discovery could be a key to unlocking anew generation of treatments that seek to head offthe disease before neurological damage becomes ir-reversible. “Our novel blood test offers the potentialto identify people at risk for progressive cognitive de-cline and can change how patients and their fami-lies, and treating physicians plan for and manage thedisorder,” said corresponding author Howard J.Federoff, MD, PhD, professor of neurology at theGUMC. Some of the same experimental treatmentsthat have thus far failed may prove to be successfulif they are given to high-risk patients much sooner.

Biomarkers of preclinical disease with high sensi-tivity and specificity are critical. Current biomarkersand methods used in attempt to detect early diseaseare very limited. For widespread use and large-scalescreening, blood-based biomarker screening would bemore attractive and may also be more effective.

The study included 525 asymptomatic partici-

pants aged 70 years and older. In the 5-year study, 74participants met the criteria either for mild AD or foraMCI. Of these, 46 were diagnosed already upon en-rollment and 28 developed aMCI or mild AD duringthe study (those of the latter group were called con-verters). In the third year, 53 participants who devel-oped aMCI/AD (including 18 converters) and 53cognitively normal matched controls were selectedfor the lipid biomarker discovery phase of the study.

Using mass spectrometry, 10 lipids were identifiedthat, if present in lower than normal blood plasma lev-els, predicted with more than 90% accuracy whetheran individual would go on to develop AD or aMCI. All10 are phospholipids. The 10-lipid panel was validatedusing the remaining 21 aMCI/AD participants (in-cluding 10 converters), and 20 controls. The lipid pan-el again distinguished with 90% accuracy.

The study also examined the APOE4 gene, anaccepted risk factor for AD, but it was not a signifi-cant predictive factor in this study.

“The ability to identify individuals who are atrisk of developing Alzheimer’s before the clinicalmanifestation of cognitive impairment has longbeen a Holy Grail of the neuromedicine communi-ty,” said lead author Mark Mapstone, PhD, neu-ropsychologist at the URSM.

Blood Test Offers Earlier Alzheimer’s Diagnosis

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fundamental problem with CA125 is that it is notcancer-specific and may be elevated in benign gy-necological conditions such as benign ovarianneoplasms and endometriosis.

Investigators at the University of Copenhagen(Denmark; http://ku.dk) based the developmentof a new assay system for glycoprofiling ovariancancer on the observation that aberrant O-glycosy-lation was an inherent and specific property ofcancer cells and could aid in differentiating cancerfrom these benign conditions, thereby improvingspecificity of the assay.

They developed a novel microarray-based plat-form for profiling specific aberrant glycoforms,

such as Neu5Acalpha2, 6GalNAc (STn) andGalNAc (Tn), present on CA125 and CA15-3.They used the assay to measure STn-CA125, ST-CA125, and STn-CA15-3 in a blinded study of acohort of patients from the United Kingdom Ovar-ian Cancer Population Study who presented withelevated CA125 levels and a pelvic mass.

Results revealed that the combined glycoformprofile was able to distinguish benign ovarian neo-plasms from invasive epithelial ovarian cancerwith a specificity of 61.1% at 90% sensitivity.

“All proteins have a type of sugar-coat – small,complicated sugar molecules that reside on a pro-tein’s surface. When cancer is present in the body,we can observe a chemical change in this sugar-

coat. It is a very complex phenomenon. Luckily, itis very simple to investigate and determine thepresence of this transformed sugar coating,” saidsenior author Dr. Ola Blixt, professor of chemistryat the University of Copenhagen. “We have decid-ed to publish these results, as opposed to taking outa patent. OK, we will not get rich, even though themarket related to ovarian cancer is worth 170 mil-lion USD annually. On the other hand, any manu-facturer is able to include this in their existing kit.And I hope that it happens soon.”

The study describing the ovarian cancer glyco-profiling assay was published in the January 29,2014, online edition of the Journal of ProteomeResearch.

Microarray Glycoprofiling Test for Ovarian Cancer


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AAsimple protein test could prove more useful inpredicting survival chances for patients with

head-and-neck cancer compared to existing methods.There is an increasing incidence of human papillo-mavirus (HPV)-positive oropharyngeal squamous cellcancers (OPSCC) mostly associated with favorableoutcomes and a tumor suppressor protein could be asurrogate marker for HPV positivity in OPSCC.

Scientists at the University of Manchester (UK;www.manchester.ac.uk) collected formalin-fixed,paraffin-embedded blocks from hospitals where217 patients were being treated for OPSCC. Oneapproach for detecting HPV-associated oral cancerrelies on finding HPV DNA in the tumor sample,but these DNA-based tests may not accurately clas-sify the tumor. Another approach is to use a mark-er of HPV rather than testing for HPV DNA direct-ly. The p16 protein usually disappears in tumors

that are not caused by HPV infection and has beenproposed as a surrogate marker of HPV.

Blocks with confirmed tumor presence were se-lected for immunohistochemistry (IHC), whichwas carried out using the CINtec histology kit(MTM Laboratories, Heidelberg, Germany;www.mtmlabs.com). Stained slides were scannedusing the Leica SCN 400 (Wetzlar, Germany;www.leica-microsystems.com). The cyclin-depend-ent kinase inhibitor protein p16 expression wasscored as positive if there was a strong and diffusebrown staining of the nucleus and cytoplasm inequal to or greater than 70% of the tumor speci-men. Of the 92% of the OPSCC originating fromtonsil and tongue base sites, 61% were p16 posi-tive.

The study was published in the November 2013issue of the journal Clinical Oncology.

Protein Test Improves Survival Prediction For Oropharyngeal Carcinoma

TThe relationship between elevated cholesteroland increased risk of Alzheimer’s disease has

been specifically linked to amyloid deposits in liv-ing human study participants.

Higher levels of high-density lipoprotein cho-lesterol (HDL-C) and lower levels of low-densitylipoprotein cholesterol (LDL-C) in the blood-stream are associated with lower levels of cerebralβ-amyloid (Aβ) plaque deposits in the brain.

Scientists at the University of California,(Davis, CA, USA; www.ucdmc.ucdavis.edu) re-cruited 74 men and women aged 70 and overwho were attending the Alzheimer’s Disease Cen-ter, stroke clinics, and community senior facilities.The group included 3 people with mild dementia,38 with mild cognitive impairment, and 33 whowere cognitively normal. All the participants hadfasting blood tests and cerebral Aβ, was measured

with carbon 11C–labeled Pittsburgh Compound B(PIB) positron emission tomography where amy-loid plaques were highlighted using a radioactivetracer that binds to them.

Elevated cerebral Aβ level was associated withcholesterol fractions in a pattern analogous to thatfound in coronary artery disease. The investiga-tors found that higher levels of LDL cholesteroland lower levels of HDL cholesterol were linkedto more amyloid plaques in the brain. The find-ings were independent of age or presence of theE4 variant of the apolipoprotein E (ApoE) gene,which has been linked to some forms ofAlzheimer’s. The mean fasting total cholesterollevel for the group was 171 mg/dL, and mean lev-els for the LDL-C and HDL-C were 92 and 54

mg/dL, respectively.Bruce Reed, PhD, the lead author said, “This

study provides a reason to certainly continue cho-lesterol treatment in people who are developingmemory loss, regardless of concerns regardingtheir cardiovascular health. It also suggests amethod of lowering amyloid levels in people whoare middle aged, when such build-up is just start-ing. If modifying cholesterol levels in the brainearly in life turns out to reduce amyloid depositslate in life, we could potentially make a significantdifference in reducing the prevalence ofAlzheimer’s, a goal of an enormous amount of re-search and drug development effort.” The studywas published on December 30, 2013, in thejournal JAMA Neurology.

Serum Cholesterol Levels Linked to Cerebral Amyloidosis

AAnew blood test is being developed that canrapidly and accurately diagnose celiac disease

without the need for prolonged gluten exposure.The diagnostic test gave a result within 24 hours

and preliminary findings indicated it could accurate-ly detect celiac disease and it is hoped that largerstudies will verify its role as a widely used tool for di-agnosing celiac disease (CD). Scientists at the Walterand Eliza Hall Institute (Melbourne, Australia;www.wehi.edu.au) recruited 27 patients being treat-ed for CD, four with CD but were untreated and 17non-CD controls. Blood for serology and cytokine re-lease assays was drawn in the morning immediatelybefore (d0) and on day six (d6) after commencinggluten challenge, or prior to commencing a gluten-free diet (GFD) in untreated CD patients.

Serum transglutaminase (tTG-IgA), deamidatedgliadin peptide (DGP)-immunoglobulin (Ig)A andDGP-IgG were evaluated with commercial kits (IN-OVA Diagnostics; San Diego, CA, USA; www.inovadx.com). Plasma interferon gamma (IFN-γ)levels on d0 and d6 were measured in triplicate byenzyme-linked immunosorbent assay (ELISA) andmeasured on an automated ELISA reader. The in-vestigators also performed the IFN-γ ELISPOT assayand spot-forming units (SFU) in individual wells

were counted using an automated AID ELISPOTreader system (Autoimmun Diagnostika GmbH;Strasberg, Germany; www.aid-diagnostika.com)and results expressed as SFU per 106 peripheralblood mononuclear cells (PBMC).

The authors concluded that the whole blood cy-tokine release assays appear to be sensitive and spe-cific potential diagnostic test for CD patients fol-lowing GFD. As an added benefit over current di-agnostic tests being performed on patients alreadyfollowing GFD, the mobilization of gluten-reactiveT-cells specific for CD into the bloodstream requiresoral gluten challenge for only three days instead ofthe weeks or months required for diagnosis basedon abnormal small bowel histology.

Jason Tye-Din, MBBS, PhD, FRACP, a gastroen-terologist and a coauthor of the study said, “A testthat simplifies diagnosis for patients is likely to sig-nificantly enhance disease detection. This new di-agnostic approach is encouraging and we hope thatlarger studies can validate these findings and estab-lish its role in the diagnosis of celiac disease, withthe possibility of avoiding intestinal biopsies for di-agnosis altogether.” The study was published onJanuary 3, 2014, in the journal Clinical & Experi-mental Immunology.

Blood Test to Simplify Celiac Disease Diagnosis

AA team of bioengineers has developed a simple,hand-held device that enables diagnosis of

asthma from a single drop of blood.The “kit-on-a-lid-assay (KOALA)” microfluidic

technology used to perform the test is the brain-child of investigators at the University of Wisconsin(Madison, USA; www.wisc.edu). KOALA capital-izes on differences in neutrophil function to dis-criminate asthma from allergic rhinitis. Neutrophilsare inflammatory immune system cells that havebeen implicated in the pathogenesis of asthma. Thehandheld KOALA device sorts neutrophils fromwhole blood within five minutes due to a gradientof chemoattractant that is generated in the device’smicrochannels when a lid with chemoattractant isplaced onto the base of the device.

KOALA technology was used in a clinical set-ting to assay 34 asthmatic (n=23) and nonasth-matic, allergic rhinitis (n=11) patients to establishdomains for asthma diagnosis based on neutrophilchemotaxis. The investigators determined thatneutrophils from asthmatic patients migrated sig-nificantly more slowly toward the chemoattrac-tant compared with nonasthmatic patients.

“What we have done in this paper is presenteddata that neutrophil cell function in some casescan predict – and in this case actually predicted

and measured – whether someoneis asthmatic or not,” said senior au-thor Dr. David Beebe, professor ofbiomedical engineering at the Uni-versity of Wisconsin. “This is one ofthe first studies to show that thisprocess could actually work in acheap, easy, and practical way.”

“The KOALA platform repre-sents the next-generation biomedical researchkit,” said Dr. Beebe. “Instead of getting a box ofmedia and staining solution and having to do alot of manual manipulation, you would get thebase for the fluid sample, the prepackagedKOALA lids, and to do any testing, just place alid (or series of lids) on the base.”

The study describing the use of KOALA waspublished in the April 7, 2014, online edition of

the journal Proceedings of the National Academyof Sciences of the United States of America(PNAS).

Image: Researchers developed the kit-on-a-lid-as-say (KOALA) microfluidic technology to detect neu-trophils using just a single drop of blood. The tech-nology is intended to offer a faster, cheaper andmore accurate tool to diagnose mild cases of asthma(Photo courtesy of the University of Wisconsin).

Five-Minute Kit-on-a-Lid Assay Detects Asthma from Whole Blood

Immunoassay DetectsSyphilis and Assists

Treatment Monitoring

AAnew syphilis immunoassay assay enables detec-tion of total antibodies to Treponema pallidum

subspecies pallidum (detection of IgG & IgM antibod-ies against TpN15, TpN17 & TpN47).

Launched by Roche (Basel, Switzerland; www.roche.com) and dubbed the Elecsys Syphilis assay, itcomplements the Mediace Rapid Plasma Reagin(RPR) and T. pallidum Latex Agglutination (TPLA)Roche assays. The Immunoassay is a diagnostic testto help detect patients infected with syphilis in rou-tine clinical practice and to make sure donated bloodis not infected with syphilis. The assay demonstrates100% sensitivity and 99.88% specificity with perfectdiscrimination of results, eliminating the need for agrey zone, and delivering confidence in all stages oftreponemal infection.

For over 25 years Roche has invested in the re-search and development of serology analyzers andassays so that, today, it offers one of the most com-prehensive infectious diseases assay portfolios avail-able on a single automated platform. With the newElecsys Syphilis assay, the Roche Diagnostics divisionstrengthens its position in the serology market andexpands its immunoassay portfolio in infectious dis-ease. Designed for the emerging needs of clinical lab-oratories for reliable and efficient detection of thisdisease, the test enhances Roche’s solutions portfolioin serology testing – the testing of antibodies formedas a response to an infection – and complements themost comprehensive offering for blood safety avail-able on the market today.






TThe automated isolation of cells directly fromwhole blood or buffy coat is based on magnet-

ic bead technology.DiaSorin (Saluggia, Italy; www.diasorin.com)

has launched LIAISON Ixt/Arrow CellSep Ad-vanced, which can isolate up to 3 cell types persample in just 32 minutes per cell type, offeringsignificant time savings compared to manual cellseparation methods.

CellSep Advanced is designed for use on theLIAISON Ixt/Arrow instrument for the separationof 1, 2, or 3 cell types in up to 12 samples at atime. Requiring minimal hands-on interventionCellSep Advanced produces good yields of puri-fied cell preparations ready for use in a wide rangeof downstream applications, including lineage-specific chimerism analysis. The Ixt/Arrow auto-mated platform also minimizes human error and

ensures reproducibility between runs for consis-tent and reliable downstream analysis.

The LIAISON Ixt/Arrow instrument can alsobe used for DNA extraction using the Ixt/ArrowBlood DNA extraction kit, ensuring streamlinedprocessing of samples and, by reducing instru-mentation requirements, saving valuable benchspace in the laboratory.

DiaSorin is a leader in the field of biotechnolo-gy. For over 40 years the company has been devel-oping, producing, and marketing reagent kits forin vitro diagnostics worldwide. With their mostrecent acquisition of the NorDiag Group, DiaSoringains access to the nucleic acid isolation and cellseparation markets. Its line of products used by di-agnostic laboratories that are part of hospital facil-ities or operate independently (private diagnosticservices laboratories) can meet the needs of the

following clinical areas: infectious diseases, car-diac markers, bone metabolism, hepatitis andretrovirus, oncology, and endocrinology.

Image: The LIAISON Ixt/Arrow CellSep Advanced,designed for the automated isolation of cells directlyfrom whole blood or buffy coat. It can isolate up tothree cell types per sample in just 32 minutes per celltype, offering significant time savings compared tomanual cell separation methods (Photo courtesy ofDiaSorin).

Automated Cell Separation Achieved For Three Cell Types Simultaneously

Blood Culture DiagnosticTechniques Compared for

Human Brucellosis

II solation of the bacteria Brucella is the gold stan-dard in the laboratory diagnosis of brucellosis and

as the organism is intracellular, the number of circu-lating bacteria is usually low. Three different bloodculture methods, the lysis concentration (LC), clotculture and conventional Castaneda blood culturetechniques, have been compared for the isolationrate and recovery time in the diagnosis of humanbrucellosis. Microbiologists at the Shri B. M. PatilMedical College (Bijapur, Karnataka, India; www.bldeuniversity.ac.in) performed blood cultures byLC, clot culture and conventional method in 169 pa-tients who had antibody titers equal to or greaterthan 160 international units by the serum agglutina-tion test (SAT).

For the conventional culture technique, the bloodspecimen was inoculated aseptically into the brothphase of Castaneda’s biphasic medium consisting ofbrain heart infusion agar and broth with Brucella se-lective supplement (Hi-Media; Mumbai, India;www.himedialabs.com). For the LC technique, themixtures centrifuged and the supernatant was dis-carded, and the sediment was inoculated in the Cas-taneda’s medium, instead of culture plate.

Provisional confirmation and biotyping of the iso-late was done by performing slide agglutination testusing B. abortus and B. melitensis monospecific antisera (Murex Biotech; Dartford, UK; www.abbott.co.uk).

The authors concluded that for the isolation ofBrucella from blood specimen, LC method is betterthan conventional Castaneda’s method as the isola-tion rate is high and the recovery time is less. Clotculture is a better option when a second blood sam-ple cannot be obtained for culture. As lysis and clotculture techniques are sensitive, simple and inexpen-sive and yield earlier results, they can be adapted inthe technically and economically deprived areaswhere automated systems are not feasible. The studywas published on March 19, 2014, in the Journal ofLaboratory Physicians.

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AAnalysis of tumor and whole blood DNA bywhole genome sequencing has identified the

fusion of two genes as the genetic driver in an ag-gressive tall cell variant of papillary thyroid cancer.

Recent advances in the treatment of cancerhave focused on targeting genomic aberrationswith selective therapeutic agents, but in radioio-dine resistant aggressive papillary thyroid cancers,there remain few effective therapeutic options.

Scientists at the Translational Genomics Re-search Institute (Phoenix, AZ, USA; www.tgen.org) obtained a blood and tumor sample fromthe patient, a 62-year-old man who underwentmultiple operations for papillary thyroid cancerand whose metastases progressed despite stan-dard treatments. Whole genome sequencing of tu-mor and white blood cell DNA was conducted bya commercial vendor according to their methodsusing photolithographically patterned high-densitynanoarrays onto which palindrome-promotedcoils of single-stranded DNA are absorbed (Com-plete Genomics; Mountain View, CA, USA;www.completegenomics.com).

Tumor paraffin blocks were submitted toARUP Laboratories (Salt Lake City, UT, USA;www.aruplab.com) for validation by reverse tran-scriptase polymerase chain reaction (RT-PCR) in aClinical Laboratory Improvement Amendments(CLIA) certified laboratory. A comparison of thetumor DNA to the patient’s normal DNA found57 mutations in 55 genes of the cancer genome.The investigators also found a rearrangement be-tween two genes. This translocation and fusion ofthe echinoderm microtubule associated proteinlike 4 (EML4) gene and the anaplastic lymphomareceptor tyrosine kinase (ALK) gene, was identi-fied as the genetic driver of the patient’s cancer.

Michael J. Demeure, MD, the study’s principalinvestigator and lead author, said, “This is the firstreport of the whole genome sequencing of a pap-illary thyroid cancer, in which we identified anEML4-ALK translocation. This is important be-cause we have a drug that can target this fusionand treat the patient. This patient’s tumor did notharbor more well-known gene mutations that areassociated with most thyroid cancers. These find-

ings suggest that this tumor has a distinct oncoge-nesis, or the genetic cause of cancer.” The studywas published on March 15, 2014, in the WorldJournal of Surgery.

Image: Tall cell variant of papillary carcinoma of thethyroid - (Photo courtesy of the PSCO).

Gene Fusion Identified as Cause of Rare Thyroid Cancer

AA study has found no significant rates of antibod-ies against Middle East respiratory syndrome

coronavirus (MERS-CoV) in one Middle East-NorthAfrica (MENA) region, suggesting that the virus hasnot been circulating in humans for long, and that themajority of the population remains susceptible to in-fection. The results of the study by Aburizaiza et al.,was published in the January 2014 issue of the Jour-nal of Infectious Diseases, (Vol. 20 (2)).

The study involved 130 blood donors and 226slaughterhouse workers from Jeddah and Makkah(Saudi Arabia) sampled during 2012. Serum sampleswere analyzed using the recommended staged sero-logical approach: screening of IgG and IgM antibod-ies by conventional IFA based on MERS-CoV-infect-

ed and non-infected cells (EUROIMMUN AG;Lübeck, Germany; www.euroimmun.de), followedup by confirmation by discriminative recombinantIFA based on viral spike proteins and plaque-reduc-tion neutralization assay. Only eight sera were posi-tive in the screening test, and these reactions weresubsequently resolved to be specific for establishedcoronaviruses. These results highlight the impor-tance of multistage serological testing. Nevertheless,the level of cross reactivity with other human coro-naviruses in the screening test is relatively low. Sig-nificantly, these results demonstrate an absence ofpopulation immunity in the region and at the sam-pling timepoint.

MERS-CoV is an emerging pathogen which is re-

sponsible for an outbreak of severe acute respiratoryillness predominantly in the Arabian Peninsula witha high number of fatalities. From September 2012 toMarch 2014, there have been 206 laboratory-con-firmed cases with 86 deaths (www.who.int). Thetransmission mechanisms of the virus remain un-known, and animal reservoirs are suspected to playa role. In a further serological study, antibodiesagainst MERS-CoV were detected in a majority ofdromedary camels sampled in the United Arab Emi-rates in 2003 and 2013 (Meyer et al., Emerging In-fectious Diseases, Vol. 20 (4), April 2014). The highantibody prevalence suggests a potential role forcamelids in the emergence and spread of the virus inhumans.

MERS-CoV Immunity Not Widespread

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HPV TESTCepheid

The Xpert HPV assay is a qualitative real-timePCR test for automated and rapid detection of Hu-man Papillomaviruses. The easy-to-use test isscalable and offers rapid turnaround time, with ac-curate and reliable results available in less than60 minutes.

AUTO-CHEMISTRY SYSTEMDirui Industrial

The CS-6400 features a cost-effective design,and an optional ISE module that provides userswith flexible choices. The throughput of each pho-tometric module is 1600 T/H (480 T/H with ISE),with a maximum of 7360 T/H with four photomet-ric modules and two units of ISE module.

PCR ANALYZERFosun Diagnostics

The LM2012 real-time analyzer features six differ-ent fluorescent channels, along with a rotary de-sign that provides high-performance thermal con-trol and uniformity. The system offers easy instal-lations and maintenance, and the system soft-ware is multifunctional for calibration and control.

AA flexible, fully automated, random access mo-lecular diagnostics system for the quantitative

and qualitative analysis of molecular targets fromclinical patient specimens has been developed.

The VERIS system developed by Beckman Coul-ter Diagnostics (Brea, CA, USA; www.beckmancoulter.com) integrates key steps in molecular diag-nostics to streamline workflow and system manage-ment, while processing critical STAT samples andensuring prompt delivery of results. By providingcontinuous access, one-step loading, and individualtest reporting, VERIS helps medical laboratory pro-fessionals advance and optimize the molecular diag-nostics lab, by providing the control and freedom togive the right answer at the right time – for patients

and physicians.The CMV assay is a polymerase chain reaction

(PCR) assay designed for the quantitative determi-nation of CMV deoxyribonucleic acid (DNA) fromhuman plasma. When used in conjunction withclinical presentation and other laboratory findings,the CMV assay aids in monitoring CMV viral load.

Beckman Coulter Diagnostics obtained the CEmarking for the VERIS MDx System and VERIS hu-man Cytomegalovirus (CMV) assay: a key mile-stone in the expansion of Beckman Coulter’s pres-ence in molecular diagnostics.

Beckman Coulter is committed to the ongoingdevelopment of assays to expand the VERIS infec-tious disease portfolio and plans to submit for CE

marking on assays for Human ImmunodeficiencyVirus (HIV), Hepatitis C virus (HCV) and HepatitisB virus (HBV) in 2014.

