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Also in this issue :

Granulomatous infections in the bone marrow Pg. 12

MSIA for quantifi cation of proteoforms Pg. 22

LC-MS/MS used in sewage-based epidemiology Pg. 26

Scalable hematology systems

Pg.32

Ultrasound tissue processor

Pg.33

Bench-top clinical chemistry analyser

Pg.34

Digital pathology - becoming indispensable? Pg.19

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

by Siemens Healthcare

by Jokoh

by EKF Diagnostics

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Frances Bushrod, Ph.D.

Th e complex neurodevelopmental condition autism spectrum disor-der (ASD), now considered to be one of the most heritable of all neu-ropsychiatric conditions, is charac-terized by impaired communication skills and diffi culties interacting socially together with limited and repetitive behaviour patterns. It has been suggested that the dramatic increase in the number of reported cases in recent decades- one out of 68 children in the US has been diag-nosed with the condition- is largely the result of changes in how and when ASD is diagnosed as well as increased public awareness. Indeed a study reported in JAMA Pediatrics last year that followed 677,915 chil-dren born in Denmark from 1980 up to 1991 concluded that 60 per-cent of the increase in ASD preva-lence could be attributed to changes in diagnostic methods, though as yet unidentifi ed environmental risk factors could also be contributing to the rise.So far there are no clinical lab tests available to facilitate diagnosis of ASD in spite of extensive research on the elevated levels of neuro-transmitters such as 5-hydroxy-tryptamine and GABA, as well as the hormonal markers dopamine and oxytocin, found in many people aff ected. Studies have also focused on the potentially higher levels of infl ammatory cytokines and autoantibodies, and the iden-tifi cation of target genes and epi-genetic changes from gene-envi-ronment interactions in people with ASD. Neuroimaging has also identifi ed activation defi cits in cer-tain areas of the brain. But currently diagnosis still relies on develop-mental screening followed by com-prehensive (and costly) evaluation if indicated, a challenging approach for the healthcare workers involved since many of the symptoms mirror those found in other developmental disorders. But ASD can be managed once the condition is diagnosed, so early and eff ective screening is essential. Appropriate and timely

behavioural and speech therapy to improve learning, communica-tion and social skills (as well as drug therapy in some cases) greatly improves the quality of life for those aff ected. And in July this year an exciting, if preliminary, study based on the sniff response to odours was

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ContentsFRONT COVERFRONT COVER

FEATURESFEATURES

[6 - 21] PATHOLOGY FOCUS

[6 - 10] Image analysis enables standardized and quantitative pathology

[12 - 15] Granulomatous infections in the bone marrow

[16 - 18] Variations in pre-analytical FFPE sample processing and bioinformatics: challenges for next generation molecular diagnostic testing in clinical pathology

[19 - 21] Digital pathology - late starter, becoming indispensable

[22 - 30] MASS SPECTROMETRY

[22 - 25] Mass spectrometric immunoassay for top-down protein analysis

[26 - 28] Use of LC-MS/MS to measure new psychoactive substances in sewage: an application of sewage-based epidemiology

[30] A three-point case for clinical labs to adopt LC-MS/MS technology

[31] CASE STUDY Workfl ow transformed: the DxN VERIS fully automated system for molecular diagnostics in the EU

REGULARSREGULARS[3] Editor’s letter

[18] Event preview

[30] Calendar of events

[32 - 34] Product news

Digital pathology is a computer-based imaging environment which enables the analysis and storage/retrieval of information from a digital slide. It is considered to be one of the most promising recent developments in diagnostic medicine, with the potential to provide better, quicker and more cost-effective diagnosis and prognosis, especially for managing diseases like cancers.

Also in this issue :

Granulomatous infections in the bone marrow Pg. 12

MSIA for quantifi cation of proteoforms Pg. 22

LC-MS/MS used in sewage-based epidemiology Pg. 26


Pg.32


Pg.33


Pg.34

Digital pathology - becoming indispensable? Pg.19

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

by Siemens Healthcare

by Jokoh

by EKF Diagnostics

For submission of editorial material, contactthe editors at [email protected]

For advertising information, go online to www.cli-online.com, simply click on ‘Magazine’

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IntroductionTh e assessment of stained tissue sections by manual observation down a microscope has been, and still is, the steadfast manner in which histopathologists observe diseased tissue architecture in order to report on a patient’s prognosis. Th e tissue, for example the tumour microenvironment, is com-plex, highly heterogeneous and heterotypic. Although specifi c stains exist to aid in the identifi cation and semi-quantifi cation of histopathological features or biomarkers, the empirical fi eld is subjective and there-fore open to observer variability. In colo-rectal cancer (CRC) this can be the case for reporting items from the minimal core clinical data set such as diff erentiation [1] or promising histopathological features such as tumour budding [2] and lymphovascu-lar invasion [3]. Similarly, in breast cancer discrepancies exist in the reproducibilty of manual reporting of human epidermal receptor protein-2 (HER2) by fl uorescence in situ hybridization (FISH) or immuno-histochemistry and the scoring of estrogen receptor (ER), both of which have predictive implications for patient treatment strategies

[4]. Some reproducibility issues may be overcome through molecular pathology and the objective automated quantifi cation of molecular biomarkers extracted from patient tissue samples. Modern methodol-ogy in quantitative pathology, spanning the classical ‘omics’ fi elds, has the ability to create a wealth of complex big data. Indeed, the fi eld of molecular pathology has seen an explosion of big data specifi cally in transla-tional genomics, transcriptomics and pro-teomics and which has the ability to map aberrant molecular pathways with direct impact on clinical decisions. Th e automated and standardized extraction of large data sets from tissue, has been termed ‘tissue datafi cation’. Th e automated quantifi cation of molecular pathology, such as next-gen-eration sequencing (NCS), gene-chip tran-scriptomics and reverse phase protein arrays may still suff er from reproducibility issues. Th ese may occur from poor and small sam-ple sizes or tissue artefacts which can stem from multiple sources: surgical ischemia, fi xation and sample preparation. Standardi-zation is therefore the key to accurate tissue datafi cation in order to report reproducible

results which translate to the clinic. Tissue heterogeneity, both inter-patient and intra-patient, poses a very real problem for the eff ective personalized treatment decisions for patients. Tissue is oft en homogenized in order to extract the DNA, RNA or pro-tein required for many molecular pathology techniques. In doing so the tissue heteroge-neity (both subpopulation and spatial heter-ogeneity) is invariably lost and a single end-point is reported from the most dominant signal within the complex sample. A patient may therefore initially respond to a targeted treatment such as cetuximab in CRC but relapse within a set time period because of the existence of resistant KRAS and BRAF mutated subpopulations within the tumour [5]. Eff ective personalized combination therapy must rely on the capture of molecu-lar end-points across the heterogeneous disease. Quantitative pathology must take into account the imperfection of the tissue sample as well as its heterogeneity in order to produce standardized and reproducible results. With the advent of digital pathol-ogy and associated image analysis solu-tions, histopathology has joined the ranks of molecular pathology with the ability to generate robust and standardized quantita-tive big data. Image analysis can also capture the heterogeneity across a patient sample by digitally segmenting the tumour subpopu-lations while extracting quantitative hier-archical morphological or biomarker data (Fig. 1). Th is review will discuss datafi cation of the tissue section through image analysis and its benefi ts as well as some of the chal-lenges within the fi eld.

Quantitative pathology through image analysisImage analysis has been well established in order to quantify in vitro cell-based assays [6, 7] but has been slow to translate to molecular pathology and histopathology. Th is is in part due to the more complex and heterogeneous nature of the tissue as well as the need for extensive validation for clinical research compared with cell culture work. Advances in both whole-slide scanners and analysis soft ware are now making the trans-lation of image analysis to clinical research a reality. Th e use of standardized and auto-mated image analysis solutions overcomes the reproducibility issues associated with manual semi-quantitative scoring of tissue

Image analysis enables standardized and quantitative pathologyThe analysis of histopathology slides is routinely performed in a manual, semi-quantitative manner which is open to observer variability. This article summarizes how technological advances in image analysis software allow the objective and standardized quantifi cation of such samples while driving pathology towards a more personalized medicine.

by Dr Peter Caie

– September 2015 Pathology focus6

Figure 1. Identifi cation and quantifi cation of heterogeneous subpopulations. (A) A colorectal cancer (CRC) tissue microarray core labelled through immunofl uorescence for nuclei (DAPI; blue), wide spectrum cytokeratin (WsCK) (epithelial cells; green) and cytokeratin 7 (CK7) (aberrant expression in cancer epithelial cells; yellow). The white box highlights two neoplastic glands within the tissue core that partially express cytokeratin 7. Cytokeratin 7 which has been associated with a more invasive phenotype in CRC. (B) This panel shows the cytokeratin 7 fl uorescence channel in grayscale. (C) Image analysis using Defi niens Tissue Studio® software allows the segmentation of all tumour glands (khaki) within the image and quantifi es the cytokeratin 7 positive subpopulations (yellow). The algorithm furthermore reports the percentage of positive cells within each neoplastic gland.

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as it negates observer variability. Image analysis has many uses within quantitative histopathology where it can report biomarker expression at sub-cellular resolution, quantify set histopathologi-cal features, identify heterogeneous subpopulations or the spatial heterogeneity of tumour and host interaction as well as identify novel histopathological features. Standardization is always the key to reproducible results and the fi eld of image analysis is no diff er-ent. Standardization and validation must be present throughout the entire process from tissue section cutting, mounting, labelling and digitizing. Th ere are a growing number of whole-slide imagers on the market but it is paramount that these allow the use of identi-cal image capture profi les and associated image quality across all the patient samples used in a study. Once the tissue is digitized in a standardized manner the image analysis algorithms themselves must be of a high enough quality in order to deal with the complex and heterogeneous tissue. Simplifi ed algorithms have their use for basic biomarker quantifi cation but may report false results or clas-sifi cations owing to heterogeneous cell populations or inter-patient heterogeneity. Autofl uorescence or non-specifi c staining in the sample may result in the reporting of false positives or inaccurate parameters when quantifying histopathological features in the com-plex tumour microenvironment. Th e image analysis workfl ow must

therefore be robust enough to take into account or build in quality control steps to negate tissue labelling artefact [8].

Image analysis can quantify biomarkersWhole-slide image analysis of molecular biomarkers labelled via antibodies or probes such as in FISH, avoids the contamination of signals from heterogeneous subpopulations that occur when the tis-sue is homogenized (Fig. 2A). Th is has advantages over destructive assays as the tissue structure, spatial orientation and sub-localiza-tion of molecules are retained [9] and heterogeneity can be com-partmentalized and quantifi ed while providing insight into cellular interactions within the tumour and its microenvironment. In order to quantify the biomarker in question the algorithm must segment the cells and nuclei within a region of interest, e.g. the tumour or stroma (Fig. 2B). Th is gives a further advantage to automated image analysis as morphometric and texture parameters may be captured and co-registered to the cell’s expression of the desired biomarker. Th is additional information can be used to identify a morphologi-cal surrogate to a biomarker or to capture a more defi nitive result that reduces false positives. When immunofl uorescence is applied to biomarker quantifi cation a continuous data capture across the dynamic range of intensity can be reported. Th e intensity of the fl uorophore signal directly correlates to the level of protein expres-sion and therefore returns a more accurate result than the classical 1+, 2+, 3+ manual scoring of chromogenic assays. Th is continuous data can be used to calculate robust cut-off points for positive and negative expression, or for patient categorization, in soft ware such as X-Tile[ 10] or TMA Navigator [11].

Image analysis can quantify histopathological featuresImage analysis may also be employed for the quantifi cation of his-topathological features. Observer variability occurs when manual semi-quantifi cation of certain set histopathological features across tissue sections stained with hematoxylin and eosin (H&E) are reported [1–3]. Automated image analysis with the aid of specifi c labels negates observer variability and introduces standardization which is applicable across heterogeneous patient cohorts. In this manner tumour buds, lymphatic vessel density and invasion were co-registered upon the same tissue section and all quantifi ed using the same algorithm across a CRC patient cohort [8]. Th is methodol-ogy allowed the computer-based algorithm to quantify small lym-phatic vessels that were invaded by up to fi ve cancer cells and which oft en go unreported because of their obscurity in H&E stained sec-tions (Fig. 3). Th e results showed that these so called ‘occult lym-phatic invasion’ events were independently predictive of poor prog-nosis in stage II CRC patients.

Similarly image analysis may be employed to quantify the host response to the tumour and not just the tumour itself; such as the lymphocytic infi ltration within the cancer microenvironment. Th e immunoscore in CRC uses image analysis to quantify CD3+ and CD8+ lymphocytes at either the invasive front or the centre of the tumour section [12]. Th e automated quantifi cation of lymphocytes and their spatial heterogeneity have also been shown to be prognos-tic in breast cancer [13].

Image analysis can identify novel featuresResearch pathologists apply their extensive experience to identify novel or signifi cant prognostic features within the tissue section. Automated segmentation of digitized tissue sections now allows the quantifi ca-tion and standardization of complex and subtle morphological fea-tures or signatures in a continuous data capture manner. Th ese features are extracted from every possible computer segmented object within


Figure 2. Biomarker quantifi cation. (A) (i) Fluorescence in situ hybridization (FISH) to assess HER2 gene amplifi cation (green probe) in a breast cancer cell (nucleus, dark blue) alongside the housekeeping gene centromere 17 (red probe). (ii) Image analysis in Defi niens Tissue Studio® segments the fl uorescence probes to quantify gene amplifi cation; maroon (HER2 probe), blue (housekeeping). (B) Ki67 proliferation marker quantifi ed through immunofl uorescence and at single cell resolution across a CRC tissue microarray core. (i) Composite image for wide spectrum cytokeratin (WsCK) (epithelial cells; green), Ki67 (proliferation marker; red) and nuclei (DAPI, blue). (ii) Defi niens machine learning Composer™ technology automatically segments background (green) and tumour tissue (purple) from stroma (turquoise). (iii) Defi niens Tissue Studio® segments individual nuclei only within the tumour gland segmented tissue and quantifi es the Ki67 expression. (iv) Using validated cut-offs across the dynamic intensity range of ki67, the algorithm categories the tumour nuclei into negative (blue), low (yellow), medium (orange), and high (red) levels of expression.

