Labmedica Intl November 2017 by Globetech

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Vol. 34 No. 7 • 11/ 2017

DAILY CLINICAL LAB NEWS

Novel Urine Test Predicts High-Risk Cervical Cancer urine test, developed for the likely emergence of cervical cancer, is highly accurate compared to other tests based on genetic markers derived directly from cervical tissue. The new test is different because it analyzes not only multiple sources of human cellular DNA altered by precancerous changes, but also DNA from

Expanded Pathogen Identification Database Gets Clearance ycobacteria, Nocardia and molds are complex organisms to identify, requiring days or weeks of specific culture conditions for appropriate growth and subsequent advanced methods for reliable identification to the species level. A newly expanded database, now offers simple, rapid, safe and reliable

identification of these medically important pathogens, providing clinicians with actionable results to better manage these infections, such as tuberculosis, lung and bone infections, and other serious organ infections. This database includes more than 15,000 distinct strains to provide extremely high accuracy and, for the

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Simple Blood Test Could Replace Biopsies in Lung Cancer Diagnosis A

Molecular Assay Identifies Colorectal Cancer olorectal cancer is one of the leading causes of cancer-related deaths worldwide, with more than 600,000 deaths each year. Among individuals undergoing treatment for the disease, recurrence occurs in 30% to 50% of all cases. The majority of these cases present in the first two to three years following initial diagnosis and treatment. A clinically validated blood test that is designed to help detect colorectal

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Test Predicts Success of Bone-Marrow Transplants enetic mutations drive the pathogenesis of the myelodysplastic syndrome (MDS) and are closely associated with clinical phenotype and therefore, genetic mutations may predict clinical outcomes after allogeneic hematopoietic stem-cell transplantation. MDS is a blood disorder in which the bone marrow does not produce enough healthy blood cells. Typical treatments include high- or

new test which can detect gene mutations in tumor DNA from blood samples, offers a major improvement over conventional biopsies, due to easier and faster diagnosis as well as improved access to life changing drugs. See article on page 4

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Clinical News . . . . . . . . . 4-60 IFCC News . . . . . . . . . . . . . 61 Product News . . . . . . . 12-60 Industry News . . . . . . . . . .64 International Calendar . . . 65

uberculosis (TB) remains a global health threat despite the development of new diagnostics and antitubercular drugs. The MTB/RIF assay was developed to improve TB and rifampin resistance (RIF-R) detection. Although the assay showed high overall sensitivity and specificity with pulmonary

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New Assay Improves Detection of Tuberculosis

recent paper described a novel diagnostic device fabricated from various types of paper to be used for point-of-care testing in areas lacking electricity and other resources. The developers at Purdue University (West Lafayette, IN, USA; www. purdue.edu) used different types of paper to fabricate Self-powered, Pa-

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per-based Electrochemical Devices (SPEDs) that were designed for sensitive diagnostics in low-resource settings. SPEDs are inexpensive, lightweight, mechanically flexible, easy to use, and disposable by burning. The top layer of the SPED was prepared from cellulose paper with patterned Cont’d on page 8

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sample availability. With clear, actionable results, the test gives clinicians the information needed to make confident treatment decisions. The Cobas EGFR Mutation Test v2 CE-IVD comes with a new feature in the report called the Semi Quantitative Index, or SQI. This number is designed to represent the percent of mutation in the sample tested. If frequently testing for the EGFR mutation using the test, tracking the SQI value and identifying a trend may lead to the understanding of tumor progression, an option not available in other tests. Phillipe Taniere, MD, PhD, a Consultant Histopathologist at UHB, said, “Identifying mutations in blood samples has historically been a challenge due to the low frequency of cancerous cells in the sample. However this new, powerful technology from Roche Diagnostics has made it possible to take a simple blood sample and rapidly identify EGFR mutations in tumors’ DNA.” Image: The¬†cobas¬†EGFR Mutation Test v2 CE-IVD identifies the epidermal growth factor receptor (EGFR) gene in the DNA from non-small cell lung cancer (NSCLC) patients (Photo courtesy of Roche Molecular Diagnostics).

New Assay Improves Detection of Tuberculosis samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIFR) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. Scientists at the Rutgers New Jersey Medical School (Newark, NJ, USA; www.njms.rutgers.edu) and their many colleagues developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance of the G4 cartridge used in Xpert (Cepheid, Sunnyvale, CA, USA; www.cepheid.com). Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or M. bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. The investigators reported that for M. tuberculosis H37Rv, the LOD was 15.6 CFU/mLof sputum for Ultra versus 112.6 CFU/mLof sputum for Xpert, and

Graham Beastall United Kingdom Claus Christiansen Denmark Hernán Fares Taie Argentina Bernard Gouget France Maurizio Ferrari Italy Jocelyn M. Hicks United States Anders Kallner Sweden Tahir S. Pillay South Africa Andreas Rothstein Colombia Dmitry B. Saprygin Russia Praveen Sharma India Rosa I. Sierra-Amor Mexico Peter Wilding United States Andrew Wootton United Kingdom

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Blood Test Provides Faster and Simpler Lung Cancer Diagnosis raditionally, lung cancer diagnosis has relied solely on lung biopsies, an invasive procedure which many patients are too unwell to tolerate. A simple test, which can detect gene mutations in tumor DNA from blood samples, is providing new choices to lung cancer patients by way of easier and faster diagnosis as well as improved access to life changing drugs. Latest statistics from Cancer Research UK show there were 46,400 new cases of lung cancer, the third most common cancer in the UK, in 2014. The new test is a true gatekeeper for molecular therapies targeting epidermal growth factor receptor (EGFR) abnormalities and identifies with fast turnaround patients eligible for targeted therapy, sparing them chemotherapy and the associated side effects. The University Hospitals Birmingham NHS Foundation Trust (UHB, Birmingham, UK; www.uhb.nhs.uk) has introduced the test to its renowned cancer diagnostics laboratory. The new testing service gives more patients access to life-saving treatments with fewer side effects than classic chemotherapy, improving quality of life for patients. The test is highly specific and its results can be used to predict a patient’s response to treatment, allowing doctors to rapidly prescribe tailored treatments in line with NHS England’s goal to ensure personalised treatment and care for everyone diagnosed with cancer. The Cobas EGFR Mutation Test v2 CE-IVD (Roche Molecular Diagnostics, Pleasanton, CA, USA; https:// molecular.roche.com) identifies the epidermal growth factor receptor (EGFR) gene in the DNA from non-small cell lung cancer (NSCLC) patients and is intended for use as an aid in selecting patients with NSCLC for therapy with an EGFR tyrosine kinase inhibitor (TKI). This innovative assay is the first to utilize plasma in addition to tissue as a sample type, thus removing common barriers to molecular testing, including surgery risks and

for M. bovis BCG, it was 143.4 CFU/mL of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert resulted in an overall sensitivity of 87.5% versus 81.0% and a sensitivity on sputum smear-negative samples of 78.9% versus 66.1%. Both tests had a specificity of 98.7% and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection. The authors conclude that that Ultra will result in greater TB case detection rates not only in subjects with paucibacillary TB, such as those with human immunodeficiency virus (HIV) coinfection, but also in pediatric patients with TB and those with extrapulmonary TB, which are known to have lower mycobacterial loads. The study was published on August 29, 2017, in the journal mBio.

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ISSN 1068-1760 Vol.34 No.7. Published, under license, by Globetech Media LLC; Copyright © 2017. All rights reserved. Reproduction in any form is forbidden without express permission. Opinions expressed are solely those of the authors, and do not represent an endorsement, or lack thereof, by the Publisher of any products or services. Teknopress Yayıncılık ve Ticaret Ltd. S¸ti. adına ˙Imtiyaz Sahibi: M. Geren • Yazı is¸leri Müdürü: Ersin Köklü Müs¸ ir Dervis¸ ˙Ibrahim Sok. 5/4, Esentepe, 34394 S¸is¸ li, ˙Istanbul P. K. 1, AVPIM, 34001 ˙Istanbul • E-mail: Teknopress@yahoo.com Baskı: Printkom Ltd. • İpkas Sanayi Sitesi 3. Etap C Blok • 34490 Başakşehir • İstanbul Yerel süreli yayındır. Yılda sekiz kere yayınlanır, ücretsiz dag˘ıtılır.

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Novel Urine Test Predicts High-Risk Cervical Cancer cont’d from cover

by precancerous changes, but also DNA from human papilloma virus (HPV) that is sexually transmitted and causes virtually all cases of the disease. An international team of scientists working with those at Johns Hopkins School of Medicine (Baltimore, MD, USA; www.hopkinsmedicine.org) tested a biomarker panel in cervical epithelium DNA obtained from 211 women evaluated in a cervical cancer clinic in Chile from 2006 to 2008. The results were verified in a prospective cohort of 107 women evaluated in a high-risk clinic in Puerto Rico from 2013 to 2015. The investigators had identified three genes associated with cervical cancer or abnormallooking cells known to become cancerous: FK506 Binding Protein 6 (FKBP6), Integrator Complex Subunit 1(INTS1) and Zinc Finger Protein 516

(ZNF516). As abnormalities progress, these genes were more likely to have a chemical methyl group attached to their DNA in certain spots. Samples were polymerase chain reaction amplified using sample-specific index primers for multiplexed sequencing on a MiSeq System (Illumina, San Diego, CA, USA; www.illumina.com). Using methylation as a value to diagnose malignancy, the three genes together showed a 90% sensitivity, meaning that their presence was able to predict a true positive cancer sample this percentage of the time and the test had an 88.9% specificity. To further improve the accuracy of the test, the investigators added a new gene marker to the test. This time,

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rather than using a human gene, they used one from the virus, HPV16-L1, which also becomes methylated in human cells as cancer develops. The team repeated the test with the four-gene combination on a new population of women from the University of Puerto Rico. The 115 women ranged in age from 21 to 49; 41 participants had healthy cervical tissue, and 74 had one of three types of precancerous cells. Using all four genes, the test now had a sensitivity of 90.9% and a specificity of 60.9%. They then tested 40 samples of paired cervical tissue, blood and urine from a subset of the patients from Puerto Rico. Using the DNA from blood, they found the test had an 85.7% sensitivity and a 60.9% specificity and using urine, they found a 75% sensitivity and an 83.3% specificity. The time to process cervical samples, blood or urine and get a test result took four days and the team plan to continue modifying the test to improve the urine sensitivity to that of the cervical tissue samples. Rafael Guerrero-Preston, DrPH, MPH, an assistant professor and lead investigator said, “Our urine test would serve as a molecular triage, at times supplementing Pap test information. In developing countries that don’t have the money, medical infrastructure or cultural approval for Pap test, our test could be used instead.” The study was published on November 8, 2016, in the journal Cancer Prevention Research. Image: The MiSeq desktop sequencer for genome sequencing (Photo courtesy of Illumina). V

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Expanded Pathogen Identification Database Gets Clearance cont’d from cover

first time, enables the safe identification of the Mycobacterium tuberculosis (TB) group, the most frequent non-tuberculous mycobacteria (NTM), Nocardia and the most medically important molds. The VITEK MS, Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry System (bioMérieux, Marcy-l’Étoile, France; www.biomerieux.com) for rapid pathogen identification, has received 510(k) clearance from the US Food and Drug Administration (FDA, Silver Springs; MD, USA; www.fda.gov) for the expanded identification of mycobacteria, Nocardia and molds. To gain new FDA clearance for

these new species, bioMérieux submitted data from a multi-center study consisting of 2,695 clinical isolates for 47 molds, 19 mycobacteria, and 12 Nocardia. The FDA clearance of Mycobacterium species was from both solid and liquid growth media. The VITEK MS system’s newly expanded database and Mycobacterium/Nocardia and Molds reagent kits are now commercially available in the USA. In cases where microorganisms are resistant to carbapenems (a very broad spectrum antibiotic class), bioMérieux has also developed and received FDA 510(k) clearance for its RAPIDEC CARBA NP test that enables the detection of carbapenemase producers within two hours. RAPIDEC CARBA

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NP helps health professionals make vital treatment decisions and facilitates implementation of infection prevention and control measures. These highly complementary solutions help improve antibiotic stewardship in the fight against antimicrobial resistance. François Lacoste, DVM, bioMérieux Corporate VP, Clinical Unit, said, “We are honored to receive the first 510(k) clearance from the FDA for the identification of mycobacteria,

Nocardia and molds on a MALDITOF system and to provide our customers in the USA with additional features for the already well adopted VITEK MS rapid identification system. As the world leader in microbiology, one of our priorities is to continuously develop high medical value solutions that enable rapid and accurate detection of important microorganisms, with the ultimate goal of improving patient care.”

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hydrophobic domains that delineated hydrophilic, wicking-based microfluidic channels for accurate colorimetric assays, and self-pipetting test zones for electrochemical detection. The bottom layer of the SPED was a triboelectric generator (TEG) made from hydrophobic paper and capable of harvesting electrical energy from the user’s interaction with the SPED. An inexpensive and rechargeable handheld potentiostat was constructed to interface with the SPED, enabling the accurate quantitative electrochemical detection of glucose, uric acid, and L-lactate. The battery powering the potentiostat could be recharged by the user, using the sequential discharge of a capacitor previously charged with the TEG built

into the SPED. A machine-vision diagnostic application was created to automatically identify and quantify each of the colorimetric tests from a digital image of the SPED, taken under a wide range of ambient light conditions, in order to provide fast diagnostic results to the user as well as to facilitate remote expert consultation. “To our knowledge, this work reports the first self-powered, paperbased devices capable of performing rapid, accurate, and sensitive electrochemical assays in combination with a low-cost, portable potentiostat that can be recharged using a paper-based TEG,” said contributing author Dr. Ramses V. Martinez, assistant professor of industrial and biomedical engineering at Purdue University.

Molecular Assay Identifies Colorectal Cancer cont’d from cover

cancer (CRC) recurrence by measuring fragments of genetic material, circulating tumor DNA (ctDNA), that leak from a tumor into the blood stream. With this test it may be possible to identify CRC recurrence in advance of symptoms before other tests indicate recurrence. In a previously published study, clinical data showed that the liquid biopsy test Colvera (Clinical Genomics, North Ryde, NSW, Australia; www.clinicalgenomics.com) detected twice the number of recurrence cases as carcinoembryonic antigen (CEA) testing, the current guidelinesrecommended blood test used for CRC recurrence monitoring. As Colvera only requires a peripheral blood sample, the test can be administered along with other CRC surveillance tests, including CEA. Colvera is a polymerase chain reaction (PCR)-based test measuring LINKXPRESS COM

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two epigenetically modified genes, hyper-methylated Branched Chain Amino Acid Transaminase 1, (BCAT1) and IKAROS Family Zinc Finger (IKZF1), in free circulating DNA which may leak from cancerous lesions into the bloodstream. Lawrence LaPointe, PhD, President and CEO of Clinical Genomics, said, “Colorectal cancer outcomes improve with early detection but currently available monitoring tests frequently fail to detect disease at a point when clinical intervention can be effective. This failure is a key reason why mortality from colorectal cancer is so high. Colvera is a new test that can detect molecular changes in circulating tumor DNA associated with cancer development. Colvera provides physicians with actionable information that can trigger further clinical assessment. The overall aim is to improve survival LabMedica International November/2017

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Markers Predict Recurrence of Atrial Fibrillation after Ablation tudy suggests that certain circulating microRNA molecules are associated with the recurrence of the abnormal erratic heartbeats in some patients after ablation procedure, potentially providing markers that may help identify such patients in advance, as ablation therapy carries some risks and is expensive. Circulating microRNAs are microRNAs (generally very stable noncoding RNAs involved in gene regulation) that have spilled out of cells into body fluids, such as blood and saliva, and can often be measured. Researchers at the Intermountain Medical Center Heart Institute (Salt Lake

City, UT, USA; www.ihc.com) have found that certain circulating microRNA could serve as markers in screening tools to help distinguish which patients with atrial fibrillation (AFib) will be more versus less likely to benefit from various therapy options. The research team examined a series of microRNA markers in blood samples to identify AFib patients whose ablations worked the first time: they compared 85 patients who had successful ablations with 55 patients whose AFib recurred within a year. The microRNAs studied involved in inflammation, fibrosis, and heart electrical activity. They found low levels of three mi-

croRNAs in patients whose AFib came back after ablation. Those molecules had previously been associated with ablation’s atrial scarring (remodeling and adverse electrical healing). Ablation cures AFib in 60-70% of patients, but multiple procedures are required for some. The researchers hope their findings will lead to development of tests that will help doctors determine which treatments are more likely to work for different pa-

tients, including those who wouldn’t benefit from ablation. The study was presented at the American College of Cardiology’s Scientific Session on March 18, 2017, in Washington, DC, USA. Image: New findings suggest that certain circulation microRNAs could serve as markers to help identify patients less likely to benefit from ablation therapy (Photo courtesy of Intermountain Medical Center).

Test Predicts Success of Bone-Marrow Transplants cont’d from cover

low-intensity ‘conditioning’ therapy, such as radiotherapy or chemotherapy, and donor stem cell transplants for patients with high risk of mortality. However, many patients can experience relapse or severe complications. A large team of scientists collaborating with those at the Dana-Farber Cancer Institute (Boston, MA, USA; www.dana-farber.org) analyzed blood cells from over 1,500 MDS patients, combined with clinical information such as age and disease status. They were able to devise a genetic profile of mutations associated with poorer patient outcomes after transplantation. They performed targeted mutational analysis on samples obtained before transplantation and evaluated the association of mutations with transplantation outcomes, including overall survival, relapse, and death without relapse. The scientists found that the most important predictor of patient prognosis was a mutation in the tumor protein p53 (TP53) gene. These patients tended to relapse sooner and survive for a shorter time after transplant. Whether patients had high- or low-intensity conditioning therapy before the transplant did not affect the outcome. Specific mutations in other LINKXPRESS COM

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genes were also linked to poorer outcomes in older patients, although only when they received low-intensity conditioning therapy. The investigators suggested that these patients may benefit from high-intensity conditioning therapy to reduce the risk. In young adults, one in 25 patients with MDS were found to have mutations associated with the rare, inherited Shwachman-Diamond syndrome, which affects the bone marrow, pancreas and skeletal system. Most of these patients were previously undiagnosed. The team found that each of these patients had acquired a TP53 mutation, indicating how MDS develops in these patients and giving insight into their poor prognosis. Robert Coleman Lindsley, MD, PhD, the lead author of the study, said, “‘In deciding whether a stem-cell transplant is appropriate for a patient with MDS, it’s always necessary to balance the potential benefit with the risk of complications. Our findings offer physicians a guide, based on the genetic profile of the disease and certain clinical factors, to identifying patients for whom a transplant is appropriate, and the intensity of treatment most likely to be effective.” The study was published on February 9, 2017, in The New England Journal of Medicine. LabMedica International November/2017

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Novel Assay Discriminates Several Aspergillus Species nvasive aspergillosis (IA) is mainly caused by Aspergillus fumigatus and when diagnosis is made early and first line therapy with voriconazole is initiated promptly, a relatively low mortality is observed. However, over the past decade, azole resistance has emerged worldwide and poses a threat as IA with azole-resistant A. fumigatus is associated with high mortality of 88%. As in vitro drug susceptibility testing is often not feasible, as cultures remain negative or sibling species fail to sporulate, molecular techniques are an option. Scientists at the Erasmus University Medical Center (Rotterdam, The Netherlands; www.erasmusmc.nl) used a multiplex real-time polymerase chain reaction (PCR) assay to detect Aspergillus species and mutations in the Cyp51A gene of A. fumigatus. They performed the assay on cultured sibling strains obtained from two clinical cases. In addition to assess the precision of the assay, a larger set of strains was tested: six A. lentulus strains and 12 A. felis species complex strains (five A. felis, four A. parafelis and three A. pseudofelis) were obtained and the three control A. fumigatus strains (one WT, one TR34/L98H mutant, one TR46/T289A/Y121F mutant). The AsperGenius multiplex real-time PCR assay (PathoNostics, Maastricht, The Netherlands; www.pathonostics.eu)

was performed on strains obtained from lung biopsy in one case and from pleural mass biopsy the second case. Both strains gave positive signals for the Aspergillus species and Aspergillus section Fumigati. The AsperGenius resistance PCR did not detect the TR34 target in A. lentulus and A. felis in contrast to A. fumigatus. Melting peaks for L98H and Y121F markers differed and those of the Y121F marker were particularly suitable to discriminate the three species. The authors concluded that the assay can be used to rapidly discriminate A. fumigatus, A. lentulus and A. felis. The study was published in the March 2017 issue of the journal Diagnostic Microbiology and Infectious Disease. Image: The AsperGenius is a multiplex real-time PCR assay which rapidly diagnoses Aspergillus infections and simultaneously identifies multiazole resistance (Photo courtesy of PathoNostics). LabMedica International November/2017

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Microarray PCR System Detects STI Pathogens automated microarray-based PCR (polymerase chain reaction) system is able to detect 11 STI (sexually transmitted infection) pathogens in parallel from the same patient sample. The EUROIMMUN AG (Luebeck, Germany; www.euroimmun.com) EUROArray platform enables PCR-based detection of both manifest and silent infections, and is, therefore, suitable for diagnosis of symptomatic patients as well as for general screening. It offers a huge time advantage over cultivation and is especially useful for detecting sexually transmitted pathogens that are difficult or impossible to grow in culture. The system works by amplifying and fluorescently labeling specific sections of pathogen DNA from pa-

tient samples in single multiplex PCR. The PCR products are then hybridized to BIOCHIP microarray slides containing immobilized complementary DNA probes and detected by their fluorescence signals. The evaluation, interpretation, and archiving of results is fully automated by EUROArrayScan software, and is thus highly standardized and objective. The EUROArray system detects and identifies Chlamydia trachomatis, Neisseria gonorrhoea, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum, Haemophilus ducreyi, Treponema pallidum, Trichomonas vaginalis, and herpes simplex viruses 1 and 2. The complete EUROArray process from sample processing to report release is IVD-validated and CE-registered.

Image: The automated EUROArrayScan PCR system reads microarrays for fast and standardized detection of STI pathogens (Photo courtesy of EUROIMMUN).

Diagnosis of CF Simplified By Sweat Sensor Device wristband-type wearable sweat sensor that measures calcium and glucose levels and transmits the data to a central laboratory is expected to simplify the current diagnostic method for cystic fibrosis (CF). Conventional methods for diagnosing cystic fibrosis require that patients visit a specialized center and wait for up to 30 minutes while electrodes stimulate sweat glands in the skin to provide enough perspiration for the test. Then a laboratory measures the level of chloride ions in the sample to determine if the subject has the disease. In contrast, the wearable sweat sensor stimulates the skin to produce small amounts of sweat, rapidly measures components in the sample, and sends the data by way of a cell phone to a laboratory that analyzes and returns the results. To demonstrate the clinical value of the platform, human subject studies were performed in the context of the cystic fibrosis diagnosis and preliminary investigation of the blood/ sweat glucose correlation. With the platform, the investigators detected the elevated sweat electrolyte content of cystic fibrosis patients compared with that of healthy control subjects. Furthermore, the results indicated that oral glucose consumption in the fasting state was followed

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by increased glucose levels in both sweat and blood. "This is a huge step forward," said contributing author Dr. Carlos Milla, associate professor of pediatrics at Stanford University (Palo Alto, CA, USA; www.stanford.edu). "The test happens all at once and in real time. CF diagnosis, as well as other kinds of diagnoses, could be done without needing a staff of skilled clinicians on duty and a well-equipped lab. You can get a reading anywhere in the world." The device was described in the April 17, 2017, online edition of the journal Proceedings of the [U.S.] National Academy of Sciences. Image: A wearable sensor that extracts sweat and analyzes its constituents could be a useful device for diagnosing and monitoring diseases (Photo courtesy of Sam Emaminejad / Stanford School of Medicine). LabMedica International November/2017

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Gene Mutations Associated With Aggressive Prostate Cancer n international study has identified inherited genomic DNA mutations in the Kallikrein 6 (KLK6) gene region that are connected to a predisposition for some men to develop aggressive prostate cancer (PC). Much like diagnostic tests developed based on association between BRCA gene mutation and risk for breast cancer in women changed the approach to prevention and treatment, discovery of the KLK6 region’s connection to aggressive PC may change the course of PC patient care. The study was led by Dr. Alexandre Zlotta of Mount Sinai Hospital and Lunenfeld-Tanenbaum Research Institute (www.lunenfeld.ca), part of Sinai Health System in (Toronto, Ontario, Canada; www.sinaihealthsystem.ca) and Dr. Paul Boutros of

Ontario Institute for Cancer Research (OICR). Early diagnosis of aggressive PC is an important unmet need. Up until now, no single test could predict severity of PC type – the current PSA test (based on KLK3, which is located near KLK6) only identifies the risk of PC, not its severity. To identify relevant mutations the researchers analyzed blood samples of 1,858 men from 3 independent cohorts in Europe and North America. The study showed a 3-fold increase in the risk of aggressive PC for men with the KLK6 mutation. The frequency of the gene variants varied from 6-14% of the population of men with PC. The KLK6 variants also independently predicted treatment failure after surgery or radiation for PC in another independent cohort of 130 men.

