News updates on | October 2015 | Volume ... · tive condition characterized by pro-gressive loss of...

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Developments in CSF biomarkers for AD Pg. 14

Colorimetric detection of HIV-1 Pg. 20

Novel confi rmation of HIV rapid testing Pg. 23

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Frances Bushrod, Ph.D.

An eventually fatal neurodegenera-tive condition characterized by pro-gressive loss of memory and cogni-tion, Alzheimer’s disease (AD) is the major cause of dementia globally. It is estimated that worldwide around 44 million people are suff ering from dementia; this has been predicted to triple by 2050 as the population ages, since AD increases expo-nentially aft er the age of 65. Much research has been carried out to elu-cidate modifi able risk factors that could prevent AD from developing in the fi rst place. And because the characteristic beta-amyloid plaques (Aβ) and neurofi brillary tangles ((NFT) eventually visible in cer-ebrospinal fl uid as well as at autopsy can begin to form up to two decades before clinical symptoms become evident, there has been a focus on much earlier diagnosis before any neuronal damage is apparent. In western Europe, however, there is good news regarding the “Dementia epidemic”. It was previously prog-nosticated, using data extrapolated from twenty years ago, that AD prev-alence would increase dramatically as a result of our ageing societies, incurring an almost insurmount-able burden for health services. But a recent analysis in Th e Lancet Neu-rology, which considered the fi nd-ings of large studies from the UK, Spain, Sweden and the Netherlands carried out between 2007 and 2013, reported a reduced prevalence at specifi c ages compared to the previ-ous generation. Th e authors suggest that this could be the result of the improved education and healthcare as well as standard of living that today’s senior citizens experienced from their early years until the pre-sent. Substantial progress has also been made in identifying modifi -able risk factors; evidence-based strategies to lower risk include abstaining from smoking, drinking alcohol in moderation, a ‘Mediter-ranean’ diet, and most importantly taking regular physical exercise (according to the Caerphilly study, which has followed the lifestyle and health of around 3000 initially

middle aged men from 1979 to the present). Recent studies have also linked serum Vitamin D defi ciency with AD, and a Mediterranean diet and endogenous synthesis through exposure to sunlight from outdoor exercise ensure optimal amounts of this vitamin.

And the bad news? Although the development of a specifi c and sensitive blood test, allowing very early diagnosis of AD based on levels of biomarkers (such as MAP kinase-activated protein kinase 5) is now on the horizon, there is still no drug that can cure AD. Th ose

available that regulate neurotrans-mitters only alleviate symptoms in some clinically diagnosed patients. Although such tests could facilitate drug discovery and development, it is questionable whether their routine use in advance of eff ective treatment would be ethical.

AD: fi rst the good news

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ContentsFRONT COVERFRONT COVER

FEATURESFEATURES

[6 - 17] ALZHEIMER’S DISEASE

[6 - 8] Alzheimer’s : biomarkers redefi ne disease

[10 - 12] Overview of biomarkers for prediciting Alzheimer’s disease

[14 - 17] Developments in cerebrospinal fl uid biomarkers for Alzheimer’s disease

[18] COMPANY PROFILE A success story for more than 35 years: Gonotec GmbH

[20 - 25] HIV TESTING

[20 - 22] Ultrasensitive colorimetric detection of HIV-1 p24

[23 - 25] Dilution testing as a novel alternative for confi rmation of HIV rapid diagnostic testing in resource-limited settings

[26] CASE STUDY Advanced automation system drives lab effi ciency while optimizing

use of staff time

REGULARSREGULARS[3] Editor’s letter

[28 - 30] Industry news

[31 - 34] Product news

[34] Calendar of events

Alzheimer’s disease (AD) biomarkers are derived from cerebrospinal fl uid (CSF) and plasma. Some bi-omarkers have been evaluated as part of regulatory guideline documents, often in Phase II drug develop-ment trials establishing safety and tolerance. Large-scale multi-centre trials are expected to validate biomarker candidates for use in Phase III studies. Nevertheless, AD biomarkers face several hurdles which they must cross before attaining their full potential.

Also in this issue :

Developments in CSF biomarkers for

Colorimetric detection of HIV-1 Pg. 20

Novel confi rmation of HIV rapid testing Pg. 23

HIV POC testing

by MP Biomedicals Pg.31


by Greiner Bio-One Pg.32

Hematology system

by BioSystems Pg.33

Alzheimer biomarkers redefine disease Pg.6

News updates on www.cli-online.com | October 2015 | Volume 39

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Challenges of timely or defi nitive diagnosis Th e timely diagnosis of AD is diffi cult because its early symptoms are shared by a variety of disorders with similar neuro-pathological features. Th ese include vas-cular dementia (VaD), frontotemporal lobe dementia (FTLD) and Lewy body dementia (LBD). Failure to distinguish AD from the others can be a challenge since clinicians are likely to select diff er-ent approaches to treatment. Still, given that AD-related molecular mechanisms precede symptoms, biomarkers have the potential to be used as early indicators and markers of pre-clinical pathological change.

One of the major, underlying challenges with AD is that defi nitive diagnosis is not possible so far in living patients. Making such a diagnosis requires clinical assess-ment of AD via biomarkers as well as post-mortem verifi cation of two main hallmarks: extracellular neuritic plaques

and neurofi brillary tangles formed by post-translational modifi ed tau protein. Th ese limitations mean that it is possi-ble to only make a probable AD diagno-sis “based on clinical criteria, including medical history, physical examination, laboratory tests, neuroimaging and neu-ropsychological evaluation.”

Beta-amyloid(1-42)Th e best known AD biomarker is beta-amyloid(1-42) in cerebrospinal (CSF) fl uid - customarily designated as CSF Aβ(1–42). Its level in AD patients has been shown to be signifi cantly reduced compared to controls, while levels of shorter Aβ(1–40) forms either remain unchanged or increase. Reduced levels of Aβ(1–42) are due to lower clearance of Aβ from the brain to CSF, as well as enhanced aggregation and plaque deposition. In addition, changes in CSF Aβ levels diff er based on the disease. Decreased levels of Aβ(1–38) correlate with frontotemporal lobe dementia (FTLD) while a reduction

in Aβ(1–37) levels is associated with Lewy body dementia (LBD). CSF Aβ levels are, however, not consid-ered to be reliable on their own. In June 2014, a meta-analysis on 14 studies and 1,349 patients by researchers at Imperial College London concluded that CSF Aß levels had “marginal clinical utility” and “cannot be recommended as an accurate test for Alzheimer’s disease.”To ensure greater accuracy in diagnosis, several studies recommend combining CSF Aβ(1–42) concentrations with Aβ42/Aβ40 ratios. However, this has not been uniformly accepted, as some researchers found no evidence of the utility of Aβ42/Aβ40 ratios.

A 42 and tTauAn alternative approach consists of com-bining CSF Aβ42 concentrations with another biomarker - total tau (tTau), which is a microtubule-associated pro-tein. One study in Germany has suggested using both CSF Aβ42 and total tau as well as the Aβ42/Aβ40 ratio for diagnosing AD.In healthy controls, levels of tTau increase with age, and some studies have sought to establish reference values for diff erent age groups. tTau levels are measurably higher in AD patients as compared with age-matched control subjects and may be a prognostic marker for conversion from mild cognitive impairment (MCI) to AD. Studies have also found high CSF tau lev-els in 90% of MCI cases progressing at a later date to AD, but not in cases with sta-ble MCI.

AD guidelines remain heterogeneous and in fl ux Th e heterogeneous and evolving nature of diagnostic choices above is also echoed in the work of professional societies. Th ere are currently two sets of guidelines for the diagnosis of asymptomatic and symptomatic Alzheimer’s disease. One was published in 2007 by an International Working Group (IWG), while the second consists of recommendations in 2011 by the US National Institute on Aging and the Alzheimer’s Association (NIA–AA). Th e NIA-AA criteria defi ne three phases in the progression of AD: preclinical AD, mild cognitive impairment due to AD and dementia due to AD. NIA-AA high-lights the need for signifi cant additional research “to validate the application of

Alzheimer’s : biomarkers redefi ne diseaseBiological markers (biomarkers) for Alzheimer’s disease (AD) assess risk as well as the presence and progression of the disease.

AD biomarkers are derived from cerebrospinal fl uid (CSF) and plasma. Some biomarkers have been evaluated as part of regulatory guideline documents, often in Phase II drug development trials establishing safety and tolerance. Large-scale multi-centre trials are expected to validate biomarker candidates for use in Phase III studies.

Nevertheless, AD biomarkers face several hurdles which they must cross before attaining their full potential.

– October 2015 ALZHEIMER’S DISEASE6

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biomarkers,” and acknowledges this is “likely to take more than a decade to fully accomplish.” Interpreting the two guidelines poses its own challenges. A comparison made by a team in the Netherlands “revealed dif-ferences in approach, terminology, and use of cognitive markers and biomarkers.” However, it found that patients “who meet the International Working Group criteria will also meet the NIA-AA criteria and vice versa,” and called for further research “to validate the criteria.

Biomarkers redefi ne ADIn 2010, one year before the NIA–AA rec-ommendations, the IWG revised its defi -nition of AD in order to provide “broader diagnostic coverage” of the disease’s clini-cal spectrum. Th e key reason for the revi-sion was “access to reliable biomarkers in vivo” which had “radically changed” the defi nition of AD.” In the coming years, it is clear that fi eld trials will be “needed to establish whether the diagnostic criteria will work eff ec-tively in clinical or research situations.”

Clinical and research requirementsTh e diff erence between clinical and research situations is an important one. A key question for both clinician and researchers is whether the new criteria “are meant for research purposes or for clinical use.” Th e former are targeted at fi elds like drug testing with their validation paving the way for (new) clinical criteria. Clinical criteria “have to pass a higher bar in terms of robustness and accuracy.”

Health authorities in the US and Europe have yet to formally qualify any AD bio-markers. Until then, most experts sug-gest that the IWG guidelines should be considered as research criteria, for use “in some drug trials and in academic medical settings that conduct clinical research.” Th e NIA/AA criteria, on their side, are published as a mixture of clinical- and research-grade, with the clinical portions of criteria meant for use in clinical set-tings, while “anything involving biomark-ers and all of the preclinical AD criteria are for research use.”

Phospho-tau provides third dimension to tTau and A (1–42)As of now, it is accepted that the best method for diagnosing AD in patients is to measure CSF levels of Aβ(1–42) and tTau, along with a third biomarker phos-pho-tau (P-tau). Several studies have sug-gested “that abnormal hyperphosphoryla-

tion of tau in the brain plays a vital role in the molecular pathogenesis of AD.” Tau is hyperphoshorylated at a poten-tial total of 39 sites in AD. Its detection at position 181 and 231 in particular are “signifi cantly enhanced in AD compared to controls.” Both have been shown to distinguish AD from controls and vascu-lar dementia (VaD), frontotemporal lobe dementia (FTLD) and Lewy body demen-tia (LBD).Th e combination of Aβ(1–42), tTau and P-Tau signifi cantly increases diagnostic validity for sporadic AD, yielding “a com-bined sensitivity of >95% and a specifi city of >85%. tTau and P-Tau have also been cen-tral to a major multi-centre trial in Europe, part of the European Alzheimer’s Disease Neuroimaging Initiative (E-ADNI).

Alternatives to CSFAD diagnosis has been largely based on collecting CSF and this is accompanied by major limitations. CSF requires lumbar puncture, which is invasive and has many potential side eff ects. Th e routine screen-ing of patients is therefore diffi cult. So too is their follow-up over several years. Given this, researchers have sought to search for AD biomarkers in other body fl uids. Saliva and urine are easily collected but “blood analysis is the gold standard.” Although the correlation of pathological changes in the brain to concentration of blood analytes remains unknown, a search for AD biomarkers in blood is likely to fi rst target “accepted CSF markers, such as Aβ and tau-related biomarkers,” and then extend to factors involved in infl am-mation, protein ageing and cell death, and cerebrovascular dysfunctions. It seems very plausible that, at some point in the next few years, the combining of diff erent blood-derived AD biomarkers leads to the

defi nition of a patient-specifi c signature. Th e pilot European trial on Alzhei-mer’s Disease Neuroimaging Initia-tives (E-ADNI) measured both CSF and plasma-derived Aβ and found that higher diagnostic accuracy was obtained with frozen rather than fresh samples. E-ADNI also confi rmed the feasibility of a mul-ticentre AD biomarker programme for future clinical trials.

Microarrays and mass spectrometryMeanwhile, researchers have been seek-ing to extend the search for other novel biomarkers. One target is to use screening technolo-gies such as microarrays and mass spec-trometry, supported by bioinformatics. Th ese would increase knowledge of dis-ease-related changes in order to uncover novel AD biomarkers, open the way to quick and inexpensive diagnosis of AD and for gauging therapeutic relevance. Some of the specifi c objectives here tar-get the measurement of Aβ oligomers, to improve diagnostic specifi city. A good example is surface-enhanced laser desorp-tion/ionization-time-of-fl ight-mass spec-trometry (SELDI-TOF-MS) which has emerged as an ideal method for the simul-taneous detection and quantifi cation of a variety of Aβ peptide cleavage products.,

Staying aheadAs disease modifying therapies based on new biomarkers are developed in clini-cal trials, it will be increasingly relevant to put the biomarkers to use. One way to do this is by accelerating the launch of interventions that arrest and (eventually) reverse AD.Th e outlook is encouraging. A study recently published by researchers at Wash-ington University in the US observes that clinicopathologic and more recent bio-marker data suggest that AD pathology “begins to accrue approximately 10 to 20 years before any cognitive signs or symp-toms”. Th is provides a window of oppor-tunity for the initiation of secondary prevention trials that aim to prevent the development of symptoms in individuals while they are still cognitively normal.Such steps were already foreseen by the NIA-AA criteria mentioned above. Th e guidelines note that if we can “defi nitively determine the risk of developing Alzhei-mer’s dementia in people who have bio-marker evidence of brain changes but are not showing outward symptoms, we will open an important window of opportu-nity to intervene with disease-modifying therapies, once they are developed.