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the image. Th is image analysis methodology quantifi es and profi les the complex phenome of the tumour’s microenvironment in an a priori ‘measure-everything big-data’ approach. Parameters extracted from single objects seg-mented across the digitized tissue section include morphometrics, texture and spatial heterogeneity. Th is is performed in an attempt to identify and quantify novel clinically rel-evant histopathological objects or predictive features from large exported image based multi-parametric big data sets. Th is emerging methodology has been termed ‘Tissue Phe-nomics’ by Gerd Binnig a Nobel Laureate and expert in image analysis. Th ese objects may represent single or combinations of morpho-metrically quantifi able histological features, which may prove too subtle to observe by eye but which could prove prognostic or predic-tive. Beck et al. demonstrated this technique in breast cancer and found the stromal micro-environment to be specifi cally relevant to prognosis [14]. Th e big data created by image analysis approaches such as these needs to be distilled in order to identify the signifi cant parameters which answer the clinical question being investigated. Bioinformatics must be applied which allows redundant parameters to be discarded and clinically relevant cut-off s to be applied to the remaining signifi cant fea-tures. Th e reduced end result of a few signifi -cant parameters from potentially thousands of captured features should form a clinically translatable test which must then be validated across multiple international cohorts.

Future developments and challenges to the fi eldTechnological advances in both image cap-ture and analysis are beginning to see the translational of automated big data from the realm of academic research to clinical tests. Further technological advances such as co-registering of tissue sections and the

ability to multiplex numerous biomarkers on a single tissue section will add greater value to the fi eld. Th is multiplexed, next-generation immunohistochemistry [15] approach coupled with automated quantifi -cation may allow whole molecular pathways to be mapped at the single cell level. Th ere are, however, challenges within the fi eld. Th e automated quantifi cation of pathology requires expensive whole-slide scanners as well as image analysis workstations along-side associated IT infrastructure to archive and keep secure the images and associated analysis. Fast Ethernet connections are also essential to recall these images in a time dependent manner. Another challenge is the acceptance of automated analysis within the clinical environment. Th is chal-lenge will need to be overcome by validat-ing the standardized and automated image analysis algorithms across multiple cohorts. Th e many applications of the fi eld, such as objective, standardized and reproduc-ible quantifi cation of biomarkers, histo-pathological features and the profi ling of a tumour’s heterogeneity hold advantages for both the pathologist and the patient. Th e negating of observer variability should increase the accuracy of patient results as should the application of clinically relevant categorical cut-off s across a continuous data set captured per patient. Th e capture of the molecular and histopathological prognostic and predictive signatures across heteroge-neous subpopulations as the potential to turn traditional population based statistics into a more personalized one which informs the optimal treatment regimen for the indi-vidual patient.

References1. Compton CC. Colorectal carcinoma: diagnostic,

prognostic, and molecular features. Mod Pathol. 2003; 16: 376–388.

2. Puppa G, Senore C, Sheahan K, Vieth M, et al. Diag-nostic reproducibility of tumour budding in colorectal cancer: a multicentre, multinational study using vir-tual microscopy. Histopathology 2012; 61: 562–575.

3. Harris EI, Lewin DN, Wang HL, Lauwers GY, et al. Lymphovascular invasion in colorectal cancer: an interobserver variability study. Am J Surg Pathol. 2008; 32:1816–1821.

4. Gown AM. Current issues in ER and HER2 test-ing by IHC in breast cancer. Mod Pathol. 2008; 21: S8–S15.

5. Baldus SE, Schaefer KL, Engers R, Hartleb D, et al. Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary colorectal adenocar-cinomas and their corresponding metastases. Clin Cancer Res. 2010; 16: 790–799.

6. Caie PD, Walls RE, Ingleston-Orme A, Daya S, et al. High-content phenotypic profi ling of drug response signatures across distinct cancer cells. Mol Cancer Th er. 2010; 9: 1913–1926.

7. Gasparri F, Mariani M, Sola F, Galvani A. Quanti-fi cation of the proliferation index of human dermal fi broblast cultures with the ArrayScan high-content screening reader. J Biomol Screen. 2004; 9: 232–243.

8. Caie PD, Turnbull AK, Farrington SM, Oniscu A, Har-rison DJ. Quantifi cation of tumour budding, lymphatic vessel density and invasion through image analysis in colorectal cancer. J Transl Med. 2014; 12: 156.

9. Kumar A, Rao A, Bhavani S, Newberg JY, Murphy RF. Automated analysis of immunohistochemistry images identifi es candidate location biomarkers for cancers. Proc Natl Acad Sci U S A 2014; 111: 18249–18254.

10. Camp RL, Dolled-Filhart M, Rimm CL. X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res. 2004; 10: 7252–7259.

11. Lubbock AL, Katz E, Harrison DJ, Overton IM. TMA Navigator: Network inference, patient strati-fi cation and survival analysis with tissue microar-ray data. Nucleic Acids Res. 2013; 41(Web Server issue): W562–568.

12. Galon J, Mlecnik B, Bindea G, Angell HK, et al. Towards the introduction of the Immunoscore in the classifi cation of malignant tumors. J Pathol. 2013; 232: 199–209.

13. Yuan Y. Modelling the spatial heterogeneity and molecular correlates of lymphocytic infi ltration in triple-negative breast cancer. J R Soc Interface 2015; 12: 20141153.

14. Beck AH, Sangoi AR, Leung S, Marinelli RJ, et al. Systematic analysis of breast cancer morphology uncovers stromal features associated with survival. Sci Transl Med. 2011; 3: 108ra113.

15. Rimm DL. Next-gen immunohistochemistry. Nat Methods 2014; 11: 381–383.

The authorPeter Caie PhDSchool of Medicine, University of St Andrews, St Andrews KY16 9TF, UK

E-mail: [email protected]


Figure 3. Quantifi cation of lymphatic vessel invasion. (A) Immunofl uorescence image showing a single cancer cell (wide spectrum cytokeratin (WsCK) labelled; green) invading a small lymphatic vessel (D2-40 labelled; red) and counterstained with DAPI (blue). (B) Shows the D2-40 captured channel so just the lymphatic vessel is visualised; automated image analysis segmented the vessel (green outline). (C) Shows the WsCK channel so just the cancer cell is visualised; automated image analysis segmented the cancer cell (red outline). (D) Automated image analysis classifi ed the event as lymphatic vessel invasion and quantifi es the invading cell (yellow) and the invaded vessel (pink).

001_011_CLI_Sep.indd 10 27/08/15 10:25

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001_011_CLI_Sep.indd 11 27/08/15 10:25

Microscopic aggregations of epithelioid histiocytes are referred to as granulomas and the respective infl ammatory process is called granulomatous. Granulomas are a well-known feature in pathology, with the fi rst description ranging back to the 17th century. Th ey show variable morphologic features including central areas of necrosis (necrotizing granulomas) or suppuration (microabsceding or suppurative granulo-mas), incorporation of foreign material or presence of giant cells due to the fusion of macrophages. Granuloma formation is usu-ally associated with local CD4+ T-cell activa-tion (and numeric increase of CD4+ T-cells at the site of granuloma formation, thus ‘con-suming’ CD4+ T-cells and leading to skew-ing of the CD4/CD8 ratio in the peripheral blood in favour of CD8) and production of interleukin 2 and 12, Interferon (IFN) γ, and tumour necrosis factor α (TNF-α) [1, 2]. In general, granulomas are provoked by agents that are diffi cult to eradicate by the enzymes of histiocytes, for example because of the diff erent phospholipid composition of the former (e.g. mycolic acids) or because of

the specifi c enzymo/immunogenetic back-ground of the host. Th e number of causative agents in granulomatous responses is rather smaller than in other infl ammatory pat-terns. Th erefore granulomas are considered to be ‘specifi c’; narrowing down the possible causative factors, their identifi cation and sub-sequent search for possible underlying agents and conditions supports the establishment of more precise clinicopathological diagnoses.

Morphology of bone marrow granulomas and etiologic considerationsGranulomatous processes involving the bone marrow (BM) can be divided into three subgroups based on morphology and clinicopathological context: (1) lipogranulo-mas, (2) infectious epithelioid granulomas, and (3) epithelioid granulomas associated with immune dysregulation. Importantly, most inborn immunodefi ciency disorders are accompanied by increased granuloma formation [3]. Examples of these disor-ders include Blau syndrome, CVID (com-mon variable immune defi ciency), RAG

(recombination-activating genes) defi ciency, XIAP (X-linked inhibitor of apoptosis) defi -ciency and chronic granulomatous disease. Depending on whether solely the BM or other organs are also involved, granuloma-tous BM processes may represent isolated fi ndings or refl ect the involvement of a sys-temic disorder. It is obvious that detection of granulomatous BM processes should be followed by an integrative diagnostic work-up considering the clinical history (travel and drug history) and presentation, but also applying imaging techniques and molecular detection methods including serology, in situ uncover techniques and PCR- and sequenc-ing-based procedures.

LipogranulomasLipogranulomas are found in up to 10% of BM samples. Th ey are not thought to be sig-nifi cant since probably not linked to specifi c underlying disorders. Yet, they may be more commonly observed in patients with acute febrile illnesses. Th ey consist of aggregates of histiocytes with variably sized lipid vacu-oles (Fig. 1A), which tend to gradually disap-pear with time resulting in a morphological appearance of lipogranulomas indistinguish-able from epithelioid granulomas. When detecting BM lipogranulomas, special atten-tion must be given to the periodic acid Schiff (PAS)/diastase-PAS stains so as not to miss involvement by Whipple’s disease, in which the foamy histiocytes stain positively (Fig. 1B); in suspect cases, the diagnosis of Whip-ple’s disease can be enhanced by additional Ziehl–Neelsen and Warthin–Starry stains as well as by PCR- and sequencing-based

Granulomatous infections in the bone marrowGranulomas are a rather uncommon, yet diagnostically helpful fi nding in trephine bone marrow biopsies. Being indicative for a rather limited number of various underlying diseases (ranging from infections to autoimmunopathies and malignant tumours), the fi nding of granulomas in the bone marrow should precipitate further analyses to uncover the underlying condition.

by Dr Thomas Menter and Dr Alexandar Tzankov


Figure 1. (A) Lipogranuloma in a patient with Escherichia coli septicemia. (B) PAS+ histiocytes in Whipple’s disease. (C) Peptoniphilus harei associated granulomas (insert Grocott-positive germ in a giant cell) in an allogeneous stem cell transplanted patient for T-PLL. (D) Sarcoid-type granuloma in a patient with sarcoidosis. (E) Granulomatous BM involvement by HL; only occasional mummifi ed Reed–Sternberg cells (Figure 1O illustrates the tumour cells of the patient stained by CD30). (F) Loose granuloma intermingled with eosinophilic granulocytes in a patient with chronic granulomatous disease. (G) Eosinophilic granuloma in a CML (chronic myelogenous leukemia) patient. (H) Giant cells in IFN-induced granulomas of a B-CLL patient, who was treated accordingly. (I) Ring-form granuloma in a patient with proven CMV infection. (J) Subchondral granuloma in an osteoporotic patient. (K) Sarcoid-type granuloma in a PTCL patient, who was given G-CSF support accompanying intensive chemotherapy. (L) Granulomatous angiitis in secondary syphilis (insert spirochete detected by immunohistochemistry). (M) Epithelioid granuloma in the centre of Castleman’s disease lesion. (N) Epithelioid granuloma in the centre of a BM infi ltration by splenic marginal zone B-cell lymphoma in a patient with chronic HCV infection, who was given IFN. (O) CD30+ Reed–Sternberg cells in granulomatous BM involvement by HL. (P) Angiocentric granulomatous appearance of a lymphomatoid granulomatosis in the BM.

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procedures. Granulomas in Erdheim–Ches-ter disease might sometimes resemble lipogranulomas [4].

Epithelioid granulomasEpithelioid granulomas are found in <1% of BM samples [5]. Th ey are more frequent in certain geographic areas and in samples from patients with immune dysregula-tion. Th ey are considered signifi cant as they are associated with various infectious (Fig. 1C), immune dysregulatory (Fig. 1D) and neoplastic disorders (Fig. 1E). Aft er clinico-pathological and molecular work-up, a spe-cifi c etiology of BM epithelioid granulomas can be attributed in up to 80% of cases. Such granulomas consist of loose [particularly in severely immunocompromised patients (the lower the CD4+ counts or the membrane-bound TNF-α and the more virulent the infectious agent, the looser the granuloma, Fig. 1F)] to cohesive clusters of epithelioid histiocytes with accompanying lymphocytes, eosinophilic (Fig. 1G) and neutrophilic gran-ulocytes, and giant cells (Fig. 1H). In patients with infectious diseases, these granulomas mostly contain organisms, which should be actively sought for and if possible visualized (e.g. mycobacteria, histoplasmata, Bartonella henselae, treponemata, Leishmania spp., toxo-plasmata) using special stains such as PAS, Ziehl–Neelsen, Fite, Grocott, May–Grün-wald–Giemsa, Warthin–Starry, etc. Immuno-histochemistry (Fig. 1L, insert) or molecular genetic methods might be necessary for etio-logic assignment. A particular CD8 predomi-nance in the BM interstitium oft en accom-panies virus infections [e.g. cytomegalovirus (CMV) and Epstein–Barr virus (EBV)] and might serve as an additional diagnostic hint [6].

