weekly news updates on neonatal ... · • high capacity, up to 660 slides simultaneously •...

Weekly news updates on www.cli-online.com | February/March 2010 | Volume 34 | Issue 1 | €15

PCR kit for the detection of dermatophytes and Trichophyton rubrum Pg.30

Surfactant Protein A ELISA Pg.31

Urinalysis test strip with infra-red ID bands Pg.32

Neonatal hypobilirubinaemia:identifying the infant at risk Pg.19

Also in this issue :

Molecular profiling in CLL Pg. 8

A new biomarker of bone metastases Pg. 22

Bacterial enteric pathogens Pg. 24

When it comes to speed and accuracy there’s no one better at getting your wheels turning.

Siemens Healthcare Diagnostics understands the constant pressure you face to streamline workflow, deliver fasterturnaround times and reduce errors. Our advanced testing systems, IT connectivity and automation solutions allowyou to work more efficiently so you can provide physicians with the critical information they need to deliver ahigher level of patient care. Get rolling today: www.siemens.com/diagnostics-streamline

A91DX-9025-A1-4A00 © 2008 Siemens Healthcare Diagnostics Inc. All rights reserved.

Answers for life.

How can I get my lab to run this smoothly?

9021_VERSANT_kPCR_Ad_ClinLabIntl_SEPT.qxd:Layout 1 12/4/08 5:13 PM

www.cli-online.com & search 24954

www.phadia.com

ImmunoCAP® ImmunoCAP Specific IgE measures the exact concentration of circulating allergen-specific IgE antibodies in human serum and plasma. By many considered the perfect tool for the diagnosis, prognosis and follow-up of patients with indications of allergy and asthma. Quantitative ImmunoCAP tests can help the physician confirm or rule out allergy with certainty.

EliA™

EliA is the premium autoimmunity diagnostic test, offering accuracy, standardization and guidance in a difficult diagnostic area. Clinically relevant and exact measurements of specific markers for several different autoimmune conditions, such as rheumatoid arthritis and celiac disease, are available.

For accuracy and efficiency, run your allergy and autoimmunity blood samples on the world’s leading test system: Phadia® Laboratory Systems. Results are precise, correct and clinically reliable. Moreover, physicians trust them.

Phadia Laboratory Systems brings accuracy, efficiency and economic value to laboratories.

Setting the standardin allergy and autoimmunity diagnostics


Editor’s lEttEr4 – issue N°1 – Feb/Mar 2010

In this year’s International

Women’s Day message,

the UN Secretary General

Ban Ki-moon reported

on the substantial prog-

ress being made towards

reaching the third of the

eight millenium develop-

ment goals, namely to promote gen-

der equality and empower women. A

bill passed in the Indian parliament

recently reserving a third of the seats

for women surely exemplifies this prog-

ress. However, Ban Ki-moon regretted

that the fifth goal - improving maternal

health - was proving the most difficult

to achieve. Over a quarter of a million

women per year still die in childbirth;

65,000 of these deaths occur in India.

Faced with such daunting figures, what

can be done to try to achieve the fifth

millenium development goal?

Haemorrhage is the main cause of

direct obstetric death, followed by

pregnancy-induced hypertension.

Obstructed labour and infection are

also significant causes. In an effort

to reduce maternal mortality, many

African and Asian countries during

the post-colonial period around forty

years ago put emphasis on the formal

training of traditional birth attendants

(TBAs). Such a strategy appeared to be

prudent: TBAs, usually older women

well-known and respected by their

communities, lived in the same ru-

ral areas, remote from urban health-

care infrastructure, as the majority of

women giving birth. It was thought

that giving TBAs some basic training

in infection control, providing them

with low-cost sterile kits and ensur-

ing that they were familiar with the

signs of potentially life-threatening

complications that needed to be re-

ferred would have a significant impact

on maternal mortality. Many studies

based in African and Asian developing

countries attempted to quantify the

effects of such training programmes.

Whilst most agreed that training TBAs

lowered neonatal mortality, at least to

some extent, there was no concensus

on the impact on maternal deaths.

However, after the WHO stated cat-

egorically that “training programmes

for traditional birth attendants have

failed to reduce maternal mortality”,

global policy shifted towards provi-

sion of skilled facility-based care. Of

course in an ideal world such care

would be available for all delivering

women. Until we reach that utopia

though, how can today’s TBAs become

more effective in reducing maternal

mortality? Many possibly self-interest-

ed professionals may not only doubt

the ability of such women to benefit

from TBA training, but may actually

resent their ‘empowerment’. Fortu-

nately however, with the drive towards

gender equality, most of the world’s

women now receive at least a primary

school education. Functional literacy

and numeracy certainly make training

programmes more likely to succeed

than in the past, and if TBA training

includes recognition of danger signs

and use (and provision) of mobile

phones to call for emergency obstetric

care when it is necessary, it will go a

long way towards reducing global ma-

ternal mortality. A small number of

urban women joining men to generate

hot air in parliaments is all very well.

But if the efforts of adequately trained

rural TBAs can stop so many women

dying in childbirth, there will really be

cause for celebration.

Improving maternal health: do traditional birth attendants still have a role to play?

Your Power for Health

Greiner Bio-One GmbH | Bad Haller Straße 32 | A-4550 KremsmünsterPhone: (+43) 75 83 67 91-0 | Fax: (+43) 75 83 63 18 | E-mail: [email protected] www.gbo.com/preanalytics

Made from polypropylene (PP).

Virtually unbreakable evacuated tube.

More safety and hygiene compared to glass tubes.

For automated and manual ESR systems.

New VACUETTE® ESR Tube


IMWG Guidelines recommend that serum free light chain (FLC)assays be used in the initial evaluation of suspected myeloma1

Guidelines published by the International Myeloma Working Group (IMWG)recommend Serum FLC for use in screening, at diagnosis, for prognosis andwhen monitoring patients with multiple myeloma and related disorders.

is a simple blood test to quantitate free kappa (κ) and free lambda (λ) immunoglobulin light chains.

Email: [email protected] for your free copy of the myeloma guidelines

1. Dispenzieri et al. Leukemia 2009:23:215-224FreeliteTM & HevyliteTM are trademarks of The Binding Site Group Ltd, Birmingham, UK.

The Binding Site Group Ltd Tel: +44 (0)121 436 1000 • Fax: +44 (0)121 430 7061 • [email protected] to www.freelite.co.uk to contact us in UK, USA, Germany, Austria, France, Spain, Canada

and 70 other countries

HevyliteTM

See what the HevyliteTM ratio offers you.

Find out more at:www.freelite.co.uk

Jon

as Berg

gren

©Fo

lio

Don’t MissThe International

Myeloma Guidelines

Golf CLI Febmarch 2010:Layout 1 11/01/2010 15:13 Page 1


ContentsFRONT COVER

FEATURES

[8-15] Leukemia

[8-11] Molecular profiling in chronic

lymphocytic leukemia (Cll)

[12-15] insights into acute myeloid leukemia

via single cell networking profiling

[16-21] HyperbiLirubinemia

[16-18] transcutaneous bilirubin measurement in neonates

[19 -21] identifying the infant at risk

of neonatal hyperbilirubinemia

[22-23] Alpha CtX as a sensitive marker for the detection

of bone metastases

[24-25] the laboratory identification of bacterial enteric pathogens:

can we stop culturing yet?

[26-28] An innovative automation solution to create an

integrated blood sciences unit

REGULARS

[4] Editor’s letter

[30] Product highlight: microbiology tests

[31-33] Product news

[34] industry news

[34] Calendar of Events

Extremely high bilirubin levels in neonates can result in permanent and serious clinical sequelae. Predischarge bilirubin screening, combined with clinical risk factors, enables the identification of infants at risk for severe hyperbilirubinemia, so that noninvasive bilirubin-reducing phototherapy can be initiated. this induces the isomerisation of bilirubin to forms that can be more readily excreted.

Weekly news updates on www.cli-online.com | February/March 2010 | Volume 34 | Issue 1 | €15

PCR kit for the detection of dermatophytes and Trichophyton rubrum Pg.30

Surfactant Protein A ELISA Pg.31

Urinalysis test strip with infra-red ID bands Pg.32

Neonatal hypobilirubinaemia:identifying the infant at risk Pg.19

Also in this issue :

Molecular profiling in CLL Pg. 8

A new biomarker of bone metastases Pg. 22

Bacterial enteric pathogens Pg. 24

For submission of editorial material, contact Frances Bushrod at [email protected]

For advertising information, go online to www.cli-online.com, simply click on ‘Magazine’ and ‘Media Information’ or contact Astrid Wydouw at [email protected]

Clinical lab professionals are entitled to receive CLI for the next 12 months completely free of charge.

To begin a new subscription or to continue your existing free subscription go to

www.cli-online.comClick on Free Subscription and follow instructions

Rue Royale 326 • 1030 Brussels, BelgiumTel. +32-2-240 26 11 • Fax: +32-2-240 26 18

www.cli-online.com

managing editorsFrances Bushrod, Ph.D.

[email protected] Barclay, Ph.D.

Contributing editorMatladi Ndlovu, Ph.D.

editorial CoordinatorAnna Hyrkäs

advertising CoordinatorJennifer Christophers

Circulation managerArthur Légerpublisher

Bernard Léger, M.D.advertising Sales manager

Astrid [email protected]

WebmasterDamien Noël de Burlin

©2010 by PanGlobal Media bvba-sprl. Production & Lay-out by Studiopress Communication, Brussels.

Circulation Controlled by Business of Performing Audits, Shelton, CT, USA.

The publisher assumes no responsibility for opinions or state-ments expressed in advertisements or product news items. The opinions expressed in by-lined articles are those of the author and do not necessarily reflect those of the publisher. No conclusion can be drawn from the use of trade marks in this publication as to whether they are registered or not.

CoMING UP IN APRIL/MAyAllergyMarkers of oxidative stressFlow cytometry

Free Subscription for Clinical lab professionals

ISSN 1373-1580

First we un derstand. Then we innovate.

Imagine ... full flexibility linked to high capacity

Dedicated to Histopathology

Sakura Finetek, again, improves the laboratory. By offering a flexible product, the histotechnologists can easily complete the various activities required. As the innovative company in histopathology, Sakura Finetek is continuously looking for possibilities to improve the laboratory... and succeeds in offering a solution for the problems found in the histopathology laboratory.

Sakura Finetek Europe B.V.The Netherlands

Phone: +31 71 589 83 00Fax: +31 71 589 84 88

[email protected]

Tissue-Tek® Prisma® & Film®

Automated Slide Stainer & Coverslipper

The Tissue-Tek® Prisma® Multiple Stainer and Film® Coverslipper offer you:• A true walk-away solution• Most flexible multiple stainer in the market• Your routine and special staining programs in one instrument• High capacity, up to 660 slides simultaneously• Unloading capacity up to 240 slides with convenient carrousel

08_NOV_SFE_5500_add-CLI-xSeries-2 2 04-11-2008 18:19:28


+31 (0)88 592 00 01Tel: +31 (0)88 592 00 00

leukemia – issue N°1 – Feb/Mar 2010 8d

isea

se f

ocu

s

CLL is increasingly diagnosed in asymptomatic patients when a lymphocytosis is found at the time of a routine blood count. Intriguingly, the increasing use of immunophenotyping led to the identification of individuals with circulating clonal B cells, often with the characteristic phenotype of CLL, but below the 5000/ µL threshold demanded by CLL guidelines. This monoclonal B cell lymphocytosis (MBL) has a prevalence in the population of 3-5% [2].

treatmentSome individuals diagnosed at an early stage may remain asymptomatic for the rest of their lives, with their lifespan unaffected by CLL, while oth-ers may progress to aggressive disease over time. Therapy should be reserved for those with advanced or symptomatic disease, with treatment being con-sidered palliative due to the incurable nature of the disease with conventional chemotherapy and the often advanced age of the patient. There has been significant improvement over the past decade in the results of treatment of CLL using combina-tion chemo-immunotherapy with the addition of therapeutic monoclonal antibodies.

Conventional criteria for measuring the success of treatment involve the microscopic examina-tion of bone marrow samples and blood to con-firm the disappearance of leukemia cells. Recently, more sensitive tests to detect minimal residual disease (MRD) have become available, specifically

multicolour flow cytometry and polymerase chain reaction (PCR) for clones defined by immu-noglobulin variable gene sequences. Patients who have no MRD demonstrated by these methods have a longer remission duration and survival [3].

Prognostic factorsIn parallel with new therapeutic manoeuvers, there has been dramatic progress in the understanding of the basic biology of CLL, and the discovery of numerous prognostic factors. Prognostic mark-ers may be clinical characteristics of the patient, features of the leukemic cells themselves, or other biological variables.

The Rai and Binet staging systems were based upon their prognostic significance, and the stage of disease remains perhaps the most useful prognostic factor in CLL. The staging systems assume that CLL cells first proliferate in the bone marrow and blood, followed by the lymph nodes, then spleen and liver, and finally anemia and thrombocytopenia develop, caused by high tumour burden in the bone marrow.

Binet stage A disease comprises 60-80% of cases and an expected survival of >10 years, close to the life expectancy of an age matched non-CLL population. However over 25% of these ‘indolent’ cases die of causes related to CLL, 40% progress to advanced stages, and 50% require treatment [4]. Therefore the staging systems of Rai and Binet are unable to predict accurately which patients among the majority early stage group will develop progressive disease.

Molecular prognostic markersAs well as clinical staging, readily available clini-cal laboratory parameters that reflect tumour bur-den have been evaluated. Absolute lymphocyte count is an independent risk factor, as is the lym-phocyte doubling time, elevated lactate dehydro-genase (LDH) [5], thymidine kinase (TK) and elevated beta-2-microglobulin (b2M) showing

positive correlation with clinical staging systems and short survival.

Cytogenetics provides one of the most useful prognostic markers, and recurrent abnormalities have been found that can now be detected using fluorescence in situ hybridisation (FISH). In a comprehensive study, chromosomal abnormali-ties were detected by FISH in 82% of CLL cases [5]. This study demonstrated convincingly that genomic aberrations in CLL are important inde-pendent predictors of disease progression and survival. The most common recurrent chromo-somal abnormalities observed include deletion of the long arm of chromosome 13 (del 13q), del 11q, trisomy 12, del 17p and del 6q.

These recurrent abnormalities point to the loci of candidate genes involved in pathogenesis. The most common abnormality is del 13q14, which occurs in 55% of cases, and has been associated with a favourable prognosis. The first report link-ing microRNAs to cancer was in CLL, where it was demonstrated that two microRNA clusters, mir-15a and mir16-1, were located within the deleted region at 13q14 [6]. The Ataxia Telangiectasia Mutated (ATM) gene is located within the mini-mal region of loss at 11q23, suggesting that altera-tions in DNA repair pathways may be involved in the pathogenesis of the disease. Although occur-ring in fewer than 10% of patients at diagnosis, del 17p is associated with rapid progression of disease, poor response to therapy and short survival. The deletion involves the p53 locus at 17p13, and it is clear that mutations in the p53 gene can contrib-ute to disease progression and alter the sensitivity of CLL cells to chemotherapy agents. Deletion of 17p and/or p53 gene mutations have been asso-ciated with failure after treatment with standard imuno-chemotherapy. Evidence is accumulating on the efficacy of anti-CD52 monoclonal anti-body therapy (alemtuzumab) in CLL with p53 gene abnormalities [7].

Molecular profiling in chronic lymphocytic leukemiaChronic Lymphocytic Leukemia (CLL) is the most frequent leukemia in the western world [1] and affects predominantly elderly individuals with only one third of patients being under the age of 60 at presentation. CLL follows a highly variable clinical course, with approximately 25% of patients requiring therapy at diagnosis. by Dr T. Butler and Prof. J. Gribben

Table 1. Diagnosis of CLL.

• Clonal expansion of abnormal B-lymphocytes in

Peripheral Blood

• At least 5 x 109 B lymphocytes/L (5000/µL)

• < 55% atypical/immature lymphoid cells

• Low density of surface Ig (IgM or IgD)

with κ or λ light chains

• B-cell surface antigens (CD19, CD20 [dim], CD23)

• CD5 surface antigen

Table 2. Binet staging system.

Binet Stage Characteristics Treat in practice?

Afewer than 3 areas of lymphadenopathy

Symptomatic patients only

B3 or more areas of lymphadenopathy

Symptomatic patients only

C

anemia Hb

<11g/dL and/or

thrombocytopenia

<100 x109/L

Yes

Our VERSANT team is ready to take your lab’s performance over the top.