Richard Creager, senior vice president, molecu-lar diagnostics business unit, and CSO at BeckmanCoulter Diagnostics, commented, “After extensiveresearch and development, Beckman Coulter hasapplied its expertise in diagnostics with ourin depth knowledge of workflow to bring molecu-lar diagnostics to the clinical laboratory. We havespent tremendous effort on understanding theneeds of molecular laboratory professionals to de-velop a system that simplifies molecular diagnostictesting, while delivering the results that patientsand clinicians need.”

Assay Quantifies CMV Deoxyribonucleic Acid from Human Plasma

AAnovel microarray-based technique uses DNAmethylation to simultaneously quantify mul-

tiple leukocyte subsets, enabling the investigationof immune modulations in both fresh blood sam-ples and archived samples that previously couldnot be used for such analysis.

Cell lineage-specific DNA methylation patternsdistinguish normal human leukocyte subsets andcan be used to detect and quantify these subsetsin peripheral blood. However, all the currentmethods for counting immune cells in a bloodsample require whole cells, with the “gold stan-dard” methods being: manual five-part differentialcount, CBC (complete blood count) with auto-mated five-part differential and fluorescence-acti-vated cell sorting (FACS).

Investigators at Brown University (Providence,Rhode Island, USA; www.brown.edu) have devel-oped an approach using DNA methylation to simul-taneously quantify multiple leukocyte subsets with-out the need for counting whole cells.

They used the Illumina (San Diego, CA, USA;www.illumina.com) Infinium HumanMethylationand VeraCode GoldenGate Methylation microarray

assays to identify cell lineage-specific DNA methy-lation signatures that distinguished among humanT-cells, B-cells, NK cells, monocytes, eosinophils,basophils, and neutrophils. They then employed abioinformatics-based approach to quantify thesecell types in complex mixtures, including wholeblood, using DNA methylation signatures at as fewas 20 CpG (cytosine and the guanine connected bya phosphodiester bond) loci.

Applying this DNA methylation-based approachto quantify the cellular components in 80 humanwhole blood samples, they verified its accuracy bydirect comparison to gold standard immune quan-tification methods that utilized physical, optical,and proteomic characteristics of the cells. They al-so demonstrated that the approach was not affect-ed by storage of blood samples, even under condi-tions prohibiting the use of gold standard methods.

“Every kind of cell has its own methylation sig-nature,” said senior author Dr. Karl T. Kelsey, pro-fessor of epidemiology at Brown University.“Once you understand the unique and really im-mutable signature that directs the differentiationof the cell, then you can use that and you do not

need the cell anymore.”The study was published in the March 5, 2014,

online edition of the journal Genome Biology.

Image: A new technique distinguishes cell types inblood by looking at signature degrees of methylationin DNA. Yellow represents no methylation, blue fullmethylation (Photo courtesy of Kelsey Lab / BrownUniversity).

Quantization of Leukocyte Subsets With DNA Methylation Microarrays

AAnother genetic factor has now been found tocontribute to an increased risk of developing

type 2 diabetes, particularly the elevated risk amongMexican and other Latin American populations.

Upon performing a study in Mexican and Mex-ican American populations, an international teamfrom Mexico and the USA – the SIGMA (Slim Ini-tiative in Genomic Medicine for the Americas)Type 2 Diabetes Consortium – has discovered arisk-gene for type 2 diabetes that had gone unde-tected in previous efforts. Carriers of one copy ofthe higher risk allele are 25% (and carriers withtwo copies 50%) more likely to have diabetes. Thehigher risk allele was found in up to50% of the population with recent Na-tive American ancestry, including LatinAmericans. It is found in about 20% ofEast Asians, and is rare in populationsfrom Europe and Africa.

“By expanding our search to includesamples from Mexico and Latin Ameri-ca, we’ve found one of the strongest ge-netic risk factors discovered to date,”said José Florez, Broad Institute (Cam-bridge, MA, USA; www.broadinstitute.org) associate member, associate profes-sor of medicine at Harvard MedicalSchool, and assistant physician at theMassachusetts General Hospital. The el-evated frequency of this risk gene in Lat-in Americans could account for as muchas 20% of the populations’ increasedprevalence of type 2 diabetes.

A description of the discovery and ini-tial characterization of the newly impli-cated gene – named SLC16A11 – waspublished online December 25, 2013, inthe journal Nature. About 9 million sin-gle-nucleotide-polymorphisms (SNPs)were analyzed in 8,214 Mexicans andother Latin Americans: 3,848 with type2 diabetes and 4,366 nondiabetic con-trols. “We conducted the largest andmost comprehensive genomic study oftype 2 diabetes in Mexican populationsto date. In addition to validating the rel-evance to Mexico of already known ge-netic risk factors, we discovered a majornew risk factor that is much more com-mon in Latin American populations thanin other populations around the world,”said Teresa Tusie-Luna, project leader atthe Instituto Nacional de Ciencias Médi-cas y Nutrición Salvador Zubirán andprincipal investigator at the BiomedicalResearch Institute, National University ofMexico.

This is also the first time SLC16A11has been highlighted as playing a role inhuman disease, and little information isavailable about its function. The paperreveals some initial clues about its possi-ble connection to type 2 diabetes. “Oneof the most exciting aspects of this workis that we’ve uncovered a new clue

about the biology of diabetes,” said David Altshuler,deputy director and chief academic officer at theBroad Institute and a Harvard Medical School pro-fessor at Massachusetts General Hospital.

Image: A map showing relative rates of type 2 dia-betes in countries in the Americas. By studying Mex-ican and Latin American populations, the SIGMAType 2 Diabetes Consortium uncovered a new ge-netic risk factor for type 2 diabetes that could ac-count for as much as 20 percent of the populations’increased prevalence of type 2 diabetes - the originsof which are not well understood (Photo courtesy ofLauren Solomon, Broad Communications, from In-ternational Diabetes Federation).

Genetic Risk Factor for Type 2 Diabetes Uncovered




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TThe need for accurate diagnosticinvestigation of cases of leish-

maniasis has made the use of molec-ular biology techniques broadly ap-plicable for identifying the proto-zoan parasite.

The reliable application of thesemethods requires highly specializedpersonnel, dedicated equipment andspace and because of these draw-backs of the molecular diagnosticapproach, has targeted several alter-natives and the most recent ofwhich is the incorporation ofnanoparticles for the detection ofspecific bio-molecules.

Laboratory scientists at the Uni-versity of Athens (Greece, www.uoa.gr) used for the assessment ofthe method’s specificity 15 culturedisolates of various Leishmaniaspecies from human and animal ori-gin. There were 40 negative con-trols selected among pathogens ge-netically related to Leishmania spp.,commonly found in clinical samples,both human and canine, as well as

some bacterial species.DNA isolation was performed on

all samples by the Nucleospin Tissuekit (Macherey Nagel; Düren, Ger-many; www.mn-net.com). Goldnanoparticles (AuNPs, Nanopartz;Salt Lake City, UT, USA; www.nanopartz.com) were conjugatedwith four oligonucleotide probes,targeting kinetoplastid minicircleDNA of Leishmania spp. The resultswere recorded by visual observationof the test tubes or by spectropho-tometry (Infinite M200, Tecan;Männedorf Switzerland; www.tecan.com). In the absence of com-plimentary DNA, AuNPs-probes pre-cipitate under acid environmentcausing a change of color from redto purple, which can be detected byvisual observation. In the presenceof target DNA the color of the solu-tion remains red.

The assay produced consistentlypositive results detectable by simplevisual observation of the test tubeswith no less than 115 ng of leishma-

nial DNA in a sample volume of 10μL and therefore the method’s mini-mum detection limit was defined as11.5 ng of target DNA per μL ofsample. Repeatability and repro-ducibility were 100% and relativesensitivity and specificity referencedto a classical polymerase chain reac-tion (PCR) method were calculatedto 92% and 100% regarding collec-tively control and clinical samples.

The authors concluded that themethod provides an easy, fast, andinexpensive way for LeishmaniaDNA detection using AuNPs conju-

gated to four single strand oligonu-cleotide-probes. This approach canbe considered an appealing diagnos-tic solution for resource-poor set-tings, especially for screening pur-poses in enzootic areas, where de-tection of very small amounts of thetargeted analyte is not a top priority.The study was published in the Jan-uary 2014 issue of the Journal ofMicrobiological Methods.

Image: The parasite Leishmania,which forms large, slow-healing soreswhen it attacks the skin (Photo cour-tesy of Dennis Kunkel Microscopy).

Novel Non-Amplification Assay Detects Leishmania

LLevels of biomarkers linked to bone metabolism(bone formation and resorption) are indicative

of survival in patients with castration-resistantprostate cancer (CRPC), and identify a small groupof patients who can benefit from treatment withthe investigational drug atrasentan.

Prior studies have found that elevated markersof bone turnover were prognostic for poor survivalin CRPC patients. The predictive role of thesemarkers relative to bone-targeted therapy has notbeen clear. To better understand the relationshipbetween bone metabolism biomarkers and cancer

outcome, investigators at the University of Califor-nia, Davis (USA; www.ucdavis.edu) analyzed bloodserum samples from 778 CRPC patients treated ona placebo-controlled phase III trial of docetaxel withor without the bone targeted endothelin-A receptorantagonist atrasentan. The serum samples weretested for biomarkers of both resorption (N-telopep-tide, pyridinoline) and formation (C-terminal colla-gen propeptide, bone alkaline phosphatase).

Results revealed that elevated baseline levels ofeach of the markers were associated with worsesurvival, and increasing marker levels by week nine

of therapy were also associated with subsequentpoor survival. Approximately 6% of patients withthe highest marker levels responded to atrasentan,an investigational drug abandoned because it hadfailed in clinical trials.

Atrasentan is an experimental drug that is underevaluation for the treatment of various types of can-cer, including non-small-cell lung cancer. It is an en-dothelin receptor antagonist selective for subtypeA. While other drugs of this type (sitaxentan, am-brisentan) exploit the vasoconstrictive properties ofendothelin and they are mainly used for the treat-ment of pulmonary arterial hypertension, atrasen-tan blocks endothelin induced cell proliferation.

“We found that patients with high levels of thesemarkers in the blood had a much shorter lifespancompared to patients with low levels,” said seniorauthor Dr. Primo Lara, associate director for transla-tional research at the University of California, Davis.“By measuring bone turnover in prostate cancer pa-tients, we can determine how well they do.”

“Atrasentan kept coming up short in random-ized trials because the drug only works for a smallgroup,” said Dr. Lara. “Because certain drugs onlysucceed in a fraction of patients, drug makers needto factor in these bone metabolism markers in theirtrial design. They need to target the patients mostlikely to benefit. I think the days of doing empiricalstudies on all comers should end. You need to havean appropriate database of patients and perform arigorous analysis to find the subset who will benefitfrom an investigational drug.”

The study on bone metabolism biomarkers waspublished in the February 24, 2014, online editionof the Journal of the National Cancer Institute.

Bone Metabolism Biomarkers Predict Prostate Cancer Survival


30LabMedica InternationalJune-July/2014130LMI-07-14LINKXPRESS COM

AAclinical study finds a new genomic test for kidney cancer highly valu-able for diagnosis, subtyping, and outcome prediction for renal can-

cers, an important complement to tissue staining based pathology.Accurately diagnosing kidney cancer using pathology alone is challeng-

ing and can delay time to first treatment, particularly if surgical resectionsare required. Cancer Genetics, Inc. (CGI; Rutherford, NJ, USA; www.cancergenetics.com), a leader in DNA-based cancer diagnostics, presentsresults of a collaborative study conducted with the Cleveland Clinic thatvalidate CGI’s novel kidney cancer microarray test. This microarray testdemonstrated 93% diagnostic sensitivity with 99% specificity. It facilitatesthe diagnosis, subtyping, and outcome prediction for kidney cancer pa-tients. The validation study was performed using samples from 188 kidneycancer patients treated at the Cleveland Clinic. The data and analysis werepresented by Dr. Magi-Galluzzi of the Cleveland Clinic at the 2014 Unit-ed States & Canadian Academy of Pathology (USCAP) 103rd annual meet-ing in San Diego (CA, USA).

“This compelling study with Cleveland Clinic,along with initial work with Memorial Sloan Ket-tering, provides significant evidence that our tech-nology has unparalleled value as a breakthroughdiagnostic for kidney cancer,” said Panna Sharma,CEO of Cancer Genetics; “These new data comeat an opportune time, since March is Kidney Can-cer Awareness month, and our test has the abilityto both subtype and help predict outcome from asmall amount of DNA taken from either paraffin-embedded or fine needle aspirate. This is veryunique and will be a tremendous aid for bothpathologists and oncologists helping patients in thebattle against kidney cancer.”

The study, “Evaluation of a decision tree in thediagnosis of renal neoplasms based on genomicaberrations detected by array-CGH,” was com-pleted in conjunction with a translational team atthe Cleveland Clinic. The study reviewed 15 tar-geted genomic regions for copy number dataacross the 188 patient samples. Samples were in-cluded from all major subtypes of renal cancer,and were initially selected for the study based onpathologic diagnosis: 62 clear cell carcinomas, 56papillary carcinomas, 34 chromophobe carcino-mas, and 36 oncocytomas.

Additional highlights include the following.Only 2 μg of DNA material was needed. Overalltumor subtyping ranged from 97% diagnostic ac-curacy for clear cell, 93% for chromophobe, 91%of papillary, and 86% in oncocytomas. Of the 188samples, 173 were correctly assigned to a tumorsubtype. Specificity of the molecular diagnosis us-ing the array ranged from 100% for chromophobe;99% for oncocytoma, and 97% for both clear celland papillary kidney cancer subtypes.

The results support implementation of the CGIkidney cancer algorithm and test in a clinical set-ting to provide highly accurate subtyping and out-come prediction for renal cancers. CGI is current-ly making the test available as a laboratory-devel-oped-test (LDT). The panel is also being trans-ferred to a Next-Generation Sequencing (NGS)platform for additional assessment of genomicaberrations.

Image: Kidney cancer cells (Photo courtesy of SPL).

High Diagnostic Accuracy Demonstrated for New Kidney Cancer Test



Click on the below to watch the video


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2014ANNUAL

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IMMUNOFLUORESCENCE ANALYZERGetein Biotechnology

The Getein 1100 portable analyzer is designed tomeasure biomarkers in human whole blood,serum/plasma or urine samples. The quantitativesystem can be utilized for tests in an existing labor at the point-of-care (POC), with results used forclinical diagnosis.

ALL-IN-ONE LAB SYSTEMHitachi Aloka Medical

The LabFLEX 2600 is a space-saving, preanalyti-cal specimen processing / aliquoting system. Asan all-in-one system, the LabFLEX 2600 handlesprocessing from arrival checks, decapping, aliqot-ing, and labeling of the daughter specimen con-tainers, through external transport processes.

POCT HbA1c METERHorron XLH Medical Electronics

The H618 is the latest maintenance-free dry sys-tem for point-of-care HbA1c testing. Key featuresof the system include a multicolor touch screen,easy operation, reduced user training, and thesystem can also produce the eAG parameter.

AAsignature immune response has been identifiedthat might help doctors identify which newly

diagnosed influenza patients are most likely to de-velop severe symptoms and suffer poor outcomes.

Children are an at-risk population for develop-ing complications following influenza infection,but immunologic correlates of disease severity arenot understood and it has been hypothesized thatinnate cellular immune responses at the site of in-fection would correlate with disease outcome.

Scientists at St. Jude Children’s Research Hos-pital (Memphis, TN, USA; www.stjude.org) con-ducted an observational cohort study with longi-tudinal sampling of peripheral and mucosal sitesin 84 naturally influenza-infected individuals, in-cluding infants. Cellular responses, viral loads,and cytokines were quantified from nasal lavagesand blood, and correlated to clinical severity. Sam-

pling began when patients sought medical atten-tion for influenza symptoms, and was repeated ondays 3, 7, 10, and 28. Along with measuring lev-els of influenza virus in the nose and sinuses, re-searchers measured 42 cytokines and antibodiesagainst circulating influenza viruses.

The testing showed children and adults wereequally successful at eliminating the virus regard-less of the subtype. In fact, based on cytokine lev-els in the blood, nose, and sinuses of patients, chil-dren mounted a more aggressive inflammatory re-sponse than adults. Adjusting for age and viralload, an innate immune profile characterized byincreased nasal lavage monocyte chemotactic pro-tein-3, interferon (IFN)-α2, and plasma inter-leukin 10 (IL-10) levels at enrollment predictedprogression to severe disease. Increased plasmaIL-10, monocyte chemotactic protein-3, and IL-6

levels predicted hospitalization.The study was published on February 15,

2014, in the American Journal of Respiratory andCritical Care Medicine.

Image: Paul Thomas, PhD, of the Department of Im-munology at St. Jude Children’s Research Hospital, iscorresponding author on a study that identifies factorsthat predict flu severity in patients and sets the stagefor more effective therapies to prevent flu complica-tions and deaths (Photo courtesy of Ann-MargaretHedges / St. Jude Children’s Research Hospital).

Immune Signature Predicts Poor Outcome in Influenza Patients

AA sensitive and robust routine diagnostic test forthe detection of rearrangements and transcrip-

tional upregulation of the ALK (anaplastic lym-phoma receptor tyrosine kinase) gene was de-scribed in a recent publication.

The ALK gene encodes a receptor tyrosine ki-nase, which belongs to the insulin receptor super-family. This protein comprises an extracellular do-main, a hydrophobic stretch corresponding to a sin-gle pass transmembrane region, and an intracellularkinase domain. It plays an important role in the de-velopment of the brain and exerts its effects on spe-cific neurons in the nervous system. This gene hasbeen found to be rearranged, mutated, or amplifiedin a series of tumors including anaplastic large celllymphomas, neuroblastoma, and non-small-celllung cancer (NSCLC).

The EML4-ALK fusion gene is responsible for ap-proximately 3%–5% of cases of NSCLC. The stan-dard test used to detect this gene in tumor samples

is fluorescence in situ hybridization (FISH), but oth-er techniques such as immunohistochemistry (IHC)and reverse transcriptase PCR (RT-PCR) can also beused to detect lung cancers with an ALK gene fusion.

Formalin fixed, paraffin-embedded (FFPE) tis-sues are the most widely available specimens forretrospective clinical studies of disease mecha-nisms. These archived materials provide a valuablesource of stable nucleic acids for gene expressionanalysis, using real-time quantitative reverse tran-scription-PCR (qRT-PCR) or microarray analysis. Asthe use of PCR technology has become more preva-lent in molecular testing, it has enhanced the clini-cal utility of FFPE tissues. However, the recovery ofquality RNA from FFPE specimens is often problem-atic, as the fixation process causes cross-linkage be-tween nucleic acids and proteins, and covalentlymodifies RNA by the addition of monomethylgroups to the bases. As a result, the molecules arerigid and susceptible to mechanical shearing, and

the cross-links may compromise the use of RNA asa substrate for reverse transcription. Therefore, inorder to utilize FFPE tissues as a source for gene ex-pression analysis, a reliable method is required forextraction of RNA from the cross-linked matrix.

Investigators at the Dr. Margarete Fischer-BoschInstitute of Clinical Pharmacology (Stuttgart, Ger-many; www.bosch-stiftung.de) recently describedthe development of a new RNA-based assay to de-tect ALK rearrangements in NSCLC. This tech-nique was based on quantitative, real-time PCR((q)RT-PCR), and included two novel features: anRNA isolation method that was optimized to re-verse formaldehyde modification and small RT-PCRamplicons to allow for the use of fragmented nucle-ic acids for efficient amplification of ALK cDNA.The test measured the expression of the 5’ and the3’ portions of the ALK transcript separately.

The study was published in the March 2014 is-sue of the Journal of Thoracic Oncology.

PCR Assay Detects Gene Rearrangements in Preserved Samples from Lung Cancer Patients

SSelf-testing for human papillomavirus(HPV), which is one of the main causes

of cervical cancer, is as effective at detect-ing cancer as a conventional cytologyscreening by smear test.

When self-testing is scaled up to testlarge populations, the number of womenreferred to clinics for follow-up tests rapid-ly increases, but the quality of clinical carethen goes down, as clinics are unable tohandle the high number of patients.

Scientists from Queen Mary University(London, UK; www.qmul.ac.uk) work-ing with colleagues in Mexico, conduct-ed a pilot study of 100,242 Mexicanwomen aged 25 to 75 and from low-in-come backgrounds. Around 11% ofwomen tested positive for HPV (10,863women). The study began in February2007 and patient follow-up ended in Ju-ly 2010. The standard Digene conicalcollection brush (Qiagen Inc.; Gaithers-burg, MD, USA; www.qiagen.com) wasused to obtain the vaginal specimens.

The High-Risk HPV DNA (hrHPVDNA) was detected with a standardcommercial kit employing in vitro nu-cleic acid hybridization and chemilu-minescent microplate-based signal am-plification. The Qiagen hybrid capture(HC2) assay was conducted in a vali-dated and accredited routine HPV test-ing laboratory. The HC2 relative lightunit/cutoff of 1 was considered a pos-itive test. The hrHPV positive womenand a randomly selected subset of3.2% of hrHPV negative participantsserving as controls were referred tocolposcopic examination for diagnosisconfirmation.

The hrHPV testing of self-collectedvaginal samples demonstrates a highnegative predictive value (NPV) for rele-vant cervical disease, highlighting thistest’s benefit as a primary screening pro-cedure in developing countries, aswomen who had a negative hrHPV re-sult have virtually no risk of cervical in-traepithelial neoplasia (CIN) 3+ lesions.Self-collected hrHPV testing may be par-ticularly attractive for increasing screen-ing intervals, and it appears to be an ac-ceptable screening option in large-scaleroutine settings. In addition, given themagnitude of the relative risk and posi-tive likelihood ratio, the findingsdemonstrate that hrHPV testing of self-collected samples is a satisfactoryscreening test for identification ofwomen with target CIN2+ lesions.

Attila T. Lorincz, PhD, a professor ofmolecular epidemiology and lead au-thor, said, “Our findings show thatlarge-scale HPV screening can be suc-cessfully implemented by home testing.

However, if a positive result is received, it’simperative that all other follow-up servicesare strengthened so they can accommodatethe big increase in screened women. This isone of the huge challenges we face in de-veloping countries like Mexico and willcontinue to work on.” The study was pub-lished on December 7, 2013, in the Inter-national Journal of Cancer.

Image: Human papilloma virus as seenthrough a colored transmission electron mi-crograph (Photo courtesy of Pasieka / SPL).

Human Papillomavirus Self-Testing Proves Effective

133LMI-07-14LINKXPRESS COM33 LabMedica International

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AAnew DNA sequencing system utilizes ad-vanced design features to generate massive

throughput and enable the world’s first USD1,000 human genome sequence.

This achievement has been reached with thenew HiSeq X Ten Sequencing System from Illu-mina (San Diego, CA, USA; www.illumina.com).The platform includes technology breakthroughsthat enable researchers to undertake populationand disease studies of unprecedented scale byproviding the throughput to sequence tens ofthousands of human whole genomes in a singleyear in a single lab. The HiSeq X Ten is theworld’s first platform to deliver high quality, high-coverage human genome sequences for less thanUSD 1,000 – inclusive of typical instrument de-preciation, DNA extraction, library preparation,and estimated labor.

Purpose-built for population-scale humanwhole genome sequencing, the HiSeq X Ten is aplatform especially suitable for scientists and insti-tutions focused on the discovery of genotypic vari-ation to enable a deeper understanding of biologyand disease. It can deliver a comprehensive cata-log of human variation within and outside of cod-ing regions. “The ability to explore the humangenome on this scale will bring the study of can-cer and complex diseases to a new level. Breakingthe ‘sound barrier’ of human genetics not only

pushes us through a psychological milestone,it enables projects of unprecedented scale.We are excited to see what lies on the otherside,” said Jay Flatley, CEO, Illumina.