The study, by Briollais L et al, was published March 14, 2017, in the Journal of the National Cancer Institute (JNCI). Image: A micrograph of prostatic adenocarcinoma (conventional, acinar type) with perineural invasion. Prostate biopsy, H&E stain (Photo courtesy of Wikimedia).

New Assay Advances Personalized Therapy for Cancer Patients recision medicine attempts to direct treatment for a patient based on molecular alterations known to exist in the patient’s disease and the treatment of patients with cancer has been at the center of the evolution for precision medicine studies. This effort addresses therapeutic efficacy across multiple tissues, but also adds data as to the clinical value of broad-based screening panels versus disease-specific assays. A novel assay tailored for these trials is highly sensitive for detecting genetic mutations from a variety of tumor tissue and, for the first time, has been reproduced with accuracy by multiple clinical laboratories, laying the groundwork for future clinical utility. A multidisciplinary team of scientists led by those at the Frederick National Laboratory for Cancer Research (Frederick, MD, USA; http://frederick. cancer.gov) tested 186 samples and 12 cell lines at four different laboratories. Steps were taken to maximize standardization, including development of standard operating procedures, use of the same commercial assay and instruments, and face-to-face discussions. The National Cancer Institute’s NCI-MATCH (Molecular Analysis for Therapy Choice) is a large, ongoing clinical trial that matches tumors to therapies based on the tumor’s genetic characteristics. The next generation sequencing (NGS) technology used in the trial was the Oncomine Cancer Panel assay and the Ion Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA, USA; www.thermofisher.com) is

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able to detect more than 4,000 pre-defined genomic variations across 143 genes, including single nucleotide variants (SNVs), insertions/deletions (indels), copy number variations (CNVs), and gene fusions. Levels of evidence were developed to select a subset of specific actionable genomic variants to be used for treatment matching. The investigators report that the assay was highly sensitive, 96.98% for 265 known mutations, with 99.99% specificity. Since one feature of the NCI-MATCH trial is the wide variety of tumors examined, including solid tumors and lymphomas that no longer respond to standard therapy, the assay used must be able to analyze specimens from different tissues. Importantly, the NCI-MATCH NGS assay was able to accurately determine genetic abnormalities in biopsies from the pancreas, melanoma, bone, and skin. Elizabeth R. Unger, PhD, MD, from the Centers for Disease Control and Prevention (Atlanta, GA, USA; www. cdc.gov) said, “The validation study reported is another step moving the field closer to the time when precision medicine will generate the expected benefits in improved clinical outcomes. Although the success of the NCIMATCH trial cannot be assured, linking precision laboratories to precision medicine trials assures that data used for drug assignment will be reliable. Further, the use of a commercial platform and integrated analysis and reporting pipeline will greatly facilitate broader translation of any successes.” The study was published on February 7, 2017, in The Journal of Molecular Diagnostics. LabMedica International November/2017

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Calibration Kit Introduced for IVD Testing of Testosterone he first CE-marked calibration kit for more sensitive testosterone measurement in men, women, and children, provides a wide analytical range and increased accuracy at low levels. Merck (Darmstadt, Germany www.merckgroup. com/en/index.html) as announced the introduction of its new testosterone calibrator kit for in vitro diagnostic (IVD) use. The kit allows users to calibrate assays and verify calibrations, and is the first of its kind to receive CE-mark approval, indicating compliance with the European Union’s Medical Device Directive. Traditional immunoassays used for measuring testosterone levels in patient samples can be inaccurate and deliver inconsistent results, especially at the low levels present in women and children. The diagnostic range of the new kit encompasses both male and female testosterone clinical reference ranges. Its broad reference range enables ample analytical sensitivity for accurate testing using new mass-spec -based methods, from the lowest levels

needed for testing of women and children to the highest levels used in testing of men. The new calibration kit delivers more accurate and consistent test results and is thus more reliable for clinical diagnosis and monitoring. The kit is manufactured and tested under the most stringent international quality assurance standards. It includes a set of 10 levels of testosterone serum concentration standards ranging from 2-2,000 ng/dL, along with a blank and internal standard. Merck’s portfolio of Cerilliant certified reference materials are used to validate analytical measurement methods and calibration, and are manufactured and authorized to ensure the highest level of accuracy. Solutions are used in quantitative massspectrometry -based applications that range from diagnostic testing and endocrinology to therapeutic drug monitoring, forensic analysis, clinical toxicology and pharmaceutical research. Merck manufac-

tures a comprehensive list of parent drug, metabolite, endogenous biomarker and internal standard “Certified Spiking Solutions” used for clinical diagnostic test applications of steroids and hormones, biogenic amines, hydroxyvitamin D, bile acids, fatty acids, vitamins, immunosuppressants, antifungals and cardiac drugs. Image: The Cerilliant certified reference materials now include the first CE-marked calibration kit to enable broad-range diagnostic testosterone testing in men, women, and children (Photo courtesy of Merck).

Positive Fecal Occult Blood Test Associated with Diabetes umerous etiologies are implicated in the complications of diabetes and a link between diabetes and the predisposition to certain cancers, including colon cancer, has been established during the last decade. The fecal occult blood test (FOBT) is a screening method used principally for detection of colon cancer or polyps; however, the relationship between FOBT and diabetes has not been explored in detail, and therefore the association between FOBT result and glycemic status, has been assessed. Scientists at the Kanagawa University of Human Services (Yokosuka, Japan; www.kanagawa-u.ac.jp) have carried out a cross-sectional study and enrolled 12,836 apparently healthy subjects (9,258 men and 3,578 women), aged 30 to 79 years who underwent a medical health checkup at the Saitama Health Promotion Corporation in 2012. Anthro-

pometric and laboratory tests were carried out after overnight fasting. Serum parameters were measured using standard methods on Hitachi autoanalyzers (Tokyo, Japan; www.hitachi.com). All subjects were classified according to their glycated hemoglobin (HbA1c) into normal (HbA1c equal to or less than 5.69%), prediabetic (HbA1c 5.7% to 6.49%) and diabetic (HbA1c equal to or greater than 6.5%) groups, regardless of whether they were being treated for diabetes. FOBT was performed automatically using a diagnostic test kit the magnetic particle agglutination method, for the detection of human hemoglobin in fecal samples (MagStream HemSp, Fujirebio, Inc., Tokyo, Japan; www.fdi.com). A positive FOBT was defined according to the two-day stool sampling method. The team found that mean age and HbA1c were significantly higher in the 1,502 in the positive

group than the 11,334 in negative FOBT groups. There were fewer men and current smokers in the positive FOBT group. Multivariate logistic regression analysis showed that, compared with HbA1c of less than 5.69%, HbA1c of greater than 6.5% was significantly associated with positive FOBT, independently of relevant confounders including age, sex, and past history of gastric/duodenal ulcers and colon cancer or polyp. The authors concluded that their results indicate a significant association between diabetes and positive FOBT in a Japanese population undergoing general health screening. This supports the position that subjects with diabetes may be predisposed towards gastrointestinal cancer or may suggest the presence of microangiopathy in the gut. The study was published in the January 2017 issue of the journal Clinical Biochemistry. LabMedica International November/2017

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Vitamin D Deficiency Predicts Poor Prognosis for IgA Nephropathy mmunoglobulin A (IgA) nephropathy (IgAN), also known as Berger’s disease is the most common form of glomerulonephritis worldwide, especially in Asia, and represents one of the main causes of the end-stage renal diseases (ESRD). Male gender, early-onset, absence of macroscopic hematuria, persistent microscopic hematuria, hypertension, proteinuria, presence of renal dysfunction at the time of diagnosis, and certain histological features of renal lesions have been identified as important risk factors for its progression. Scientists working in a renal unit recruited a total of 105 patients with newly diagnosed, biopsyproven primary IgAN from the First Affiliated Hospital of Guangxi Medical University (Nanning, China; www.gxmu.edu.cn) between 2012 and 2015. IgAN was diagnosed with mesangial depositions of IgA under immunofluorescence microscope and

with electron-dense mesangial deposits under electron microscope. Blood samples were collected before the kidney biopsy. All kidney tissue specimens were obtained by percutaneous kidney biopsy, and examined using light microscope, immunofluorescent, and electron microscope. All histological slides were evaluated by an experienced renal pathologist. Serum 25-hydroxy-vitamin D (25(OH)D) was measured using electrochemiluminescent immunoassay (ECLIA) with an Elecsys 10100/201 system (Roche Diagnostics, Risch-Rotkreuz, Switzerland; www.rochediagnostics.ch). All measurements were performed in a blind manner and in duplicate. Vitamin D deficiency is defined as 25-hydroxy-vitamin D of less than 15 ng/mL. The investigators reported that 101 patients (96.2 %) had 25 (OH) vitamin D levels less than 30 ng/mL, and 51 patients (48.6.5 %) had 25 (OH) vitamin D less than 15 ng/mL, which are the thresholds for 25 (OH) vitamin D insufficiency and deficiency, respectively. They also showed that

compared with those showing a higher 25(OH)D level, those with a 25(OH)D deficiency were significantly associated with a lower estimated Glomerular Filtration Rate (eGFR) and a higher proteinuria level. The risk for reaching the primary endpoint was significantly higher in the patients with a 25(OH)D deficiency compared to those with a higher level of 25(OH)D. The association between the plasma 25(OH)D level at the time of initial diagnosis and the severity of histologic lesions, and found that the plasma 25(OH)D level was associated with the percentage of interstitial fibrosis/tubular atrophy. The authors concluded that 25(OH)D deficiency is significantly correlated with a poorer renal function and more severe renal pathological features, and is strongly associated with increased risk of renal progression in IgAN patients, making a 25(OH)D deficiency a good prognostic marker to predict the severity and clinical outcome in IgAN patients. The study was published on November 2, 2016, in the journal BMC Nephrology.

Gene Sets Identified May Predict Response to RA Therapies blood biomarkers search has identified 3 gene expression signatures that helped predict which patients were more likely to respond to tumor necrosis factor inhibitors (TNFi) or B-cell depletion therapies in patients with moderate to severe rheumatoid arthritis (RA). The findings were presented at the 2016 ACR/ARHP Annual Meeting (Washington, DC, USA) of the American College of Rheumatology (Atlanta, GA, USA; www.rheumatology.org) and its Association of Rheumatology Health Professionals. Drawing on data from the ORBIT study, a randomized, controlled trial of RA patients in the UK, researchers looked for gene expression markers that would help predict responses to either TNFi drugs or the B-cell therapy rituximab, or both. The ORBIT data “showed that patients who have seropositive RA are just as likely to respond to rituximab therapy when compared to anti-TNF therapy,” said co-lead-author Duncan Porter, MD, honorary associate professor at Queen Elizabeth University Hospital (Glasgow, Scotland; www.nhsggc.org.uk), “However, a significant proportion of patients failed to respond to their first biologic drug, but responded when they were switched to the alternative. If we could identify markers in the blood that predicted which drug patients were most likely to respond to, that would allow us to choose the best treatment for that patient at the start, rather than rely on a trial-and-error approach.” Dr. Porter and fellow researchers sequenced the RNA from the peripheral blood of 241 RA patients recruited for the ORBIT study, after first depleting ribosomal and globin RNA. They used 70% of the samples to develop response-prediction models, and reserved 30% for validation. Clinical response to the therapies was defined as a drop in DAS28-ESR (disease activity score) of 1.2 units between the baseline and at 3 months. They used multiple machine-learning tools to predict general responsive-

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ness and differential responses to TNFi and rituximab. They also used 10-fold cross-validation to train the models for responsiveness, and then tested these on the validation samples as well. Using support vector machine recursive feature elimination, the researchers identified 3 gene expression signatures that predicted therapy responses: 8 genes predicted general responsiveness to both TNFi and rituximab, 23 genes predicted responsiveness to TNFi, and 23 genes predicted responsiveness to rituximab. The researchers also tested their prediction models on the validation set, and this resulted in ROC (receiver operating characteristic) plot points with an AUC (area under the curve) of 91.6% for general responsiveness, 89.7% for TNFi response, and 85.7% for rituximab response. “There are indeed gene expression markers that predict drug-specific response,” said Dr. Porter, “If confirmed, this will allow stratification of patients into groups more likely to respond to one drug rather than another. This would lead to higher response rates, and reduced likelihood of receiving a trial of an ineffective drug. Because ineffective treatment is associated with pain, stiffness, disability, and reduced quality of life, this will lead to better patient care.” “The findings need to be confirmed using targeted RNA sequencing, or internal validation, and then tested in a new cohort of patients, or external validation. Ultimately, a commercial testing kit would be developed to allow clinicians to test patients before they receive treatment,” said Dr. Porter. V

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Comparison of Vitamin Concentrations in Tears and Serum esults obtained during a recent study suggest that tears could be used as specimens to monitor nutritional health, since they are easily obtained, sufficiently mirror blood serum data, and enhance the speed of deficiency diagnoses. Investigators at Michigan Technological University (Houghton, USA; www.mtu.edu) obtained matched samples of tears and serum from 15 family pairs, each pair consisting of a four-month-old infant – fed either liquid formula or mother’s milk – and one parent. Several water-soluble and fat-soluble vitamins were detected and their levels were quantified by liquid chromatography/mass spectrometry. Vitamin concentrations were compared between tears and blood serum for individual subjects, between infants and parents, and against self-reported dietary intakes. They investigators reported that water-soluble vitamins B1, B2, B3 (nicotinamide), B5, B9 and fatsoluble vitamin E (alpha-tocopherol) were routinely detected in tears and blood serum while fat-soluble vitamin A (retinol) was detected only in blood

serum. Water-soluble vitamin concentrations measured in tears and blood serum of single subjects were comparable, while higher concentrations were measured in infants compared to their parents. Strong positive correlations were found between tear and blood serum concentrations of vitamin E from infants and parents and vitamin B3 concentrations from parents, while slight positive correlations were detected for infants B3 and parents B1 and B2 concentrations. Correlations between infants and parents were found for the concentrations of B1, B2, B3, and E in tears, and the concentrations of B2, A, and E in blood serum. Stronger vitamin concentration correlations were found between infants and parents for the breast-fed infants, while no significant difference was observed between breast-fed and bottle-fed infants. “Our goal was to seek the viability of establishing measurable units of tears for nutritional assessments,” said first author Dr. Maryam Khaksari,

mass spectrometry research specialist at Michigan Tech University. “Your body cannot manufacture vitamins, and vitamins reflect available food sources in your body. That is what makes them good indicators of nutritional health. Since tears contain vitamins, they might have real potential to replace other clinical tests.” The study was published in the December 23, 2016, online edition of the journal Experimental Eye Research. Image: Only a few microliters of tears are needed to analyze the vitamins in them (Photo courtesy of Sarah Bird, Michigan Technological University).

Novel Biosensor Selectively Measures Cancer Patient p53 Autoantibodies team of Spanish cancer researchers has developed a disposable electrochemical biosensor for the specific and sensitive determination of p53-specific autoantibodies, which are biomarkers for certain types of cancers with p53 gene mutations. The 10 to 40% of all cancer patients with aberrantly mutated p53 have cancer cells that multiply without control, and their immune systems generate autoantibodies against the p53 protein. To more effectively determine levels of p53 autoantibodies, investigators at Universidad Complutense de Madrid (Spain; www.ucm.es) developed a disposable electrochemical biosensor. This specific and sensitive biosensor was based on magnetic microcarriers (MBs) modified with covalently

immobilized HaloTag fusion p53 protein as solid supports for the selective capture of specific autoantibodies. HaloTag is a modified bacterial enzyme designed to covalently bind to a synthetic ligand of choice and fuse to a protein of interest. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the electronic signal generated by the hydroquinone/ H2O2 system was correlated to the levels of p53autoantibodies in the sample. The biosensor was used to analyze sera from 24 patients with high-risk of developing colorectal cancer and six from patients already diagnosed with colorectal (four) and ovarian (two) cancer. The biosensor was able to determine p53 autoantibodies with

sensitivity higher than that of a commercial standard ELISA using a simpler protocol with less sample volume. The biosensor can be easily miniaturized and developed into a cost-effective diagnostic tool. “Our immune system produces these cancer autoantibodies even three years before the first symptoms appear,” said contributing author Dr. Susana Campuzano, associate researcher in analytical chemistry at Universidad Complutense de Madrid. “Its simplicity of handling, portability, and time to complete the full procedure make it suitable for application in clinical routine.” The biosensor was described in the November 26, 2016, online edition of the journal Analytical Chemistry. LabMedica International November/2017

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Intestinal Fungi Contribute to Alcoholic Liver Disease hronic liver disease with cirrhosis is the 12th leading cause of death in the USA, and alcoholic liver disease accounts for approximately half of all cirrhosis deaths. Chronic alcohol consumption is associated with intestinal bacterial dysbiosis, yet little is known about the contribution of intestinal fungi, or mycobiota, to alcoholic liver disease. Intestinal fungi may contribute to the development of alcoholic liver disease (ALD), which encompasses a broad range of liver diseases, from simple steatosis (fatty liver) to end-stage liver disease, or cirrhosis (liver cell death). Patients with cirrhosis are either frequently exposed to fungal products or develop fungal infections, with high mortality. Scientists at the University of California, San Diego (La Jolla, CA, USA; www.ucsd.edu) and the J. Craig Venter Institute (Rockville, MD, USA; www.jcvi.org) used both a mouse model and human samples of serum and feces in their study. They analyzed human serum samples data collected from 28 patients with alcoholic liver cirrhosis from July 1, 2010, through October 31, 2010. The human fecal samples were from eight healthy individuals without chronic disease (controls), 10 alcohol-dependent patients without evidence of progressive liver disease (non-progressive liver disease), six patients with alcoholic hepatitis (alcoholic hepatitis) and four patients with alcoholic liver cirrhosis (alcoholic cirrhosis). Primary human hepatocytes, Kupffer cells, and hepatic stellate cells were isolated and RNA extraction and quantitative reverse transcriptase polymerase chain reaction (PCR) were performed. The scientists observed that alcohol-dependent patients displayed reduced intestinal fungal diversity and Candida overgrowth. Compared with healthy individuals and patients with non–alcohol-related cirrhosis, alcoholic cirrhosis patients had increased systemic exposure and immune response to mycobiota. Moreover, the levels of extraintestinal exposure and immune response correlated with mortality. There were concomitant decreases in Epicoccum, unclassified fungi, Galactomyces, and Debaryomyces. The authors concluded that alcohol-associated fungal dysbiosis in humans is

characterized by a large increase in Candida, which is susceptible to amphotericin B. The effects of this drug should be tested in patients with alcohol-related liver disease, who are in urgent need of new therapeutics. Manipulation of the intestinal mycobiome might be an effective strategy for attenuation of alcohol-related liver disease. Bernd Schnabl, MD, an associate professor of gastroenterology and senior author of the study, said, “Not only is this the first study to associate fungi and liver disease, we might be able to slow the progression of alcoholic liver disease by manipulating the balance of fungal species living in a patient’s intestine.” The study was published on May 22, 2017, in the Journal of Clinical Investigation. Image: A new study determined intestinal fungi may contribute to the development of alcoholic liver disease (Photo courtesy of Shutterstock).

Elevated Blood Platelet Count Predicts Cancer he most common route to cancer diagnosis follows the development of symptoms, and definitive diagnosis by biopsy and access to specialist care often rely on a primary care physician to recognize the possibility of cancer. It has been discovered that having

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a high blood platelet count is a strong predictor of cancer and should be urgently investigated to save lives. Around 2% of people over the age of 40, which is up to half a million people in the UK, have a raised blood platelet count, also known as thrombocytosis.

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Scientists at the Medical School at University of Exeter (Exeter, UK; www.exeter.ac.uk) examined the incidence of cancer in a cohort of patients with thrombocytosis, to determine how clinically useful this risk marker could be in predicting an underlying malignancy. The 1-year incidence of cancer was compared between two cohorts: 40,000 patients aged equal to or more than 40 years with a platelet count of greater than 400 × 109/L (thrombocytosis) and 10,000 matched patients with a normal platelet count. In the study of study of 40,000 patient records the team found that more than 11% of men and 6% of women over the age of 40 with thrombocytosis went on to be diagnosed with cancer within a year. This rose to 18% of men and 10% of women if a second elevated platelet count was recorded within six months. They calculated that if only a conservative estimate of 5% of patients with cancer have thrombocytosis before a cancer diagnosis, one third of them have the potential to have their diagnosis expedited by at least three months by the identifica-

tion of this risk marker, equating to 5,500 earlier diagnoses annually. Willie Hamilton, MD, a professor of primary care diagnostics and lead investigator, said, “The UK lags well behind other developed countries on early cancer diagnosis. In 2014, 163,000 people died of cancer in this country. Our findings on thrombocytosis show a strong association with cancer, particularly in men, far stronger than that of a breast lump for breast cancer in women. It is now crucial that we roll out cancer investigation of thrombocytosis. It could save hundreds of lives each year.” The study was published on May 22, 2017, in the British Journal of General Practice. V

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New Technique Generates High-Res Images with Conventional Microscopes esearchers have developed a technique for molecule with a fluorescent probe, take an image, obtaining high-resolution images of tissue and then wash the probe away. This can then be resamples with confocal microscopes, at a peated many times, each time using the same colors fraction of the cost of other techniques that offer to label different molecules. similar resolution. This level of resolution allowed The researchers hope to achieve a third round of them to see, for example, proteins that cluster toexpansion, which could, in principle, enable resolugether in complex patterns at brain synapses. tion of about 5 nanometers. However, the resoluThe new technique, developed by researchers of tion is also limited by the size of the labeling antiMassachusetts Institute of Technology (Cambridge, bodies (~10-20 nanometers), which would need to MA, USA; http://web.mit.edu), relies on expandbe overcome. The study, by Chang JB et al, was ing the tissue before imaging. The team previously published in the April 17, 2017, issue of Nature showed it was possible to expand tissue volumes Methods. 100-fold, resulting in an image resolution of about 60 nanometers. This was “useful Visit us at for many scientific questions but couldn’t come close the performance of the highMEDICA est-resolution imaging methods,” said the 2017 study’s senior author Ed Boyden, associHall 3-A74 ate professor at MIT. This approach could also be used to image many other phenomena. Resolution of ~25 nanometers is similar to that achieved by high-resolution techniques such as stochastic optical reconstruction microscopy (STORM). However, expansion microscopy is much cheaper and simpler to perform because no specialized equipment or chemicals are required. The method is also much faster and thus compatible with large-scale, 3D imaging. The resolution does not yet match that of scanning electron microscopy (about 5 nanometers) or transmission electron microscopy (about 1 nanometer). However, electron microscopes are very expensive and not widely available, and with those microscopes it is difficult to label specific proteins. To expand tissue samples, the researchers embed them in an expandable gel made of the very absorbent material polyacrylate. Before the gel is formed, they use antibodies to label the cell proteins they want to image. These antibodies bear DNA “barcodes”, attached to cross-linking molecules that bind the gel polymers. The researchers then break down the proteins that normally hold the tissue together, allowing the DNA barcodes to expand away from each other as the gel swells. These enlarged samples can then be labeled with fluorescent probes that bind the DNA barcodes, and imaged with a confocal microscope, whose resolution is usually limited to hundreds of nanometers. “My hope is that we can, in the coming years, really start to map out the organization of these scaffolding and signaling proteins at the synapse,” said Prof. Boyden. Combining iterative expansion microscopy with a new tool called temporal multiplexing should help to achieve that, he added. Currently, only a limited number of colored probes can be used to image different molecules in a tissue sample. With temporal multiplexing, researchers can label one

Image: High-resolution imaging with conventional confocal microscopes: by expanding brain tissue twice, researchers were able to obtain high-resolution images of neurons in the hippocampus (Photo courtesy of MIT).

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Novel Device Reduces Blood Draw Contamination novel device can significantly reduce contamination of blood cultures, potentially reducing risky overtreatment and unnecessary use of antibiotics for many patients. This approach could also substantially reduce healthcare costs. Thousands of patients get their blood drawn every day for blood cultures in order to diagnose serious infections such as sepsis, which can be a deadly condition. A small but significant percentage of the blood cultures are contaminated, due in part to skin fragments containing bacteria that are dislodged during a blood draw. Scientists at the University of Nebraska Medical Center (Omaha, NE, USA; www.unmc.edu) conducted a prospective, controlled study with 904 patients and 1,808 blood cultures and compared the standard procedure to an initial specimen diversion device (ISDD) to determine whether blood culture contamination was reduced. The sterile blood collection system diverts and sequesters the first 1.5 to 2 mL of blood, which often carries contaminating

skin cells and microbes and this part of the blood is discarded. The team used the SteriPath initial specimen diversion device (ISDD, Magnolia Medical Devices, Seattle, WA, USA; www.magnolia-medical. com) and were able to decrease the false positive rate significantly through use of this device, from 1.78% down to 0.2%, which represents an 88% reduction. Contamination rates routinely range from 0.6% to 6% in health care institutions the USA. Costs associated with blood culture contamination per patient case ranged from USD 1,000 in 1998 to USD 8,700 in 2009. A more recent study in the USA observed excess charges of USD 8,720 per contamination event. Mark Rupp, MD, professor and lead author of the study, said, â&#x20AC;&#x153;A lot of people think this is a minor problem. However, contaminated blood cultures are a big deal. Physicians can be led astray and patients may be harmed by additional tests and un-

necessary antimicrobial therapy. What is important about this device is it can greatly limit the blood culture from being contaminated, so physicians are rarely fooled by false-positive results. It gives clinicians confidence that results are accurate.â&#x20AC;? The study was published on in the journal Clinical Infectious Diseases. Image: The SteriPath initial specimen diversion device reduces blood culture contamination (Photo courtesy of Magnolia Medical Devices).