The Siemens VERSANT® family of products delivers unmatched precision, proven accuracy, and versatility so you can perform molecular testing with more confidence. Our molecular diagnostic systems provide vital information to physicians, assisting them in monitoring and determining optimal therapies for HIV and hepatitis patients. Go above and beyond: www.siemens.com/diagnostics-molecularadvantage

A91DX-9027-A1-4A00 © 2010 Siemens Healthcare Diagnostics Inc. All rights reserved.VERSANT is a trademark of Siemens Healthcare Diagnostics Inc.

Answers for life.

Is there a molecular testing supplierwho can really give us an edge?

Siemens_VERSANT_Family_Ad_CLI:CLI 3/18/10 1:39 PM Page 1


An important advance in the understanding of CLL was made with the demonstration that 50% of CLL cases have somatic hypermutation (SHM) in the variable regions of the immunoglobulin heavy chain (IgHV) genes and that this has prognostic signifi-cance. The levels of somatic hypermutation in par-ticular B cells are evaluated by comparison of the sequence of the rearranged variable region gene with germline sequences.

Sequences with less than 98% homology to germ-line are considered to have undergone somatic hypermutation. UM-CLL cells are presumed to have not passed through a germinal centre in the generation of an antibody response (naïve), or alternatively to have responded to T cell inde-pendent antigens. The finding that CLL cases can be divided into mutated (M) and unmutated (UM) groups implied that the two groups may have dis-eases that arise from different normal cellular counterparts [8].

As well as informing us about the possible patho-physiology of the disease, it has become clear that the mutation status of a particular case of CLL is one of the most powerful predictors of prog-nosis, with UM-CLL cases typically progressing rapidly and having shorter survival times than M-CLL [9,10].

Other data indicate the centrality of the immu-noglobulin B Cell Receptor (BCR) to the biol-ogy of CLL. Analysis of variable region sequences demonstrated that CLL cells utilise a biased rep-ertoire of V genes characterised by over-represen-tation of selected Ig gene segments, in particular IGHV1-69, IGHV4-34, IGHV3-7, and IGHV3-21 [11]. These findings have led some authors to suggest a microbial antigen or autoantigen as a stimulus for CLL growth, and the immunoglobu-lin expressed by some CLL clones has been shown to have autoantibody activity [12].

It is not possible to perform IgHV mutational sta-tus on a routine basis in clinical laboratories and attempts have therefore been made to identify surro-gate markers for mutational status and its powerful role as a prognostic tool. In particular, expression of two proteins, 70kDa zeta-associated protein (ZAP70) and CD38 have been examined.

Most mutated cases are ZAP-70 negative and unmu-tated cases are ZAP70 positive. ZAP70 expression can be measured by a number of conventional meth-ods including western blotting, reverse transcriptase-PCR, immunohistochemistry and flow cytometry.

Levels of expression are higher in T cells and NK cells than in CLL cells, and it is important that effec-tive multiparametric gating strategies are used to ensure that expression is being measured in the CLL cells. The relationship of ZAP-70 positivity and UM-CLL is not absolute, however, with discrepant cases ranging from 8-25% [13]. Difficulty with standardi-sation has plagued studies measuring ZAP-70, and impaired its translation into a standard clinical tool.

CD38 is a surface marker associated with CLL, and is easily determined using standard flow cytomet-ric methods. It was initially found to correlate with IgHV mutation status [10], however the relation-ship is not absolute, and there is a concern that CD38 expression may vary over time [14]. The field is somewhat confused by a variety of cut-offs being used to define a case as being CD38 positive (5%, 7%, 20% or 30% in different series) [15]. Originally valued as mere surrogates for mutation status, it may be that ZAP-70 and CD38 status are ultimately accepted as complementary prognostic markers with independent value.

Other surrogates of mutation status have been sug-gested including expression of many other serum enzymes. MicroRNA arrays have revealed a 13 gene signature found to correlate with ZAP-70 status, UM-CLL and disease progression [6] and two of the most differentially expressed micro RNAs (miR15a and miR 16) are those located at the 13q locus com-monly deleted in CLL. Epigenetic alterations in can-cer are well described, and aberrant methylation has been demonstrated in CLL, both globally and for

leukemia – issue N°1 – Feb/Mar 2010 10

V7

D19

J5

V1 V2 V3 V 3 D1 D 2 J1 J6

V1 V2 V3 V 3

D19

J5 μ

μ

μ J5 D19 V7

Germline DNA

Splicing produces IgH mRNA

Light chain

Heavy chain (μ)

Translation of heavy chain into protein and combination with light chains produces complete B cell Receptor

Somatic recombination

Figure 1 Variation in the antigen-binding region of the B Cell Receptor is generated by random DNA recombination of V, D and J gene segments. Further diversity is produced by introduction of mutations in the V, D and J genes during the germinal centre reaction.

Antigen binding regions

Somatic hypermutation

Figure 1. VDJ recombination.

B cell progenitor

Acute Lymphoblastic Leukemia

Chronic Lymphocytic Leukemia (Unmutated)

Chronic

Lymphocytic

Leukemia (Mutated)

Diffuse Large

B Cell

Lymphoma

Follicular Lymphoma

Mutiple Myeloma

Mantle Zone Lymphoma

GERMINAL CENTRE REACTION:

Proliferation and Somatic

Hypermutation

Germinal Centre B Cell

Naïve B Cell

Plasma Cell

Memory B Cell

Figure 2: B Cell development and the cell of origin hypothesis of lymphomagenesis. The developmental stages of the

normal B cell are shown, with passage through the germinal centre and antigenic stimulation resulting in Somatic

Hypermutation (SHM). The presence or absence of SHM and other markers can indicate the life history of an individual cell.

The cell of origin hypothesis implies that B cell lymphomas and leukaemias arise from a B cell at a particular stage of

development that has acquired mutations that convert a healthy B cell into a cancerous one. The characteristics of the

lymphoma cell are similar to the cell of origin, including the presence of SHM. The existence of CLL cells with and without

SHM implies different cells of origin for these two subgroups, which also differ in terms of disease aggressiveness.

= tumorigenic mutation

Figure 2. Cell of origin hypothesis.

specific genes [16]. Whilst array-based analyses of different subgroups may well lead to novel insights into biology, their routine use in the clinical setting is currently not possible.

ConclusionsIt is obvious that the diagnostic methods and prognostic tools relating to CLL have expanded in accompaniment to knowledge of the under-lying pathophysiology of the disease. However, this has not translated into straightforward rec-ommendations as to when and how to treat an individual patient. What we do know is that the molecular profile of CLL provides insight into the underlying pathogenesis of the disease and pro-vides predictors of time to progression, response to therapy and overall survival. Once patients become symptomatic, or fulfill the criteria for need for treatment, there are not yet sufficient data to suggest that the detection of any of these markers should alter which therapy is offered with the exception of those patients who present with del 17p or mutations of p53. Molecular profiling has led to great insights into the biology of this leukemia, yet while various markers have pow-erful prognostic value, they have as yet provided little help regarding how an individual patient should be treated. This view may change in the future with the results of ongoing clinical tri-als that are examining the use of these markers inguiding treatment.

references1. Kristinsson SY et al. Improved survival in chronic

lymphocytic leukemia in the past decade: a popu-lation-based study including 11,179 patients diag-nosed between 1973-2003 in Sweden. Haematologica 2009;94:1259-1265.

2. Landgren O et al. B-cell clones as early markers for chronic lymphocytic leukemia. N Engl J Med 2009;360:659-667.

3. Provan D et al. Eradication of polymerase chain reac-tion-detectable chronic lymphocytic leukemia cells is associated with improved outcome after bone marrow transplantation. Blood 1996;88:2228-2235.

4. Dighiero G et al. Chlorambucil in indolent chronic lymphocytic leukemia. French Cooperative Group on Chronic Lymphocytic Leukemia. N Engl J Med 1998;338:1506-1514.

5. Dohner H et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 2000;343:1910-1916.

6. Calin GA et al. Frequent deletions and down-regula-tion of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2002;99:15524-15529.

7. Gribben JG, Hallek M. Rediscovering alemtuzumab: current and emerging therapeutic roles. Br J Haematol 2009;144:818-831.

8. Kuppers R et al. Cellular origin of human B-cell lym-phomas. N Engl J Med 1999;341:1520-1529.

9. Hamblin TJ et al. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic

lymphocytic leukemia. Blood 1999;94:1848-1854.10. Damle RN et al. Ig V gene mutation status and CD38

expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999;94:1840-1847.

11. Fais F et al. Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J Clin Invest 1998;102:1515-1525.

12. Chu CC et al. Chronic lymphocytic leukemia anti-bodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA. Blood 2008;112:5122-5129.

13. Rassenti LZ et al. ZAP-70 compared with immu-noglobulin heavy-chain gene mutation status as a pre-dictor of disease progression in chronic lymphocytic leukemia . N Engl J Med 2004;351:893-901.

14. Hamblin TJ et al. CD38 expression and immu-noglobulin variable region mutations are independ-ent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 2002;99:1023-1029.

15. Seiler T et al. Risk stratification in chronic lym-phocytic leukemia. Semin Oncol 2006;33:186-194.

16. Raval A et al. Epigenetics in chronic lymphocytic leukemia. Semin Oncol 2006;33:157-166.

the authorsDr T. Butler and Prof. J.GribbenMedical OncologyBarts and the London School of Medicine and Dentistry,London, UK

– issue N°1 – Feb/Mar 201011

42 Linus Allain Avenue • Gardner, MA 01440 USAToll Free: 800/253-3684 • Tel: 978/632-2555 Fax: 978/632-2524 • E-mail: [email protected]

BIOMEDICALPOLYMERS, INC.

BMP Cytology Funnelswith New Patent PendingDisposable Funnel Clips

BMP Cytology Funnelswith New Patent PendingDisposable Funnel ClipsDisposable Plastic Clips Eliminate Timeand Labor Required to Sanitize Metal ClipsAfter Each Use� Complete system fully compatible for use with the

Shandon CytoSpin® Centrifuge

� Disposable clips work with both of our single and double sample chamber cytology funnels

� The new patent pending disposable clip is priced to be a cost effective alternative to other disposable cytologyfunnel systems

BMP Cytology Funnels are the functional equivalentof the Thermo Shandon CytoFunnels® and are fullycompatible with the Shandon CytoSpin Centrifuge.

For more information and the name of the distributornearest you call 1-800-253-3684 or visit our websitewww.biomedicalpolymers.com.

CytoSpin® and CytoFunnels® are registered trademarks of the Thermo Electron Corporation

plastic products for laboratory & medical specimen handling

3203 BioP 8.125x5.1875:Layout 1 3/15/10 10:34 AM Page 1


leukemia – issue N°1 – Feb/Mar 2010 12d

isea

se f

ocu

s

single Cell Network Profiling (sCNP)SCNP is an approach for analysing and interpreting post-translational protein modifications (e.g. phospho-rylation, acetylation etc.) at the sin-gle cell level. This technology is able to simultaneously characterise the range of critical cellular processes within AML, such as growth (cell cycle), viability (apoptosis), DNA damage, presence and function of drug transporters, and in vitro effects of therapies on signalling networks. Using viable cells, measurements are made on endogenous proteins before and after exposure to extracellular modulators such as growth factors,

cytokines or drugs. The modulators are meant to mimic the stimuli that the cell encounters in the body and are chosen to evoke a response from the cell that echoes how the signal-ling system is normally, or abnor-mally, patterned. The proteomic rea-dout in the presence or absence of a specific modulator is termed a “sig-nalling node”. Signalling nodes are evaluated within cells from samples that have associated relevant clini-cal information regarding response to the therapy of interest. Multi-variate analysis can then be per-formed to create predictive models that can be validated in subsequent independent studies.

SCNP is relatively straightforward [Figure 1]. Within seconds to min-utes from cell exposure to specific modulators (depending on the per-turbant and determined by experi-mental design and the state-specific proteins being studied), a fixative such as paraformaldehyde is applied to the cell mixture that rapidly, and covalently, cross-links proteins within the cell. This ‘freezes’ the cell signalling processes and prevents the functional signal from decaying or being lost. The cells are then permea-bilised allowing access to subcellular compartments, such as the nucleus, for binding of fluorophore-conju-gated antibody staining reagents.

Concomitantly, cell surface markers are identified by other fluorophore-conjugated antibodies. The samples are then examined by flow cytom-etry for quantitative determination of fluorophore levels associated with each antibody. This technology has been shown to efficiently and repro-ducibly allow for the interrogation of intracellular signalling pathways within heterogeneous populations of primary cells, with potential applications in diagnostic testing, statistical mapping of signalling net-works and high-throughput drug screening [1-3]. Use of sCNP in AMl Prediction of response to induction therapyFor more than three decades, cytara-bine-based combination regimens have constituted the chemother-apy backbone used to induce dis-ease remission in newly diagnosed adult patients with AML [4]. While these chemotherapeutic regimens result in complete disease response (i.e. the level of response shown

Insights into acute myeloid leukemia via single cell network profilingSingle Cell Network Profiling (SCNP) uses multi-parameter flow cytometry to simultaneously measure resting and evoked intracellular signalling molecules and pathways in distinct cell subsets within a complex tissue sample. This review focuses on the utility of SCNP and its potential applications for chemotherapy selection in Acute Myeloid Leukemia (AML).

by Dr A. Kornblau, Dr S.M. Minden, Dr D. Hogge, Dr A. Cohen, and Dr A. Cesano

Figure 1. Single Cell Network Profiling (SCNP) technology. Cells of interest are first challenged by modulators (e.g. cytokines, drugs) in vitro to reveal signalling pathways. Proteins are then fixed, permeabilised and stained with both surface antibodies and state-specific antibodies. Cells of interest are then identified by surface/lineage markers and light scatter properties during analysis by flow cytometry. Surface/lineage antibodies discern sub-populations of interest whose signalling networks are revealed at the single cell level where basal/unmodulated signalling nodes can be compared to “evoked”/modulated signalling nodes.

Figure 2 A. Predicting response to induction chemotherapy in AML.A. Modulators and networks assessed. Schematic of the cell signalling pathways probed using SCNP. Signalling node is defined as a combination of a specific proteomic read out in the presence or absence of a specific modulator. Text in blue indicates assay readouts. Biological categories of signalling pathways and nodes are indicated by colour: 1) Chemokine, Cytokine, Growth factor Signal Induction (green), 2) Functional Apoptosis (pink), 3) Phosphatase Regulation /Reactive Oxygen Species (ROS) Stress(purple) and 4) Surface Markers/Drug Transporters (orange).


to be associated with long-term survival) in the majority of adult patients younger than 60 years of age, in two thirds of these patients the disease will eventually relapse and lead to patient death [5-7]. In addition, approximately 40-50% of AML patients older than 60 years are refractory to standard induction chemotherapy [5-8].

Currently, the most valuable prog-nostic factors in AML are patient age, the presence of de novo versus secondary AML, and cytogenetic analysis (either alone or in com-bination with molecular mark-ers) [8-10]. While these factors offer prognostic information at the population level there is currently no validated means to predict the disease response to standard AML induction chemotherapy at the individual patient level.

SCNP was applied to two independent sequential training cohorts of AML samples (N = 34 and 88) to predict the likelihood of response to stand-ard induction chemotherapy. Modu-lators relevant to AML biology were used to evoke signalling networks [Figure 2A]. Intracellular signalling profiles showed high reproducibil-ity and univariate analysis identified multiple signalling nodes relevant to myeloid biology that correlated with disease response. Younger patients with chemotherapy sensitive leuke-mia displayed intact communication between a DNA damage response and the apoptotic machinery after in vitro exposure to chemotherapeutic agents [Figure 2B]. By contrast, in older patients or those with secondary AML, lack of response to induction chemo-therapy was associated with increased phosphorylation of PI3 kinase (PI3K) related proteins after FLT3 ligand (FLT3L) stimulation (data not shown). Importantly, combining age with some predictive signalling node/metrics improved predictive value of response to chemotherapy of age alone, show-ing that SCNP adds information beyond known prognostic factors [Figure 2C].

identification of chemotherapy-resistant AMl cell populationsThe idea that subgroups of malignant cells, including the possibility of a cancer

stem cell, exist within a given cancer is not a new concept [11], but it supports the paradigm that a negative clinical outcome may be associated with the outgrowth of a subclone of cells with a distinct signalling signature. This has clear implications for early detection of negative prognostic indicators, and could inform on drug dosing versus efficacy, as well as detection of chemotherapy- resistant disease. SCNP was performed on three paired diagnostic/relapse samples to

determine whether intracellular sig-nalling profiles dominant at relapse could be identified in subpopulations of cells present at diagnosis and pre-dict for disease relapse [manuscript in preparation]. Chemo-resistant subpopulations, such as those shown using Flt3 ligand as a modulator, were more pronounced at relapse than at diagnosis [Figure 3]. Nota-bly, a small population of leukemic cells characterised by simultane-ous SCF-mediated increase in the levels of phosphorylated (p-) Akt

and p-S6 was identified in all three relapse samples studied. This func-tionally defined leukemic cell sub-population was present at a much lower frequency in the diagnostic samples. When an independent set of diagnostic AML samples were examined for blast subpopula-tions that signalled with p-Akt and p-S6 in response to SCF, a group of patients was identified who subse-quently experienced disease relapse (data not shown). These data show that leukemic cell populations differ

Sunquest delivers

Interoperability

We deliver innovative connectivity with your referring GPs.