Different granuloma morphotypesTh ere are diff erent granuloma morphotypes, which should be recognized because they may give a clue to the underlying disorder [7]. Caseating granulomas (i.e. granulomas with central necrosis) are usually caused by infectious agents such as mycobacteria, his-toplasmata, Francisella tularensis, Yersinia pes-tis or brucellaceae. Ring-form granulomas (Fig. 1I) can be observed in acute virus infec-tions (e.g. CMV), brucellosis, leishmaniasis and those appearing as ‘doughnut rings’ in Q-fever. Foreign body granulomas are rarely seen, but can be encountered in patients aft er repeated BM sampling (e.g. containing dis-placed keratin), or in patients with degen-erative and debilitating disorders, in whom subchondral epithelioid clusters may raise diff erential diagnostic concerns of metastatic carcinomas (Fig. 1J). Occasionally detached giant osteoclasts in

patients receiving long-term bisphosphonate therapy may be conventionally indistinguish-able from foreign body giant cells, but immu-nohistochemistry for tartrate-resistant acid phosphatase (TRAP) can be helpful, as oste-oclasts are intensively positive. Sarcoid-type granulomas (Fig. 1K) consist of compact epithelioid collections and are less specifi c as they can accompany genuine sarcoidosis and autoimmune disorders such as, for example, rheumatoid arthritis [8].

The role of giant cells in granulomasAnother clue to the etiology in a granuloma-tous infl ammation might be the type of giant cells present around the epithelioid histio-cytes [9]. Langhans giant cells are character-istically seen in tuberculosis, and appear with nuclei arranged in a horseshoe-like fashion at the periphery of the cell below the cell mem-brane. In contrast, in Touton giant cells that are typically observable in areas of fat necro-sis, the nuclei form a complete ring, and the cytoplasm is rather foamy than eosinophilic. In foreign body giant cells, the nuclei do not show a particular order but are rather haphazardly distributed. Besides, the foreign material incorporated in these cells (a clue to this diagnosis) is oft en visible by conven-tional or polarized light, being either pig-mented or birefringent.

A particular vascular association of granu-lomas should raise suspicion of vasculitis, either primary like granulomatous polyangi-itis (formerly known as Wegener’s disease), or secondary/infectious such as syphilis (Fig. 1L).

Granulomas and malignanciesImportantly, detection of BM granulomas does not exclude an underlying malignant process; on the contrary, BM granulomas may accompany various lymphoproliferative processes such as Castleman’s disease (Fig. 1M), B- and T-cell (so called ‘non-Hodgkin’) as well as Hodgkin lymphomas (HL) [10, 11], but also the BM spread of solid tumours such as lobular breast cancer [12]. Th ere is a signifi cant association between BM granu-lomas and non-Hodgkin lymphoma spread to the BM. Granulomas due to IFN therapy can be encountered in lymphoma patients [especially patients suff ering from splenic marginal zone B-cell lymphoma and diff use large B-cell lymphoma (DLBCL)] treated for underlying chronic hepatitis B- or C-virus (HBV, HCV) infections – both viruses are known to increase the risk of these lympho-mas (Fig. 1N). Occasionally, granulomatous reactions might obscure lymphoma. Th is may particularly apply to HL, in which on the one hand granulomatous reactions can occur

independently of BM infi ltration by HL (not worsening patients’ prognosis; indeed 5% of HL patients have BM granulomas without BM involvement by lymphoma), and on the other hand BM involvement by HL is usually granu-lomatous with only a handful Reed–Sternberg cells. Th erefore step-sections supported by immunohistochemistry (CD15, CD30) are warranted if BM granulomas are encoun-tered in patients suff ering from HL (Fig. 1O). Patients with lymphomas are at an increased risk of developing infections, which may also lead to granulomas and should therefore raise awareness for the diff erential work-up for infectious agents as described above. Finally, patients suff ering from sarcoidosis are at increased risk of lymphoma (odds ratio 2) and incipient lymphomas [especially Burkitt lym-phomas, DLBCL, small lymphocytic B-cell lymphomas, lymphoplasmacytic lymphomas and peripheral T-cell lymphomas (PTCL)] may provoke sarcoid-like reactions summa-rized in the so called ‘lymphoma-sarcoidosis syndrome’. To be comprehensive, apart from IFN, several other treatment compounds such as hematopoietic growth factors like G-CSF (Fig. 1K), TNF-α blockers, BCG vaccination, allopurinol, amiodarone, antipsychotics, phe-nytoin, sulfonamides, etc., can lead to BM granuloma formation [13].

Further etiologies of granulomas in bone marrow biopsiesSeveral other neoplastic and non-neoplas-tic conditions [like systemic mastocytosis, (Langerhans cell) histiocytoses], genuine histiocytic and metabolic/storage disorders (like Erdheim–Chester, Rosai–Dorfman and Gaucher’s disease), sea blue histiocy-toses, hemophagocytic lympho-histiocytosis, T-cell and histiocyte-rich B-cell lympho-mas or lymphomatoid granulomatosis (Fig. 1P) involving the BM can morphologically mimic granulomas and should be distin-guished from the latter by means of ancillary studies.

Technical considerationsA comprehensive review on technical han-dling of BM biopsies to obtain optimal immunohistochemical results has been pub-lished recently [14]. Trephine BM biopsies are best taken from the iliac crest. For further analysis, they should be sent to the pathol-ogy institutions in 10% buff ered formalin (fi nal formaldehyde concentration 4%) in order to prevent autolytic changes and to allow for an optimally preserved morphol-ogy. For decalcifi cation, chelate binders such as EDTA should be used. We discourage the use of other decalcifi cative agents such as for-mic acid or mercury containing agents such as SUSA-fi xation because of the alteration of


0012_025_CLI_Sep.indd 14 27/08/15 10:51

proteins and destruction of DNA as well as for health issues for the laboratory staff with regard to mercury exposure. Standard spe-cial stains of BM biopsies include H&E, PAS, Giemsa and Gömöri stains. Applying the PAS and the Gömöri stain can highlight fungi, but in the case of artefacts caused by BM fi bro-sis, other than Gömöri silver, stains such as a Grocott stain are helpful. Th e Giemsa stain is useful in the context of leishmaniasis and tox-oplasmosis. Other histochemical stains used in the context of infectious diseases include Fite or Ziehl–Neelsen stains (for less or more acid-fast bacteria) and the Warthin–Starry stain. Besides which, many infectious agents including parasites, bacteria and viruses can be detected using immunohistochemistry or in situ hybridization methods. In the con-text of lymphoma or suspicion of carcinoma, additional immunohistochemical stains are recommended including CD3, CD5, CD15, CD20, CD30 and pan-cytokeratin.

Take home messages1. 0.5% of BM biopsies display epi-

thelioid granulomas, up to 10% lipogranulomas.

2. 80% of such patients with epithelioid granulomas are symptomatic and in 80% of them a specifi c integrative diagnosis is possible, mostly infectious

diseases (30–50%), achievable by applying:

• special stains• fi eld studies (travelling, ethnicity, clinical

exam, drug exposure)• serology• PCR-based molecular genetic studies.3. Detection of BM granulomas does

not exclude an underlying malignant process, in fact this possibility must be actively sought for and, if needed, excluded.

4. Genuine neoplastic and non-neoplastic histiocytic disorders represent impor-tant diff erential diagnoses to granu-lomatous BM process.

References1. Zumla A, James DG. Clin Infect Dis. 1996; 23(1):

146–158.2. Helming L, Gordon S. Trends Cell Biol. 2009; 19(10):

514–522.3. Rosé CD, Pans S, Casteels I, et al. Rheumatology 2014;

54: 1008–1016.4. Kim NR, Ko YH, Choe YH, et al. Int J Surg Pathol.

2001; 9(1): 73–79.5. Brackers de Hugo L, Ffrench M, Broussolle C, et al.

Eur J Intern Med. 2013; 24(5): 468–473.6. Blanco P, Viallard JF, Parrens M, et al. Lancet 2003;

362: 1224.7. Eid A, Carion W, Nystrom JS. West J Med. 1996;

164(6): 510–515.8. Rao DA, Dellaripa PF. Rheum Dis Clin North Am.

2013; 39(2): 277–297.9. Brodbeck WG, Anderson JM. Curr Opin Hematol.

2009; 16(1): 53–57.10. Brunner A, Kantner J, Tzankov A. J Clin Pathol.

2005; 58(8): 815–819.11. Brunning RD, McKenna RW. Pathol Annu. 1979; 14

Pt 1: 1–59.12. Kettle P, Allen DC. J Clin Pathol. 1997; 50(2):

166–168.13. Bhargava V, Farhi DC. Hematol Pathol. 1988; 2(1):

43–50.14. Torlakovic EE, Brynes RK, Hyjek E, et al. Int J Lab

Hematol. 2015; 37: 431–449.

The authorsTh omas Menter MD, Alexandar Tzankov* MDInstitute of Pathology, Basel, Switzerland*Corresponding authorE-mail: [email protected]

AcknowledgmentTh is article is based on the presentation ‘Granuloma-tous infection (and reactions) in the bone marrow other than mycobacteria’ by Dr Tzankov at the 26th European Congress of Pathology in London, UK, 2014. A PDF of this presentation is available on the CLI website (www.cli-online.com).

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0012_025_CLI_Sep.indd 15 27/08/15 10:51

IntroductionMolecular diagnostics within cellular pathology have been performed since the late 1990s and have developed to include a range of techniques including short tandem repeat (STR) identity analysis, classifi cation of tumours and clonality determinations in hematopathology. More recently, with the introduction of qPCR and more recently of next generation sequencing (NGS) as shown in Figure 1, precision medicine test-ing for targeted therapies has rapidly gained access to daily practice and become a chal-lenge for molecular biologists and patholo-gists to provide the most accurate and

relevant information. As part of this testing process we discuss two major challenges which have developed, these are:• Firstly, pre-analytical processing of

formalin-fi xed paraffi n-embedded (FFPE) tissue, has shown to be a criti-cal determinant in the accuracy of downstream molecular testing in spe-cialities such as mutational screening for targeted therapies.

• Secondly, bioinformatics has become a bottleneck in data processing and inter-pretation, with the processing, analysis and reporting of the data shown vari-ability between diff erent laboratories.

Th is article looks to raise the awareness of these issues and presents possible areas for consideration to aid in their resolution.

Variation in pre-analytical sample processing of FFPE samples may lead to discrepancies in muta-tional testing of actionable genesWithin cellular pathology, the majority of molecular diagnostic clinical sample testing is now carried out on FFPE samples. Gen-erally the tissue is screened using hematox-ylin and eosin stained sections to estimate the tumour content before the preparation process of material for subsequent molecu-lar testing, as shown in Figure 2.

Recent studies have shown that variations in pre-analytical processing of samples lead to discrepancies in downstream molecu-lar diagnostic testing [1–3]. Th e variations using singleplex mutational screening were largely due to the DNA extraction system used [2, 3], quantitation using spectropho-tometry and training of laboratory staff as one study showed that pre-analytical vari-ation was signifi cant even among experi-enced laboratories [3]. In addition both DNA quantitation and integrity measure-ments play important roles in the accuracy of downstream multiplex testing using NGS.

In order to resolve some of those issues it is important to include control series of diag-nostic samples, prepared according to the diagnostic operating procedures of the labo-ratory with a variety of known mutations comprising missense mutations, simple and complex deletions and insertions. Assay con-trol using known representative DNA sam-ples from the FFPE tissue is also essential to ensure that the process of DNA extraction, quantitation and integrity measurements are performed correctly and consistently. Th is is important as DNA quality has a major eff ect on NGS performance, i.e. poor quality DNA causes a higher error rate [3].

Variations in pre-analytical FFPE sample processing and bioinformatics: challenges for next generation molecular diagnostic testing in clinical pathology

Advances in cellular pathology techniques will improve diagnostic medicine. However, such improvements have to overcome many challenges including variations in pre-analytical sample processing, bioinformatics data analysis and clinical interpretation of data. In order to resolve such challenges, bioinformatics needs to become more tightly coupled to the experimental methodology development.

by Dr Rifat Hamoudi, Dr Joshua Kapp, Sevgi Umur and Michael Gandy


Figure 1. Historical development of sequencing technology.

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In addition, diff erences in quantitation meas-urements need to be accounted for, since the diff erent instruments used have diff er-ent ways of measuring the concentration of DNA. For example, variations can be seen between systems such as Nanodrop spectro-photometry and Qubit fl uorometry. Meas-urement of DNA integrity is also important and most labs use assays such as BIOMED [4, 5] or qPCR as the ‘gold standard’ measure.

Also European external quality assurance (EQA) programmes for mutation detection of solid tumours such as European Society for Pathology (ESP, www.esp-pathology.org), European Molecular Genetics Qual-ity Network (EMQN, www.emqn.org), and United Kingdom National External Qual-ity Assessment Scheme UK NEQAS for Molecular Pathology (www.ukneqas.org.uk and www.ukneqas-molgen.org.uk) may consider including pre-analytical (e.g. pre-PCR) component in their assessment for mutation detection from FFPE samples.

Discrepancies in variant-calling pipelines and high-throughput sequencing clinical interpretationMost diseases such as cancer and inherited dis-eases are driven by genomic alterations. Recent advances in high-throughput sequencing technologies have enabled the identifi cation of somatic mutations at very high resolution. However, accurate somatic mutation-calling using high-throughput sequence data remains one of the major challenges in genomics. For somatic mutation-calling, one looks for a site in which a variant allele exists in the tumour sample but not in the normal sample. Even with the sequence data from a normal sample, variant-calling in high-throughput sequencing data is challenging due to the multiple poten-tial sources of errors. For example, artefacts occurring during PCR amplifi cation or tar-geted capture (e.g. exome-capture), machine sequencing errors, and incorrect local align-ments of reads are all well documented sources of error [6–8]. Tumour heterogeneity and

normal contamination contribute additional challenges for the tumour samples [9].