Enzyme Deficiency Diagnosed with Rapid Tests lucose-6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of the pentose phosphate pathway and is closely associated with the hemolytic disorders among patients receiving anti-malarial drugs, such as primaquine. G6PD deficiency (G6PDd) is an impending factor for radical treatment of malaria, which affects the clearance of gametocytes from the blood and subsequent delay in the achievement of malaria elimination. About 8% of the people who are exposed to malaria have an inherited disorder that impairs G6PD, leaving them vulnerable to develop clinical consequences. Scientists at the World Health Organization regional office (Katmandu, Nepal; www.searo.who.int) conducted a cross-sectional prevalence study during

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from April to December 2013. Volunteers who were between five and 60 years, without known chronic diseases, and consenting/assenting for voluntary participation were enrolled for the study. Equal proportions of equal to or more than 200 participants from each of six malaria endemic district were enrolled considering ethnic and demographic representation of the population groups. Whole blood specimens from the1,341 enrolled volunteer participants were collected and tested immediately using Care-Start G6PD RDT (Access Bio, Inc., Somerset, NJ, USA; http://accessbio.net) and BinaxNOW G6PD (Alere, Waltham, MA, USA; www.alere.com) test kits in the field settings. The Care-Start RDT format is a qualitative enzyme chromatographic test, based on the reduction of color-

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less nitroblue tetrazolium dye to dark colored formazan. Samples with normal G6PD activity produced a distinct purple color in the result window, while no color change was observed for samples with G6PDd subjects. The BinaxNOWG6PD test is a qualitative enzyme chromatographic test (ECT) for detecting G6PD activity. The test device contains a lateral flow test strip comprised of a white sample pad and a reaction pad. When no change in the red color of the sample front was observed at the test read time, the sample was presumed to be deficient in G6PD enzyme activity. The team reported that out of total 1,341 blood specimens collected from six districts, the overall prevalence of G6PDd was 97/1,341 (7.23%) on BinaxNOW and 81/1,341 (6.0%) on the Care Start test. A higher prevalence was observed in male than females; male 10.2% versus female 5.4% with BinaxNOW. G6PDd was higher in two ethnic groups and two districts. The authors noted that Care-Start kits were designed for one step, with all available peripherals, while BinaxNOW had multiple steps, which included the additional step of sample mixing with buffer in the supplied tube. The result window of Care-Start test was clearly visible with distinct purple color appearance for normal cases and transparent (white background) in G6PD deficient cases. The authors concluded that the prevalence of G6PD deficiency in Nepalese population varies in ethnic groups, strongly recommending need of G6PDd testing before the start of primaquine for each confirmed Plasmodium vivax case. Knowing the G6PDd status gives leverage to use 14 days primaquine in G6PD normal patients, while weekly primaquine under close clinical monitoring/medical supervision with ready access to blood transfusion services in G6PD deficient cases. The study was published on May 23, 2017, in the Malaria Journal. LabMedica International November/2017

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Circulating TB Antigens Allow Rapid Diagnosis of Active Disease rapid and quantitative blood-based assay with high sensitivity and specificity for active Mycobacterium tuberculosis (Mtb) infections has been developed that can be used to monitor responses to anti-mycobacterial therapy. Tuberculosis (TB) is a major global health threat, resulting in an urgent unmet need for a rapid, non–sputum-based quantitative test to detect active Mtb infections in clinically diverse populations and quickly assess Mtb treatment responses for emerging drug-resistant strains. A team of scientists led by those at the Arizona State University’s Biodesign Institute (Tempe. AZ; USA; www.asunow. asu.edu) have identified Mtb-specific peptide fragments and developed a method called NanoDisk-MS, to rapidly quantify their serum concentrations, using antibody-labeled and energy-focusing porous discoidal silicon nanoparticles (nanodisks) and high-throughput mass spectrometry (MS) to enhance sensitivity and specificity. The scientists found that NanoDiskMS diagnosed active Mtb cases with high sensitivity and specificity in a casecontrol study with cohorts reflecting the complexity of clinical practice. It does so by accurately detecting minute blood levels of two proteins: 10-kDa culture filtrate protein (CFP-10) and 6-kDa early secretory antigenic target (ESAT-6) that Mtb release only during active infections. Similar robust sensitivities were obtained for cases of culture-positive pulmonary TB (PTB; 91.3%) and extrapulmonary TB (EPTB; 92.3%), and the sensitivities obtained for culture-negative PTB (82.4%) and EPTB (75.0%) in human immunodeficiency virus (HIV)positive patients significantly outperformed those reported for other available assays. NanoDisk-MS also exhibited high specificity (87.1% to 100%) in both healthy and high-risk groups. Absolute quantification of serum Mtb antigen concentration was informative in assessing responses to anti-mycobacterial treatment. Tony Ye Hu, PhD, an associate professor and lead author of the study said, “In the current frontlines of TB testing, coughed-up sputum, blood culture tests, invasive lung and lymph biopsies, or spinal taps are the only way to diagnose TB. The results can give false negatives, and these tests are further constrained because they can take days to weeks to get the results. We are particularly excited about the ability of our high-throughput assay to provide rapid quantitative results that can be used to monitor treatment effects, which will give physicians the abil-

LabMedica International November/2017

ity to better treat worldwide TB infections.” The study was published on March 27, 2017, in the journal Proceedings of the National Academy of Sciences. Image: The newly developed NanoDiskMS assay, could significantly improve TB diagnosis and management because it is the first test that can measure the severity of active TB infections (Photo courtesy of Jason Drees, Biodesign Institute at Arizona State University).

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Organ Cells Derived from Stem Cells May Predict Drug Sensitivity Testing the drug pazopanib on liver-like cells derived from patient-specific stem cells, researchers have shown first proof-of-concept for a new approach that could lead to development of personalized drug-toxicity assays for patients. Different people react differently to the same drug, yet it is difficult to predict the side effects for an individual in advance. Now, researchers from Singapore’s Institute of Bioengineering and Nanotechnology (IBN) of A*STAR and the National Cancer Centre Singapore (NCCS; www.ibn.a-star.edu.sg) have developed an approach to screen for severe side-effects by first testing a drug on stem cells made from the patient’s blood. “Adverse side-effects from drugs are a major clinical concern, which could and should be preventable. Knowing whether an individual is susceptible to a particular medicine will improve health-

care and treatment outcome,” said Prof. Jackie Y. Ying, executive director at IBN. In the study, the researchers used induced pluripotent stem cells (iPSCs) to create liver-like cells from the blood of 5 kidney cancer patients. The liver cells were then exposed to the cancer drug pazopanib, which can cause liver damage. Of the 5 patients: 3 were known to react badly to pazopanib, while the other 2 had no side effects. The results showed that each patient’s iPSC-derived liver cells exhibited the same sensitivity to the drug when compared with their post-treatment data from liver biopsies. Further, using these stem cells, the researchers were able to analyze how the drug caused liver damage, which was previously unknown. The project was led by Dr. Min-Han Tan and Prof. Hanry Yu of IBN. Researchers at NCCS recruit-

ed the kidney cancer patients and provided clinical data and analysis. “Our hypothesis was that liver cells made from the individual’s blood might show similar sensitivity or resistance to pazopanib. This study is the first proof-of-concept that our approach can predict drug-induced liver damage for an individual. Importantly, we were able to figure out how the drug works from the way they react to the liver cells,” said Dr Min-Han Tan. Prof Hanry Yu added, “Currently, new drugs are tested for toxicity using generic liver cells, which cannot model patient-specific reaction. By personalizing liver cells from the blood of individual patients, we can help doctors to prescribe safer and more effective therapies.” “We are very excited that this study demonstrates an approach that could transform how drug toxicities are evaluated. It also sheds light on the mechanism of a particular side effect of pazopanib, which may lead to ways to overcome it. We are already planning formal clinical trials on this,” said Dr Ravindran Kanesvaran, consultant at NCCS. This validation in patients suggests that it would be possible to screen personalized stem cells comprising a range of liver, lung, kidney, and heart cells to predict whether the patient would get side-effects from taking a particular drug. The research team will conduct further studies on drugs that affect other types of organs, and hope to work with industry partners to make this technology widely available. The study, by Choudhury Y et al, was published January 25, 2017, in the journal Scientific Reports. Image: A transmission electron micrograph (TEM) of a mesenchymal stem cell displaying typical ultrastructural characteristics (Photo courtesy of Robert M. Hunt).

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Molecular Testing Evaluated For Thyroid Nodules hyroid nodules are a common clinical concern and increasing use of diagnostic imaging likely explains a large part of the increased incidence of thyroid nodules and the subsequent diagnosis of thyroid cancer that has been observed during the last three decades. Prior to molecular assays, most patients with indeterminate cytology were referred for a diagnostic lobectomy or total thyroidectomy, based on other risk factors for cancer or the presence of contralateral nodularity, immediately or after another biopsy demonstrating persistently indeterminate cytology results. However, most of the nodules that fall into an indeterminate category are benign on resection. Pathologists at the Duke University Medical Center (Durham, NC, USA; www. dukehealth.org) performed a retrospective analysis of cytology and all in-house thyroid fine needle aspirations (FNAs) sent for molecular testing from September 2013 to March 2015. Each FNA was performed by palpation or with ultrasound guidance by board-certified radiologists, endocrinologists, surgeons, and cytopathologists. Immediate assessments for adequacy were performed with each biopsy. The study cohort comprised 115 thyroid nodules from 110 patients, including 86 females (78%) and 24 males (22%). The ages of the patients ranged from 16 to 87 years, with a mean age of 56.5 years at the time of FNA. The scientistsâ&#x20AC;&#x2122; objective was to report their experience at a tertiary thyroid referral center with the Afirma Gene Expression Classifier (Veracyte, San Francisco, CA, USA; www.veracyte.com) in repeat fine-needle aspirations of thyroid nodules with a previous indeterminate cytological result. The surgical pathology results were correlated with the FNA and Afirma GEC findings by matching the biopsied nodule to the surgically resected nodule, which served as the gold standard. The fine-needle aspiration diagnostic categories for the115 nodules were 100 (87%) Bethesda III, 10 (9%) Bethesda IV, 3 (2%) Bethesda II, 1 (1%) Bethesda V, and 1 (1%) Bethesda I. Afirma results for 52 (45%) of the nodules were benign, 57 (50%) were suspicious and 6 (5%) specimens yielded no result because of low messenger RNA content. Three of the benign nodules (6%) were treated surgically, and all were benign on final surgical pathology. Forty-six (81%) of the suspicious nodules were treated surgically; final surgical pathology revealed 30 (65%) were benign and 16 (35%) malignant, yielding a positive predictive value of 35%. The authors concluded that 50% of the indeterminate nodules were classified as suspicious by Afirma, with a 35% rate of malignancy in these nodules at surgical resection, in comparison with a historical rate

LabMedica International November/2017

of malignancy at their institution of 11% for Bethesda III nodules and 23% for Bethesda IV. Their experience at a tertiary referral center was that when reserved for use in repeat-indeterminate nodules, the test has similar performance to that published at initial biopsy, thus avoiding the need to collect large numbers of additional passes for Afirma GEC testing at first biopsy, while also keeping the benefit of potentially reducing the number of operations performed for benign nodules. The study was published in the July 2017 issue of the journal Archives of Pathology & Laboratory Medicine.

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Image: The Afirma Gene Expression Classifier kit for thyroid FNA Analysis reduces thyroid cancer surgery treatment costs (Photo courtesy of Veracyte).

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POC Assay Developed for Reliable Blood Grouping apid and accurate blood grouping plays a critical role in multiple scientific disciplines, particularly in the biological and medical sciences and especially for pregnancy, blood transfusion, and bone marrow transplantation. A fast, accurate, and versatile paper-based blood test has been developed that could be performed without the need for specialized equipment providing a more cost-effective strategy for blood grouping. The blood typing assay is based on the color change that occurs when a common pH indicator dye reacts with blood. Medical scientists at the Third Military Medical University, Chongqing, China (www.dapinghospital. com) assayed 3,550 venous and finger prick blood samples on a paper-based test using bromocresol green. Blood groups were primarily identified by a diagnostic laboratory using the BioVue gel-card assay (Ortho Clinical Diagnostics Inc., Raritan, NJ, USA; www.orthoclinical.com). The paper-based assay used immobilized antibodies and bromocresol green dye for rapid and reliable blood grouping, where dye-assisted color changes corresponding to distinct blood components provide a visual readout. ABO antigens and five major Rhesus antigens could be detected within 30 seconds and simultane-

ous forward and reverse ABO blood grouping using small volumes (100 L) of whole blood was achieved within two minutes through on-chip plasma separation without centrifugation. A machine-learning method was developed to classify the spectral plots corresponding to dye-based color changes, which enabled reproducible automatic grouping. Using optimized operating parameters, the dye-assisted paper assay exhibited comparable accuracy and reproducibility to the classical gel-card assays in grouping 3,550 human blood samples. When translated to the assembly line and low-cost manufacturing, the proposed approach may be developed into a cost-effective and robust universal blood-grouping platform. The authors concluded that the assay not only provides a new strategy for blood grouping but can also be used in time- and resource-limited situations, such as war zones, in remote areas, and during emergencies. Characterized by an intensified and streamlined workflow capability, the proposed blood-grouping assay may be further developed into highly compact and fully automatic platforms that are highly efficient and economical, making

large-scale manufacturing possible. The study was published on March 15, 2017, in the journal Science Translational Medicine. Image: ABO grouping for A, B, AB, and O as indicated by the presence of teal color in the observation zone (red dashed box). Images of multiplexed assays testing (C) ABO/Rh and (D) rare blood systems (Photo courtesy of Third Military Medical University).

Urine Biomarkers May Identify Down Syndrome Patients with OSA panel of urine biomarkers has been identified that may allow clinicians to easily diagnose Down syndrome patients with obstructive sleep apnea (OSA). OSA occurs when an individual's airway is restricted or blocked during sleep by some physical feature. Affected individuals may briefly stop and then resume breathing. In addition to disturbing sleep, the reduction in oxygen supply can cause cardiovascular problems – including hypertension and arrhythmias – and metabolic issues. The effects of OSA are exacerbated in those with Down

syndrome because of their physical and cognitive differences. Currently polysomnography (PSG) is the method used to diagnose OSA. This is a comprehensive recording of the biophysiological changes that occur during sleep. The PSG monitors many body functions including brain (EEG), eye movements (EOG), muscle activity or skeletal muscle activation (EMG) and heart rhythm (ECG) during sleep. After the identification of the sleep disorder sleep apnea in the 1970s, the breathing functions respiratory airflow and respiratory effort indicators Visit us at MEDICA® 2017

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were added along with peripheral pulse oximetry. Since the PSG process can be disruptive and disturbing for Downs syndrome patients, investigators at Massachusetts General Hospital (Boston, USA; www.mgh.harvard.edu) compared urinary biomarkers in individuals with Down syndrome with and without OSA to those of age- and sexmatched neurotypical developing healthy controls (HC). They further investigated whether it was possible to predict OSA in individuals with Down syndrome using these biomarkers. To perform the study, the investigators collected urine samples were from 58 individuals with Down syndrome the night before or the morning after their scheduled overnight polysomnogram or both. Concentrations of 12 neurotransmitters were determined by enzyme-linked immunosorbent assay. Results revealed differences between the urinary biomarkers of participants with Down syndrome who did and did not have OSA – with a combination of four neurotransmitters most clearly distinguishing between the two groups. In addition, there were significant differences between the biomarker signatures of all participants with Down syndrome and those of the control participants, regardless of the presence or absence of OSA. Dr. Skotko stressed that the results of this study required confirmation in larger groups before biomarker screening can be used to screen for the presence of OSA. "Our findings are not yet ready for prime time," said Dr. Skotko. "Before they can be used in clinical practice, we will need to validate these findings in a new group of patients with Down syndrome, which we are working on right now." The study was published in the March 7, 2017, online edition of the journal Sleep Medicine. LabMedica International November/2017

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Mass Spectrometry Detection for the Masses esearchers are developing a plasmabased technology to enable generalized use of mass spectrometry (MS) with new instruments that can analyze a much broader range of molecular species than current technology allows. Current MS instruments are bulky, expensive, and typically specialize in one class of chemicals, discouraging widespread use outside of a specialized lab setting. Better technology is needed to make more flexible instruments. Research being conducted at Rensselaer Polytechnic Institute (Troy, NY, USA; www.rpi.edu) uses an atmospheric-pressure glow discharge plasma – a partially ionized gas that can be made stable at room temperature and pressure – to probe samples for elemental and molecular species, and could lead to user-friendly MS analyses with broad capabilities. “Ideally we want one system that can detect everything, and we want to be able to take that system into the field to test materials on site,” said Prof. Jacob Shelley of Rensselaer Polytechnic, “We’re trying to make a more flexible instrument that will allow us to detect many things simultaneously.” The hitch is that current instruments can only analyze molecules that are in gas state and ionized, which means that most samples must first be processed. Current MS relies on a variety of timeconsuming processing methods that separate and ionize molecules prior to analysis. And depending on the method, samples (e.g. tissues, pharmaceuticals, or foods) may be destroyed during processing. The biggest challenge to developing a generalized processing method is the chemistry needed to ionize the molecule. Most methods rely on specific chemistries that favor ionization of one class of molecules over another. Prof. Shelley team is developing a method that takes advantage of the unusual properties and chemistries of plasmas, which are rich in free-moving ions and electrons, and therefore highly interactive. Although the most commonly known plasmas are extremely hot (at nearly 10,000 degrees Kelvin, some plasmas rival the sun’s temperature), the team is working with more recently developed glow discharge plasmas that are stable at room temperature and atmospheric pressure. In his lab, Prof. Shelley demonstrates an experimental instrument so benign it can test samples ionized from a fingertip, and so versatile it can detect molecular species from small amounts of metals to large labile biomolecules like peptides and proteins. In developing the technology, the team has used the instrument to detect counterfeit honey, to quantify harmful toxins in freshwater algal blooms, and to screen the raw materials

LabMedica International November/2017

used in nutritional supplements. “The plasma is useful as an ionization source because it makes a diverse range of chemistries available,” said Prof. Shelley, “It may make it possible to ionize a broad class of molecules, which could lead to more generalized instruments.” This research is enabled by the New Polytechnic vision, a transformative emerging paradigm for higher education, which recognizes that even the most talented person working alone cannot adequately address global challenges and opportunities. It helps Rensselaer serve as a crossroads for collaborations to address some of the world’s most pressing technological challenges.

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Image: Research being conducted uses an atmospheric-pressure glow discharge plasma to probe samples for elemental and molecular species, and could lead to user-friendly MS analyses with broad capabilities (Photo courtesy of the Rensselaer Polytechnic Institute).

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The Granulated Culture Media combines the high throughput technology of granulation and production of dehydrated culture media. The effective media has similiar qualitative attributes to that of powdered dehydrated culture media with several added benefits in physical parameters.

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The immunoblot strip assay can process up to 44 samples per run from sample processing to the final result. Designed to be more convenient, stable and user-friendly, it can be customized for various applications of strip-based assays such as allergy, autoimmune and infectious diseases.

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Lab-on-a-Chip Nanotechnology Tracks Multiple Markers esearch engineers have invented a technology for nanoelectronic barcoding of microparticles that could be used in wearable or hand-held devices to analyze body fluids, such as sweat or blood, for various biomarkers or pollutants simultaneously. The team, led by Mehdi Javanmard, assistant professor at Rutgers University-New Brunswick (New Brunswick, NJ; www.rutgers.edu), invented the biosensor technology to better monitor health and exposure to pathogenic microorganisms as well as to pollutants. “This is really important in the context of personalized medicine or personalized health monitoring,” said Prof. Javanmard, “Our technology enables true labs on chips. We’re talking about platforms the size of a USB flash drive or something that can be integrated [into a fitness watch].”

In recent decades, research on biomarkers has revealed the complex nature of the molecular mechanisms behind human disease. That has heightened the importance of testing bodily fluids for numerous biomarkers simultaneously, the authors said. “One biomarker is often insufficient to pinpoint a specific disease because of the heterogeneous nature of various types of diseases,” said Prof. Javanmard, “To get an accurate diagnosis and accurate management of various health conditions, you need to be able to analyze multiple biomarkers at the same time.” Bulky optical instruments are the state-of-the-art technology for detecting and measuring biomarkers, but are too large to add to a portable device. Electronic detection of microparticles allows for ultracompact instruments needed for wearable devices.

The researchers’ technique for barcoding particles to identify them is, for the first time, fully electronic, allowing biosensors to be shrunk to the size of a wearable band or a microchip, the authors said. The technology was greater than 95% accurate in identifying tested biomarkers, and fine-tuning is underway to make it 100% accurate, said Prof. Javanmard. The team is also working on portable detection of microrganisms, including pathogenic bacteria and viruses. “Imagine a small tool that could analyze a swab sample of what’s on the doorknob of a bathroom or front door and detect influenza or a wide array of other virus particles,” he said, “Imagine ordering a salad at a restaurant and testing it for E. coli or Salmonella bacteria.” This form of the tool could be commercially available within about two years, while health monitoring and diagnostic tools could be available within about five years, he said. The study, by Xie P et al, was published April 28, 2017, in the journal Lab on a Chip. Image: An artist’s rendition of microparticles flowing through a channel and passing through electric fields, where they are detected electronically and barcode-scanned (Photo courtesy of Ella Marushchenko and Alexander Tokarev, Ella Maru Studios).

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LAMP-based Assay Launched for Detection of Malaria new molecular diagnostic kit for the detection of the malaria parasite has been described as being up to 80,000 times more sensitive at detecting the malaria parasite than current testing options. The illumigene Malaria test was developed by Meridian Bioscience, Inc. (Cincinnati, OH, USA; www.meridianbioscience.com). It uses loop mediated isothermal amplification (LAMP) technology, and is the tenth item on Meridian's list of illumigene assays for the diagnosis of infectious diseases, which includes C. difficile, whooping cough, Group A and B Streptococcus, mycoplasma, and herpes simplex virus. LAMP is a single tube technique for the amplification of DNA. In contrast to the polymerase chain reaction (PCR) technology in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler. In LAMP, the target sequence is amplified at a constant temperature of 60–65 degrees Celsius using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. Typically, four different primers are used to identify six distinct regions on the target gene, which adds highly to the specificity. An additional pair of "loop primers" can further accelerate the reaction. Due to the specific nature of the action of these primers, the amount of DNA produced in LAMP is considerably higher than PCR based amplification. Detection of amplification product can be determined via photometry for turbidity caused by an increasing quantity of magnesium pyrophosphate precipitate in solution as a byproduct of amplification. illumigene Malaria is user-friendly, does not require special training or capital investment, yields results in under one hour, and the testing materials can be stored at room temperature. “This is a major step forward in the fight to bring better care to those infected with malaria and to stop its spread,” said John A. Kraeutler, CEO of Meridian Bioscience. “Malaria is a devastating disease and we are proud to work with all the talented and dedicated individuals around the world in the fight to eliminate it.”

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Image: The illumigene Malaria is a diagnostic test for malaria that uses Loop-Mediated Isothermal Amplification (LAMP) technology to amplify DNA and detect the presence of the malaria parasite (Photo courtesy of Meridian Bioscience).