Building on the proven performance of Il-lumina sequencing-by-synthesis (SBS) tech-nology, HiSeq X Ten utilizes a number of ad-vanced design features to generate massivethroughput. Patterned flow cells (which con-tain billions of nanowells at fixed locations)combined with a new clustering chemistrydeliver a significant increase in data density(6 billion clusters per run). Using state-of-the artoptics and faster chemistry, HiSeq X Ten canprocess sequencing flow cells more quickly thanever before – generating a 10x increase in dailythroughput when compared to current HiSeq2500 performance. The HiSeq X Ten is sold as aset of 10 or more ultra-high throughput sequenc-ing systems, each generating up to 1.8 terabases(Tb) of sequencing data in less than 3 days or upto 600 gigabases (Gb) per day, per system.

Initial users of the transformative HiSeq X TenSystem include Macrogen (Seoul, Republic of Ko-rea) and its CLIA laboratory (Rockville, MD,USA), the Broad Institute (Cambridge, MA, USA),and the Garvan Institute of Medical Research(Sydney, Australia).

“The sequencing capacity and economies of

scale of the HiSeq X Ten facility will also allowGarvan to accelerate the introduction of clinicalgenomics and next-generation medicine in Aus-tralia,” said Prof. John Mattick, Executive Direc-tor of the Garvan Institute of Medical Research.

Eric Lander, founding director of the Broad In-stitute and professor of biology at MIT, said, “TheHiSeq X Ten should give us the ability to analyzecomplete genomic information from huge samplepopulations. Over the next few years, we have anopportunity to learn as much about the genetics ofhuman disease as we have learned in the historyof medicine.”

Image: The HiSeq X Ten is composed of 10 HiSeq XSystems, and enables population-scale projects ongenotypic variation to understand and improve hu-man health (Photo courtesy of Illumina).

System Breaks Technology and Cost Barriers for High-Throughput Large-Genome Sequencing

TTwo new diagnostic instruments, which recent-ly have been certified for marketing in Europe

(CE marked), will bring an unprecedented level ofautomation and efficiency to laboratories thatprocess large and very large numbers of specimenson a daily basis.

The Roche Molecular Diagnostics (Pleasanton,CA, USA; http://molecular.roche.com) Cobas

6800/8800 system has automated PCR technologyfor virology, blood screening, HPV (human papillo-mavirus and CT/NG (Chlamydia trachomatis andNeisseria gonorrhea) testing. The system comes intwo models – large (6800) and very-large (8800)throughput capacity – and was developed to deliverincreased automation and throughput, with mini-mal user intervention and improved quality control.

The instruments are equipped with an onboardrefrigerator to allow for significantly longer reagentstorage capacity and less loading and unloading ofreagents. The instrument design provides greaterergonomics in terms of the user-instrument inter-face, reducing the number of required user interac-tions. Once the instrument is loaded, the systemcan run continuously for eight or four hours for theCobas 6800 and Cobas 8800 system, respectively.This translates into a throughput of 300 and 1000results in eight hours, respectively. Once the instru-ment begins processing samples, the first 96 resultscan be reported within three-and-a-quarter hours.Thereafter, the Cobas 6800 and 8800 instrumentsprovide up to 96 results every 90 or 30 minutes, re-spectively.

This level of efficiency was obtained by com-pletely automating the processing of primary andsecondary tubes all the way through to the patientsample result for any combination of three differenttests at the same time. This parallel sample to resultprocess is accomplished using a universal samplepreparation, PCR profile, and reagent setup process.

During the first six months of the systems’ CElaunch, several assays will be available for virologylabs (HIV, HCV, HBV, and CMV) and for bloodscreening labs (combined HIV/HCV/HBV, WNV,and combined Parvovirus B19/HAV). In the nearfuture, the system will support additional assays in-cluding HPV, CT/NG, and a utility channel foruser-defined applications.

New Instruments Automate PCR Diagnostic Applications



AA scanning acoustic microscope(SAM) uses ultrasound to image

an object by plotting the speed-of-sound (SOS) through tissues onscreen, and because hard tissues re-sult in great SOS, SAM can providedata on the tissue elasticity.

Scanning acoustic microscopy us-es ultrasound at 120 MHz with al-most the same resolution of approx-imately 12.5 μm as the low magnifi-cation of a light microscope (LM),and it had been found useful in gen-erating useful information on thelung, stomach, and lymph node le-sions.

Scientists at Hamamatsu Universi-ty School of Medicine (Japan; www.hama-med.ac.jp) selected and exam-ined formalin-fixed, paraffin-embed-ded blocks that were flat-sectioned in10 μm thick sections from patientswith inflammatory non-neoplasticand neoplastic thyroid lesions. Speci-mens were randomly selected fromthe computer database of pathologi-cal sections and typical lesions of thy-roid diseases were identified basedon hematoxylin and eosin sections.

The formalin-fixed, paraffin sec-tions were scanned with a 120 MHztransducer using the SAM modelAMS-50AI (Honda Electronics Co,Ltd; Toyohashi, Japan; www.honda-el.co.jp). SOS through each area wascalculated and plotted on the screento provide histological images, andSOS of each lesion was comparedand statistically analyzed.

High-concentrated colloids, redblood cells, and collagen fibersshowed pronounced SOS while low-concentrated colloids, parathyroids,lymph follicles, and epithelial tissuesincluding carcinomas demonstratedlower SOS. SAM clearly discriminat-ed structure of thyroid componentscorresponding to low magnificationof light microscopy. Thyroid tumorswere classified into three groups byaverage SOS: the fast group consistedof follicular adenomas/carcinomasand malignant lymphomas; the slowgroup contained poorly differentiat-ed/undifferentiated carcinomas; andthe intermediate group comprisedpapillary/medullary carcinomas.

The authors concluded that SAMimaging has the following benefits:precise images were acquired in afew minutes without special stain-ing; structural irregularity anddesmoplastic reactions, which indi-cated malignancy, were detected;images reflected tissue elasticity,

which was statistically comparableamong lesions by SOS; and tumorclassification was predictable by SOSbecause more poorly differentiatedcarcinomas had a tendency to showlower SOS. The study was publishedon February 11, 2014, in the journalPathology and Laboratory MedicineInternational.

Image: The AMS-50SI scanning acous-tic microscope system (Photo courtesyof Honda Electronics).

Thyroid Lesions Examined by Scanning Acoustic Microscope




141LMI-07-14LINKXPRESS COM138LMI-07-14LINKXPRESS COM

TThe diagnosis of lung cancer can involve a num-ber of tests, but specific compounds in exhaled

breath have been discovered that may be used to di-agnose the disease in its early stages. Of all cancers,lung cancer is the biggest killer in both men andwomen it causes more deaths than colon, breast andprostate cancer combined according to the AmericanLung Association (Chicago, IL, USA; www.lung.org).

Scientists at the University of Louisville (KY,USA; www.louisville.edu) used a silicone micro-processor and mass spectrometer to test exhaledbreath of patients with suspected lung cancer forspecific volatile organic compounds (VOCs) knownas carbonyls. These included aldehydes and ke-tones, organic compounds with carbon double-bonded to oxygen, which are at very low concen-

trations and produced by the human body.The team developed the silicone microprocessor,

which was coated with an amino-oxy compoundthat binds to carbonyl compounds found in exhaledbreath. Measuring the levels of carbonyls in ex-haled breath has provided the investigators with away to accurately identify early lung cancer. Afterremoving malignant nodules in certain patients, theteam found that these elevated carbonyl concentra-tions returned to normal. The scientists made thediscovery when they were examining patients with“suspicious” lung lesions.

Michael Bousamra, MD, an associate professorat the University of Louisville, said when discussingthe potential for use of this technique as a standardtest. “Instead of sending patients for invasive biop-

sy procedures when a suspicious lung mass is iden-tified, our study suggests that exhaled breath couldidentify which patients may be directed for an im-mediate intraoperative biopsy and resection. Thenovelty of this approach includes the simplicity ofsample collection and ease for the patients.”

Professor Bousamra added, “Although the dataare preliminary, we found that patients with an ele-vation of three or four cancer-specific carbonyl com-pounds was predictive of lung cancer in 95% of pa-tients with a pulmonary nodule or mass. Converse-ly, the absence of elevated VOC levels was predic-tive of a benign mass in 80% of patients.” The studywas presented at the 50th Annual Meeting of TheSociety of Thoracic Surgeons, held January 25–29,2014, in Orlando (FL, USA; www.sts.org)

Exhaled Breath Compounds Identify Early Lung Cancer

IInnovative single-cell DNA library preparationkits can now be used with Illumina sequencing

platforms. Rubicon Genomics (Ann Arbor, MI,USA; www.rubicongenomics.com) now offers itsPicoPLEX DNA-seq kits for use with Illuminanext-generation sequencing (NGS) platforms. Thekits are among Rubicon’s sample-specific librarypreparation products used in clinical and researchtesting on NGS, microarray, and quantitative poly-merase chain reaction (qPCR) platforms. Pi-coPLEX’s excellent robustness and reproducibilityoffer excellent single-cell DNA amplification formicroarray and PCR-based preimplantation genet-ic screening and diagnosis. PicoPLEX DNA-seqnow enables clinicians and researchers to accessPicoPLEX technology for analyses conducted onIllumina NGS systems.

PicoPLEX DNA-seq kits amplify DNA to yield ahighly reproducible NGS-ready library from a singlecell with input concentrations of six picograms orless. PicoPLEX DNA-seq employs the same technol-ogy currently used by in vitro fertilization (IVF) clin-

ics to detect chromosomal aneuploidies, copy num-ber variations, and single-gene disorders in repro-ductive cells. It is also used for the genetic charac-terization of other single-cell samples such as circu-lating tumor cells. The easy-to-use, three-stepprocess is performed in a single tube or well in lessthan 3 hours, thereby reducing error and contami-nation, speeding time to results, and reducing costs.

James Koziarz, PhD, CEO of Rubicon Ge-nomics, commented, “PicoPLEX DNA-seq em-phasizes our commitment to increasing the ro-bustness and reproducibility of DNA analyseswhile also delivering greater speed, efficiency andcost savings. This launch represents another mile-stone in our ongoing strategy to make RubiconGenomics’ proprietary library preparation technol-ogy available to users of all sequencing platforms.”

Dr. Brian Mariani, chief scientist and scientificdirector of the Genetics and IVF Institute, helpedtest the PicoPLEX DNA-seq kits. He commented,“Remarkably, the sequencing data from the Pi-coPLEX DNA-seq libraries of embryo DNA clearly

identified a male balanced translocation that hadnot been detected by previous microarray andFISH [fluorescence in-situ hybridization] analyses.This is a significant example of how PicoPLEX se-quencing data can expose important structural re-arrangements missed by other approaches.”

At the 15th annual Advances in Genome Biol-ogy and Technology meeting (AGBT 2014; Febru-ary 12–15, 2014, Marco Island, FL, USA), Rubi-con CSO Dr. John Langmore presented studieshighlighting PicoPLEX DNA-seq and discussed“Preimplantation genetic screening and diagnos-tics (PGS/PGD) at low NGS coverage for aneu-ploidy, CNV, and single-gene disorder detectionfor in vitro fertilization.”

Image: The PicoPLEX DNA-seq kits, designed foruse with Illumina NGS platforms (Photo courtesy ofRubicon Genomics).

Superior Single-Cell Library Preparation Technology Now Available for Additional Platforms

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TThe molecular diagnosis of malaria by nu-cleotide amplification requires sophisticated

and expensive instruments, typically found onlyin well-established laboratories. Loop-mediatedisothermal amplification (LAMP) has provided anew platform for an easily adaptable moleculartechnique for molecular diagnosis of malaria with-out the use of expensive instruments.

Scientists from the International Center for Diar-rheal Disease Research (Dhaka, Bangladesh; www.icddrb.org) working with colleagues in NorthAmerica collected blood samples included 106 mi-croscopy-positive P. falciparum infections and 105microscopically negative samples. All the subjectshad febrile illness and were suspected of malaria.

Routine microscopy was performed by experiencedmicroscopists on thick and thin smear slides.

DNA was extracted and real-time polymerasechain reactions (RT-PCR) were performed usingInvitrogen SYBR Green I supermix UDG (LifeTechnologies Corporation; Grand Island, NY,USA; www.lifetechnologies.com). A new primerset was designed targeting the 18S ribosomal ri-bonucleic acid (rRNA) gene for the detection of P.falciparum in whole blood samples. The efficacyof LAMP (Eiken Chemical Co. Ltd.; Tokyo, Japan;www.eiken.co.jp) using the new primer set wasassessed in this study in comparison to that of apreviously described set of LAMP primers. Mi-croscopy and real-time PCR were used as refer-ence methods for detecting P. falciparum. Pre-ad-dition of hydroxy napthol blue (HNB) in theLAMP reaction caused a distinct color change,thereby improving the visual detection system.

The new LAMP assay was found to be 99.1%

sensitive compared to microscopy and 98.1% whencompared to real-time PCR and the specificity was99% and 100% in contrast to microscopy and real-time PCR, respectively. The augmented LAMPmethod can detect at least 5 parasites/μL of infect-ed blood within 35 minutes, while the other LAMPmethod tested in this study, could detect a mini-mum of 100 parasites/μL of human blood after 60minutes of amplification.

The authors concluded that the new LAMPmethod is highly sensitive and specific for the di-agnosis of symptomatic falciparum malaria. Thismethod can be an alternative molecular diagnos-tic tool to PCR and might become a standardmethod for wider use. Furthermore, this methodhas immense potential to become a tangible toolfor point-of-care diagnosis of malaria and treat-ment monitoring in healthcare and epidemiologi-cal studies. The study was published on March 5,2014, in the journal Acta Tropica.

Improved LAMP Test Diagnoses Symptomatic Falciparum Malaria

Leukocyte Ratio InvestigatedFor Familial Mediterranean

Fever Patients

BB lood neutrophil-to-lymphocyte (N/L) ratio is asimple marker of inflammation that can be easi-

ly obtained from the differential leucocyte count andhas been used to determine disease activity and diag-nosis in patients with ulcerative colitis (UC) andacute appendicitis.

Familial Mediterranean Fever (FMF) is a recur-rent, autosomal recessive autoinflammatory diseasecharacterized with fever and serositis, which is ac-companied by pain in the abdominal area, chest, andjoints and the disease, is common among Mediter-ranean communities including Turks, Armenians,Jews, and Arabs.

Scientists at the Bozok University Medical School(Yozgat, Turkey; www.bozok.edu.tr) enrolled 115patients and controls in the study. The cases in thestudy were categorized as FMF with attack, FMFwith attack-free period, and controls. There were 79patients with FMF and 36 control subjects. Therewere 29 females and 50 males in the FMF group and12 females and 24 males in the control group.

The median disease duration in FMF patients was10 years and age and sex were similar in the FMFand control groups. The ESR was significantly higherin FMF patients during the attack phase. The CRP in-creased during attack but this was not statisticallysignificant and WBC counts did not differ amonggroups. The mean N/L ratios of the controls was1.63 (range: 1.41 to 2.33), for FMF patients whowere attack- free the ratio was 1.83 (range 1.21 to2.23), and for FMF patients during attack it was 2.95(range: 1.91 to 3.46). The serum N/L ratios of FMFpatients during attack were significantly higher thanthose of attack-free FMF patients and controls.

The authors concluded that the N/L ratio, an in-dicator of the overall inflammatory status of thebody, is higher in active FMF patients than in FMFpatients in remission and healthy controls. A cut-offratio value of 2.63 can be used to identify patientswith active FMF. N/L ratio is not related with dis-ease duration and inflammatory markers such asCRP and ESR in FMF patients. It is an inexpensiveand readily available measure that could in combi-nation with other markers help identify patientswith active disease. The study was published onMarch 28, 2014, in the Journal of Clinical Labora-tory Analysis.

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AAmultibiomarker-based approach has been de-veloped to estimate mortality risk in adults

with septic shock and tested on blood samplesfrom critically ill patients.

Septic shock is a severe systemic infection andmajor cause of death for the old and young alike,but testing new drug regimens to stop the infec-tion is confounded because clinical trials includepatients who are either too sick to be saved by ex-perimental therapies or not sick enough to war-rant the treatments.

An international team led by scientists at theCincinnati Children’s Hospital Medical Center(OH, USA; www.cincinnatichildrens.org) studied882 adults in intensive care units in medical cen-ters located in the USA, Finland and Canada. Thestudy included three different groups of patientsto develop, test and retest the tool. This helps tomake sure it works in different patient mixes, of-ten a limiting factor in large clinical trials. Theteam selected 12 candidate biomarkers from 117gene probes previously shown to have predictivestrength for poor outcomes in microarray-basedstudies involving children with septic shock.

The plasma concentrations of the candidate bio-markers were measured using a multiplex magnet-

ic bead platform, the MILLI-PLEX MAP (EMD MilliporeCorporation; Billerica, MA,USA; www.millipore.com)and a Luminex 100/200multiplex analyzer system(Luminex Corporation; Aus-tin, TX, USA; www.luminexcorp.com). Maximum accu-racy was achieved with fiveof the 12 candidate stratifi-cation biomarkers: C-C chemokine ligand 3(CCL3), heat shock protein 70 kDa 1B (HSPA1),interleukin-8 (IL8), granzyme B (GZMB), and C-Cchemokine ligand 4 (CCL4). Serum lactate con-centration at study entry, age, and presence ofchronic disease further improved predictive accu-racy.

The data was then used in a mathematicalmodel to combine the information into a singletool that separates high- and low-risk patients. Theinvestigators found that the tool accurately deter-mined that patients who tested positive had lessthan a 50% chance of surviving, but patients whotested negative had more than a 90% chance ofsurviving. More detailed analyses allowed the au-

thors to identify patients with a more than a 98%chance of surviving, and those with more than75% chance of dying.

Hector R. Wong, MD, a lead investigator anddirector of Critical Care Medicine said, “Substan-tial resources are invested in trying to find newtreatments for septic shock, but the vast majorityof them fail when they get to clinical trials. Thereare many reasons for this, but a consistent one isthat the baseline mortality risk varies widely inseptic shock patients, which muddies the water.”The study was published in the April 2014 issueof the journal Critical Care Medicine.

Image: The Model 100/200 multiplex analyzer sys-tem (Photo courtesy of Luminex).

Blood Test Identifies Septic Shock Risk Biomarkers

AAnewly developed method has correctly identi-fied pancreatic cancer potential with higher ac-

curacy than cytology and cyst fluid carcinoembryon-ic antigen (CEA) testing, and could be used to im-prove prevention and reduce unwarranted surgery.

Current prognosis for pancreatic cancer (PCa) issuch that only about 5% of patients survive 5 yearsafter diagnosis. Pancreatic cystic lesions (PCLs) al-most invariably reflect an underlying inflammatoryor neoplastic condition, ranking among the mostimportant incidentalomas to have emerged with ra-diological advances. This offers an opportunity forpreventive intervention against pancreatic cancer(ductal adenocarcinoma) because a substantial pro-

portion of cystic tumors can be considered precur-sor lesions – of malignant potential (premalignantor malignant tumors). Although PCLs (found in10% of the population above age 70 years, andcommon also in younger people) can now be dis-covered with CT or MRI, imaging alone cannot de-termine which cysts are at risk for developing intocancer. It often becomes necessary to puncture thecyst and test for tumor markers in the fluid; howev-er these analyses are not reliable. Removing thecyst by surgery, knowing that it may turn out to bebenign is also problematic, since the operation isextensive with considerable risks for the patient.

Much better diagnostic tools are required for pa-

tients to benefit from PCL detection. Researchers atThe Sahlgrenska Academy (Gothenburg, Sweden;www.sahlgrenska.gu.se/english) have now devel-oped a mass spectrometry (MS) based method thatidentified PCa precursor cysts with 97% certainty(of 79 examined cysts). The method detects thepresence of mucus protein, mucins, in the cysticfluid. The PCL proteomic mucin profiling accuracywas nearly identical (96.6% vs. 98.0%) betweenthe discovery (n = 29) and validation (n = 50) co-horts. “This is an exceptionally good result for a di-agnostic test, and we are very hopeful that themethod will enable more instances of early discov-ery of pancreatic cancer, at a stage when the cancercan be treated or prevented,” said Dr. Karolina Jab-bar, PhD student at The Sahlgrenska Academy andphysician at The Sahlgrenska University Hospital.

The method was also tested in a blinded analy-sis of existing tumors and determined with about90% certainty, which tumors have already devel-oped into cancers. Therefore, the method could al-so be used to determine which patients require im-mediate surgery and which can wait while undercontinued monitoring.

Prof. Gunnar C. Hansson, who initiated thestudy with senior physician and Assistant ProfessorRiadh Sadik, is convinced that MS-based pro-teomics will soon be introduced in health care:“The technique has been developed, and we cannow measure biomarkers both quickly and exactly.Moreover, the method requires minimal biomateri-al, in this case 25 times less cyst fluid than conven-tional tumor marker analyses. I am certain thatwithin 5 years the mass spectrometers will havemoved into the hospital corridors,” he said.

Jabber et al. describe the method in the Journalof the National Cancer Institute, February 2014.

Proteomic Profiling Enables Earlier Diagnosis of Pancreatic Cancer



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AAnovel genetic defect has been identi-fied among patients with bone marrow

failure, which could reveal its underlyingcause. Bone-marrow-failure syndromes,such as fanconi anemia, dyskeratosis con-genita, and aplastic anemia, are a heteroge-neous group of life-threatening disorderscharacterized by the inability of the bonemarrow to make an adequate number ofmature blood cells.

Scientists at the Queen Mary Universityof London (UK; www.qmul.ac.uk) and theircolleagues chose three genetically un-characterized index cases from theirrepository of bone-marrow-failure case.They performed exome sequencingwith the aim of identifying variants in acommon gene. The three had trilineage,erythroid, myeloid, and megakaryocyt-ic, bone marrow failure and came fromconsanguineous families. All three alsohad developmental delay characterizedby learning disability, and two out of thethree cases also had microcephaly.

Peripheral-blood samples were ob-tained from the three patients and DNAextracted. Various other techniqueswere used in the study including, histol-ogy and immunohistochemistry, im-munocytochemistry, imaging, and colo-calization studies, real time polymerasechain reaction (PCR) and immunoblot-ting densitometry and small interferingribonucleic acid RNA (siRNA) studies.For the evaluation of basal reactive oxy-gen species (ROS), cells were labeledwith ROS marker dihydroethidium andthe fluorescence was measured by flowcytometry. Real-time changes in ROSfluorescence after treatment were meas-ured with the use of a FLUOstar Optimaplate reader (BMG Labtech; Ortenberg,Germany; www.bmglabtech.com).

Exome sequencing revealed that twoof the three cases studied with bonemarrow failure and developmental de-lay had homozygous truncating variantsin the gene Excision Repair Cross-Com-plementing Rodent Repair Deficiency,Complementation Group 6-Like 2 (ER-CC6L2). This ERCC6L2 mutation linksa distinct bone-marrow-failure syn-drome to DNA repair and mitochondri-al function. The findings mean it is nowpossible to carry out a reliable genetictest, including antenatal testing, inthese families and get an accurate diag-nosis. In the long term, with furtherstudies, the findings could lead to thedevelopment of new treatment for thisspecific gene defect.

Inderjeet Dokal, MD, a professor ofpediatrics and child health, and the sen-ior author of the study said, “New DNAsequencing technology has enabled us to

identify and define a new gene defect whichcauses a particular type of bone marrow fail-ure. This is a promising finding, which wehope one day could lead to finding an effec-tive treatment for this type of gene defect.Clinicians treating patients with bone mar-row failure should now include analysis forthis gene in their investigation.” The studywas published on February 6, 2014, in theAmerican Journal of Human Genetics.

Image: The FLUOstar Optima plate reader(Photo courtesy of BMG Labtech).