New biomarkers could help identify asymptomatic patients at risk of developing Alzheimer’s, allowing them to be treated with yet-to-be-developed therapies.

001_013_cli_oct_2015.indd 8 2/10/15 13:17


001_013_cli_oct_2015.indd 9 2/10/15 13:17

IntroductionMild cognitive impairment (MCI), which commonly arises as a result of underlying neurodegenerative pathology, is a clini-cal syndrome characterized by insidious onset and gradual progression. Recently, much research has focused on delineating a set of biomarkers that provide evidence of such neurodegenerative pathology in living individuals and increasing atten-tion has been devoted to combine the information from imaging, genetic, clini-cal, behavioural and fl uid data to predict the conversion from MCI to AD. In this article, we fi rst review some of the litera-ture on MCI-to-AD conversion and their limitations. Subsequently, we review the results in Kong et al. [1]. According to the best of our knowledge, it is the fi rst paper that addresses the question of how to combine the whole genome single nucleo-tide polymorphism (SNP) data and high-dimensional whole-brain imaging data to

off er predictive values to identify subjects at risk for progressing to AD.

Methods for predicting progres-sion from MCI to ADTh ere are several studies assessing the rela-tive importance of diff erent modalities in predicting the diagnostic change from MCI to AD by using a small subset of biosigna-tures [2–6]. For example, Cui et al. simul-taneously examined multiple features from diff erent modalities of data [2]. In particular, they combined structural magnetic reso-nance imaging (MRI) morphometry, cer-ebrospinal fl uid biomarkers and neuropsy-chological measures to access an optimal set of predictors of conversion from MCI to AD. Th eir fi ndings suggested that structural changes within the medial temporal lobe (MTL), particularly the hippocampus, and the performance on cognitive tests that rely on MTL integrity provided strong prediction for MCI-to-AD conversion.

Recent studies focused on the analy-sis of longitudinal data to assess the dynamic changes of various biomarkers associated with the MCI-to-AD con-version. A prominent neural correlate of MCI–AD is volume loss within the MTL, especially within the hippocam-pus and entorhinal cortex [7, 8], with increasing atrophy in these structures from normal aging to MCI to AD [9, 10]. The importance of assessing MTL changes in tracking the progression of MCI to AD has been highlighted in vari-ous longitudinal studies of individuals with MCI-AD conversion. For instance, an increased likelihood of progressing to clinical dementia has been linked with documented diminished baseline hip-pocampal and entorhinal volumes in several studies [11, 12].

The aforementioned studies focused on the question of whether the MCI subjects progress to AD or not, i.e. treating the conversion as a binary response. How-ever, an important question remains, namely, how can we predict the time to conversion in MCI individuals, as well as determine the early markers of conver-sion? In Tarbert et al., the authors used 148 MCI subjects to identify the most predictive neuropsychological meas-ures [13]. In Li et al., the authors used 139 MCI subjects from the Alzheimer’s disease neuroimaging initiative phase 1 (ADNI-1) to evaluate the prediction power of brain volume, ventricular vol-ume, hippocampus volume, apolipopro-tein E (APOE) gene status, cerebrospinal fluid (CSF) biomarkers, and behavioural scores [14]. They found that baseline volumetric MRI and behavioural scores were selectively predictive, and their model can achieve a moderately accu-rate prediction with the value of an area under the curve of 0.757 at 36 months. In Da et al., the authors used 381 MCI subjects from ADNI-1 to evaluate how several biomarkers for predicting MCI-to-AD conversion including spatial pat-terns of brain atrophy, Alzheimer’s dis-ease assessment scale-cognitive subscale (ADAS-Cog) score, APOE genotype, and cerebrospinal fluid (CSF) biomarkers [15]. They have found that a combina-tion of spatial patterns of brain atrophy

Overview of biomarkers for predicting Alzheimer’s diseaseThe two aims of this article are to review some current methods of early diagnosis of Alzheimer’s disease (AD) and to discuss a new integration method proposed in Kong et al. [1]. We focus on how to combine the whole genome single nucleotide polymorphism (SNP) data and high-dimensional whole-brain imaging data to offer predictive values to identify subjects at risk for progressing to AD.

by Dr Dehan Kong, Prof. Kelly S. Giovanello, Eunjee Lee, Prof. P. Murali Doraiswamy and Prof. Hongtu Zhu


Figure 1. A plot of the hippocampus surface.

001_013_cli_oct_2015.indd 10 2/10/15 13:17

and ADAS-Cog score offers a good pre-dictive power of conversion from MCI to AD.

To the best of our knowledge, none of the previous studies have leveraged both genome-wide association study (GWAS) SNP data as well as high-dimensional whole-brain imaging data to exam-ine their combined value in identifying subjects at greatest risk for progressing to AD.

Predicting AD using combined imaging–whole genome SNP dataIn Kong et al., the authors focused on the MCI patients and combined information from whole brain MR imaging and whole genome data to predict the time to onset of AD in a 48-month national study of subjects at risk [1]. This study consid-ered 343 subjects with MCI enrolled in ADNI-1. The patients were followed over 48 months, with 150 participants pro-gressing to AD. The data can be treated as time-to-event data before those MCI subjects without conversion are censored data. One of the most popular models for the time-to-event data is the Cox propor-tional hazards model. The authors used

this model to account for the covariates that are associated with the time of the events, i.e. conversion from MCI to AD.

The candidate covariates include demo-graphic covariates (age, gender, handed-ness, mean education length, retirement percentage, and three dummy variables for the marital status), the APOE4 geno-type, the AD assessment scale-cognitive subscale (ADAS-Cog) score, the hip-pocampus surface data, the region of interest (ROI) volume data, the chromo-some-wise information and the signifi-cant SNP information. For each subject,

the radial distance was obtained from the baseline hippocampal surfaces data for each subject, which yielded two sets of 15 000 dimensional vectors denoting the surface data from both parts of the hippocampus. For better illustration, we have plotted the hippocampus surface in Figure 1.

In Kong et al., the authors treated each part of the hippocampi as a functional predictor, and applied functional prin-cipal component analysis, and selected seven functional principal component scores for each functional predictor, which explain approximately 70% of the variance [1]. These functional princi-pal component scores were taken as the summary measures for the hippocampus surface and put into the Cox regression model as predictors. For the chromo-some-wise information, they extracted the top two principal components of the SNP data along each chromosome as predictors. For the significant SNP infor-mation, the top 101 significant SNPs were picked up using a kernel machine method, and then their top five principal components (PCs) were calculated and been used as predictors.

– October 201511

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Specifi cally, they considered three candi-date models. Th e fi rst model is to fi t a Cox regression model with demographic, clin-ical and ADAS-Cog score as predictors as well as APOE. Th is model did not include any other imaging and genetic data. Th e second model is to fi t a Cox regression model with demographic, imaging and chromosome-wise predictors, but with-out the ADAS-Cog score and signifi cant SNPs information. As a comparison, they also considered the genetic information from genome-wide association analysis (GWAS). Th e third model is to fi t a Cox regression model with demographic, imaging and signifi cant SNP information, but without the ADAS-Cog score and chromosome-wise information.

Th ey compared the predictive value of the fi rst and second model, and receiver operating characteristic (ROC) analysis indicated that the fi rst model had a lower predictive value at 48 months than the second model. For the fi rst model, they only identifi ed APOE4 and ADAS-Cog score as the signifi cant predictors. For the second model, they found that combining full genetic SNP and high-dimensional imaging data had a much higher predic-tive value. In particular, SNPs on chro-mosomes 2, 10, 11, 15, 17 and 18, APOE4 genotype, surface morphology data of both hippocampi and volumes of hip-pocampus, amygdala and thalamus con-tributed signifi cantly. Th e fi ndings support those from previous MRI studies of volu-metric hippocampal changes in prodro-mal AD and extend them by fi nding that the possible prognostic value of combin-ing information from high-dimensional imaging and genetics may be superior to that provided by routine clinical cognitive testing data. Th e fi ndings also confi rm the association between APOE4 status and AD, and identify additional new markers on chromosomes 2, 10, 11, 15, 17 and 18 as signifi cant predictors for conversion. For the third model, they found that the predictive value is lower than that of the second model. Th is fi nding indicates that using the chromosome-wise information instead of the traditional signifi cant SNPs information would be favoured. Th ey sug-gest it may be due to the pitfalls of predic-tion using signifi cant SNPs [16].

Th ere are some limitations to their analysis. First, there are no test data for this study, and their fi ndings are based on the inter-val cross validation. Second, they did not include measures of pathology in the mod-els, as cerebrospinal fl uid and amyloid-PET

were available only in a small subset of indi-viduals in ADNI-1. It would be benefi cial to combine the data from ADNI-GO and ADNI-2 for future research.

SummaryIn this article, we fi rst reviewed some cur-rent methods of diagnosis of AD, and dis-cussed their limitations. Th en we reviewed the method and fi ndings in Kong et al. and discussed the novelty and advantages of their study and how their proposal can be used for early diagnosis of AD by combined imaging–whole genome SNP data [1].

AcknowledgementsTh is material was based upon work partially supported by the NSF grant DMS-1127914 to the Statistical and Applied Mathematical Science Institute. Th e research of Dr Zhu was supported by NSF grants SES-1357666 and DMS-1407655 and NIH grants MH086633, T32MH106440, and 1UL1TR001111. Th e content is solely the responsibility of the authors and does not necessarily represent the offi cial views of the NIH and NSF.

References1. Kong D, Giovanello KS, Wang Y, Lin W, Lee E, Fan

YD Doraiswamy PM, Zhu H, Alzheimer’s Neuroim-aging Initiative. Predicting Alzheimer’s disease using combined imaging-whole genome SNP data. J Alz-heimer’s Dis. 2015; 46(3): 695–702.

2. Cui Y, Liu B, Luo S, Zhen X, Fan M, Liu T, Zhu W, Park. M, Jiang T, Jin SE. Identifi cation of conversion from Mild Cognitive Impairment to Alzheimer’s dis-ease using multivariate predictors. PLoS One 2011; 6: e21896.

3. Davatzikos C, Bhatt P, Shaw LM, Batmanghelich KN, Trojanowski JQ. Prediction of MCI to AD conver-sion, via MRI, CSF biomarkers, and pattern classifi ca-tion. Neurobiol Aging 2011; 32: 2322.e19–2322.e27.

4. Dickerson BC, Wolk DA, Alzheimer’s Disease Neuroimaging Initiative. Biomarker-based pre-diction of progression in MCI: comparison of AD signature and hippocampal volume with spinal fl uid amyloid-β and tau. Front Aging Neurosci. 2013; 5: 1–9.

5. Young J, Modat M, Cardoso MJ, Mendelson A, Cash D, Ourselin S. Accurate multimodal probabilistic prediction of conversion to Alzheimer’s disease in patients with mild cognitive impairment. Neuroim-age Clin. 2013; 2: 735–745.

6. Zhang D, Shen D. Predicting future clinical changes of MCI patients using longitudinal and multimodal biomarkers. PLoS One 2012; 7(3): e33182.

7. Dickerson BC, Goncharova I, Sullivan MP, Forchetti C, Wilson RS, Bennett DA, Beckett LA, deToledo-Morrell L. MRI-derived entorhinal and hippocampal atrophy in incipient and very mild Alzheimer’s dis-ease. Neurobiol Aging 2001; 22: 747–754.

8. Xu Y, Jack CR, O’Brien PC, Kokmen E, Smith GE, Ivnik RJ, Boeve BF, Tangalos RG, Petersen RC.

Usefulness of MRI measures of entorhinal cortex versus hippocampus in AD. Neurology 2000; 54: 1760–1767.

9. Du AT, Schuff N, Amend D, Laakso MP, Hsu YY, Jagust WJ, et al. Magnetic resonance imaging of the entorhinal cortex and hippocampus in mild cognitive impairment and Alzheimer’s disease. J Neurol Neuro-surg Psychiatry. 2001; 71: 441–447.

10. Pennanen C, Kivipelto M, Tuomainen S, Hartikainen P, Hanninen T, Laakso MP, et al. Hippocampus and entorhinal cortex in mild cognitive impairment and early AD. Neurobiol Aging. 2004; 25: 303–310.

11. Jack CR, Petersen RC, Xu YC, O’Brien PC, Smith GE, Ivnik RJ, Boeve BF, Waring SC, Tangalos EG, Kokmen E. Prediction of AD with MRI-based hippocampal volume in mild cognitive impairment. Neurology 1999; 52: 1397–1403.

12. Killiany RJ, Gomez-Isla T, Moss M, Kikinis R, San-dor T, Jolesz F, et al. Use of structural magnetic reso-nance imaging to predict who will get Alzheimer’s disease. Ann Neurol. 2000; 47:430–439.

13. Tabert MH, Manly JJ, Liu X, Pelton GH, Rosenblum S, Jacobs M, Zamora D, Goodkind M, Bell K, Stern Y, Devanand DP. Neuropsychological prediction of conversion to Alzheimer disease in patients with mild cognitive impairment. Arch Gen Psychiatry. 2006; 63(8): 916–24.

14. Li S, Okonkwo O, Albert M, Wang M-C. Variation in variables that predict progression from MCI to AD dementia over duration of follow-up. American J Alzheimer’s Dis. 2013; 1: 12–28.