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Cancer DNA Biomarkers Detected by Lab-On-A-Chip Technique iniaturized lab-on-chip approaches are prime candidates for developing viable diagnostic tests and instruments because they are small, need only limited test volumes, and can be cost-effective. Cancer is the second leading cause of death in the USA, making early, reliable diagnosis and treatment a priority for doctors. Genomic biomarkers offer great potential for diagnostics and new forms of treatment, such as immunotherapy. A team of scientists and engineers from the University of California, Santa Cruz (CA, USA; www. ucsc.edu) and Brigham Young University (Provo, UT, USA; www.byu.edu) developed an optofluidic analysis system that processes biomolecular samples starting from whole blood and then analyzes and identifies multiple targets on a silicon-based molecular detection platform. Instead of transferring relatively large (micro- to milliliters) samples between test tubes or using bulky analytical equipment, samples and reagents are handled on chip-scale devices with fluidic microchannels. This requires much smaller test volumes, and multiple functions can be integrated on a single device, improving speed, reliability and portability of these laboratory processes. The scientists demonstrated blood filtration, sample extraction, target enrichment, and fluorescent labeling using programmable microfluidic circuits. They detected and identified multiple targets using a spectral multiplexing technique based on

Image: A microfluidic chip for sample preparation and an optofluidic chip for optical detection of individual molecules (Photo courtesy of Joshua W. Parks).

wavelength-dependent multi-spot excitation on an antiresonant reflecting optical waveguide chip. Specifically, they extracted two types of melanoma biomarkers, mutated cell-free nucleic acids, BRAFV600E and NRAS, from whole blood. They detected and identified these two targets simultaneously using the spectral multiplexing approach with up to a 96% success rate. Holger Schmidt, PhD, a professor of electrical engineering and senior author of the study, said,

“Our approach uses optofluidic chips where both fluid processing and optical sensing are done on a chip, allowing for further miniaturization and performance enhancements of the chip system. In the near term, we hope to build new diagnostic instruments for molecular diagnostics with applications in oncology and infectious disease detection, both viruses and (drug-resistant) bacteria.” The study was published in the December 2016 issue of the journal Biomicrofluidics.

Kidney Biomarkers Investigated to Track Lupus

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upus is a chronic autoimmune disease in which the body’s immune system becomes hyperactive and attacks normal, healthy tissue. This results in symptoms such as inflammation, swelling, and damage to joints, skin, kidneys, blood, the heart, and lungs. The most useful tests to aid a diagnosis identify certain autoantibodies often present in the blood of people with lupus. For example, the antinuclear antibody (ANA) test is commonly used to look for autoantibodies that react against components of the nucleus of the body’s cells. Scientists at the Centers for Disease Control and Prevention (Atlanta, GA, USA; www.cdc.gov) working with their colleagues at the University of Michigan School of Public Health (Ann Arbor, MI, USA; www.med. umich.edu) measured the urinary epidermal growth factor in patients with lupus. They had previously showed this protein to be a promising, noninvasive biomarker of kidney disease progression. Their team found a decrease in urinary epidermal growth factor protein was an indication of diminishing kidney function in people

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with chronic kidney disease. In the latest study, they extended these findings to show that levels of epidermal growth factor in the urine of 394 lupus patients provided improved ability over standard markers, such as protein-to-creatinine ratio, to distinguish those with kidney involvement from those without. Emily Somers, PhD, ScM, an associate professor of internal medicine and a senior author of the study said, “Lupus patients have a high risk of kidney involvement, which can lead to endstage renal disease requiring dialysis or transplant. In addition, there is a great need for biomarkers to detect early kidney involvement and to monitor progression. Validating this biomarker as a way to monitor lupus severity and progression is an exciting step in piecing together the complexity of lupus. Ultimately we aim to enhance our ability to identify and treat those affected sooner, before the disease has caused even more complications.” The study was presented at the American Society of Nephrology Kidney Week 2016 meeting held October 31 to November 5, 2016, in New Orleans, LA, USA. LabMedica International November/2017

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Genetic Markers Discovered for COPD hronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease characterized by long-term poor airflow and the main symptoms include shortness of breath and cough with sputum production and typically COPD worsens over time. Chronic obstructive pulmonary disease is the third leading cause of death in the USA, yet there are no effective medicines that improve mortality from the disease and while smoking remains the single most important risk factor for COPD, genetics also play an important role.

Biomarker Assay Evaluates Efficacy of Novel Breast Cancer Drug

A large team of scientists led by those at Brigham and Women’s Hospital (Boston, MA, USA; www.brighamandwomens.org) conducted a genome-wide association study of risk for chronic obstructive pulmonary disease (COPD) in a large, multi-ancestry cohort including 15,256 cases and 47,936 controls. This type of study allows investigators to look across a comprehensive set of genetic variants in different individuals to see if any variant is associated with disease and the most important findings from this study were replicated in a second cohort. In addition to identifying 13 new genetic regions associated with COPD, the scientists also discovered four genetic regions that were not previously associated with any lung function trait. Nine of the genetic regions have been identified as playing an important role in lung function. Two have previously shown an association with pulmonary fibrosis;

however, the specific forms of these genetic variants that increase risk for COPD decrease risk for pulmonary fibrosis. All analyses accounted for the effects of age, gender, and cigarette smoking on disease risk. They also identified genetic correlation between COPD and asthma. The findings highlight new loci associated with COPD, demonstrate the importance of specific loci associated with lung function to COPD, and identify potential regions of genetic overlap between COPD and other respiratory diseases. Michael Cho, MD, MPH, one of the senior authors of the study, said, “While it is extremely important that patients not smoke for many health reasons, including the prevention of COPD, we know that smoking cessation may not be enough to stave off the disease.” The study was published on February 6, 2017, in the journal Nature Genetics.

reast cancer is the most common form of cancer among women today, affecting approximately 362,000 individuals in the European Union (EU) and 233,000 in the USA each year. Around 1,600 new cases are diagnosed every day and 136,000 deaths occur annually (EU+USA). Palbociclib, a drug for the treatment of estrogen receptor (ER)-positive and HER2-negative breast cancer, was FDA-approved in February 2015. In the drug’s first year on the US market, more than 20,000 women were prescribed the medicine, whose sales are estimated to exceed USD 2 billion in 2016. In November 2016 palbociclib was approved in the EU. Scientists at Washington University School of Medicine (St Louis, MA, USA; https://medicine. wustl.edu) investigated 50 women with clinical stage II or III ER- positive, HER2 negative breast cancer, treated with anastrozole in combination with palbociclib prior to surgery. DiviTum (Biovica, Uppsala, Sweden; https://biovica.com) was used to measure levels of thymidine kinase (TK) activity, an enzyme closely linked to cell proliferation rate, in blood samples collected before and after treatment. DiviTum is a highly sensitive assay for measuring cell proliferation. Since one of the most fundamental characteristics of cancer is uncontrolled and increased cell growth, DiviTum enables valuable prediction capability and monitoring of compounds regulating cell proliferation and the cell cycle. The results demonstrated a highly significant correlation between the anti-proliferative effect of palbociclib and the reduction in TK levels measured by DiviTum post two weeks of adding palbociclib and at the time of surgery. The assay may thus serve as an early indicator of treatment response by cyclin-dependent kinase (CDK) 4/6 inhibitors like palbociclib. Dr. Cynthia Ma, MD, PhD, an Associate Professor of Medicine and the lead investigator on the study said, “Our study provides the first clinical evidence of a method, DiviTum, for palbociclib treatment effect in breast cancer. The results are very promising and support future studies of DiviTum to evaluate and identify patients for response to CDK 4/6 inhibitors.” The study was presented at the San Antonio Breast Cancer Symposium, held December 6-10, 2016, in San Antonio, TX, USA.

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LabMedica International

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Gene Drives Deadly Brain Cancer lioblastoma is the most common and aggressive brain cancer in adults and over 70% of patients with glioblastoma die within two years of diagnosis, though a gene has been identified that is overactive in a deadly form of brain cancer. A new study suggests that inhibiting that gene may improve the outlook for glioblastoma patients. Nicotinamide adenine dinucleotide (NAD+) plays a pivotal role in cancer cell metabolism, but how NAD+ impacts functional signaling events in glioblastoma is not well understood. A large team of scientists from Washington University School of Medicine (St. Louis, MO, USA; https://medicine.wustl.edu) provided clinical evidence that high expression of nicotinamide phosphoribosyltransferase (NAMPT), the ratelimiting step in NAD+ biosynthesis, in glioblastoma tumors is associated with poor overall survival in patients, and demonstrated NAMPT and NAD+ are required for the maintenance of patient-derived

glioblastoma stem-like cells (GSCs). High NAMPT expression in tumors correlates with decreased patient survival. Pharmacological and genetic inhibition of NAMPT decreased NAD+ levels and GSC self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs. Regulatory network analysis of RNA sequencing data using GSCs treated with NAMPT inhibitor identified transcription factor E2F2 as the center of a transcriptional hub in the NAD+-dependent network. The team demonstrated that E2F2 is required for GSC selfrenewal. Downstream, E2F2 drives the transcription of members of the inhibitor of differentiation (ID) helix–loop–helix gene family. Albert H. Kim, MD, PhD, an assistant professor of neurological surgery, and senior author of the study said, “If you target the NAD+ pathway, you can disrupt the ability of the cancer stem cells to self-renew, and you can also make them more sensitive to radiation treatment. In a patient, that could mean that if you

suppress the pathway, the same dose of radiation may be more effective at destroying the tumor.” The study was published on November 14, 2016, in the journal Proceedings of the National Academy of Sciences.

Image: A histopathology of cerebral glioblastoma showing marked cellularity, with marked hyperchromatism and pleomorphism. Note the prominent vascularity as well as the area of necrosis at the left with neoplastic cells palisading around it (Photo courtesy of KGH).

Evidence Shows Nosocomial Infections Linked to Hospital Sink Bacteria nvestigators working with a unique hospital sink laboratory demonstrated that noso comial infections, including those of multiple drug resistant organisms, are spread by bacterial colonies in the waste pipes that migrate up to the sink strainers where they are spread to the surface of the sink, faucets, and nearby counters. To better understand how patients become infected with bacteria in the hospital setting, investigators at the University of Virginia (Charlottesville, USA; www.virginia.edu) built a unique sink lab containing five identical sinks, modeled after the most common sinks in the University of Virginia’s hospital in Charlottesville. Escherichia coli bacteria that had been labeled with green fluorescent protein (GFP) were allowed to colonize the sinks. The investigators found that the bacteria established colonies in the elbows of the drainpipes. Subsequently,

a biofilm spread upward over seven days to reach the strainer. Running water into the sink then resulted in droplet dispersion to the surrounding areas (within about 30 inches) during faucet operation. “Our study demonstrates that bacterial spread from drainpipes to patients occurs via a staged mode of transmission,” said senior author Dr. Amy Mathers, associate professor of medicine and pathology at the University of Virginia. “We wanted to better understand how transmission occurs, so that the numbers of these infections could be reduced. This type of foundational research is needed to understand how these bacteria are transmitted so that we can develop and test potential intervention strategies that can be used to prevent further spread.” The study was published in the February 24, 2017, online edition of the journal Applied and Environmental Microbiology.

Image: New research shows nosocomial infections may be spread through bacteria in drains that migrates up into hospital sink drains (Photo courtesy of the University of Virginia). LINKXPRESS COM

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Cause of NMO Illuminated by High-Tech Microscope euromyelitis optica (NMO), also known as Devic’s disease or Devic’s syndrome, is a heterogeneous condition consisting of the simultaneous inflammation and demyelination of the optic nerve (optic neuritis) and the spinal cord (myelitis) and it can be monophasic or recurrent. Determining the spatial relationship of individual proteins in dense assemblies remains a challenge for super-resolution nanoscopy. A unique microscope capable of illuminating living cell structures in great detail has been used to find clues into how this destruc-

Device Simplifies Identification of Leishmania Hosts esearchers have developed a novel device for obtaining essentially painless, minimally invasive microbiopsies (MBs) that mimic sand fly bites and meals, providing a convenient sampling method for identifying asymptomatic, potentially infectious carriers of Leishmania donovani in endemic areas. Leishmania donovani species, parasites that cause visceral leishmaniasis (VL), are transmitted to humans by phlebotomine sandflies infected from a blood (+skin) meal taken upon biting an infected reservoir host animal – mainly humans (India and East Africa) or dogs (Latin America, Europe, Middle East, and North Africa). The majority of infected individuals remains asymptomatic, but serve as parasite reservoirs for transmission of the disease via sandflies. The gold standard for assessing infectiousness of human hosts to biting vector insects is xenodiagnosis – scoring infection rates among insectary-reared insects that had fed on humans suspected of being infected. An international research team from Australia, Ethiopia, Israel, and USA, led by Alon Warburg, professor at Hebrew University Hadassah Medical School (Jerusalem, Israel; https://medicine.ekmd. huji.ac.il), has developed and tested minimally invasive microbiopsy (MB) devices designed to penetrate the skin to a depth of 200μm and absorb blood as well as skin cell lysates, mimicking the “pool-feeding” mode by which the sandflies acquire blood meals. Using the devices, MBs taken from 137 of 262 volunteers in endemic VL foci in Ethiopia detected Leishmania parasites that could potentially be imbibed by sandflies upon feeding. Although the volume of MB samples was 10-fold smaller than fingerprick blood samples, Leishmania DNA detection rates from MBs were significantly higher, implying that skin, more often than blood, was the main source of parasites. Volunteers with histories of VL were almost as likely as healthy volunteers to test positive by MBs, suggesting the importance of asymptomatic people as reservoir hosts. These MB devices could enable to reliably and efficiently assess both L. donovani infection rates among large numbers of asymptomatic carriers and their infectiousness to blood-feeding sandflies. The study, by Kirstein OD et al, was published April 26, 2017, in the International Journal for Parasitology.

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tive autoimmune disease works, setting the stage for more discoveries in the future. Biophysicists at the University of Colorado Anschutz Medical Campus (Aurora, CO, USA; www. ucdenver.edu) used a custom Stimulated Emission Depletion (STED) microscope built at CU Anschutz; they were able to actually see clusters of antibodies atop astrocytes, the brain cell target of the autoimmune response in NMO. They imaged secondary antibody labeling of monoclonal aquaporin-4- immunoglobulin G (AQP4-IgGs) with differing epitope specificity bound to isolated tetramers (M1-AQP4) and large orthogonal arrays of AQP4 (M23-AQP4). Imaging secondary antibodies bound to M1-AQP4 allowed the team to infer the size of individual AQP4IgG binding events. This information was used to model the assembly of larger AQP4-IgG complexes on M23-AQP4 arrays. A scoring algorithm was gener-

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ated from these models to characterize the spatial arrangement of bound AQP4-IgG antibodies, yielding multiple epitope-specific patterns of bound antibodies on M23-AQP4 arrays. The authors concluded that their results delineate an approach to infer spatial relationships within protein arrays using stimulated emission depletion nanoscopy, offering insight into how information on single antibody fluorescence events can be used to extract information from dense protein assemblies under a biologic context. Jeffrey Bennett, MD, PhD, a professor and senior author of the study, said, “We discovered that we could see the natural clustering of antibodies on the surface of target cells. This could potentially correspond with their ability to damage the cells. We know that once antibody binds to the surface of the astrocyte, we are witnessing the first steps in the disease process.” The study was published on April 25, 2017, issue of the Biophysical Journal.

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Lab-On-A-Chip Designed to Minimize Preterm Births n the USA, a half million babies are born preterm; worldwide, the number is an estimated 15 million and complications associated with preterm birth are the number one cause of death for children under five, and those who live often face a range of health problems. There presently are no known current biomarker-based diagnostics for preterm births, and doctors typically only pay attention to women who have other clear risk factors. However with the help from a palm-sized plastic rectangle doctors are hoping to minimize the problem of premature deliveries. The chip is designed to predict, with up to 90% accuracy, a woman's risk for a future preterm birth. Scientists at Brigham Young University (Provo, UT, USA; https://byu. edu) have developed a microfluidic device that uses pH-mediated solid phase extraction (SPE) for the enrichment and elution of preterm birth (PTB) biomarkers. Furthermore, this

SPE module was integrated with microchip electrophoresis for combined enrichment and separation of multiple analytes, including a PTB peptide biomarker (P1). The team used a reversed-phase octyl methacrylate monolith that was polymerized as the SPE medium in polyethylene glycol diacrylate modified cyclic olefin copolymer microfluidic channels. Eluent for pH-mediated SPE of PTB biomarkers on the monolith was optimized using different pH values and ionic concentrations. The scientists obtained a nearly 50-fold enrichment that was observed in single channel SPE devices for a low nanomolar solution of P1, with great elution time reproducibility. The monolith binding capacity was determined to be 400 pg (0.2 pmol). A mixture of a model peptide (FA) and a PTB biomarker (P1) was extracted, eluted, injected, and then separated by microchip electrophoresis in our integrated device with approximately 15-fold enrichment. Adam T. Woolley, PhD, a chem-

istry professor and senior author of the study, said, “Among other benefits, the device is cheap, small and fast: once fully developed, it will help make detecting biomarkers a simple, automated task. Some peg the annual costs associated with preterm birth just in the USA at close to USD 30 billion, so one clear perk of such a screening tool, is economic. More significantly, there are a lot of preterm babies who don't survive: if we could

get them to survive and thrive, it would be a huge gain to society.” The study was published on March 8, 2017, in the journal Electrophoresis. Image: Scientists loading the integrated electrokinetically driven microfluidic device with pH-mediated solid-phase extraction coupled to microchip electrophoresis for preterm birth biomarkers (Photo courtesy of Nate Edwards, Brigham Young University).

CTC Protein Expression Uses Microfluidic Western Blotting irculating tumor cells (CTCs) are rare tumor cells found in the circulatory system of certain cancer patients and the clinical and functional significance of CTCs is still under investigation. Circulating tumor cells have been isolated from the blood of breast cancer patients and, then microscale physics has been used to design a precision test for protein biomarkers, which are indicators of cancer. Scientists at the University of California (Berkeley, CA, USA; www. berkeley.edu) recruited 12 patients with advanced breast cancer and blood was drawn and processed with in five hours after collection. White blood cells were prepared by lysing the red blood cells. Single-cell resolution western blots (scWB) were used to measure a panel of proteins in single CTCs isolated from patients with primary estrogen receptor-positive (ER+) breast cancer. A commercially available microfluidic tool (Vortex Biosciences, Menlo Park, CA, USA; www.vortexbiosciences.com) was used for label-free isolation of circulating cancer cells in both the cell line spiking and cancer patient blood experiments. Flow cytometry analysis

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was performed and cells were analyzed on a Guava flow cytometer (MilliporeSigma, Billerica, MA, USA; www.merckmillipore.com). The scientists found that the precision handling and analysis revealed a capacity to assay sparingly available patient-derived CTCs, a biophysical CTC phenotype more lysis-resistant than breast cancer cell lines. A capacity to report protein expression was demonstrated on a per CTC basis and two statistically distinct glyceraldehyde 3phosphate dehydrogenase (GAPDH) subpopulations within the patient-derived CTCs. By sorting and probing the protein targets, the test is more selective than existing pathology tools. Amy E. Herr, PhD, a professor and senior author of the study said, “Microfluidic design was key in this study. We were able to integrate features needed for each measurement stage into one process. Systems integration allowed us to do every single measurement step very, very quickly while the biomarkers are still concentrated. If not performed exceptionally fast, the cell’s proteins diffuse away and become undetectable.” The study was published on March 23, 2017, in the journal Nature Communications. LabMedica International November/2017

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Prognostic Tool Stratifies Risk of Parathyroid Carcinoma esearchers have identified key predictors of parathyroid cancer (PTC) recurrence after surgery, and developed a 3-point scoring system to more reliably identify patients at the highest risk of recurrence. Consequently, the tool can also help determine optimal postoperative strategy. The best chance for cure of PTC is early diagnosis then surgical removal of all tumor cells. However, over 50% of patients develop a recurrence after the first surgical procedure, said study first author Angelica Silva-Figueroa, MD, oncological surgeon at RedSalud Avansalud Clinic, Chile, “What is needed is a prognostic staging system for PTC. We do not know which group of patients has an increased risk of relapse.” The study team, including fellows of American College of Surgeons (ACS; Chicago, IL, USA; www. facs.org), examined data on patients treated for PTC between 1980-2016 at the University of Texas MD (UTMD) Anderson Cancer Center (Houston, TX, USA). From a sample size of 68 patient records, 26 patients developed recurrent disease after a median follow up of 4.6 years. Rather than using the traditional parameter called disease-free survival (DFS), which evaluates effectiveness of a therapy over time, the investigators assessed recurrence-free survival (RFS). After the initial operation to remove the tumor, the RFS rates were 85% at 1 year, 67% at 2 years, and 51% at 10 years. “The data points used to determine RFS are quantitative and can predict disease recurrence in the first 2-3 years after disease resection,” said senior author Nancy D. Perrier, MD, FACS (fellow of ACS), UTMD Anderson Cancer Center, “This [approach] offers a means to stratify patients and consider more aggressive adjuvant treatment for those at higher risk of recurrence.” Patients with PTC typically have significantly elevated levels of calcium in their blood as well as other abnormal parathyroid hormone levels. In the multivariate analysis, the team identified 3 adverse characteristics as key prognostic indicators of PTC recurrence: serum calcium level greater than 15 mg/dL, age over 65, and invasion of the tumor into blood vessels. From there they developed a simple predictive tool by combining these three variables. Patients were stratified into 3 risk groups – low, moderate, and high – based on the number of adverse characteristics each one had, from 0 to 3. The study found that the 2-year RFS rate after parathyroidectomy was 93% in those with 0 adverse characteristics (low risk), 72% in those with 1 adverse characteristic (moderate risk), and 27% in those with 2 adverse characteristics (high risk). The study also showed that, although the risk of recurrence is greater within 2 years after the initial surgery, this risk continues to increase over the next 10 years and beyond in the moderate risk group. With this combination of measurable and available information, a patient’s risks can be assessed. For individuals at elevated risk, additional surgery or other adjuvant therapies could be used early on to

LabMedica International November/2017

control cancer recurrence. “We believe that this scoring system is the first step in personalized cancer care,” Dr. Silva-Figueroa said, “The system may help physicians predict the clinical progression of this disease, reliably aid immediate postoperative treatment decisions, and guide clinical monitoring for progression.” It is currently being validated at 4 centers across the USA. Once these prospective studies have been completed and results are published, the tool can be made available for public use. This approach “should be employed for other rare tumors where data are insufficient to generate prognostic stages,” said David Winchester, MD, FACS, medical director of Cancer Programs at ACS, “It’s a good model to pave the way for future studies.”

The study, by Silva-Figueroa AM et al, was published April 19, 2017, in the Journal of the American College of Surgeons. Image: Researchers have identified key predictors of PTC recurrence after surgery, and developed a system to identify patients at the highest risk of recurrence (Photo courtesy of HealthTap).

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PRODUCT NEWS HEMOGLOBIN ANALYZER

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COAGULATION ANALYZER

CENTRIFUGE

DAS

Rayto Life and Analytical Sciences

Drucker Diagnostics

The HB Analyzer Plus is designed for the fast and accurate determination of hemoglobin using the cyanmethemoglobin method. It immediately displays the direct reading, expressed in concentration (g/l, g/dl, mmol/l), and automatically performs the zero (autozero).

The RAC 050 random access, fully automated coagulation analyzer uses the clotting, chromogenic, and immunologic measuring methods. It can perform high-throughput routine assays and features a user-friendly touch screen interface, which is simple and easy to operate.

The 853VES can produce platelet poor plasma in three minutes, and offers versatility for sensitive urine and fertility samples. Its design ensures the highest quality separations, while digital controls make it easy to adjust settings and the internal memory allows for common settings to be stored.