Gene Discovery Illuminates Cause of Bone Marrow Failure



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SLIDE DRYING BENCHKarl Hecht

The Assistent bench with heating foil accepts upto 48 slides (76 mm x 26 mm) to speed up slidepreparation. Slides can be placed in any position(on bars, against bars, or flat on heating foil), withtemperatures capable of reaching up to +100 °C.

ELISA RANGEMP Diagnostics

The BloodScreen ELISA range is designed to en-sure the safety of the blood supply, with each kithighly sensitive and specific. Key features includeready-to-use reagents that reduce preparationtime, and short assay times for fast, accurate, andreliable test results.

BENCHTOP SYSTEMOrion Diagnostica

The GenRead is a flexible benchtop system con-sisting of ready-to-use kits and the GenRead in-strument that can be used in labs of various sizesand settings. The system is designed for easy, ac-curate, and fast nucleic acid testing, with an ad-justable number of tests from one to 12.

TThe prognosis of a patient suffering frommelanoma can be determined by the presence

of cancer cells in sentinel lymph nodes. Cancer cellsspread to the sentinel node – the lymph node towhich cancer cells are most likely to spread from aprimary tumor – is a risk factor for melanoma death.

The prognosis of melanoma patients depends onthe number of disseminated cancer cells per millionlymphocytes in the sentinel node. Even very lownumbers were found to be predictive for reducedsurvival. Anja Ulmer, Christoph Klein, and col-leagues from the Universities of Tübingen (Ger-many; www.uni-tuebingen.de) and Regensburg(Germany, www.uni-regensburg.de) studied 1,834sentinel lymph nodes from 1,027 patients withmelanoma and followed them for five years.

The scientists labeled disseminated cancer cells

(DCCs) in the lymph nodes with a marker formelanoma cells, counted them, and calculated DCCdensity. They determined whether DCC density wasrelated to a patient’s survival and found that patientswith high DCC density in the lymph nodes weremore likely to die from melanoma within 5 years. A10-fold increase in DCC density nearly doubled therisk of death. A model was created based on tumorthickness, tumor ulceration, and lymph-node DCCdensity that provided survival prediction superior tothat of a model based on the current standard stag-ing recommendations. The investigators demonstrat-ed that their new model predicted patients’ progno-sis more accurately. It classified 13% of patients inthis cohort correctly as high risk for progression,which the standard model did not. This group of pa-tients could potentially have benefitted from more

aggressive treatments. The new model also correctlyidentified a group of low risk patients who had excel-lent long-term outcomes, whereas the standard mod-el overestimated their risk of death.

The study was published in the February 2014edition of PLoS Medicine. However, the resultsneed to be validated in an independent study, in or-der to establish how this methodology could be usedin a clinical setting. The authors said, “Our studyshows that the extent of metastatic disseminationlargely determines the disease courses of patients.The better we are able to predict the risk of patientsto die from melanoma the better can we balancecost and benefit of potentially toxic therapies. Forearly melanoma, this might become even more im-portant as novel drugs to prevent lethal metastasisare currently under investigation.”

Cancer Cells in Sentinel Node Indicates Melanoma Risk

TThe determination of the precise lifespan of ahuman T-cell is challenging due to inability of

standard techniques to distinguish between divid-ing and dying cells. The duration of in vivo persist-ence can be measured by following a pool of T-cellsthat were “naturally” labeled with a single integrat-ed clone of a replication-incompetent human im-munodeficiency virus (HIV-1), called a provirus.

Scientist from the National Institute of Allergyand Infectious Diseases (Bethesda, MD, USA;www.niaid.nih.gov) have discovered a novelmethod for tracking cluster of differentiation 4(CD4+) T-cells in people infected with HIV. CD4+

T-cells are critical for immune defense against anarray of pathogens and are a primary target of HIV.They utilized a combination of techniques to se-quence/map an integration site of a uniqueprovirus with a stop codon at position 42 of theHIV-1 protease. The defective virus had integratedinto the genome of a single CD4+ T-cell.

In vitro reconstruction of this provirus into aninfectious clone confirmed its inability to repli-cate. By combing cell separation and integrationsite-specific polymerase chain reaction (PCR)

techniques, they were able to follow the fate ofthis single provirus in multiple T-cell subsets overa 20-year period. As controls, a number of addi-tional integrated proviruses were also sequencedand analyzed with the ABI PRISM 3130xl Genet-ic Analyzer (Applied Biosystems; Foster City, CA,USA; www.appliedbiosystems.com).

This innovative method allows scientists to distin-guish dividing cells from dying ones, something thathas not been possible with existing labeling tech-niques, but is essential for studying how immunecells survive HIV infection. The replication-incompe-tent HIV-1 provirus was solely contained in the poolof effector memory (EM) CD4+ T-cell for 17 years.The percentage of the total EM CD4+ T-cells con-taining the replication-incompetent provirus peakedat 1% with a functional half-life of 11.1 months. Inthe process of sequencing multiple proviruses, theyalso observed high levels of lethal mutations in theperipheral blood pool of proviruses.

The scientists also observed in the blood cells ofpatients a higher frequency of defective HIVproviruses than what has been reported in previouswork. Although these defective variants cannot

produce an infectious virus, many retain the abilityto generate small pieces of HIV, leading the investi-gators to speculate that these “foreign materials”within CD4+ T cells may play a key role in the on-going immune activation that is characteristic ofHIV infection, including in patients with “unde-tectable” virus in their blood. The study was pub-lished on January 31, 2014, in the journal AIDS.

Image: A scanning electron micrograph (SEM) show-ing HIV particles attacking a T-cell (Photo courtesy ofThomas Deerinck / National Center for MicroscopyImaging Research).

Novel Method Tracks T-Cells in HIV Patients




Click on the below to watch the video

BB iotech and other life science researchers canincrease their productivity by selecting one or

more new laboratory balances from an innovativenew line.

Sartorius (Gottingen, Germany; www.sartorius.com) recently introduced the new Entris, a bal-ance that focuses on the most commonly usedweighing applications and dispenses with costlyadditional functions.

The line of Entris balances comprised 15 ana-lytical and precision balances that cover a weigh-ing capacity from 60 grams to 8.2 kilograms witha readability of 0.1 milligram to 0.1 gram.

The logical key assignment on the Entris andits clearly structured user interface make daily

routine weighing mucheasier. Most models ofthe new Entris family areequipped with a mono-lithic weighing systemfeaturing external adjust-ment or an internal, mo-torized adjustment func-tion. This guaranteesconsistently accurateweighing results, which are displayed on the easy-to-read backlit, high-contrast display.

The Entris system, which can display informa-tion in German, English, French, Italian, Span-ish, Russian and Polish, is equipped with applica-

tions for density determination, animal weigh-ing, counting, conversion, and weighing in per-cent. The easily removable draft shield and stain-less steel parts render the Entris quick and easyto clean.

Gene Profile Predicts Risk ofBladder Cancer Recurrence

AAgenomic study has pinpointed several markersthat identify bladder cancer patients at risk of

recurrence and that may be indicators of overall sur-vival status.

Nearly half of patients with bladder cancer expe-rience recurrences, so reliable predictors of this re-current phenotype are needed to guide surveillanceand treatment. To identify genetic variants thatmodify bladder cancer prognosis, investigators atDartmouth Medical School (Lebanon, NH, USA;www.dartmouth.edu) focused on genes involved inmajor biological carcinogenesis processes (apoptosis,proliferation, DNA repair, hormone regulation, im-mune surveillance, and cellular metabolism).

The investigators analyzed genes from 563 blad-der cancer patients to identify genetic variants thatmodify time to bladder cancer recurrence and pa-tient survival. Patients were followed for a medianof 5.4 years during which about half of them expe-rienced at least one recurrence.

Results revealed that those patients with variantsin the ALDH2 (aldehyde dehydrogenase 2) gene,which encodes an enzyme involved in alcohol me-tabolism, were likely to experience bladder cancerrecurrence shortly after treatment. Time to recur-rence was also shorter for patients who had a vari-ant in the VCAM1 (vascular cellular adhesion mol-ecule 1) gene, which encodes a glycoprotein in-volved in the development of lymphoid tissues, andwere treated with immunotherapy. Patients whohad noninvasive tumors and a variant in the DNArepair gene XRCC4 tended to live longer than pa-tients who did not have the variant.

“The genetic markers that we found could po-tentially be useful for individually tailoring surveil-lance and treatment of bladder cancer patients,”said first author Dr. Angeline S. Andrew, assistantprofessor of community and family medicine atDartmouth Medical School.

The study that identified biomarkers linked tobladder cancer recurrence was published in theMarch 26, 2014, online edition of the journal BJUInternational.

New Line of Laboratory Balances Focuses on Most Often Used Weighing Tasks

Image: Models from the Entris line oflaboratory balances (Photo courtesyof Sartorius Stedim Biotech).

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HEMATOLOGY ANALYZERRayto Life and Analytical Sciences

The Hemaray 86 features a 5-part differentialmenu, and re-exam rule customization. With ahigh-test speed of 60 tests per hour, and a high-automation system, the analyzer serves as acompact workstation that streamlines workflow inmedium-sized hematology labs.

CLIA SYSTEMSNIBE

The Maglumi 800 features a ready-to-use inte-grated kit, as well as RFIF sample loading for upto 40 sample positions and nine reagent positions.The system also offers a wide, easy-to-use testmenu, with an enhanced throughput of up to 180tests per hour.

IMAGE ANALYSIS SYSTEMWest Medica

The Vision Hema Assist is a cost-effective bloodcell identification and preclassification solution de-signed for small-sized labs. The system auto-mates and simplifies blood smear analysis, offer-ing improved quality, increased productivity, an ef-ficient use of time, and correct ergonomics.

DDetermination of mRNA signatures in urine sam-ples may replace needle biopsy for diagnosing

kidney dysfunction following transplant surgery.Investigators at Weill Cornell Medical College

(New York, NY, USA; www.med.cornell.edu) mea-sured absolute levels of 26 pre-specified mRNAs inurine samples collected from kidney graft recipi-ents at the time of needle biopsy for acute allograftdysfunction and investigated whether differentialdiagnosis of acute graft dysfunction would be feasi-ble using urinary cell mRNA profiles. They profiled52 urine samples from 52 patients with biopsyspecimens indicating acute rejection (26 acute Tcell–mediated rejections and 26 acute antibody-

mediated rejections) and 32 urine samples from 32patients with acute tubular injury without acute re-jection.

Results of stepwise discriminant mRNA analysisidentified a linear combination of mRNAs forCD3epsilon, CD105, TLR4, CD14, complementfactor B, and vimentin that distinguished acute re-jection from acute tubular injury. Among patients di-agnosed with acute rejection, a similar statistical ap-proach identified a linear combination of mRNAs forCD3epsilon, CD105, CD14, CD46, and 18S rRNAthat distinguished T cell–mediated rejection fromantibody-mediated rejection.

“Using statistical methods we have combined the

mRNAs to yield a diagnostic signature,” said seniorauthor Dr. Thangamani Muthukumar, assistant pro-fessor of medicine at Weill Cornell Medical College.“Our study shows that when the creatinine level iselevated in the blood of a kidney transplant recipi-ent, use of our urine test would differentiate thecommon causes of kidney dysfunction that led tothe elevation in creatinine, hence benefiting manypatients by allowing them to avoid the need for aninvasive needle biopsy.”

The study showing the correspondence betweenmRNA signatures and needle biopsy results waspublished in the March 7, 2014, online edition ofthe Journal of the American Society of Nephrology.

Urinary RNA Assay May Replace Needle Biopsy for Detection of Kidney Transplant Rejection

GGenetic mutations have been identified that fa-cilitate screening of men for prostate cancer,

particularly men with a family history of the disease,to identify those who are at higher risk of aggressiveforms and in need of lifelong monitoring.

Scientists at The Institute of Cancer Research,(ICR; London, UK; www.icr.ac.uk) found 13 muta-tions in known cancer genes that predicted the de-velopment of the prostate cancer (PrCa). The find-ings demonstrate not only that some men have a ge-netic profile that puts them at higher risk of PrCa,but that particular genetic profiles match to a high-er risk of advanced, invasive disease. “The minefieldof PrCa diagnosis is one of the biggest hurdles facingtreatment of the disease today. Current tests fail todifferentiate between aggressive cancers that couldgo on to kill and cancers that may never cause anyharm. This lack of clarity means that too often menand their doctors are left having to make incrediblydifficult decisions on whether to treat the disease ornot,” said Dr. Iain Frame, Director of Research atProstate Cancer UK.

In the study, published by Leongamornlert etal. in the British Journal of Cancer, online ahead

of print February 20, 2014, the researchers exam-ined men with a history of three or more cases ofPrCa in their close family, in order to mirror useof family history as a criterion for current genetesting programs in breast cancer. Blood samplesfrom 191 men with PrCa at several different UKcenters were analyzed. New “second generation”DNA sequencing technologies were used to assessmutations in 22 different known cancer genes si-multaneously, opening up, for the first time, theprospect of rapid genetic screening for PrCa for awide range of mutations.

The results showed 13 loss-of-function muta-tions among 8 DNA-repair genes. The eight geneswere BRCA1 and BRCA2 (already routinely testedfor in women with a strong family history of breastor ovarian cancer) plus ATM, CHEK2, BRIP1, MU-TYH, PALB2, and PMS2. Men with ANY of these13 mutations were much more likely than thosewithout to develop an advanced, invasive form ofcancer, which spread to the lymph nodes or otherparts of the body, and to die from the disease.

“Our study shows the potential benefit of put-ting PrCa on a par with cancers such as breast

cancer when it comes to genetic testing. Althoughours was a small, first-stage study, we proved thattesting for known cancer mutations can pick outmen who are destined to have a more aggressiveform of PrCa,” said study co-leader Prof. Ros Ee-les, professor of Oncogenics at the ICR. Fellowstudy co-leader Dr. Zsofia Kote-Jarai, senior staffscientist at the ICR, added, “One of the importantmessages to come out of our study is that muta-tions to at least eight genes – and probably manymore – greatly increase the risk of aggressivePrCa. Any future screening program would needto assess as many of these genes as possible.”

Image: Micrograph of prostate cancer. Researchershave found 14 gene mutations indicating life-threat-ening disease (Photo courtesy of Steve Gschmeiss-ner / SPL).

Genetic Screening Can Identify Men at Higher Risk of Aggressive Prostate Cancer




BBacterial pathogens, such as methicillin-resistantStaphylococcus aureus (MRSA), cause disease in

part due to toxicity, or the bacterium’s ability to dam-age a host’s tissue.

The spread of the antibiotic-resistant pathogen re-mains a concerning public health problem, especial-ly among doctors trying to determine appropriatetreatment options for infected patients.

Microbiologists at the University of Bath (UK;www.bath.ac.uk) and a team of international scien-tists used whole genome sequences from 90 MRSAisolates to identify over 100 genetic loci associatedwith toxicity. Bacterial adhesion to human fi-bronectin and fibrinogen was assessed and adherentbacteria were calculated by using the crystal violetmethod and absorbance measured at A595 using a

microtiter plate reader. The toxicity of individual iso-lates was assayed in three ways.

The identification of genetic variation in the clini-cal isolates was studied using unique index-tagged li-braries created for each sample, and up to 12 sepa-rate libraries were sequenced in each of eight chan-nels in the Genome Analyzer GAIIx cells (Illumina;San Diego, CA, USA; www.illumina.com) with 75-base paired-end reads.

The authors found that by using whole genomesequences from 90 MRSA isolates they were able toidentify over 100 genetic loci associated with toxici-ty and despite belonging to the same ST239 clone,the isolates varied greatly in toxicity. Importantly, thehighly toxic isolates shared a common genetic signa-ture. By looking for this signature in the MRSA

genome, the investigators were able to predict whichisolates were the most toxic and thus more likely tocause severe disease when used to infect mice.

Ruth C. Massey, PhD, the lead author of thestudy, said, “As the cost and speed of genome se-quencing decreases, it is becoming increasingly feasi-ble to sequence the genome of an infecting organism.In a clinical setting, sequencing may be useful for de-ciding the course of MRSA treatment. For example,a clinician may treat a highly toxic infection more ag-gressively, including prescribing certain antibioticsknown to reduce toxin expression. The patient alsomay be monitored more closely for complicationsand isolated from others to help control the spread ofinfection.” The study was published on April 9,2014, in the journal Genome Research.

Genome Sequencing of MRSA Infection Predicts Disease Severity

TThe genetic cause of a rare type of ovarian can-cer known as small cell carcinoma of the

ovary, hypercalcemic type (SCCOHT), that mostoften strikes girls and young women has been re-vealed.

This type of cancer, SCCHOT, usually is not di-agnosed until it is in its advanced stages and itdoes not respond to standard chemotherapy, and65% of patients die within two years. It can affectgirls as young as 14 months, and women as old as58 years, with a mean age of only 24 years old.

An international team led by the TranslationalGenomics Research Institute (TGen; Phoenix, AZ,USA; www.tgen.org) analyzed the genetic etiologyof SCCOHT by performing next-generation se-quencing on a series of tumors and germline sam-ples from 12 SCCOHT cases. This included nine tu-mors with four matched germline samples andthree additional germline samples, and on the

SCCOHT cell line BIN-67. DNA from tumor andblood specimens was analyzed using whole-genome sequencing and whole-exome sequencing.

Genomic DNA from each sample was frag-mented to a target size of 300 to 350 base pairs(bp). After ligation, samples were run on a gel toseparate products and the products were quanti-fied using the High-Sensitivity DNA chip on anAgilent 2100 Bioanalyzer (Santa Clara, CA, USA;www.genomics.agilent.com). A tissue microarray(TMA) representing nine SCCOHT cases was fab-ricated at TGen for the study. Protein blot analysiswas performed on whole cell extracts.

The scientists identified frequent germline andsomatic gene SWItch/Sucrose NonFermentable(SWI/SNF) Related, Matrix Associated, Actin De-pendent Regulator of Chromatin, Subfamily A,Member 4 (SMARCA4) mutations and SMARCA4protein loss in SCCOHT. The loss of SMARCA4

protein expression is extremely specific to SCCO-HT and can facilitate the differential diagnosis ofSCCOHT.

The study was published on March 23, 2014,in the journal Nature Genetics.

Image: Small cell carcinoma of the ovary, hypercal-caemic type (SCCOHT), the most common undiffer-entiated ovarian cancer to strike women under 40(Photo courtesy of TGen).

Genetic Cause Found for Rare Aggressive Ovarian Cancer




PROTEIN ANALYZERAgappe Diagnostics

The Mispa i3 is a cartridge-based, automatedspecific-protein analyzer that offers the benefits ofa fully automated analyzer. The system supportsthe clinical management of diseases such as CVrisk, rheumatoid diseases, inflammation, kidneydisease, and other specific protein assays.

LIQUID AMIES TRANSPORT SYSTEMPuritan Medical Products

The Liquid Amies Transport Systems are unique-ly engineered to provide superior performanceand ease of use in the collection and transport ofclinically significant bacteria. The systems are de-signed to use HydraFlock swabs to provide themost efficient absorption and elution available.

FOB RAPID TESTApacor

The FOB Rapydtest is an immunochemical deviceintended for the qualitative detection of fecal oc-cult blood levels. The test is designed as a usefulaid to detect bleeding caused by a number of gas-trointestinal disorders such as diverticulitis, colitis,polyps, and colorectal cancer.

FF luorescent in situ sequencinghas potential applications, such

as new diagnostics that spot the ear-liest signs of disease.

Fluorescent in situ RNA sequenc-ing (FISSEQ), could lead to earliercancer diagnosis by revealing molec-ular changes that drive cancer inseemingly healthy tissue. It cantrack cancer mutations and their re-sponse to modern targeted thera-pies, and uncover targets for saferand more effective ones. Themethod could also help biologistsunderstand how tissues change sub-tly during embryonic development –and even help map the maze of neu-rons. The researchers described themethod in the February 2014 onlineedition of Science.

The Wyss Institute of BiologicallyInspired Engineering at HarvardUniversity and Harvard MedicalSchool (Boston, MA, USA; http://wyss.harvard.edu) in collaborationwith the Allen Institute for BrainScience (Seattle, WA, USA; www.alleninstitute.org) developed thenew method that allows scientists topinpoint thousands of mRNAs andother types of RNAs in intact cells,while determining the sequence ofletters. Messenger RNAs (mRNAs)are positioned throughout living tis-sues, and their location often helpsregulate how cells and tissues growand develop. Until today, in order toanalyze many mRNAs simultane-ously, scientists have had to grindcells to a pulp, which left themwithout a method to pinpoint their

location within the cell.“By looking comprehensively at

gene expression within cells, wecan now spot numerous importantdifferences in complex tissues likethe brain that are invisible today,”said George Church, a professor ofgenetics at Harvard Medical Schooland a core faculty member at theWyss Institute. “This will help usunderstand like never before howtissues develop and function inhealth and disease.”

Healthy human cells typicallyturn on nearly half of their 20,000genes at any given time, and theychoose those genes carefully to pro-duce the desired cellular responses.Moreover, cells can dial gene ex-pression up or down, adjusting toproduce anywhere from a few work-ing copies of a gene to several thou-sand. Prof. George Church and JeHyuk Lee, a research fellow at Har-vard Medical School and the WyssInstitute wanted to pinpoint the cel-lular location of the mRNAs. Theywanted to simultaneously deter-mine the sequence of those RNAs,which identifies them and often re-veals their function.

The team first treated the tissuechemically to fix the cell’s thou-sands of RNAs in place. Then theyused enzymes to copy those RNAsinto DNA replicas, and copy thosereplicas many times to create a tinyball of replica DNA fixed to thesame spot. They managed to fix andreplicate thousands of the cell’sRNAs at once – but then the RNAs

were so tightly packed inside thecell that even a tricked-out micro-scope and camera could not distin-guish the flashing lights of one indi-vidual ball of replica DNA fromthose of its neighbors.

To solve that problem the re-searchers assigned the cell a uniqueaddress: the sequence of “letters,”or bases, in the RNA molecule itself.They figured they could read the ad-dress using methods akin to next-generation DNA sequencing, a set ofhigh-speed genome sequencingmethods Prof. Church helped devel-op in the early 2000s.

The scientists sought to fix RNAin place in the cell, make a tinyball with many matching DNAreplicas of the RNAs, then adaptnext-gen DNA sequencing so itworked in fixed cells. The fourflashing colors would reveal thebase sequence of each replica

DNA, which would tell them thebase sequence of the matchingRNA from which it was derived.And those sequences would in the-ory provide an unlimited numberof unique addresses – one for eachof the original RNAs.

The method, called fluorescentin situ RNA sequencing (FISSEQ),could lead to earlier cancer diagno-sis by revealing molecular changesthat drive cancer in seeminglyhealthy tissue. It could track cancermutations and how they respond tomodern targeted therapies, and un-cover targets for safer and more ef-fective ones.

Image: To develop fluorescent in situsequencing, scientists first fix in placethousands of RNAs – including work-ing copies of genes called messengerRNAs – in cells, tissues, organs orembryos. Here, RNAs are labeled redin a mouse brain (Photo courtesy ofHMS / Wyss).

Fluorescent In Situ SequencingHelps Diagnose Sick Tissues Early

AAcomprehensive genomic analysis ofcervical cancer in two patient popu-

lations has been completed and recurrentgenetic mutations have been identifiednot previously found in cervical cancer.

Cervical cancer is the second mostcommon cancer in women and is respon-sible for approximately 10% of cancerdeaths in women, particularly in develop-ing countries where screening methodsare not readily accessible and almost allcases of the disease are caused by expo-sure to human papillomavirus (HPV).

An international team of scientistsand led by those from the Broad Insti-tute (Cambridge, MA, USA; www.broadinstitute.org) performed wholeexome sequencing, which examinesthe genetic code in the protein-codingregions of the genome, on samples from115 cervical cancer patients from Nor-way and Mexico. Genomic DNA and ri-bonucleic acid (RNA) were extractedfrom tumors found by frozen section in-vestigations to have greater than 40%malignant epithelial cell component.