W e D e l i v e r

Sunquest’s Integrated Clinical

Environment™ (ICE) provides web-based

services and comprehensive software

applications for use in primary and

secondary care NHS Trusts.

Communication systems enable

seamless integration with all installed

PAS, LIMS, RIS, and departmental

systems.

Features provide positive patient

identification, automating and tracking

specimen movement throughout the

enterprise, and connecting clinicians and

remote care locations to the laboratory.

This best-of-suite portfolio allows diverse

systems to work together, increasing

efficiencies and positively impacting the

bottom line.

For more information about Sunquest

Integrated Clinical Environment (ICE),

call +44 (0) 1603 819600 or visit

www.sunquestinfo.com.

Sunquest _Clin Lab Intl 03-10.indd 1 2/11/2010 1:51:18 PM www.cli-online.com & search 24967

quantitatively and qualitatively before and after in vivo therapeutic pressure in AML, and that SCNP offers a novel approach for identifying chemotherapy- resistant subpopulations.

targeted AMl therapiesGiven the complexity of AML as well as the availability of newly approved and investigational therapeutic agents, a greater understanding of disease biology is needed to chart disease progression and assist in the optimal selec-tion of therapeutic agents on an individual patient basis. One such agent recently approved in the United States for second line treatment of AML in the elderly is Gemtuzumab Ozogamicin (GO, Mylotarg). GO is a monoclonal antibody conjugated with the cytotoxic antibiotic calicheamicin that targets the cellular surface antigen CD33. Using SCNP, pathways can be evaluated that are related to cell cycle, DNA damage, apoptosis and functional drug transporter activity, all of which have been shown to be important in GO’s mechanism of action and relevant to the mechanism of action of many other drugs. Interrogation of these pathways in appropriate clinical samples with known response to GO will aid in the development of a diagnostic with the ability to predict which subgroup of AML patients will respond to GO. Initial feasibility experiments in CD33+ cell lines and AML patient samples exposed to GO in vitro assessed drug responsiveness and revealed interesting disease biology. We found that AML samples fell into one of three groups based on GO-induced DNA damage response (as measured by increased p-Chk2 and p-H2AX) and apoptosis (as measured by cleaved Caspase and cleaved PARP). Further studies aimed at understanding why an AML sample responds dif-ferentially to GO and what that response means clinically are underway. The ultimate goal of these studies is to develop and subsequently validate a model for the prediction of GO response.

ConclusionAML classifications, based on clinical, laboratory or molecular features, have been shown to be associated with disease-free and overall survival, and can offer predictive information on disease outcomes. However, a more valuable

leukemia – issue N°1 – Feb/Mar 2010 14

Figure 2 B. Apoptosis pathway distinguishes CR in younger patients with AML. Functional apoptosis assays can stratify NR and CR responses in diagnostic AML leukemia samples. Shown in the side panel is a schema for functional apoptosis after DNA damage. In this example the modulator is etoposide and the readouts are cleaved PARP and p-Chk2. The upper left panel shows the AUC of the ROC (0.81) for the signalling node etoposide → p-Chk2, cleaved PARP+. The upper right panel shows the box and whisker plots with standard deviation shown. Circles represent individual patient samples percentage of PARP positive and p--Chk2 negative cells. The frequency of p-CHK2-, Cleaved PARP+ (c-PARP+) apoptotic cells (upper left quadrant of the flow cytometry plots) after overnight exposure to Etoposide was used to quantify apoptosis.

Figure 2 C. Signalling nodes add value to age alone for induction response stratification. PMA induced p-CREB readout improves upon age as a classifier for response to induction therapy. Significant p-values (slope = 0) for the signalling node were found after accounting for age in the logistic regression model. The ROC plot illustrates CR and NR stratification using the model incorporating the signalling node (PMA → p-CREB) with age.

Figure 3. Chemo-resistant subpopulations increase at relapse. Flow plots are shown of p-Akt (vertical axis) and p-S6 (horizontal axis) basal levels(left) and in response to modulator FLT3L (right) in paired diagnostic (upper panels) and first relapse (lower panels) AML samples. Percent double positive for p-Akt and p-S6 is indicated above the pink box. Note the enrichment of basal and FLT3L-induced phospho-proteins at relapse versus diagnosis.

clinical tool would be a biological method that predicts disease response to therapy with higher specificity and sensitivity at the individual patient level. SCNP is a biological method that allows for an evaluation of dynamic protein responses to external stimuli in specific cell subpopulations (such as leukemic stem cells) that are present in a heterogeneous population of AML cells from bone marrow or peripheral blood.

Currently there are no measures to indicate why patients with similar appearing disease have dif-ferent responses to therapy and no way of making real time adjustments of treatment. A closer rela-tionship between the hypothesis of disease deter-mination and the efficacy of treatment choice is needed. SCNP allows for a more efficient deline-ation of the normality or pathology of diseased cells. Studies that test models for prediction of induction response, relapse risk and response to novel therapies in AML are forthcoming.

references1. Irish JM, Hovland R, Krutzik PO et al. Single cell profil-

ing of potentiated phospho-protein networks in cancer cells. Cell 2004; 118: 217-228.

2. Kotecha N, Flores NJ, Irish JM et al. Single-cell profiling identifies aberrant STAT5 activation in myeloid malig-nancies with specific clinical and biologic correlates. Cancer Cell 2008; 14: 335-343.

3. Parkinson DR, Cesano A. Patient-specific classifications of human malignant disease. Curr Opin Mol Ther 2009; 11: 252-259.

4. Tallman MS, Gilliland DG, Rowe JM. Drug therapy for acute myeloid leukemia. Blood 2005; 106: 1154-1163.

5. Appelbaum FR, Kopecky KJ. Long-term survival after chemotherapy for acute myeloid leukemia: the experi-ence of the Southwest Oncology Group. Cancer 1997; 80: 2199-2204.

6. Bennett JM, Young ML, Andersen JW et al. Long-term survival in acute myeloid leukemia: the Eastern Coop-erative Oncology Group experience. Cancer 1997; 80: 2205-2209.

7. Schiffer CA, Dodge R, Larson RA. Long-term follow-up of Cancer and Leukemia Group B studies in acute mye-loid leukemia. Cancer 1997; 80: 2210-2214.

8. Appelbaum FR, Gundacker H, Head DR et al. Age and acute myeloid leukemia. Blood 2006;107: 3481-3485.

9. Mrozek K, Radmacher MD, Bloomfield CD et al. Molec-ular signatures in acute myeloid leukemia. Curr Opin Hematol 2009;16: 64-69.

10. Schlenk RF, Dohner K, Krauter J et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med 2008; 358: 1909-1918.

11. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primi-tive hematopoietic cell. Nat Med 1997; 3: 730-737.

the authorsKornblau, Steven M., MDMD Anderson Cancer CenterUniversity of Texas1515 Holcombe Blvd, Box 448Houston, TX 77030-4095, USA

Minden, Mark D., MD, PhDDept of Medical Onc/HemPrincess Margaret Hospital610 Univ. Ave. Rm 5-126

Toronto, ON M5G 2M9, CanadaHogge, Donna, MD, PhDBC Cancer Research Center675 W. 10th AveVancouver, BC V5Z 1L3, Canada

Cohen, Aileen C, MD, PhDNodality201 Gateway BlvdSouth San Francisco, CA 94080, USA

Corresponding author: Alessandra Cesano, MD, PhD201 Gateway Blvd., South San Francisco, CA 94080, USAEmail : [email protected]: +1 650 827 8017Fax: +1 650 827 8001


Genetic link to leukemias with an unknown origin

Although leukemia is one of the best studied cancers, the cause of some types is still poorly understood. Now a newly found mutation in acute myeloid leukemia patients could account for half of the remaining cases of adult acute leukemia with an unknown origin. Using sam-ples from a Penn tissue bank of acute myeloid leukemia (AML), a team lead by Dr Craig B. Thompson, director of the Abramson Cancer Center of the University of Pennsylvania, USA found that AML patients have increased levels

of the protein 2-hydroxyglutarate (2HG). The increased amounts of 2HG stem from IDH1 or IDH2 mutations.

Screening for elevations in 2HG in the tissue bank, the team found that IDH1 and IDH2 mutations were observed in over 23 percent of the AML patients studied. A shared feature of cancer-related IDH mutations is increased pro-duction of 2HG. The IDH gene mutations are the first known cancer mutations that result in the creation of a protein with a new enzy-matic activity. Most cancer-causing mutations make the mutated protein either overactive or inactive in performing its normal function. In contrast, the mutations in the IDH proteins give these enzymes the blueprint to create a new molecule not normally produced by cells. Interestingly, the researchers also found that IDH2 mutations are more common than IDH1 mutations in AML. The results suggest that blocking the production of 2HG might reverse the ability of the mutant genes to maintain the leukemic cells. penncancer.org

CellaVision AB, Lund, Sweden • +46 46-286 44 00 [email protected] • www.cellavision.com

You can’t do that on a microscope ...

NEW

Denna går i CLi aug.09s

... but you can do it on the CellaVision® DM1200. It auto-

matically locates and pre-classifies blood cells in peripheral blood

— and it allows slides to be reviewed from any location on

your network.

CELLAVISION®

CELLAVISION®

for medium-sized laboratories

DM1200DM1200for medium-sized laboratories


Metabolic disorders – issue N°1 – Feb/Mar 2010 16la

b t

echn

olo

gy

BackgroundNeonatal jaundice is extremely common. Virtu-ally all newborn infants will have a total serum bilirubin level of >1 mg/dL (17.1 µmol/L), which is the upper limit of normal in adults. Up to 60% of infants have clinically evident jaundice in the first week of life. Newborns are affected due to increased bilirubin production, decreased bilirubin clearance and increased enterohepatic circulation. For most infants, this “physiological jaundice” resolves without long-term sequelae. Rarely, hyperbilirubinemia leads to bilirubin-induced neurological dysfunction, which includes acute bilirubin encephalopathy and its permanent sequelae, kernicterus. Chronic bilirubin encepha-lopathy can manifest as cerebral palsy, hearing loss, dental enamel dysplasia, upward gaze paralysis and rarely intellectual or other handicaps. The morbidity

and mortality rates associated with kernicterus are at least 70% and 10%, respectively. The National Quality Forum in the United States considers kernicterus to be one of 28 serious pre-ventable adverse events referred to as “never events”. In the United States, the American Academy of Pedi-atrics recommends that all infants be assessed for risk of significant hyperbilirubinemia before hospital discharge, either through risk factor assessment or bilirubin measurement [1]. The Canadian Paediat-ric Society recommends that all infants receive total serum bilirubin (TSB) or transcutaneous bilirubin (TcB) measurement between 24 and 72 hours of life [2]. An hour-specific nomogram, published in 1999, is commonly used to determine risk of hyperbiliru-binemia [3]. This tool stratifies infants into one of four risk zones based on their serum bilirubin levels; from this, clinicians can base decisions about follow-up. Interlaboratory variability must be considered when interpreting the results. Early identification of infants at risk for significant hyperbilirubinemia increases the likelihood that noninvasive bilirubin-reducing intervention will be successful. For infants with high bilirubin levels, phototherapy is provided. Phototherapy induces the isomerisation of bilirubin to forms that can be more readily excreted [Figure 1]. It is estimated that 6-10 infants with TSB levels ≥ 15 mg/dL (257 µmol/L) need to be treated to prevent the TSB of one infant from rising to above 20 mg/dL (342 µmol/L) [4]. For those infants for whom phototherapy fails, or who show signs of bilirubin encephalopathy, exchange transfusion is generally performed.

Recently, the recommendation for universal screen-ing for hyperbilirubinemia has been questioned. The United States Preventive Services Task Force

concluded that the evidence is insufficient to assess the benefits and harms of screening for hyperbiliru-binemia to prevent chronic bilirubin encephalopathy [4]. The clinical impact of this is not yet known.

screeningVisual inspection is often used to assess jaundice. However, studies have shown clinical assessment to be an inaccurate predictor of hyperbilirubinemia, especially in infants with darker skin tones. Addi-tionally, interrater reliability is low, even among experienced health care providers.

serum bilirubin measurementBilirubin measurement has traditionally been per-formed through blood sampling. Blood for bilirubin measurement can be of capillary (ie: heel lance), venous or arterial origin. Measurements are obtained via a chemical reaction (the Diazo method), direct spectrophotemetry of whole blood (blood gas ana-lysers or bilirubinometers), spectrophotemetry of separated serum or plasma on an automated chemis-try system (the Vitros method), or high performance liquid chromatography (HPLC). HPLC, although suffering from fewer interferences than most labo-ratory methods, is not practical for routine use. The Diazo method is widely used to estimate bilirubin concentrations, but (depending upon manufacturer formulation) may have interferences with hemoglobin and other intracellular compounds; this can be prob-lematic as blood from neonates is often hemolyzed due to the difficulty of neonatal blood collection.

Bilirubin sampling is one of the most common reasons for neonatal venipuncture. Phlebotomy in infants has many downsides, including pain and stress for infants and parents, potential risk of infec-tion, delay in obtaining results and the need for ancillary support, both by phlebotomy staff and the laboratory. Studies comparing heel lancing to veni-puncture have shown the former to be more painful for infants, and heel lancing may rarely be associated with osteomyelitis.

transcutaneous bilirubin measurementTranscutaneous bilirubin measurement has recently gained popularity as a noninvasive way to meas-ure bilirubin levels in neonates. Measurements are obtained using multiwavelength spectral reflect-ance from the skin surface. The meter’s optic head is pressed against the infant’s skin, usually the fore-head or chest, generating a light [Figure 2]. The light passes through the infant’s subcutaneous tissue, and the reflected light returns to the spectrophotometer.

Transcutaneous bilirubin measurement in neonatesNeonatal jaundice is common. Though generally benign, hyperbilirubinemia occasionally leads to neurological dysfunction. Many healthcare experts advocate universal screening for neonatal hyperbilirubinemia before hospital discharge. Serum bilirubin measurement is the standard method of assessing bilirubin levels. Transcutaneous bilirubin measurement provides a noninvasive way of approximating serum bilirubin concentrations. At levels up to 15 mg/dL (257 µmol/L), transcutaneous levels correlate well with serum levels, making this method ideal for screening.

by Dr A. C. Wickremasinghe, Dr W. J. Cook and Dr B. S. Karon

Figure 1. Phototherapy is provided for infants with high bilirubin levels.

Diagnostica Stago S.A.SRCS Nanterre B305 151 4099, rue des Frères Chausson92600 Asnières sur Seine (France)Tél. +33 (0)1 46 88 20 20Fax +33 (0)1 47 91 08 [email protected]

Boost your Quality approach

The answer for regulatory requirements and accreditation

The reference Haemostasis EQA programme, with thousandsof participants worldwide

A wide range of parameters covering the entire working ranges

A customized programme

Simple and rapid interpretation of results

A user-friendly web interface

Qualiris by Stago, your new International Haemostasis EQA Programmew

ww

.epi

cea.

com

- ©

2009

Dia

gnos

tica

Sta

go -

All

right

rese

rved

- 1

0/20

09

AnnonceQualiris206x276-GB:- 26/10/09 18:28 Page 1


A number displayed on the device indicates the intensity of the yellow colour of the reflected light.

The benefits of TcB measurement are based on its non-invasiveness: decreased pain, stress, skin injury and potential for infection. Additionally, TcB measurement provides results more quickly than venipuncture and does not require phlebotomy or laboratory support. However, TcB measurements may not be as reliable after phototherapy and may be affected by sunlight exposure. Measurements cannot be made on skin that is bruised, birthmarked or covered with hair. The relationship between TcB and TSB is linear. Studies evaluating the correlation between TSB and TcB have produced varied results, with some find-ing TcB to overestimate TSB, and others finding it to underestimate TSB. TcB is generally thought to rea-sonably approximate TSB for values <15 mg/dL (257 µmol/L) [1]. Additionally, having blood collected in an amber tube, which protects the sample from light, versus a clear tube, may provide a higher correlation between TSB and TcB values [5].