15. Da X, Toledo JB, Zee J, Wolk DA, Xie SX, Ou Y, Shacklett A, Parmpi P, Shaw L, Trojanowski JQ, Davatzikos C, Alzheimer’s Neuroimaging Initiative. Integration and relative value of biomarkers for pre-diction of MCI to AD progression: Spatial patterns of brain atrophy, cognitive scores, APOE genotype and CSF biomarkers. Neuroimage Clin. 2014; 4: 164–173.

16. Wray NR, Yang J, Hayes BJ, Price AL, Goddard ME and Visscher PM. Pitfalls of predicting complex traits from SNPs. Nat Rev Genet 2013; 14(7): 507–15.

The authorsDehan Kong1 PhD, Kelly S. Giovanello2,3

PhD, Eunjee Lee4 MS, P. Murali Doraiswamy5 MD, Hongtu Zhu*1,3,6 PhD1 Department of Biostatistics, University of North Carolina (UNC), NC, USA2 Department of Psychology, UNC, NC, USA3 Biomedical Research Imaging Center, UNC, NC, USA4 Department of Statistics, UNC, NC, USA5 Departments of Psychiatry and Duke Institute for Brain Sciences, Duke Univer-sity, Durham, NC, USA6 Department of Radiology, UNC, NC, USA

*Corresponding authorE-mail: [email protected]


001_013_cli_oct_2015.indd 12 2/10/15 13:17

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IntroductionAlzheimer’s disease (AD) is a neurode-generative disease that leads to cognitive impairment and ultimately dementia. The majority of the 45 million demen-tia patients world-wide have dementia due to AD [1]. In terms of brain pathol-ogy, AD is characterized by progressive accumulation of extracellular deposits of β-amyloid (Aβ) peptides in plaques and intracellular deposits of tau pro-teins in neurofibrillary tangles. The abnormal metabolism of Aβ and tau is believed to lead to impaired brain func-tion, loss of synapses and neurons, and cognitive decline. The cognitive domain that is most severely impaired in most AD patients is episodic memory, but other domains such as language, visuo-spatial performance, behaviour and executive function may also become affected. In a subset of patients, deficits in non-memory domains are the domi-nating (early) features, as we will further discuss below.

Stages of Alzheimer’s diseaseThere is an increasing awareness among researchers and clinicians that AD starts many years before the onset of demen-tia. The first stage is thought to be asymptomatic, when pathologies accu-mulate silently in the brains of affected individuals for years or even decades before clinical symptoms emerge [2]. This is followed by an early clinical stage, which is characterized by objec-tive cognitive impairment but still pre-served function. This is often called mild cognitive impairment (MCI) due to AD, or prodromal AD [3]. The final stage is the classic stage, where AD has caused sufficient functional impairment

for the patient to qualify for a dementia diagnosis.

Previously the procedure to diagnose AD was based solely on clinical examination and neuropsychological testing of the patient and interviews with proxies and caregivers. These methods are hampered by low sensitivity in the early stages of the disease and low specificity in the late stages of the disease. With the develop-ment of novel imaging and biochemistry technologies it is now possible to diag-nose AD with the aid of direct evidence of relevant molecular pathologies in the brain. This is a conceptual leap that has revolutionized clinical AD research and is rapidly transforming clinical practice and clinical trial design. In this article we will discuss this development, with a focus on cerebrospinal fluid biomark-ers for AD. Key points are summarized in Table 1.

The fi rst stepsTh e amino acid sequences of Aβ and tau were identifi ed in the mid-1980s [4, 5]. Early on it was suggested that a test for

AD could be developed based on serum measurements of Aβ [4], but it was the discovery in the early 1990s that Aβ is secreted into the CSF that boosted the modern development of biochemi-cal AD markers [6]. In the mid-1990s immunoassays (ELISAs) were devel-oped for CSF Aβ1-42 (the prominent Aβ peptide isoform in Aβ plaques, typically reduced in AD patients com-pared to controls [7]), total-tau (T-tau, increased in AD patients compared to controls [8, 9]), and phosphorylated-tau (P-tau, increased in AD patients com-pared to other neurological diseases and controls [8]).

A window into the brainTraditionally, AD could only be diag-nosed at the dementia stage, and defi nite diagnosis was only possible through post-mortem analysis of brain tissue. Th e CSF biomarkers Aβ1-42, T-tau and P-tau (and imaging technologies not covered in this article) have made it possible to approach identifi cation of AD brain pathology in living patients. Several studies have found that CSF Aβ1-42 is strongly related to the presence of brain Aβ pathology, quanti-fi ed at autopsy [10] or in vivo using posi-tron emission tomography imaging with tracers specifi c for fi brillar Aβ [11]. Like-wise, although with less strong associa-tions, CSF P-tau correlates with neocorti-cal tangle pathology [12, 13], whereas CSF T-tau is more non-specifi cally increased in a number of neurological diseases, with the magnitude of the increase cor-relating with the size of the damaged tissue and the clinical outcome [14, 15]. CSF biomarkers enable detection of AD

Developments in cerebrospinal fl uid biomarkers for Alzheimer’s diseaseAlzheimer’s disease (AD) is the most common cause of dementia. It is characterized by accumulation of -amyloid (A ) peptides and tau proteins, loss of neurons and cognitive decline. It is diffi cult to diagnose AD in the early stages by clinical examination. Cerebrospinal fl uid (CSF) biomarkers can be used to overcome this and could be useful in clinical practice, research and in trials of novel treatments.

by Philip Insel, Dr Rik Ossenkoppele and Dr Niklas Mattsson


Disease stage

Asymptomatic Early clinical (MCI) Late clinical (dementia)

Clinical practice – Enable AD diagnosis prior to dementia

Increase diagnostic accuracy at the dementia stage

Research

Characterize AD pathology in cognitively-

normal people

Characterize AD pathology in mildly impaired people

Characterize AD pathology in demented people

Study disease mechanisms (relationships between different disease hallmarks)

Clinical trials

Screening for inclusion in very early prevention

trials

Enrich MCI trials with people with symptoms

likely caused by underlying AD

Exclude clinically misdiagnosed AD dementia

patients from trials

Monitor longitudinal effects of drugs on AD processes

Table 1. Uses of cerebrospinal fl uid biomarkers for Alzheimer’s disease (AD). MCI, mild cognitive impairment.

014_027_cli_oct_2015.indd 14 2/10/15 13:18

pathology in patients in early stages of the disease, when only mild symptoms or no clinical symptoms are present. As clini-cal diagnosis alone is inadequate in those early disease stages, biomarkers may be critical for an accurate diagnosis. CSF bio-markers may also increase the diagnostic accuracy of the diagnosis in advanced clinical stages, by providing biological evidence of AD related pathology. Th is helps in identifying the clinical syndrome and diff erentiating it from other neuro-logical diseases.

ChallengesTh e fi eld is rapidly advancing to overcome some hurdles that have prevented wide-spread implementation of CSF biomark-ers. Problems with between-laboratory and between-assay variability in meas-urements have been noticed [16] and are being tackled by the development of certifi ed reference procedures (based on selected reaction monitoring mass spectrometry [17]) and certifi ed refer-ence materials (created by the Institute of Reference Materials and Methods [18]). Development of fully automated assays will further bring down the variability and facilitate implementation of CSF

biomarkers outside of expert centres (see www.neurochem.gu.se/TheAlzAssQC-program for updated comparisons of dif-ferent assay systems in a global quality control programme). In some countries a remaining obstacle is the unwilling-ness of medical practitioners to perform lumbar punctures, especially outside of highly specialized clinics. More training is needed to increase the familiarity of doc-tors with this procedure, and more educa-tion is needed to inform staff and patients that the procedure is safe. Headache is the only common complication (2–5 % incidence), but this is usually benign and treatable by common analgesics. Severe complications are extremely rare.

Alzheimer’s disease variantsCSF biomarkers of Aβ and tau may be particularly helpful to assist the diagnos-tic process in patients with a non-amnes-tic presentation of AD who may show substantial clinical overlap with patients experiencing non-AD types of demen-tia. Recently, there has been an increased awareness of these atypical presentations such as posterior cortical atrophy (PCA, ‘visual variant AD’ [19]), logopenic variant primary progressive aphasia (‘language

variant AD’ [20]), and the behavioural/dysexecutive variant of AD [21]. As previ-ous studies with small sample sizes have yielded confl icting results, we performed a study in 176 patients selected for abnor-mal CSF Aβ biomarkers to assess whether CSF T-tau and P-tau diff er between atypi-cal variants of AD [22]. Bootstrapping showed that the prevalence of abnormal T-tau and P-tau was ~80–90%, roughly equally distributed across AD phenotypes. Th is suggests that CSF T-tau and P-tau are equally useful in all clinical phenotypes of AD, which is compatible with current National Institute on Aging–Alzheimer’s Association (NIA-AA) and International Working Group for New Research Cri-teria for the Diagnosis of AD (IWG-2) diagnostic criteria.

Biomarkers in clinical trialsBiomarker measurement is a recent addi-tion to AD clinical trials. Th e use of bio-markers is thought to improve trial design both in terms of subject selection and measurement of disease progression. It becomes particularly important in trials of disease-modifying treatments to recruit only those subjects with the target pathol-ogy of the therapy. Several recent failed

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– October 201517ALZHEIMER’S DISEASE

trials may have been hindered by the inclu-sion of subjects without the underlying pathology [23]. Biomarkers are also less aff ected by measurement error compared with clinical outcomes and thus off er cer-tain advantages in measuring progression over time, especially in early stages of dis-ease [24]. In these early stages of the dis-ease, before the onset of clinical symptoms, the ability of biomarkers to predict future pathology and cognitive decliners will aid in identifying those most in need of treat-ment. Th is will become especially impor-tant if intervention in the earliest stages of the disease, prior to substantial neu-rodegeneration, off ers the best chance of a treatment to be eff ective.

Early treatment trials of anti-Aβ thera-pies employ thresholds to ensure recruit-ment of subjects with a minimal level of Aβ pathology. Th is threshold is frequently taken to be the level of amyloid pathology that most accurately distinguishes cases of AD from cognitively-normal controls [25]. However, if earlier treatment has a higher likelihood of success, identify-ing subjects with normal amyloid levels who are likely to have elevated levels in the future may be a further step toward early intervention. A recent study dem-onstrated that amyloid-negative subjects with low levels of CSF Aβ1-42 were much more likely to become amyloid-positive in the near term [26]. Individuals with CSF Aβ1-42 levels in the low normal range may be optimal candidates for early inter-vention trials aimed at halting further Aβ accumulation.

ConclusionsCSF biomarkers have helped to trans-form the diagnosis of AD from a clini-cal diagnosis to a biomarker-informed diagnosis based on molecular evidence of the underlying neuropathology. Th is has implications for research, where CSF bio-markers enable researchers to characterize subjects at all levels of cognitive function, in clinical practice, where CSF biomark-ers aid doctors in diagnosis of AD versus other causes of cognitive impairment, and in the design of clinical trials, where CSF biomarkers may be used to enrich study populations and construct sensitive meas-ures of outcomes to increase study power.

References1. Alzheimer’s disease International: World Alzhei-

mer Report 2015 (http://www.alz.co.uk/research/world-report-2015).

2. Jansen WJ, Ossenkoppele R, Knol DL, Tijms BM, et al. Prevalence of cerebral amyloid pathology

in persons without dementia: a meta-analysis. JAMA 2015; 313: 1924–1938.

3. Albert MS, DeKosky ST, Dickson D, Dubois B, et al. Th e diagnosis of mild cognitive impairment due to Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement. 2011; 7: 270–279.

4. Glenner GG, Wong CW. Alzheimer’s disease: ini-tial report of the purifi cation and characterization of a novel cerebrovascular amyloid protein. Bio-chem Biophys Res Commun. 1984; 120: 885–890.

5. Grundke-Iqbal I, Iqbal K, Quinlan M, Tung YC, et al. Microtubule-associated protein tau. A compo-nent of Alzheimer paired helical fi laments. J Biol Chem. 1986; 261: 6084–6089.

6. Seubert P, Vigo-Pelfrey C, Esch F, Lee M, et al. Isolation and quantifi cation of soluble Alzheimer’s beta-peptide from biological fl uids. Nature 1992; 359: 325–327.

7. Motter R, Vigo-Pelfrey C, Kholodenko D, Barbour R, et al. Reduction of beta-amyloid peptide42 in the cerebrospinal fl uid of patients with Alzhei-mer’s disease. Ann Neurol. 1995; 38: 643–648.

8. Blennow K, Wallin A, Agren H, Spenger C, et al. Tau protein in cerebrospinal fl uid: a biochemical marker for axonal degeneration in Alzheimer dis-ease? Mol Chem Neuropathol. 1995; 26: 231–245.

9. Vandermeeren M, Mercken M, Vanmechelen E, Six J, et al. Detection of tau proteins in normal and Alzheimer’s disease cerebrospinal fl uid with a sensitive sandwich enzyme-linked immunosorb-ent assay. J Neurochem. 1993; 61: 1828–1834.

10. Strozyk D, Blennow K, White LR, Launer LJ. CSF Abeta 42 levels correlate with amyloid-neuro-pathology in a population-based autopsy study. Neurology. 2003; 60: 652–656.

11. Fagan AM, Mintun MA, Mach RH, Lee SY, et al. Inverse relation between in vivo amyloid imag-ing load and cerebrospinal fl uid Abeta42 in humans. Ann Neurol. 2006; 59: 512–519.