DNA from 115 tumor-normal pairedsamples was subjected to Sure-SelectHuman All Exon v2.0 based hybrid se-lection (Agilent; Santa Clara, CA, USA;www.agilent.com) followed by exomelibrary construction for sequencing (Il-lumina; San Diego, CA, USA; www.illumina.com), and 14 pairs for wholegenome library construction. Comple-mentary DNA (cDNA) from 79 sam-ples was subjected to transcriptome li-brary construction, according to stan-dard methods.

The study identified 13 mutationsthat occurred frequently enough acrossthe samples to be considered signifi-cant in cervical cancer. Eight of thesemutations had not been linked to thedisease previously, and two had notpreviously been seen in any cancertype. Among the most notable findingswere somatic point mutations in the V-Erb-B2 Avian Erythroblastic LeukemiaViral Oncogene Homolog gene (ERBB2),which was found in a small, but signifi-cant subset of the tumors. Mutations inthis gene had not been previously beenlinked to cervical cancer, but it is aknown oncogene common in breastcancer. The team also identified a novelmutation in the mitogen-activated pro-tein kinase 1 (MAPK1) gene and anoth-er key finding was the prevalence of mu-tations in genes affecting the immunesystem.

Hugo Alberto Barrera Saldaña, PhD,a professor of biochemistry and coau-thor of the study said, “The outstandingfindings of our successful collaborative

international research is giving us, here inMexico, very powerful arguments in favorof the benefit of speeding up the adoptionof molecular methods for screening HPV,followed by colposcopy to detect cancer atits earliest phases when it is curable; not tomention the possibility of new targetedtherapies that our discoveries open up.”The study was published on December 25,2103, in the journal Nature.

Image: The Genome Analyzer (Photo cour-tesy of Illumina).

Cervical Cancer GenomicStudy Completed



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2014ANNUAL

MEETING

LabMedicaLabMedica

AAutomated quantitative reading of fluorescenceintensity has been evaluated for clinical rele-

vance, which allows for value-added reporting oftest results.

Antinuclear antibodies (ANA) are important di-agnostic markers for systemic rheumatic diseases(SRD), such as systemic lupus erythematosus (SLE),systemic sclerosis (SSc), primary Sjögren’s syn-drome (SS), mixed connective tissue disease(MCTD), and, to a lesser extent, polymyositis(PM), and dermatomyositis (DM).

Medical laboratory scientists at the UniversityHospitals Leuven (Belgium; www.uzleuven.be) col-lected blood samples from 434 controls, which in-cluded 150 healthy blood donors, 150 chronic fa-tigue syndrome, and 134 diseased controls, and252 samples obtained at diagnosis from patientswith SRD. This latter group of 59 males and 193 fe-males with a mean age of 46, range 15 to 85 years,included 32 PM/DM patients, 15 with MCTD, 10cutaneous lupus cases, 83 diagnosed with SLE, 36with SS, and 76 SSc patients.

The antinuclear antibodies (ANA) were detectedusing NOVA Lite HEp-2 ANA kit (Inova Diagnostics,Inc.; San Diego, CA, USA; www.inovadx.com). Theslide processing was carried out on Inova’s QUAN-TA-Lyser 2 and read using their NOVA View digitalIndirect Immunofluorescence (IIF) microscope soft-

ware. NOVA View is an automated fluorescent mi-croscope programmed to acquire, archive and man-age digital images of fluorescent stained slides. Thesystem encloses an Olympus 1×81 inverted fluores-cent microscope (Olympus Belgium N.V.; Aartselaar,Belgium; www.olympus.be). Likelihood ratios werecalculated for fluorescence intensity result intervals.

The investigators found a significant correlationbetween end-point titer and fluorescence intensity.Likelihood ratios for a systemic rheumatic diseaseincreased with increasing fluorescence intensity.The likelihood ratio for a systemic rheumatic dis-ease was 0.06, 0.18, 0.51, 5.3, and 37.5 for a flu-orescence intensity of equal to or greater than 66,67 to 150, 151 to 300, 301 to 1,000, greater than1,000, respectively. A range of 31% to 37% of thepatients with Sjögren’s syndrome, systemic sclero-sis or systemic lupus erythematosus had fluores-cence intensities greater than 1,000.

The authors concluded that estimations of fluo-rescence intensity by automated antinuclear anti-body analysis offers clinically useful information.Likelihood ratios based on fluorescence intensitytest result intervals aid with the interpretation ofautomated antinuclear antibody analysis and allowvalue-added reporting. The study was published onApril 1, 2014, in the journal Clinical Chemistryand Laboratory Medicine.

Antinuclear-Antibody Testing Analyzed by Automated Indirect Immunofluorescence

AAnovel method for distinguishing differenttypes of bowel disease using the stool sam-

ples of patients has been created which is an ide-al noninvasive testing method for the diagnosisof gastro-intestinal diseases. The method worksby analyzing the chemical compounds emittedfrom the samples and could provide cheaper,quicker and more accurate diagnoses, at thepoint of care, for a group of diseases that have upuntil recently been very hard to differentiate.

Scientists at the University of the West of Eng-land (Bristol, UK; www.uwe.ac.uk) obtained 182stool samples from patients with inflammatorybowel disease (IBD) and irritable bowel syn-drome (IBS) between October 2010 and October2011. IBS samples were obtained from patientswith diarrhea predominant (IBS-D), constipationpredominant (IBS-C), and the alternating be-tween the two syndrome (IBS-A). IBD sampleswere obtained from patients with both ulcerativecolitis (UC) and Crohn’s disease (CD). Controlsamples were collected from healthy patients.

The test was based on the volatile organiccompounds (VOCs) emitted from their stool sam-ples, which act as a proxy for conditions in thegastrointestinal tract and provide a unique pro-file, or fingerprint, for the different bowel dis-eases. The investigators developed the methodusing an SRI 8610C gas chromatograph (SRI In-struments, Torrance, CA, USA; www.srigc.com)coupled to a metal oxide sensor system with pat-tern recognition software.

The results showed that patients with IBDcould be distinguished from healthy controls with

an accuracy of 79%. The method was able to dis-tinguish IBD from IBS with an accuracy of 76%.Differentiating patients with IBS from healthy con-trols using VOCs appeared to be more difficult andcould only be achieved with an accuracy of 54%.The reasons could be because IBS is a functionaldisorder as opposed to a structural disorder, so thechanges in composition of VOCs in the stool sam-ples would not be as great, producing a very simi-lar pattern to healthy controls.

The study was published on March 27, 2014,in the Journal of Breath Research.

Image: The Model 8610C gas chromatograph, usedto help develop markers for bowel disease (Photocourtesy of SRI Instruments).

Stool Samples Provide Marker for Bowel Disease

148LMI-07-14LINKXPRESS COM48LabMedica International

June-July/2014




TThe human eye using a microscope can be moreaccurate than automation when it comes to

analyzing urine samples for signs of kidney damage.An automated urine analysis system can miss

signs of kidney damage that can easily be identifiedby a trained observer looking through a compoundmicroscope at the urine sample.

A team of scientists at the University of Arizona(Phoenix, AZ, USA; www.arizona.edu) investigat-ed how well each approach, microscopy and auto-mated analysis, was able to identify granular andmuddy brown casts, tiny tube-like structures madeof protein secreted by the kidney. Casts in the set-

ting of kidney damage can help identify the under-lying problem such as inflammation or infection,and counting them and identifying their character-istics and composition can provide important diag-nostic and prognostic information.

Each of the 10 samples analyzed were from pa-tients with acute kidney injury. A single person an-alyzed each urine sample, first by examining it un-der the microscope, and then with the automatedIRIS 200 urinary analysis system (Iris Diagnostics;Chatsworth, CA, USA; www.irisdiagnostics.com).The IRIS 200 system coded 70% of the samples ashaving no casts. All of the samples were classifiedas having few casts using manual microscopy. Thesamples that were automatically coded on IRIS 200as having many casts were also coded as few bymanual microscopy.

Natasha Sharda, MD, the lead investigator, said,“The automated system underreported the value ofgranular casts in our patient cohort of acute kidney

injury. The automated system still has utility as ascreening test, but manual microscopy should bedone in all cases of abnormal kidney function, asaccurate quantification of casts could have someprognostic benefit to patients. Even though the au-tomated urinalysis system has many benefits, itshould not replace direct observation, but rather beused as augmentation.”

Thomas Manley, director of scientific activitiesat the National Kidney Foundation (New York,NY, USA; www.kidney.org) added, “Technologicaladvances in medicine have been extremely valu-able in improving outcomes for patients, but insome cases the human eye is still superior to ma-chines. In this study manual microscopy was su-perior to an automated urinalysis in evaluatingurine sediment properties.” The study was pre-sented at the National Kidney Foundation’s 2014Spring Clinical Meetings held April 22–26, 2014,in Las Vegas (NV, USA).

Classical Microscopy Outperforms Automated Urine Analysis

Hybridization-Based Solid Tumor Panel Delivers

Consistent, Reliable Results

AAnew 60-gene next generation sequencing (NGS)hybridization-based enrichment panel offers sci-

entists accurate and reliable solid tumor profiling forboth known and novel variants.

The SureSeq solid tumor panel was launched bythe Oxford Gene Technology (OGT; Begbroke, Ox-fordshire, United Kingdom; www.ogt.com) at the As-sociation for Clinical Genetic Science (ACGS) meetingat Birmingham (UK), on April 29–30, 2014. The pan-el was fully validated on formalin-fixed, paraffin-em-bedded (FFPE) samples.

The content of the panel has been defined by can-cer experts, covering key genes for a range of cancertypes including breast, prostate, ovarian, lung, and col-orectal. All exons of these genes are fully covered, in-cluding mutation hotspots, enabling both detectionand discovery of known and novel variants respective-ly.

The hybridization-based SureSeq Solid Tumor Pan-el minimizes polymerase chain reaction (PCR) biasand duplications commonly associated with alterna-tive enrichment methodologies, enabling greater run-to-run consistency. This is particularly important in sit-uations where there is a limited sample or where theability to detect minor allele frequencies is required,such as in heterogeneous tumor samples. Such sampletypes require a highly uniform and sensitive enrich-ment and OGT’s expert bait design ensures this byproviding efficient and improved uniformity of cover-age of the targeted regions, enabling all variants to becalled with maximum confidence.

Providing easy access to meaningful data, the Sure-Seq Solid Tumor Panel comes with OGT’s VariantAnalysis Report, equipping researchers with the free-dom to explore and retrospectively interrogate datawith additional or new selection criteria, without theneed for additional in-house bioinformatics resource.Using the report, data can be easily filtered by numer-ous parameters, including gene, depth of coverage, so-matic variants and predicted effect on the protein. Inaddition, all variants are fully annotated with links tovarious databases.

OGT’s Cytocell, CytoSure and Genefficiency rangeof fluorescence in situ hybridization (FISH), microar-ray and next generation sequencing (NGS) productsand services deliver high-quality genetic analysis, en-abling accurate identification and confirmation of thecausative variation underlying genetic disease.

AAn ultra-sensitive RT-PCR ap-proach using breakthrough

DNA-enrichment technology hasnow enabled low-level DNA muta-tions in cancer patients to be detect-ed from whole blood samples.

A breakthrough achievementwith the DNA-enrichment technolo-gy used in “PointMan” from EKFDiagnostics (Cardiff, UK; www.ekfdiagnostics.com) paves the wayfor its potential use in cancer patienttesting, monitoring, and treatmentby enabling blood sampling insteadof tissue biopsies for assessment ofcancer gene-mutation status. Thefirst successful results of collabora-

tion between EKF Diagnostics andthe Institute of Life Sciences (ILS) atSwansea University have demon-strated the detection of gene muta-tions in blood from samplesarchived in the Wales Cancer Bank.EKF’s PointMan technology wasused to analyze the whole blood ofskin cancer patients diagnosed withmetastatic melanoma, enabling theidentification of gene mutations as-sociated with response to drug treat-ment.

Crucially, the results observed formutations in the gene BRAF wereconsistent with the formalin fixedparaffin embedded (FFPE) tissue

samples; FFPE being the standardmethod to prepare biopsy samplesfor pathology review in order to di-agnose the cancer. These resultshave been confirmed by DNA se-quencing, which had failed to iden-tify the mutations prior to sampleenrichment through EKF’s Point-Man technology.

PointMan is a real-time PCRtechnology that provides reliableand extremely sensitive detectionfor cancer mutations. It is highly ef-ficient in amplifying the target se-quence of interest, while suppress-ing amplification of the wild type.The resulting sample is effectivelyenriched for the mutation, therebyhaving the potential to offer highsensitivity in a wide variety of sam-ple types, including whole blood.This is demonstrated in the ILSSwansea study.

“This is a major step forward notjust for the company but also for thefuture testing of cancer patients,where we hope that less-invasive

testing will become routine usingour PointMan technology. We arelooking forward to continuing towork with ILS Swansea to continueto build the evidence base. Furtherevidence will be generated fromother collaborations and I look for-ward to providing further updatesduring 2014,” said Julian Baines,CEO of EKF.

“Current collaborations focus onthe unmet requirements for patientmonitoring from a peripheral sam-ple, thereby negating the require-ment for a surgical procedure to ob-tain a tissue biopsy, and screeningfor early cancer diagnosis.” Dr. Ri-cardo Del Sol, Senior Lecturer, ILSSwansea University, added.

EKF’s portfolio of PointMan DNAenrichment products include thegenes for: BRAF, KRAS, EGFR,NRAS, and JAK2.

Image: The PointMan technology en-ables detection of low-level DNA mu-tations from whole blood samples(Photo courtesy of EKF Diagnostics).

DNA-Enrichment Technology May Help Replace Biopsies forCancer Mutation Testing




TEST READERDIALAB

The ELX 808IU features a wavelength range of340-900 nm, and can accommodate standard 96-well, flat- or round-bottom plates. The unit offersELISA reading speeds of 8-12 seconds in singlewavelength, and 13-20 seconds in dual wave-length, and is temperature controlled to 50 °C.

ELISA SYSTEMDYNEX Technologies

The DSX is a fully automated, four-plate ELISAprocessing system that is capable of performingmultiple assays per plate. Key features such asflexible and reliable sample in/result out process-ing provide true walkaway automation, and en-sure the quality and security of results.

HELICOBACTER PYLORI TESTGold Standard Diagnostics

The H. pylori EIA (IgG, IgA, IgM*) is designed toimprove sensitivity and specificity for the mostdangerous strains of H. pylori. Key features in-clude a unique antigen purification process thatmaintains CagA and VacA, no specialized equip-ment or training required, and no patient fasting.


MMobile zinc is an indispensablecomponent of prostate physi-

ology and the prostate containsmore zinc than any other soft tissuein the body, and there is a clear cor-relation between total prostatic zinclevels and cancer.

A new optical sensor that cantrack zinc in the body’s cells hasbeen described and how the sensorfluoresces when it binds to zinc canbe targeted to a specific organellewithin a cell, enabling the establish-ment of where the zinc is most con-centrated.

Scientists at the MassachusettsInstitute of Technology (Cambridge,MA, USA; www.mit.edu) developeda sensor that relies on Zinpyr1(ZP1), a molecule originally devel-oped more than 10 years ago. ZP1 isbased on a dye called fluorescein,but in the sensor, this is modified tofluoresce only when it binds withzinc. High-resolution mass spectrawere resolved using a mass spec-trometer (Bruker Daltonics; Billeri-ca, MA, USA; www.bruker.com).Fluorescence spectra were recordedon a Quanta Master scanning spec-trofluorometer (Photon TechnologyInternational; Birmingham, NJ,USA; www.pti-nj.com).

The investigators could track thelocation of zinc within cells andgained a better understanding of the

role the mineral plays in cancerouscells. The scientists made twochanges to the sensor’s design. First,they installed a zinc-reacting pro-tecting ring, which changed itsphysical properties and made it easi-er to target. They also attached an“address tag” to the ZP1, directing itto the mitochondria. This tag, a de-rivative of triphenylphosphonium, isboth positively charged and hy-drophobic. The resulting sensor eas-ily entered the cells, which allowedthem to visualize pools of mobilezinc within the mitochondria.

Inside the mitochondria of ep-ithelial prostate cells, zinc is knownto inhibit the metabolic enzyme,aconitase. The scientists believethat by blocking aconitase, zincshortens the citric acid cycle, whichare the series of reactions needed toproduce adenosine triphosphate(ATP). Most ATP production occursin the mitochondria, and the MITteam theorized that when prostatecells become cancerous, they ban-ish zinc from there, allowing thecancer cells to produce the extraenergy they need to grow and di-vide. The scientists found that al-though the cancerous prostate cellsabsorbed the zinc, it did not collectin the mitochondria.

Robert Radford, PhD, the seniorauthor of the paper said, “We can

use these tools to study zinc traffick-ing within prostate cells, bothhealthy and diseased. By doing so,we’re trying to gain insight into howzinc levels within the cell changeduring the progression of prostatecancer.” The study was published

on December 12, 2013, in the jour-nal Proceedings of the NationalAcademy of Sciences of the UnitedStates of America (PNAS).

Image: The QuantaMaster scanningspectrofluorometer (Photo courtesy ofPhoton Technology International).

Sensor Tracks Zinc in Cells For Prostate Cancer Diagnosis

TThe concentration of circulatingpeptides produced by CPN (car-

boxypeptidase N) cleavage reflectsthe CPN activity in tumors, andanalysis of these biomarkers demon-strates potential for the noninvasiveand early diagnosis of breast cancer.

CPN is important in regulating va-soactive peptide hormones, growthfactors, and cytokines by specificallycleaving their C-terminal basicresidues. Investigators at Weill CornellMedical College (New York, NY, USA;www.med.cornell.edu) investigatedwhether circulating peptides specifi-cally cleaved by CPN in the tumor mi-croenvironment could be stage-specif-ic indicators of breast cancer.

The investigators identified pep-tide fragments produced by CPN us-ing an ex vivo peptide cleavage assay.They incubated a synthesized C3fpeptide (His6-C3f_S1304-R1320-His6) in interstitial fluids taken frombreast tumors and adjacent normalbreast tissues in mice with orthotopic

implantation of the human breastcancer cell line MDA-MB-231. Thenature and extent of peptide cleavageby CPN was investigated by fragmentprofiling using nanopore fractionationand mass spectrometry.

Results revealed that generation ofthe C3f_R1310-L1319 cleavage pep-tide specifically correlated with theCPN expression level. In both themouse and clinical patient samples,CPN was clearly increased in tumortissues compared with normal breasttissue, whereas corresponding CPNabundance in blood remained con-stant. Concentrations of six CPN-cat-alyzed peptides predominantly in-creased in sera taken from the miceat two weeks after orthotopic implan-tation. Six homologous peptides dis-played significantly higher expressionin the patients’ plasma as early as thefirst pathologic stage of breast cancer.

The study was published in theJanuary 2014 issue of the journalClinical Chemistry.

Determination of Circulating CPN Cleavage Peptides Enables Early Diagnosis of Breast Cancer







LAB STORAGE SYSTEMGrifols

The GRI-FOLDER is a lab folder specially de-signed for storing, freezing, and transportingblood, plasma or similar lab bags. The folder istransparent for easy visual ID and barcode read-ing, can be handled at any temperature, and isstackable both before and after use.

CLINICAL CHEMISTRY ANALYZERKehua Laboratory System

The ZY-260 features high precision microsam-pling, along with an auto-dilution function and au-to-probe cleaning. The system offers 12 wave-lengths, enhanced reliability, low maintenance,and a throughput of up to 200 tests per hour (400T/H with ISE).

MEDICAL SAPPHIRE OPTICSMeller Optics

The custom sapphire optics are designed for usein high-value medical/lab instruments that comeinto frequent contact with skin, blood, and harshchemicals. The optics are impervious to chemi-cals, chlorine and fluorine gas, and blood, and areavailable in various sizes and shapes.

DD isease-free ticks have been used to detect Ly-me disease bacteria in people who continue to

experience symptoms such as fatigue or arthritis af-ter completing antibiotic therapy.

The technique, called xenodiagnosis, attemptsto find evidence of a disease-causing microorganismindirectly, through use of the natural disease-carrier,in this case, ticks of the Ixodes genus.

Scientists at the Tufts Medical Center (Boston,MA, USA; www.tuftsmedicalcenter.org) and theircolleagues from other institutions enrolled 36 adultvolunteers in the study at locations in Maryland,Connecticut, and Massachusetts. Participants in-cluded 10 people with post-treatment Lyme diseasesyndrome (PTLDS); 10 who had high levels of anantibody against Borrelia burgdorferi after antibiot-ic treatment; 5 who had erythema migrans, a bull’s-eye rash typical of Lyme disease, and had received

antibiotic treatment in the past; 1 with erythemamigrans who began antibiotic therapy at the time oftick placement; and 10 healthy volunteers.

Participants consented to have up to 30 labora-tory-bred, pathogen-free, larval ticks I. scapularis,placed under a dressing. When possible, the tickswere placed near areas where a rash had been ob-served or near affected joints.

Ticks were tested for B. burgdorferi by poly-merase chain reaction (PCR), culture, and/orisothermal amplification followed by PCR and elec-trospray ionization mass spectroscopy. In addition,attempts were made to infect immunodeficient miceby tick bite or inoculation of tick contents. Xenodi-agnosis was repeated in seven individuals. Ticks andskin biopsies were extracted using Qiagen DNEasycolumns from the DNEasy Blood and Tissue Kit (Qi-agen; Valencia, CA, USA; www.qiagen.com).

Not all of the placements yielded enough blood-engorged ticks to perform xenodiagnosis, but 23 vol-unteers with Lyme disease had at least one tick test-ed and of these, 19 people tested negative. Two peo-ple had indeterminate results, thought to be due tolaboratory contamination. Xenodiagnosis was posi-tive for B. burgdorferi DNA in the person with ery-thema migrans who underwent xenodiagnosis earlyduring therapy, and in a volunteer with PTLDS.

Adriana Marques, MD, the lead author, said,“Xenodiagnosis using ticks to detect B. burgdorferihas been used previously in animal studies, but thisis the first time it has been tried in people. Our pri-mary goals in this initial trial were to develop pro-cedures for tick xenodiagnosis and to determine itssafety in humans.” The study was published onFebruary 11, 2014, in the journal Clinical Infec-tious Diseases.

Xenodiagnosis Tested for Persistent Lyme Disease

LLactobacilli play a key role in promoting vagi-nal health and depletion of these bacteria is

associated with bacterial vaginosis (BV), the mostcommon vaginal disorder. A novel single-tubemultiplex quantitative polymerase chain reaction(qPCR) assay for the identification and quantita-tive assessment of the four major vaginal Lacto-bacillus species has been developed.

Microbiologists at the Femeris Women’s HealthResearch Center (Hamilton, NJ, USA; www.femeris.com) assessed 60 vaginal swab specimensas part of a study on BV pathogenesis. Sampleswere shipped frozen in Universal Transport Medi-um (UTM-RT) (Copan Italia SpA; Brescia, Italy;www.copanitalia.com) from August to October of2012. DNA from clinical samples was extractedusing the QIAamp Mini Kit (Qiagen; Valencia, CA,USA; www.qiagen.com) in combination with pre-treatment with proteinase K and mechanical ho-mogenization. Multiplex and uniplex TaqManqPCRs were performed using primers and probes

from Integrated DNA Technologies (Coralville, IA,USA; www.idtdna.com) and Qiagen’s RotorGeneQ instruments were used for all qPCR assays.