The two transcutaneous bilirubinometers that have been studied most extensively are the Air-Shields Jaundice Meter (Konica Minolta) and the BiliChek (Respironics, Inc). Both are thought to be accept-able for screening [6]. The Jaundice Meter uses two wavelengths (450 nm and 550 nm) to measure bilirubin. The present version, the JM-103, shows better correlation with TSB than did its predeces-sors. It needs daily calibration, and may not be as accurate in African Americans as in Caucasians [7]. The BiliChek device uses multiple wave-lengths over the entire spectrum of visible light for spectral reflectance. It corrects for the effects of melanin, hemoglobin and dermal thickness. One study showed its correlation with HPLC to be stronger than that of standard laboratory meas-urements [8]. However, it requires that a clean, disposable tip be used with each measurement, adding to its cost.

Of other devices studied, Bilitest BB77 (Bertocchi SRL Elettromedicali) underestimated TSB at val-ues ≥ 12 mg/dL (205 µmol/L), BiliMed (Medick SA) was less accurate than the BiliChek, and the Colormate III (Chromatics Color Sciences Inter-national Inc) requires a baseline measurement to be done for each infant and is no longer manu-factured [9,10,11]. Icterometers have been used in the past, but are less reliable as they depend on observer visualisation of the yellow colour of the skin.

Quality issues need to be considered when using transcutaneous bilirubinometers. The mean intradevice coefficients of variation were 1.5% for the JM-102 and near 9% for the BiliChek [6]. The interdevice precision for the difference between two readings on the BiliChek was 2.2 mg/dL (37 µmol/L) [6]. In order to obtain accurate results, staff must be trained on the proper use of the devices.

Some advocates of universal screening consider TcB measurement to be the optimal method of screen-ing before hospital discharge. Though there has been concern that implementing universal screening for neonatal hyperbilirubinemia will increase serum blood draws, the evidence does not support this. A study evaluating the effects of universal screening with TcB found no increase in serum blood draws and fewer hospital readmissions (1.8 vs. 4.5%) after initiation of the screening protocol; however, more initial phototherapy (7.7 vs 5.9%) was performed [12]. This study estimated that implementing uni-versal screening with TcB would cause a small but statistically insignificant increase in cost [12].

There is a paucity of studies examining TcB in the outpatient setting. One study showed TcB to system-atically underestimate TSB in outpatients [13]. In this population, it is suggested that the chest is a bet-ter location for measurement than the forehead, as it is exposed to less light. Further studies are needed to better determine the optimal use of TcB after hospital discharge.

ConclusionsTranscutaneous bilirubin measurement can be used to screen neonates for hyperbilirubinemia before hospital discharge, as it reasonably approximates low to medium serum bilirubin levels. Its nonin-vasive nature allows for less discomfort for infants. It is important that transcutaneous bilirubin levels are adjusted to match local laboratory serum val-ues. Further studies should be performed before transcutaneous bilirubin is routinely used in outpa-tients. The long-term cost/benefit ratio of universal screening has yet to be elucidated.

references1. American Academy of Pediatrics Subcommittee on

Hyperbilirubinemia. Management of Hyperbilirubine-mia in the Newborn Infant 35 or More Weeks Gestation. Pediatrics 2004; 114(1): 297-316.

2. Canadian Paediatric Society, Fetus and Newborn

Committee. Guidelines for Detection, Management and Prevention of Hyperbilirubinemia in Term and Late Pre-term Newborn Infants (35 or More Weeks’ Gestation). Paediatr Child Health 2007;12(5):1B-12B.

3. Bhutani VK, Johnson L, Sivieri EM. Predictive Ability of a Predischarge Hour-Specific Serum Bilirubin for Sub-sequent Significant Hyperbilirubinemia in Healthy Term and Near-Term Newborns. Pediatrics 1999;103(1):6-14.

4. US Preventive Services Task Force. Screening of Infants for Hyperbilirubinemia to Prevent Chronic Bilirubin Enceph-alopathy: US Preventive Services Task Force Recommen-dation Statement. Pediatrics 2009; 124(4): 1172-7.

5. Karon BS, Teske A, Santrach PJ, Cook WJ. Evaluation of the BiliChek Noninvasive Bilirubin Analyzer for Predic-tion of Serum Bilirubin and Risk of Hyperbilirubinemia. Am J Clin Pathol 2008; 130: 976-982.

6. Wong CM, van Dijk PJ, Laing IA. A Comparison of Tran-scutaneous Bilirubinometers: SpectRx BiliCheck Versus Minolta AirShields. Arch Dis Child Fetal Neonatal Ed 2002; 87(2): F137-40.

7. Maisels MJ, Ostrea EM Jr, Touch S, Clune SE, Cepeda E et al. Evaluation of a New Transcutaneous Bilirubinometer. Pediatrics 2004;113(6):1628-35.

8. Rubaltelli FF, Gourley GR, Loskamp N, Modi N, Roth-Kleiner M et al. Transcutaneous Bilirubin Measurement: A Multicenter Evaluation of a New Device. Pediatrics 2001;107(6):1264-71.

9. Bertini G, Pratesi S, Cosenza E, Dani C. Transcutaneous Bilirubin Measurement: Evaluation of Bilitest. Neonatol-ogy 2008;93(2):101-5.

10. De Luca D, Zecca E, Corsello M, Tiberi E, Semeraro C et al. Attempt to Improve Transcutaneous Bilirubinom-etry: A Double-Blind Study of Medick BiliMed Versus Respironics BiliCheck. Arch Dis Child Fetal Neonatal Ed 2008;93(2):F135-9.

11. El-Beshbishi SN, Shattuck KE, Mohammad AA, Petersen JR. Hyperbilirubinemia and Transcutaneous Bilirubi-nometry. Clinical Chemistry 2009; 55 (7): 1280-7.

12. Petersen JR, Okorodudu AO, Mohammad AA, Fern-ando A, Shattuck KE. Association of Transcutaneous Bilirubin Testing in Hospital with Decreased Readmis-sion Rate for Hyperbilirubinemia. Clinical Chemistry 2005; 51(3): 540-544.

13. Engle WD, Jackson GL, Stehel EK, Sendelbach DM, Manning MD. Evaluation of a Transcutaneous Jaun-dice Meter Following Hospital Discharge in Term and Near-Term Neonates. Journal of Perinatology 2005; 25(7):486-90.

the authorsAndrea C. Wickremasinghe MD1

Walter J Cook MD1 Brad S Karon MD, PhD2

1 Department of Pediatric and Adolescent Medicine, and

2 Department of Laboratory Medicine and Pathology Mayo Clinic,

Rochester, MN 55905, USA

Metabolic disorders – issue N°1 – Feb/Mar 2010 18

Comments on this article?Feel free to post them at

www.cli-online.com/comment/Tcb

Figure 2. Infant undergoing transcutaneous bilirubin measurement on the sternum.

– issue N°1 – Feb/Mar 201019la

b techno

log

yMetabolic disorders

An increased level of serum bilirubin compared to normal adult values is almost universally found in newborn infants and its clinical manifesta-tion, jaundice, occurs in at least two out of three of normal full-term newborns. Following birth, the total serum bilirubin (TSB) in the neonate increases steadily and, depending on the gestation [1], usually peaks between 72-96 hours. Depend-ing on the infant’s race, gestation, method of feed-ing and other epidemiological factors, the average peak TSB level in infants > 35 weeks of gestation varies from about 5-12 mg/dL (86-205 µmol/L) [2]. Recent studies in large populations report mean peak TSB values between 8-10 mg/dL (137-17 µmol/L) [3,4] with a 95th percentile of 15-17 mg/dL [3-6]. On rare occasions, however, and for reasons that are not always clear, extreme levels of hyperbilirubinemia (that is with serum levels

>25-30 mg/dL [428-513 µmol/L], develop and can result in acute bilirubin encephalopathy and its chronic permanent manifestation, kernicterus [7,8]. Although originally a pathological diagno-sis characterised by bilirubin staining of the brain stem nuclei and cerebellum, the term kernicterus is now used to describe the chronic syndrome of bilirubin encephalopathy – a choreo-athetoid form of cerebral palsy, auditory neuropathy and sensorineural hearing loss, gaze palsies and dental dysplasia [2].

Although kernicterus is a rare condition, it is still being reported in North America and Western Europe where the incidence ranges from about 1 in 40,000 to 1 in 150,000 live births [9-11]. In less developed parts of the world, kernicterus is probably much more common although no good

incidence figures are available [12-14]. In con-trast to the original case descriptions, most of the infants who now develop kernicterus are not those with Rh hemolytic disease and they often have no documented evidence of hemolysis. Rather, they are term and late preterm infants who have been discharged from the nursery as “healthy newborns” yet have returned to a pediatrician’s office, clinic or emergency department with extreme hyperbi-lirubinemia and have gone on to develop the clas-sic neurodevelopmental findings associated with kernicterus [7,15].

GuidelinesThe American Academy of Pediatrics (AAP), in 2004 [16] and the Canadian Paediatric Society in 2007 [17] published guidelines on the management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation and, although a recent commentary has suggested some important modi-fications to these guidelines [18], the basic prin-ciples still apply. Risk assessment and follow-up are the two essential elements of these guidelines that form the core of our approach to preventing hyperbilirubinemia and kernicterus.

risk assessmentClinical risk factorsThe important clinical and laboratory risk factors for the development of severe hyperbilirubinemia are well documented [16,17] and shown in Table 1 [18]. In the absence of overt hemolytic disease, the most important factors that help to predict the risk of hyperbilirubinemia are the infant’s gesta-tional age and the TSB level or a transcutaneous bilirubin level (TcB), measured before the newborn is discharged [19-21].

Neonatal hyperbilirubinemia – how to identify the infant at riskExtremely high levels of bilirubin (>430 µmol/L) in the neonate are relatively rare but can have serious and permanent clinical sequelae, such as kernicterus. This article describes the factors involved in the risk assessment of neonatal hyperbilirubinemia and presents algorithms and procedures for management and follow-up.

by Dr J Maisels

Table 1. Important risk factors for severe hyperbilirubinemia. TSB= Total Serum Bilirubin, TcB = Transcutaneous bilirubin.

Predischarge TSB or TcB measurement in the high-risk or high-intermediate-risk zone (Figure 1)

Lower gestational age

Exclusive breastfeeding, particularly if nursing is not going well and weight loss is excessive

Jaundice observed in the first 24 h

Isoimmune or other hemolytic disease (eg. G6PD deficiency, congenital spherocytosis)

Previous sibling with jaundice

Cephalhematoma or significant bruising

East Asian race

Figure 1. Nomogram for designation of risk in 2840 well newborns at >36 weeks’ gestational age with birth weight of >2000 g or >35 weeks’ gestational age and birth weight of >2500 g based on the hour-specific serum bilirubin values. Note that because of sampling bias, this nomogram does not describe the natural history of neonatal hyperbilirubinemia [34] although it functions well as a predictive tool. Reproduced with permission from [5].

Presdischarge measurement of total serum bilirubin and transcutaneous BilirubinBhutani and associates were the first to demonstrate a strong relationship between the hour-specific predis-charge TSB level and the likelihood of the subsequent development of hyper-bilirubinemia [Figure 1], [5]. Several other studies have confirmed these observations [3, 19, 20, 22-24]

Transcutaneous bilirubin (TcB) meas-urements are now being used with increasing frequency in hospital nurs-eries [19,20] and outpatient settings [25-27] and there is good evidence that these measurements provide excellent estimates of the TSB level although they

are not a substitute for TSB values [28].The TcB is a measurement of the yellow colour of the blanched skin and subcu-taneous tissues, not the serum bilirubin, and should be used as a screening tool to help determine whether the TSB should be measured. In a recent study [20], 39% of infants who had a predis-charge TcB above the 95th percentile [Figure 1] subsequently developed a TSB level that exceeded or was within 1 mg/dL (17 µmol/L) of the AAP recom-mended phototherapy level, compared with only 0.4% of infants with a pre-discharge TcB below the 75th percen-tile (relative risk 97.5). Combining the predischarge TSB or TcB with clinical risk factors significantly improves the prediction of the risk of subsequent hyperbilirubinemia [19-21]. When combined with a bilirubin risk zone, the factors that are most predictive of hyperbilirubinemia risk are lower ges-tational age and exclusive breastfeeding. [18-21] [Figure 2].

It is also recognised that, although jaun-dice is the key clinical manifestation of hyperbilirubinemia, using the intensity or extent of jaundice to estimate the TSB level can be misleading [29-31] and experts now recommend that a TSB or TcB should be performed on every newborn infant, after age 18 hours and prior to discharge from the nursery [18]. Figure 3 provides an algorithm for the

management and follow up of newborns according to the predischarge bilirubin measurement, the gestational age, and certain risk factors for subsequent hyper-bilirubinemia [Table 2]. Recent data suggest that this type of predischarge screening can reduce the incidence of

a TSB level >25 mg/dL (428 µmol/L) [32,33] perhaps by increasing the use of phototherapy prior to discharge [32].

Follow-upFigure 3 provides recommenda-tions for follow up based on the AAP

Metabolic disorders – issue N°1 – Feb/Mar 2010 20

Figure 2. Probability of the bilirubin level at any time after birth exceeding or within 1 mg/dL (17 µmol/L) of the hour-specific AAP recommended phototherapy level. An increase in gestation of just 1 week produces a striking decrease in the risk of developing significant hyperbilirubinemia. See Figure 1 for the bilirubin percentiles. [Reproduced with permission from 20].

Table 2. Other risk factors for severe hyperbilirubinemia to be considered with the gestational age and the pre-discharge TSB or TcB level.

Exclusive breastfeeding particularly if nursing is

not going well and/or weight loss is excessive

(>8-105)

Isoimmune or other hemolytic disease (eg.

G6PD deficiency, hereditary spherocytosis)

Previous sibling with jaundice

Cephalohematoma or significant bruising

East Asian race

The gestational age and the predischarge TSB

or TcB level are the most important factors that

help to predict the risk of hyperbilirubinemia.

The risk increases with each decreasing week of

gestation from 42-35 weeks [see Figure 2]

Figure 3. Algorithm providing recommendations for management and follow-up according to predischarge bilirubin measurements, gestation, and risk factors for subsequent hyperbilirubinemia.• Provide lactation evaluation and support for all breastfeeding mothers.• Recommendation for timing of repeat TSB measurement depends on age at

measurement and how far the TSB level is above the 95th percentile [Figure 1]. Higher and earlier initial TSB levels require an earlier repeat TSB measurement.

• Perform standard clinical evaluation at all follow-up visits.• For evaluation of jaundice see 2004 AAP guidelines [16].

Explanatory symbols. In the above algorithms a = See Table 2; b = See Figure 1; c = In hospital or as outpatient; d =Follow-up recommendations can be modified according to level of risk for hyperbilirubinemia. In infants at low risk, later follow-up can be considered, depending on the circumstances.[Reproduced with permission from 18].

1

1

Figure 3. Algorithm Providing Recommendations for Management and Follow-up according to Predischarge Bilirubin Measurements, Gestation, and Risk Factors for Subsequent Hyperbilirubinemia

Gestational Age 35-37 6/7 weeks plus other hyperbilirubinemia risk factors a

Predischarge TcB/TSB

Assign bilirubin risk zone b

High High-Intermediate Low-Intermediate Low

Evaluate for

phototherapy

TSB in 4-8 h c

Evaluate for

phototherapy

TSB/TcB in 4-24 h c

If discharging < 72 hr, follow up within 2 days

If discharging <72 hr,

follow up within 2 days

Consider TSB/TcB at follow-up

2

1

Gestational Age 35-37 6/7 weeks and, no other hyperbilirubinemia risk factors

OR Gestational Age 38 weeks plus other hyperbilirubinemia risk factors




Evaluate for

phototherapy

TSB in 4 – 24 h c

Evaluate for

phototherapy TcB/TSB within 24 h c

If discharging <72 hr, follow up within 2 days

If discharging < 72

hr, follow up within 2-3 days

3

1

2

3

4

Gestational Age 38 weeks and no hyperbilirubinemia risk

factors a




Evaluate for phototherapy

TSB in 4-24 h c

Follow-up within 2 days Consider TcB/TSB at

follow-up

If discharging <72 hr, follow up within 2-3 days

If discharging < 72 hr, time follow up according to age

at discharge or concerns

other than jaundice (e.g. breastfeeding) d

guideline with additional modifica-tions depending on the bilirubin risk zone, gestational age and other risk factors. A general rule is that any infant discharged at age < 72 hours should be seen within two days of discharge unless there is little risk of subsequent hyperbilirubinemia, in which case a later follow up is appro-priate [Figure 3].