Various studies have shown low concord-ance between diff erent variant callers and bioinformatics analysis pipelines. Wang et al. [10] compared six variant callers on whole exome sequencing melanoma sample and matched blood of 18 lung tumour–nor-mal pairs and seven lung cancer cell lines carried out on the Illumina HiSeq 2000. Th e results showed discordance between the six variant callers, and the top two performing callers could only detect 86% and 71% of validated mutations respectively. O’Rawe et al. [11] compared the analysis of fi ve diff er-ent Illumina alignment and variant-calling pipelines on 15 exome sequencing data carried out using Illumina HiSeq 2000 and Agilent SureSelect version 2 capture kit at 120X mean coverage. Results showed vari-ant-calling concordance of 57.4% between the fi ve diff erent Illumina pipelines across all 15 exomes with the authors urging more caution when analysing individual genomes in genomic medicine. In addition, com-parison of the two most prominent cancer genome sequencing databases; catalogue of somatic mutations in cancer (COSMIC) [12] and Cancer Cell Line Encyclopaedia (CCLE) [13] revealed marked discrepan-cies in the detection of missense mutations in identical cell lines (57.4% conformity), where the main reason for such discrepancy is inadequate sequencing of GC-rich areas of the exome [14].

In addition to the above, various studies have shown discrepancies in the interpreta-tion of genomic data between the clinician and diagnostic laboratory. Shashi et al. [15] tried to follow up the results of 93 patients who underwent exome sequencing. Th ey investigated how the clinical interpretation of the lab results changed the diagnosis and its conformity with it. Overall, the results showed that in 25% of patients (24/93), exome sequencing showed a positive result

and in 80% (19/24) of cases, the clinicians agreed with the molecular diagnosis of the lab. However, in 20% of patients reported to be positive by the diagnostic lab, the cli-nicians thought that the suggested molecu-lar diagnosis was not correct. In addition, 5% of patients that were considered nega-tive by the exome lab or had a lower con-fi dence diagnosis, were eventually found to be positive when the exome data was reviewed by clinicians. In summary the results showed 20% false positives and 5% false negatives when comparing the inter-pretation of genomic data between diff er-ent healthcare staff .

However, it is worth noting that all the above studies used samples with high molecular weight DNA from cell lines, fresh frozen tis-sue or blood and carrying out the same studies above using FFPE samples has the potential to lead to further discrepancies due to the degraded DNA inherent to those samples increases the variation at the pre-analytical steps resulting in downstream discrepancies in mutational profi l-ing. Th is crates it a big challenge in the devel-opment of bioinformatics pipelines required to produce consistent clinically reliable data.

One way to resolve some of the bioinfor-matics related issues is to exchange the raw datasets between laboratories that prefer-entially use diff erent soft ware as part of the soft ware validation process to establish the ability of the various laboratories to detect identical gene mutations. In addition, new soft ware updates need to be validated by analysis of prior NGS datasets covering simple and complex mutations. Finally, raw NGS datasets need to be included in EQA programmes as in silico assessment.

ConclusionAlthough the above discussion very briefl y surveys the current landscape in cellular pathology, the future of molecular diag-nostics will undoubtedly develop to include integrated RNA expression analysis, DNA amplifi cation and epigenetics. Each meth-odology will have its own idiosyncrasies and will require the development of new clinically validated bioinformatics pipeline. Additionally, the need for a novel bioinfor-matics system to support integrative analy-sis will become essential. Although previ-ously attempted [16], new systems need to be developed to support integrative high-throughput sequencing analysis.

However, before novel bioinformatics soft ware solutions can be devised for big data, concerns about bioinformatics soft ware development need to be addressed. A potential starting point to


Figure 2. Formalin-fi xed paraffi n-embedded (FFPE) tissue preparation for molecular pathology testing.

0012_025_CLI_Sep.indd 17 27/08/15 10:51

address this is via supporting new bioinformatics courses that use soft ware engineering, computer programming and mathematical modelling of biological complexity at their core, supporting the education of future bioinformaticians in the art of bioinformatics soft ware development. Th is will help support a change in the current para-digm where much of the current bespoke bio-informatics soft ware today has been developed by local institutions in relative isolation, oft en in conjunction within the framework of a specialist area experimental research program [17].

Th e future landscape highly likely see the validation of wet chemistries (laboratory and clinical based) and dry (computational based) experiments carried out in more tightly coupled format than is currently performed, supporting clinical product development in the commer-cial market. Also, the future will see more focus on the development of more effi cient adaptive algorithms that address the clinical questions, leading to faster analysis and improving the clarity in the interpretation of the data.

In conclusion, within cellular pathology the incremental development of pre-analytical processing from FFPE samples coupled with more effi cient adaptive bioinformatics algo-rithms implementation are key areas of focus and crucial to the further advancement of next generation molecular pathology.

References1. Carrick DM, Mehaff ey MG, Sachs MC, Altekruse S, et al.

Robustness of Next Generation Sequencing on older formalin-fi xed paraffi n-embedded tissue. PLoS One 2015; 10: e0127353.

2. Heydt C, Fassunke J, Kunstlinger H, Ihle MA, et al.

Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequenc-ing in routine diagnostics. PLoS One 2014; 9: e104566.

3. Kapp JR, Diss T, Spicer J, Gandy M, et al. Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial. J Clin Pathol. 2015; 68: 111–118.

4. Johnson NA, Hamoudi RA, Ichimura K, Liu L, et al. Appli-cation of array CGH on archival formalin-fi xed paraffi n-embedded tissues including small numbers of microdis-sected cells. Lab Invest. 2006; 86: 968–978.

5. van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobu-lin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Con-certed Action BMH4-CT98–3936. Leukemia 2003; 17: 2257–2317.

6. Meacham F, Boff elli D, Dhahbi J, Martin DI, et al. Iden-tifi cation and correction of systematic error in high-throughput sequence data. BMC Bioinformatics 2011; 12: 451.

7. Nakamura K, Oshima T, Morimoto T, Ikeda S, et al. Sequence-specifi c error profi le of Illumina sequencers. Nucleic Acids Res. 2011; 39: e90.

8. Nielsen R, Paul JS, Albrechtsen A, Song YS. Genotype and SNP calling from next-generation sequencing data. Nat Rev Genet. 2011; 12: 443–451.

9. Gerlinger M, Rowan AJ, Horswell S, Larkin J, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012; 366: 883–892.

10. Wang Q, Jia P, Li F, Chen H, et al. Detecting somatic point mutations in cancer genome sequencing data: a com-parison of mutation callers. Genome Med. 2013; 5: 91.

11. O’Rawe J, Jiang T, Sun G, Wu Y, et al. Low concordance of multiple variant-calling pipelines: practical implica-tions for exome and genome sequencing. Genome Med. 2013; 5: 28.

12. Forbes SA, Beare D, Gunasekaran P, Leung K, et al. COSMIC: exploring the world’s knowledge of somatic mutations in human cancer. Nucleic Acids Res. 2015; 43: D805-D811.

13. Barretina J, Caponigro G, Stransky N, Venkatesan K, et al. Th e Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012; 483: 603–607.

14. Hudson AM, Yates T, Li Y, Trotter EW, et al. Discrepan-cies in cancer genomic sequencing highlight opportuni-ties for driver mutation discovery. Cancer Res. 2014; 74: 6390–6396.

15. Shashi V, McConkie-Rosell A, Schoch K, Kasturi V, et al. Practical considerations in the clinical application of whole-exome sequencing. Clin Genet. 2015; doi: 10.1111/cge.12569.

16. Watkins AJ, Hamoudi RA, Zeng N, Yan Q, et al. An inte-grated genomic and expression analysis of 7q deletion in splenic marginal zone lymphoma. PLoS One 2012; 7: e44997.

17. Prins P, de Ligt J, Tarasov A, Jansen RC, et al. Toward eff ective soft ware solutions for big biology. Nat Biotech-nol. 2015; 33: 686–687.

The authorsRifat Hamoudi*1 PhD, Joshua Kapp1 MBBS, Sevgi Umur2 BSc and Michael Gandy3 MSc1Division of Surgery and Interventional Sci-ence, University College London, London, UK2Genonymous Sciences, Küçükbak-kalköy, Defne Sokak, Flora Residence Istanbul,Turkey3Health Services Laboratories, 60 Whitfi eld Street, London, UK

*Corresponding authorE-mail: [email protected]


POCT and preanalytics to be the themes of Labquality Days 2016

Th e Labquality Days Congress will be held at the Messukeskus Expo and Convention Centre in Helsinki on 11th-12th of February 2016. Labquality Days is one of the largest annual congresses in Scandinavia focused on quality and laboratory medicine. Th e con-gress inspires clinical chemistry, laboratory medicine professionals, researchers, healthcare

experts, users of point-of-care devices, medical staff working with quality issues, managers and higher level personnel administra-tion of social- and or healthcare sectors. Th e 2016 congress themes are now announced: Point-of-Care Testing (POCT) and pre-analytics. POCT has already a major role in healthcare workfl ow. Test sensitivity or specifi city, price, speed and patient convenience are some heavily discussed topics in scientifi c meetings. In preanalytics, various disciplines such as microbiology, clinical chemistry and hematology have their own characteristic variables. Individual analyses have some unique factors that should also be taken into account in order to obtain reliable results. Labquality Days will bring together leading international speakers and opinion leaders. Th e programme consists of scientifi c lectures and panel discussions. During the congress participants have the opportunity to meet colleagues, share ideas and experience the vast clinical laboratory exhibition.

www.labqualitydays.com

EVENT PREVIEW

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Whole slide imagingA key technology driver of digital pathol-ogy is whole slide imaging (WSI), some-times also described as whole slide digital imaging. WSI scans and converts specimen glass slides into digital images, which are made accessible by soft ware for display on a computer monitor. Digitized slides allow analysis via computer algorithms, which

automate the counting of structures and quantitatively classify tissue condition. Th is task is otherwise performed, painstak-ingly, by pathologists using a microscope to view, analyse and stain tissue slides. Th e pace of development in virtual micros-copy has accelerated. Recent advances in scanning technology allow for achiev-ing over 100,000 dpi resolutions, in other

words approaching the level of optical microscopes.

Cancer staging versus grading: the role of pathologyDigital pathology off ers particular prom-ise in the grading of tumours. Tumour ‘grade’ is diff erent from the ‘stage’ of a cancer. Cancer stage refers to the size of a tumour and whether or not cancer cells have spread in the body.Pathologists play a role in providing stag-ing information, alongside physical exam-ination, imaging and lab tests. Physicians select diff erent combinations of each of these modalities for staging.By contrast, grading is principally a pathologist’s area of expertise.

Grading scalesTh e grade describes a tumour and the likelihood of its growth and spread, based

Digital pathology - late starter, becoming indispensableDigital pathology is a computer-based imaging environment which enables the analysis and storage/retrieval of information from a digital slide. It is considered to be one of the most promising recent developments in diagnostic medicine, with the potential to provide better, quicker and more cost-effective diagnosis and prognosis, especially for managing diseases like cancers.

Digital pathology is closely associated with the term ‘virtual’ microscopy, since images are viewed remotely, without a microscope or slides, after being transferred over a hospital network or the Internet.

– September 201519Pathology focus


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on the abnormality of tumour cells as seen under a microscope.Tumours are typically graded on a 1-4 scale. A lower grade indicates better prognosis, while higher grades tend to grow and spread more quickly, thus requiring more aggressive treat-ment. Grade 1 tumours are usually described as “well-diff erentiated” with tumour cells and tissue appearing close to normal. Grade 3 and 4 tumours look least like normal cells and tis-sue. Th ey are oft en described as “poorly dif-ferentiated” or “undiff erentiated,” and tend to grow and spread faster than tumours with a lower grade.

The pathologist and visual perspectivesOne of the strongest arguments in favour of digital pathology is that microscopes require pathologists to always possess keen eyesight to determine the ‘diff eren-tiation’ required for assigning a grade to a tumour. Like many other healthcare pro-fessionals, pathologists are also burdened by heavy workloads. Th is, in turn, can impact on visual acuity and interpretation.

Digitized images, in contrast, can quantify the diff erentiation (and grading) process via algorithms, reduce the risk of human error and improve accuracy.

Glass slides and inconsistent interpretationTh e challenge of consistency in pathology has been a vexing issue for some time. In March 2015, the ‘Journal of the American Medical Association’ (JAMA) published

the results of a study to “quantify the mag-nitude of diagnostic disagreement among pathologists.” Th e study focused on pathol-ogists interpreting breast biopsies from glass slides in eight US states and noted that although a breast pathology diagnosis provided “the basis for clinical treatment and management decisions,” its accuracy was “inadequately understood.” Among its fi ndings - one in four cases did not show consonance of individual pathologists’ interpretations with expert consensus. Disagreement with the reference diagnosis was statistically signifi cant among patholo-gists who interpreted lower weekly case vol-umes or worked in smaller practices - con-fi rming the observation about the inverse correlation between workload on the one side, and quality of eyesight on the other.

Digital pathology and second opinionsTo address such variations in diagnoses, second opinions have become common-place. However, for glass slides, a second opinion entails long lead times and com-plexities in a pathologist’s workfl ow (from glass packaging and transport, and at the other end, unpacking materials, verifying sample/reference case, registering the case in a laboratory information system etc.). Many pathologists are forced to cope qui-etly with a diffi cult decision - weighing up the value of a second opinion against the extra waiting time for a patient.Digital pathology streamlines access to second opinions, enabling quicker and more accurate delivery of diagnoses. Both

these correlate strongly to successful treat-ment outcomes. Telemedicine has taken explicit note about this potential. In 2014, the American Telemedicine Association published draft guidelines on the use of digital pathology in telemedicine.