12. Tapiola T, Alafuzoff I, Herukka SK, Parkkinen L, et al. Cerebrospinal fl uid {beta}-amyloid 42 and tau proteins as biomarkers of Alzheimer-type pathologic changes in the brain. Arch Neurol. 2009; 66: 382–389.

13. Seppala TT, Nerg O, Koivisto AM, Rummu-kainen J, et al. CSF biomarkers for Alzheimer disease correlate with cortical brain biopsy fi nd-ings. Neurology. 2012; 78: 1568–1575.

14. Hesse C, Rosengren L, Andreasen N, Davidsson P, et al. Transient increase in total tau but not phospho-tau in human cerebrospinal fl uid aft er acute stroke. Neurosci Lett. 2001; 297: 187–190.

15. Otto M, Wiltfang J, Tumani H, Zerr I, et al. Ele-vated levels of tau-protein in cerebrospinal fl uid of patients with Creutzfeldt-Jakob disease. Neu-rosci Lett. 1997; 225: 210–212.

16. Mattsson N, Andreasson U, Persson S, Carrillo MC, et al. CSF biomarker variability in the Alz-heimer’s Association quality control program. Alzheimers Dement J Alzheimers Assoc. 2013; 9: 251–261.

17. Leinenbach A, Pannee J, Dülff er T, Huber A, et al. Mass Spectrometry-Based Candidate Refer-ence Measurement Procedure for Quantifi ca-tion of Amyloid-β in Cerebrospinal Fluid. Clin Chem. 2014; 60: 987–994.

18. Mattsson N, Zetterberg H. What is a certifi ed ref-erence material? Biomark Med. 2012; 6: 369–370.

19. Crutch SJ, Lehmann M, Schott JM, Rabinovici GD, et al. Posterior cortical atrophy. Lancet Neu-rol. 2012; 11: 170–178.

20. Gorno-Tempini ML, Hillis AE, Weintraub S, Kertesz A, et al. Classifi cation of primary pro-gressive aphasia and its variants. Neurology 2011; 76: 1006–1014.

21. Ossenkoppele R, Pijnenburg YAL, Perry DC, Cohn-Sheehy BI, et al. Th e behavioural/dysex-ecutive variant of Alzheimer’s disease: clinical, neuroimaging and pathological features. Brain J Neurol. 2015; 138: 2732–2749.

22. Ossenkoppele R, Mattsson N, Teunissen CE, Barkhof F, et al. Cerebrospinal fl uid biomarkers and cerebral atrophy in distinct clinical vari-ants of probable Alzheimer’s disease. Neurobiol Aging 2015; 36: 2340–2347.

23. Karran E, Hardy J. Antiamyloid therapy for Alz-heimer’s disease—are we on the right road? N Eng J Med. 2014; 370: 377–378.

24. Hendrix SB. Measuring clinical progression in MCI and pre-MCI populations: Enrichment and optimizing clinical outcomes over time. Alzhei-mer’s Res Th er. 2012; 4: 24.

25. Shaw LM, Vanderstichele H, Knapik�Czajka M, Clark CM, et al. Cerebrospinal fl uid biomarker signature in Alzheimer’s disease neuroimag-ing initiative subjects. Ann Neurol. 2009; 65: 403–413.

26. Mattsson N, Insel PS, Donohue M, Jagust W, et al. Predicting reduction of cerebrospinal fl uid β-amyloid 42 in cognitively healthy controls. JAMA Neurol. 2015; 72: 554–560.

The authorsPhilip Insel1,2,3 MSs; Rik Ossenkoppele4,5

PhD; Niklas Mattsson*1 MD, PhD1 Clinical Memory Research Unit, Fac-ulty of Medicine, Lund University, Lund, Sweden2 Center for Imaging of Neurodegenerative Diseases, Department of Veterans Aff airs Medical Center, San Francisco, CA, USA3 Department of Radiology and Biomedi-cal Imaging, University of California, San Francisco, CA, USA4 Alzheimer Center and Department of Neurology, Neuroscience Campus Amster-dam, VU University Medical Center, Amsterdam, the Netherlands5 Memory and Aging Center, University of California San Francisco, San Francisco, CA, USA


014_027_cli_oct_2015.indd 17 2/10/15 13:18

It all started with a cryoscopic osmom-eter for clinical application: the OSMO-MAT 030. At the first exhibitions he vis-ited, Klaus Noack was surprised by the high level of interest generated by the instrument. There was no other way than to expand production to cope with the increasing demand for this osmom-eter, which offered a complete ease of handling, previously unattained, resulting in a growing number of sales.Inspired by this success, a new osmometer was developed by Klaus Noack to complete the osmometer line for medical application: the col-loid osmometer OSMOMAT 050. Even though the market for this instrument,

basically used in intensive care units, was not as big as for the OSMOMAT 030, the product also contributed to the further success of GONOTEC GmbH.In the middle of the 80’s, develop-ment started on a new range of instru-ments, namely the chemical osmom-eters. The aim was the development of instruments for the determination of molar masses based on osmotic parameter for chemical applica-tion that also offer easy handling for the user.The three osmometers, vapor pres-sure osmometer OSMOMAT 070, membrane osmometer OSMOMAT 090 and cryoscopic osmometer OSMOMAT 010 complement one another due to their different meas-uring methods in the determination of the range of molar masses up to 2,000,000 Dalton.In 2001 the general management of GONOTEC was taken over by Jan Celinsek, who worked already with GONOTEC since 1991.In 2003 a new model in the osmom-eter family was launched into the market: the OSMOMAT auto, which is also characterized by extreme reli-ability and easy handling, thus fitting perfectly into the already well known GONOTEC osmometer line.In 2009 GONOTEC moved to new premises with lots of space for new ideas! In the same year, the chlorid-meter CM20 was launched, followed in 2013 by the next generation of

osmometers: the OSMOMAT 3000 and the OSMOMAT 3000 basic, both replacing the well known OSMOMAT 030.To this day, GONOTEC is no large, anonymous concern but still a medium-sized, private company, owned by Klaus Noack, the founder. However, we became a global player with cus-tomers in more than 60 countries.One of the most valuable resources GONOTEC always had was its perma-nent staff. Once people start working for GONOTEC they stay with the com-pany as they are proud of their work. The same applies to the numerous number of dealers all over the world. The cooperation between the agents and GONOTEC is like in a family; con-stant trainings at the company head-quarters improve this special relation-ship between agent and manufacturer. GONOTEC products do deserve the description “Made in Germany”, as the whole production is in one location. It is easy for external persons visiting the company to see an osmometer being manufactured from the very beginning to its finishing and perfect functioning. Since GONOTEC was able to export the company’s philosophy by means of the highest quality standards and competence as well as constant assis-tance to customers and agents, it is looking optimistically into the future. Our company’s philosophy is a prom-ise to all our customers and potential customers.

A success story for more than 35 years: Gonotec GmbHWhen founding the company GONOTEC GmbH in 1979, electronics engineer Harald Göritz and chemist Klaus Noack could not possibly imagine that their target to develop, produce and market analytical measuring instruments for medical and chemical application would be as successful as it turned out to be. Both founders of the company could already look back to decades of experience in this field. Klaus Noack, founder and owner of Gonotec GmbH

“We are the osmometer people.”

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BackgroundDuring the window period between infec-tion with human immunodefi ciency virus type 1 (HIV-1) and the appearance of detectable antibodies to HIV-1, the infec-tion cannot be diagnosed. Attempts to shorten this period have been made using a fourth-generation immunoassay that detects both HIV-1/2 IgG/M and HIV-1 p24 antigens [1]. However, most of the commercially available detection systems for fourth-generation immunoassays use chemiluminescent measurement and thus require specialized, highly expensive auto-mated measurement equipment. For this

reason, fourth-generation immunoassays are performed only at diagnostics com-panies and hub hospitals. To overcome this limitation and to test many samples simultaneously, there is need of an immu-noassay with increased sensitivity for the HIV-1 p24 antigen that nonetheless uses a common enzyme and does not require any specialized instruments.

In 2010, French health authorities man-dated a limit of detection of at least 2 IU/mL of HIV-1 p24 antigen for a Con-formité Européenne (CE)-marked HIV antigen/antibody assay [2]. According

to this mandate, commercially available assay kits were manufactured to detect p24 antigen with limits of detection ranging from 0.505 to 1.901 IU/mL and from 11.9 to 33.5 pg/mL [2]. Units of pg/mL are used for the Société Française de Tranfusion Sanguine (SFTS) standard (i.e. recombinant proteins), versus IU/mL for the WHO (World Health Organiza-tion) standard. As 1 IU/mL is estimated to be equivalent to 10 pg/mL and MW = 24 000 for p24, the best sensitivity in these kits is 0.505 IU/mL, which is ~2 × 10−16 moles/mL.

To date, numerous methods have been proposed for the detection of p24 antigen. However, the limit of detection of p24 anti-gen is not expected to overcome the sensi-tivity of 10−17 to 10−18 moles/mL. In addi-tion, we have to note that HIV testing of many samples requires not only ultrasensi-tive HIV-1 p24 detection but also rapidity, a reasonable cost, and a simple protocol with-out the requirement of special equipment.

Ultrasensitive colorimetric detection of HIV-1 p24To reduce the window period for HIV-1 infection, a method for detecting trace amounts of HIV-1 p24 in blood is needed. We developed a simple de novo ultrasensitive colorimetric ELISA by adding a thio-NAD cycling solution to the standard ELISA. The limit of detection for p24 was 0.005 IU (i.e. attomoles) per assay by the ultrasensitive colorimetric ELISA.

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– October 2015 HIV TESTING20

Figure 1. Mechanism of ultrasensitive detection of HIV-1 p24 antigen by an ELISA coupled with thio-NAD cycling using alkaline phosphatase, androsterone derivatives, and 3 -hydroxysteroid dehydrogenase and its coenzymes.

014_027_cli_oct_2015.indd 20 2/10/15 13:18

In the present review, we intro-duce a de novo ultrasensitive colorimetric enzyme-linked immunosorbent assay (ELISA) for HIV-1 p24 [3].

Mechanism of ultrasensitivecolorimetric ELISAWatabe and colleagues devel-oped an ultrasensitive ELISA to measure trace amounts of proteins by combining a con-ventional ELISA with thion-icotinamide-adenine dinu-cleotide (thio-NAD) cycling [4]. Th eir rationale was that although proteins cannot be amplifi ed by polymerase chain reaction (PCR) in the man-ner of nucleic acids, a detect-able signal for proteins can be amplifi ed. Th us, their ultrasen-sitive ELISA (Fig. 1) employs a sandwich method using a pri-mary and a secondary antibody for antigens. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phos-phate with alkaline phosphatase linked to the secondary anti-body. Th is 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α-hydroxysteroid dehydro-genase (3αHSD) with a cofac-tor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumu-lates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors.

Th is method enables the detection of a target protein with ultrasensitivity (10−19 moles/assay) by measuring the cumulative quantity of thio-NADH by a colorimet-ric method without the use of any special instruments for the measurements of fl uorescence, luminescence or radio iso-topes [4]. Further, we should note that this ultrasensitive method will allow a techni-cian to detect trace amounts

of proteins simply by applying thio-NAD cycling reagents to the conventional ELISA sys-tem. We therefore applied this ultrasensitive ELISA to the detection of HIV-1 p24 anti-gen in blood [3].

Sensitivity and stabil-ity of the ultrasensitive colorimetric ELISA for HIV-1 p24A typical linear calibration curve for HIV-1 p24 antigen

provided by the ultrasensi-tive ELISA coupled with thio-NAD cycling was y = 0.27x + 0.019, R2 = 0.99 in the range of 0.1‒1.0 IU/mL. Th e limit of detection of p24 was 0.0055 IU/assay (i.e. ~2 × 10−18 moles/assay). Th ese fi ndings indicate that the ultrasensitive col-orimetric ELISA succeeds in detecting p24 at the attomole level [3]. Because this meas-urement system employs a 50 μL solution for each assay, the

detection limit corresponded to 0.1 IU/mL, or 10−17 moles/mL. Th erefore, even in terms of the concentration per mL, our detection limit is less than one-tenth of that required by the French health authorities [2]. Th e coeffi cient of variation was 8% for 1 IU/mL.

Spike-and-recovery test using serumWe attempted to perform spike-and-recovery tests

– October 201521

MP Biomedicals Europe, Tel: 00800 7777 9999 • email: [email protected]


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in which the HIV-1 p24 antigen was added to the control serum. Because our results demonstrated that the ratio was about 100% for 0.5 IU/mL of HIV-1 p24, which was less than the value (2 IU/mL) required for a CE-marked HIV antigen/antibody assay (see Back-ground), the ultrasensitive method was judged to sufficiently detect IV-1 p24 antigen in human blood obtained from patients in the very early period after infection.

Detection of HIV-1 p24 in the early stages of infectionIt is important to diagnose primary HIV-1 infection and begin antiretrovi-ral treatment as early as possible. Most HIV-1/2 antibody diagnostic tests detect the antibodies for the antigens of HIV-1 gp41 and HIV-2 gp36, which are highly conservative transmembrane proteins. These tests are quick and easy, and thus have been widely used in many clinics and public health cen-tres. However, when only the antibody diagnostic tests are used, there is a long delay (generally a 28-day window period) before diagnosis is possible [5]. Further, HIV-1/2 antibody tests in children younger than 18 months tend to be especially inaccurate as a result of the continued presence of maternal antibodies [6]. To shorten the delay and to validate HIV tests, the HIV-1 p24 antigen, the concentration of which is expected to increase before antibodies emerge, should be detectable in trace amounts. HIV-1 p24 in blood emerges transiently in the very early period after infection, and then its concentra-tion quickly returns to the basal level [5]. An HIV-1 p24 test is, therefore, very useful as a screening test in the early stage of infection.