The assay utility was evaluated by the analysisof lactobacilli in noncultured clinical vaginal swabspecimens collected from BV patients and healthyindividuals. As confirmed by the assay, L. crispa-tus, L. jensenii, and to a lesser extent L. gasseri,are common in the vagina of healthy women,whereas L. iners dominance is associated with BV.In order to confirm multiplex TaqMan qPCR re-sults, bacterial cultures were isolated from vaginalsamples and 11 swab specimens yielded isolatesof Lactobacillus species identified in them by themultiplex TaqMan qPCR assay. A total of 11 indi-vidual Lactobacillus strains were obtained: threeL. crispatus, two L. gasseri, two L. jensenii, andfour L. iners. Their species assignments were con-firmed by both multiplex TaqMan qPCR and uni-plex SYBR Green qPCRs with 100% concordance.

The authors concludes that their assay had

been validated using bacterial cultures of lacto-bacilli and other microbial commensal and patho-genic species populating the microflora of the low-er female genital tract, human chromosomalDNA, synthetic plasmid controls, and clinicalvaginal swab specimens. The multiplex qPCR as-say is an advance in the detection and quantita-tion of the major vaginal lactobacilli, potentiallyfacilitating the molecular diagnosis of BV and post-therapy restoration of the vaginal microflora. Thestudy was published online on January 18, 2014,in the journal Diagnostic Microbiology and Infec-tious Disease.

Image: The Rotor-Gene Q cycler, designed to carry outPCR and data analysis (Photo courtesy of Qiagen).

Multiplex Molecular Assay Identifies Major Vaginal Lactobacilli

TThe first and only molecular diag-nostic test to detect and differ-

entiate influenza A and B virus inless than 15 minutes is now avail-able in Europe.

Rapid diagnostics with increasedsensitivity are essential for enablinghealthcare professionals to makemore effective decisions. The “Alerei Influenza A & B” test from Alere(Waltham, MA, USA; www.alere.com) provides highly accurate, rapidmolecular results via the simple,user-friendly “Alere i” platform.

“Alere i is a transformational plat-form that allows healthcare profes-sionals to make a rapid influenza di-agnosis and effective patient manage-ment decisions in a clinically mean-ingful timeframe, whether the pa-tient is in the physician office, emer-gency department, or urgent careclinic,” said Avi Pelossof, Alere Glob-al President of Infectious Disease.“Alere i also significantly expandsscreening opportunities by makinginnovative, rapid molecular testingtechnology available at the point ofcare as well as in laboratory settings.”

The proprietary technology uti-lizes isothermal nucleic acid amplifi-cation technology (iNAT), which,

unlike polymerase chain reaction(PCR) testing, does not require tem-perature cycling and can thereforedeliver results more quickly (“Mole-cular-In-Minutes” (MIM)) and to abroader range of settings.

Alere i Influenza A & B deliversactionable, lab-accurate results. Itsclinical performance was establishedin a multicenter, prospective studyconducted at 8 US trial sites duringthe 2012-2013 respiratory season. Atotal of 571 prospective nasal swabspecimens, collected from patientspresenting with influenza-like symp-toms and representing a wide rangeof ages, were evaluated with Alere-iInfluenza A & B and compared to vi-ral culture. All discrepant sampleswere tested on an FDA-cleared RT-PCR assay at a central testing labora-tory to confirm influenza status. Per-formance vs. culture discrepant re-sults resolved by RT-PCR were: In-fluenza A: resolved sensitivity –99.3%, resolved specificity – 98.1%;Influenza B: resolved sensitivity –98.9%, resolved specificity – 99.6%.

Alere i Influenza A & B is nowcommercially available in Austria,France, Spain, Switzerland, Ger-many, Italy, and the UK. It is cur-

rently not available in the USApending completion of regulatory re-

view by the US Food and Drug Ad-ministration (FDA).

TThe discovery that breast cancerpatients who experienced quick

relapses tended to have low levels ofthe microRNA miR-21 and high lev-els of the microRNA miR-205 in theirtumor tissues suggests that measure-ment of these two biomarkers couldidentify women at risk of metastasis.

The stability of microRNAs (miR-NAs) – snippets of about 20 nu-cleotides that block gene expressionby attaching to molecules of messen-ger RNA (mRNA) in a fashion thatprevents them from transmitting theprotein synthesizing instructionsthey had received from the DNA –in formalin-fixed paraffin-embedded(FFPE) tissues enables their reliableanalysis in archived FFPE tissue sam-ples. Such samples are an invaluablesource in the search for novel bio-markers. This is especially true inthe case of breast cancer, for whichlate relapses occur in many cases.Thus, analysis of miRNAs in FFPEbreast tumor samples holds great po-tential, as this type of testing canlead to the discovery of novel bio-markers suitable for future routineclinical diagnostics.

Investigators at the University ofAthens (Greece; www.uoa.gr) inves-tigated the prognostic significance ofsix metastasis-related miRNAs thatregulate various stages of migrationand invasion and play critical roles inthe multistep metastatic process.They quantified the expression ofmiR-21, miR-205, miR-10b, miR-210, miR-335, and let-7a by reverse-transcription quantitative PCR in FF-PE tissues from 84 patients with ear-ly breast cancer and a long follow-up,and 13 cancer-free breast tissue FFPEsamples that were used as the controlgroup. They further correlated indi-vidual miRNA over- or under-expres-sion with the disease-free interval(DFI) and overall survival (OS).

Results published in the January2014 issue of the journal ClinicalChemistry revealed that that bothmiR-21 and miR-205 were signifi-cantly associated with DFI and onlymiR-205 with OS. Multivariate analy-sis demonstrated that miR-205 andmiR-21 were independent factors as-sociated with early disease relapse,whereas only miR-205 overexpres-sion was associated with OS.

MicroRNAs Predict Likelihood Of Breast Cancer Metastasis

Image: The rapid Alere i Influenza A & B test is designed for the Alere i platform (Photo courtesy of Alere).

First Test to Accurately Detect Influenza A and B in Under 15 Minutes







DATA MANAGEMENT SYSTEMSiemens Healthcare

The RAPIDComm system (V5.0) centrally man-ages in vitro diagnostics analyzers and operatorsat the POC. A recent upgrade includes support forthe system’s Web app that enables user manage-ment of blood gas analyzers from an iPad, as wellas interface to a PEP administrator.

URINE ANALYZERUnited Diagnostic Industry

The Uditest-50 features up to 13 parameters onstrips, two test modes and speeds for selection,and data transfer capability via RS 232 port. Theunit offers a throughput of 60 tests per hour in nor-mal mode, 120 T/H in continuous mode, and hasa memory capacity for 1,000 test results.

AUTOMATED ANALYZERYD Diagnostics

The UriScan Super+ features a touch screenLCD, double-sealed cassette for on-board stabili-ty, and various interfaces for easy integration.Other benefits include a barcode reader for sam-ple ID, STAT position for urgent test samples, andthe capacity for loading 100 samples at once.

AApreliminary study has indicatedthat a blood test for infection

by the prion that causes variantCreutzfeldt-Jakob disease (vCJD)has sufficient sensitivity and speci-ficity to justify a large study compar-ing vCJD prevalence in the United

Kingdom with a bovine spongiformencephalopathy-unexposed popula-tion.

vCJD is a fatal degenerative braindisorder thought to be caused by amisfolded protein (prion) in thebrain and contracted most common-

ly through eating beef infected withBSE (bovine spongiform en-cephalopathy). Up to three millioncattle in the United Kingdom mayhave been infected with BSE, andestablishing accurate prevalence es-timates through screening for vCJDinfection would guide public healthinitiatives.

Investigators at University Col-lege of London (United Kingdom;www.ucl.ac.uk) employed a proto-type test (now in clinical diagnosticuse) that captured, enriched, anddetected disease-associated prionprotein from whole blood samplesusing stainless steel powder to de-termine the presence of vCJD infec-tion in 5,000 blood samples fromUS donors, 200 samples fromhealthy United Kingdom donors,352 samples from patients withnon-prion neurodegenerative dis-ease, 105 samples from patients inwhom a prion disease diagnosis waslikely, and 10 samples from patientswith confirmed vCJD.

Results revealed that the assay’sspecificity among the presumed neg-ative American donor samples was

100%, which was confirmed in thehealthy United Kingdom cohort(100% specificity). Of potentiallycross-reactive blood samples frompatients with non-prion neurode-generative diseases, no samples test-ed positive. The test identified aspositive 71.4% of the patients withvCJD.

The authors concluded that,“Most importantly, the prototypevCJD assay has sufficient perform-ance to justify now screening a largeUnited Kingdom population sampleand at-risk groups to produce an ini-tial estimate of the level of prione-mia [prions in the blood] in the Unit-ed Kingdom blood donor popula-tion. A blood prevalence studywould provide essential informationfor policy makers for deciding if rou-tine vCJD screening is needed forblood, tissue, and organ donationsand patients prior to high-risk surgi-cal procedures.”

The study was published in theMarch 3, 2014, online edition of theJournal of the American Medical As-sociation Neurology (JAMA Neurolo-gy).

Highly Specific Blood Test For Creutzfeldt-Jakob Disease

Image: A new blood testhas accurately screened

for infection with theagent responsible for

variant Creutzfeldt-Jakob disease (vCJD),

a fatal neurological disease (Photo courtesy

of the University College of London).


Laboratory medicineis evolving rapidly

and is playing an evermore important role in modern healthcare.National and interna -tional structures to

support the evolving labo ratory medicine are not al-ways able to accom modate this change. In May 2013the IFCC Executive Board launched a one yearconsulta tion entitled ‘Shaping the Futureof Laboratory Medicine’ which sought tostimulate discussion at national and inter-national level. This article brings togetherthe major points of discussion ahead of‘The Great Debate’ at the IFCC Councilmeeting in Istanbul in June 2014.

Central Role of Laboratory Medicine It is generally accepted that a high per-centage of all clinical decisions are influ-enced by labora tory medicine results ata small overall cost to the healthcarebudget. This places great respon sibilityon laboratory medicine specialists to po -sition themselves at the center of themultidis ciplinary team that is responsiblefor all aspects of healthcare from well-ness screening through to monitoringthe response to therapy.

Laboratory Medicine under Review Despite, or perhaps because of its cen-tral role laboratory medicine servicesare currently under review in a largenumber of countries around the world.The exact terms of these re views maydiffer but there three components tothe review:

• Improving quality across the spec-trum from analytical quality, to quality as-surance to quality management to labo-ratory ac creditation. Different countriesare on different rungs of the ‘quality lad-der’ but the direction of travel is clear;

• Improving clinical effectiveness bytargeting the use of laboratory medicineto improve clinical outcomes. The timelypresentation of results is one componentas is a clinical in terpretive and advisoryservice. Recognizing the growing impor-tance of patient-focused medicine is an-other requirement;

• Improving cost effectiveness by do-ing more at equal or higher quality for alower total cost. Laboratory medicinehas a unique record of achievement inthis area but the trend will continue. Theappropriate use of the labora tory anddemonstrating value for money aregrowing facets of cost effectiveness.

Mega-Trends in Global Healthcare Laboratory medicine needs to adapt tothe changing shape and delivery ofhealthcare. Ex perts in the businesscommunity have highlight ed 12 mega-trends in future healthcare:

An aging population with increasing chronic dis-ease; Technological advance supporting personal-ized medicine; Innovation and increasing demand,espe cially in developing countries; Evidence-based medicine and the adoption of clinical prac-tice guidelines; Environmental challenges – e.g.,air, water, food, climate, congestion; Global pan-demics – e.g., pandemic influenza; Monitoringhealthy people to prevent dis ease and keep themwell; Greater devolution of aspects of healthcare to

trained nonmedical professionals; Philanthropy tospeed up advances in healthcare in developingcountries; Intelligent and informed patientsinfluenc ing decisions on their healthcare; Medicaltourism to get the best quality or value healthcarein another country; Rising costs and inadequatehealth budgets.

Laboratory medicine contributes to virtually all ofthese mega-trends by facilitating improved clinicaleffectiveness and/or cost effectiveness.

MESSAGE FROM THE PRESIDENTby Dr. Graham Beastall, President, IFCC

Shaping the Future of Laboratory Medicine: The Great Debate

Edited by Tahir Pillay MBChB, PhD, FRCPath(Lon), FCPath(SA)IFCC members may send news to: Tahir Pillay MBChB, PhD, Head, Dept of Chemical Pathology, Faculty of Health Sciences, University of Pretoria, Private Bag Bag x323, Arcadia, 0007, South AfricaTel: (27) 12 319-2116; Fax: (27) 328 6000; Email: [email protected] NEWS


Cont’d on page 56

Laboratory Medicine - Future Priorities It follows from the discussion to date that the future pri-orities for laboratory medicine must lie in three areas:

Continuous laboratory quality improvement; Im-provement in clinical outcomes; Improvement in effi-ciency and cost effec tiveness.

One way of looking at this is by adding value to ahigh quality service. To deliver these priorities will re-quire laboratory medicine specialists to work outsideas well as inside laboratories.

Drivers for Change in Laboratory Medicine There are many drivers for change in laboratory med-icine. It is convenient to divide these into five majorcategories to aid understanding:

• Globalization; • Technological advance;• Smarter working;• Integrated diagnostics;• Adding value to improve outcomes.Each of these topics merits a detailed report. For the

purposes of this review each will get a short paragraph. Globalization. We live in a world of instant com -

munication. This provides an opportunity to share in-formation on an international scale on a wide range oftopics including:

• Quality standards;• Laboratory practice;• Clinical applications.Through sharing we can more rapidly meet the re-

quirements of patients, clinicians and other health-care interests.

Technological Advance. We are in the middle of atechnological revolution. Advances in technol ogy en-able us to achieve higher quality, more rapidly and ona smaller scale. Sometimes, but not always this isachieved at a lower cost. There are many examplesof technological advance in laboratory medicine in-cluding:

Nanotechnology and point of care testing (POCT);Automation including robotics, platforms and integrat-ed systems; Mass spectrometry on the bench topacross all of laboratory medicine; Bioinformatics tomake sense of the huge volumes of data now avail-able; Genomics informing greater understanding anddriving personalized medicine; Proteomics andmetabolomics facilitating new biomarkers.

Rapid technological advance has implications forknowledge and skills training.

Smarter Working. The combination of an agingpopulation, medical advances and rising work loadscombine to put unsustainable pressure on healthcarebudgets, whether they are funded by the state or theindividual. The response of the laboratory medicineprofession has been ad mirable but it will need to con-tinue. Improved efficiency, workload managementand shared resources are just some examples ofsmarter working but these will impact on staffing lev-els and the skills mix amongst staff.

Integrated Diagnostics. Laboratory medicine, im-aging and endoscopy all contribute diagnos tic patientdata. Through integration and incor poration into pa-tient pathways this data can be converted into knowl-edge which can be used to bring about faster and bet-ter clinical outcomes. One consequence of integrateddiagnostics is an erosion of the traditional boundarieswithin lab oratory medicine and between the diagnos-tic specialties, with consequences for education,training and future job roles.

Adding Value to quality laboratory medicine servicescomprises a wide range of opportuni ties to go beyond asimple request-result serv ice. A simple tool to explain

the complexities of adding value is the mnemonic SCI-ENCE, which breaks down adding value into: • Standardization or harmonization of meth ods and

practices;• Clinical effectiveness improvement through greater

involvement with users;• Innovation in methods, clinical settings and serv-

ice delivery;• Evidence-based medicine and clinical prac tice

guideline implementation;• Novel applications exemplified by the shift from re-

active to ‘P4’ medicine;• Cost effectiveness and value for money; • Education of others to better understand the role

of laboratory medicine. This is a professional responsibility. More detail

may be found in Clin Chem Lab Med 2013; 51: 221-28.

‘Divisions’ in Laboratory Medicine We have an identity problem in laboratory medicineat national, regional and international level. Wehave many different names for our profession. Wehave different grades of staff working in the labora-tory (e.g., medical doctors, scientists, technologists)not always harmoni ously. We have several subspe-cialties in labo ratory medicine (e.g., clinical chem-istry, haema tology, microbiology, and genetics) withblurred boundaries and variable interpretationacross the world. We deliver our services in a widerange of clinical settings (e.g., public, private, hospi-tal, clinic, POCT) often in an unconnected way. Thisis not good for the patient and it is not good for theprofession. Within the profes sion we are confusedby these ‘divisions’ and it is hardly surprising thatthose outside do not re ally understand who we areand what we do.

The solution to this identity problem is to be moreinclusive and to put aside professional issues in theinterest of the patient, who must be the primary con-sideration. The laboratory medicine specialist shouldbe a central part of the clinical team supported by aninclusive and integrated team of staff working in acoordinat ed group of laboratory subspecialties,deliver ing ‘joined up’ laboratory medicine services.For most of us this destination is a long way off withmany barriers in the way but the journey should start,driven by the quality standards and serv ice specifica-tion needed by the patient.

‘Shaping the Future of Laboratory Medicine’ Against this complex and dynamic background thereare opportunities for laboratory medicine specialists.As part of its strategic plan the IFCC Executive Boardlaunched a consultation docu ment in May 2013,which challenges all involved in laboratory medicineto consider the implica tions of the future for the serv-ice they deliver. The consultation would last a yearand culmi nate in a debate (‘The Great Debate’) at theIFCC Council meeting in June 2014.

The aims of the consultation are twofold • To stimulate IFCC Members to discuss how best

to support the changing face of labora tory medi-cine at local and national level;

• To consider how IFCC may position itself to en-hance its global leadership role.I am aware that many IFCC Members have been

having local discussions and I look forward to hearingtheir plans for the future.

Opportunities for IFCC A more inclusive and more broadly based IFCC canbe brought about through expanded mem bership.

This will enable IFCC to develop further its globalleadership through: • Increased influence with the World Health Organi-

zation; • More global standardization and harmonization

initiatives; • More global practice standards and guide lines; • A more effective global voice for laboratory medicine; • Increased focus on added value and clinical out-

comes;• Increased credibility with global clinical organiza-

tions; • An improved range and quality of service for IFCC

Members; • Increased collaboration for Full and Corpo rate

Members. There is a barrier to IFCC being more inclusive.

This is IFCC Statute 4.1.1, which effectively limits IFCCFull Membership to one society per coun try. ThisStatute dates back 60 years to a very different periodfor laboratory medicine when IFCC was dedicatedsolely to the developing field of Clinical Chemistry.

A November 2013 survey of IFCC Full Memberssocieties revealed that: • 100% are active in clinical chemistry;• >70% are active in immunology and hematology; • >60% are active in microbiology, molecular pathology; • >50% are active in genetics and virology; • <50% are active in transfusion, transplanta tion, in-

formatics;• Only 2 IFCC Full Members are active in ana tomic

pathology.From the results of this survey IFCC concludes: • Laboratory medicine and anatomic pathol ogy are

generally organized and delivered separately andso IFCC should not include anatomic pathology;

• There is considerable scope for IFCC to be moreinclusive of all areas of laboratory medicine. Thiscould be achieved through expanded membership.

Accordingly, the IFCC Executive Board wishes to pro-pose to the IFCC Council that it should consider: • Amending Statute 4.1.1 that restricts IFCC Full

Membership to one society per country;• Opening IFCC Membership to any properly consti-

tuted society that is active in labora tory medicine;• Facilitating Full Membership from societies active

in microbiology, genetics, transplan tation, bioinfor-matics etc.;

• Adopting a similar inclusive approach to CorporateMembership to expand the range of company in-terest in laboratory medicine.

‘The Great Debate’ The IFCC Council will debate the future of labo ratorymedicine from 13.45-15.45h on Sunday 22 June 2014in the Congress Center in Istanbul (Turkey). This is anopen meeting that any inter ested person may attendand contribute. The key points in the debate will be:

Drivers for change in laboratory medicine; Divi-sions in laboratory medicine; ‘Shaping the future oflaboratory medicine’; Opportunities for IFCC.

No formal vote will be taken by Council but the de-bate will inform the proposals to be put to IFCC Mem-bers for voting at a later date.


IFCC OFFICE

Via Carlo Farini 81, 20159 Milan, ITALYTel: (39) 02-6680-9912 • Fax: (39) 02-6078-1846E-mail: [email protected] • Web: www.ifcc.orgOffice Hours: 9.00-13.00 and 14.00-18.00Staff Members: Paola Bramati, Silvia Cattaneo,Silvia Colli-Lanzi

News from the World of the International Federation of Clinical Chemistry and Laboratory Medicine

Visit www.ifcc.org for more informationNEWSShaping the Future of Laboratory Medicine: The Great Debate

Cont’d on page 56

The 9th Uruguayan Congress ofClinical Biochemistry “The Clinical

Laboratory. Research, Science andTechnology in the service of healthand wellness” took place on Novem-ber 7-9, 2013, at TelecommunicationsTower, Montevideo, Uruguay. High-lights of the event included:• The following IFCC visiting lecturersattended the meeting: Dr. GrahamBeastall (UK) IFCC President; Dr.Mario Plebani (Italy); Dr. Edward Chan(USA);• The President of the Latin AmericanConfederation of Clinical Biochemistry(COLABIOCLI), Dr. Carlos Navarro(Argentina) and Dr. Roberto Garcia(President of Argentinean Biochem-istry Foundation) also attended theevent, and other distinguished col-leagues from USA, Paraguay, Argenti-na, and Brazil as well; • There were 400 participants in total;• Sixteen symposia, seven plenary lec-tures, and two pre-congress courseswere conducted;• During the congress, an agreementbetween Latin American Confedera-tion of Clinical Biochemistry (CO-

LABIOCLI) and IFCC was signed; • Committee for Standardization andQuality Control (CECC), the Uru-guayan EQAS, inaugurated its newheadquarters on the 9th of May at theFaculty of Chemistry. At the sameevent were honored its founders: Prof.Dr. Raul Somma (1979) and Q F. OlgaBorrat (1985).

Activities scheduled for year 2014by Uruguayan Association of Bio-chemistry (ABU): • Course on biological fluids; • Course on superficial mycoses.

Both activities will be performed incollaboration with Wiener Foundation.

Regional Activities of UruguayanAssociation of Biochemistry (ABU): • Uruguay was elected as the place tohold the COLABIOCLI congress in2017; • Uruguayan Association of Biochem-istry (ABU) was elected financialheadquarters of COLABIOCLI; • ABU and Argentinean Association ofBiochemistry (ABA) signed an agree-ment, for collaboration, assistanceand cooperation in academic, scientif-ic and cultural areas.

9th Uruguay Congress Attended byIFCC and COLABIOCLI Leaders

by Ana Piana


News from the World of the International Federation of Clinical Chemistry and Laboratory MedicineVisit www.ifcc.org for more information NEWS


Photo: (From left to right) Dr. Daniel Mazziotta, (Argentina), Dra. Ana Lena, (Pres-ident of the Uruguayan Congress), Dr. Carlos Navarro, (President of COLABIO-CLI), Dr. Graham Beastall, (President of IFCC), Stella Raymondo (IFCC NationalRepresentative of Uruguay).

In 2013 the IFCC Board of Directors ap-pointed a Historian for the Federation

(Dr. Peter Wilding, Philadelphia, USA)who is charged with improving thearchives of the Federation. We believethat an important component of thearchives will be to document the historyof member associations so that their rolein our world-wide profession is docu-mented.

You are invited to prepare a briefdocument that highlights the founding ofyour association, the key individuals (pi-oneers) that created it, and majorachievements in your history.

Attached are two examples of docu-mented collected by Dr. Wilding for arti-cles in the Newsletter of the AACC His-tory Division.

We sincerely hope that you will pro-vide this information so that the IFCCArchives will record the important contri-butions of your association to the profes-sion of clinical chemistry. You can send

the document tothe IFCC Historianc/o Silvia C-L,IFCC Office ([email protected]), byJuly 18, 2014.

Copies of ALLthe collected docu-ments will be avail-able via the IFCC Webpage.

As a further step, if in your NationalSociety someone is interested in or re-sponsible for the history of your society,you could perhaps invite him/her to joinan informal network of international ‘his-torians.’ Please pass contact details toSilvia C-L as above.

Dr. Wilding, who will be present at theIstanbul Congress, invites you to visitthe IFCC booth, and look at the first ‘His-tory’ posters that will be displayed there.Also, your visit would provide a nicepossibility to learn more about IFCC andto add your contribution.