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Mobile Device Rapidly Detects Zika and Related Viruses team of biologists and engineers have developed a smartphone-controlled, battery-operated, low-weight diagnostic technology that costs as little as USD 100 and can detect Zika, dengue, and chikungunya viruses within 30 minutes. The same platform can also be adapted to detect other human or animal pathogens. Accurate testing for these viruses currently requires a laboratory with expensive instruments, making local testing unrealistic for limited-resource clinics where the viruses are prevalent. The team, from Sandia National Laboratories (Livermore, CA, USA; www.sandia.gov), used smart-

phone technology as a key feature: “In addition to creating an app that serves as a simple interface to operate the device, we were able to adapt smartphone camera sensors to replace traditional laboratory sample analysis tools, allowing for unprecedented mobility,” said lead author Aashish Priye. The “laboratory in a box” device is based on the loop-mediated isothermal amplification (LAMP) diagnostic method, which eliminates PCR thermal cycling as well as the need to process the biological sample (e.g. blood or urine) before testing. LAMP copies viral DNA/RNA but without the heating/cooling cycles of PCR, so

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a heavy-duty power source is not needed. The addition of a few carefully designed biochemical reagents allows a LAMP box to test a sample that is heated only once for 30 minutes. “We’ve demonstrated that the chemistry we’re using can amplify viral RNA directly from raw, unprocessed samples,” said project lead Robert Meagher, “That is the ideal for a point-of-care testing scenario because you don’t want to have extra equipment for isolating DNA or RNA.” Meagher and team previously developed a method to combine LAMP with an additional detection technique so that multiple viruses could be tested simultaneously. This other technique, named quenching of unincorporated amplification signal reporters (QUASR), involves using synthetic virus-based primers tagged with fluorophores for the sample-DNA/RNA amplification step. For the Zika project, Meagher’s team developed a novel algorithm that allows a smartphone sensor to

act as a fluorimeter, detecting QUASR LAMP light signals if they appear. The user need only place the smartphone on top of the LAMP box and open an app that turns on the heater to initiate the LAMP reaction, after which the smartphone photographs the sample. The app then employs a novel image analysis algorithm to determine the color and brightness of the fluorescence emitted from the LAMP reaction. Zika, dengue, and chikungunya are spread via the same mosquito vector and have similar early symptoms. The team’s prototype diagnostic tool could enable care providers to test quickly for all three at the same time, preventing misdiagnoses. The study, by Priye A et al, was published online March 20, 2017, in the journal Scientific Reports. Image: A prototype of the mobile diagnostic device developed by Sandia scientists and engineers for rapid detection of Zika and related viruses (Photo courtesy of Sandia National Laboratories / Randy Wong). LabMedica International November/2017

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LabMedica International

New Test May Predict Gestational Diabetes Better estational diabetes is a type of diabetes that occurs during a woman’s pregnancy, increasing the mother’s risk of delivering a large-for-gestational-age baby, which can lead to pre-term birth, fetal injury, perinatal mortality and cesarean delivery. Gestational diabetes is also a risk factor for preeclampsia and gestational hypertension. Since treatment of gestational diabetes can lessen the risk of adverse pregnancy outcomes, practice guidelines recommend screening all non-diabetic, pregnant women for the disease. Scientists at Brigham and Women’s Hospital (Boston, MA, USA; www.brighamand womens.org) conducted a case-control study of 1,000 pregnant women who were receiving standard prenatal care at the hospital. Included in the study were 500 women who had a normal glucose challenge test (control subjects) and 500 women who failed the glucose challenge test and required a subsequent oral glucose tolerance test (case patients). The team’s primary goal was to assess the accuracy of the diabetes biomarker, plasma glycated CD59 (pGCD59), in predicting the results of the standard of care glucose challenge test used to screen for gestational diabetes. They assessed whether pGCD59 could predict the following: the results of the glucose challenge test (GCT) for screening of gestational diabetes mellitus (GDM) (primary analysis); and the diagnosis of GDM and prevalence of large for gestational age (LGA) newborns (secondary analyses). The scientists found that, when compared with the control subjects, the median plasma GCD59 value was 8.5-fold higher in the patients who failed the glucose challenge test and 10-fold higher in the subset of these patients who met diagnostic criteria for gestational diabetes in the subsequent oral glucose tolerance test. They also found that higher plasma GCD59 levels at gestational week 24-28 were associated with higher prevalence of large-for-gestationalage newborns, with the higher the level, the higher the risk (4% higher risk for patients in the lowest quartile of GCD59 plasma levels, and 14% in the highest quartile). Out of the 58 large-for-gestational-age babies born to mothers that failed the glucose challenge test in this study, 80% were born to mothers who did not meet oral glucose tolerance test criteria for gestational diabetes, but had median plasma GCD59 levels 7fold higher than control women with a normal glucose challenge test. These findings are consistent with other studies showing that women who fail the glucose challenge test, but do not meet criteria for gestational diabetes, are still at a higher risk of abnormal pregnancy outcomes, including delivering large for gestational age babies. The team concluded that as non-enzymatic glycation inactivates the complement inhibitor CD59, forming glycated CD59 (GCD59), they could therefore use a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for GCD59 in blood, and they showed that plasma GCD59 levels are significantly higher in individuals with type 2 diabetes and independently predict the response to the oral glucose tolerance test. Jose A. Halperin, MD, a hematologist and senior author of the study said, “This is the first study to demonstrate that a single measurement of plasma GCD59 can be used as a simplified method to identify women who are at risk for failing the glucose challenge test and are at higher risk for developing gestational diabetes. These results suggest that a single measurement of plasma GCD59 during weeks 24-28 may also help stratify the risk for delivering larger infants among women with gestational glucose intolerance.” The study was published in the April 2017 issue of the journal Diabetes Care.

LabMedica International November/2017

Image: A new blood test for pregnant women may predict gestational diabetes better than existing methods (Photo courtesy of the APA).

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PRODUCT NEWS ESR ANALYZER

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CHEM/IMMUNOASSAY SYSTEM

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The Monitor-100 random access ESR analyzer offers a processing time of one hour with results in 30 minutes and two hour processing with results in one hour. It requires a sample volume of 1.28 ml with vacuum tube and can be adapted for bar code and auto labeling.

The Alinity ci-series consists of compact, clinical chemistry and immunoassay systems that help high performing laboratories run at their best. The innovative testing solutions allow labs to run more tests in less time, reduce human error and increase testing productivity.

The ichroma II is designed for measuring the concentration of analytes contained in blood, urine, or other samples, in quantitative or semi-quantitative ways. An improved user interface/display and an advanced optical system, provide the user with a wide range of disease test options.

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Listeria May Pose Serious Threat Early in Pregnancy rom their study of how Listeria affects the fetus, researchers suggest that infection with Listeria monocytogenes likely poses a greater risk of miscarriage in the early stages of pregnancy

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than has been appreciated. “Listeria has been associated with adverse outcomes in pregnancy, but particularly at the end of pregnancy,” said senior author Ted Golos, of University of Wisconsin-Madison (Madi-

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son, WI, USA; www.wisc.edu), “What wasn’t known with much clarity before this study is that it appears it’s a severe risk factor in early pregnancy.” “The problem with this organism is not a huge number of cases. It’s that when it is identified, it’s associated with severe outcomes,” said coauthor Charles Czuprynski, professor at UW-Madison. Pregnant women are warned to avoid many of the foods that can harbor Listeria, and severe outcomes have resulted in a zero-tolerance regulatory policy for Listeria in ready-to-eat foods. But Listeria infection in pregnancy may go unnoticed; the few recognizable symptoms are nearly indistinguishable from the discomfort most newly pregnant women feel. Yet “it has a profound impact on the fetus,” said Prof. Golos, “That’s familiar now because we’ve been talking about the same difference in Zika virus.” For the study, Sophia Kathariou, professor at North Carolina State University, provided a strain of Listeria that caused miscarriage, stillbirth, and premature delivery in at least 11 pregnant women in 2000. Four pregnant rhesus macaques at the Wisconsin National Primate Research Center were fed doses of the Listeria comparable to what one might encounter in contaminated food. Lead author Bryce Wolfe, a UW-Madison graduate student, monitored the speed and progression of Listeria’s spread. “What’s particularly striking about the work Bryce did is the detailed information we now have about the organism,” said Prof. Czuprynski, “she tracked it being shed in feces and showing up in the bloodstream. They did ultrasound analysis of the fetus, and could then show events in terms

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of where the organism was preceding fetal demise.” None of the monkeys showed obvious signs of infection before their pregnancies came to abrupt ends. But in tissue samples taken after each monkey experienced intrauterine fetal death, Wolfe found Listeria had invaded the placenta. “In that region there’s a rich population of specialized immune cells, and it is exquisitely regulated,” said Wolfe, “When you introduce a pathogen into the midst of this, it’s not very surprising that it’s going to cause some sort of adverse outcome disrupting this balance.” The researchers suggest the inflammation caused by the maternal immune response affects the placenta, preventing it from protecting the fetus. “It should be a barrier,” said Prof. Golos, “But we’re hypothesizing that the maternal immune system’s attempt to clear the bacteria actually results in collateral damage to the placenta that then allows the bacteria to invade the fetus.” The results suggest Listeria (and perhaps other pathogens) may be the culprit in some miscarriages that usually go without diagnosed cause, but the bacteria’s stealth and speed may still make it hard to control. “There are effective antibiotics available. It is treatable,” said Wolfe, but “the fetus may be infected by the time anyone realizes the mother was infected.” The researchers plan to further investigate progression of infection and the maternal immune response to intracellular pathogens in pregnancy, in hope that such basic knowledge would help battle this and similar dangers, such as Zika virus. The study, by Wolfe B et al, was published February 21, 2017, in the journal mBio. LabMedica International November/2017

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Genetic Test Identifies Risk of Anal Cancer nal cancer is mostly caused by human papillomavirus (HPV), which is the same virus that causes cervical cancer. Diagnosis presents many challenges as full biopsies are painful, and taking a small sample of cells for cytology is problematic because lesions can be hidden. A new genetic test has been developed that could be an accurate and inexpensive way to find and treat those at highest risk of anal cancer, a disease with growing incidence in women, men who have sex with men (MSM) and people with human immunodeficiency virus (HIV). Scientists at the Queen Mary University of London (UK; www.qmul.ac.uk) and their colleagues obtained a set of anal and perianal biopsy specimens from 148 patients (116 men, 31 women, and one person of unrecorded gender) of whom 94 were HIV positive, 40 were HIV negative and 14 had not been tested for their HIV status. Most of the biopsies showed morphological changes varying from slight to severe which distinguished them from the surrounding non-acetowhite normal epithelium. For HPV genotyping the team used hematoxylin and eosin stained sections which were processed and the samples were tested using the PapType High Risk HPV Detection and Genotyping kit (Genera Biosystems Ltd, Scoresby, Australia; http:// generabiosystems.com). The kit is able to detect 13 high-risk HPV types, a possible high-risk type (HPV66) and two low-risk types (HPV6 and HPV11). Bisulfite conversions of DNA extracts were done using EZ DNA Methylation Kit (Zymo Research, Irvine CA, USA; www.zymoresearch. com). The polymerase chain reaction (PCR) products were pyrosequenced using a PyroMarkQ96 ID instrument (Qiagen, Hilden Germany; www. qiagen.com). The scientists found that the most prevalent HPV type was HPV16, detected in 54% of the 30 benign biopsies, 33% of the 43 low-grade anal intraepithelial neoplasia (lgAIN), 82% of the 59 high grade AIN (hgAIN) and four of the five anal cancers. A methylation score was developed which had increasing values with severity of disease: the mean was 8.1% in benign, 13.2% in lgAIN, 22.3% in hgAIN and 49.3% in cancers. The methylation score as a triage classifier at a cut-off of 8.8 gave a sensitivity of 90.6%, specificity of 50.7%, and area under the curve of 0.82 for separating hgAIN and cancer from benign and lgAIN biopsies. The authors concluded that that all of the anal cancers showed the presence of specific epigenetic markers on the patients' Erythrocyte Membrane Protein Band 4.1 Like 3 gene (EPB41L3), a tumor suppressor gene and also on certain regions of their viral HPV genome. Attila T. Lorincz, PhD, a professor and lead author of the study said, “We believe this new set of biomarkers goes a long way to indicating which men and women are at risk of developing anal cancer. Now that we can identify those at risk, and conversely, those not at risk, we hope to see a big improvement, by making sure that anoscopies and laser or chemical surgery are only given to those who need it.” The study was published on May 18, 2017, in the journal Oncotarget.

LabMedica International November/2017

Image: Early research found that a genetic test could lead to a reduction in painful procedures and minimize the over-treatment of people at low risk for anal cancer (Photo courtesy of Queen Mary University of London).

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Mass Spectrometry Identifies Bacteria Resistant to Colistin method using mass spectrometry has been successfully developed to quickly and accurately diagnose drug-resistant bacterial infections, as well as to distinguish between the two main mechanisms of resistance. The research was a collaboration led by Dr. Laurent Dortet, from South Paris University (Paris, France), and Dr. Gerald Larrouy-Maumus and Prof. Alain Filloux from Imperial College London (London, UK; www3.imperial.ac.uk). The team tested Escherichia coli and Klebsiella pneumoniae, both members of Enterobacteriacae. Some strains have become resistant to nearly all available antibiotics. Colistin often remains the only treatment option for these multidrug resistant bacteria, but some strains have now also developed colistin resistance. Previous research has shown that chromosome-encoded and plasmid-encoded colistin resistance has arisen. Plasmid-encoded resistance is considered more dangerous because it can be passed on from one type of bacteria to other types. “This plasmid-encoded resistance is particularly worrying because it has the potential to spread quickly and easily and, if that happens, last resort drugs like colistin could also become obsolete,” said Dr. Dortet, "If, on the other hand, we are able to rapidly identify bacteria that have this type of resistance, we can take measures to stop its spread. This might include isolating the patient in a separate room where they are treated by dedicated medical staff.” The researchers tested 134 different colonies of bacteria using a mass spectrometer. They found that it was possible to distinguish not only between colistin resistant and nonresistant bacteria, but also which bacteria have plasmid-encoded resistance. Also, the test can be carried out in around 15 minutes and would cost less than 1 USD per sample. “The exciting thing about this technique is that it relies on technology that is already available in most hospitals. This means that it could be rolled out quickly and cheaply, and potentially have a rapid impact on tackling drug resistance,” said Dr. Larrouy-Maumus. The researchers are now working with Imperial Innovations, Imperial College London’s technology transfer office, to patent the technique and develop it for clinical use in hospital laboratories. The test could also be useful for screening veterinary samples, where levels of colistin-resistance are known to be high. It might also be used for testing whether new drugs are able restore bacteria’s vulnerability to colistin. “The rapid detection of colistin resistance will be of enormous value in healthcare. It will enable early interventions to control transmission, protect patients, and improve individual patient management,” said Prof. Alison Holmes, of Imperial, “Outside healthcare, it will also have enormous potential in farming and meat production. The ability to discriminate the different mechanisms of colistin resistance will also generate a greater understanding.” The study was presented at the 27th European Congress of Clinical Microbiology and Infectious Diseases (April 22-25, 2017, Vienna, Austria).

Image: Researchers tested bacterial colonies in the method they developed for using a mass spectrometer to rapidly distinguish between colistin resistant and nonresistant bacteria, as well as to identify which bacteria have plasmid-encoded resistance (Photo courtesy of Imperial College London).

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Early Biomarker Found for Sepsis-Induced Kidney Injury epsis is a worldwide health care problem and a leading cause of death of patients in intensive care unit. On the other hand, acute kidney injury (AKI) is a clinical syndrome of an abrupt renal dysfunction leading to uremic toxins accumulation and several organs dysfunction. An early sepsis-induced acute kidney injury (sepsis-AKI) biomarker is currently needed and urinary neutrophil gelatinaseassociated lipocalin (uNGAL) is a candidate of sepsis-AKI biomarker. Urinary exosomal activating transcriptional factor 3 (uATF3) has also been mentioned as an interesting biomarker. Scientists at the Chulalongkorn University (Bangkok, Thailand; www.chula.ac.th) collected spot urine samples from 139 patients that were included in study and 79 of these patients were diagnosed with sepsis-AKI implied the importance of AKI in sepsis. Subsequently, all urine samples from patients who underwent the new onset of sepsis-AKI during the first week of admission were analyzed in comparison with 40 sepsis-non-AKI. Of note, there were 17 and 11 patients of sepsis-AKI and non-sepsis-AKI, respectively, transferred to intensive care unit. Serum creatinine (Scr) and uNGAL were measured by enzyme-linked immunosorbent assay (ELISA) assay (R&D Systems, Minneapolis, MN, USA; www. rndsystems.com) and enzymatic based automated analyzer, respectively. Western blot analysis was performed to quantitate urinary exosomal ATF3. For the visualization, the blot was incubated with appropriate horseradish peroxidaselinked secondary antibodies and visualized for the chemiluminescence with SuperSignal (Thermo Scientific, Waltham, MA, USA; www.fishersci.com) and CDiGit Blot Scanner (Li-Cor BioTech, Lincoln, NE, USA; www.licor.com). The team reported that the analysis showed higher Scr, uNGAL and uATF3 in patients with sepsis-AKI in comparison with patients with sepsis-non-AKI and healthy volunteers. In sepsis-AKI and sepsis-non-AKI groups, uNGAL were 367 ± 43 ng/mL and 183 ± 23 ng/mL, respectively; and uATF3 were 19 ± 4 ng/mL and 1.4 ± 0.8 ng/mL, respectively. Although both uNGAL and uATF3 correlated with Scr levels, uATF3 provided the better differentiation between sepsis-AKI and sepsis non-AKI. The authors concluded that they had identified ATF3, a transcriptional factor; in urine exosome as an interesting sepsisAKI biomarker. Since uATF3 could not be detected in most of the patients with sepsis-non-AKI, the qualitative test of uATF3

LabMedica International November/2017

might be adequate for detecting sepsis-AKI. In contrast, with the baseline of uNGAL, the debates on the cut-off values of uNGAL for sepsis-AKI are still ongoing. Our results were also a proof of concept that urine exosomes were an interesting source of urine biomarkers. The study was published on January 7, 2017, in the journal BMC Nephrology. Image: The C-DiGit blot scanner (Photo courtesy of Li-Cor BioTech).

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Miniature Technology May Improve Disease Detection esearchers have developed an improved microcontact printing technology to create an optimal disease diagnostic device of bioreceptor arrays for multiplexed microfluidic assays. Since efficiency of microfluidic bioassay devices relies on how intact and functional the bioreceptors are, immobilizing these bioreceptors without causing damage has proved daunting. Over the last two decades, microcontact printing, which uses a rubber stamp to immobilize the bioreceptors, has been established as a robust method to create a variety of assays with multiple applications. Yet this method also has its flaws, particularly when utilized at the nano scale of proteins and DNA. At this scale, the techniques currently used compromise resolution, whether by deforming the stamp or damaging the bioreceptors, thus yielding data somewhat unman-

ageable for diagnostics or other applications. Now researchers at Okinawa Institute of Science and Technology Graduate University (OIST; Okinawa, Japan; www.oist.jp) have developed a sequence of printing steps that have rectified these issues. For microcontact printing “you need a stamp, an ink, and a surface, and then you create your pattern on your surface. It’s as simple as that,” said paper first author Shivani Sathish, OIST PhD student. The stamp is made of polydimethylsiloxane (a flexible solid similar to the rubber used in everyday stamps), the ink is (3aminopropyl)triethoxysilane (APTES; a solution composed of silicon- and oxide-containing molecules), and the surface is glass.

After coating the stamp with the ink, the stamp is pressed onto the glass, and then removed after a short incubation. The result is a patterned layer of APTES on the glass – a checkerboard of regions with or without APTES. Next, a microfluidic device (which contains microchannels configured to guide fluid through specified pathways) is sealed over the patterned glass. Finally, the bioreceptors are chemically linked to the APTES regions within the microfluidic channels. The system is now ready for use as a diagnostic assay by delivering a body fluid sample through the microfluidic device attached to the glass. The APTES solution has convenient chemistry. “Depending on your bioreceptor of interest, you just have to choose the appropriate chemistry to link the molecule with the APTES,” said Ms. Sathish. One stamp can be used to prepare an assay with the ability to immobilize a variety of different bioreceptors for multiplexing. Thus one stamp allows for multiple tests and diagnoses on a single surface. This feature would be advantageous for diagnosing complex diseases such as cancer, which relies on tests that can detect multiple markers to improve the diagnosis. Ms. Sathish and colleagues first patterned nanoscale features of APTES using ink made of APTES in water, as opposed to harsh chemicals, which eliminated the stamp-swelling issue. Then, they immobilized the bioreceptors onto the surface as the very last step of the process, after patterning the APTES and attaching the microfluidic device. By attaching the bioreceptors as the final step, the researchers avoided exposing them to extreme and damaging conditions. They then demonstrated the efficacy of the final device by running an assay to capture the biomarkers interleukin 6 and human creactive protein, which are often elevated during inflammation. “The final goal is to create a point-of-care device,” said OIST Professor Amy Shen, who headed the study. “If you get your bioreceptors pre-immobilized within microfluidic devices, you can then use them as diagnostic tools as and when required,” Ms. Sathish continued, “[Eventually] instead of having a whole clinical team that processes your sample…we’re hoping that the patients can do it themselves at home.” The study, by Sathish S et al, was published April 5, 2017, in the journal Analyst. Image: Microfluidic bioassay devices are currently the preferred diagnostic tools. They measure concentration of disease biomarkers within a patient sample, such as blood, which is passed across a surface containing immobilized bioreceptors to capture the biomarker. They can indicate the likelihood of a disease based on presence/absence or based on comparison of the biomarker concentration in the sample relative to the normal body level (Image courtesy of Okinawa Institute of Science and Technology Graduate University).

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New Immunoassay Developed For Zika Virus Detection he new assay can improve patient care by enabling differential diagnostics according to the World Health Organization (WHO) test algorithm. It is capable of detecting Zika virus during the acute phase of infection, approximately a few days after the onset of symptoms. The CE-marked Novagnost Zika virus IgM μ-capture assay, developed by Siemens Healthineers of Siemens Healthcare (Erlangen, Germany, and Tarrytown, NY, USA; www.siemens.com/healthineers), is now commercially available in many countries (currently not available in the US). The assay is user-friendly, utilizing the same dilution and reagents as other Novagnost assays. It is validated for use on plasma and serum, and can be performed on the BEP III and BEP 2000 Advance Systems (currently not available in the US). Together with the recently announced real-time molecular VERSANT Zika RNA 1.0 Assay (kPCR), Siemens Healthineers thus now offers Zika assays both for immunoassay and molecular detection. The VERSANT Zika assay is currently intended for research use only (RUO) in regions outside of the US. In July 2016, it received Emergency Use Authorization (EUA) from the US Food and Drug Administration (FDA), allowing for unapproved medical products or unapproved uses of approved medical products to be used in an emergency to diagnose, treat, or prevent serious or lifethreatening diseases or conditions caused by chemical, biological, radiological, and nuclear (CBRN) threats when there are no adequate, approved, and available alternatives. “As the Zika virus continues to rise as a global public health concern, there is an

Image: An illustration of the Zika virus in blood (Photo courtesy of iStock).

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increased focus on detecting the Zika virus during the acute phase of infection,” said Franz Walt, president, Laboratory Diagnostics, Siemens Healthineers, “With the introduction of the Novagnost Zika Virus IgM μ-capture Assay, Siemens Healthineers completes laboratories’ virus testing menu by delivering assays for both immunoassay and molecular detection.”

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Blood Levels of Specific MicroRNA Predict Likelihood of Metastasis he microRNA (miRNA) that regulates the activity of the ubiquitin ligase enzyme SOCS2 (Suppressor of Cytokine Signaling 2) has been linked to the ability of prostate cancer to metastasize. MicroRNAs (miRNAs) are a small noncoding family of 19- to 25-nucleotide RNAs that regulate gene expression by targeting messenger RNAs (mRNAs) in a sequence specific manner, inducing translational repression or mRNA degradation, depending on the degree of complementarity between miRNAs and their targets. Many miRNAs are conserved in sequence between distantly related organisms, suggesting that these molecules participate in essential processes. In fact, miRNAs have been shown to be involved in the regulation of gene expression during development, cell proliferation, apoptosis, glucose metabolism, stress resistance, and cancer.

Investigators at the University of Adelaide (Australia; www.adelaide. edu.au) had demonstrated previously that a high level of the microRNA miR-194 in a patient’s blood was associated with rapid relapse of prostate cancer following surgical removal of the tumor. In the current study, which was published in the December 23, 2016, online edition of the journal Cancer Research, the investigators reported that miR-194 was a driver of prostate cancer metastasis. Prostate tissue levels of miR-194 were associated with disease aggressiveness and poor outcome. Ectopic delivery of miR-194 stimulated migration, invasion, and epithelial-mesenchymal transition (EMT) in human prostate cancer cell MEDICA® 2017

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lines, and stable overexpression of miR-194 enhanced metastasis of tumor xenografts. Conversely, inhibition of miR-194 activity suppressed the invasive capacity of prostate cancer cell lines in vitro and in vivo. At the molecular level the investigators found that the ubiquitin ligase SOCS2 was a direct, biologically relevant target of miR-194 in prostate cancer. Low levels of SOCS2 – caused by overexpression of miR-194 – correlated strongly with disease recurrence and metastasis in clinical specimens. Senior author Dr. Luke A. Selth, senior research fellow at the University of Adelaide, said, “This new work explains why miR-194 is associated with a poor outcome, and in the process reveals a completely novel pathway regulating prostate cancer metastasis. Importantly, measuring miR-194 in a patient’s blood at the time of diagnosis could become a test for the likelihood of metastasis. Patients with high levels of miR-194 in their blood could receive more aggressive treatment to reduce the chance of the cancer spreading to other parts of the body. There are currently no drugs that effectively inhibit the spread of prostate cancer. We propose that inhibiting miR-194 could reduce rates of metastasis in patients with aggressive disease, but the development of a drug to achieve this goal is still a long way off.” Image: MiR-194 drives prostate cancer metastasis by suppressing SOCS2 activity (Photo courtesy of Dr. Luke Selth, University of Adelaide).