Closing the gap on PCR-based nucleic acid testing (NAT)Generally, the gold standard for diag-nosing HIV-1 is PCR-based nucleic acid testing (NAT) [7], but this method is expensive and has infrastructure requirements, a long measuring time, and high complexity, thereby limit-ing its usefulness for large numbers of samples. There is also the issue that much of the world lacks access to reliable NAT, and thus in many geo-graphic regions the policy is to simply wait until symptoms develop. Use of ultrasensitive detection of HIV-1 p24 antigen for early diagnosis would be a simple and reasonable alternative to

NAT, such as for monitoring HIV treat-ment and protecting the blood supply. Accordingly, it is time to reconsider whether NAT should be the gold stand-ard for diagnosing HIV-1. Barletta et al. claimed that the target protein (i.e. HIV-1 p24 antigen) is present in the virion in much higher numbers than viral RNA copies (approximately 3000 HIV-1 p24 antigen molecules ver-sus 2 RNA copies per virion) [8]. The 10−18 moles/assay value in our present results corresponds to 106 protein mol-ecules/assay, or ~103 RNA copies/assay. Although under laboratory conditions a real-time PCR (i.e. NAT) can detect on the order of 101 RNA copies/assay, the limitation of detection is usually in the order of 102 RNA copies/assay [9]. Hence, the ultrasensitive ELISA cou-pled with thio-NAD cycling for HIV-1 p24 is closing in on the detection limit obtained by NAT, with a margin of dif-ference of only one order of magnitude.

ConclusionTh e ultrasensitive ELISA coupled with thio-NAD cycling is a very convenient method for the early testing of HIV-1 infection because it requires only the addition of a thio-NAD cycling solu-tion to the usual ELISA without the use of any specialized measuring equipment. Consequently, the pre-sent method could be widely used as a powerful tool to test many samples simultaneously.

References1. George CRR, Robertson PW, Lusk MJ, Why-

bin R, Rawlinson W. Prolonged second diagnostic window for human immunode-ficiency virus type 1 in a fourth-generation immunoassay: Are alternative testing strat-egies required? J Clin Microbiol. 2014; 52: 4105–4108.

2. Ly TD, Plantier JC, Leballais L, Gonzalo S, Lemée V, Laperche S. The variable sensi-tivity of HIV Ag/Ab combination assays in the detection of p24Ag according to geno-type could compromise the diagnosis of early HIV infection. J Clin Virol. 2012; 55: 121–127.

3. Nakatsuma A, Kaneda M, Kodama H, Mori-kawa M, Watabe S, Nakaishi K, Yamashita M, Yoshimura T, Miura T, Ninomiya M, Ito E. Detection of HIV-1 p24 at attomole level by ultrasensitive ELISA with thio-NAD cycling. PLoS One 2015; 10: e0131319.

4. Watabe S, Kodama H, Kaneda M, Morikawa M, Nakaishi K, Yoshimura T. Ultrasensi-tive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the

thio-NAD cycling method. BIOPHYSICS. 2014; 10: 49–54.

5. World Health Organization (WHO). HIV/AIDS Fact sheet No 360. WHO 2015; http://www.who.int/mediacentre/factsheets/fs360/en/

6. Zijenah LS, Tobaiwa O, Rusakaniko S, Nat-hoo KJ, Nhembe M, Matibe P, Katzenstein DA. Signal-boosted qualitative ultrasensi-tive p24 antigen assay for diagnosis of sub-type C HIV-1 infection in infants under the age of 2 years. J Acquir Immune Defic Syndr. 2005; 39: 391–394.

7. Patel P, Mackellar D, Simmons P, Uniyal A, Gallagher K, Bennett B, Sullivan TJ, Kowal-ski A, Parker MM, LaLota M, Kerndt P, Sulli-van PS; Centers for Disease Control and Pre-vention Acute HIV Infection Study Group. Detecting acute human immunodeficiency virus infection using 3 different screening immunoassays and nucleic acid amplifica-tion testing for human immunodeficiency virus RNA, 2006-2008. Arch Intern Med. 2010; 170: 66–74.

8. Barletta JM, Edelman DC, Constantine NT. Lowering the detection limits of HIV-1 viral load using real-time immuno-PCR for HIV-1 p24 antigen. Am J Clin Pathol. 2004; 122: 20–27.

9. Wagatsuma A, Sadamoto H, Kitahashi T, Lukowiak K, Urano A, Ito E. Determina-tion of the exact copy numbers of particu-lar mRNAs in a single cell by quantitative real-time RT-PCR. J Exp Biol. 2005; 208: 2389–2398.

The authorsAkira Nakatsuma1 PhD, PhC; Mugiho Kaneda1 BAgr; Hiromi Kodama1 MAgr; Mika Morikawa1,2 BASc; Satoshi Watabe3 BPha; Kazunari Nakaishi2; Masakane Yamashita4 PhD; Teruki Yoshimura5 PhD, PhC; Toshiaki Miura6 PhD, PhC; Masaki Ninomiya1 PhD, PhC; Etsuro Ito*1 PhD

1 Kagawa School of Pharmaceutical Sci-ences, Tokushima Bunri University, Sanuki, Japan2 TAUNS Laboratories, Inc., Izunokuni, Japan3 BL Co., Ltd., Numazu, Japan4 Faculty of Science, Hokkaido University, Sapporo, Japan5 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan6 Graduate School of Pharmaceutical Sci-ences, Hokkaido University, Sapporo, Japan


– October 2015 HIV TESTING 22

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BackgroundTh e diagnosis of HIV infection in devel-oped countries is based on initial screen-ing for HIV antibodies, and if detected, confi rmation with nucleic acid testing (NAT) [1]. Th is ensures high sensitiv-ity and specifi city. However, the cur-rent World Health Organization (WHO) HIV Testing Services Guidelines do not include specifi c confi rmation testing for the diagnosis of HIV across large popu-lation groups in resource-limited settings [2]. Instead WHO recommends that diag-nosis be made on the basis of rapid diag-nostic tests (RDTs) only (or equivalent

enzyme immune assay tests) requiring a minimum of two positive test results, using test devices from diff erent sources, for a positive diagnosis (or three in low prevalence settings) [2]. Although the WHO strategy has enabled life-saving scale-up of HIV diagnosis the signifi cant compromise is that without confi rmation there is a risk that patients/clients can be falsely diagnosed as HIV positive [3]. Th is is also well demonstrated in the study discussed now in this article where the WHO RDT algorithm resulted in 6.8% false positive results (n = 2897). Incor-rect HIV diagnosis can have devastating consequences for the individual as well as wasting oft en over-stretched resources required for treatment and care.

Médecins Sans Frontières (MSF) has strongly advocated for the use of serologi-cal HIV confi rmation testing in resource-limited settings when it is impractical to perform NAT [4–6]. Commercial con-fi rmation kits are available that detect individual specifi c HIV antibodies, such as gp40, gp120, p24 and p32, that signifi -cantly increase the accuracy of testing at a considerably lower cost than NAT, and this type of confi rmation testing can be performed by non-specialized labora-tories. Th e downside, however, is that commercial confi rmation kits neverthe-less add cost, albeit reduced compared to NAT, and logistical complications that restrict their widespread use.To address this, MSF has recently pub-lished a simplifi ed confi rmation approach based on antibody dilution requiring only the use of an additional routinely used RDT test device [7]. Th is study has been published as a ‘proof of concept’ paper

and requires further testing across diff er-ent settings for refi nement before it can be generally recommended.

Causes of false positive HIV anti-body detectionAs with all tests, false positive HIV RDTs can be caused by user error (clerical mis-takes, incorrect test performance, misin-terpretation and cross-contamination). Other causes for HIV tests include non-specifi c IgG binding [2], cross reactiv-ity [2, 5], contaminating proteins [2] and pseudo-antigens created during the manufacturing process [5]. However, a key additional vulnerability for HIV anti-body detection testing is that all com-monly available HIV RDTs share a com-mon gp41 detection antigen. Th erefore, a cross-reactive antibody interacting with gp41 will act as a pan-cross-reactive anti-body across multiple test devices [5].

Th e WHO algorithm is based on the assumption that HIV RDTs that use diff er-ent antigen preparations are independent and, therefore, by requiring two positive tests (at a prevalence >5%) before report-ing HIV positivity, the algorithm assumes that the second test confi rms the result of the initial screening test [2]. However, in one MSF published study 50% of false positive samples had cross-reactive anti-gp41 activity, identifi ed by Western blot (WB), that was the likely cause of the double false positive reactions with the two independent RDTs used in the testing algorithm [4].

MSF has proposed that early-immune-response broad-specifi city polyclonal B-lymphocyte antibodies are a potential source of HIV RDT cross-reactive inter-fering antibodies [4]. Th ese antibodies are likely to have increased frequency and intensity in resource-limited settings because of the higher prevalence of con-comitant infections [8–10]. Additionally, displaced populations and individuals, such as caused by oppression, confl ict and famine, are likely to have a greater vulner-ability to cross-reacting infections than stable communities.

Dilution testing as a novel alternative for confi rmation of HIV rapid diagnostic testing in resource-limited settingsRapid diagnostic testing enables life-saving scale up of HIV diagnosis but is vulnerable to false positive results. Confi rmation testing can be impractical or cost prohibitive in resource-limited settings. Retesting a diluted blood sample is evaluated and proposed, at a proof of concept level, as a simple cost-effective HIV confi rmation methodology.

by Derryck Klarkowski and Dr Erwan Piriou

– October 201523HIV TESTING

Figure 1. HIV test performance by Médecins Sans Frontières

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Theoretical basis for dilution methodologyConfirmation by dilution is based on the established sensitive/less sensitive (S/LS) methodology developed to identify recent HIV infection for the purposes of incidence surveys [11–15]. This method-ology is based on the principle that HIV antibody titres increase over a period of several months after initial infection. Samples are initially tested using a high sensitivity HIV enzyme immunoassay (EIA) and if reactive are then further tested by the same EIA assay but using a diluted sample and reduced incubation time to reduce sensitivity. Samples test-ing positive on the sensitive (S) test but negative on the less sensitive (LS) test are designated a recent infection. The meth-odology has been successfully extended to the use of RDTs [13–15]. Confirma-tion by dilution adapts the S/LS principle to differentiate between high titre true HIV antibodies and low titre cross-react-ing antibodies.

One postulated source of cross-reactive antibodies are broad spectrum anti-bodies produced in the early immune response to a wide range of infectious disease antigens, and these antibodies can cause nonspecific cross reactivity in HIV serological testing [5]. In proposing dilution as a methodology to confirm HIV infection, we postulate that cross-reacting antibodies will have a low titre relative to specific HIV antibodies.

Cross-reacting antibodies can gener-ally be expected to have low avidity, as has been demonstrated by work in blood donors [16] and in MSF findings [4]. This will result in weakly positive results that can provide an alert for the tester; however, manufacturers generally state that any positive test line independent of strength should be interpreted as a positive result. Cross-reactive antibod-ies can also have high avidity as shown

in a previous MSF publication where 7 of 24 (29.2%) false positive samples (total sample size 229) had strongly positive test lines in two RDTs but had a low titre relative to the confirmed true HIV anti-bodies [4].

The use of dilution as a supplementary confirmatory test by using antibody rela-tive titres has been previously reported by Urwijitaroon et al. [17]. In another study, 41 samples were found positive using the HIV RDT Determine™ and 23 were negative on dilution [18]. Only 1 of these 23 samples was confirmed to be positive using serological confirmation (INNO-LIA™).

Field testingA study was conducted at two sites in north western Ethiopia in programmes covering both residents and seasonal migrant workers. Seasonal workers are transient and, as postulated by MSF, may potentially have a higher risk of false positivity caused by cross-reacting anti-bodies [4, 5].

The study recruited 2897 individuals, and 265 (9.1%) samples tested as posi-tive using two HIV RDTs from differ-ent manufacturers and would have been interpreted as HIV positive using the WHO algorithm [2]. Of the negative samples, 229 (approximately every 11th sample) were selected as a control. All algorithm-positive and negative control samples were further tested by dilution in situ, and additional confirmation test-ing performed by reference laboratories using WB and NAT for indeterminate WB samples.

All negative samples were confirmed as negative (100% sensitivity). However, 18/265 (6.8%) algorithm ‘positive’ sam-ples were identified as HIV negative (false positive) by either WB or NAT.

Dilution testing was performed by titrat-ing the patient’s plasma using confirmed seronegative plasma from healthy blood donors using a micropipette. Ten micro-litres of patient plasma was first diluted 1 : 10 in 90 μL of negative plasma fol-lowed by a serial 4-fold dilution from 1:40 to 1 : 10,240. Testing was performed using Determine™ HIV-1/2 (Alere Labo-ratories, Japan) following manufactur-er’s instructions. Tests were interpreted as positive if there was any colouration of the test line and the highest dilution that gave a positive result was recorded. Where the lowest dilution (1 : 10) was negative, the sample was reported as negative.

Findings and conclusionIn this study, based on a specific popu-lation group over a specific time period, repeating the RDT test using the sample diluted 1 : 160 identified all false posi-tive results and misidentified one true positive (see Table 1). However, there is a safety net that any sample with a reactive HIV RDT test that is not resolved as a true positive at the time of testing is not reported as negative but as inconclusive [2]. The patient/client is advised that testing has been inconclusive and testing should be repeated at a later time; WHO recommends retesting after 14 days. This allows time for true HIV antibodies to increase in titre.

The discriminatory threshold dilution may vary between different settings. In an earlier MSF study, a dilution of 1 : 1000 differentiated 229 true HIV posi-tive from 27 HIV false positive samples (unpublished data, for further details see Klarkowski et al. [4]).