Peter Wilding Appointed IFCC Historianby Dr. Graham Beastall, President, IFCC

Guangzhou Wondfo Biotech Co. Ltd.was founded in 1992 as a research-

based company in the campus of SouthChina University in Guangzhou, Guang-dong Province, China. In 2010, Wondfomoved to a new site, located at Scientif-ic City, Luogang District, Guangzhou.The operation quickly grew beyond re-search and purposed towards manufac-

turing qual-ity medicalproducts and biochemical reagents, inparticular point-of-care testing kits anddevices. Wondfo has obtained the ISO13485:2003 certificate. Its productshave been cleared by the US FDA, Chi-nese FDA, and received the CE mark-ing. Website: www.wondfo.com.cn

New IFCC Corporate Member:Guangzhou Wondfo Biotech Co. Ltd.

Dr. Peter Wilding

News from the World of the International Federation of Clinical Chemistry and Laboratory Medicine

Visit www.ifcc.org for more informationNEWS


In September 1993 some fifty peoplemet in Bardejov Spa to found a tra-

dition of an event with internationalparticipation “LABKVALITA.” Anniver-saries are a time for celebration andrecognition, but they are also a timefor reflection. Since its foundation theinitiative has been taken to organizethe biennial congress for clinical lab-oratory scientists seeking quality inmedical laboratories. For twentyyears, thanks to sustainable sponsor-ship supported by diagnostic compa-nies, the Slovak Society of ClinicalBiochemistry has been able to organ-ize this Conference for medical labo-ratory specialists (biochemists, mi-crobiologists, haematologists, immu-nologists, pathologists), laboratorydirectors, quality managers, technol-ogists, clinical laboratory scientists,medical technologists, medical labo-ratory technicians, medical laborato-ry assistants, hospital administrators,referring physicians, and allied pro-fessionals from diagnostics industry,and those involved in laboratory ac-creditation and regulatory issues andpolicy makers. Nearly 2,000 partici-pants have taken part in the Confer-ence, from 1993 to 2014.

Labkvalita is a meeting of profes-

sionals especially from Slovakia, butalso from neighboring countries ofEastern and Central Europe. Theshort period of the Slovak Republicmembership in the European Unionhas proven that mainly those, whoare capable, prepared and active, aresuccessful in the Community of Euro-pean countries, regardless ofwhether they are small or large. Slo-vakians wish and therefore should belike that. The strengthening of the in-

ternational cooperation in the field isinevitable, even more among the Eu-ropean Union member states.

Labkvalita became the challengeof motivation, our passion for workand service, the alignment of peopleand resources; developing relation-ships and understanding the forcesthat drive clinical laboratories. Duringthe past 20 years, a series of choices,several which put the Labkvalitaevent at risk, have finally resulted in asuccess. A couple of volunteers, whohave held events on different loca-tions, have been also able to givesomething back to the Slovak Societyof Clinical Biochemistry. The activitiesof the Slovak colleagues have beenbased on a great amount of enthusi-asm and on good reading of the situ-ation, tradition, experience and theability to react quickly and accuratelyin time and space. I cordially wish tosay THANKS to each of them for be-ing part of the “Labkvalita” story whichis only beginning to unfold and maycontinue for the next 20 years.

The LABKVALITA vision is to setthe educational direction and stan-dard in a variety of settings for clinicallaboratory. The Conference educateson behalf of laboratory professionalsand plays a leadership role in en-hancing the quality issues in medicallaboratories.

The main objective of the confer-ence is to systematically review thecore knowledge that must be mas-tered by medical laboratory providingaffordable, accessible experiencesresonate with participants.

Beyond learning and sharing high-quality educational programs andstrategies the Conference drew lec-turers, educators including renownedscientists and distinguished speakersfrom all over the world: James O.Westgard, Callum G. Fraser, MarekDominizcak, Christopher P. Price,Per-Hyltoft Petersen, Michael Mayer,David Bullock, Linda Thienpont,

Jean- Claude Libeer, Dietmar Stöckl,Ian Wilkinson, Harald Schlebusch,Walter Hübl, Jerzy Naskalski, Andrea-Rita Horvath, Vladimir Palicka,Alexander Lapin, Raija Pikkarrainen,Mauri Keinanen, Risto Heikinen, Tor-ben Orntoft, Adam Uldall, Ulf Orne-mark, Alexey Moshkin, Jerzy Roguls-ki, Mieczyslaw Wozniak, Josef Kra-tochvila, Bedrich Friedecky, TomasZima, and many others.

The scientific program of the Con-ference is developing towards beinghighly interactive with actual issues inthe field. The LABKVALITA hostslinks between quality assurance,quality management, quality controland quality improvement affectingthe quality of laboratory practice onpatient outcome.

The conference goals are focusedon the variety of legal issues that canarise in laboratory medicine. Theserange from the recommendations onlaboratory cost containment and prac-tices to the improvement of cost-effec-tiveness to monitor the total testingprocess. There are still unresolved top-ics: what quality is acceptable today?How much conformity to goals is need-ed? What is medically relevant qualitycontrol? Do the current QC techniquesestimate an error in patient results?What do we need more: strong theoryand beautiful ideas or practical guide-lines? How to implement up to dateanalytical techniques to reduce labora-tory errors? Is quality built by the man-ufacturers or by the medical laborato-ry? Is modern instrumentation so med-ically reliable and foolproof that we canstop worrying about analytical QC?

The ongoing Labkvalita contribu-tions of top lecturers from the Euro-pean Federation of Clinical Chemistryand Laboratory Medicine and the Eu-ropean Association for Predictive,Preventive and Personalized Medi-cine will enrich the program throughenhancing the diversity of perspec-tives and content it presented.

Photo: (From left to right) Katarina Lepejova, SSCB Secretary; Marko Kapalla, current Labkvalita Coordinator; Jan Balla,Labkvalita Founder & Former Coordinator.

Slovak Society Marks 20th Biennial Congressby Jan Balla, National Representative of the Slovak Society of Clinical Biochemistry, Labkvalita founder & long-term coordinator


As part of an increasingly interconnected world,the European drug market is facing major

changes happening at a very fast pace. The old di-chotomy between a relatively small number of veryproblematic drug users—often using intravenousdrugs, and a large number of users taking drugs

recreationally or experimentally is disappearing, tobe replaced by a more complex and nuanced situ-ation. In its annual report entitled “European DrugReport 2014: Trends and Developments” pub-lished late May 2014, the European MonitoringCenter for Drugs and Drug Addiction (EMCDDA)

explains that stimulants, synthetic drugs, Cannabisand pharmaceutical products are increasingly be-coming more commonly consumed than heroinand cocaine. The reemergence of ecstasy pow-ders and pills is also causing concern. The reportconfirms the trend toward global stabilization ofdrug use, but with a more complex drug market,and new challenges continue to appear given theincreasing number, variety and availability of newpsychoactive substances.

The trend to lower use of heroin concerns useand availability. The number of users enteringtreatment for the first time has dropped from amaximum of 59,000 in 2007 to 31,000 in 2012. Incontrast, stimulants, synthetic drugs, Cannabis,and pharmaceutical products are increasing in im-portance. The increase in the number, variety andavailability of new psychoactive substances is on-going. In 2013, 81 new drugs, including 29 synthet-ic cannabinoids, were reported for the first time bymeans of a European Union early alert system,which brings the number of new substances moni-tored to 350.

The internet plays a key role in market struc-ture. In 2013, the EMCDDA identified some 650websites selling these substances aimed at Euro-peans. It also reported the purchase of new or tra-ditional drugs via “darknets” (clandestine onlinenetworks permitting anonymous communication),which is a “new challenge for law enforcement.”New harmful substances such as 25I-NBOMe, AH-7921, MDPV and methoxetamine are sold to re-place the drugs that they are designed to imitate,i.e., LSD, morphine, cocaine, and ketamine.

The EMCDDA points out several issues consid-ered to be particularly concerning. In particular,they report disturbing localized and/or national epi-demics of synthetic cathinone injection. This prac-tice is a problem in groups of high-risk drug usersin countries such as the Czech Republic, Ger-many, Ireland, Spain, Austria, Poland, Finland,Sweden and the United Kingdom. In Romania, thisinjection is a more generalized practice. In Hun-gary, one study showed that in 2012, cathinoneswere the main drug injected for 36% of users.

An increasingly worrisome behavior has alsoappeared among homosexuals: the injection of acocktail of illicit drugs at “chem sex parties." Thuswe see a reversal of the HIV epidemic situationamong drug users. The most recent results showHIV outbreaks recently observed among drugusers in Greece and Romania and in some Balticcountries, jeopardizing the long-term decline in thenumber of new HIV cases diagnosed in Europe.

If these drugs are spreading, this is not an acci-dent: for the most part, they are much easier to pro-duce than conventional drugs. They are difficult todetect and classify (overdoses) and [contribute to]the transmission of AIDS in some countries. Andfrom this advantage arises another. Consumed invery small quantities, these stimulants and synthet-ic drugs have powerful effects. They become diffi-cult to detect and classify, which explains an in-crease in overdoses, the growth in which is a realpublic health problem on the continental scale.

Engaging in the fight against drugs and addic-tive behaviors is the responsibility of any healthprofessional. A broadened conception of preven-tion should be fostered, educating the public andthe scientific community and integrating aware-ness of all the risks, including behavioral influ-ences related to the internet and social networks,especially in young people.

by Dr. Bernard GougetSFBC-EFLM Representative; IFCC Treasurer;Secretary General, International Francophone Federation of Clinical Biology and Laboratory Medicine (FIFBCML)

Understanding Drug Useand Problems in Europe

European Federation of Clinical Chemistry and Laboratory Medicine

EFLM CORNEREdited by Dr. Bernard Gouget



EFLM CORNER


On March 18-19, 2014, the UK’s 3rd Diag nosticsForum was held at the Uni versity of Oxford, UK,

in the beautiful surroundings of Magdalen College.The meeting was sponsored by the UK TechnologyStrategy Board, the British In-Vitro Diagnostics As-sociation, the UK National Institute for Health andCare Excellence, and the University of OxfordNuffield Department of Prima ry Health Care’s Cen-ter for Monitoring and Diagnosis. These DiagnosticsFora form a special series of conferences, bringingto gether over 100 attendees of which 40% are fromindustry, 40% from aca demia, and 20% from NICE,from the NHS, and other health care organiza tions.This mixed audience makes it a special and inter-esting event and the format of the conference allowsboth communities to mix and mingle.

The focus of this year’s conference – for the firsttime in a two-day format – was on the generation ofevidence to support the introduction of novel IVD,and on government support for the di agnostics in-dustry in doing so. Overall, there were a few pre-sentations and posters on the generation of evi-dence and the methods for doing so (from JonDeeks and Elisabeth Adams, amongst others), butby and large the main focus of the meeting this yearwas on funding opportunities, in particular UK initia-tives to stimulate innovation and collaboration indeveloping and evaluating diagnostics.

Penny Wilson, from the Technology StrategyBoard, one of the conference’s main sponsors, in-troduced the Precision Medicine Catapult, an-nounced in August 2013 and targeted at the devel-opment of diagnostics for stratified medicine. Instratified medicine, treatment decisions for sub-groups are based on specific markers: marker pos-itives would be given one treatment; marker nega-tives another form of treatment. Catapults are tech-nology and innovation centers where UK business-es, scientists and engineers should work side byside on R&D, transforming ideas into new productsand services to generate economic growth.

Several other organizations and programs werein vited to introduce themselves these two days.The National Institute for Health Research (NIHR)aims at transforming research in the NHS, to in-crease the volume of applied health research forthe benefits of patients and the public. NIHR hasfunded several initiatives, such as biomedical re-search centers & units, the Collaborations for Lead-ership in Applied Health Research and Care(CLAHRCs), the NHR clini cal research network,and Academic Health Science Networks (AHSNs).

The NIHR has also—very recently—providedGBP 4 mil lion funding to four Diagnostic EvidenceCoopera tives (DECs), for a four-year period, start-ing Sep tember 1, 2013. These DECs are organiza-tions that are expected to act as catalysts for thegeneration of evidence for commercially-suppliedIVDs. As such, they should foster collaboration be-tween compa nies involved in the CE marking andmarketing of IVDs and other parties.

The four DECs presented themselves at themeeting as well: Dr. John Simpson talked about theNewcas tle DEC, Dr. Michael Messenger presentedthe Leeds DEC, Dr. George Hanna introduced theLondon DEC at Imperial College, and MathewThompson spoke about the plans of the Oxford DEC.

More programs were presented at the meeting.Dr. Mehdi Tavakoli introduced the audience toHealth KTN, which has a Stratified Medicine Innova-

tion Platform. This platform seeks to build on theUK’s strength within the global healthcare industriesby working in partnership with 6 other organizations,who together will invest around GBP 200 million over5 years in the area of stratified medicine. The invest-ment will go into areas such as improved tumor pro-filing in cancer, novel biomarkers and the uptake ofcom panion diagnostics in the NHS.

Other speakers presented a perspective from in-dustry. David Horne (Alere) described the incrediblycomplicated healthcare landscape, where a citizenor patient can access the health care system inmany different ways (such as the internet phoning111, High St retailer, pharmacy, walk in clinic, GP,A&E, alternate practitioner, patient associationsetc), and where IVD companies face an incredibleamount of bodies and acronyms. The UK govern-ment, he felt, could simplify things and remove bar-riers. Government should also support SME andlarger enterprises, using competitive tax rates, forexample. Industry should change as well, by in-creasing transparency, generating better and moreevidence, learning to partner, and by demonstratingvalue, rather than being fixated by price.

The very rich funding alphabet soup served atthis conference (with DECs, CLAHRCs, KTNs, AH-SNs, and more) figured also in the final words ofMatthew Thompson, one of the organizers of theconference. There seem to be many initiatives,many opportu nities for funding, but the landscapeis quite frag mented, and it may be daunting for thehealthcare professional to find the way through themyriad of acronyms, organizations and options.

The Diagnostics Forum has a website, whereone can download reports of previous DiagnosticFora and the presentations and the list of attendeesof the 2014 event: www.oxford.dec.nihr.ac.uk/diag-forum/2014-uk-diagnostic-forum

3rd UK Diagnostics Forum Held at Magdalen Collage, Oxford

by Patrick MM Bossuyt; EFLM WG-Test Evaluation, Professor of Clinical Epidemiology, University of Amsterdam, The Netherlands

Johannes Zander Winner of 2014 Walter Guder

Preanalytical Award

The Walter Guder Preanalytical Award 2014, presentedby EFLM and sponsored by Becton Dickinson, has

been granted to Dr. Johannes Zander for the article: Effect of biobanking conditions on short-term stabil-

ity of biomarkers in human serum and plasma. Jo-hannes Zander, Mathias Bruegel, Alisa Kleinhempel,Susen Becker, Sirak Petros, Linda Kortz, JulianeDorow, Jürgen Kratzsch, Ronny Baber, Uta Ceglarek,Joachim Thiery and Daniel Teupser; Inst. of Lab. Med-icine, Clinical Chemistry and Molecular Diagnostics,University Leipzig, Leipzig, Germany; LIFE – LeipzigResearch Center for Civilization Diseases, UniversityLeipzig, Leipzig, Germany; Institute of Laboratory Med-icine, Ludwig-Maximilians-University Munich, Munich,Germany; Medical ICU, University Leipzig, Leipzig,Germany

The Walter Guder Preanalytical Award is given tothe best published paper, as judged by an independentpanel of experts, which demonstrates a significant con-tribution to the improvement of the preanalytical phase.The award is intended to achieve wider recognition ofthe importance of high quality research in the field ofthe preanalytical phase among laboratory profession-als in Europe. The Award will be presented to Dr. Zan-der, as submitting author, during the EuroLabFocusCongress in Liverpool (October 7-10, 2014). The awardconsists of a certificate and a monetary award of EUR5,000, to be shared with coauthors, in addition to thecost of the participation to attend the Congress in Liv-erpool.

Please consider that the Walter Guder PreanalyticalAward will be awarded biannually, so prepare yourselffor the next edition!


VISIT US AT:

Booth: 1367

2014ANNUAL

MEETING

EFLM CORNER European Federation of Clinical Chemistry and Laboratory Medicine



The 21st Congress of RSML took place on June4-7, 2014, in Sibiu, Romania. What was the new

concept of the event? The Romanian laboratoryspecialists tried to make of their Congress a multi-disciplinary scientific event, inviting as speakers,along with laboratory specialist, clinicians, pharma-cists, laboratory technicians, in a word, the wholehealthcare team, considering the laboratory as theturnaround point in the medical diagnostic, prog-nostic and follow up of patients’ treatment.

The Congress was divided into several ses-sions. First day the lecturers were Romanian spe-cialists in laboratory Medicine, hematology, pedi-atrics, internal medicine, infectious diseases, der-matology, balneo-physiotherapy, rheumatology, gy-necology, urology, who spoke about their experi-ence in using lab tests in their diagnostic fields.

The second day the international speakersmade a general overview in subjects like: “Bonemarrow report”- Dr. Adrian Padurean (Wisconsin,USA); “How we use lab tests in endocrinology”-Prof. Dr. Maria Fleseriu (Oregon, USA); “The im-portance of Vit. D determination in the medicalmanagement of elderly persons”- Dr. RoxanaFournier (Amiens, France); “The stem cells in thetreatment of neurologic diseases”- Prof. Cristina

Iftode (New Jersey, USA); “The importance of inter-nal Quality control in laboratory”- Anne Vassault(Paris, France); “The transposition of EU directivesinto national laws-what are the problems?”- SimoneZerah (Paris, France).

The next two days was the Regional Meeting ofThe International Association of Therapeutic DrugMonitoring and Clinical Toxicology, InternationalScientific Society who agreed to join the RomanianNational Congress in order to spread their knowl-edge about personalized medicine, the future oflab diagnostic using new technologies like LC-MSin the follow up of medical treatment and ClinicalToxicology. Romanian laboratory is at the begin-ning of using these new methods in laboratory, butthe idea of the organizers was “If we cannot doeverything it is known, at least know everything itis done.” In addition, the lecturers covered a largevariety of subjects: “Future of Laboratory Medi-cine,”- Prof. Michael Oellerich (Goettingen, Ger-many); “How to organize a TDM laboratory”- Prof.Pierre Wallemacq (Brussels, Belgium); “Im-munoassays in TDM”- Dr. Eberhard Wieland(Sttutgart, Germany); “Methods of testing drug ofuse”- Dr. Hans Maurer (Homburg, Germany); “Per-sonalized pharmacotherapy for cancer”- Prof.

Yusuke Tanigawara (Tokyo, Japan); “TDM inepilepsy”- David Berry (London, UK); “Alcohol anddrug of use - a deadly combination”- Manuela Neu-mann (Toronto, Canada); “ Medicine in digital era”-Prof. Liviu Iftode (Rutgers, USA).

The audience was very much interested in allthe subjects and appreciated the nice mixt betweenspecialties, having as central point the improve-ment of laboratory diagnostics in the benefit of pa-tients. We hope that the motto of the Congress“Good medical science, better practice” has beentransposed into practice and will stimulate futurecollaboration between specialists and scientific as-sociations.

We also have to remark the beauty of Sibiu – anice town in the center of Romania, known afterForbes classification as “the 8th most idyllic placeto live,” who was hosting, during this period, the21st edition of the International Theatre Festival, abig cultural event in this “city of culture, city of cul-tures,” who contributed to the general atmosphere,appreciated by all the participants.

21st Romanian National Congress Builds Multidisciplinary Scientific Appeal

by Dr. Camelia Grigore, President of RSML

We are glad to inform you that the 14th EFLMPostgraduate Continuous Course in Clinical

Chemistry and Laboratory Medicine “New Trends inDiagnosis and Management of Diabetes Mellitus:Diabetes Mellitus Revisited 14 Years After the FirstDubrovnik Course” is now open for registration(www.dubrovnik-course.org/registration).

The Course will be held at the Inter-UniversityCenter Dubrovnik on October 25-26, 2014, withthe goal to bring a lot of new information docu-menting the progress in diagnosis and manage-ment of Diabetes Mellitus in the last 14 years. Mostimportantly, this event brings together Europe’sspecialist trained in Laboratory Medicine andphysicians focused on Diabetes Mellitus and itscomplications, one of the most frequent diseasesworldwide. It will be an opportunity not only, to lis-ten to the renowned expert in the field, but also foractive participation of attendees with poster pres-entation. These results presented as posters, willreflect attendees contribution in the care of Dia-betes Mellitus across Europe.

We would like to draw your attention to the dateof the registration fees: EUR 150 before, and EUR250 after August 1, 2014. More information can befound on the web (www.dubrovnik-course.org).

For the poster session, the participants are invit-ed to submit their posters before August 1, 2014.Several interesting posters will be chosen for shortoral presentation. The best poster chosen by Sci-entific and Organizing Committee will be awarded.

EFLM will announce bursaries for young partici-pants (<35 year). Applications should be submittedby July 1, 2014, electronically. All applicants will benotified about the results by e-mail, latest by theend of July 2014. Further detailed information willfollow in May. The course will be accredited by Na-tional Society rules.

We look forward to welcoming you in Dubrovnikwhere you will have the opportunity for an interac-tive discussion during the course as well as duringsocial events

Preliminary ProgramSaturday, October 25, morning- Introduction- Part I : Epidemiology, prevalence, the major com-

plications of DM• The diabetes epidemic – prevalence and clas-

sification• Complication of diabetes – strategies for re-

ducing the risk of long term complications• The role of diabetes registries to monitor the

treatment and complications of diabetes• Poster presentation

- Part II: The role of testing in the diagnosing andmanagement of DM• Guidelines and recommendations for testing in

diagnosis of DM: The role of HBA1c• HbA1c analyzing – challenges for the laborato-

ry – internal and external QC• Post-analytical factors – how should HbA1c re-

sults be communicated to clinicians• Poster presentations

Saturday, October 25, afternoon- Part III: Do we have markers for early diagnosis

of diabetes?• Early recognition of gestational diabetes (Intro-

duction of new guidelines and practice) – how

should the routines be?• How to diagnose the pre-diabetes• Diabetes in children – Impact of obesity on de-

velopment of diabetes• Poster presentations

Sunday, October 26, morning- Part IV: Can good management prevent the dia-

betic complication• POC testing instruments for diagnosing and

monitoring diabetes in clinical settings• The impact of preanalytic factors of glucose

measurement

• Self measurement of glucose – how useful is itand how can it be done

• Poster presentations• Break, poster walk, exhibition

- Part V: Can good management prevent the dia-betic complication• Obesity and Diabetes. The role of Laboratory

Medicine• Pros and Cons of Incretin therapy in Type 2 di-

abetes• The practical issues in patient management –

pharmacogenetics



EFLM CORNER

Registration Underway for the 14th EFLM Postgraduate Course in Dubrovnikby Elizabeta Topic, EFLM Education and Training Committee Faculty

of Pharmacy and Biochemistry University of Zagreb, Croatia

Dubrovnik Harbour

Liverpool, UK • 7-10 October, 2014

EFLM CORNER European Federation of Clinical Chemistry and Laboratory Medicine


One of the foremost features in theevolution of laboratory medicine

has been the pursuit of quality in theperformance of analytical methods.Highlights include the creation of in-ternational reference materials andreference methods1, 2 alongside theunderlying concepts of how thesecan be translated into the creation ofcalibration materials that can be em-ployed in the methods routinely usedin the laboratory, and at the point ofcare3. These achievements havebeen complemented by develop-ments in quality control and externalquality assurance4. More recently wehave seen the call for harmonizationof analytical methods5, as it has be-come obvious in recent years that theunderlying principles of the analyticalmethods can also impact on day today performance. All of this activityhas been primarily driven by the pur-suit of analytical and scientific excel-lence, which is traceable to a robustpoint of reference6.