The use of universal predischarge bilirubin screening, when combined with clinical risk factors (of which gestational age and exclusive breast-feeding are the most important), is a systems approach that is easy to implement and understand, and provides a method of identifying infants at either high or low risk for the development of severe hyperbi-lirubinemia. When combined with a targeted follow-up, this approach could play an important role in preventing acute bilirubin encepha-lopathy although this has yet to be demonstrated [18].

references1. Maisels MJ, Kring E. Pediatrics 2006;

117:1169-1173.

2. Maisels MJ. Jaundice. In: MacDon-ald MG, Seshia MMK, Mullett MD, editors. Avery’s Neonatology. Phila-delphia, PA: Lippincott Co., 2005: 768-846.

3. Stevenson DK et al. Pediatrics 2001; 108:31-39.

4. Fouzas S et al. Pediatrics 2010; 125: e52-e57.

5. Bhutani VK, Johnson L, Sivieri EM. Pediatrics 1999; 103:6-14.

6. Newman TB et al. Pediatrics 1999; 104:1198-1203.

7. Johnson L et al. J Perinatol 2009; 29: S25-S45.

8. Maisels MJ. Early Hum Dev 2009; 85(11):727-732.

9. Sgro M et al. http://www.cps.ca/english/surveillance/CPSP/index.htm.

10. Manning D et al. Arch Dis Child Fetal Neonatol Ed 2007; 92:342-346.

11. Ebbesen F. Acta Paediatr 2000; 89: 1213-1217.

12. Banerjee TK et al. Indian J Pediatr 2009; 76:139-146.

13. Mezaal MA et al. The Open Neuro J 2009; 3:24-26.

14. Slusher TM et al. J Pediatr 1995; 126:102-108.

15. Maisels MJ, Newman TB. Pediatrics 1995; 96:730-733.

16. American Academy of Pediatrics. Pedi-atrics 2004; 114:297-316.

17. Canadian Paediatric Society. Paediatr Child Health 2007; 12:1B-12B.

18. Maisels MJ et al. Pediatrics 2009; 124(4):1193-1198.

19. Maisels MJ et al. J Perinatol 2009; 29:612-617.

20. Keren R et al. Pediatrics 2008; 121http://www.pediatrics.org/cgi/content/full/121/1/e170:e170-e179.

21. Newman T, Liljestrand P, Escobar G. Arch Pediatr Adolesc Med 2005; 159:113-119.

22. Alpay F et al. Pediatrics 2000; 106:E16.23. Carbonell X et al. Acta Paediatr 2001;

90:166-170.24. Kaplan M et al. Pediatrics 2000;

105:533-537.25. Maisels MJ, DeRidder J, Kring E. E-PAS

2007; 618441.5.26. Engle W et al. J Perinatol 2005; 25:

486-490.27. Maisels MJ et al. E-PAS 2009;

2842.398.28. Maisels MJ. Neoreviews 2006;

7:e217-e225.29. Davidson LT, Merritt KK, Weech AA.

Am J Dis Child 1941; 61:958-980.30. Moyer VA, Ahn C, Sneed S. Arch

Pediatr Adolesc Med 2000; 154:

391-394.31. Keren R et al. Arch Dis Child Fetal

Neonatol Ed 2009; 94:F317-F322.32. Kuzniewicz MW, Escobar GJ, New-

man TB. Pediatrics 2009; 124(4): 1031-1039.

33. Eggert L et al. Pediatrics 2006; 117:e855-e862.

34. Maisels MJ, Newman TB. Pediatrics 1999; 103:493-495.

the authorM. Jeffrey Maisels, M.B., B.Ch., DS.c.Department of PediatricsOakland University William Beaumont School of Medicine and Division of NeonatologyBeaumont Children’s Hospital3601 W. 13 Mile RoadRoyal Oak, Michigan 48073, [email protected] +1 248-551-0412Fax: +1 248-551-5998



Comments on this article?

Feel free to post them at

www.cli-online.com/comment/kernictus

oncology – issue N°1 – Feb/Mar 2010 22A

naly

te o

f th

e M

ont

h

Bone metastases and their consequencesCancers have the ability to metastasise to organs distant from the site of the primary tumour. Breast, prostate and lung cancers are primary tumours that frequently metastasise to the skeleton. Metabolically and hormonally active bone metastases (BMs) exert profound effects on surrounding bone cells. The con-sequence is an alteration of the natural processes of bone resorption, formation, or both, which affects the maintenance of bone integrity [1]. Chronic BM often results in complete pathological remodelling of the affected bone compartment making affected bones vulnerable to several complications, including patho-logical fractures, spinal cord compression, hypoc-alcaemia, and severe bone pain. All of these reduce the patient’s quality of life and worsen prognosis [1]. Therefore, early diagnosis and adequate treatment of BM is critically important for the clinical manage-ment of patients with cancer, particularly those with breast, prostate and lung tumours.

Since biochemical markers of bone turnover can be assessed non-invasively, they could prove clinically practical in providing additional systemic information of bone turnover[2]. A panel of biomarkers of bone resorption, formation, and osteoclastogenesis has been evaluated for the ability to detect BM in patients with lung, breast or prostate cancer [2-11]. Although the general perception is that markers of bone turnover are elevated in blood and urine of patients with metastatic bone disease, different studies reveal notable differ-ences in the indicative value of these markers. Figure 1 is a schematic diagram illustrating the interactions between tumour cells, osteoblasts and osteoclasts in the so-called “vicious cycle”[11] resulting in the release of biomarkers. The rate of formation of bone matrix can be measured using markers of enzymatic activity or generated matrix components, whereas the rate of resorption can be quantified by measuring different peptide fragments released during the degradation of type I collagen molecules. Due to coupling between these events, biomarkers of formation and resorption

can be elevated simultaneously, reflecting a higher rate of bone turnover. Importantly, bone turnover markers merely reflect the interaction between an invasive and hormonally active malignant tissue and the surround-ing bone cells, and are not able to indicate the primary disease, i.e., the type of cancer.

Alpha CtXProteins containing an aspartate (D) residue linked to a low-molecular-weight amino acid, such as glycine (G), can undergo spontaneous non-enzymatic isom-erisation [2]. This isomerisation introduces a kink in the conformation of the molecule, as the peptide backbone is redirected from the a-carboxyl group in

the native newly synthesised form to the side chain β-carboxyl [3]. The alpha 1 chain of type I collagen undergoes an isomerisation in the DG motif within the C-telopeptide of collagen type I (CTX-I) sequence (1207EKAHDDGR1214) of its C-terminal telopeptide. This transformation has been shown to occur in vitro when bone matrix is incubated at 37°C and has also been documented as occurring in vivo in humans [3,5]. The Alpha form of the epitope is released dur-ing degradation of newly synthesised type I collagen (young bone), whereas the Beta epitope is released from matured collagen type I (old bone). The resorp-tion rate of newly synthesised collagen type I can be assessed by specific immunoassays detecting cross-linked non-isomerised Alpha CTX in urine samples. The degradation rate of matured collagen can be esti-mated by another specific assay targeting cross-linked isomerised Beta CTX in both urine and serum sam-ples. Since biochemical markers are measured system-ically, these levels represent resorption of bone matrix as the sum of all sites of bone remodelling.

Clinical application of Alpha CtX for the detection of bone metastasesThe relationship between skeletal tumour load and elevations in serum or urine levels of Alpha CTX and seven other biomarkers related to bone turno-ver has been investigated in a pooled group of breast and prostate cancer patients [10], who were stratified according to the Soloway score: Score 0 =0 BM; Score 1= <6 BM; Score 2= 6-20 BM; Score 3= >20 BM; Score 4= Superscan: >75 % ribs, vertebrae and pelvic bone infected. A strong linear association was observed between BM and all biomarkers except osteoprote-gerin (OPG) and receptor activator of nuclear fac-tor κB ligand (RANKL) [Figure 2]. All six remaining markers were significantly elevated in patients with Soloway score 1. The relative percent increases due to the presence of bone metastases were most pro-nounced for Alpha CTX-I, being more than 600% elevated at Soloway score 3, followed by bone specific alkaline phosphatase (BSAP) and N-telopeptide of collagen type I (NTX), which were elevated 470% and 440% at Soloway score 3, respectively. This finding was supported by observations in prostate cancer patients showing that of seven different biomarkers, Alpha CTX-I was the most sensitive marker for BM [4]. The higher sensitivity of Alpha CTX-I could be explained by the fact that this epitope is release from sites of high bone remodelling, where collagen fibrils do not have time to mature and undergo β-isomerisation. Fur-thermore, the Alpha CTX epitope has been located in adjacent sections of bones invaded by metastases from breast cancer or prostate cancer [8-9]. Immunostain-ing for the Alpha CTX epitope revealed the presence

A sensitive biomarker for the detection of bone metastasesBelonging to the group of molecules that contain neo-epitopes, Alpha CTX is formed by post-translational modification of the C-telopeptide of collagen Type I (CTX-I). This article describes the evidence suggesting that Alpha CTX may become the most useful biomarker for the detection of bone metastases (BM) in breast and prostate cancer patients and for the monitoring of the efficacy of anti-resorptive treatment in such patients.

by D.J. Leeming and Prof. M. A. Karsdal

Figure 1. Interactions between osteoblasts and osteoclasts in the proximity of tumour cells in a bone metastasis. Tumour cells may stimulate bone cells with predominantly osteolytic or osteoblastic mediators. Increased activity of osteoblasts and osteoclasts results in the release of different biomarkers (enzymes, bone matrix components, and degradation peptides from collagen type I), which can be detected in the serum/plasma and/or urine. At sites of BMs, bone remodelling is accelerated and hence collagen molecules are in an immature state. Biomarkers such as non-isomerised cross-linked C terminal telopeptide of collagen type I (CTX) that are able to reflect the source (i.e., young bone) represent promising markers for detecting changes in bone turnover attributable to metastatic bone disease [12].

enzymes, matrix components, degradation peptides osteoclast regulatory proteins

Cancer cells

Osteoblasts OsteoclastRANK-L, M-CSF


of this epitope with more intensive staining at the sites of high bone remodelling.

Finally, Alpha CTX has been proven to be more use-ful for the evaluation of BM in a longitudinal study of prostate cancer patients than prostate specific antigen (PSA) and total alkaline phosphatase (tALP) [9]. PSA was elevated in both lymph node negative and positive patients compared to controls, while Alpha CTX was elevated only in lymph node positive patients. tALP levels were similar across the groups. In a second arm of this study patients were treated with docetaxel alone or docetaxel and zoledronic acid combined. PSA and tALP levels decreased from baseline values in patients with and without BM who received either treatment regimen, indicating that docetaxel or docetaxel/zoledronate treatment had similar effects on these markers. In contrast, Alpha CTX did not decrease with docetaxel treatment in the BM negative group compared to baseline while it decreased significantly with docetaxel/zoledronate treatment in the BM posi-tive group. This suggest that Alpha CTX is superior to PSA and tALP for identifying patients at high risk of metastatic disease and for monitoring progression of BM in prostate cancer patients during treatment.

Future perspectives Biochemical markers of tissue turnover are increas-ingly used in both basic and clinical research, for diagnosis, prognosis and effectiveness of therapy [1, 7]. In addition, such markers may provide additional information for understanding the pathogenesis and pathology of disease. A novel class of markers is emerg-ing with special focus on neo-epitopes from post-translational modifications (PTM). Protein degrada-tion fragments such as CTX-I, in combination with other PTMs, may be used to identify other pathology-specific biochemical markers. More research groups

are working on such neoepitope platforms that show promise in multiple disease areas [1,6]. ConclusionThe Alpha CTX biomarker may be the most useful marker for the detection of BM in breast and pros-tate cancer patients. Further research should deter-mine the ability of Alpha CTX to detect early phase BM and to monitor efficacy of anti-resorptive treat-ment in patients with BM.

references1. Bauer DC et al. Osteoarthritis Cartilage 2006; 14:

723-727.2. Cloos PA and Christgau S. Biogerontology 2004; 5:

139-158.3. Fledelius C et al. J Biol Chem 1997; 272: 9755-9763.4. Garnero P et al. Br J Cancer 2000; 82: 858-864.5. Garnero P et al. J Bone Miner Res 2002; 17: 826-833.6. Karsdal MA et al. Biomarkers 2009; 14: 181-202.7. Karsdal MA et al. Biomarkers 2009; 14: 181-202.8. Leeming DJ et al. Cancer Epidemiol Biomarkers Prev

2006; 15: 1392-1395.9. Leeming DJ et al. Cancer Epidemiol. Biomarkers Prev

2008; 17: 1269-1276.11. Leeming DJ et al. Cancer Epidemiol Biomarkers Prev

2006; 15: 32-38.12. Mundy GR. Pathophysiology of bone metastases.

Rubens RD, Mundy GR. Cancer and the skeletons. 2000. London (UK), Martin Dunitz Ltd.

13. Tanko LB et al. Cancer Metastasis Rev 2006; 25: 659-668.

the authorsDJ Leeming1 and Dr MA Karsdal1,2

1 Nordic Bioscience, Herlev, Denmark 2 Dept. of Molecular Medicine, University of Southern Denmark.

0 1 2 3

0

100

200

300

400

500

600

700

CTX

NTx

BSAP

CTX

TRAP5b

ICTP

OPG

RANKL

Soloway Score

Marker level

(% of Soloway score

Figure 2. Relative increases in bone resorption, bone formation and osteoclastogenesis marker levels as a function of the extent of skeletal involvement, assessed in 132 patients with breast or prostate cancer. Relative increases are expressed as a percentage of levels in patients with a Soloway score 0 [10].

www.genzymediagnostics.com

H. pylori TestAt Genzyme, we believe diagnosis is key in controlling healthcare costs.The choice of testing also affectsthe course of treatment. Our NEWOSOM® H. pylori Test provides theperfect starting point to a diagnosticpathway which may prevent costly,invasive procedures. The test is accurate, convenient, and simple torun. With a sensitivity of 95.9% versusbiopsy/ histology and using wholeblood, serum, or plasma, this testprovides an ideal solution to detectH. pylori antibodies.

To add this exciting new product toyour portfolio or to find out moreabout our many other point of careproducts, please contact us at [email protected]

SEE US AT

ECCMID

Vienna

APRIL 10-13, 2010

CLI_ad_d3_alt_Layout 1 3/9/10 6:20 PM Page 1


Microbiology – issue N°1 – Feb/Mar 2010 24

infectious gastroenteritisInfectious gastroenteritis is a significant cause of morbidity and mortality worldwide. There are many causative agents, including a variety of bacteria, viruses and protozoan parasites. However, the labo-ratory investigation of faecal samples from a patient suffering from gastroenteritis usually focuses on the detection of Campylobacter spp., Salmonella enter-ica, Shigella spp. and verotoxigenic Escherichia coli, which, except for shigella, are zoonotic.

The most frequently isolated of these pathogens is campylobacter, primarily C. jejuni subsp. jejuni (other species include C. coli, C. upsaliensis, C. hyoin-testinalis and C. fetus). All of these species, except for C. fetus, grow best between 37 and 43ºC. Most infections are sporadic, though outbreaks can occur, and the most common sources of infection include poultry, water and milk [1].

The genus Salmonella comprises two species, namely S. enterica and S. bongori, which have more than 2,500 serotypes or serovars, differentiated on the basis of their somatic (O) and flagellar (H) anti-gens by the White-Kauffmann-Le Minor Scheme[2]. Most salmonella infections are confined to the gastrointestinal tract.

There are four species in the genus Shigella; Sh. son-nei (1 serotype), Sh. flexneri (14 serotypes), Sh. boydi (18 serotypes) and Sh. dysenteriae (13 serotypes), all of which are characterised serologically on the basis of the O antigen only, as Shigella spp. lack the H anti-gen [3]. Shigella dysentery is confined to humans and primates, and transmission, which is most com-mon in developing countries, is human-to-human or via contaminated water or food.