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Platform Speeds Biomolecule Detection Using Magnetic Web esearchers have used micromagnetophoretic network patterns that resemble spider webs to develop an improved diagnostic and treatment-monitoring technology. By overcoming the diffusion-based transport limitation, their lab-on-a-chip (LOC) platform has achieved 20 times faster detection capability than existing biosensors. A research team from Daegu Gyeongbuk Institute of Science and Technology (DGIST; Daegu, Korea; http://en.dgist.ac.kr) led by Professor CheolGi Kim developed the platform, which increased the ability to collect low-density biomolecules by attracting biomolecules labeled with the superparamagnetic particles to the sensing area. "The existing biosensors require long time to detect low-density biomolecules and result in poor sensing efficiency as they only depend on diffusion. The magnetic field based biosensor platform improves the collection capability of biomolecules and increases the speed and sensitivity of the biomolecules movement,â&#x20AC;? said Prof. Kim, â&#x20AC;&#x153;We are planning to use this platform for early diagnosis as well as recurrence diagnosis of diseases such as cancer." The sensing capability of a biosensor is determined by the resolution of the sensor itself and the movement and reaction rate of molecules. Many research groups have been improving the resolution through the development of nanomaterials but there has been a limitation to improving sensor sensitivity due to the low diffusion transport of biomolecules (e.g. proteins, DNA) toward the sensing region. The team used a magnetic field in order to overcome the drawback that the biomolecules movement is slow when transport depends only on diffusion. The biomolecules labeled with superparamagnetic particles and the use of an external magnetic field enabled the movement of the biomolecules to be easily controlled and detected with an ultra-sensitive magnetic sensor. First author Byeonghwa Lim, PhD student at DGIST, added: "We placed a spider web-shaped micro-magnetic pattern, which was designed to move the superparamagnetic particles toward the center of the biosensor, and a high-sensitivity biosensor on the platform. When a rotating magnetic field is applied to a spider web-shaped magnetic pattern, it can attract biomolecules labeled with superparamagnetic particles faster to the sensor. The speed of the movement is very fast and it can detect the subject 20 times faster than the diffusion method." The team also succeeded in monitoring the biomolecules, conjugated to the

LabMedica International November/2017

superparamagnetic particles, at a distance from the sensing area. In addition, they found that the superparamagnetic particles not only play the role in biomolecular cargo for transportation, but also act as labels for the sensor to indicate the location of biomolecules. The study, by Lim B et al, was published March 31, 2017, in the journal NPG Asia Materials. Image: A diagram of a biosensor platform using magnetic patterns resembling a spider web (Photo courtesy of DGIST).

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Optofluidic Device Quantifies CTCs in Blood esearchers have developed a portable device that rapidly detects and counts circulating tumor cells (CTCs) in blood samples. It can help provide early diagnosis and help assess a patient’s level of health with convenient, inexpensive, effective testing for monitoring patients with cancer. These patients need to be constantly monitored during treatment to assess disease progression, particularly if their cancer has metastasized. Monitoring is currently done using imaging techniques and biopsies, which are invasive and not always possible. In contrast, the new device is highly sensitive and requires no surgery or treatment involving radiation, thus improving patient quality of life. The device was developed by a team of researchers and clinicians led by Universitat Rovira i Virgili (Tarragona, Catalonia, Spain; www.urv.cat/ en) professors Francesc Díaz, Ramon Álvarez

Puebla, and Jaume Masons and by HM Torrelodones University Hospital’s Dr. Eduardo García–Rico. It counts the number of tumor cells in a blood sample in real time, and has been successfully tested on patients in various stages of breast cancer. It can be adapted for use to determine the presence of other tumors by analyzing for different antibodies in the blood sample. The patented device has been licensed for commercialization to Medcom Science, a company engaged in research and development of technologies for diagnosing and treating cancer. The study, by Pedrol E et al, was published June 16, 2017, in the journal Scientific Reports. Image: The device employs two systems in miniature: a flow system and an optical system. The ratio of the two systems provides a quantitative indication about how the cancer is progressing (Photo courtesy of the Universitat Rovira i Virgili).

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PCR Test for Vaginal Pathogens Approved for Use molecular diagnostic test that identifies the three pathogens most commonly associated with vaginitis has been approved for sale as a diagnostic tool in the United States. The test employs real-time polymerase chain reaction (PCR) methodology to amplify large amounts of specific DNA sequences from the three most common causes of vaginitis (bacterial vaginosis, vulvovaginal candidiasis, and trichomoniasis) from patient samples, then reads either a positive or a negative result based on whether enough DNA was present to indicate infection. Investigators at Johns Hopkins University (Baltimore, MD, USA; www.jhu.edu) developed the test, which was subsequently licensed for marketing by BD Diagnostics (Franklin Lakes, NJ, USA; www. bd.com) under the name BD MAX Vaginal Panel for use with the BD MAX System. The BD MAX System offers an efficient path to improved clinical outcomes by combining and automating extraction and thermocycling into a single platform capable of running both FDA-cleared and open system assays. A study was designed to evaluate the clinical accuracy of the investigational test for vaginal swabs collected by patients (self) or clinicians. The women collected a vaginal swab, sheathed, and then handed it to the clinician. These swabs were to evaluate how self-collected swabs compared with cliniciancollected swabs. The clinician collected an investigational test swab and reference test swabs. Cliniciancollected and self-collected vaginal swabs from 1,740 symptomatic patients were evaluated by the molecular test and six other tests. Results from self-collected swabs were similar to clinician-collected swabs. Further analysis of the results showed that the molecular-based test using vaginal swabs collected by clinicians or patients could accurately diagnose most common bacterial, fungal, and protozoan causes of vaginitis. These results contributed to the decision by the [U.S.] Food and Drug Administration to issue 510(k) approval for marketing the test as a diagnostic tool in the United States. “Diagnostic tests traditionally used to distinguish among the causes of vaginitis are archaic, quite subjective, and time-intensive, plus they require extensive training for those reading the results,” said first author Dr. Charlotte Gaydos, professor of medicine at Johns Hopkins University. The sample collection study was published in the June 8, 2017, online edition of the journal Obstetrics & Gynecology.

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Yeast-Based Tool Developed For Pathogen Detection he monitoring of global pathogen burden has been traditionally limited to a small number of specialized centers, but more effective detection could be performed in real time by making accurate diagnostics accessible at the point of care. A tool has been created that is an extremely low-cost, low-maintenance, onsite dipstick test that will aid in the surveillance and early detection of fungal pathogens responsible for major human diseases. The emerging field of synthetic biology has the potential to provide novel diagnostic platforms to overcome global health challenges – much like advances in molecular biology gave rise to antibody diagnostics. Scientists at Columbia University (New York, NY, USA; www.columbia.edu) and their colleagues swapped out naturally occurring cell surface receptors of Saccharomyces cerevisiae, or baker’s yeast, with pathogen-specific receptor proteins. They started by building a biosensor for the detection of Candida albicans, a human fungal pathogen, that occurs naturally in the human gut, but can cause serious medical problems and even death if the population gets out of control. After replacing S. cerevisiae natural receptor with that of C. albicans, the team then altered its DNA to enable production of lycopene, the pigment responsible for the red coloring of tomatoes. This allowed the engineered yeast to turn red when in the presence of a target molecule, in this case, C. albicans fungus pheromones. The scientists successfully tested their assay for the ability to detect ten additional major pathogens, including Paracoccidioides brasiliensis, a fungus responsible for a progressive tropical disease affecting the mucosa in the nose, sinuses and skin. In each case, the test functioned accurately without sacrificing any of the sensitivity and specificity attainable with other, significantly more expensive tests. Virginia Cornish, PhD, a chemist and the principle investigator of the study, said, “We can now alter the DNA of S. cerevisiae to give it new functions that make it useful for a variety of applications. The prospect of using this technology in rural communities with little access to high-tech diagnostics is particularly compelling. The possibilities, as we see it right now, are limitless. We’ve just opened the door to this exciting new technology. It’s the beginning of a journey rich with potential.” The study was published on June 28, 2017, in the journal Science Advances.

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Image: The dipstick device, a single-component, customizable, inexpensive biosensor uses live cells to detect pathogens worldwide (Photo courtesy of Columbia University).

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Immunoassay Device Based on Acoustic Vortex Nanoparticle Enrichment n inexpensive acoustic transducer is the key to a novel immunoassay that may eventually be combined with a smartphone camera to form a platform for the rapid detection of diagnostic proteins in blood, urine, or saliva samples. Investigators at Duke University (Durham, NC, USA; www.duke.edu) developed an acoustic-fluidic chip capable of generating single vortex acoustic streaming inside a glass capillary through using low-power acoustic waves (only five volts was required). The single vortex acoustic streaming that was generated, in conjunction with the acoustic radiation force, was able to enrich submicrometer- and nanometer-sized particles in a small volume. Numerical simulations were used to clarify the mechanism of the single vortex formation and were verified experimentally, demonstrating the focusing of silica and polystyrene particles ranging in diameter from 80 to 500 nanometers. In a proof-of-principle study, the acoustic-fluidic chip was used to perform an immunoassay in which nanoparticles that captured fluorescently labeled biomarkers were concentrated in a long, thin glass vial to enhance the emitted signal. “Diagnosis impacts about 70% of healthcare decisions,” said senior author Dr. Tony Huang, professor of mechanical engineering and materials science at Duke University. “If we can improve the quality of diagnostics while reducing its costs, then we can tremendously improve the entire healthcare system. My goal is to create a small diagnostic device about the size of a cell phone that can autonomously separate biomarkers from samples. With this vortex technology, the biomarkers could then be concentrated enough to see with a simple camera like the ones found in today’s cellular phones.”

Image: A sample of 500 nanometer particles in solution. In the top image, the acoustic whirlpool device was turned off. The bottom image shows that when the device was turned on, the nanoparticles were concentrated to the point of becoming visible as a dark line down the center of the chamber (Photo courtesy of Duke University).

Cerebrospinal Fluid Lens-Free Microscopy Used to Diagnose Meningitis he performance of the cytological analysis of the cerebrospinal fluid (CFS) and enumerating leukocytes and erythrocytes is a routine first step in the laboratory diagnosis of meningitis. CSF cytology and cell counting is routinely performed by optical microscopy. Optical microscopy observation is an operator-dependent task, with both counting itself and the subsequent reporting being subject to variability; this may indeed result in the erroneous classification of the CSF specimen as meningitis or non-meningitis. Scientists at Aix Marseille University (Marseille, France; www.univ-amu.fr) conducted a prospective blind inter-operator variability study was for two months. It consisted of optical microscopy cell counting conducted by five different operators on 35 consecutive and independent CSF specimens. The cell counts were performed on Glasstic 10 counting slides

with grids (KOVA, Garden Grove, CA, USA; www. kovaintl.com), performing the count of leukocytes and erythrocytes in the nine cell segmentations corresponding to the count per mm3. The optical microscope used in the study was an Olympus CX41 (Tokyo, Japan; www.olympus-lifescience.com). To compare the microscope results the team, used a lensfree microscopy algorithm adapted for counting cerebrospinal fluid cells and discriminating leukocytes from erythrocytes was modified step-by-step in the prospective analysis of 215 cerebrospinal fluid specimens. The acquisition was sequential, and each red, green and blue LED was lit up one after the other, while the three corresponding wide-field holograms were acquired by the complementary metal-oxide semiconductor (CMOS) sensor (red, green, and blue). In the first step, prospective optical microscopy counts of leukocytes done by five different operators

yielded an overall 16.7% misclassification of 72 cerebrospinal fluid specimens in meningitis/non-meningitis categories using a 10 leukocyte/ L cut-off. In the second step, the definite algorithm yielded a 100% sensitivity and an 86% specificity compared to confirmed diagnostics. In the third step, a blind lens-free microscopic analysis of 116 cerebrospinal fluid specimens, including six cases of microbiology-confirmed infectious meningitis, that yielded a 100% sensitivity and 79% specificity. The authors concluded that adapted lens-free microscopy is thus emerging as an operator-independent technique for the rapid numeration of leukocytes and erythrocytes in cerebrospinal fluid. In particular, this technique is well suited to the rapid diagnosis of meningitis at point-of-care laboratories. The study was published on January 3, 2017, in the journal Scientific Reports. LabMedica International November/2017

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LabMedica International

Pumps Achieve High-Speed Sorting of Large Cells he sorting of individual cells is necessary for many medical applications, including the isolation of specific cell types from cell suspensions. A fluorescence-activated cell sorting (FACS) has been used for high-throughput cell sorting. A FACS of larger cells requires the samples to be processed under low pressure through wider nozzles to prevent damage and therefor sorting is limited to low-level throughput. Lasers are used to excite auto-fluorescence or tagged-fluorescence of cell included in droplets, and then the droplets are diverted into different containers depending on their characteristics. This technique is a concern owing to sample infections due to aerosols generation. Scientists at Nagoya University (Nagoya, Japan; www.nagoya-u.ac.jp) investigating cell sorting used a microfluidic chip to prevent sample infection. This chip has microchannels into which cell suspensions are introduced for sorting. The group integrated two externally driven on-chip pumps into the microfluidic chip for high-speed flow control.

New Biomarkers Provide Diagnostic Tool for AD lzheimer’s disease (AD) is an age-related neurodegenerative disorder that results in the gradual deterioration of specific brain regions that hinders the person’s ability to think, recall memories, learn, and perform daily tasks. Currently, AD is diagnosed using the “evaluate and eliminate” approach. With this strategy, patient history, physical exams, laboratory tests, imaging scans, and neurophysiological assessments are examined by doctors as a means to diagnose AD and determine its progression. Scientists at Ohio State University (Columbus, OH, USA; www.osu.edu) and their colleagues analyzed 34 cerebrospinal fluid (CSF) samples from 24 patients with AD and from 10 healthy individuals serving as controls. They analyzed 30 serum samples from 22 patients with AD and from eight healthy individuals serving as controls. The processed CSF samples were combined with rabbitderived primary antibodies anti-A (1–42) antibody) and anti-tau) and goat-derived anti-rabbit secondary antibody (Abcam, Cambridge, UK; www.abcam. com) in immunofluorescence assays. A CSF sample of 10 L was dried on a slide glass surface for atomic force microscopy (AFM) nanomechanics characterization (MFP-3D SPM, Asylum Research, Santa Barbara, CA, USA; www. asylumresearch.com). Hyperspectral microscope imaging (HMI, CytoViva, Auburn, AL, USA; https:// cytoviva.com) was used for particle visualization and analysis and using the system, images of micro/nanoscale structures and micro-/nanoparticles were captured. Coated with gold and antiA (1–42) antibody, AFM tips were functionalized to specifically detect the A -embedded proteins inside human serum. Serum or anti-A (1–42) antibody was used to coat the substrates, respectively. The study was published on July 28, 2017, in the journal Science Advances.

LabMedica International November/2017

Using a high-speed actuator as the driving source of pump, they succeeded in producing a flow with 16 microseconds for cell sorting. Although various methods of on-chip cell sorting have been proposed, high-throughput sorting of large cells remains hampered by the difficulty of controlling high-speed flow over a wide sorting area. To overcome this problem, the team proposed high-speed local-flow control using dual membrane pumps driven by piezoelectric actuators placed on the outside of a microfluidic chip. They evaluated the controllability of shifting the flow profile by the local-flow. The technique allows them to sort not only large but also small cells with high speed, high purity, and high viability. The method was tested on microalgae as an example of large cells, around 100 μM in size, and achieved 95.8% purity, 90.8% viability, and a 92.8% success rate. As a model

small cell type, they used a cancer cell whose size is around 24 μM, and achieved 98.9% purity, 90.7% viability, and a 97.8% success rate. The study was first published on June 14, 2017, in the journal Lab Chip. Image: The developed microfluidic chip enables sorting of cells at high speed of 16 microseconds. The enlarged view shows a demonstration of onchip cell sorting of a Euglena gracilis cell (Photo courtesy of Nagoya University).

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ELISA INSTRUMENT

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Cypress Diagnostic

DIALAB

The WR-5000 fully automated microplate reader features an 8- channel reading system and allows reading in four user-selectable wavelengths. It can be programmed with analytical protocols of several types of ELISA tests with user-friendly software for easy understanding.

The CYANStart requires limited consumables and a minimum reaction volume, resulting in decreased costs per test. It allows for easy tracking of all results from each patient through a unique identifier and offers 180 programmable open channels to allow for other user-defined tests.

The DIAReader ELX808IU uses the absorbance detection mode and offers the endpoint, kinetic, and linear scanning reading methods. Other features include 96-well plates microwell, four-zone temperature control to 50 degrees centigrade, and low, medium, high and variable shaking speeds.

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Serum MiR-122 Levels Associated with Histopathological Features of NAFLD on-alcoholic steatohepatitis (NASH) can only be diagnosed by the presence of histopathological components, such as steatosis, lobular inflammation, ballooning, and fibrosis and therefore there is a need for non-invasive surrogate markers of histopathological features. Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide across different ethnicities and associated is with serious healthcare issues including NASH, which may lead to liver cirrhosis, hepatocellular carcinoma (HCC), and liver failure. Scientists at the Toranomon Hospital (Tokyo, Japan; http://jp-jmhc.com) diagnosed a total of 321 Japanese patients with NAFLD based on histopathological examination of liver biopsies between 1980 and 2016. Of these, 39 patients underwent at least two liver biopsies and were evaluated in detail clinically over time. The need for repeated liver biopsies was determined by the attending physician. Of the 39 patients, 36 did not develop HCC during the period from the first to the second biopsy (median: 4.6 years, range: 0.5-19.0 years).

Liver histopathology was performed and NAFLD activity score represented the sum of steatosis, lobular inflammation, and hepatocyte ballooning scores. Serum samples were obtained at least twice a year after the time of histopathological diagnosis of NAFLD. Circulating microRNA was extracted from serum samples using the QIAGEN miRNeasy serum-plasma kit (Qiagen KK, Tokyo, Japan; www. qiagen.com). Serum miR-122 was amplified using primers and probes provided by Applied Biosystems (Foster City, CA, USA; www.appliedbiosystems. com). The serum miR-122 ratio was represented serum miR-122 level at second biopsy to that at first biopsy. In patients who showed improvement of histopathological scores (steatosis, ballooning, and stage), serum miR-122 levels were significantly lower at second biopsy than first biopsy. In patients who showed no improvement, the changes at second biopsy were not different from those at first biopsy. There were significant and strong associations between serum miR-122 ratio level at second biopsy to that at first biopsy and changes in histopathological

scores (of steatosis, lobular inflammation, and stage). There were also significant and strong associations between serum miR-122 ratio and changes in other clinical parameters, including aspartate aminotransferase and alanine aminotransferase. The authors concluded that longitudinal examination of serial liver biopsies showed significant association of serum miR-122 with histopathological features of NAFLD in patients free of HCC. The study was published on December 7, 2016, in the journal BMC Gastroenterology. Image: The miRNeasy serum-plasma kit is designed for purification of cell-free total RNA (Photo courtesy of Qiagen).

Bioinformatics Tool for Evaluation of Cancer Genes esearchers offer a new software tool being developed to help assess computational algorithms used in methods to identify mutant genes that drive cancer. The tool could thereby lead to more precise diagnostics and targeted treatments for patients. In the search for new ways to tackle cancer, many scientists use genome sequencing to hunt for mutations that facilitate tumor cell growth. To aid in this search, some researchers have developed new bioinformatics methods that each claim to help pinpoint the cancer driver mutants. But a question remains: Among the numerous new tactics, which produce more accurate results? To help solve this puzzle, a team of Johns Hop-

kins University (Baltimore, MD, USA; www.jhu. edu) computational scientists and cancer experts devised the new tool to help assess how well current strategies work in identifying cancer-promoting mutations and distinguishing them from benign mutations in cancer cells. “Identifying the genes that cause cancer when altered is often challenging, but is critical for directing research along the most fruitful course,” said co-author Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, “This paper establishes novel ways to judge the techniques used to identify true cancercausing genes and should considerably facilitate advances in this field in the future.” Lead author Collin J. Tokheim, doctoral stu-

dent in the laboratory of senior author Rachel Karchin, associate professor at Johns Hopkins, said one of the challenges the team faced was the lack of a widely accepted consensus on what qualifies as a cancer driver gene. “People have lists of what they consider to be cancer driver genes, but there’s no official reference guide, no gold standard,” said Tokheim. Nevertheless, the team was able to develop a machine-learningbased method for driver gene prediction and a framework for evaluating and comparing other prediction methods. The study, by Tokheim CJ et al, was published December 13, 2016, in the journal Proceedings of the National Academy of Sciences. LabMedica International November/2017

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LabMedica International

A First: Using Graphene to Detect Cancer Cells sing Raman spectroscopy in developing a new technology that could improve diagnosis and monitoring of cancer, researchers have, for the first time, successfully used graphene to distinguish cancer from healthy cells. The system could potentially also be used to distinguish between various other cell types or cell activities. By interfacing brain cells onto graphene, researchers at the University of Illinois at Chicago (Chicago, IL, USA; www.uic.edu) have shown they can differentiate a single hyperactive cancerous cell from a normal cell, pointing the way to developing a non- or less invasive tool for early cancer diagnosis. “This graphene system is able to detect the level of activity of an interfaced cell,” said Vikas Berry, associate professor at UIC, who led the research along with Ankit Mehta, assistant professor at UIC College of Medicine. “Graphene is the thinnest known material and is very sensitive to whatever happens on its surface,” added Prof. Berry. The nanomaterial is composed of a single layer of carbon atoms linked in a hexagonal

chicken-wire pattern, and all the atoms share a cloud of electrons moving freely about the surface. “The cell’s interface with graphene rearranges the charge distribution in graphene, which modifies the energy of atomic vibration as detected by Raman spectroscopy,” he explained. Raman spectroscopy is routinely used to study graphene. The atomic vibration energy in graphene’s crystal lattice differs depending on whether it’s in contact with a cancer cell or a normal cell because the cancer cell’s hyperactivity leads to a higher negative charge on its surface and the release of more protons. The electric field around the cell pushes away electrons in graphene’s electron cloud, which changes the vibration energy of the carbon atoms. Recently, Prof. Berry and other coworkers had introduced nanoscale ripples in graphene, causing it to conduct differently in perpendicular directions, useful for electronics. They wrinkled the graphene by draping it over a string of rod-shaped bacteria, then vacuum-shrinking the germs. “We took the earlier work and sort of flipped it over,” said Prof.

Berry, “Instead of laying graphene on cells, we laid cells on graphene and studied graphene’s atomic vibrations.” The new study examined cultured human brain cells, comparing normal astrocytes to their cancerous counterpart, the highly malignant brain tumor glioblastoma multiforme. The technique is now being studied in a mouse model of cancer, with results that are “very promising,” said Prof. Berry. Experiments with patient biopsies would be further down the road. “Once a patient has brain tumor surgery, we could use this technique to see if the tumor relapses,” said Prof. Berry, “For this, we would need a cell sample we could interface with graphene and look to see if cancer cells are still present.” The same technique may also work to differentiate between other types of cells or the activity of cells. “We may be able to use it with bacteria to quickly see if the strain is Gram-positive or Gram-negative,” said Prof. Berry, “We may be able to use it to detect sickle cells.” The study, by Keisham B et al, was published November 14, 2016, in the journal ACS Applied Materials and Interfaces.

Abnormal Immune Cells Involved in Causing Fibrosis sing a mouse model, researchers have discovered a form of atypical monocyte as well as a committed progenitor that are involved in development of fibrosis. Fibrosis is a form of scarring that, if uncontrolled, can cause deleterious thickening of tissues. Although it is known that an activated immune system can lead to fibrosis, which specific cells are responsible continuous to elude researchers. Scientists at the WPI Immunology Frontier Research Center (IFReC) of Osaka University (Osaka, Japan; www.osaka-u.ac.jp/en) have now identified a subgroup of cells, a class of monocytes with strange morphology, involved in causing fibrosis. “The cells had a bi-lobed segmented nuclear shape

LabMedica International November/2017

and many cytoplasmic granules. We therefore called them ‘Segregated nucleus atypical monocytes (SatM)’,” said Prof. Shizuo Akira, head of the Host Defense Laboratory at IFReC. To identify this subgroup, the researchers examined immune cell subpopulations that predominantly appeared in fibrosis and found that “These cells were regulated by C/EBP ,” said Prof. Akira. Detailed examination of immune cells showed that C/EBP mutant mice, unlike normal mice, produced no SatM, whereas no other observed immune cell population was changed. The mice were also significantly more resistant to fibrosis. On the other hand, when the mutant mice were exposed to SatM, their susceptibility to fibrosis rose.

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Although Prof.Akira, first author Dr. Satoh, and colleagues describe SatM as a subset of monocytes, SatM showed characteristics that suggested they may be hybrids of different immune cells. Gene analysis of SatM “showed granulocyte markers, but SatM are definitely not granulocytes. [This] cell type is one of monocyte,” said Prof. Akira. Additional study found the progenitor cells responsible for producing SatM: adoptive transfer of these progenitors into mutant mice unable to produce SatM resulted in a SatM population, and C/EBP was found to be essential for maintaining the progenitors. The study, by Satoh T et al, was published online December 21, 2016, in the journal Nature.

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The GEMweb Plus 500 software provides complete control of the POCT process from a single dashboard. It offers enhanced mobility with accessibility from tablet devices, in addition to any networked analyzer or PC, with support for integration with hospital info systems, LIS and EMRs.

The Evidence MultiSTAT enables on-site simultaneous detection of up to 21 classical, prescription and synthetic drugs from a single sample. The multi-analyte testing platform provides a complete immunoassay profile within minutes and requires minimal sample preparation.