Radiology and pathology: collaborative cousinsPathology is involved in almost all cancer diagnoses. Digital pathology is being seen as both a catalyst and enabler for more collabora-tion across specialties, beginning with radiology - one of the fi rst fi elds to be digitized. A September 2012 ‘BMC Medicine’ article titled ‘Integrating Pathology and Radiol-ogy Disciplines: An Emerging Oppor-tunity?’ argues for an end to traditional pathology-radiology workfl ows where the two specialties “form the core of cancer diagnosis” but remain “ad hoc and occur in separate ‘silos’, even though “the oppor-tunity for pathology-radiology integration to improve patient care is great, and more importantly, the tools to achieve this exist.”

DICOM and HIPAAUntil recently, digital pathology was ham-pered by a lack of standards for storing and transferring images, among other things, to be more in line with modern PACS systems storing radiology images. However, this was successfully addressed by DICOM (Digital Imaging and Com-munications in Medicine) supplement for digital pathology (No. 145), which was released in July 2010. According to a May 2011 report in the ‘Journal of Pathology Informatics’, the DICOM supplement standard was hailed by “everyone involved in the fi eld of digi-tal pathology” since it made it easier for hospitals “to integrate digital pathology into their already established systems without adding too much overhead costs.”

Besides, it was seen to enable diff erent vendors developing scanners “to upgrade their products to storage systems that are common across all systems.”Th ere is already suffi cient integration between digital pathology systems (DPS) and anatomic pathology laboratory information systems (APLIS) to provide pathologists with access to images and image analysis data from either, and input it to a Patient Report. On the regulatory front, DPS vendors are also well placed to support HIPAA compliance by encrypt-ing protected health information (PHI) metadata such as slide labels, hospital,


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patient and specimen information, etc. A March 2007 issue of ‘Neuroimage’ points to another major attribute of digital pathology, namely the capacity for data mining.”

Digital pathology in medical educationSo far, the key application of digital pathology has been in teach-ing. As the University of Minnesota observes, “virtual microscopes can transform traditional teaching methods by removing the reli-ance on physical space, equipment, and specimens to a model that is solely dependent upon computer-internet access. Th is rich data-base is enhanced with patient clinical presentations, laboratory data, comprehensive slide interpretations, and diagnoses.”Also in the US, a partnership between Oklahoma University Medical Center (OUMC), the Children’s Hospital, and the Uni-versity of Oklahoma College of Medicine, observes that digital pathology promotes effi ciency and cost-eff ectiveness as a teach-ing tool as well as in using digital slides for consultation with patients referred to OUMC from other hospitals.

Barriers to digital pathologyBarriers to the more widespread implementation of digital pathology have also been recently assessed. Th ese concern eco-nomics (mainly return on investment) and consistency and methodological robustness in WSI.

ROIUnlike digital radiology which has a longer legacy and a stronger case for ROI (return on investment) - principally in terms of replacing fi lm, the arguments for digital pathology are less obvious. A study by a Swedish hospital found the following justifi cations for digital pathology: savings of time in administrative tasks (13%), slide review (6%) and supervision (3.1%), alongside an increase in effi ciency of administrative tasks (100%), supervision (33%) and slide review (16%).In terms of productivity per pathologist, the gain attained by digital pathology was 10%, while overall time savings were 24%.

Consistency in digital pathology interpretationsTh e second challenge facing digital pathology has been to deter-mine the diff erence of interpretation of whole-slide images from glass-slide interpretation in diffi cult surgical cases, and the impact of such diff erences. Th is issue has been the subject of a study, with an article on the fi ndings published in the December 2009 issue of the ‘Archives of Pathology & Laboratory Medicine’.

Overall concordance between digital whole-slide and standard glass-slide interpretations was 91%, with agreement among digi-tal, glass, and reference diagnoses in 85% of cases. 9% of digital cases were discordant with both reference and glass diagnoses. Th is was due to incorrect digital whole-slide interpretation, mainly because of issues such as fi ne resolution and navigating ability at high magnifi cation.

FDA approvalOne of the biggest obstructions to the growth of digital pathology has been the absence of approval by the US Food and Drug Admin-istration (FDA) for primary diagnosis. Several EU countries allow pathologists to use WSI for primary diagnosis, with some fl ex-ibility. For instance, in Sweden, slides are digitally scanned but also physically delivered to a consulting pathologist who has the choice to review the slides on screen, in the microscope, or both. However, much of the technology development in digital pathol-

ogy - as well as vendor interest - has originated in the US, and it is evident that freeing up digital pathology for primary diagnosis in that country would galvanize use worldwide.US manufacturers have so far been able to market digital pathol-ogy technology for Research Use Only (RUO). Several vendors have also received one or more FDA 510 (k) clearances, with a key justifi cation being manual and/or quantitative analysis of immunohistochemistry and/or in situ hybridization.More developments are in the pipeline.In February 2015, the FDA issued draft guidance for the techni-cal performance assessment of digital pathology WSI devices. Th is followed an FDA Hematology and Pathology Devices Panel meet-ing six years previously to obtain industry feedback on replacing glass slides and conventional microscopy with whole slide images (WSI) for the purpose of rendering surgical pathology diagnosis. On its part, the Digital Pathology Association (DPA) expects the draft guidance to lead to follow-on guidance and clarify the FDA’s expectations for WSI regulatory submissions, enabling increased access and adoption of digital pathology for clinical use.



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The role of mass spectrometry in protein analysesMass spectrometry (MS) has proven suc-cessful in the clinical laboratory for the analysis of small molecules, but is on the rise as an emerging methodology for pep-tides and proteins [1]. Currently, a hand-ful of MS-based protein assays have been adapted in the routine clinical analyses and used for in vitro diagnostic (IVD) testing [2, 3]. MS-based methodologies are the assays of choice because they can overcome the limitations of immunoassays (i.e. nonspe-cifi c binding, cross-reactivity of analytes,

etc.). In order to be clinically applicable, all MS-based assays should comply with the well-established ‘fi t-for-purpose’ approach and be fully validated and characterized [4]. Also, working protocols must be prac-tical (in terms of sample preparation), as well as cost effi cient, so they are price-competitive with current immunoassays. Although overcoming these requirements is still a challenge, one inevitable advantage that makes MS-based protein assays indis-pensable, is their unique ability to address protein heterogeneity.

Th e majority of clinically adapted MS-based methodologies for protein profi ling are the single/multiple reaction monitor-ing liquid chromatography MS (SRM/MRM LC-MS) assays [5, 6] and mass spec-trometric immunoassays (MSIA) [7, 8]. MRM assays are ‘bottom-up’ assays and use isotopically labelled peptides as inter-nal reference standards for surrogate pro-tein quantifi cation via chosen, enzymati-cally generated peptides. Because SRM/MRM LC-MS assays detect only specifi c peptides, important information about novel proteoforms or post-translational modifi cations with potential clinical impli-cations can be overlooked. MSIAs, on the other hand, follow a ‘top-down’ approach, having intact proteins as primary targets. As a result of the immunoaffi nity capture of a targeted protein(s), and the ‘soft ’ ioni-zation in MALDI-TOF (matrix-assisted laser desorption/ionization–time of fl ight) MS, MSIA enable for detection of post-translationally modifi ed proteoforms as well as other changes in protein structure without the harsh enzyme digestion. Lit-erature data show that post-translationally modifi ed proteins have the potential to be used as biomarkers [9]. Having that in mind, the proteoform detection adds a whole new dimension to the way we look at proteins.

Mass spectrometric immuno-assay for analysis of RANTES proteoformsHere we review a mass spectrometric immunoassay (MSIA) for quantifi cation of the chemokine RANTES proteoforms in human plasma samples. RANTES (Regulated on Activation, Normal, T-cell Expressed and Secreted), is a member of the CC chemokine family (hence its alter-native name – CCL5) and is essential in the initiation and maintenance of infl am-mation [10]. RANTES has been studied extensively in clinical context, in associa-tion with autoimmune diseases, arthritis, diabetes, obesity and metabolic syndrome, some types of cancer and viral infections [11–13]. In addition, RANTES proteo-forms have been associated with athero-sclerosis and cardiovascular diseases [14].

Th ere are several types of commercially available, as well as in-house devel-oped immunoassays for total RANTES

Mass spectrometric immunoassay for top-down protein analysisMass spectrometry-based methods hold great promise for addressing protein heterogeneity. As a result of post-translational processing, proteins can exist in vivo as multiple proteoforms. The added information contained in the protein profi le can be important in physiological and pathological states. Presented here is an overview of a mass spectrometric immunoassay (MSIA) for quantitative determination of the chemokine RANTES proteoforms. MSIA offers protein quantifi cation and profi ling in a high-throughput and time-effi cient manner. Across a cohort of ~300 human plasma samples, a total of 11 different RANTES proteoforms were quantifi ed in less than 3 hours.

by Dr O. Trenchevska, N. D. Sherma, Dr P. V. Reaven, Dr R. W. Nelson and Dr D. Nedelkov

– September 2015 Mass Spectrometry22

Figure 1. Example of MALDI-TOF mass spectra obtained in the MSIA. Presented in the full-range form and zoomed in the inlets are the detected RANTES proteoforms – native full-length RANTES and met-RANTES (the internal reference standard), and truncated (3-68), (4-68), (4-66), (3-66), M-RANTES, (4-65) and (4-64) proteoforms. The signal labelled (*) is non-specifi cally bound.

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0012_025_CLI_Sep.indd 23 27/08/15 10:51

quantifi cation [15]. Th ese assays, however, are not tailored for detecting and quantifying the numerous proteoforms associated with RANTES. In previous work, we have addressed RANTES heterogeneity by qualitative and quantitative MSIA [16, 17]. In developing the quantitative MSIA for RANTES, we took on the approach of using RANTES standard and a homologous RANTES derivative – met-RANTES as an internal reference standard (IRS) for quantifi cation. Met-RANTES is a recombinant derivative of RANTES (therefore not found in humans) and has a molecular weight (MW) of 7979.2 Da, which is in close proximity to that of full-length human RANTES (MW=7847.9 Da). Another advan-tage of using the RANTES/met-RANTES pair was the ability of a single anti-RANTES antibody to capture both proteins from the biological samples.

Th e immobilization of the anti-RANTES antibody was onto acti-vated surfaces of affi nity pipettes as previously described [17]. Th e quantity of the anti-RANTES antibody (7.5 μg Ab/tip) was optimized to be enough that variable RANTES concentrations in the samples could be truly quantifi ed with the assay. Due to low plasma RANTES physiological concentration (in the ng/mL level), undiluted plasma was used for the analyses. In the analyti-cal samples, met-RANTES was spiked at a constant concentra-tion (V=250 μL at c=50 ng/mL), in order to produce a constant signal in the mass spectra. Following sample preparation and affi nity pipette derivatization, the antibody-coated pipettes were mounted onto the head of an automated 96-channel pipettor and initially rinsed with PBS/0.1% Tween buff er. Next, the pipettes were immersed into a microplate containing the analytical sam-ples and 500 aspirations and dispense cycles were performed (100 μl volumes each) allowing for affi nity capture of RANTES proteo-forms and met-RANTES. Th e pipettes were then rinsed with assay buff er water to remove non-specifi cally bounded proteins. Cap-tured proteins were eluted directly on a 96-well formatted MALDI target using sinapic acid. Five-thousand laser shots of mass spec-tra were acquired from each sample spot on a Bruker’s Ultraf-lex III MALDI-TOF/TOF mass spectrometer. Th e mass spectra were externally and internally calibrated with protein standard

mix and the singly and doubly charged met-RANTES signals before analysis.

In the mass spectra, several RANTES proteoforms can be detected. As shown in Figure 1, most abundant are signals rep-resenting full-length, native RANTES (1-68) and met-RANTES, along with the N-terminally cleaved RANTES proteoforms (3-68) [MW=7,663.7; missing the ‘SP’ N-terminal dipeptide, product of dipeptidyl peptidase IV (DPP IV) enzyme cleavage] and (4-68) (MW=7,500.6; missing ‘SPY’ N-terminal tripeptide). RANTES proteoforms missing N-terminal tripeptide and C-ter-minal dipeptide, (4-66) (MW=7,282.3) completed the dominant signals (Figure 1, top right inlet). Additional RANTES proteo-forms were identifi ed, in lower abundance and frequency: (7-66) (MW=6993.1; missing six N-terminal and two C-terminal amino acids), (4-64) (MW=7040.1; missing three N-terminal and four C-terminal amino acids), (4-65) (MW=7153.2; missing three N- and three C-terminal amino acids) and (3-66) (MW=7445.5; miss-ing two N- and two C-terminal amino acids). Th e signal labelled M-RANTES with MW=7413.5 has multiple N- and C-terminal truncation possibilities, and has not been specifi cally assigned. Th e assignation of these signals was done using the observed m/z values and the program Paws, and was in accordance with previ-ously published qualitative results [16].

All identifi ed RANTES proteoforms were quantifi ed using an eight-point standard curve, in the range from 1.56 to 200 ng/mL. Th e standard curve was constructed from the ratio of the peak intensities of the RANTES standard and the met-RANTES IRS (y-axis) versus the RANTES standard concentration (x-axis). For the analytical samples, fi rst, the RANTES/met-RANTES peak intensity ratios for each proteoform were determined and summed up. Using the generated standard curve equation, these ratios were used to determine the total RANTES concentration in the analysed plasma sample. Th en, the concentration of the individual RANTES proteoforms was calculated based on their percentage of the total RANTES. Th e assay was validated through several standard procedures. Th e intra- and inter-assay precision experiments yielded coeffi cients of variation of <10%. Linearity and spiking-recovery experiments produced results between 92 and 112% (observed vs expected concentration). In a fi nal test, the results of the RANTES MSIA were compared with those obtained with commercially available ELISA using Altman–Bland plot. A good correlation, with slight positive bias (11.3%) was obtained with the native RANTES [17].