Strains of E. coli that produce a protein toxic to Vero cells are termed verotoxigenic E. coli (VTEC), and are capable of causing two types of disease, namely haemorrhagic colitis and haemolytic uraemic syn-drome [4]. Serogroup O157 is the most common cause of these illnesses, but at least 150 serotypes

have been identified as verotoxigenic [5]. The two toxins, VT1 and VT2, were also termed “shiga-like”, or “STEC”, due to their similarity to the toxin of Sh. dysenteriae [5].

laboratory culture and identification methodsTraditionally, the laboratory methods used to detect and identify these four pathogen groups combine culture with a range of biochemical tests and serol-ogy, using antisera raised against O and, in some cases, H antigens. Campylobacter spp. are exceptions to this threefold approach to identification.

Campylobacter bacilli are small, curved, motile, Gram-negative and microaerophilic, and require a controlled atmosphere for growth. There is a range of culture media available selecting for the growth of the relatively slow-growing campylobacter over faster-growing competitors. The identification of campylobacter is commonly performed to genus level only in the clinical laboratory. Furthermore, in many laboratories, campylobacter is identified on the basis of colonial morphology, microscopic appearance using Gram or another staining method, and a positive oxidase reaction.

Salmonella, shigella and verotoxigenic E. coli are members of the family Enterobacteriaceae, and grow well aerobically, typically within a 24-hour period at 37ºC. Salmonella is routinely identified to serovar level. First, the faecal sample may be enriched for the selective growth of salmonella by incubation overnight in selenite broth. A sub-culture of this suspension is made to a selective medium, which requires a second overnight incu-bation step. Following incubation, colonies show-ing typical morphology expected for salmonella are investigated further. One approach at this point is to rule out the presence of proteus and pseu-domonas, by testing for the production of urease and oxidase, respectively. Colonies negative for these tests may be subjected to a serological analy-sis, using two polyvalent antisera to detect O and

H antigens of salmonella. A positive result requires further analysis using monovalent antisera. Moreo-ver, a colony showing agglutination with both the polyvalent antisera may not be confirmed as Sal-monella spp., as it is not uncommon to find shared antigens among Enterobacteriaceae. Furthermore, the detection of agglutination with the polyvalent H antiserum without a corresponding O agglutina-tion may indicate the presence of S. enterica Typhi or Dublin, whereby the somatic Vi antigen masks any other somatic antigen. For these reasons, it is necessary to confirm the identity of the suspected salmonella isolate using biochemical tests.

The identification of shigella requires a similar approach to that used for salmonella. The com-bination of isolation on a selective medium and serology also requires an additional biochemical profile, to confirm the presence of the pathogen, because of the potential for non-specific agglu-tination with other Enterobacteriaceae. Routine investigation may only include antisera against Sh. sonnei and Sh. flexneri unless there is a history of foreign travel to areas in which the other species are more commonly found.

Finally, the laboratory isolation of VTEC requires its differentiation from other strains of E. coli. Most E. coli O157 isolates do not ferment sorbitol, so the incorporation of sorbitol into a medium such as MacConkey agar (SMAC) is often used to iso-late VTEC. Suspect colonies are investigated sero-logically and biochemically to confirm the identity of the isolate. The toxigenic status of the isolate must be established before generation of the final diagnostic report.

The laboratory detection of bacterial enteric pathogens: can we stop culturing yet ?Laboratory detection of the bacterial enteric pathogens Campylobacter spp., Salmonella enterica, Shigella spp., and E. coli o157 is traditionally conducted using culture methods, combined with biochemical confirmatory tests and serology. This article discusses an alternative approach to diagnosis whereby gene targets specific for each pathogen are used to screen samples.

by Dr B. Lucey

Figure 1. EntericBio line-blot hybridisation results for a positive control (denoted by P), and a negative control (denoted by N).


disadvantages of conventional culture methodsThe challenges regarding tradi-tional laboratory isolation methods include the time to diagnosis. For the four pathogens outlined above, this is likely to take between 48 and 96 hours. Furthermore, given the normally heavy bacterial load in the large bowel, overgrowth of the pathogen by commensal flora is fre-quent, particularly where collection of a faecal sample has been delayed until the patient is recovering. When individual biochemical reactions are used to select for the pathogen under investigation, it is also possible that not every strain within the species or serotype shares the selected bio-chemical characteristic. For example, in the case of E. coli O157, there is a subset of strains that ferment sorbi-tol [6] and therefore strains will be missed using SMAC as a screening tool, as will strains other than sero-type O157. To compound these dif-ficulties, the relatedness of different genera within the family Enterobac-teriaceae may include sharing the same surface antigens; for exam-ple many E. coli O antigens cross-react serologically with shigella O antigens [3].

The investigation of faecal samples for bacterial enteric pathogens is relatively labour-intensive, requiring a range of culture media, serological approaches and biochemical tests. The best that the routine diagnostic laboratory can provide is the “best-fit” method, whereby most strains are detected.

the molecular diagnosis of bacterial enteric infectionAn alternative approach to the use of culture is to detect genes unique to the pathogens being investigated. Papers describing a number of such assays have been published includ-ing, for example, the detection of multiple pathotypes of E. coli [7] and the detection of S. enterica and Campylobacter spp. [8]. To date, only one commercial mul-tiplex system is available for the detection of the four pathogens. This is the EntericBio system (Serosep Ltd., Limerick, Ireland). It combines overnight broth enrichment with

PCR amplification and detection by hybridisation [Figure 1]; results can be available within 32 hours of receipt of a specimen in the labora-tory. A comparison of conventional culture methods with this system for the detection of the four pathogens yielded combined positivity rates of 5.4% and 7.6%, respectively, suggest-ing an increased sensitivity by the molecular method over culture [9]. Using an expanded gold standard to include alternative individual molec-ular assays to augment the sensitivity of culture, the calculated sensitivity, specificity, positive predictive value and negative predictive value for the EntericBio system were 100%, 99.3%, 91.5% and 100%, respectively. In addition, the system was less labour-intensive than culture methods. The already decreased turnaround time for this system over culture may, in the future, be decreased further by using real time PCR, allowing detec-tion in real time rather than requiring the visualisation of conventional PCR products through through techniques such as hybridisation.

One disadvantage of using such a molecular method is its increased cost over culture, at least when using a commercial system. It may be argued however, that the earlier detection of pathogens allows infection control measures to be implemented sooner, thereby limiting the spread of out-breaks. Alternatively, the non-detec-tion of these pathogens means that hospital isolation protocols may be reviewed for individual patients ear-lier, which is particularly significant in healthcare facilities having multi-bedded wards. A disadvantage of the EntericBio system is the specific detection of E. coli O157, rather than the detection of verotoxins associ-ated with pathogenicity. Given that there are multiple serotypes associ-ated with verotoxin production, it may be argued that the detection of genes associated with disease is more important than the micro-bial epidemiology of the infection, while additionally, missing the less common serotypes altogether.

Finally, allowing for the potential plasticity of bacterial genomes, it may be necessary when using molecular methods for diagnosis, to consult the international databases regularly for

evidence of changing genetics and updated taxonomy.

the continuing need for culture of bacterial enteric pathogensFor epidemiological purposes, the iden-tification of a salmonella strain to sero-var level is used universally, and the sub-typing of a serovar is often performed, using bacteriophage analysis or a variety of molecular-based typing methods after DNA extraction from culture. Shigella isolates are speciated and typed using antisera, and may be subtyped. A con-firmed E. coli O157 isolate is commonly phage typed. These characterisation tests require culture, as does most antimicro-bial susceptibility testing. For now, the most useful diagnostic approach appears to be the molecular detection of patho-gens, with retrospective culture-based analysis of positive results.

references1. Moore JE et al. Campylobacter.Vet Res 2005;

36: 351-382.2. Grimont PAD, Weill F-X. Antigenic for-

mulae of the salmonellae serovars (9th ed.) 2007; http://www.pasteur.fr/sante/clre/cad-recnr/salmoms/WKLM_2007.pdf.

3. Li Y et al. Molecular detection of all 34 dis-tinct O-antigen forms of Shigella. J Med Microbiol 2009: 58: 69-81.

4. Mora A et al. Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003. BMC Microbiol 2007; 7: 13.

5. Gould LH et al. Recommendations for diag-nosis of shiga toxin-producing Escherichia coli

infections by clinical laboratories. MMWR Recomm Rep 2009; 58 (RR12): 1-14.

6. Karch H, Bielaszewska M. Sorbitol-fer-menting Shiga toxin-producing Escherichia coli O157:H(-) strains: epidemiology, phe-notypic and molecular characteristics, and microbiological diagnosis. J Clin Microbiol 2001; 39; 2043-2049.

7. Aranda KR et al. Single multiplex assay to identify simultaneously enteropatho-genic, enteroaggregative, enterotoxigenic, enteroinvasive and Shiga-toxin-producing Escherichia coli strains in Brazilian children. FEMS Microbiol Letts 2007; 267: 145-150.

8. Schuurman T et al. Feasibility of a Molecular Screening Method for Detection of Salmo-nella enterica and Campylobacter jejuni in a Routine Community-Based Clinical Micro-biology Laboratory. J Clin Microbiol 2007; 45: 3692-3700.

9. O’Leary J, Corcoran D, Lucey B. Compari-son of the EntericBio multiplex PCR system with routine culture for detection of bac-terial enteric pathogens. J Clin Microbiol 2009; 47: 3449-3453.

the authorBrigid Lucey FAMLS, Ph.D.,Head of Molecular Diagnostics and ResearchDepartment of MicrobiologyCork University HospitalWilton Cork Republic of Ireland

DETECTION OF DERMATOPHYTES BY MULTIPLEX PCR

■ Identification of fungi causing nail infections

■ Higher sensitivity than conventional methods

■ Results within 5 hours

Statens Serum Institut, SSI DiagnosticaTel: +45 4829 9178E-mail: [email protected]


Comments on this article?

Feel free to post them at

www.cli-online.com/comment/enteric_pathogens

lab automation – issue N°1 – Feb/Mar 2010 26

In the last five years, the diagnostics service at the El Poniente Hospi-tal Public Trust (El Ejido, Almeria, Spain) has seen its workload increase by more than 200 percent. In spite of this, its productivity is now amongst the highest in Andalusia, with the laboratory handling approximately 5.4 million individual tests annually. By 2009, the laboratory’s productiv-ity and workflow had even overtaken that of many of Spain’s leading urban hospitals. This has been achieved by automating 90 percent of manual processes, delivering a consistent high-speed throughput and trans-forming the laboratory service into a fully integrated blood sciences diagnostic unit, which was officially opened a year ago.

For El Poniente, this was a pioneer-ing project, recognising that the most suitable systems were offered not by one, but two companies, and invit-ing them to work together to deliver a “best of breed” total automation solution. This led to a unique col-laboration between Beckman Coul-ter, Inc. – leaders in total labora-tory automation – and the former

Olympus Diagnostics, which has since been acquired and integrated into the global company [See text box].

The ever-expanding workload at the El Poniente laboratory in part reflects the economic prosperity of the region. Almeria is a rapidly expanding tourist area, with one of the fastest growing populations in Spain. In addition, El Poniente hos-pital itself has special responsibility within the modernised healthcare system being introduced by the regional government of Andalusia. Five years ago, the regional govern-ment began a multi-million Euro programme to transform medical services. This included the establish-ment of 22 ‘high resolution’ hospital centres. ‘High resolution’ is defined as a comprehensive, fast-track medical service in a centre of excellence.

El Poniente’s new role was to man-age a network of five of these high resolution hospital centres. The hospital laboratory was required to provide the network with a cen-tralised diagnostics service as part of this service. Being able to deliver

a fast and consistent throughput for high-volume workloads was an immediate requirement.

Complex testing demandsTwo of the five new hospital centres managed by El Poniente have already opened with the laboratory provid-ing an analytical results service to more than 240 different medical teams spread across the three hospi-tals and their catchment areas. The laboratory is able to carry out almost all of this activity in-house. Analysis of its laboratory’s 2009 activity for three of the hospital centres shows that it handled 1,300,000 sample tubes (approximately 5,400 daily)

with an average of 13 test requests per patient [Figures 1, 2].

These requests come from more than 800 different healthcare profession-als, working in the three accident and emergency departments, hospital inpa-tient and outpatient services, as well as the network’s widely dispersed GP and primary healthcare centres. These make significant demands on the lab’s workload, with test requests coming in from more than 100 different primary care sources. In addition, there are more than 60 extraction points within the hospital, and the laboratory also provides a decentralised anticoagulant service to all centres.

An innovative automation solution creates a flagship integrated blood sciences unitAn innovative automation solution is described that has enabled the El Poniente hospital lab in El Ejido, Almeria, Spain to fulfil its obligations to the region of Andalusia. The laboratory has established an efficient network, transforming the diagnostics service to patients.

by Dr C. Avivar

1

Five year increase in workloads, 2004 to 2009

0

1m

2m

3m

4m

5m

6m No: Parameters

200

9

200

8

200

7

200

6

200

5

200

4

Year

Microbiolog

y

Hematology

Biochemistr

y

Tota

l

Figure 2. Five year increase in workloads, 2004 to 2009.

Figure 1. Number of requests treated in 2009 by the El Poniente laboratory.

Unique collaboration

In 2009, Olympus Diagnostics’ clinical chemistry products were formally acquired by

Beckman Coulter and are now rebranded under that name. El Poniente laboratory

was one of a number of laboratories in Europe where the two companies had been

collaborating prior to the acquisition.

When Beckman Coulter acquired Olympus Diagnostics, it also acquired a manufacturing

base in Mishima, Japan, further extending its worldwide capabilities. It has also acquired

the previous company’s flagship European research and development centre in Munich,

Germany. Globally, this means the company has a new and expanded infrastructure —

with manufacturing and R&D capabilities as far afield as the US, Japan, Germany and

County Clare, Ireland.


By 2008, the increase in activity, together with the organisational demands of managing diagnostic services for the existing three hospi-tal centres, required the laboratory to carry out a fundamental review of its service delivery. The laboratory also wanted to plan how it would handle further pressure on its workloads when the remaining two hospitals were added to the network. It set two prime goals - and the solution had to fit both equally well. First, the lab knew that it had to auto-mate and streamline the majority of its manual processes to maintain a consistent workflow. Second, an analysis of the requirements of the medical professionals confirmed that the preferred solution would bring together the separate areas of clinical analysis, biochemistry, haematology and haemotherapy, microbiology and pathology into one, combined blood service facility. When assessing likely automation partners, key selection criteria pri-oritised the following: analyser speed, technology that delivered innovation and robustness, chemistry and immu-noassay reagent quality, plus a high-speed, high-throughput pre-analytic capability. In addition, the lab had to demonstrate an overall improvement in organisational efficiency that would facilitate future expansion. Improving sample management and laboratory processes without increasing running costs was also critical.

Winning staff cooperationAutomating the different laboratory processes and disciplines and consoli-dating them into one integrated unit can be particularly stressful for staff, requiring a more flexible working approach from everyone. Reducing the time spent on unproductive man-ual tasks has the potential to offer new challenges and an improved working environment. However, new skills must be acquired and staff have to be willing to adopt multitasking across several disciplines. The solution therefore had to involve investment in retraining, to ensure the staff could understand and adapt quickly to this new model for laboratory work. High-speed sample sortingFrom the moment samples enter the laboratory, technology auto-

mates their handling and sorting. This provides a fast and consistent response to test requests, regardless of fluctuating workload pressures or the differing requirements from hospital or external centres.

The high-speed AutoMate 2550, with aliquot capability, can handle 1,200 samples per hour. It provides the crucial first step in helping the lab successfully achieve its current per-formance levels. A second sorter, the AutoMate 1250, provides additional

support when needed. With aliquot capability, the 1250 can handle 800 samples an hour.

Pre-analytical processes such as decapping, preparation of selected aliquots and the sorting and dis-tributing of samples to the appro-priate work area or analyser, have been automated. Staff are no longer required to handle samples, which helps to eliminate potential hazards associated with manual handling.Once sorted, most of the samples

are sent to the fully automated core lab. This is where the Beck-man Coulter Power Processor sys-tem progresses the samples along a continuously moving track to the interconnected chemistry and immunoassay platforms.

PrepLink, the computer manage-ment software for track automa-tion, controls all functions —auto-matically sharing information with the laboratory information sys-tem (LIS). The software tracks the

HeadquartersPartec GmbH | Am Flugplatz 13 | D-02828 Görlitz | GermanyFon +49 3581 8746 0 | Fax +49 3581 8746 70 | [email protected]

Partec has subsidiaries and distributors covering 100 countries worldwide.Details can be found at the Partec website: www.partec.com

Let’s flowon May 8-12, 2010At the XXV ISAC Congress in Seattle Partec presents a new dimension in Flow Cytometry

www.partec.com/newdimension

Special Partec Congress TutorialDate: May 10, 2010 Time: 12:30 - 13:30

ww

w.c

yclo

s-de

sign

.de

PAR_CyFlowCLI_154x206_16-03-10_RZ.indd 1 17.03.2010 16:05:26 Uhr www.cli-online.com & search 24827

progress of the sample as it moves through the pre- and post-analytical stages, in real time.