The Alinity c is a compact system that maximizes throughput of photometric and potentiometric assays. Designed with a universal, intuitive interface, it can run more tests in less space, generate test results faster and minimize human errors.

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Vaginal Microbes Denote Early Detection of Endometrial Cancer ndometrial cancer triggers remain elusive, but given the typical inflammatory profile in these cases, microbes in the uterine environment are suspected to play a role in the development of this disease. The microbial partners along the female reproductive tract have been long known to play an important role in health and disease along the woman’s reproductive tract. Lactic acid producing microbes have a strong role in determining the microbial community membership of the vaginal microbiome and have been shown to protect against infection. Medical scientists at the Mayo Clinic (Rochester, MN, USA; www.mayoclinic.org) studied 31 Caucasian women undergoing hysterectomy. Of those, 10 women were diagnosed with a benign gynecologic condition, four women were diagnosed with endometrial hyperplasia, and 17 women were diagnosed with endometrial cancer. All diagnoses were made based on the final surgical pathology following hysterectomy. Vaginal, cervical, Fallopian, ovar-

ian, peritoneal, and urine samples were collected aseptically both in the operating room and the pathology laboratory. All genomic DNA extractions were performed by using the MoBio PowerSoil Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA; https://mobio. com) and the DNA content was measured using High Sensitivity Qubit (Life Technologies Corporation, Carlsbad, CA, USA; www.thermofisher.com). The V3-V5 region of the 16S rDNA was then amplified through a polymerase chain reaction (PCR) and the products of the amplification were verified by a TapeStation D1K Tape (2200 TapeStation Instrument, Agilent Technologies, Santa Clara, CA, USA; www.agilent.com). The microbiome sequencing (16S rDNA V3-V5 region) revealed that the microbiomes of all organs (vagina, cervix, Fallopian tubes, and ovaries) are significantly correlated and that there is a structural microbiome shift in the cancer and hyperplasia cases, distinguishable from the benign cases. Several taxa were found to be significantly enriched in

samples belonging to the endometrial cancer cohort: Firmicutes (Anaerostipes, Dialister, Peptoniphilus, Ruminococcus, and Anaerotruncus), Spirochetes (Treponema), Actinobacteria (Atopobium), Bacteroidetes (Bacteroides and Porphyromonas), and Proteobacteria (Arthrospira). Of particular relevance, the simultaneous presence of Atopobium vaginae and an uncultured representative of the Porphyromonas sp. (99 % match to P. somerae) were found to be associated with disease status, especially if combined with a high vaginal pH (>4.5). The authors concluded that the detection of A. vaginae and the identified Porphyromonas sp. in the gynecologic tract combined with a high vaginal pH is statistically associated with the presence of endometrial cancer. Given the documented association of the identified microorganisms with other pathologies, these findings raise the possibility of a microbiome role in the manifestation, etiology, or progression of endometrial cancer that should be further investigated. The study was pub-

Liquid Chromatography Analyzer Evaluated for Hemoglobinopathy he quantification of HbA2 and HbF, and the detection and quantification of hemoglobin variants, is an essential tool in the diagnosis of hemoglobinopathies such as thalassemia or sickle cell syndromes. The narrow separation between normal and pathological HbA2 values, strict analytical quality of HbA2 measurement is an essential requirement for accurate diagnosis, particularly for genetic counseling when couples at-risk must be identified. A hematologist at the Hospital Galdakao – Usansolo (Vizcaya, Spain; www.osakidetza.euskadi.eus) evaluated the analytical performance and quality of results obtained from a fully automated high-pressure liquid chromatography (HPLC) analyzer for routine estimation of HbA2 and the screening of β thalassemia. Blood specimens were obtained from patients whose diabetic control or hemoglobinopathy were being assessed.

The scientist measured HbA2 values with the fully automated Glycohemoglobin analyzer based on HPLC ADAMS A1c HA-8180 (Arkray, Inc, Kyoto, Japan; www.arkray.co.jp) on Thalassemia mode and were compared with those obtained with their current method, the established HPLC ADAMSTM A1c HA-8160. Comparison was performed by running 400 samples from healthy subjects, 30 alpha and 80 beta carriers. Hemoglobin was measured on a XN counter (Sysmex Corporation, Kobe, Japan; www.sysmex.com). Different common variant hemoglobins were analyzed: HbS, HbC, HbD, HbJ, Hb Lepore, HbSC, Hb E, Hb O, and β thalassemia carriers with raised HbF. Twenty samples of heterozygous HbS were measured with capillary electrophoresis in the reference laboratory to discard the spurious increment of HbA2. The mean difference between HbA2 results of

ADAMS A1C HA-8180T and capillary electrophoresis, in samples of heterozygous HbS, was 0.2 %. The hematologist found an ample gap between healthy persons and carriers. A potential bias is that they have not included carriers of mutations producing borderline values of HbA2, which was absent in the area, and this fact must be considered in regions with high prevalence of those patients. The author concluded that the ADAMS A1c HA-8180T provided a rapid and reliable separation of HbA2. The measurement is accurate and reproducible, which is needed because of the slight difference between normal and pathological values. The gap in HbA2 values between normal subjects and β-thalassemia carriers makes this an appropriate method for rapid screening for carriers. The study was published in the December 2016 issue of the International Journal of Laboratory Hematology. LabMedica International November/2017

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LabMedica International

Test Markers Identify Cancer DNA and Pinpoint Tumor Location team of molecular geneticists has developed a diagnostic tool that identifies circulating cancer DNA and determines the organ in which the tumor is located. Investigators at the University of California San Diego (USA; www. ucsd.edu) based their method on the presence of methylation haplotype blocks in the genome. A haplotype is a set of single-nucleotide polymorphisms (SNPs) on one chromosome that tend statistically to always occur together. It is thought that identifying these statistical associations and few alleles of a specific haplotype sequence can facilitate identifying all other such polymorphic sites that are nearby on the chromosome. The investigators focused on a systematic search and investigation of regions in the full human genome that showed highly coordinated methylation. They defined 147,888 methylation haplotype blocks of tightly coupled CpG sites. CpG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in

the linear sequence of bases along its 5’ to 3’ direction. CpG is shorthand for 5’-C-phosphate-G-3’, that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA. Each tissue in the body can be identified by its unique signature of methylation haplotypes. Using methylation haplotype data, the investigators compiled a database of the complete CpG methylation patterns of 10 different normal tissues (liver, intestine, colon, lung, brain, kidney, pancreas, spleen, stomach, and blood). They then analyzed tumor and blood samples from cancer patients to assemble a database of cancer-specific genetic markers. Using this database the investigators demonstrated quantitative estimation of tumor load and tissue-oforigin mapping in the circulating cell-free DNA of 59 patients with lung or colorectal cancer. “Knowing the tumor’s location is critical for effective early detection,” said senior author Dr. Kun Zhang, pro-

fessor of bioengineering at the University of California, San Diego. “This is a proof of concept. To move this research to the clinical stage, we need to work with oncologists to further optimize and refine this method.” “We made this discovery by accident. Initially, we were taking the conventional approach and just looking for cancer cell signals and trying to find out where they were coming from. But we were also seeing signals from other cells and realized that if

we integrate both sets of signals together, we could actually determine the presence or absence of a tumor, and where the tumor is growing,” said Dr. Zhang. The method was described in detail in the March 6, 2017, online edition of the journal Nature Genetics. Image: Bioengineers at UCSD have developed a new blood test that could detect cancer and locate where in the body the tumor is growing (Photo courtesy of iStock).

Colorectal Cancer Predicted by Merging Risk Factors team of Spanish cancer researchers has devised a method for predicting risk of developing colorectal cancer (CRC) that combines life style choices, family background, and genetic data. Investigators at IDIBELL-Bellvitge Biomedical Research Institute (Barcelona, Spain; www.idibell.cat) set out to elaborate a model to stratify the risk of CRC by merging environmental information and single nucleotide polymorphisms (SNPs) data. To achieve this end, they conducted a case-control study that included 1336 CRC cases and 2744 controls. Subjects were interviewed on lifestyle factors, family, and medical history. In addition, 21 CRC susceptibility SNPs were genotyped. Results revealed that the environmental risk model, which included alcohol consumption, obesity, physical activity, red meat and vegetable consumption, and nonsteroidal anti-inflammatory drug use, contributed to CRC with an average OR (odds ratio) factor of 1.36. Family history of CRC contributed an OR of 2.25, and each additional SNP contributed an OR of 1.07. The risk of subjects with more than 25 risk alleles (fifth quintile) was

LabMedica International November/2017

82% higher than subjects with less than 19 alleles (first quintile). Thus, environmental factors had more weight than the genetic score, which should be considered to encourage patients to achieve a healthier lifestyle. The investigators did stress that this study only included 21 risk SNPs, while more than 60 have already been identified. More studies will be needed to determine the generalizability, usefulness of information, and the cost-effectiveness of applying individual genotyping in a CRC screening program. “A risk model is a mathematical tool that allows us to predict who is most likely to suffer from a particular disease, in this case colon cancer,” said senior author Dr. Victor Moreno, professor of medicine and health sciences head at IDIBELL-Bellvitge Biomedical Research Institute. “Today, screening for colon cancer in patients with no family history is based solely on age. If we include information about lifestyle and genetics, we could classify the population into groups of greater or lesser risk, which would allow us to offer a more personalized follow-up.” The study was published in the February 24, 2017, online edition of the journal Scientific Reports. LINKXPRESS COM

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COAGULATION ANALYZER

HEMOGLOBIN ANALYZER

DAS

EUROLyser Diagnostica

EKF Diagnostics

The ANALYZER 90 is a semi-automated, clinical chemistry 10 mm flow cell photometer with seven optical filters and wavelength of 340-400/700 nm. It offers both mono and bichromatic reading systems, and its operating software can run and store up to 200 tests.

The Smart 700/546 is designed to perform coagulation monitoring, thrombosis analysis, examine inflammation status, assess cardiologic risk, diabetes status, iron deficiency and screening of colorectal cancer risk. The easy-to-operate analyzer is ideal for a wide range of clinicians and labs.

The HemoPoint H2 DMS provides users with the ability to quickly and securely manage, access and analyze hemoglobin test results. The DMS software allows seamless linking to a nearby computer, aiding in the accuracy of patient records, future billing, and potential statistical analysis.

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Increased Triglyceride Content Associated with Plasma Lipoprotein Lipase on-fasting plasma triglyceride (TG) reflect a higher risk for cardiovascular disease (CVD) than TG in the fasting plasma and this the postprandial increase of TG is the most common form of hyperlipidemia which is associated with increased remnant lipoproteins (RLP) as a risk factor for CVD. It has been reported that the fasting TG concentration was not an independent cardiovascular risk factor, while RLP-cholesterol (RLP-C) was an independent risk factor in the fasting plasma in women. Therefore, it is necessary to investigate the difference between fasting and postprandial TG and RLP. An international team of scientists working with those at Gunma University Graduate School of Medicine (Maebashi, Japan; www.gunma-u.ac.jp) recruited 54 volunteers (30 males and 24 females, aged 28 to 63 years) who were without evidence of

CVD, diabetes or other chronic disease for the oral fat load test. TG and RLP-TG together with other lipids, lipoproteins and lipoprotein lipase (LPL) in both fasting and postprandial plasma were determined in generally healthy volunteers and in patients with coronary artery disease (CAD) after consuming a fat load or a more typical moderate meal. RLP-C and RLP-TG were determined using an immunoseparation method (JIMRO Co., Gunma, Japan; www.jimro.com) and Small Dense Low Density Lipoprotein cholesterol (LDL-C) was also determined by a commercial kit (Denka-Seiken, Tokyo, Japan; http://denka-seiken.jp). The team found that RLP-TG/TG ratio (concentration) and RLP-TG/RLPC ratio (particle size) were significantly increased in the postprandial plasma of both healthy controls and CAD patients compared with those in fasting plasma. LPL/RLP-TG ratio demonstrated the interaction

correlation between RLP concentration and LPL activity The increased RLP-TG after fat consumption contributed to approximately 90% of the increased plasma TG, while approximately 60% after a typical meal. Plasma LPL in postprandial plasma was not significantly altered after either type of meal. The authors concluded that non-fasting TG concentrations to be stronger predictor for the risk of CVD than fasting TG, because of the higher concentration of a larger sized RLP particles expressed as higher RLP-TG along with a small amount of LPL in the postprandial plasma compared to the fasting plasma. Therefore, non-fasting TG measurements performed 3 to 6 hours after food intake may be able to take the place of the direct measurement of RLP-TG for the assessment of cardiovascular disease risk. The study was published in the February 2017 issue of the journal Clinica Chimica Acta.

Dysbiosis of Urinary Microbiota Positively Correlated with Diabetes ype 2 diabetes mellitus (T2DM) accounts for 90% of diabetes, and T2DM is not due to insufficient use of insulin but due to insufficient insulin secretion and insufficient insulin action. Hospitalization rate for urinary tract infection (UTI) caused by diabetes is over twice as much as those caused by other factors. Damage to the genitourinary system caused by diabetic neuropathy results in bladder dysfunction, and increases the probability of UTI. High levels of urine glucose (UGLU) can favor a proper microenvironment for UTI due to increased bacterial overgrowth and female patients are known to have higher prevalence of UTI than males, which may be associated with the anatomical and structural differences in the urethra between genders. Scientists at Zhejiang University (Hangzhou, Zhejiang, China; www.zju.edu.cn) investigated alterations of urinary microbiota in Chinese female T2DM patients, and explored the associations between urinary microbiota and a patient’s fasting blood glucose (FBG), urine glucose (UGLU), age,

menstrual status, and body mass index (BMI). They collected the modified mid-stream urine (MMSU) and asymptomatic bacteriuria is defined as the presence of two consecutive MMSU specimens with isolations of the same bacterial strain at more than 105 CFU/mL. The matched case-control study enrolled 70 patients with T2DM patients and 70 healthy controls (HCs) from June 2015 to January 2016. Total DNA was extracted from the pellet of urine from Tubes 2 and 3, and 40 mL of urine was aspirated from each tube, separated into three sections, and injected into three 15 mL sterile centrifuge tubes. Magnetic bead isolation of genomic DNA from bacteria was performed and the concentration of extracted DNA was determined by using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA; www.thermofisher.com). Microbial diversity and composition were analyzed using the MiSeq sequencing platform (Illumina, San Diego, CA, USA; www.illumina.com) by targeting the hypervariable V3-V4 regions of the 16S rRNA gene. The investigators found that found that bacterial

diversity was decreased in T2DM patients. Increased Actinobacteria phylum was positively correlated with FBG, UGLU, and BMI; Lactobacillus abundance decreased with age and menopause; and increased Lactobacillus correlated positively with FBG and UGLU. Decreased Akkermansia muciniphila was associated with FBG and UGLU. Escherichia coli abundance did not differ between the two cohorts. Carbohydrate and amino acid metabolism was reduced in T2DM patients, which were associated with bacterial richness indices. The authors concluded that microbiota dysbiosis may be associated with T2DM. Secondly, the relative abundance of some key bacteria in T2DM patients was different than in the HCs, and the relative abundancies were affected by the patients’ characteristics. Lastly, there was interdependency between urine microbiota and the patients’ metabolism. Future studies should focus on how the urinary microbiota affects patient’s characteristics such as FBG and UGLU. The study was published on December 19, 2016, in the journal Oncotarget. LabMedica International November/2017

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Breast Cancer Diagnosed by Measuring Circulating Tumor DNA n unintended consequence of a recent drug study was the discovery that measurement of circulating tumor DNA (ctDNA) could be used to diagnose a type of breast cancer with an unamplified HER2 mutation. HER2 is a member of the human epidermal growth factor receptor family. Amplification, over-expression, or a mutation of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. In recent years the protein has become an important biomarker and target of therapy for approximately 30% of breast cancer patients. Investigators at Baylor College of Medicine (Houston, TX, USA; www.bcm.edu) conducted a single arm phase II trial to assess the clinical benefit rate of the drug neratinib, a dual inhibitor of Her2 and epidermal growth factor receptor (EGFR) in HER2-mutated non-amplified metastatic breast cancer. Neratinib blocks the action of its target proteins by covalently binding to a cysteine side chain. To conduct the study, the investigators required biopsy material from the tumor in order to determine the presence of the HER2 mutation. However, this proved to be a major difficulty, as 20 to 30% of the patients could not provide sufficient material to make the diagnosis.

In contrast, baseline ctDNA sequencing identified the same HER2 mutation in 11 of 14 tumor positive cases and correctly assigned 32 of 32 informative negative cases. Additionally, ctDNA HER2-mutant variant allele frequency decreased in nine of 11 paired samples at week four, followed by an increase upon progression. “To assist in our ability to identify patients with HER2 mutation-positive tumors, we conducted circulating tumor DNA analysis,” said senior author Dr. Matthew Ellis, professor of oncology at Baylor College of Medicine. “The tumor’s DNA is released into the human bloodstream, and we were able to determine the presence of the mutation in blood samples from the patients. Importantly the circulating tumor DNA results were highly concordant with the tumor sequencing results, and they were much easier to determine. Notably, the blood test was sensitive enough that we could use it as a tool to determine eligibility for the clinical trial. A circulating tumor DNA-based blood test also could therefore be potentially used to monitor tumor progression and to determine whether patients are responding or not to treatment after just one month of therapy.” The study was published in the July 5, 2017, online edition of the journal Clinical Cancer Research.

Image: A cluster of circulating tumor cells (CTCs, shown in red) originated from the blood of a breast cancer patient (Photo courtesy of the NIH).

Pre-Op Cholesterol Level Predicts RCC Patients Survival enal cell carcinoma (RCC) is the most frequently diagnosed renal malignancy. The constant advances of modern imaging technologies and the percentage of incidentally detected renal tumors has constantly increased during the last couple of decades, and a good percentage of patients are still diagnosed with metastatic renal cell carcinoma (mRCC). Lipid metabolism is one of the important tumor metabolic mechanisms that are essential to tumor survival and progression. Since cholesterol is an essential cellular component that plays a crucial role in lipid metabolism, preoperative serum cholesterol level (PCL) may have significant correlation with prognosis in RCC patients. A large team of scientists led by those at the Seoul National University Bundang Hospital, (Seongnam, South Korea; www.snuh.org) retrospectively analyzed the data of 244 patients diagnosed with mRCC and initially treated with nephrectomy at multiple centers of South Korea. Every patient was initially evaluated using chest computed tomography (CT) (or simple radiography), abdominal CT, and bone scan. The PCL was

LabMedica International November/2017

included in the routine chemistry panels, which were performed as a part of preoperative anesthetic risk evaluation within 4 weeks preceding the surgery. Patients were stratified into two groups according to the PCL cut-off level of 170 mg/dL. There were 88 patients in the high PCL group and 156 patients in the low PCL group. The low PCL group showed significantly lower hemoglobin level and higher platelet level than the high PCL group, but no significant differences were noted in the other clinical characteristics or pathological outcomes between the two groups. The median age was 59.0 years; median tumor diameter was 8.0 cm, median PCL was 156.0 mg/dL (interquartile range (IQR), 132.3–173.8 mg/dL), and median follow-up time was 13.0 months. The authors concluded that decreased preoperative serum cholesterol level was significantly correlated with worse survival outcomes in patients with metastatic renal cell carcinoma treated with cytoreductive nephrectomy. The underlined mechanism is still uncharted and requires further investigation. The study was published on May 25, 2017, in the journal BMC Cancer. LINKXPRESS COM

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Bacteria Links Crohn’s Disease to Spondyloarthritis eripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with active inflammatory bowel disease (IBD) characterized by inflammatory enthesitis, dactylitis, or synovitis of nonaxial joints. Patients with Crohn’s disease, a type of IBD that causes abdominal pain and diarrhea can also experience joint pain. In Crohn’s disease, which affects about 800,000 Americans, the immune system can attack not only the bowels, but the musculoskeletal system as well, leading to spondyloarthritis, a painful condition that affects the spine and joints. A large team of scientists working in conjunction with those at Weill Cornell Medicine College (New York, NY, USA; www.weill.cornell.edu) used fecal samples from patients with IBD to identify bacteria in the gut that were coated with antibodies called immunoglobulin-A (IgA) that fight infection. Using flow cytometry, in which fluorescent probes are used to detect IgA-coated bacterial species, the team discovered that IgA-coated Escherichia coli were abundant in fecal samples from patients with both Crohn’s disease and spondyloarthritis.

The investigators used both patient samples and mouse models and then linked these bacteria to cells that help regulate inflammation, known as Th17 cells, in people with autoimmune disorders. The investigators found that patients with Crohn’s disease and spondyloarthritis had higher levels of Th17 cells, and that a protein called interleukin-23 (IL-23) triggers their activity. E. coli isolates from CD-SpA–derived IgA-coated bacteria were similar in genotype and phenotype to an adherent-invasive E. coli (AIEC) pathotype. By modeling the increase in mucosal and systemic TH17 immunity they observed in CD-SpA patients, colonization of interleukin-10–deficient or K/BxN mice with CDSpA–derived E. coli lead to more severe colitis or inflammatory arthritis, respectively. Randy Longman, MD, PhD, a gastroenterologist and mucosal immunologist and a principal investigator of the study said, “In IBD therapy, this is a step toward precision medicine; to be able to clinically and biologically characterize a subtype of disease

and then select the medicine that would best fit the patient with this type of inflammation. The results of this innovative study will start to inform our decision of which of our available medications will give the best chance of helping the individual patient.” The study was published on February 8, 2017, in the journal Science Translational Medicine. Image: Escherichia coli bacteria (red), which are abundant in the immunoglobulin-A-coated microbiota of patients with a Crohn’s disease-associated condition called spondyloarthritis, promote systemic inflammation. The blue circular structures depict the nuclei of cells called epithelial cells (Photo courtesy of Dr. Kenneth Simpson, Cornell University).

Liquid Filtration System Captures Circulating Tumor Cells stand-alone lab-on-a-disc system captures from the blood 95% of circulating tumor cells (CTCs), which can be analyzed for early detection of cancer metastasis and for monitoring response to various cancer treatments. Investigators at Ulsan National Institute of Science and Technology (Republic of Korea; www. unist.ac.kr) used the Fluid Assisted Separation Technology (FAST) system to detect CTCs in the blood of 142 patients with various cancers and 50 healthy control subjects. The FAST system is based on a series of antifouling membranes with liquid-filled pores.

These pores enable clog-free, highly sensitive, selective, rapid, and label-free isolation of viable CTCs from whole blood without prior sample treatment. Numerical simulation and experiments showed that this method provided uniform, clog-free, ultrafast cell enrichment with pressure drops much less than in conventional size-based filtration. Results obtained with the FAST system showed that CTCs isolated from the blood of patients with lung cancer, contained the same genetic information as found in histologic examination. This indicated that the FAST technology

could be used for molecular diagnosis or customized medical treatment. “This technology can be directly used by hospitals because it uses small equipment and is very simple to use,” said senior author Dr. Yoon-Kyoung Cho, professor of biomedical engineering at Ulsan National Institute of Science and Technology. “This will enable early diagnosis of metastatic cancer as well as patient-tailored cancer treatment.” The FAST system was described in detail in the January 2017 issue of the journal Analytical Chemistry. LabMedica International November/2017

Edited by Tahir Pillay MBChB, PhD, FRCPath(Lon), FCPath(SA)

NEWS

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 Africa Tel: (27) 012-319-2114; Fax: (27) 012-328-3600; Email: enews@ifcc.org

IFCC Regional Representatives Elected for 2018-2020 Term he IFCC Nominations Committee is happy to announce the IFCC Regional Representatives for 2018-2020. We warmly congratulate the newly elected IFCC Regional Federation Representatives to the IFCC Executive Board for the period January 1st, 2018 until December 31th 2020. We wish the new EB the best success to ensure the promotion of clinical chemistry and laboratory medicine world-wide, promoting the 'added value' of laboratory medicine to healthcare, enhancing international clinical collaboration and education and management support to developing countries, and communicating good laboratory medicine practices and patient safety. The result of the ballot required to confirm (or reject) the IFCC Regional Federation Representatives’ nominations, for the term 2018-2020, was concluded on September 30th. The IFCC Executive Board for 20182020, will be composed as follows: President: Prof Howard Morris (Australia); Past President: Prof Maurizio Ferrari (Italy); Secretary: Dr David Kinniburgh (Canada); Treasurer: Prof Tomris Ozben (Turkey); Regional Federation Representatives: African Federation of Clinical Chemistry (AFCC), Prof Adekunle Bashiru Okesina (Nigeria); Arab Federation of Clinical Biology (AFCB), Prof Abderrazek Hedhili (Tunisia); Asia-Pacific Federation for Clinical Biochemistry and Laboratory Medicine (APFCB), Dr Sunil Sethi (Singapore); European Federation of Clinical Chemistry and Laboratory Medicine (EFLM), Prof Sverre Sandberg (Norway); Latin-American Confederation of Clinical Biochemistry (COLABIOCLI), Dr Rosa Sierra-Amor (Mexico); North American Federation of Clinical Chemistry and Laboratory Medicine (NAFCC), Dr Ann Gronowski (USA); Corporate Representative: Dr. Rolf Hinzmann (Roche Diagnostics). The confirmation of the results has been officially presented at the Council on Sunday 22 October in Durban. On behalf of the Nomination Committee, we would like to thank the candidates who campaigned and took actively part in the IFCC electoral process as well as for their steadfast commitment to serving IFCC. We congratulate warmly the newly elected IFCC Regional Federation Representatives. Best wishes to the new Executive Board for a productive term 2018-2020!