Th e developed MSIA for RANTES proteoforms was applied to a cohort of 297 human plasma samples. Th e analyses were performed on an automated platform, which enabled for a high-throughput analysis of 96 samples in a single run. Among the samples, we were able to determine the concentration and frequency of 11 RANTES proteoforms (Figure 2). Th e total average concentration of RANTES was found to be 44.9 ng/ml (2.15–163 ng/mL). In majority of sam-ples, the main proteoform was the full-length, native RANTES [c(RANTES(1-68))avg=37.4 ng/mL; 1.92–132 ng/mL], followed by RANTES (3-68), [c(RANTES(3-68))avg =6.64 ng/mL; 0.138–34.4 ng/mL]. Th e other truncated RANTES proteoforms were present in variable frequencies in the samples, albeit at much lower concentra-tions (<10% of the total RANTES). Figure 2 summarizes the distri-bution and frequency of all 11 RANTES proteoforms. Even though majority of RANTES proteoforms were detected in only a handful of samples and in low quantities, they should be given full attention. Cleaved proteoforms have the potential to be used as indicators of


Figure 2. Quantitative distribution of RANTES proteoforms across a cohort of 297 human plasma samples. Box represents 25th–75th percentile; solid line, median concentration; dashed line, mean concentration; error bars, 10th and 90th percentile; N, number of samples expressing the specifi c RANTES proteoforms.

0012_025_CLI_Sep.indd 24 27/08/15 10:51

an enzymatic activity, and, in turn, of changes in the meta-bolic homeostasis [18]. Th e information that this MSIA pro-vides puts a new perspective of RANTES quantitative analysis and can be a good starting point for looking at RANTES hetero-geneity in clinical context.

Concluding remarksTh e assay described above uses MALDI-TOF-MS to fully quantify RANTES pro-teoforms, and it is one of just a handful of such MALDI-based assays in existence today. Th e assay’s two-step approach is similar to that of well-estab-lished immunoassays, with the added benefi t of MS detection as an enabling factor in dif-ferentiating the multiple pro-teoforms. Th e MALDI target is designed to accept the elu-ates from 96 tips at the same time, therefore making it high-throughput and time effi cient (total time for RANTES assay is ~1 hour). Th e assay is per-formed on an automated plat-form, which limits the errors that can occur during assay execution. In review of previ-ous and ongoing work, MSIA for RANTES performs well and introduces a new prospect and capacity for potential clin-ical applications in the fi eld of biomarker discovery/rediscov-ery and diagnostics.

References1. Strathmann FG, Hoofnagle AN. Am

J Clin Pathol. 2011; 136: 609–616.2. Agger SA, Marney LC, Hoofna-

gle AN. Clin Chem. 2010; 56: 1804–1813.

3. Kiernan UA, Phillips DA, Trenchevska et al. PLoS One 2011; 6: e17282.

4. Carr SA, Anderson L. Clin Chem. 2008; 54: 1749–1752.

5. Anderson NL, Anderson NG, Haines LR, et al. J Proteome Res. 2004; 3: 235–244.

6. Yocum AK, Chinnaiyan AM. Brief Funct Genomic Proteomic. 2009; 8: 145–157.

7. Nelson RW, Krone JR, Bieber AL, et al. Anal Chem. 1995; 67: 1153–1158.

8. Trenchevska O, Kamcheva E, Ned-elkov D. Proteomics 2011; 11:

3633–3641.9. Jin H, Zangar RC. Biomark Insights

2009; 4: 191–200.10. Youn BS, Mantel C, Broxmeyer HE.

Immunol Rev. 2000; 177: 150–174.11. Lit LC, Wong CK, Tam LS, et al.

Ann Rheum Dis. 2006; 65: 209–215.12. Matter CM, Handschin C. Circula-

tion 2007; 115: 946–948.13. Azenshtein E, Luboshits G, Shina

S, et al. Cancer Res. 2002; 62: 1093–1102.

14. Winnik S, Klingenberg R, Matter CM. Eur Heart J. 2011; 32: 393–395.

15. Kaburagi Y, Shimada Y, Nagaoka T, et al. Arch Dermatol Res. 2001; 293: 350–355.

16. Oran PE, Sherma ND, Borges CR, et al. Clin Chem. 2010; 56: 1432–1441.

17. Trenchevska O, Sherma ND, et al. J Proteomics 2014; 116C, 15–23.

18. Lim JK, Lu W, Hartley O, et al. J Leukoc Biol. 2006; 80: 1395–1404.

The authorsOlgica Trenchevska*1, Nisha D. Sherma1, Peter V. Reaven2,

Randall W. Nelson1, Dobrin Nedelkov1

1Molecular Biomarkers, Th e Biodesign Institute at Arizona State University, Tempe, AZ, USA2Phoenix Veterans Aff airs Health Care System, Phoenix, AZ, USA



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0012_025_CLI_Sep.indd 25 27/08/15 10:51

IntroductionSewage-based epidemiology (SBE) is an alternative method of monitoring popula-tion drug use by the analysis of excretion products of drugs in sewage (Fig. 1). SBE has been applied since 2005 as a complementary approach to classical investigation meth-ods, such as interviews with users, medical records, population surveys, and crime statis-tics for estimating illicit drug use in commu-nities [1–3]. Data obtained from SBE provide information on drug use in a direct, quick and objective way.

New psychoactive substances (NPS) are sub-stances that are not controlled by the 1961 United Nations Single Convention on Nar-cotic Drugs or the 1971 Convention on Psy-chotropic Substances and that may pose a threat to public health [4, 5]. Th ese compounds mimic eff ects of illicit drugs like cocaine, can-nabis and amphetamines and are produced to evade law enforcement by introducing slight modifi cations to chemical structures of con-trolled substances [6]. Currently, more than 450 NPS are being monitored by the Euro-pean Monitoring Centre for Drug and Drug

Addiction (EMCDDA) with 101 new sub-stances reported for the fi rst time in 2014 to EU Early Warning System (EWS). Synthetic cannabinoids and synthetic cathinones are the largest groups in the NPS scene [7]. NPS are easily acquired through online vendors and in smart shops where they are sold with mislead-ing information about their eff ects and safety [8]. Th ey are considered a growing problem in many communities and are responsible for numerous fatal intoxications [9]. SBE has the potential to be usefully applied for the detec-tion and quantifi cation of NPS to document their occurrence to appropriate authorities. Being an emerging issue, only a few stud-ies have applied SBE for the analysis of NPS [10–13]. In this contribution, the optimiza-tion, validation and application of an ana-lytical method using liquid chromatography coupled to positive electrospray tandem mass spectrometry (LC–ESI-MS/MS) for the deter-mination of seven NPS in sewage: methoxe-tamine (MXE), butylone, ethylone, methylone, methiopropamine, 4-methoxymethampheta-mine (PMMA), and 4-methoxyamphetamine (PMA) is described together with a critical evaluation of the methodology.

LC-MS/MS methodologyAn LC-MS/MS method was developed and validated using a Phenomenex Luna HILIC (hydrophilic interaction liquid chromatog-raphy) 200A (150 x 3 mm, 5 μm) column, with a mobile phase composed of A) 5 mM ammonium acetate in ultrapure water and B) acetonitrile. Th e mass spectrometer com-pound dependent parameters, fragmentor voltage and collision energy, were optimized to acquire two multiple reaction monitoring (MRM) transitions (qualifi er and quantifi er) for each compound, and one MRM for the internal standards (IS). Th e method was vali-dated, assessing accuracy and precision, using blank sewage (samples collected prior to 2009 in which NPS have not been detected). A lin-ear range with lower limits of quantifi cation (LLOQ) of 0.5 ng/L (MXE and methylone) and 2 ng/L (all other compounds) and upper limits of quantifi cation (ULOQ) of 200 ng/L was achieved for investigated compounds. Th e limit of detection (LOD) was between 0.02 and 0.2 ng/L for all compounds.

Sample collection and preparation24-h composite infl uent sewage samples were collected from diff erent wastewater treatment plants (WWTPs) in Belgium and one WWTP in Zurich. Before sample extrac-tion, 50 mL sewage was fi ltered through a 0.7 μm glass fi lter to remove solid particles. Aft er fi ltration, the samples were brought to pH 2 using a 6 M HCl solution and spiked with deuterated IS at a concentration of 100 ng/L. Th ereaft er the solid-phase extraction (SPE) procedure was performed using a mixed-mode strong cation exchange sorbent-Oasis MCX (Fig. 2).

Application of the procedureTh e method could reliably diff erentiate the analytes and IS from endogenous compo-nents. MXE, methylone and ethylone could be detected. Th e method revealed the pres-ence of MXE in sewage from fi ve urban cen-tres within two counties in Belgium. Methyl-one was detected and quantifi ed in only two samples from Switzerland at levels slightly higher than LLOQ (Fig. 3). Th e compounds that were not detected could be absent in the sewage or present in the form of metabolites which were not targeted in the present study.

Use of LC-MS/MS to measure new psychoactive substances in sewage: an application of sewage-based epidemiology

This contribution describes the possibility of applying liquid chromatography coupled to tandem mass spectrometry for analysing sewage in order to track down the use of new psychoactive substances.

by J. Kinyua, Prof. A. Covaci, Prof. A. L. N. van Nuijs


Figure 1. Illustration of a sewage-based epidemiology approach. WWTP, wastewater treatment plant.

0026_036_CLI_Sep.indd 26 27/08/15 10:40

CHANGES EVERYTHINGShimadzu’s new LCMS-8060 makes a real differ-ence to working better and faster. The LCMS-triple-quadrupole combines all UF technologiesand pushes the limits of LC-MS/MS quantitationfor applications requiring highest sensitivity androbustness.

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*2,400 samples of femtogram levels of alprazolam spiked into protein-precipitated human plasma extracts over a 6 dayperiod (over 400 samples were injected each day).


0026_036_CLI_Sep.indd 27 27/08/15 10:40

Advantages/limitations of the SBE methodologyPhenylethylamine-based compounds (syn-thetic cathinones and amphetamine-like substances) form a large group of NPS and they are very polar. Hydrophilic interaction was found to be a good and robust LC sta-tionary phase to obtain retention for these high-polarity compounds. Furthermore, we showed for the fi rst time in SBE that the use of a more realistic matrix for method devel-opment, such as real sewage, can help in over-coming challenges associated with matrix eff ects in MS detection. Th e results from these samples demonstrate the importance of developing highly sensitive analytical meth-ods that can detect and quantify NPS at very low concentrations (<10 ng/L).

Limitations of the present methodsIt is diffi cult to determine if the low drug concentrations in sewage are related to low popularity of the NPS or due to the presence

of an unknown form of the parent drug in sewage, urinary metabolites or transforma-tion product from other in-sewer processes. SBE requires a specifi c, reliable and stable biomarker for the NPS of interest. Further studies on the metabolism and in-sewer transformation processes (which may aff ect stability of drug residues) of NPS needs thus to be carried out to provide SBE with infor-mation regarding additional biomarkers of NPS parent drugs.

Future of SBE in NPS analysisConcentrations of NPS in sewage may be low depending on the area served by the WWTP and on the prevalence of its use [14]. Th ere-fore, pooled urine analysis would be useful in detecting the occurrence of NPS before dilu-tion into sewage [15]. It would be a valuable approach to combine pooled urine analysis and SBE to track down the actual use of NPS in communities.

ConclusionIn conclusion, SBE can help in revealing the occurrence of NPS within catchment areas of urban centres and showed the need to develop very sensitive analytical methods to detect NPS in sewage.

References1. Bijlsma L, Sancho JV, Pitarch E, et al. Simultaneous

ultra-high-pressure liquid chromatography-tandem mass spectrometry determination of amphetamine and amphetamine-like stimulants, cocaine and its metabolites, and a cannabis metabolite in surface water and urban wastewater. J Chromatogr A 2009; 1216: 3078–3089.

2. Boleda MR, Galceran MT, Ventura F. Trace determi-nation of cannabinoids and opiates in wastewater and surface waters by ultra-performance liquid chroma-tography-tandem mass spectrometry. J Chromatogr A 2007; 1175: 38–48.

3. Huerta-Fontela M, Galceran MT, Ventura F. Ultrap-erformance liquid chromatography-tandem mass

spectrometry analysis of stimulatory drugs of abuse in wastewater and surface waters. Anal Chem 2007; 79: 3821–3829.

4. United Nations Offi ce on Drugs and Crime (UNODC). Global synthetic drugs assessment. (United Nations publication, Sales No. E.14.XI.6), 2014. http://www.unodc.org/documents/scientifi c/2014_Global_Syn-thetic_Drugs_Assessment_web.pdf

5. King LA, Kicman AT. A brief history of ‘new psycho-active substances’. Drug Test Anal. 2011; 3: 401–403.

6. Dargan PI, Wood DM. Novel psychoactive substances classifi cation, pharmacology and toxicology. Elsevier/Academic Press, 2013. ASIN: B00FK8HYY2.

7. European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). New psychoac-tive substances in Europe. An update from the EU Early Warning System, 2015. http://www.emcdda.europa.eu/publications/2015/new-psychoactive-substances

8. EMCDDA. EMCDDA–Europol 2013 Annual Report on the implementation of Council Decision 2005/387/JHA, 2014. http://www.emcdda.europa.eu/publications/implementation-reports/2013

9. Vevelstad M, Øiestad E.L, Middelkoop G, et al. Th e PMMA epidemic in Norway: Comparison of fatal and non-fatal intoxications. Forensic Science Interna-tional 2012; 219: 151–157.

10. Kinyua J, Covaci A, Maho W, et al. Sewage-based epidemiology in monitoring the use of new psycho-active substances: validation and application of an analytical method using LC-MS/MS. Drug testing and analysis 2015 ( In press).

11. Reid M.J, Derry L, Th omas K.V. Analysis of new classes of recreational drugs in sewage: Synthetic cannabinoids and amphetamine-like substances. Drug Test Anal. 2014; 6: 72–79.

12. Van Nuijs ALN, Gheorghe A, Jorens PG, et al. Opti-mization, validation, and the application of liquid chromatography-tandem mass spectrometry for the analysis of new drugs of abuse in wastewater. Drug Test Anal. 2014; 6: 861–867.