High-volume analysers deliver workflow efficiencyThe Power Processor automates rou-tine, time-consuming manual tasks such as initial sample entry onto the system (via an inlet module), cen-trifugation and cap removal — pre-senting samples to the analysers, and then transferring them for the next processing step. Two high-speed centrifugation systems are linked to

the track, each with 40-tube capaci-ties, capable of spinning 450 tubes an hour.

The automated track connects two Beckman Coulter UniCel DxI 800 immunoassay analysers, able to run up to 400 tests an hour for high-speed chemiluminiscent analysis. Workflow is maintained because rea-gents can be added while the analys-ers are running. The DxI 800 has an extensive menu, including anaemia and cardiac tests, hormone profiles, tumour markers and an expanded

range of routine immunoassay tests that are required daily. The lab has specific high-volume clinical chemistry requirements. It needs an overall system with a mod-erate footprint that is still capable of delivering an hourly maximum throughput of approximately 8,000 tests when all the analysers are connected to the Power Processor. The AU5420 and AU2700 clinical chemistry analysers more than meet these requirements and link seam-lessly to the Power Processor. As a result of the acquisition of Olympus Diagnostics, these systems are now part of the Beckman Coulter family of products. An automated refrigeration unit is an efficient way of storing sam-ples for high-volume laboratories. As test requests are completed and tubes automatically recapped, the track directs them without manual intervention to Beckman Coulter’s 3,060-capacity refrigerated storage unit. Once stored, samples needed for add-ons, reruns and reflex test-ing are easily located by PrepLink. It automatically identifies the patient

via a bar code, and retrieves the sam-ple within seconds so that it can be directed to the appropriate analyser.

ConclusionThe innovative collaboration between Beckman Coulter and Olympus Diag-nostics has been instrumental in sup-porting the El Poniente laboratory in the fulfilment of its obligations to the region of Andalusia. It has ena-bled the laboratory to establish an efficient network, transforming the diagnostics service to patients as well as the professional teams caring for them. Alongside this high standard of technology, the teamwork of labo-ratory staff and the efficiency of the new organisational structure deliver a framework for further expansion of the lab’s new integrated blood sciences service.

the authorDr Cristóbal Avivar Director of the Integrated Biotechnology Management Area El Poniente Hospital El Ejido, Almeria Spain www.cli-online.com & search 25024

28 lab automation –issue N°1 – Feb/Mar 2010

What automation delivers - Reduced turnaround time (TAT)

- Elimination of most manual tasks

- Minimisation of sample handling errors

- Simplification of processes

- Consolidation of parameters

- Improved quality and safety

- Ability to locate samples faster & efficiently

- Increased analytical productivity and coordination of work

- Enhanced working practices and staff job satisfaction

We’re better together.

B l o o d B a n k i n g C e n t r i f u g a t i o n C h e m i s t r y F l o w C y t o m e t r y H e m a t o l o g y H e m o s t a s i s I m m u n o a s s a y I n f o r m a t i o n S y s t e m s L a b A u t o m a t i o n M o l e c u l a r D i a g n o s t i c s R a p i d D i a g n o s t i c s

More solutions for your lab than ever before.Beckman Coulter now offers an unprecedented product portfolio for your laboratory, including the broadest line ofchemistry analyzers and automation systems in the world.

Focused solely on the laboratory, we deliver complete diagnostics solutions to meet your evolving needs. You candepend on us to help you achieve faster, more reliable results in every testing discipline. Now we are even bettertogether with you – our laboratory partners.

For more information, contact your Beckman Coulter sales representative or visit us on the web.

www.beckmancoulter.com/tls

© Copyright 2010 Beckman Coulter, Inc. Beckman Coulter and the stylized logo are registered trademarks of Beckman Coulter, Inc.

7333_TotalSolutionsAd_11_CLI.qxd:Layout 1 3/15/10 8:53 AM Page 1


PCr kit for detection of dermatophytes and Trichophyton rubrum

Nail infections are caused by dermatophytes, mainly by Trichophyton rubrum and Trichophyton menta-grophytes. As onychomycosis requires long-term sys-temic antifungal treatment, the correct identification of causal fungi is mandatory. Traditionally the time required for species identification by culture may vary from two to four weeks. The Multiplex PCR based in vitro diagnostic method from Staten Serum Institut can detect dermatophytes in general, and T. rubrum specifically, within five hours. A study has shown that this method increases the detection of pan-dermatophyte by 4.3% and T. rubrum by 18.6% compared to conventional methods. In-troduction of the PCR-based methodology will as a result increase specificity, simplicity and speed of diagnosis, and potentially even reduce cost. The multiplex PCR kit is CE marked and certified. Staten Serum InStItuteHillerod, Denmark www.cli-online.com & search 25031

Antimicrobial products adapted to EUCAst

The recently launched Oxoid range of products for antimicrobial susceptibility testing (AST) includes all products recommended by EUCAST (the European Committee on Antimicrobial Sus-ceptibility Testing) for use in the new, harmo-nised European disc diffusion method. Other products in this range include Mueller-Hinton Agar in dehydrated form, or a variety of pre-pared media formats (plain or with defibrinated horse blood and NAD). In addition, there are ergonomically-designed disc dispensers for the accurate placement of discs onto a variety of agar plate sizes (90, 120 and 140mm), the Turbidom-eter for accurate preparation of 0.5 McFarland inocula. A wide range of QC organisms is avail-able, with all EUCAST-recommended strains, in

convenient Culti-Loop format for quick, easy and safe preparation of standardised cultures.

OxOIDBasingstoke, Hants, uK www.cli-online.com & search 24993

Monoclonal antibodies against West Nile Virus (WNV)

A member of the flavivirus group, the West Nile Virus infects humans as well as animals, including birds and horses. Most human infections are asymp-tomatic, but some can result in serious neuroinva-sive complications such as encephalitis. ViroStat has released five new monoclonal antibodies to WNV, all specific for the viral envelope glycoprotein (E). They function in ELISA and IFA, and cross reactivities to other flaviviruses can be found on the data sheet that can be downloaded from the company website.

VIrOStat IncPortland, me, uSa www.cli-online.com & search 24986

immunoblots for confirmation of borreliosis

Two novel immunoblots for the second, confirmatory step in the serological diagnosis of Lyme disease combine the advantages of Western blots (higher sensitivity) and line blots (easier evaluation). Sera identified by the initial screen-ing step can be confirmed with ease and efficiency. The Anti-Borrelia EUROLINE-WB con-sists of ready-prepared Western blot strips that are additionally fitted with membrane chips containing purified native or recombinant antigens. Thus, the complete antigen spectrum

of the Borrelia cell extract is available together with important additional antigens such as VlsE, which is not produced in sufficient quantities in cultured cells. In the Anti-Borrelia EUROLINE-RN-AT, recom-binant antigens are placed side by side with antigens purified from Western blot as a line blot for simpler evaluation. All antigens have been carefully selected for their diagnostic value, and each individual antigen preparation is optimised to yield maximum sensitiv-ity and specificity. For example, antigens such as p83,

p39 and OspC are most specific in their native form, while other antigens such as VlsE can only be pro-duced efficiently in recombinant form. Many of the recombinant antigens used have been enhanced by molecular manipulation to increase specificity. Fur-thermore, innovative antigens, such as the company’s immunoreactive membrane lipids, are present in the test system, increasing both sensitivity and specificity. The entire immunoblot procedures from incubation to evaluation and archiving of results can be automated. The EUROBlotMaster provides standardised process-ing of immunoblots, allowing higher precision and better reproducibility. Incubated strips are digitalised using a special scanner or camera module, and sub-sequently evaluated and archived using the program EUROLineScan. These two immunoblot products are part of a complete package for borreliosis diag-nostics, which includes an Anti-Borrelia-plus-VlsE ELISA, indirect immunofluorescence tests, ELISA for CSF diagnostics, avidity tests and comprehensive automation concepts.

eurOImmun aG Luebeck, Germany www.cli-online.com & search 25029

ElisA-based tests for detection of gastrointestinal protozoan parasites

Unlike the traditional laboratory detec-tion of gastrointes-tinal parasites that rely on microscopic examination of stool samples and water concentrates, the innovative concept of Savyon Diagnos-tics uses a panel of ELISA-based tests for detection of com-

mon gastrointestinal protozoan parasites. The panel includes the following five organisms: Blastocystis, Giardia lamblia, Cryptosporidium spp., Entamoeba histolytica and Dientamoeba fragilis. This new all-in-one well platform allows the simultaneous detection for screening purposes of all five parasites; the dis-tinctive tests can be used for confirmatory differen-tial diagnosis as well as stand-alone tests. The panel, thus, provides the means for accurate, rapid, efficient and economical diagnostic workup.

SaVyOn DIaGnOStIcashdod, Israel www.cli-online.com & search 24997

Adenovirus stool antigen microwell ElisA kitUnlike the traditional electron microscopy com-monly used to detect viruses associated with gas-troenteritis, the DAI Adenovirus Antigen Detection

ProdUCt HiGHliGHt MiCroBioloGy tEsts – issue N°1 – Feb/Mar 2010 30

ELISA is a sensitive, specific and rapid in vitro diagnos-tic method for the qualitative deter-mination of enteric adenoviruses. The kit only requires

1 gram of stool sample and has 100% specificity and 95.5% sensitivity; the total time for the test is approximately 100 min. It is a double antibody (sandwich) ELISA using a polyclonal anti-adenovi-rus antibody to capture the antigen from the stool supernatant. A second anti-adenovirus monoclonal antibody is then added, which binds to the complex and “sandwiches” the antigen. The third incubation attaches horseradish peroxidase to the sandwich and after washing to remove unbound enzyme, the reaction is visualised by the addition of anti-mouse antibodies conjugated to peroxidase. The resulting blue colour, following the addition of the chromogen and peroxide, indicates the presence of adenovirus antigens. The use of a positive and negative control allows easy validation of the kit stability. The kit has a shelf life of 12 months.

DIaGnOStIc autOmatIOn, Inc. calabasas, ca, uSa www.cli-online.com & search 24990

Whole blood neonatal bilirubin testing

Siemens RAPIDLab 1200 series blood gas analysers offer neonatal bilirubin (nBili) testing that detects elevated lev-els of bilirubin with a 60 second turna-

round time. If undetected, elevated bilirubin can lead to a variety of health issues in newborn infants, from jaundice to neurological disorders and, in severe cases, brain damage. The nBili test provides fast, accurate results without increasing monthly operat-ing costs. The test requires only a 100µL sample of whole blood, has a measuring range of 2 – 30 mg/dL, and can be conducted as part of a neonatal test panel that includes blood gas, pH, electrolytes, metabolites, total hemoglobin and CO-oximetry. No additional reagents or sample preparation are needed.

SIemenS meDIcaL SOLutIOnS DIaGnOStIcSDeerfield, IL, uSa www.cli-online.com & search 25032

surfactant Protein A ElisASurfactant associated protein-A (SP-A) belongs to the family of innate host defence proteins termed collectins, of which SP-A is the most abundant

pulmonary sur-factant protein. SP-A is an impor-tant component of host defence against respiratory patho-gens and allergens. Besides pulmonary

host defence, SP-A is also involved in the formation of pulmonary surfactant, as it is essential for the structure of tubular myelin. An increased serum SP-A level has been recently reported as a strong and independent predictor of early mortality among patients with idiopathic pulmonary fibrosis. Reduced levels of SP-A in alveolar fluid have been reported in other pulmonary diseases, e.g. acute respiratory distress syndrome. Elevated levels of SP-A in the lungs have been observed in pulmo-nary alveolar proteinosis. BioVendor’s ELISA kit has enhanced the company’s pulmonary infection or inflammation diagnostic test portfolio. The assay has been validated to measure SP-A in serum, cit-rate and heparin plasma, bronchoalveolar lavage and amniotic fluid. The 96 well-microtitre plate format allows use on standard ELISA equipment or robotic systems.

BIOVenDOrmodrice, czech republic www.cli-online.com & search 25046

Monoclonal antibody to human thrombomodulin Cd141 for flow cytometryA monoclonal antibody is available for the detec-tion of human thrombomodulin, a surface receptor for thrombin present on endothelial cells. Thrombin bound to thrombomodulin activates the antico-agulant, activated protein C. Thrombomodulin is thought to play a protective role in many carci-noma types, with elevated expression correlating with lower metastasis and a positive prognosis. The anti-CD141 mAb is a mouse monoclonal for use in immunohistochemistry, immunoblotting and FACS applications.

Qna InternatIOnaL Pty LtDmelbourne, australia www.cli-online.com & search 25030

iVF centrifuge and cryopreservation tubes

The success of in vitro fer-tilisation (IVF) depends to a great extent on the qual-ity and reliability of all material used in the pro-cedure. Nunc IVF centri-fuge and cryopreservation tubes are MEA (mouse embryo assay) and HSSA

(human sperm motility assay) tested and certi-fied. Compliance with these standards maintains cell viability before and after long-term storage. Made of polystyrene, the IVF centrifuge tubes are HSSA tested so as not to impact on sperm motil-ity. Furthermore, the cryopreservation tubes are produced in accordance with a proven design for the safe cryogenic storage of sperm, eggs, embryos or tissue samples. These consumables are certified free of toxins that would impair viability during the centrifugation and cryogenic freezing proc-esses. Furthermore, the entire range is CE marked as medical devices for IVF and thoroughly tested against the highest available standards to ensure safety and reproducibility for in vitro fertilisation (IVF) procedures.

tHermO FISHer ScIentIFIcWinchester, Va, uSa www.cli-online.com & search 24999

– issue N°1 – Feb/Mar 201031ProdUCt NEWs

BioVendor‘s Range of Pulmonary Disorder BiomarkersARDS, Asthma, Cancer, COPD, Interstitial Pulmonary Fibrosis, Lung Inflammation and Infection, Sarcoidosis...

Surfactant Protein A ELISA KL-6 ELISA

Surfactant Protein D ELISA Clara Cell Protein ELISA

• serum, plasma, BALF, amniotic fluid • superior assay validation• simple, robust, user-friendly• excellent technical support

BioVendor – Laboratorni Medicina a.s.Evropska 873, 664 42 Modrice, Czech RepublicPhone: +420–549 124 185, Fax: +420–549 211 460, E-mail: [email protected], www.biovendor.com

used & recognized

new & unique


reagents for total and direct bilirubin measurement

The two Synermed near-infrared clinical chemistry reagents for total and direct bilirubin are based upon the conversion of bilirubin to cholecyanin, which can be detected at 700 nm in the near-infrared region. Both reagents are ready-to-use mono-reagent liquids which are stable for up to two years at room temper-ature. because opf their use of near-infrared detec-tion, the Synermed methods are substantially free from interference from even high levels of haemo-lysis and show minimal interference from lipaemia. The direct bilirubin method is carried out as a fixed-time kinetic reaction at 700 nm and is highly specific for conjugated bilirubin even in the presence of high levels of unconjugated bilirubin. The total bilirubin uses end-point detection at 700nm and may use a bichromatic correction.

QBcBolton, Lancs, uK www.cli-online.com & search 24996

Urinalysis test strip with infra-red id bands

In order to improve the workflow and clinical infor-mation for busy clinics and lab-oratories, Sie-mens urinalysis test strips with infra-red iden-tification (ID)

bands offer important automatic checks when used on the CLINITEK Status analysers with software version 2.0 or more. The new technol-ogy enables automated identification of the strip type, removing the need to enter the information manually. Detection over-exposure to humidity minimises false positive results. Common sample interferences are assessed and if any are present the clinicians are alerted and notes provided for results that may be affected.

SIemenS meDIcaL SOLutIOnS DIaGnOStIcStarrytown, nJ, uSa www.cli-online.com & search 25026

oligonucleotide arrays for detecting disease and syndrome-related aberrationsIn addition to offering whole genome coverage, the new 8 x 60k, 4 x 180k and 4 x 44k CytoSure ISCA formats also provide powerful arrays focusing on disease and syndrome-associated genome regions. Using a proprietary 60-mer probe design and mul-tiple rounds of optimisation, aCGH arrays ensure exceptional reliability and confident detection of genetic aberrations with high signal-to-noise ratios. Each array is accompanied with user-centric Inter-pret analysis software, a powerful and easy-to-use tool that ensures effortless translation of data into meaningful results. Intuitive features include the “Accelerate” workflow, for customisable automa-tion of routine data analysis, and full integration with existing data and publicly available cytogenet-ics databases including the Database of Genomic Variants (DGV, Canada) and DECIPHER (Sanger Institute, UK). These features have ensured that the arrays are already used at a number of key facilities where they were evaluated as part of an ongoing multi-platform aCGH comparison.