IFCC OFFICE Via Carlo Farini 81, 20159 Milan, ITALY Tel: (39) 02-6680-9912 • Fax: (39) 02-6078-1846 E-mail: ifcc@ifcc.org • Web: www.ifcc.org Office Hours: 8.30-13.00 and 13.30-17.30 Staff Members: Paola Bramati, Silvia Cardinale, Silvia Colli-Lanzi

LabMedica International November/2017

Adekunle Bashiru OKESINA African Federation of Clinical Chemistry (AFCC)

Abderrazek HEDHILI Arab Federation of Clinical Biology (AFCB)

Sunil SETHI Asia-Pacific Federation for Clinical Biochemistry and Laboratory Medicine (APFCB)

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North American Federation of Clinical Chem. and Laboratory Medicine (NAFCC)

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

NEWS VIEWPOINT

Vaccination: A Public Health Issue by Dr. Bernard Gouget Counselor for Public Health FHF; Chair-Human Health Care Committee-COFRAC; IFCC Chair-Nominations Committee; General Secretary of the International Francophone Federation Of Clinical Biology and Laboratory Medicine (FIFBCML)

or many of us, it will soon be ‘flu season. ‘Flu vaccines are the first step to take before winter, without forgetting other procedures that can reduce propagation. ‘Flu vaccination prevented 39% of hospitalizations for confirmed ‘flu in the elderly for the winter of 2015-2016 in Europe. More particularly, vaccination reduced the ‘flu hospitalization rate of diabetics by 62%, people with lung disease by 60%, and those with heart disease by 36%. Nevertheless, the low efficacy of the ‘flu vaccine, particularly in the elderly, seems to call into question the strategies deployed for several years. Two hypotheses could explain it: the appearance of mutations or a reduction of immune response brought about by vaccina-

tion. Analysis of data according to the date of vaccination shows that vaccine efficacy versus A(H3N2) was null four months after vaccination. Efficacy versus A(H1N1) remained stable throughout the entire season and there was only a slight decrease at the end of the season versus influenza B. In response to a request of the European Medicines Agency to provide efficacy data by type of vaccine and by brand, it was shown that adjuvanted vaccines appeared to be more effective. Even though, with the current vaccines, the reallife efficacy is rarely greater than 50%, we should continue to assess vaccine efficacy for purposes of diversified prevention, based, in particular, on vaccination, hand hy-

giene, wearing masks, and in some situations, prophylactic antivirals. A recent study showed that in France alone, a quarter of the healthcare professionals are "covered" with very strong disparities between the medical and paramedical professions. There is an issue of the credibility of public action. The efficacy of vaccination in people responsible for the frail elderly in institutions remains controversial. But the benefit of this so-called altruistic vaccination of healthcare professionals to prevent the transmission of ’flu to residents shows, according to a Cochrane metaanalysis, that health professional vaccination reduces all-cause mortality in residents by 30%. As a health care professional, one must be extremely clear and as factual as possible, by saying that immunization primarily serves to protect oneself. It would be a shame not to set an example as a caregiver, given that several indicators raise concerns about a reduction in the number of children properly vaccinated. There is a non-negligible risk of seeing the return of serious diseases, such as diphtheria or tetanus. Lower vaccine coverage would also allow apparently milder pathogens to spread more widely during epidemics. Also, the Prof. Agnès Buzin, new French Minister of Health, is going to extend mandatory vaccinations for young children. Today to be admitted into nursery school, kindergarten or elementary school, three vaccines are necessary (against diphtheria, tetanus and poliomyelitis). In 2018, eleven will be mandatory. Vaccines for pertussis, Haemophilus influenzae b, measles, mumps, rubella, meningococcus C, pneumococcus and hepatitis B will be added to the three above. This is a shock measure, planned for a limited duration, maybe for five to ten years. The objective is to improve coverage, especially for measles; Europe has been observing a resurgence since 2008 (24,000 cases). Italy has gone further, making 12 vaccines mandatory in June and Germany, without establishing mandatory vaccination, now re-

quires proof of a pediatric consultation on vaccination to enroll a child in school. Although complications remain rare, as soon as the first thousand people are infected, drama is inevitable, especially among the most frail populations, such as those whose immune defenses are reduced or infants who have not yet been vaccinated. This is true. But is mandatory vaccination really the solution? In Europe, only twelve countries have mandatory requirements for at least one disease and fifteen confine themselves to recommended vaccines, for coverage that varies greatly depending on the country and the disease concerned. For the 90 years that aluminum has been used as a vaccine additive, it has become the ideal scapegoat: autism, MS, it is suspected of being a neurotoxin and triggering autoimmune reactions in a small part of the population, although the causal relationship is not resolved. In the country of Pasteur, we talk about vaccines with devotion or hatred, but factual discourse is non-existent. On both sides, we are asked to believe, while we want to understand. It is time we free ourselves of the myths and fears, and abandon simplified and generalized discourse on vaccination. Each vaccine has different issues and distinct advantages and disadvantages. Faced with uncertainty, we are more easily manipulated, especially via the Internet. The lack of information leads to over-interpretation of the risks that exist, similar to all medicines. But they remain very low: on the individual scale, there is little to fear from vaccines. Adverse reactions are much less frequent than those reported for conventional medicines. Vaccination is still one of the pillars of our societies today: it allows us to benefit from this global microbial celebration that is life. In combination with hygiene and antibiotics, it has increased our life expectancy by thirty years and, in one century, reduced by 25-fold the risk of seeing children die before their first birthday. It deserves better than polarized and cartoonish debates. However, with regard to the benefit for the general population, it is necessary to continue to study each vaccine meticulously, one by one, without preconceived ideas, with only the tools of reading scientific articles as well as epidemiological, pharmacovigilance, sociological and economic data.

LabMedica International November/2017

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

NEWS

IFCC Welcomes a New Member: Panama he IFCC is happy to announce that the “Colegio Nacional de Laboratorístas Clínicos de Panamá” (CONALAC) joined the Federation as its 92th Member. The “Colegio Nacional de Laboratorístas Clínicos de Panamá” is a Clinical Chemistry and Laboratory Medicine professional organization. Members relationships are excellent and this allows CONALAC Panama to develop activities for the benefit of health; the Colegio Nacional de Laboratorístas Clínicos de Panamá is well recognized by the community, by other professionals of the sector and by authorities. Its mission is to strengthen the process of unity of Clinical Laboratories in a continuous way to reach, ensure and maintain the most profound principles of integrity and union improvement in order to contribute to the health of the community.

Photo: CONALAC Executive Board members 2017 2020

New Issue of eJIFCC Available JIFCC Vol 28, n°3 is now available on the IFCC website (ifcc@ifcc.org). In this issue you will find the following articles: "Postprandial dyslipidaemia: pathophysiology and cardiovascular disease risk assessment"; "e-Learning: a model to support ongoing education"; "Compendium of Terminology and Nomenclature of Properties in Clinical Laboratory Sciences”; "Correlation of HbA1C levels with body mass index in newly diagnosed polycystic ovary syndrome"; "Factors affecting quality of laboratory services in public and private health facilities in Addis Ababa, Ethiopia"; and "Retrospective approach to evaluate interferences in immunoassay". The issue also features a "Case report: biliary pancreatitis with acute cholangitis in a patient under anticoagulant treatment with dabigatran". Subscribe to eJIFCC free mailing list sending an email to ifcc@ifcc.org

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Industry News

Top Trends Impacting Global POC Testing Market he global point-of-care (POC) HbA1C testing market is projected to grow at a CAGR of almost 9% from 2017-2021, driven mainly by the unhealthy lifestyles of people resulting in increased prevalence of diabetes and obesity, easy patient accessibility to POC tests and faster availability of results as compared to conventional laboratory testing procedures. These are the latest findings of Technavio Research, (London, UK; www.technavio.com), a global technology research and advisory company. According to Technavio, the following three factors are contributing to the growth of the global POC HbA1C testing market:

Rising equipment rental agreements: The growth in online marketing has helped end-users who demand time-efficient tests to obtain the required products within a short time. It is estimated that about 70% of the US population used online shopping portals to purchase various products in 2015. “Vendors are using promotional strategies such as online marketing to offer better accessibility of their products to end-users, which, in turn, will increase their product sales,” said Srinivas Sashidhar, a lead in-vitro diagnostic research analyst at Technavio. “Online marketing strategies also help the vendors in minimizing business setup, distribution, and op-

Thermo LC-MS Solutions Receive ANVISA Registration in Brazil hermo Fisher Scientific’s (Hampton, NH, USA; www. thermofisher.com) liquid chromatography mass spectrometry (LCMS) in vitro diagnostic (IVD) systems, which enable the quantitation of analytes in complex matrices, have received registration by Brazil’s National Health Surveillance Agency (ANVISA), allowing clinical laboratories in the country to now screen and quantify drugs efficiently. The Thermo Scientific Endura MD mass spectrometer (MS), Prelude MD high performance liquid chromatography (HPLC) system and ClinQuan MD software have been recognized by ANVISA as capable of meeting its Good Manufacturing Practice (GMP) requirements. This will allow clinical laboratories in

Brazil to leverage the high sensitivity and quantitative accuracy of Thermo Fisher Scientific’s LC-MS IVD systems for developing and validating their own methods for the analysis of a large range of drug analytes. Both Prelude MD and Endura MD combine the capabilities of HPLC with Thermo Scientific TurboFlow technology to enable automated in-line sample cleanup, thus eliminating the need for error-prone manual tasks. Both the systems are powered by the ClinQuan MD software, which is designed to safely store, retrieve and process data from laboratory-developed IVD tests. In line with the Clinical Laboratory Improvements Amendments (CLIA), the software generates time-stamped audit trails to ensure the integrity of results is preserved at all times.

erational costs. They help end-users by providing information about devices and their types.” Increased diabetic prevalence: Providers of POC HbA1C testing and glucose meters are focused mainly on enabling quick measurements and improving optical sensitivity, such as infrared spectroscopy, for the development of non-invasive POC testing meters. Hence, the efforts are being focused on collaborating HbA1c testing with other tests, such as those for cholesterol. “Combining HbA1c testing with urine analysis is also an effective solution for measuring microalbuminuria and nephropathy,” said Srinivas. “These expanded menus will provide a wider scope of test menus on a single platform along with efficient workflow for diagnostic laboratories.” The increasing prevalence of diabetes and the diagnosis of pre-diabetes are also driving the sales of POC HbA1C testing kits among hospitals. The shift among hospitals from conventional diagnostic procedures for glucose monitoring to improved methods is resulting in faster delivery of results and quicker initiation of treatment procedures for patients. The increase in diabetes HbA1C testing is also fueling the demand for consumables. Hospitals also avail lucrative discounts on bulk purchase of diagnostic kits and consumables, thus helping them controls their expenditure on laboratory supplies and consequently, helping the end-users to reduce costs. High adoption rate of molecular POC testing: The introduction of

Companies with Lab-Developed Tests Raise USD 2 Billion ompanies with laboratorydeveloped tests (LTDs), tests performed by laboratories owned by IVD companies and others, are attracting increasing investor interest and raised over USD 2 billion between January and August 2017. These are the latest findings of Kalorama Information, (New York, NY, USA; www.kaloramainformation.com), an independent medical market research firm. LDTs, also called as “home-brew tests” or “in-house tests” were historically low-volume, simple and wellcharacterized tests for low-risk diagnostic applications. However, in recent years, there has been increased focus and attention on the emergence and growing use of complex LDTs based on technologies such as polymerase

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POC diagnostics has brought about a change in the way digital platforms function. The diabetic population monitors their clinical conditions by using personal health records, such as electronic health records (EHRs), which helps vendors to focus on developing mobile apps for the remote use of portable diagnostic devices. A number of non-profit organizations and governments are supporting the ongoing researches on digital healthcare platforms. Regarding the competitive landscape, the POC HbA1C testing market is comprised of several regional and global players who compete against each other on the basis of pricing, product differentiation, technological advancements, and strength in distribution channel. Currently, vendors are making increased investments for acquiring advanced technologies and are also diversifying their product portfolios. The intense competition among existing players makes it difficult for new players to enter the market. Region-wise, the adoption of POC HbA1C testing is high in the Americas due to growing patient awareness, sophisticated healthcare infrastructure and quick adoption of advanced technologies in the region. Growing patient awareness is expected to provide a boost to the application of POC hemoglobin A1c testing devices in home care settings. The implementation of the patient protection and affordable care act (PPACA) is expected to help the POC HbA1C testing market in the Americas continue recording growth during the forecast period.

chain reaction (PCR), microarrays, next generation sequencing, or other complex technology. High-risk, complex tests have now been developed as LDTs in order to provide clinical results to physicians and their patients. Presently, LDTs are being used for complex testing which smaller laboratories are not equipped or do not choose to handle and are used for a variety of medical conditions. There are all types of LDTs, including histology and molecular testing to immunoassays; or equipment-intensive testing such as mass spectrometry or flow cytometry. The largest segments of the LDTs market are oncology, genetic (inherited) disorders, and infectious disease, although these tests can be developed and used for virtually all types of disorders. LabMedica International November/2017

International Calendar For a free listing of your event, or a paid advertisement in this section, contact:

International Calendar, LabMedica International P.O.Box 802214, Miami, FL 33280-2214, USA Fax: 1-954-893-0038 • E-mail: info@globetech.net

JANUARY 2018 The British Fertility Society (BFS) Annual Meeting. Jan 4-6; Liverpool, UK; Web: http:// fertilityconference.org 2018 Winter Conference on Plasma Spectrochemistry. Jan 8-13; Fernandina Beach, FL, USA; Web: http://icpinformation.org Gordon Research Conference & Seminar Origins of Life. Jan 14-19; Galveston, TX, USA; Web: www.grc.org CBRD 2018 – 3rd Caribbean Biomedical Research Days. Jan 16-18; Rodney Bay, Saint Lucia; Web: www.stressandbehavior.com

FEBRUARY 2018 SLAS 2018 – Society for Laboratory Automation and Screening. Feb 3-7; San Diego, CA, USA; Web: www.slas2018.org MEDLAB 2018. Feb 5-8; Dubai, UAE; Web: www.medlabme.com Labquality Days 2018. Feb 8-9; Helsinki, Finland; Web: www.labqualitydays.fi 13th Annual Biomarkers Congress. Feb 1516; Manchester, UK; Web: www.biomarkerscongress.com Pittcon Conference and Expo 2018. Feb 26Mar 1; Orlando, FL, USA; Web: www.pittcon.org 21th Annual Mayo Clinic Endocrine Update 2018. Feb 26-Mar 2; San Juan, PR, USA; Web: https://ce.mayo.edu

MARCH 2018 Global Engage’s 4th Biologics Congress. Mar 5-6; Berlin, Germany; Web: www.global engage.co.uk KIMES 2018. Mar 15-18; Seoul, South Korea; Web: www.kimes.kr Medical Fair India. Mar 16-18; Mumbai, India; Web: http://medicalfair-india.com The Endocrine Society Meeting. Mar 17-20; Chicago, IL, USA; Web: http://2018endo.org ArabLab 2018. Mar 18-21; Dubai, UAE; Web: www.arablab.com LabTechMed/ExpoMed Eurasia 2018. Mar 22-25; Istanbul, Turkey; Web: www.expomedistanbul.com AIUM Annual Convention 2017 – American Institute of Ultrasound in Medicine. Mar 2428; New York, NY, USA; Web: www.aium.org

APRIL 2018 MEDLAB Asia Pacific. Apr 2-4; Singapore; Web: www.medlabasia.com World Vaccine Congress 2018. Apr 3-5; Washington DC, USA; Web: www.terrapinn.com KOREA LAB 2018. Apr 17-20; Seoul, South Korea; Web: www.korealab.org EuroPrevent 2018. Apr 19-21; Ljubljana, Slovenia; Web: www.escardio.org ECCMID 2018 – 26th European Congress of Clinical Microbiology and Infectious Diseases. Apr 21-24; Madrid, Spain; Web: www.eccmid.org

MAY 2018 AAI Immunology 2018 – American Association of Immunologists. May 4-8; Austin, TX, USA; Web: www.aai.org Kenya Laborum 2018. May 10-12; Kenya; Web: http://kenyalaborum.com ISLH 2018 International Society of Laboratory Hematology. May 10-12; Brussels, Belgium; Web: www.islh.org ICC 2018 – 20th International Congress of Cytology. May 14-15; London, UK; Web: www.cytologyjapan2016.com SEACare 2018 – Southeast-Asian Healthcare Show. May 14-16; Kuala Lumpur, Malaysia; Web: www.expocheck.com AACE 2018 – 27th Annual Meeting and Clinical Congress of the American Association of Clinical Endocrinologists. May 16-20; Boston, MA, USA; Web: www.aace.com European Congress of Endocrinology. May 19-22; Barcelona, Spain; Web: www.esehormones.org Molecular Diagnostics Europe. May 22-24; Lisbon, Portugal; Web: www.moleculardx europe.com HOSPITALAR 2018. May 22-25; Sao Paulo, Brazil; Web: www.hospitalar.com EuroPCR 2018 – European Association of Percutaneous Cardiovascular Interventions

LabMedica International November/2017

Congress. May 22-25; Paris, France; Web: www.europcr.com 25th Biennial International Congress on Thrombosis. May 23-26; Venice, Italy; Web: www.thrombosis2016.org EEACI 2018 – European Association of Allerology and Clinical Immunology. May 2630; Munich, Germany; Web: www.eaaci.org ESPID 2018 – European Society for Paediatric Infectious Diseases. May 29-Jun 2; Malmo, Sweden; Web: http://lp.www2.kenes.com

JUNE 2018 ISBT 2018 – 35th International Society Blood Transfusion Congress. Jun 2-6; Toronto, Canada; Web: www.isbtweb.org 2018 BIO International Convention. Jun 4-7; Boston, MA, USA; Web: http://convention.bio.org European Congress of Cytology. Jun 10-13; Madrid, Spain; Web: www.cytology2018.com 14th Annual Biomarkers & Immuno-Oncology World Congress. Jun 11-13; Boston, MA, USA; Web: www.biomarkerworldcongress.com ESHG 2018 – European Human Genetics Conference. Jun 16-19; Milan, Italy; Web: www. eshg.org International Congress of African Society for Blood Transfusion (AfSBT). Jun 19-22; Arusha, Tanzania; Web: www.afsbt.org FOCIS 2018 – Federation of Clinical Immunology Societies. Jun 20-23; San Francisco, CA, USA; Web: www.focisnet.org

JULY 2018 ESHRE 2018 – European Society for Human Reproduction and Embryology Annual Meeting. Jul 1-4; Barcelona, Spain; Web: www.eshre.eu AACC 2018 – Annual Meeting of American Association for Clinical Chemistry. Jul 29-Aug 2; Chicago, IL, USA; Web: www.aacc.org

AUGUST 2018 FIME 2018 – Florida International Medical Exhibition. Aug 7-9; Orlando, FL, USA; Web: www.fimeshow.com

SEPTEMBER 2018 Eurotox 2018 – 54th Congress of the European Societies of Toxicology. Sep 2-5; Brussels, Belgium; Web: www.eurotox-congress.com ESP 2018 – 30th European Congress of Pathology. Sep 9-12; Bilbao, Spain; Web: www.esp-pathology.org ESCV 2018 – 21th Annual Meeting of the European Congress of Virology. Sep 23-26; Athens, Greece; Web: www.escv.org MEDLAB Europe 2018. Sep 25-27; Barcelona, Spain; Web: www.medlabeurope.com ESPE 2018 – 57th Annual Meeting of European Society of Paediatric Endocrinology. Sep 27-29; Athens, Greece; Web: www.espe2016.org BSACI 2018 – British Society of Allergy & Clinical Immunology Annual Meeting. Sep 30-Oct 2; Telford, UK; Web: www.bsaci.org

OCTOBER 2018 ASHI 2018 – 44th Annual Meeting of the American Society for Histocompatibility and Immunogenetics. Oct 1-5; Baltimore, MD, USA; Web: www.ashi-hla.org ASCP 2018 – American Society for Clinical Pathology. Oct 3-5; Baltimore, MD, USA; Web: www.ascp.org BCLF 2018 – Meeting of Balkan Clinical Laboratory Federation. Oct 3-5; Skoplje, Macedonia; Web: www.bclf.info 5th Joint EFLM-UEMS Congress Laboratory Medicine at the Clinical Interface. Oct 10-13; Antalya, Turkey; Web: www.ifcc.org ASHG 2018 – The American Society of Human Genetics. Oct 16-20; San Diego, CA, USA; Web: www.ashg.org ANALYTICA CHINA 2018. Oct 31-Nov 2; Shanghai, China; Web: www.analyticachina.com

NOVEMBER 2018 Association for Molecular Pathology (AMP) Annual Meeting. Nov 1-3; San Antonio, TX, USA; Web: www.amp.org Medica 2018. Nov 12-15; Dusseldorf, Germany; Web: www.medica.de

LabMedica International Inq.No.

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Inq.No.

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Inq.No.

Vol. 34 No.7 • 11/ 2017 Advertiser

Page

108 77 Elektronika . . . . . . . . . . .8

110 DxGen . . . . . . . . . . . . . . . .10

– MEMBS 2017 . . . . . . . . . .62

– AACC . . . . . . . . . . . . . . . . .61

164 Dymind . . . . . . . . . . . . . . . .64

115 Mindray . . . . . . . . . . . . . . .15

– ACBICON 2017 . . . . . . . . .65

112 EKF . . . . . . . . . . . . . . . . . .12

130 NG Biotech . . . . . . . . . . . .30

146 Agappe . . . . . . . . . . . . . . .46

102 ELITech Group . . . . . . . . . . .2

111 Nova Biomedical . . . . . . . .11

134 Alcor . . . . . . . . . . . . . . . . . .34

125 Erba . . . . . . . . . . . . . . . . . .25

117 Quantimetrix . . . . . . . . . . .17

122 Brand . . . . . . . . . . . . . . . . .23

127 Erba . . . . . . . . . . . . . . . . . .27

105 Randox . . . . . . . . . . . . . . . .5

120 Biohit . . . . . . . . . . . . . . . . .20

114 Euroimmun . . . . . . . . . . . .14

142 Rayto . . . . . . . . . . . . . . . . .42

129 Biokit . . . . . . . . . . . . . . . . .29

– ExpoMedical 2018 . . . . . . .65

119 Sekisui . . . . . . . . . . . . . . . .19

144 Boule . . . . . . . . . . . . . . . . .44

155 Hecht, Karl . . . . . . . . . . . . .55

136 SFRI . . . . . . . . . . . . . . . . . .36

132 Caretium . . . . . . . . . . . . . .32

128 HiMedia . . . . . . . . . . . . . . .28

103 Siemens Healthineers . . . . .3

167 Cellavision . . . . . . . . . . . . .67

109 Instrumentation Laboratory .9

107 Siemens Healthineers . . . . .7

147 Chemclin . . . . . . . . . . . . . .47

– KIMES 2018 . . . . . . . . . . .49

131 SNIBE . . . . . . . . . . . . . . . .31

– CMEF 2018 . . . . . . . . . . . .53

162 Labex . . . . . . . . . . . . . . . . .63

168 SNIBE . . . . . . . . . . . . . . . .68

124 Coris BioConcept . . . . . . . .24

148 Lee Company, The . . . . . .48

157 Sugentech . . . . . . . . . . . . .57

151 Denka Seiken . . . . . . . . . .51

133 Liftronik . . . . . . . . . . . . . . .33

159 Sugentech . . . . . . . . . . . . .59

135 Diagnostica Stago . . . . . . .35

– LabMedica.com . . . . . . . . . .6

– TradeMed.com . . . . . . . . . .50

137 Diagnostica Stago . . . . . . .38

145 Maccura . . . . . . . . . . . . . . .45

139 VEDA.LAB . . . . . . . . . . . . .39

143 Diagon . . . . . . . . . . . . . . . .43

140 Mast Group . . . . . . . . . . . .40

163 Vicotex . . . . . . . . . . . . . . . .63

141 Diasys . . . . . . . . . . . . . . . .41

113 Mayo Clinic . . . . . . . . . . . .13

138 Vircell . . . . . . . . . . . . . . . . .38

126 DRG . . . . . . . . . . . . . . . . . .26

116 Medix Biochemica . . . . . . .16

121 Zivak . . . . . . . . . . . . . . . . .21

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