One strength of this MSF study is that NAT testing was available to resolve indeterminate WB samples which made it possible to rule out early seroconver-sion as a potential cause of false positive results. Th e limitation is that the fi nd-ings are restricted to a single cohort with a single RDT and should be viewed as a ‘proof of concept’. More experience is needed in diff erent settings and by dif-ferent workers before the dilution meth-odology can be considered for potential scale up. It is proposed that the method-ology has potential for use as a supple-mentary test in a confi rmatory algorithm, whereby double positive RDT results are tested by dilution, with positive results above a determined threshold confi rm-ing HIV infection. Dilution results below

– October 2015 HIV TESTING24

DilutionSamples positive

following dilution

False positive

% False positive

False negative

% False negative

Neat 265 18 6.8% (3.8–9.8) 0

1:10 259 12 4.6% (2.1–7.1) 0

1:40 249 2 0.8% (0.3–1.9) 0

1:160 246 0 1 0.4% (0–1.2)

1:640 244 0 3 1.2% (0–2.6)

1:2560 240 0 7 2.8% (0.7–4.9)

1:10,240 221 0 26 10.5% (6.7–14.3)

Table 1. Dilution results compared to the reference laboratory confi rmed result. 265 samples tested positive by HIV rapid test algorithm; 247 samples true HIV positive by nucleic acid testing (NAT) or Western blot (WB).

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the threshold would require further testing, such as repeat testing at a later time or NAT, to rule out false negative results either due to seroconversion or misclassifi cation by the lower sensitivity dilution test.

References1. Centers for Disease Control and Prevention

and Association of Public Health Laboratories. Laboratory testing for the diagnosis of HIV infection: updated recommendations. 2014; http://stacks.cdc.gov/view/cdc/23447.

2. World Health Organization. Consolidated guidelines on HIV testing services. 2015; http://www.who.int/hiv/pub/guidelines/hiv-testing-services/en/.

3. Johnson C, Fonner V, Sands A, Tsui S, Ford N, Wong V, Obermeyer C, Baggaley R. Annex 14 A report on the misdiagnosis of HIV sta-tus. In: World Health Organization. Consoli-dated Guidelines on HIV Testing Services. 2015; http://www.who.int/hiv/pub/guidelines/hiv-testing-services/en/.

4. Klarkowski DB, Wazome JM, Lokuge KM, Shanks L, Mills CF, O’Brien DP. The evalua-tion of a rapid in situ HIV confirmation test in a programme with a high failure rate of the WHO HIV two-test diagnostic algorithm. PLoS One 2009; 4(2): e4351.

5. Klarkowski D, O’Brien DP, Shanks L, Singh KP. Causes of false positive HIV rapid diagnostic test results. Expert Rev Anti-infect Ther. 2013; 12(1): 49-62

6. Shanks L, Klarkowski D, O’Brien DP. False positive HIV diagnoses in resource limited settings: operational lessons learned for HIV programmes. PLoS ONE 2013; 8(3): e59906.

7. Shanks L, Siddiqui MR, Abebe A, Piriou E, Pearce N, Ariti C, Masiga J, Muluneh L,

Wazome J, Ritmeijer K, Klarkowski D. Dilu-tion testing using rapid diagnostic tests in a HIV diagnostic algorithm: a novel alternative for confirmation testing in resource limited settings. Virol J. 2015; 12: 75.DOI 10.1186/s12985-015-0306-4

8. Messele T, Abdulkadir M, Fontanet AL, Petros B, Hamann D, Koot M, Roos MT, Schellekens PT, Miedema F, Rinke de Wit TF. Reduced naive and increased activated CD4 and CD8 cells in healthy adult Ethiopians compared with their Dutch counterparts. Clin Exp Immunol. 1999; 115(3): 443–50.

9. Clerici M, Butto S, Lukwiya M, Saresella M, Declich S, Trabattoni D, Pastori C, Piconi S, Fracasso C, Fabiani M, Ferrante P, Rizzardini G, Lopalco L. Immune activation in Africa is environmentally-driven and is associated with upregulation of CCR5. Italian-Ugandan AIDS Project. AIDS 2000; 14(14): 2083–2092.

10. Clerici M, Declich S, Rizzardini G. African enigma: key player in human immunodefi ciency virus pathogenesis in developing countries? Clin Diagn Lab Immunol. 2001; 8(5): 864–866.

11. World Health Organization Technical Work-ing Group on HIV Incidence Assays. When and how to use assays for recent infection to estimate HIV incidence at a population level. 2011; http://www.who.int/diagnostics_labo-ratory/hiv_incidence_may13_final.pdf 2011.

12. Constantine NT, Sill AM, Jack N, Kreisel K, Edwards J, Cafarella T, Smith H, Bar-tholomew C, Cleghorn FR, Blattner WA. Improved classification of recent HIV-1 infection by employing a two-stage sensitive/less-sensitive test strategy. J Acquir Immune Defic Syndr. 2003; 32: 94–103.

13. Soroka SD, Granade TC, Candal D, Parekh BS. Modification of rapid human immunode-ficiency virus (HIV) antibody assay protocols

for detecting recent HIV seroconversion. Clin Diagn Lab Immunol. 2005; 12: 918–21.

14. Kshatriya R, Cachafeiro AA, Kerr RJS, Nel-son JA, Fiscus SA. Comparison of two rapid human immunodeficiency virus (HIV) assays, Determine™ HIV-1/2 and OraQuick Advance Rapid HIV-1/2, for detection of recent HIV seroconversion. J Clin Microbiol. 2008; 46(10): 3482–3483.

15. Girardi SB, Barreto AM, Barreto CC, Proi-etti AB, Carvalho SM, Loureiro P, Sabino EC. Evaluation of rapid tests for human immu-nodeficiency virus as a tool to detect recent seroconversion. Braz J Infect Dis. 2012; 16(5): 452–456.

16. Bouillon M, Aubin E, Roberge C, Bazin R, Lemieux R. Reduced frequency of blood donors with false-positive HIV-1 and -2 anti-body EIA reactivity after elution of low-affin-ity nonspecific natural antibodies. Transfu-sion 2002; 42(8): 1046–1052.

17. Urwijitaroon Y, Barusrux S, Romphruk A, Puapairoj C, Thongkrajai P. Anti-HIV Antibody Titer: An Alternative Supple-mentary Test for Diagnosis of HIV-1 Infec-tion. Asian Pac J Allergy Immunol. 1997; 15:193–198.

18. Duedu KO, Hayford AA and Sagoe KW. Mis-classification of recent HIV-1 seroconversion in sub-Saharan Africa using the sensitive/less sensitive technique. Virol J. 2011; 8: 176.

The authorsDerryck Klarkowski* MAppSc, Erwan Piriou PhDMédecins Sans Frontières, Amsterdam, The Netherlands


– October 201525

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National Health Service (NHS) TaysideNHS Tayside serves a population of 480,000 through a network of 22 hospitals/infi rmariesand 69 general-practice sites that rely on two laboratories. Th e Blood Sciences Laboratoryis located at the 900-bed Ninewells Hospital in Dundee, one of the UK’s major teaching hospitals. Here, Aptio Automation merges the three former individual labs onto a sin-gle track, providing a full complement of pre- and post-analytical sample-processing modules along with comprehensive analyt-ics. Th e effi ciencies gained have empowered the Ninewells Hospital laboratory to take on 73% of the testing that historically had been conducted at the 260-bed Perth Royal Infi r-mary (PRI), enabling the smaller PRI labora-tory to focus exclusively on acute admissions and inpatient testing. Ninewells now handles 100% of the general practice testing in the entire region.“Underpinning all of our actions is the com-mitment to reduce waste and variation—and most of all, to prevent harm to patients,” says Dr. Bill Bartlett, Tayside’s joint clinical director of diagnostics. “Aptio Automation is helping us enable our vision of cost-eff ective, patient-focused care.” Using Aptio Automation, Tayside now pro-cesses 7,000 tubes per day—a 20% increase in the workload of its main laboratory with no additional staff . Decreased TAT across the board drove a 61% improvement in the TAT for add-on tests - all with the high-quality results derived from the consistency and standardization enabled by automation. Increased capacity has even enabled Tay-side to introduce new testing protocols that improve the quality of care and can save the hospital money.While Tayside staff had ideas about what they needed and wanted to do, Siemens gave them data-driven information to guide their deci-sion making. “Siemens’ expertise and consul-tative approach was paramount to the success of this project, from beginning to end,” says Dr. Bartlett. “We relied on them to evaluate workloads from a variety of locations and torecommend the optimal mix of instruments to support peak loads. Th ey devised the fi nal

track layout for the new space and helped optimize the use of automation to best man-age the workfl ow.”

Carlos Haya Hospital MalagaRising along Spain’s Mediterranean coast, Malaga has a population of approximately 600,000 through tourism, construction, and technology services. Th e healthcare needs of locals and tourists are met by the 1,100-bed Universitario Carlos Haya Hospital. Part of the government-run Andalusian Public Health System, Carlos Haya is a regional institution with four hospitals: the Gen-eral Hospital, Civil Hospital, CARE Joseph Estrada, and Hospital Materno Infantil. Th e latter, in addition to providing healthcare for mothers and infants, houses the core labo-ratory that serves as the reference lab for all four hospitals. Th e Carlos Haya General Hos-pital also operates a biochemistry lab and its own emergency lab.Hospitals in Spain are classifi ed into three tiers, depending on the complexity of the dis-eases they can treat. Materno Infantil holds the highest rating, Tier 3, which means it handles the most diffi cult cases. Its core lab provides testing across a wide spectrum of disease states, performing seven million tests a year for approximately 660,000 patients.Th e great leap forward occurred in 2012, with the implementation of Siemens Aptio Automation integrated with the Siemens CentraLink Data Management System. Th e solution delivers extensive automation, cus-tomization, and traceability, while eliminating the need for third-party informatics soft ware. “Aptio Automation strengthened our preana-lytical and analytical phases, giving us more capacity, more versatility, and overall, more possibility,” says Dr. Manuel Rodriguez, who is responsible for biochemistry and automation labs. “CentraLink, meanwhile, makes it much easier to manage quality. It facilitates sample follow-up and management of repetitions. With CentraLink control over instrument alarms, we gain comprehensive information on the situation of a particular sample. What’s more, the solution is simple, problem-free, and easy to use.”

Hospital Clinic de Barcelona (CDB)In 2001, the Hospital Clinic de Barcelona in Spain was among the fi rst healthcare provid-ers in the world to create an automated core laboratory. Today the laboratory is gaining even greater effi ciencies with Aptio Automa-tion connecting analytical systems across all four core laboratory disciplines: clinical chemistry, immunoassay, hematology, and hemostasis.Over the course of its 13-year journey with Siemens, the laboratory has been able to con-solidate instruments, integrate and automate STAT testing, reallocate staff to higher-value responsibilities, and save upwards of €600,000 in tube costs - all while increasing clinicians’ trust in the laboratory to support excellence in patient care.In 2000, Hospital Clinic de Barcelona estab-lished the Biomedical Diagnostic Centre (CDB) to provide high-quality, comprehen-sive service in all areas of laboratory medi-cine and to be a reference for excellence in the related specialties. Th e CDB uses a client-focused model to optimize the use of resources while ensuring advanced tech-nological development in healthcare and research.Th e CDB is divided into fi ve specialty depart-ments and an operative core laboratory where high-volume automated testing is performed. In total, the CDB is composed of 100 staff specialists from the various laboratory areas along with 300 professionals representing pathology, biochemistry, molecular genetics, hemotherapy and hemostasis, immunology, and microbiology specialties.Th e CDB recently began a project to create a Molecular Biology Core Laboratory, which will integrate the most frequently tested molecular technologies. “Economic pressure is an ongoing fact of life,” says Dr. Aurea Mira, director of the CDB. “Lab automation allows us to improve workfl ows, optimize human and technology resources, and save money. It also raises hospital awareness of the lab as a provider of fast, accurate testing that supports good clinical outcomes.”