13. Kankaanpää A, Ariniemi K, Heinonen M, et al. Use of illicit stimulant drugs in Finland: a wastewater study in ten major cities. Sci Total Environ. 2014; 487: 696–702.

14. Archer JRH, Dargan PI, Lee HMD, et al. Trend analysis of anonymised pooled urine from portable street urinals in central London identifi es variation in the use of novel psychoactive substances. Clinical Toxicol (Phila). 2014; 52: 160–165.

15. Archer JRH, Dargan PI, Hudson S, et al. Analysis of anonymous pooled urine from portable urinals in central London confi rms the signifi cant use of novel psychoactive substances. QJM 2013; 106: 147–152.

The authorsJuliet Kinyua MSc, Adrian Covaci PhD, Alex-ander L.N. van Nuijs* PhDToxicological Center, University of Antwerp, Belgium



Figure 2. Solid-phase extraction procedure for sewage using Oasis MCX cartridge. HILIC, hydrophilic interaction liquid chromatography.

Figure 3. Chromatogram with quantifi er transitions from an infl uent sewage sample from a Swiss city. The measured concentrations of MXE and methylone were 2.5 ng/L. Ethylone was detected with concentration below lower limit of quantifi cation (LLOQ).

0026_036_CLI_Sep.indd 28 27/08/15 10:40

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25-OH Vitamin D2/D3 in Human Serum Samples

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Quantitative Amino Acids in Biological Fluids Samples

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0026_036_CLI_Sep.indd 29 27/08/15 10:41

There are three compelling reasons for clinical labs to incorporate LC-MS/MS solutions into their routine operations:

1. Quality from improved specifi city2. Workfl ow effi ciency3. Meeting market demand

Testing quality Direct measurement technology is more specifi c and can address the limitations inherent to immunoassay testing. In par-ticular, for small-molecule analyte testing, immunoassay results can be elevated due to the presence of metabolites from other drugs with core structures that are similar to the targeted analyte.

Workfl ow effi ciency As LC-MS/MS solutions are typically found in specialty laboratories, most clinical labs must outsource certain tests. Transporting samples adds com-plexity, cost, and time to the testing pro-cess. In drugs of abuse testing for exam-ple, patients are initially screened using immunoassay and then confi rmed using LC-MS/MS. Having this capability within the laboratory can provide quick turna-round times for faster diagnosis and treat-ment for patients. LC-MS/MS methods can also test for multiple analytes simul-taneously where immunoassay methods require a separate test for each analyte.

Meeting market demandTh e market demand that LC-MS/MS addresses arises from trends such as the growing use of opiates and increas-ingly more stringent regulations. On-site LC-MS/MS testing can deliver both qualitative and quantitative accuracy and precision to help clinicians understand

the actual consumption of abused and or prescription drugs.

While the reasons for adopting LC-MS/MS are compelling, there are logistical and regulatory barriers to entry rooted in the current state of LC-MS/MS auto-mation. LC-MS/MS processes are auto-mated to some degree, but the entire process must be improved. Workfl ows still require many manual steps, includ-ing sample preparation and data entry into LIMS systems. Th is takes labour and time and can lead to errors, all of which are unacceptable to regulatory bodies and laboratory managers. Th e industry recognizes that innovative solutions are required to address these analytical chal-lenges; however, only when LC-MS/MS achieves the rigorous engineering and quality developments required for regu-latory approval will more labs be allowed to adopt this gold standard technology and make a meaningful advancement in diagnostic testing.

A three-point case for clinical labs to adopt LC-MS/MS technologyLiquid chromatography-mass spectrometry (LC-MS/MS) is one of the most promising diagnostic technologies in the in-vitro diagnostics industry, but it is not yet widely adopted by mainstream laboratories. An estimated fi ve percent of LC-MS/MS instruments reside in truly clinical diagnostic settings while the majority are deployed in research and reference laboratories.

by Dr Bori Shushan


Bori Shushan, Ph.D. is an Analytical Chem-ist with over 25 years experience in the application of mass spectrometry to screen-ing and clinical diagnostics. He currently is President of Clinical MassSpec Consultants, a consultancy based in Toronto.

CALENDAR OF EVENTS

Sept 3-5, 2015Biotech ChinaNanjing, Chinawww.biotechchina-nj.com

Sept 4-6, 2015British Society for Allergy & Clinical ImmunologyTelford, UKwww.bsaci.org

Sept 5-9, 2015European Congress of PathologyBelgrade, Serbiawww.esp-congress.org

Sept 9-12, 201518th Annual Meet-ing of the ESCV (European Society for Clinical Virology)Edinburgh, Scotland, UKwww.escv2015.com

Sept 13-16, 2015Eurotox 2015 - 51st Congress of the EuropeanSocieties of ToxicologyPorto, Portugalwww.eurotox2015.com

Sept 17-20, 2015MEMBS (Middle East Molecular Biology Society)Istanbul, turkeywww.membs.org

Sept 20-23, 201539th European Con-gress of CytologyMilan, Italywww.cytology2015.com

Sept 28-Oct 2, 2015ASHI (American Society for Histo-compatability & Immunogenetics)Savannah, GA, USAwww.ashi-hla.org

Oct 1-3, 201554th Annual ESPE MeetingEuropean Society for Paediatric Endocri-nologyBarcelona, Spainwww.espe2015.org

Oct 7-9, 2015ESPT 2015 – Integra-tion of Pharmacoge-nomics in clinical decision supportBudapest, Hungarywww.esptcongress.eu

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

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

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

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

Feb 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 11-13, 201619th SE Asian Health-care & Pharma showKula Lumpur, Malaysiawww.abcex.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/

0026_036_CLI_Sep.indd 30 27/08/15 10:41

Andrew Williams, (Nexus Global Solu-tions) explained how the company con-ducted a multi-site time/motion workfl ow analysis study comparing Beckman Coulter’s DxN VERIS Molecular Diagnostics System to existing batch and semi-automatic molec-ular diagnostic platforms. During two 4-day studies conducted in Sheffi eld (UK) and Bar-celona (Spain) in May 2015, the systems were run in tandem, and the study focused on key areas including time to result, hands on time, and maintenance requirements.

Jordi Vila, (Barcelona) identifi ed how his laboratory needed workfl ow improve-ments to reduce waste, increase effi ciency, maximize use of personnel and equip-ment, and to reduce the potential for errors. “Currently molecular diagnostics are undertaken on three platforms, with specifi c assays only run on certain days of the week,” Vila commented. “DxN VERIS would take up less space in our cramped laboratory and allow up to 20 assays to be run at any time. Batching would not be required as samples can be added as they arrive, and results are available as sam-ples are running. Th is is a great advantage; using existing systems we have to wait until the end of the run.”

“Th e VERIS simplifi ed workfl ow, reduced the number of steps required from sample preparation to result from 29 to just 11,” Vila continued, “we also made savings in maintenance time and reagent/consumables. Assay reagents are stored on board for up to 14 days, and, as shown in fi gure 1, only four consumables are required, comparable systems need up to 20 or more.” “We reduced hands-on time for HIV-1 testing; DxN VERIS took approximately half the time to result against other sys-tems. We experienced workfl ow advan-tages via continuous loading; use of universal tube racks; true single sample random access; the ability to add urgent samples, and test multiple target viruses at any time. We could save space with the need for only one instrument, together with more economical use of laboratory staff because no pipetting is required,” confi rmed Vila. Duncan Whittaker, (Sheffi eld, UK) explained that the study undertaken at his site involved comparison of DxN VERIS with three other systems. “Work-loads within our department have increased by 57% in the last 3 years”, Whittaker reported. “Faced with this challenge, together with competition from other private and public labora-tories and the loss of experienced staff , workfl ow improvements and effi cient utilization of both staff and resources are key”.

“Currently we use three systems, housed in diff erent rooms, so a lot of staff time is spent moving from one instrument to another. Complexity of use was studied and with these systems we found that 29

or 30 steps were required, but only 11 steps are needed with VERIS”, Whittaker continued (fi gure 2).

“When you combine this with the ease of use – it only took 20 minutes to train staff to use the equipment – and rapid time to result and the ability to run multiple targets on one system, we could off er signifi cant benefi ts to clini-cians, allowing them to deliver better patient management. Just to cite HBV as an example, we would normally do 3-4 extractions over a couple of days before batching on to the next system, so results would take several days. With VERIS we can off er same day results.”

“Feedback from renal transplant and renal dialysis departments has shown that the true single sample random access mode and rapid time to result would greatly ben-efi t the way patients are seen and treated in clinics. For example, with current systems, dialysis patients returning from abroad need to attend two hospital visits to con-fi rm negative status, but with VERIS we could reduce this to one visit – an imme-diate benefi t for the patient who may have had to travel many miles to get to us,” Whit-taker concluded.

For further information about these studies, DxN VERIS Molecular Diagnostics System and the DxN VERIS assays currently availa-ble, please contact: Tiff any Page, Senior Pan European Marketing Manager Molecular Diagnostics, Email: [email protected] or visit:www.beckmancoulter.com/moleculardiagnostics

Workfl ow transformed : the DxN VERIS fully automated system for molecular diagnostics in the EUDelegates at EuroMedLab 2015, held in Paris 21st-25th June, attended the Beckman Coulter Molecular symposium ‘Workfl ow Transformed’. Chairman Jacques Izopet (Toulouse) introduced the importance of molecular diagnostics in providing faster, reliable results, enabling improved patient management while saving laboratory time allowing redistribution of staff and resources for research and innovation.

Figure 1.

Figure 2.

– September 201531CASE STUDY

Hospital Clinic, Barcelona

0026_036_CLI_Sep.indd 31 27/08/15 10:41

Multisample cell disrupterTh e new Soni-Beast cell disrupter quickly and com-pletely disrupts all types of cellular samples, from fungi,

bacteria and spores to animal and plant tis-sues. Using a new technology, the SoniBeast is capable of processing small samples in sealed 0.5 ml PCR tubes. Th e tubes, fi lled with sample, extraction media and ceramic beads, are agitated at an unprecedented 21,000 oscillation per minute – some 5 to 10x faster than current machines. Th e ultra-high energy disrupter can process up to 12 samples, is operated with a simple one touch control and, importantly, isolates nucleic acids, proteins and other intracellular com-ponents in a few seconds rather than min-utes. Th ere is no programming, no clean up and no cross-contamination between samples, making it an ideal cell disrupter for PCR methodologies.

BIOSPEC www.cli-online.com & search 27043

Diagnostic antisera for bacterial identifi cation

MAST ASSURE Bacterial Agglu-tinating Antisera products are a com-prehensive range of polyvalent and monovalent diag-nostic antisera for

bacterial identifi cation. Bacterial sero-type is easily determined according to the expression of fl agella (H) and somatic (O) antigens. Mast Antisera products are used globally for antigenic analysis of clini-cally signifi cant bacteria, including MAST ASSURE Salmonella Antisera for iden-tifi cation of the species within the genus Salmonella using the Kauff mann-White Scheme for classifi cation. Th e MAST ASSURE product range features simple slide and test tube agglutination methods and easy to read results. All antisera are derived from immunized rabbits, adsorbed to remove cross agglutinins and fi lter steri-lized. Presented in 2ml dropper bottles, the reagents are user friendly and ensure mini-mal laboratory wastage. An extensive range of antisera is available from stock with a large range of sera manufactured to special order.

MAST GROUP www.cli-online.com & search 61645

High-performance water purifi cation

Th e AFS 40E / 80E / 120E and 150E water purifi cation systems have been developed to provide clinical laboratories with an economical and reliable water puri-

fi cation solution for daily water volumes of up to 3000 L. Th ey rely on two power-ful technologies to produce water quality meeting Clinical and Laboratory Standards Institute (CLSI) clinical laboratory reagent water standards. State-of-the-art, proven Elix electrodeionization (EDI) technol-ogy ensures constant water quality with low and predictable running costs, while unique E.R.A. (Evolutive Reject Adjust-ment) technology takes feed water qual-ity into account in order to automatically optimize water recovery - and reduce water usage costs. Th e AFS 40E / 80E / 120E and 150E systems also off er users powerful 24/7 real-time monitoring and remote control over their water purifi cation systems, as well as a new generation of enhanced ser-vices. Th ese advanced monitoring features, along with an unprecedented level of ser-vice, also help maximize water purifi cation system and analyser uptime. Th e systems have been designed to provide quick and precise remote diagnostics and off er pro-active service in order to avoid downtime. Service starts with feed water analysis by a certifi ed Merck Millipore fi eld service engi-neer prior to system installation. Th en, over time, users can ensure the best upkeep of their AFS E water purifi cation system with a customized Watercare Pact service plan. With its large new touch screen, the system is designed for intuitive operation and for supporting the user with easy step-by-step instructions during routine maintenance. For increased fl exibility, the system inter-face can also be accessed from another location, using a PC, tablet, or smart phone through a web browser. Mobile and cus-tomizable, the new range of systems is designed to make the best use of lab space. Th e quiet, compact systems are mounted on wheels, and can be moved around the lab - or to another location - depending on cur-rent lab requirements. AFS E system users can choose from a number of options and accessories to match their specifi c require-ments, including an online Total Organic Carbon (TOC) monitor, degassing option, and sanitary sampling valve, among others.