OxFOrD Gene tecHnOLOGyOxford, uK www.cli-online.com & search 24984

large particle flow cytometersSamples that are too large or too fragile for traditional flow cytometers can now be analysed, sorted and dis-pensed, while maintaining their viability, through the new BioSorter platform. Utilising the patented soft-touch air-jet diverter technology, a variety of samples are analysed one by one in a continuously flowing stream and can be sorted based on size, optical den-sity and up to four fluorescent parameters. Typical samples include delicate large cells/cell clusters (adi-pocytes, embryoid bodies, pancreatic islets, duct cells, hepatocytes) and beads (cells growing in or on beads, combi-chem libraries, bead based assays).

unIOn BIOmetrIca IncHolliston, ma, uSa www.cli-online.com & search 25027

live cell imaging and analysis system

The innovative Cell-IQ2 platform builds on the suc-cess of the original Cell-IQ system, offering fully automated imaging and analysis for the study of live cell behaviour in an optimised and stable envi-ronment. This second generation system provides increased usability and precision for users, offer-ing advanced features for cell biology, functional genomics, drug discovery, in vitro fertilisation, in vitro toxicology and regenerative medicine applica-tions. The system’s intuitive user interface has also been updated, with the addition of the new Analyser Pro-Write software for greater functionality. The sys-tem allows accurate and reproducible quantitation of results for live cell imaging.

cHIP-man tecHnOLOGIeS LtDPangbourne, Berks, uK www.cli-online.com & search 24988

Automated and simplified image capture software

Bio-Rad’s new tool for Molecular Imager Gel Doc XR+ or ChemiDoc XRS+ systems provides an easy-to-use, fully automated image capture and analy-sis system that delivers high quality images. Image Lab sofware automatically calibrates these systems at initial set up such that when a sample is imaged for a given application, the picture is always in focus — at any sample height, at any zoom level and in any user’s hands. An important aspect to optimis-ing images, allowing for reliable quantification, is a proprietary process called “flat fielding” that adjusts images for optical and illumination artifacts result-ing from lens curvature and minor lighting vari-ances. Once flat fielding is completed at initial set up, it can be consistently applied to every image capture for improved data quality and reproducibility. Flat fielding improves image uniformity by 45 percent for chemiluminescent applications and 95 percent for experiments using fluorescence.

ProdUCt NEWs – issue N°1 – Feb/Mar 2010 32

Users can set up imaging protocols with the click of a mouse and the system will store the entire workflow, from image capture to result, in a pro-tocol file, removing the need to reset the imager each time a new gel or blot is loaded. Once saved, the protocols can be edited, re-saved, re-applied and exchanged between users. This enables identical or similar experiments to be performed by different users and repeated accurately and reproducibly. Custom data reports are also created as well as images that can be easily exported to various programs includ-ing Adobe Acrobat, and Microsoft PowerPoint, Excel and Word. Users can opt to manually analyse data when needed. The software is com-patible with all current Mac and PC operating systems

BIO-raDHercules, ca, uSa www.cli-online.com & search 24987

Cytokine multiplex ElisA arrays

Allowing researchers a better under-standing of the simultaneous relation-ship between multiple cytokines, new high-throughput interferon detec-tion kits combine various cytokine antibodies within multiplex arrays. Examination of these relationships reveals key information that can help predict clinical outcomes based on several cytokine and growth factor biomarkers. These include the assess-ment of cytokine profiles at various points throughout the course of dis-ease, correlation of cytokine profile alterations with patient responses to therapies and determination of drugs’ off-target effects. The Q-Plex quan-titative ELISA-based tests include six human and four mouse cytokine arrays and contain up to 16 distinct capture antibodies in each well of a 96-well plate. Each spot is well defined and represents a distinct antibody population. Up to 84 different samples of less than 30 µL can be assayed for all 16 unique cytokines in less than 2.5

hours. Cytokines can be detected with a lower limit of detection (LLOD) from 30 pg/mL to less than 1 pg/mL. Assay variability is low — about 8% intra-assay and 13% in inter-assay. The kit is compatible with serum, plasma, cell culture supernatant, homogenates, lysates, nasal lavage, tears and urine.

PBL InterFerOnSOurce Piscataway, nJ, uSa www.cli-online.com & search 24998

A virtual slide system for multiple applications

Olympus has introduced a new improved version of its highly suc-cessful dotSlide virtual slide system. The new VS110 combines microscopy seamlessly with imaging to create a virtual slide that is an exact copy of the real specimen. Generating a high reso-lution image of the whole specimen, this can be viewed and analysed from the overview image at low magnifica-tion up to maximum magnification by simply zooming in. All samples can be instantly and simultaneously evaluated around the world, as they are stored electronically on a central server. This is ideal for use in a number of areas including pathology, research and education. In addition, the Net Image Server SQL together with the VS110 platform is a real client-server-based data management system and storage facility and helps to keep information clearly structured for easy manage-ment and efficient sharing of images and data.

OLymPuS DIaGnOStIca GmBHHamburg, Germany www.cli-online.com & search 24985

Cancer biomarker discovery platform with 58 additional new sequences An updated version of Febit’s Gen-iom Biochip, containing 58 newly discovered sequences in addition to

all of the human miRNAs from miR-Base version 14 (www.mirbase.org) is now available for cancer research. The new sequences were found by deep sequencing in a miRNA discovery study performed on an Applied Bio-systems SOLiD 3 sequencing system. All 58 miRNAs have been validated using the ABI TaqMan miRNA qRT-PCR assay and are now available for further studies using Febit’s microar-ray technology for miRNA-profiling.Several studies investigating the role of miRNAs have shown evidence for their influence in cell develop-ment processes. Recently, promising biomarkers for the future diagnosis and differentiation of lung cancer and multiple sclerosis have been found. The results of studies demonstrated

that miRNA biomarkers in blood reliably distinguish between affected and healthy individuals. Each miRNA of the Geniom Biochip, from either miRBase 14 or the SOLID sequenc-ing-studies, can thus be a biomarker candidate or a therapeutically rel-evant molecule.

FeBItHeidelberg, Germany www.cli-online.com & search 25033

– issue N°1 – Feb/Mar 201033ProdUCt NEWs

Human Sclerostin can now be measured with a conventional 96-well format ELISA, containing 6 ready to use standards and 1 control in only 20ul of serum or plasma. Areas of interest are cancer induced bone diseases, osteoporosis and therapy monitoring of anabolic treatment.

More information on the Sclerostin ELISA is available onwww.bmgrp.com/or contact Biomedica’s customer service by e-mail: [email protected]: +43/1/29107-45fax: +43/1/29107-6389

SCLEROSTIN A NEW REGULATORY MARKER OF BONE TURNOVER

Canonical Wnt-signaling plays an important role in the regulation of bone homeostasis by promoting the development of osteoblasts. Negative regulators of the Wnt pathway are important new therapeutic targets for the treatment of diseases with enhanced bone resorption. One of these molecules is Sclerostin, the product of the SOST gene, a 22.5 kD secreted glycoprotein. It antagonizes the canonical Wnt signaling by binding to LRP 5/6 receptors, thus inhibiting bone formation. Sclerostin is nearly exclusively produced in osteocytes and is therefore considered to be a clinical marker providing high bone specifi city.

Modifi ed from: Piters E. et al., Arch Biochem Biophys. 2008;473(2):112-116

Extracellular

Cytoplasm

NucleusTCF

WntLRP5/6

SclerostinZF

No Bone Formation

β-catenindegradation

ad jennifer.indd 1 22/03/10 14:04:48 www.cli-online.com & search 25043

Binding site sponsors 6th serum Free light Chain symposium

The 6th International Symposium on the Clinical Applications of Serum Free Light Chain analysis (and Hevylite) will be held in the historic city of Bath, UK on September 23-24, 2010, hosted by Professor A.R Bradwell, Secretariat Chairman, Department of Immunology & Immunity, University of Birming-ham, UK and sponsored by Binding Site.Symposium sessions will include measurements in monoclonal gammopathies, myeloma kidney and inflammatory diseases. Data from the multi-centre, Phase III, randomised control trial on extended haemodialysis with the Gambro protein-leaking HCO 1100 dialyser will be shown and there will be presentations on Hevylite (intact immunoglobulin kappa/lambda ratios) in a variety of clinical situations. The programme will focus on results from a number of recent studies, with a distinguished panel of speak-ers including Robert Kyle, Brian Durie, Herve Avet-Loiseau, Angela Dispenzieri, Ola Landgren, Philip Hawkins and Heinz Ludwig. Delegates can claim 10 CPD points from the Royal College of Pathologists.Visitors to the conference website can see the prelimi-nary programme, register to attend, book accommo-dation and, if interested, submit an abstract. www.bath2010.com & www.bindingsite.co.uk

Company wins two strategic new lis clientsSunquest, a leading provider of healthcare diagnos-tic information technology and outreach solutions, has recently won contracts for two new clients in the mission-critical and competitive laboratory information market space. Both health systems will implement the Sunquest diagnostic IT software solu-tions suite to optimise clinical effectiveness, improve operational capabilities, and enhance patient safety for their laboratory and health systems.According to Richard Atkin, Sunquest President and CEO, a major new operation in Dallas, Texas, USA has signed a business agreement to implement the Sunquest laboratory information management, molecular diagnostics, and anatomic pathology solutions. Reliability and scalability of the Sunquest solutions were paramount in the decision process, as was certainty of implementation and the quality of support. Additionally, a prestigious New York City-area healthcare delivery network has selected Sun-quest to replace their existing LIS software vendor in all of their facilities in order to improve the cost effectiveness of the laboratory operations, positively impact the quality of care and enhance the safety of the patients they serve.According to Kathryn Jehle, Vice President of Finance and CFO for Sunquest Information Sys-tems, the company recognises the significant finan-cial commitment these organisations are mak-ing to implement new laboratory software and is pleased that two new customers have expressed confidence in its solutions in order to build a long-term relationship.www.sunquestinfo.com

iNdUstry NEWs – issue N°1 – Feb/Mar 2010 34

Book rEViEWClinical Applications of Mass spectrometry Methods and ProtocolsEdited by Uttam Garg and Catherine A. Hammett-Stabler Published by Humana Press 2009, 540pp, £81

As mass spectrometric methods now offer a level of specificity and sensitivity unrealised by spectrophoto-metric- and immunoassay-based methods, mass spec-trometry has entered the clinical laboratory where the technique is now being

used for a wide range of applications. In Clinical Applications of Mass Spectrometry: Methods and Protocols (Volume 603 in the Methods in Molecular Biology series), expert researchers provide detailed step-by-step procedures for the analysis of a number of analytes of clinical importance. This versatile and expansive volume covers mass spectrometry methods for analytes

including a variety of drugs, hormones and met-abolic compounds spanning the disciplines of toxicology, therapeutic drug monitoring, endo-crinology and paediatric metabolism. Written in the highly successful series format, chapters include brief introductions to the analytes, lists of the necessary materials and reagents, readily reproducible analytical protocols and detailed notes on troubleshooting and avoiding known pitfalls. Comprehensive and dependable, this book offers biochemists, laboratory scientists, pharmacologists, toxicologists and endocrinol-ogists a wide array of valuable methods for both experienced mass spectrometric labs that are looking to introduce new analyses and for those laboratories currently considering the addition of this resourceful and vital technology.

Humana PreSS, an ImPrInt OF SPrInGer neW yOrK Secaucus, nJ, uSa www.cli-online.com & search 25035

April 10-13, 2010ECCMID 2010 – 20th Euro-pean Congress of Clinical Microbiology & Infectious DiseaseVienna, Austriatel. +41 61 686 77 11 Fax +41 61 686 77 88 e-mail: [email protected]/ecc-mid2010/

April 18-21, 201063rd CMEF Spring 2010shenzen, Chinatel. +86 10 6202 8899 ext 3825Fax +86 20 6235 9314e-mail: [email protected]://en.cmef.com.cn/

May 9-13, 2010Focus 2010 Glasgow, Uk tel. +44 141 434 1500Fax +44 141 434 1519 e-mail: [email protected]

May 10-12, 2010ISLH 2010 - XXIII Inter-national Symposium on Technological Innovations in Laboratory HematologyBrighton, Ukwww.islh.org/islH_2010

May 13-15, 2010The European Congress on Rare Diseaseskrakow, Polandtel. +33 1 5653 5263 Fax +33 1 5653 5215e-mail: [email protected]

May 25-28, 2010Hospitalar 2010sao Paolo, Braziltel. +55 11 3897 6199Fax +55 11 3897 6191e-mail: [email protected]/ingles/

June 5-9, 2010EAACI 2010 – 29th Congress of the European Academy of Allergy and Clinical Immunologylondon, Uktel. +46 8 4596600Fax +46 8 6619125e-mail: [email protected]

June 10-13, 201015th Congress of the European Hematology AssociationBarcelona, spaintel. +31 70 3455563Fax +31 70 3923663e-mail: [email protected]

June 15-16, 2010PerMediCon - Personalized Medicine ConventionCologne, Germanytel. +49 221 821 3427www.permedicon.com

July 18-23, 2010International AIDS Confer-ence (AIDS 2010)Vienna, Austriawww.aids2010.org

July 25-29, 20102010 AACC Annual MeetingAnaheim, CA, UsAwww.aacc.org/events/2010am/

September 15-17, 2010Medical Fair Asia 2010suntec singaporetel: + 65 6332 9620Fax: +65 6332 9655 / 6337 4633e-mail: [email protected]

October 13-16, 2010Laboratory Medicine in Health Care – First European Joint Congress of EFCC and UEMSlisbon, Portugaltel. +351 217 583 325Fax +351 217 583 324 e-mail: [email protected]

November 3-5, 2010Journèes Internationales de Biologie (JIB)Paris, France www.jib-sdbio.fr

November 17-20, 2010MEDICAdüsseldorf, Germanye-mail: [email protected]

CAlENdAr oF EVENts

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

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

How far would you go to protect your samples?

Researchers worldwide protect more than two billion samples inside

Thermo Scientific cold storage equipment. With +4°C refrigerators

to -196°C cryogenic freezers and Thermo Scientific Nalgene and Nunc

consumables, you’re free to concentrate on your work without worrying

about your valuable samples.

Thermo Scientific Revco high-performance laboratory refrigerators and

freezers are engineered for the laboratory, providing:

• Security – Our microprocessor-based control system manages setpoint and actual conditions, providing the highest level of security

• Efficiency – Advanced defrost sensors minimize frost built-up, maintaining coil efficiency

• Versatility – A variety of sizes from undercounter to upright models is available to meet your space requirements

So relax – and choose Thermo Scientific Revco laboratory

refrigerators and freezers. Visit www.thermo.com/cold

© 2

010

Ther

mo

Fish

er S

cien

tific

Inc.

All

right

s re

serv

ed. A

ll tra

dem

arks

are

the

prop

erty

of T

herm

o Fi

sher

Sci

entif

ic In

c. a

nd it

s su

bsid

iarie

s.

Moving science forward

Thermo Scientific Revco refrigerators and freezers are available for general and specialized applications includingpharmacy, blood banking and chromatography.

RevcoFrigFreez_CLI_0310:Thermo 2/17/10 4:41 PM Page 1


First we un derstand. Then we innovate.

Imagine ... innovating the science of histopathology

Dedicated to Histopathology

Sakura Finetek, again, improves the laboratory. By offering products to automate manual procedures and smoothen the workflow, the histotechnologists can easily complete the other activities required and eliminate potential risks. As the innovative company in histopathology, Sakura Finetek is continuously looking for possibilities to improve the laboratory... and succeeds in offering solutions for the problems found in the histopathology laboratory.

Sakura Finetek offers you the only concept to achieve:• An efficient, manageable, continuous workflow• Same day results by real reduction of turn-around time• Higher productivity resulting in a higher morale• Consistent high quality; sample by sample• Improved health and safety standards in your laboratory

Sakura Finetek Europe B.V.The Netherlands

Phone: +31 71 589 83 00Fax: +31 71 589 84 88

[email protected]

08_009_SFE_5500_add-xSeriesA&P_D1 1 04-11-2008 16:15:29


+31 (0)88 592 00 01Tel: +31 (0)88 592 00 00

weekly news updates on neonatal ... · • high capacity, up to 660 slides simultaneously •...

Documents