Advanced automation system drives lab effi ciency while optimizing use of staff timeFollowing the trend in the US, pressure is mounting on big European hospital labs to consolidate their operations and boost workfl ow performances while at the same time making better use of their staff. The Aptio Automation system from Siemens Healthcare Diagnostics goes a long way to fulfi ll these objectives. Here we take a look at three specifi c examples of hospitals that have integrated the system and how it has helped both their clinical and operational effectiveness

CASE STUDY – October 2015 26

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INDUSTRY NEWS – October 2015 28

Puritan Liquid Amies Transport System changes name to Opti-Swab™

Formerly known as “Puritan Liquid Amies Transport System” for aerobic & fastidi-ous bacteria, this combination of Puritan liquid Amies medium and HydraFlock® swabs is now marketed under a new brand name, Opti-Swab™. Th e Opti-Swab™ name and fresh new label assure the end user rapid and easy product recognition and confi rmation that they’ve made the opti-mal choice. Opti-Swab™ incorporates 1ml of liquid Amies medium and is available in 3 con-fi gurations - with one standard, one mini-tip, or one ultrafi ne tip HydraFlock® swab for specimen collection. Vial and cap, medium and swab – all components have been engineered and produced by Puritan. Opti-Swab™ Transport Systems was inde-pendently confi rmed by a complete valida-tion study on Copan’s Wasp®: Walk-Away Specimen Processor (WASP).www.puritanmedproducts.com

Beckman Coulter and COPAN extend distribution agreement for automated sample processing systems and digital microbiology

Beckman Coul-ter Diagnostics and COPAN Group have entered into an amendment to their distribu-

tion agreement that expands their rela-tionship into new products and geo-graphic territories. Additional products include COPAN’s WASPLab automated, front-end robotic specimen processing, full lab automation and digital micro-biology system. Th e amendment grants Beckman Coulter distribution rights in 21 global markets, including a number of territories in North America, Asia (including, among other territories, Japan and China), Europe, South Amer-ica and many emerging markets.“We’re very excited to expand our rela-tionship with COPAN to off er clinical microbiology laboratories an extended portfolio of products to help provide phy-sicians with the critical information they need regarding bacteria resistance,” said

Arnd Kaldowski, president, Beckman Coulter Diagnostics. “COPAN’s products complement our newly acquired Micro-Scan brand of microbiology solutions and off er hospitals and private laborato-ries a complete solution which stream-lines workfl ow—demonstrating Beck-man Coulter’s commitment to growth and investment in this new area of our business.”Stefania Triva, COPAN Group’s CEO said, “Th is is a new step in the collabo-ration between the two companies and we look forward to continue working together to expand our reach.”Norman Sharples, Executive VP and co-founder of COPAN Diagnostics, Inc. added, “COPAN has always been committed to provide open platform solutions to our customers and this partnership is further evidence and rein-forcement of our ability to integrate and interface with important platforms in microbiology.”In addition to WASPLab, the amend-ment allows Beckman Coulter to dis-tribute COPAN’s CTracer, SYNAPSEPro MINI and MALDI-Trace products.www.beckmancoulter.com www.copaninnovation.com

Ortho Clinical Diagnostics is emerging stronger than ever since becoming an independent company in 2014, when it was pur-chased by Th e Carlyle Group, and has made tremendous pro-gress in developing a range of new assays. Company researchers presented data from fi ve assays currently under development at this year’s American Association for Clinical Chemistry (AACC) meeting.

“We are investing in our business to better serve the mod-ern clinical lab with state-of-the-art solutions,” said Ted Far-rell, Vice President Business Field Assays. “We continue to enhance the quality of our products and expand our new product development pipeline for our Clinical Laboratory business.”

Th e assays presented at AACC address a range of important areas for clinical lab testing including HIV detection and cardiac event monitor-ing. Following is a quick overview:

• Ortho Clinical Diagnostics is developing a fourth generation assay to detect both HIV 1 antigen genotypes and HIV 1 & 2 antibody subgroups for use on its random access VITROS® systems. Th e assay demon-strated seroconversion sensitivity consistent with a com-mercially available fourth generation assay and was more sensitive than a third generation assay.

• A rapid, fully automated, high sensitivity assay is under devel-opment for the measurement of cardiac troponin I (cTnl) and is designed to be more analytically sensitive than contempo-rary cTnl and cTnT assays.

• Preliminary performance data showed that OCD’s prototype VITROS® Insulin Assay has excellent precision, cross-reactivity with pro-insulin and c-peptide as well as good correlation with two methods that are already commercially available.

• Th e current VITROS® Cl- Slide is FDA cleared for use with serum and plasma, but not in urine. Testing of urine samples using the current calibration and the proper testing protocol for plasma and serum resulted in impres-sive performance, reproducibility and linearity.

Ortho Clinical Diagnostics is focused on bringing targeted solu-tions like these to its clinical laboratory customers aimed at addressing unmet clinical needs and driving improvements to quality care. It con-tinues to press the boundaries of what’s possible in its quest for new and better assays. www.orthoclinical.com

Ortho Clinical Diagnostics researchers present data on pipeline of assays

Schematic representation of Chloride Slide

028_036_cli_oct_2015.indd 28 2/10/15 13:48

DIAsource 25OH Vitamin D ELISA CFDA-cleared and launched in the Chinese market

DIAsource ImmunoAssays has received China FDA clearance for its 25OH Vita-min D ELISA assay based on proprietary (Patented) monoclonal antibodies. Th e test is successfully launched in the Chi-nese market in collaboration with a local Chinese distributor and its sub-distribu-tors. Th e assay which has the CE mark and is FDA cleared, is characterized by a very simple protocol with an extremely effi -cient pretreatment solution directly in the ELISA well. Its extreme user-friendliness makes it a popular assay among manual ELISA users as well as in laboratories that need high throughput using open ELISA instruments. Th e company provides vali-dated protocols for the most common ELISA automates in the market. Th is assay is one of the various assays for determina-tion of Vitamin D (25OH Vitamin D, 1,25 (OH)2 Vitamin D and free 25OH Vitamin D) that DIAsource has available (IVD and RUO versions) in its product portfolio either in an ELISA and/or RIA format. www.vitamin-d-diagnostics.com (DIAsource dedicated Vitamin D website)

Binding Site launches dedicated website for clinical specimens to support IVD manufacturers & researchers

Binding Site has announced that its Immunologicals Group has just launched a brand-new website, www.thespecimen-bank.com, which has been expressly cre-ated and designed to serve as a resource for those in-vitro diagnostic (IVD) man-ufacturing companies and clinical/life-science research facilities in search of a high-quality source of clinical specimens.

Clinical specimens from human patients are essential and frequently overlooked components within the IVD industry. Required for ensuring the validity and integrity of products prior to, and aft er market introduction, they are also nec-essary for a host of other critical clinical studies, including assay performance test-ing, validation studies, trouble-shooting, and for the data and documentation required for regulatory approval and clearance. Historically, both the availabil-ity and sourcing of high quality clinical specimens has been problematic, until now. Th rough utilization of a network of various collection sites, Binding Site is able to provide those interested organiza-tions with human clinical specimens to meet their specifi c testing criteria. We can provide human patient specimens in sin-gle or multiple matrices (serum, plasma, urine, CSF, etc.), with or without patient information, and/or consent forms. All can be characterized by individual ana-lyte, age, and gender, along with the quan-titative and/or qualitative test result(s). www.thespecimenbank.com features a broad off ering of clinical specimens avail-able, along with other vital details on additional services available within this product line off ering, including quality assurance, regulatory information, and documentation.www.thebindingsite.com

Rheonix receives patent for device and process that will improve workfl ow and lower costs of molecular diagnostic testing

Rheonix Inc., a developer of fully auto-mated molecular diagnostics solutions, has been granted patent 9,132,398, “Inte-grated Microfl uidic Device and Methods,” for the Rheonix CARD® cartridge, which enables assays to be performed on the company’s EncompassMDx® and Encom-pass Optimum™ instruments. Th e CARD, which stands for Chemistry and Reagent Device, will make molecular diagnostics simpler and easier to perform through an innovative and functional design that delivers a fully automated molecular assay at a fraction of the cost of other options. All assay steps are performed within the

– October 201529INDUSTRY NEWS

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fully enclosed cartridge, thus eliminating the potential for con-tamination, reducing user error, and streamlining workfl ow.Th e CARD’s design enables adoption of advanced molecular technology by laboratories of all types, from small community hospital labs to highly complex, centralized laboratories. Th e design also facilitates implementation across a wide range of market opportunities, including next-generation sequencing (NGS) sample prep, research-use-only testing, food and bever-age industry applications, and in vitro diagnostics. Th e ’398 patent allows researchers and clinicians to quickly, eas-ily, and cost-eff ectively run several samples through a fully inte-grated and automated nucleic acid amplifi cation test, from raw sample input through detection, with no user intervention. Each CARD allows for simultaneous testing of four diff erent samples and can handle a broad range of sample types, such as fresh tissue, urine, whole blood, serum, saliva, swabs, and formalin-fi xed, paraffi n-embedded (FFPE) tissue. Th e Rheonix CARD performs multiple molecular techniques, including sample prep-aration, such as chemical and enzymatic lysis and DNA purifi ca-tion; amplifi cation, such as endpoint polymerase chain reaction (PCR), reverse transcriptase PCR, and quantitative PCR; and detection on a low-density microarray or lateral fl ow strip.“The ’398 patent recognizes the groundbreaking achievement we have reached with the Rheonix CARD. From the device’s hardware to its process, it will help make molecular diagnos-tics a reality in laboratories worldwide,” said Tony Eisenhut, president of Rheonix. “With the lowest cost of ownership of any molecular platform, the patent confirms the novelty of the Rheonix approach to molecular diagnostics. Where other systems have traditionally emphasized either multiplex or

throughput, Rheonix has designed single-use cartridges that do both and can perform sophisticated functions with a sim-ple design. This lowers laboratory costs by eliminating waste in time, equipment and consumables, and reduces the amount of highly skilled labour. Rheonix is helping bring powerful molec-ular tools to laboratories that could not previously afford to purchase or run them.” Th e dual-layer design of the Rheonix CARD automatically manipulates reagents internally with its active fl uidic network of pumps, valves and channels. Th e upper surface of the CARD contains reservoirs that hold reagents used in the extraction, purifi cation, amplifi cation and detection process and any result-ing liquid waste. Th e channels and pumps located on the lower surface of the CARD are used to transport and mix reagents and move wastes into the reservoirs on the top. By actively pumping fl uids from reservoir to reservoir within the CARD, molecular diagnostic tests can be performed automatically. www.rheonix.com

Population-based screening study of asymptomatic persons to start in China using GastroPanel bio-markers to identify gastric cancer riskA gastric cancer risk screening study will be organized in Chi-nese healthcare centres by the China Health Promotion Founda-tion. Th e foundation is a public organization, managed by the Chinese Ministry of Health.Th e multi-centre study will be conducted by fi fty to one hundred primary healthcare units. Th e screening of about half a million 40-80-year-old asymptomatic persons will be tested with Gas-troPanel biomarkers, delivered by Biohit Oyj. Th e parties have agreed not to disclose the value of the contract. Data collection and analysis, including evaluation, are planned to be fi nalized at the end of 2016. Th e sample collection has started in the summer of 2015. GastroPanel is a non-invasive blood test for stomach health. Th e test diagnoses Helicobacter pylori infection and atrophic gastri-tis, caused by H. pylori infection or autoimmune disease. Th ese results can be used to assess whether asymptomatic patients have an increased risk of gastric or esophageal cancer, peptic ulcer disease or risk of vitamin B12-, calcium-, magnesium- and iron malabsorption and if further examinations or treatments are needed. According to CEO Liu Feng, Biohit Biotech (Hefei) Co., Ltd, ’Th e most important risk factors for stomach cancer are H. pylori infection and atrophic gastritis, which oft en are asymptomatic, and can be accurately detected by GastroPanel biomarkers used for this population-based screening. Early detection of risk groups is important for the eff ective prevention of gastric cancer.’CEO Semi Korpela, Biohit Oyj said: ‘Th is is an outstanding opening for GastroPanel biomarkers in the screening of asymp-tomatic subjects to identify the risk groups for gastric cancer and vitamin B12 malabsorption among other things. Gastric cancer is the leading cause of cancer related mortality in China. Th e use of the very informative GastroPanel for the screening of gastric cancer risk off ers the possibility of prevention and early detec-tion of stomach cancers. Based on correct diagnosis, screening reduces sick leaves and loss of labour input, as well as self-medi-cation with its associated risks. Early detection of risk conditions for gastric cancer and vitamin and mineral defi ciencies saves healthcare costs and human suff ering as well.’[email protected]

INDUSTRY NEWS – October 2015 30

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Risk management software Bio-Rad Mission: Control is the fi rst objective risk management soft ware to help laboratories identify the best QC rules and the right QC frequency to develop a customized quality control plan to deliver reliable test results. Bio-Rad Mission: Control uniquely quanti-

fi es a laboratory’s risk tolerance, going beyond traditional subjec-tive risk assessments. It allows a lab to establish a QC plan that is consistent with the CLSI EP23-A guideline and better assess the risk of reporting incorrect patient test results back to the physician.

BIORAD www.cli-online.com & search 27076

Fully automated histology tissue embedderThe AutoTEC a120 is a second-generation, fully automated tis-sue embedder that eliminates the labour intensive need to manually orient and embed tissue specimens and form tissue or cell paraffin blocks. The AutoTEC technology, combined with the Paraform Sec-

tionable Cassette System ensure that the orientation of speci-mens is locked from grossing to microtomy for all routine tis-sue types, thereby eliminating the risk of orientation mistakes and tissue loss for increasing patient safety. The new AutoTEC a120 with the Paraform Sectionable Cassettes remains the industry’s first and only fully automated embedding system. Enhanced features include the incorporation of a cassette bar-code reader for LIS connectivity and traceability of specimen

blocks, and the SMARTair technology to optimize block qual-ity. A larger 15 inch touch screen monitor and the enhanced, intuitive operating system both deliver user convenience. Based on customer feedback, the input and output doors, the paraffin reservoir and access to the base molds are redesigned providing improved ergonomics while requiring minimal user maintenance and further increasing up-time. When used in conjunction with the Tissue-Tek Xpress x120 Rapid Tissue Processors the AutoTEC a120 offers a high throughput of up to 120 cassettes per hour and enables laboratories to reallocate valuable personnel now manually working at embedding sta-tions, and to conveniently level the daily workload in a lean continuous workflow. These SMART Automation systems pro-vide high quality and safe tissue processing, consistent clean blocks, and all important same-day diagnosis.