MERCK MILLIPORE www.cli-online.com & search 27048

PRODUCT NEWS – September 2015 32


Th ree scalable hematology systems, the ADVIA 360 System, ADVIA 560 System, and ADVIA 560 AL System, off er state-of-the-art hematology testing capabilities for small to mid-sized laboratories and may also be used as back-up systems for larger laboratories. Engineered for safety and ease-of-operation, the ADVIA hematology sys-tems help optimize and manage workfl ow through several convenient features, such as the choice of open- or closed-tube sampling and customizable result printing. Automatic anti-clogging and cleaning procedures are employed to ensure results reliability. Along with automated maintenance, this reduces manual procedures and bio-hazard expo-sure. Th e systems off er fast, high quality CBC testing, running up to 60 samples per hour. Other system features include: the option for manual or automatic calibration proce-dures, a cap-piercing function for accurate and safer sampling, a multilingual operating menu and bidirectional LIS communication for easy patient data transfer between labo-ratories and minimized paper-based work lists. Th ese lower capacity systems address major diff erences in hematology testing volumes from laboratory to laboratory and network to network. With a compact foot-print, the ADVIA 360 System allows smaller laboratories to effi ciently generate reliable and accurate results while conserving pre-cious benchtop space. Th is system provides a three-part white cell diff erential and stor-age capacity for 10,000 results. An integrated ticket printer streamlines results reporting. Th e ADVIA 560 System can serve as the pri-mary hematology analyzer in small to mid-sized laboratories and as a backup for the company’s ADVIA 2120i System in larger laboratories. It provides a fi ve-part white cell diff erential and storage capacity of 100,000 results. Also, two scattergrams and two his-tograms per result help aid interpreting disease-state information. Th e ADVIA 560 AL System off ers automatic sampling with an optional autoloader that simply plugs into the side of the system for even greater workfl ow effi ciency and true “walk-away” capability.

SIEMENS HEALTHCARE www.cli-online.com & search 27042

0026_036_CLI_Sep.indd 32 27/08/15 10:41

Digital pathology systemSectra’s solution for storing, view-ing and sharing digital pathol-ogy images has received the CE

mark for primary diagnostic use. Th e sys-tem enables more effi cient cancer care by enhancing collaboration and sharing of images between pathologists and radi-ologists. Pathologists will now be able to review cases digitally on a computer screen, enabling them to eliminate the microscope. Th e digital format brings new tools to the pathologists’ fi ngertips and also allows sam-ples to be sent easily to other pathologists to share the workload or for specialist con-sultations, thereby facilitating quicker and more accurate diagnosis. Pathology and radiology are at the centre of cancer diag-nosis and their collective fi ndings are the basis for patient treatment and its outcome. Both disciplines are highly dependent on adequate clinical information in order to provide accurate and useful reports for the clinician, surgeon, oncologist or patient. Despite their joint responsibility for can-cer diagnosis, the exchange of information between the two groups is oft en very lim-ited as the radiology and pathology work-fl ows are usually completely diff erent. Sec-tra’s system for digital pathology is built on the same platform as its radiology PACS for managing radiology images. Th e common technical platform enables closer collabo-ration between the two specialties, some-times referred to as integrated diagnostics, facilitating, for example, more effi cient multi-disciplinary team meetings. Focus-ing on end-user experience and workfl ow effi ciency, the system provides a platform where images from diff erent hardware ven-dors can be viewed and archived, and where histology or cytology images can be shown together with macro images. Furthermore, it allows full case overview including inte-gration with other image systems, enabling access to radiology or dermatology data and images in the same workstation. Sec-tra digital pathology workstation, IDS7/px, consists of a dedicated image window for pathology images and the regular IDS7 information window. Th e information win-dow includes worklists, full patient infor-mation, with possibility to access images from other specialties such as mammog-raphy, radiology and dermatology and a reporting window with support for speech recognition. Th e possibility to reach dif-ferent types of images, such as pathology and radiology from the same workstation is especially useful for multidisciplinary

teams (MDT) meetings. Th e image window supports the pathologist during review, preparation of MDTs and education and it contains for example tools for text anno-tations, distance and area measurements. Images from the same block are registered as such and if similar enough are automati-cally synchronized by the soft ware enabling effi cient comparison of the same structure stained with diff erent techniques.

SECTRA


Serological diagnosis of Chikungunya virus infections

A n t i b o d i e s against chikun-gunya virus (CHIKV) can be detected with high sensitiv-ity and specifi c-ity using new

ELISA and indirect immunofl uorescence test (IIFT) systems from EUROIMMUN. Serological analysis is an important sup-plement to direct pathogen testing in the diagnosis of CHIKV infections, especially given the short viremic phase. Antibodies against CHIKV are detectable from 6 to 8 days aft er onset of clinical symptoms, when direct detection is generally no longer eff ective. Th e determination of CHIKV antigens or anti-CHIKV IgM antibod-ies is, moreover, of major importance in the screening of blood reserves. Th e Anti-Chikungunya Virus ELISA (IgM) is a fully automatable test for the detection of acute infections. Th e assay is based on recombi-nant structural proteins from CHIKV and shows 100% sensitivity, as demonstrated with clinically characterized samples. Th e Anti-Chikungunya Virus ELISA (IgG) is based on the same antigen and is used to demonstrate seroconversion following infection with the virus. A four-fold IgG titre increase between acute and convales-cence samples taken at least 14 days apart indicates an acute infection. Th e Anti-Chi-kungunya Virus IIFT (IgM or IgG) utilizes virus-infected cells for detection of specifi c antibodies. In the Arboviral Fever Mosiac 1 IIFT, the CHIKV substrate is combined with other arbovirus substrates for the dif-ferential diagnosis of CHIKV infections from e.g. dengue virus or Japanese enceph-alitis virus infections, which oft en show similar clinical symptoms.

EUROIMMUN www.cli-online.com & search 27045

– September 201533PRODUCT NEWS


Developed for his-topathology applica-tions, the Histra-GT utilizes ultrasound to allow rapid tissue processing and diag-nosis in one day. Th e proper use of ultra-

sonic energy ensures uniform radiation of the tissue, eliminating uneven processing and distorted cell morphology. Th e Histra-GT is a rapid processor that requires only 80 minutes to process any tissue less than 3mm thick in one batch processing with 100-cassette capacity, and can also process larger specimens in a shorter time than when using conventional methods. Th ere are 3 modes to processing - rapid process-ing mode, normal processing mode and rapid preprocessing mode – enabling the instrument to address variations in daily workload. Using the rapid processing mode for biopsy samples, only 80 min-utes are required from fi xation to paraffi n infi ltration (including the time for liquid supply and drainage). Th e normal pro-cessing mode enables the user to set the required processing time, including over-night processing. Th e rapid preprocessing mode allows delipidation of fat tissue and rapid refi xing of the half-fi xed tissue. A large touch key pad makes for easy opera-tion. A dual safety system is also featured. Volatilized gas does not leak because of active carbon. Th e ultrasonic technology not only performs rapid processing of tissue specimens, but also allows high quality staining. Th e Histra-DC is a processor for recalcifi cation, delipida-tion and fi xation that shortens the process-ing time to one fi ft h or one sixth of the time needed for conventional methods, using ultrasonic technology and temperature con-trol. Specimen sizes can vary with the use of the optional specimen cassette basket. Optimized for use with formalin-fi xed, paraffi n-embedded human breast can-cer tissue samples, the Histra HER2 FISH kit detects amplifi cation of the HER2/neu gene via fl uorescence in situ hybridization. Th is advanced kit, which utilizes pretreatment of the samples with hydrochloric acid, is very easy to use and involves only six steps, providing very clear results via a strong fl uorescence.

JOKOH www.cli-online.com & search 27044

0026_036_CLI_Sep.indd 33 27/08/15 10:41

S. pneumoniae and L. pneumophila urinary antigen test

Th e ImmuView S. pneumoniae and L. pneumophila Uri-nary Antigen Test is a combined rapid lateral fl ow test for qualitative detection of S. pneumoniae and

L. pneumophila serogroup 1 in human urine samples. Th e test is eff ective in presumptive diagnosis of Legionella infection (Legion-naires’ Disease) caused by L. pneumophila serogroup 1 or pneumococcal pneumonia caused by S. pneumoniae in conjunction

with culture and other methods. Correct and early treatment is vital for the prognosis of both diseases and therefore rapid methods to confi rm both diseases in the initial phase are very important in order to initiate the proper antibiotic treatment as soon as possible. Th e target groups for this test include hospital departments in respiratory medicine, infec-tion control, infectious diseases and intensive care. Th e combination of Pneumococcus and Legionella in one test results in laboratories saving time by performing two tests in one – test result in 15 minutes – and money, as the price for the combined test is lower than what is normally paid for two separate tests. Testing for S. pneumoniae and L. pneumoph-ila is recommended in guidelines in several countries. ImmuView S. pneumoniae and L. pneumophila Urinary Antigen Test kit con-tains 1 tube with 22 test strips, 0.5 mL posi-tive control, 0.5 mL negative control, 2.5 mL running buff er, 1 tweezer, 22 transfer pipettes, 22 test tubes, 1 cardboard test tube holder and a quick guide. Package insert is also included.

SSI DIAGNOSTICA www.cli-online.com & search 61624

Glucose meter system receives FDA clearance for use with critically ill patients

Last May, the U.S. Food and Drug Administration (FDA) cleared Nova Bio-medical’s StatStrip Xpress Glucose Hospital Meter System for use through-out all hospital and all professional healthcare settings, including criti-

cally ill patients. StatStrip Glucose and Stat-Strip Xpress Glucose are now the only two hospital blood glucose meters to be cleared by the FDA for use with critically ill patients. Use of all other glucose meters with critically ill patients is considered off -label by the FDA and high complexity testing under the Clini-cal Laboratory Improvement Amendments. High complexity testing requirements are so stringent that to use a glucose meter other than StatStrip Glucose and StatStrip Xpress Glucose with critically ill patients is not a practical alternative. StatStrip Xpress Glu-cose utilizes the same test strip measurement technology as StatStrip Glucose, which was cleared in 2014 aft er an extensive, four-year study conducted at fi ve major university med-ical centres. Th e study included 1,698 criti-cally ill patients with over 257 medical condi-tion subcategories as designated by the World Health Organization. Over 8,000 medications were investigated for potential interference

to StatStrip Glucose measuring technology. StatStrip Glucose demonstrated excellent agreement compared to central laboratory reference methods and no clinical interfer-ences were found. In addition to the study submitted to the FDA, 138 other independent studies over the last eight years—including 53 critical care studies—have found no clinically signifi cant interferences for StatStrip Glucose measuring technology.

NOVA BIOMEDICAL www.cli-online.com & search 27019

Automated molecular diagnostics system

E L I Te c h G r o u p Molecular Diag-nostics and Preci-sion System Science, Co., Ltd (PSS), a Japan-based global

provider of laboratory instruments for nucleic acid extraction, jointly announced the upcoming CE-IVD marking of ELITe InGenius for IVD applications in Europe and the upcoming availability of the instrument as Laboratory Use Equipment in the US.ELITe InGenius automatically performs all the steps of molecular diagnostics including extraction, amplifi cation and result analy-sis. 1 to 12 samples can be processed with greater fl exibility off ering the possibility to potentially mix any kind of sample matrices within the same run and perform multiple and independent PCR even with completely diverse thermal profi les simply on demand. In addition, each of the 12 real-time PCR thermal cyclers is equipped with 6-optical channels, supplemented when appropriate with melt-curve analysis to provide extended multiplexing capability. Th e system is enabled with a unique real-time PCR menu based on ELITechGroup’s proven MGB technology, providing a rapid introduction of CE-IVD infectious disease menu for transplant moni-toring and Healthcare Acquired Infections. Th e versatile system can also be used as an open platform running laboratory devel-oped tests or other tests of interest. Th e ELITe InGenius combines unique capabilities not available on other sample-to-result systems, like universal extraction, storage of extracted nucleic acid and multiple and independently controlled RT PCR with multiplexing capa-bilities, off ering to the laboratory an unprec-edented fl exibility and assay menu possibility. ELITechGroup has more than 12 assays in the pipeline for infectious disease testing.

ELITECHGROUP www.cli-online.com & search 27049

PRODUCT NEWS – September 2015 34


Th e new Altair 240 clinical chemistry analyser fulfi lls an international market need for a fully automated bench-top plat-

form supported by the Stanbio chemis-try reagent menu. Th e fully automated system with LIS bi-directional connec-tivity is capable of performing up to 480 tests per hour. In addition to being sup-ported by EKF’s broad menu of Stanbio ready-to-use, bar coded, liquid reagents for routine and special chemistries, the Altair 240 also provides the fl exibility to confi gure open channel applications for many esoteric assays. EKF will expand the international presence of its clinical chemistry range with the Altair 240, as its customers have long asked for a cost-eff ective platform designed with Stanbio Chemistry products in mind. Built on 54 years of clinical chemistry expertise, this new platform enables EKF to off er a cost-eff ective solution, as either a main or back-up analyser, to improve produc-tivity in both the hospital and physician offi ce laboratory environments. Because it runs on Windows 7, operators can eas-ily navigate its intuitive, touch screen menu. Features such as the capability to use bar coded, primary sample tubes, auto-rerun, auto-dilution and STAT interruption, all function to maximize the system’s overall effi ciency.

EKF DIAGNOSTICS www.cli-online.com & search 27047

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A BREAKTHROUGH IN SPECIALTY TESTING. IT’S ABOUT TIME. AND ACCURACY. The ACL AcuStar brings full automation to specialty hemostasis testing that, until now, has required time-consuming manual processes. Advanced chemiluminescent technology automates and standardizes highly specialized assays and enhances routine testing. The ACL AcuStar is changing today’s hemostasis lab, combining testing accuracy and efficiency in ways never before possible. The result is greater savings and no waiting. Automated specialty testing is here. Break through with IL.

For more information, please contact your local Werfen sales representative/distributor or visit www.werfen.com.

AUTOMATING COMPLEX ASSAYS.

THE WAIT IS OVER.

*Not currently 510(k) cleared. I **In development.

ACL AcuStar HemosIL® Test MenuComprehensive line of high-performance chemiluminescent assays, with more in development.

D-Dimer HIT-IgG(PF4-H)*HIT-Ab(PF4-H)*

aCL IgGaCL IgMaß2GPI IgG aß2GPI IgMaß2GPI Domain I*

VWF:RCo*VWF:Ag*VWF:CB**


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