In more recent times there hasbeen a parallel evolution in evidence-based laboratory medicine, whichhas a different point of reference,namely the impact of a test result onclinical decision making and healthoutcomes7. In this evolution it soonbecame clear that the evidence basewas poor, and furthermore the gener-

ation of the required evidence waschallenging8. A test result, of itself,will not have an impact on a healthoutcome; it requires appropriate clin-ical decision making and action9. Itwas inevitable that, at some point,the impact of analytical performanceon the delivery of health outcomeswould be questioned. This has beenaddressed in terms of desirable ana-lytical performance, by reference tothe biological variation of the analyteand its impact on the quality of deci-sion making10. An alternative ap-proach has been to employ modellingof the clinical decision pathway. Anearly example was the modellingwork of Bruns and Boyd on the im-pact of blood glucose measurementimprecision of insulin dosing11.

A recent paper by Langlois andcolleagues reporting a collaborativeproject, sponsored by the EuropeanFederation of Clinical Chemistry andLaboratory Medicine and the Euro-pean Atherosclerosis Society, investi-gating the impact of the method biasin routine HDL and LDL cholesterolmethods on cardiovascular risk strat-ification, in hypertriglyceridemia12.The authors used the results from theDutch National External Quality As-surance program to study the risk ofmisclassification due to method vari-ability. Data from three normotriglyc-

eridemic pools and two hypertriglyc-eridemic pools were selected on thebasis that their mean concentrationswere close to the high risk cut-pointsfor HDL and LDL cholesterol. The tar-get values were assigned by a LipidReference Laboratory. They simulat-ed the risk classification using HDL –and sex-specific SCORE multipliersapplied to two fictitious moderate riskpatients with an initial SCORE of 4%(male) and 3% (female). In additionthey examined the concordance ofclassification into treatment groupsusing the relative numbers of LDLcholesterol concentrations above thehigh risk cut-point for each of themethods contributing to the qualityassurance scheme. The authors ob-served biases in hypertriglyceridemicsera beyond the recommended limitswhich impacted on the proportion ofhigh risk classifications, and whichvaried between methods. The errorsin the HDL cholesterol measure-ments also impacted on the calculat-ed LDL cholesterol values and theassignment of treatment goals.Clearly method performance wasshown to have the potential to impacton clinical decision making and sub-sequent treatment actions.

There are important messages inthis paper, as well as indicating theway that laboratory professionals andmanufacturers of diagnostic technolo-gies should be appraising the outputof the services they provide. There arethree important conclusions from thisstudy (i) the choice of method em-ployed to generate the evidence onthe use of the analyte for clinical deci-sion making should be clearly stated;it should be a method with traceablecalibration, and with minimal risk ofbias (ii) the impact of variation inmethod imprecision and bias and theirimpact on clinical decision making andoutcomes should be modelled wher-ever possible, for the routine methodsused in the laboratory practice, and(iii) clinicians should be made awareof the risks of poor method perform-ance on clinical decision making andhealth outcomes. A similar approachshould also be considered when usingsemiquantitative methods for rule in orrule out decisions.

This study is to be welcomed as ithighlights an important tool for thelaboratory medicine professional toemploy in both quality improvementand innovation. Furthermore it pro-vides a means of demonstrating howthe laboratory professional plays acrucial role in the work of the clinicalteam.

References1. Tietz NW. A model for a comprehensive

measurement system in clinical chem-istry. Clin Chem. 1979;25:833-9.

2. Klee G, Westgard JO. Quality manage-ment. In (eds Burtis CA, Ashwood ER,Bruns DE) Tietz Textbook of ClinicalChemistry and Molecular Diagnostics.Fifth edition, Philadelphia, Elsevier,2012: 163-203.

3. Grubb A, Blirup-Jensen S, Lindström V,Schmidt C, Althaus H, Zegers I; IFCCWorking Group on Standardization ofCystatin C (WG-SCC). First certified ref-erence material for cystatin C in humanserum ERM-DA471/IFCC.. Grubb A,Blirup-Jensen S, Lindström V, SchmidtC, Althaus H, Zegers I; IFCC WorkingGroup on Standardization of Cystatin C(WG-SCC).

4. Ceriotti F. The role of External Quality As-sessment Schemes in Monitoring and Im-proving the Standardization Process. ClinChim Acta. 2014 Jan 2. pii: S0009-8981(13)00527-5. doi: 10.1016/j.cca.2013.12.032. [Epub ahead of print].

5. Tate JR, Johnson R, Barth J, Pante-ghini M. Harmonization of laboratory testing - Current achievements and fu-ture strategies. Clin Chim Acta. 2013 Aug 31. pii: S0009-8981(13)00331-8. doi: 10.1016/j.cca.2013.08.021. [Epubahead of print].

6. Vesper HW, Thienpont LM. Traceabilityin laboratory medicine. Clin Chem.2009;55:1067-75.

7. Price CP. Evidence-based laboratorymedicine: is it working in practice? ClinBiochem Rev. 2012;33:13-9.

8. Sackett DL, Haynes RB. The architec-ture of diagnostic research. BMJ 2002;324:539-41.

9. Ferrante di Ruffano L, Hyde CJ, McCaf-fery KJ, Bossuyt PM, Deeks JJ. Assess-ing the value of diagnostic tests: aframework for designing and evaluatingtrials. BMJ. 2012 Feb 21;344:e686. doi:10.1136

10. Fraser CG. Test result variation and thequality of evidence-based clinical guide-lines. Clin Chim Acta. 2004;346:19-24.

11. Boyd JC, Bruns DE. Monte Carlo simu-lation in establishing analytical qualityrequirements for clinical laboratory testsmeeting clinical needs. Methods Enzy-mol. 2009;467:411-33.

12. Langlois MR, Descamps OS, van derLaarse A, Weykamp C, Baum H, PulkkiK, von Eckardstein A, De Bacquer D,Borén J, Wiklund O, Laitinen P, Ooster-huis WP, Cobbaert C; EAS-EFLM Col-laborative Project. Clinical impact of di-rect HDLc and LDLc method bias in hy-pertriglyceridemia. A simulation study ofthe EAS-EFLM Collaborative ProjectGroup. Atherosclerosis. 2014;233:83-90.

Impact of Analytical Method Performance on Clinical Decision Making and Health Outcomes

by Christopher P Price, Visiting Professor in Clinical Biochemistry, Department of Primary Care Health Sciences, University of Oxford, UK

TThe new collaboration betweenBiocartis (Biocartis NV; Meche-

len, Belgium; www.biocartis.com)and Abbott Laboratories (AbbottPark, IL, USA; www.abbott.com) isexpected to fuel new pharmaceuticalpartnerships for companion diagnos-tics programs.

Under the agreement, the compa-nies will develop and commercializemultiplex companion diagnosticstests, and will leverage Biocartis' mo-lecular diagnostics system "Idylla"and Abbott's regulatory, scientific,and commercialization expertise."Much of the current molecular diag-

nostic practice involves a series ofspecialized, labor intensive, and time-consuming steps," said Rudi Pauwels,CEO and chairman, Biocartis.

In partnership with pharmaceuti-cal companies, Biocartis and Abbottwill create various biomarker panelsfor use on the Idylla system, whereeach marker in the panel has aknown clinical significance. Multi-plexing is important for pharmaceuti-cal partners that need to analyze mul-tiple biomarkers in their clinical trialsand ultimately helps physicians ob-tain more useful information fromlimited patient samples.

Abbott and Biocartis in Research Alliance For Multiplex Companion Diagnostics

SSequenom (San Diego, CA, USA;http://laboratories.sequenom.com)

has sold its Bioscience business forup-to-USD 35.8 million to Agena Bio-science (San Diego, CA, USA; www.agenabioscience.com), which said itplans to expand clinical use of theMassarray System for mass spectrom-etry-based detection of nucleic acids.

Agena agreed to pay SequenomUSD 31.8 million cash up front, plusup to USD 4 million in contingentconsideration tied to achieving undis-closed regulatory and sales mile-stones. Agena assumed the lease forthe Bioscience business in San Diego,as well as liabilities related to Bio-science – but which does not includeany portion of Sequenom¹s bank debtor convertible notes outstanding, thebuyer said.

Achieving that expansion will bethe job of Agena’s new chairman andinterim CEO John Lillig, a senior lifesciences and diagnostics industry ex-ecutive with more than 30 years ofexperience in clinical diagnostics, aswell as the life science instrumentsand reagents markets.

Agena said it will improve theMassarray System with expandedhardware automation, software func-tionality, high value assay content,and resources to support growing usein basic, agricultural, and clinical re-search. Massarray has been used inmore than 2,000 published researchstudies, Agena said. It incorporatesMALDI-TOF mass spectrometry forhighly sensitive and specific interro-gation of tens to hundreds of multi-plexed genetic biomarkers.

Sequenom Sells Bioscience Unit to Agena

TThe growth in personalized medi-cine has led to increased interest

in companion diagnostics (CDx) tests,or tests that match the right therapy tothe patient. Over the past few years,market participants have been enter-ing pharmaceutical and diagnosticpartnerships in order to develop andcommercialize CDx tests for drugsthat already exist in the market. Cur-rently, the oncology segment is themain focus in the CDx market, but itis envisioned that non-oncology seg-ments – such as central nervous sys-tem, infectious disease, and cardiovas-cular conditions – will receive a greatdeal of attention in the years to come.

A new analysis from Frost & Sulli-van (London, UK; www.frost.com)finds that the CDx market earnedrevenues of USD 457 million in 2013and estimates this to reach USD1,295.1 million in 2018, at a com-

pound annual growth rate of 23.2 %.The study covers the oncology areassuch as breast cancer, colorectal can-cer, and lung cancer panel segmentsand non-oncology areas such as infec-tious, CNS, and cardio vascular dis-eases for Western Europe, includingthe United Kingdom, Germany, Italy,France, Spain, Benelux, and Scandi-navia.

Intense competition from labora-tory-developed test (LDT) manufac-turers – who offer tests through refer-ence laboratories across Europe andthe rest of the world – is hamperingmarket growth. The entry of foreignLDT companies into the marketthrough partnership with local com-panies, as well as the inclination ofcompanies across Western Europe topromote the use of LDT over manu-factured and approved CDx tests, areexacerbating the situation.

Fewer Regulatory Barriers in Europe Benefit Companion Diagnostics Sector

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FEBRUARY 2015SLAS 2015 - Society of Laboratory Automa-tion and Screening. February 7-11; Washington,DC, USA; Web: www.slas2015.org

MARCH 2015KIMES 2015. Mar 5-8; Seoul, Korea; Web:www.kimes.kr MEDLAB Asia Pacific. Mar 18-20; Singapore;Web: www.medlabasia.comMEDICAL FAIR INDIA. Mar 21-23; New Del-hi, India; Web: http://medicalfair-india.comARABLAB 2015. Mar 23-26; Dubai, UAE; Web:www.arablab.comExperimental Biology 2015. Mar 28-Apr 1; Bos-ton, MA, USA; Web: http://experimentalbiology.org

APRIL 20153rd EFLM-BD European Conference on Pre-analytical Phase. April; Porto, Portugal; Web:www.preanalytical-phase.orgSEACARE 2015 – 18th Southeast-Asian Health-care & Pharma Show. Apr 6-8; Kuala Lumpur,Malaysia; Web: https://abcex.com/default.aspxWorld Vaccine Congress 2015. Apr 7-9; Wash-ington, DC, USA; Web: www.terrapinn.comECCMID 2015 – 25th Eur. Cong. of Clin. Mi-crobiology & Infectious Diseases. Apr 25-28;Copenhagen, Denmark; Web: www.congrex.ch4th Congress of the African Federation ofClinical Chemistry (AFCC). Apr 28-30; Victo-ria Falls, Zimbabwe; Web: www.afccafrica.org

MAY 201517th European Congress of Endocrinology. May3-7; Dublin, Ireland; Web: www.ece2015.orgBiomarkers & Diagnostics World Congress2015. May 5-7; Philadelphia, PA, USA; Web:www.biomarkerworldcongress.com EuroPRevent – European Society of Cardiolo-gy. May 14-16; Lisbon, Portugal; Web: www.escardio.orgISLH 2015 – International Society of Labora-tory Hematology. May 19-21; Chicago, IL; Web:www.islh.orgDiagnostica / Hospitalar 2015. May 19-22; SaoPaulo, Brazil; Web: www.hospitalar.comEuroPCR 2015 – European Association of Per-cutaneous Cardiovascular Interventions Con-gress. May 19-22; Paris, France; Web: www.europcr.com

SEPTEMBER 2014Eurotox 2014 – 50th Congress of the EuropeanSocieties of Toxicology. Sep 7-10; Edinburgh,Scotland; Web: www.eurotox2014.com10th EFLM Symposium for Balkan Regionand 19th Congress of Medical Biochemists ofSerbia. Sept 9-13; Belgrade, Serbia; Web:www.dmbj.org.rsMEMBS 2014 – 1st Middle East Molecular Bi-ology Congress & Exhibition. Sep 14-17;Dubai, UAE; Web: www.membs.org34th Nordic Congress in Clinical Chemistry. Sep16-19; Göteborg, Sweden; Web: www.nfkk2014.seAACC Symposium: Critical and Point-of-CareTesting: Real World and Emerging Applica-tions for Improved Clinical Outcomes. Sep 17-20; San Diego, CA, USA; Web: www.aacc.org12th Baltic Congress in Laboratory Medicine(BALM). Sep 18-20; Riga, Latvia; Web: www.balm2014.info53rd Annual ESPE Meeting – European Socie-ty of Paediatric Endocrinology. Sep 18-20;Dublin, Ireland ; Web: www.espe2014.orgUnipath 2014 - 54th Annual Pathology Con-gress of the Federation of South African Soci-eties of Pathology. Sep 19-21; Pretoria, SouthAfrica; Web: www.pathconference.comAnalytica China. Sep 24-26; Shanghai, China;Web: www.analyticachina.comDKLM 2014 - German Congress for Laborato-ry Medicine. Sep 24-27; Mannheim, Germany;Web: http://laboratoriumsmedizin2014.de7th Santorini Conference “Sytems Medicine,

Personalized Health and Therapy”. Sep 25-27;Santorini, Greece; Web: www.santorini2014.org17th Annual Meeting of the ESCV – EuropeanCongress of Virology. Sep 27-Oct 1; Prague,Czech Republic ; Web: www.escv.org38th European Congress of Cytology. Sep 27-30; Geneva, Switzerland; Web: www.cytologygeneva2014.comBSACI – British Society of Allergy & ClinicalImmunology Annual Meeting. Sep 28-30;Shropshire, UK; Web: www.bsaci.org

OCTOBER 2014ExpoMedical 2014. Oct 1-3; Buenos Aires, Ar-gentina; Web: www.expomedical.com.ar17. Congreso Chileno de Tecnología Médica.Oct 2-4; Concepción, Chile; Web: www.congresotm.clIFBLS 2014 – 31st World Congress of Biomed-ical Laboratory Science. Oct 3-7; Taipei, Tai-wan; Web: www.ifbls2014.org3rd EFLM-UEMS Congress, LaboratoryMedicine at the Clinical Interface. Oct 7-10;Liverpool, UK; Web: www.eurolabfocus2014.orgJIB 2014 – Journées Internationales de Biologie.Oct 8-10; Paris, France; Web: www.jib-sdbio.frASCP 2014 – American Society for ClinicalPathology. Oct 8-11; Tampa, FL, USA; Web:www.ascp.org7th Spanish National Congress of Clinical Labo-ratory. Oct 15-17; Seville, Spain; Web: www.labclin2014.es40th Annual Meeting of the American Societyfor Histocompatibility and Immunogenetics.Oct 20-24; Denver, CO, USA; Web: www.ashi-hla.org14th EFLM Continuous Postgraduate Coursein Clinical Chemistry. Oct 24-25; Dubrovnik,Croatia; Web: www.dubrovnik-course.orgIFCC Specialized Conference Biomarkers inNeuropsychiatric Disorders. Oct 24-25; Toron-to, Canada; Web: www.ifcc.org52nd Annual Scientific Conference of the Aus-tralasian Association of Clinical Biochemists –AACB. Oct 27-30; Adelaide, Australia; Web:www.aacb.asn.auESID 2014 – 16th Biennial Meeting of the Eu-ropean Society for Immunodeficiencies. Oct29-Nov 1; Prague, Czech Republic; Web: www.

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INTERNATIONAL CALENDARINTERNATIONAL CALENDAR

V I S I T

ADVERTISING INDEXInq.No. Advertiser Page Inq.No. Advertiser Page

– 18 Cong. Chileno de Quim. Clin. .64157 77 Elektronika . . . . . . . . . . . . . . . .57

– AACC . . . . . . . . . . . . . . . . . . . . . .55133 AB Sciex . . . . . . . . . . . . . . . . . . . .33131 Advanced Instruments . . . . . . . . .31143 Advanced Instruments . . . . . . . . .43163 Alcor . . . . . . . . . . . . . . . . . . . . . . .63130 AVE Science . . . . . . . . . . . . . . . . .30111 Autobio Diagnostics . . . . . . . . . . .11122 Bioron . . . . . . . . . . . . . . . . . . . . . .22159 Brand . . . . . . . . . . . . . . . . . . . . . .59136 Caretium . . . . . . . . . . . . . . . . . . . .36168 Cellavision . . . . . . . . . . . . . . . . . . .68

– CHINA MED 2015 . . . . . . . . . . . . .65– CMEF 2014 . . . . . . . . . . . . . . . . . .62

112 D-Tek . . . . . . . . . . . . . . . . . . . . . . .12110 DAS . . . . . . . . . . . . . . . . . . . . . . . .10140 DiagCor . . . . . . . . . . . . . . . . . . . . .40121 Diagnostico Stago . . . . . . . . . . . . .21108 DiaSource . . . . . . . . . . . . . . . . . . . .8139 DiaSys . . . . . . . . . . . . . . . . . . . . . .39125 DIRUI . . . . . . . . . . . . . . . . . . . .24-25147 Drucker . . . . . . . . . . . . . . . . . . . . .47162 EKF . . . . . . . . . . . . . . . . . . . . . . . .62102 ELITech Group . . . . . . . . . . . . . . . .2158 Emperor . . . . . . . . . . . . . . . . . . . .58109 Erba . . . . . . . . . . . . . . . . . . . . . . . .9

– EuroLabFocus 2014 . . . . . . . . . . .61– EuroMedLab Paris 2015 . . . . . . . .60

116 Fluid Metering . . . . . . . . . . . . . . . .16153 Fosun Diagnostics . . . . . . . . . . . .53145 Hecht, Karl . . . . . . . . . . . . . . . . . .45115 Hitachi Aloka Medical . . . . . . . . . .15113 HORIBA Medical . . . . . . . . . . . . . .13

– JIB 2014 . . . . . . . . . . . . . . . . . . . .67– LabMedica.com . . . . . . . . . . . . . . . .6

128 Lee Company, The . . . . . . . . . . . .26151 Medix Biochemica . . . . . . . . . . . . .51119 Mindray . . . . . . . . . . . . . . . . . . . . .19141 NG Biotech . . . . . . . . . . . . . . . . . .38138 Operon . . . . . . . . . . . . . . . . . . . . .38148 Peripheral Visions . . . . . . . . . . . . .48118 Puritan . . . . . . . . . . . . . . . . . . . . . .18105 Randox . . . . . . . . . . . . . . . . . . . . . .5150 Rayto . . . . . . . . . . . . . . . . . . . . . . .50117 Sekisui . . . . . . . . . . . . . . . . . . . . .17103 Siemens Healthcare . . . . . . . . . . . .3124 Siemens Healthcare . . . . . . . . . . .23134 SNIBE . . . . . . . . . . . . . . . . . . .34-35137 Socorex . . . . . . . . . . . . . . . . . . . . .37127 Thermo Scientific . . . . . . . . . . . . .27107 Tokyo Boeki . . . . . . . . . . . . . . . . . . .7

– TradeMed.com . . . . . . . . . . . . . . .41129 VEDA.LAB . . . . . . . . . . . . . . . . . .29161 Vircell . . . . . . . . . . . . . . . . . . . . . .54126 West Medica . . . . . . . . . . . . . . . . .26149 West Medica . . . . . . . . . . . . . . . . .49

– World IVD Congress China . . . . . .65

LabMedica International Vol. 31 No. 4 • 6-7/2014


25th Annual Meeting and Clinical Congress ofthe American Association of Clinical Endocri-nologists (AACE). May 25-29; Orlando, FL,USA; Web: www.aace.com

JUNE 2015

European Human Genetics Conference 2015.Jun 6-9; Glasgow, UK; Web: www.eshg.orgEAACI 2015 – 31st Eur. Cong. of Allergologyand Clin. Immunology. Jun 6-10; Barcelona,Spain; Web: www.eaaci2015.comEuropean Society for Human Reproductionand Embryology Annual Meeting – ESHRE2015. Jun 14-17; Lisbon, Portugal; Web: www.eshre2015.eu2015 BIO International Convention. Jun 15-18; Philadelphia, Pennsylvania; Web: http://convention.bio.org/2015/EuroMedLab 2015 – 21th IFCC-EFLM Euro-pean Congress of Clinical Chemistry and Lab-oratory Medicine / JIB 2015. Jun 21-25; Paris,France; Web: www.paris2015.orgEuroMedLab 2015 Satellite Meeting. HbA1cand Management of Diabetes Mellitus in the21st Century. Jun 26; Reims, France; Web:www.paris2015.org

JULY 2015

AACC 2015 - Annual Meeting & Clinical LabExpo. Jul 26-30; Atlanta, GA, USA; Web:www.aacc.org

AUGUST 2015

FIME 2015 – Florida International MedicalExhibition. Aug 5-7; Miami, FL, USA; Web:www.fimeshow.com/exhibit.cfm

SEPTEMBER 201534th ISBT – International Society of BloodTransfusion Congress. Sep 4-8; Dubai, UAE;Web: www.isbtweb.orgESP 2015 – 27th European Congress of Pathol-ogy. Sep 5-9; Sava Centar, Belgrade, Serbia;Web: www.esp-pathology.org

18th Annual Meeting of the ESCV – EuropeanCongress of Virology. Sep 9-12; Edinburgh,Scotland; Web: www.escv.orgEurotox 2015 – 51st Congress of the EuropeanSocieties of Toxicology. Sep 13-16; Porto, Portu-gal ; Web: www.eurotox2015.comBSACI – British Society of Allergy & ClinicalImmunology Annual Meeting. Sep 20-22; Lon-don, England; Web: www.bsaci.org39th European Congress of Cytology. Sep 20-23; Milan, Italy; Web: www.cytology2015.comESPT 2015 - 3rd Conference on Integration ofPharmacogenomics in Clinical Decision Su-port. Sep 24-26; Budapest, Hungary; Web:www.esptcongress.eu41st Annual Meeting of the American Societyfor Histocompatibility and Immunogenetics(ASHI). Sep 28-Oct 2; Savannah, GA, USA;Web: www.ashi-hla.org

OCTOBER 2015

54th Annual ESPE Meeting – European Socie-ty of Paediatric Endocrinology. Oct 1-3; Barce-lona, Spain ; Web: www.espe2015.orgASCP 2015 – American Society for ClinicalPathology. Oct 28-31; Long Beach, CA, USA;Web: www.ascp.orgCOLABIOCLI 2015 – 22. Congreso Lati-noamericano de Bioquímica Clinica. Oct 29-31; Quito, Ecuador; Web: www.sebiocli-ec.org

NOVEMBER 2015

MEDICA 2015. November; Dusseldorf, Ger-many; Web:www.medica-tradefair.comArabMedLab 2015 - 14th Arab Congress ofClinical Biology (AFCB). November; Khartoum,Sudan; Web: www.ifcc.orgAssociation for Molecular Pathology (AMP)Annual Meeting 2015. Nov 5-7; National Austin,TX, USA; Web: www.amp.orgCongress on Pathology and Laboratory Medi-cine. Nov 18-21; Cancun, Mexico; Web: www.pathologycancun2015.org


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