SAKURA FINETEK www.cli-online.com & search 27078

– October 201531PRODUCT NEWS

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HIV POC testing

Developed for use with the MULTISURE HIV test, the ASSURE Palm Reader is a soon to be CE-marked professional small instrument for rapid and automate analysis of HIV. Th is combination is ideal for meet-ing the needs of healthcare professionals for point-of-care testing. Th e MP Diagnos-tics MULTISURE HIV Test is a qualitative

immunochromatographic assay for the rapid in vitro detection and diff erentiation of antibodies to HIV-1 and HIV-2 in human serum, plasma or whole blood. Th is MULTISURE HIV test is the only screening product with 4 individual tests markers to detect antibodies to gp120 (HIV-1), gp41 (HIV-1), p24 (HIV-1/2) and gp36 (HIV-2) and is also equipped with MP Diagnostics’ patented reverse fl ow technology which gives the user stronger visual identi-fi cation, easier interpretation of results and reduced accordance of intermediates. Th e MP Diagnostics ASSURE Palm Reader is a fully integrated handheld instrument with soft ware and barcode technol-ogy that make for easy operation. Th e key advantages of the com-bination of ASSURE Palm reader and MULTISURE HIV are result interpretation, documentation and ease of use in point-of-care test-ing for HIV in only 15 minutes.

MP BIOMEDICALS www.cli-online.com & search 27080

028_036_cli_oct_2015.indd 31 2/10/15 13:48

ELISAs for Alzheimer’s disease Determination of the biomarkers amyloid beta (Aβ) 1-40 and 1-42 and total tau in cer-ebrospinal fl uid (CSF) is increasingly impor-tant for the diagnosis of Alzheimer’s disease, especially in the early stages. Measurement

of these highly sensitive and specifi c analytes aids the classifi ca-tion and prognosis of dementia. Patients with Alzheimer’s disease show a signifi cantly decreased level of Aβ 1-42, which is already detectable 5-10 years before the start of cognitive changes. Aβ 1-40 remains unchanged. Th e determination of the ratio Aβ 1-42 to Aβ 1-40 increases the effi ciency of early diagnostics and might help to discriminate Alzheimer’s disease from vascular dementia. Th e concentrations of total tau and phosphorylated tau increase when patients show advanced neurodegeneration and cognitive impair-ment. It is thus possible to discriminate Alzheimer’s patients from healthy persons by means of CSF analyses. Th e analytes Aβ 1-42, Aβ 1-40 and total tau can be measured precisely and reproducibly using a new generation of test systems. Th e ELISAs are based on well-characterized capture antibodies. Lyophilized calibrators and controls provide convenient test performance, high precision and clinical accuracy. Th e procedures are highly standardized and can be automated on EUROIMMUN analysers and other open ELISA platforms. Th e assays were developed by EUROIMMUN in col-laboration with ADx Neurosciences. Th e EUROIMMUN Beta-Amyloid (1-42), Beta-Amyloid (1-40) and Total-Tau ELISAs are CE-certifi ed.

EUROIMMUNMEDICA Hall 01 / A08


Fastest 25OH Vitamin D ELISA assayTh is new 25OH Vitamin D ELISA assay allows the determination of 25OH Vitamin D in serum and plasma in 90 minutes. Th is assay has all the features of the original FDA-approved 25OH Vitamin D ELISA that DIAsource launched in 2012 but is

by far the fastest 25OH Vitamin D ELISA in the market. Th e Vitamin D ELISA assay is typically used in smaller and mid-size laboratories either on an automated ELISA platform or manu-ally. Also for these laboratories it has become more important to off er results as quickly as possible. Th e actually launched assay is a direct answer to these requests in the market. With the addi-tion of this unique 25OH Vitamin D Total ELISA 90’ assay to its product line the company completes its Vitamin D product port-folio. Vitamin D is commonly known for its importance in bone metabolism. It promotes calcium absorption ensuring adequate bone mineralization. Recent studies have shown additional roles in the body, including modulation of cell growth, neuromuscular and immune function, and reduction of infl ammation. Mount-ing evidence suggests that Vitamin D defi ciency could be linked to several chronic diseases, including cardiovascular disease and cancer. Production of Vitamin D is induced in the skin through exposure to sunlight, and it can be found in food like fi sh and liver oil. Th e overall prevalence of Vitamin D defi ciency however is extremely high.

DIASOURCEMEDICA Hall 03 / B59


PRODUCT NEWS – October 2015 32

®



For many healthcare workers, needlestick injuries carry a high risk of infection with blood-borne pathogens such as hepatitis C and HIV, with recent studies revealing

signifi cantly higher levels of affl iction amongst people in hospitals than in the wider population. Given the trauma experienced by those aff ected as well as the substantial consequential costs, Greiner Bio-One has added another new product to its range of safety solu-tions in an attempt to eliminate this risk as far as possible. Th e new VACUETTE CLIX Safety Hypodermic Needle can be used for tak-ing venous blood samples and for giving injections. Th is versatile product comes with a wide range of needle thicknesses and lengths, easily distinguishable thanks to their colour-coded safety shields. Its intuitive use requires only minimal training. Th e needle has an inte-grated safety mechanism, which can be activated using a solid sur-face or the user’s thumb. When the users hear a click, they know that the safety shield is positioned securely around the needle and they can proceed without any risk of a needlestick injury. Th is product has been designed to be used with the VACUETTE HOLDEX tube holder, which has an eccentric luer fi tting for a fl atter puncture angle and thus increased comfort for the patient and user.

GREINER BIOONEMEDICA Hall 03 / G60 www.cli-online.com & search 27079

028_036_cli_oct_2015.indd 32 2/10/15 13:49

Ultra-low temperature freezer reduces energy usageTh is ultra-low temperature freezer is designed to off er laboratories a greener solution: less power consumption, less noise and higher effi ciency without compromising the integrity of samples. Th e Th ermo Scientifi c TSX ultra-low tempera-ture freezer features natural refrigerants for lower environmental impact and higher cooling effi -

ciency. Due to its intuitive design, the TSX freezer uses up to 50 percent less energy than conventional refrigerant ultra-low freez-ers and delivers temperature uniformity that continuously adapts to a laboratory’s environment. Conventional ultra-low tempera-ture freezers use single-speed compressors that continually cycle on and off , resulting in poor temperature recovery following door openings. Th e TSX ultra-low temperature freezer is equipped with the unique V-Drive technology. When conditions are stable, the V-Drive is designed to operate at a low speed to reduce energy consumption while maintaining a uniform temperature. When dealing with frequent door openings or when samples are added to the freezer, the control system detects the activity and increases the drive speed to bring temperatures quickly back to the set point. Th e V-Drive also helps limit sound output to 46 db(A), making the TSX freezer up to 20 times quieter, comparable to a conventional refrigerator. For busy labs, the constant, disruptive noise created by compressors can compromise communications and create a less–than-ideal working environment. Th e freezer also utilizes water-blown foam insulation, which eliminates the off -gassing typical of urethane-insulated freezers. Additional product features include a 600-box sample capacity to maximize storage within a 1.06m2 footprint, intuitive touch screen interface for access to vital freezer information, and on-board computer and USB port for data storage and exchange. Th e TSX freezer is now available in Europe in a 50 Hz version.

THERMO FISHER SCIENTIFICMEDICA Hall 03 / K59


NGS panel molecular test for colon cancer EKF subsidiary, Selah Genomics’ newly introduced PrecisionPath next genera-tion sequencing (NGS) tech-nology cancer test is the first commercially available NGS panel molecular test priced under US$1,000 (€880).

Recently launched in partnership with Greenville Health sys-tems (GHS), the test paves the way for accessible, low-cost personalized medicine in colon cancer. PrecisionPath can help oncologists select the most effective treatment options for a patient based on the specific molecular profile of their tumour.

EKF DIAGNOSTICS MEDICA Hall 03 / C70


– October 201533PRODUCT NEWS

THE FASTEST 25OH VITAMIN D ELISA ASSAY ON THE MARKE T

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Hematology system

BioSystems off ers a complete hematology system based on an HA3 three part-diff erential and specifi c reagent, mostly suitable for routine laboratories, medi-

cal offi ces and small hospitals. Th is system establishes a new standard in blood count analysis, providing remarkable labo-ratory savings in space, time and operating costs. Th e HA3 hematology analyser utilizes the volumetric impedance method and incorporates microfl uidic technology to reduce reagent consumption, providing optimal determination of the parameters. Th e analyser provides 22 parameters with a speed of 60 tests/hour, using a 2.4uL processed sample volume. Fea-tures of the instrument include a 10-inch LCD touch screen, 2 USB, Ethernet and LIS connection ports and a storage capac-ity of 100,000 results including histograms.

BIOSYSTEMSMEDICA Hall 01 / B12 www.cli-online.com & search 27077

028_036_cli_oct_2015.indd 33 2/10/15 13:49

Automated urine analyserDesigned for uri-nalysis testing with the Mission urinaly-sis reagent strips, the updated Mis-sion U500 analyser

includes over 15 strip combinations, 8 built-in languages, and the ability to input colour and clarity all accessible through the new LCD colour touch screen. Th e Mission U500 urine analyser tests up to 500 tests/hour to fi t medium/large volume sample testing. Th e analyser handles 14 diff erent parameters including Microalbumin, Creatinine, and Calcium. Th e results are delivered via print-outs with the ability to store up to 2,000 test results in the analyser’s memory searchable by test number, patient ID, date, STAT results, and QC results. Th e Mission U500 urine ana-lyser off ers professional accuracy combined with trusted technology, delivering confi -dence in results in any clinical setting.

ACON LABORATORIESMEDICA Hall 02 / A17 www.cli-online.com & search 27085

‘Sign & seal’ durable laboratory labelsL a b o r a t o r y labelling experts CILS have developed high p e r f o r m a n c e

‘sign & seal’ durable laboratory labels for situations where the users do not have the facility to print labels using a ther-mal transfer or laser printer. The new CILS ‘sign & seal’ labels allows the user to handwrite information and then pro-tect the label with the attached, easy-to-apply, ‘wipe clean’ over-laminate layer. The durable labels shown protect hand-written data against solvents (xylene, DMSO, etc.), extreme temperatures (-80ºC to +155 ºC), alcohol wipes, auto-clave cycles, repeated handling, humid-ity and moisture, etc. CILS durable labels are manufactured to any size, shape, for-mat and design for all laboratory applica-tions and are easily transportable - ideal for ‘off-site’ clinical trials.

CILS www.cli-online.com & search 27014

Coagulation systemsSclavo Diagnos-tics International now off ers a complete system for coagulation

named KOS. Sclavo KOS 1 is a single chan-nel coagulation analyser, while KOS 2 is a dual channel system. Th ey can be used with all of the company’s coagulation rea-gents which have the applications ready for both instruments. KOS 1 & KOS 2 support clotting, chromogenic or immunoturbi-dimetric assays and are easy to operate. KOS 1 uses microvolumes (75 μl total) and is a compact, light weight instrument. KOS 2 allows double determination and PT+APTT profi le testing. It also features a magnetic reagent stirrer and a dual stop-watch function. Th e Sclavo coagulation reagents are a complete line for diagnostic screening and special tests with applica-tions developed for automated systems.

SCLAVO DIAGNOSTICSMEDICA Hall 03 / A26


PRODUCT NEWS – October 2015 34

CALENDAR OF EVENTS

Oct 18-21, 2015CMEF AutumnWuhan, Chinawww.cmef.com.cn

Oct 28-30, 2015ASCPLong Beach, CA, USAwww.ascp.org

November 16-19, 2015MedicaDüsseldorf, Germanywww.medica.de

January 25-28, 2016MEDLAB at Arab Health 2016Dubai, UAEwww.arabhealthonline.com/en/Medlab

February 11-12, 2016LabQuality Days CongressHelsinki, Finlandwww.labquality.fi

March 15-17, 2016EuroLab ExpoWarsaw, Polandwww.targieurolab.pl

March 22-24, 2016Medlab Asia Pacifi cSingaporewww.medlabasia.com

April 9-12, 2016ECCMIDIstanbul, Turkeywww.eccmid.org

April 11-13, 2016SEACare 2016Southeast Asian Healthcare & Pharma ShowKuala Lumpur, Malaysiawww.abcex.com

April 17-20, 2016CMEF Spring 2016Shanghai, Hong Kongwww.cmef.com.cn/g1225.aspx

Dates TBCFocus 2016Warwick, UKwww.focus-acb.org.uk

June 21-23, 2016JIB Journées Françaises de BiologyPariswww.jib-sdbio.fr/

July 31-August 4, 2016AACCAtlanta, GA, USAwww.aacc.org

Dates TBCMSACL EUSalzburg, Austriawww.msacl.org

September 21-24, 2016EFLM-UEMS CongressWarsaw, Polandwww.efl m-uems.warsaw2016.eu

September 25-30, 2016European Congress of PathologyColognewww.esp-congress.org/2016

Location and date TBCCMEF Autumn 2016www.cmef.com.cn/g1250.aspx

November 14-17, 2016MEDICADüsseldorf, Germanywww.medica.de

January 23-26, 2016MEDLAB at Arab HealthDubai, UAEwww.arabhealthonline.com

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

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

028_036_cli_oct_2015.indd 34 2/10/15 13:49

25-28 January 2016

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The Aptima HIV-1 Quant Dx assay

leads the hunt for HIV-1 diagnosis

and viral load monitoring.

The Aptima HIV-1 Quant Dx assay combines excellent assay performance with the full, sample-to-result automation of the Panther® system.

Panther has new preyNow CE Certified

visit AptimaVirology.com

ADS-01111-001 Rev. 001 ©2014 Hologic, Inc. All rights reserved. Hologic, Aptima, Panther and associated logos are trademarks and/or registered trademarks of Hologic, Inc., and/or its subsidiaries in the United States and/or other countries. This information is intended for medical professionals and is not intended as a product solicitation or promotion where such activities are prohibited. Because Hologic materials are distributed through websites, eBroadcasts and tradeshows, it is not always possible to control where such materials appear. For specific information on what products are available for sale in a particular country, please contact your local Hologic representative or write to [email protected].

The Aptima HIV-1 Quant Dx assay is not approved in the United States.


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