Protein profiling of arterial thrombosis in acute MI Pg.12

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Protein profiling of arterial
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Weekly news updates on | October 2009 | Volume 33 | Issue 5
Protein profiling of arterial
thrombosis in acute MI
Turbidimetric NGAL test for
automated systems
The fine details of the molecular and cellular mechanisms underlying plaque rupture and thrombus formation in acute myocardial infarction are not yet fully
elucidated. Profiling of cellular and soluble proteins from
the arterial thrombus site may identify local effectors
that amplify the vascular occlusion process illustrated on
the front cover.
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Also in this issue :
Reagent for measuring
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chemistry analysers
α thalassaemia
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NMR in clinical
microbiology Pg 16
Markers of bone
remodelling Pg 26
Now availa
Rapid and accurate a-thalassaemia screening
using quantitative real-time PCR
[12-14] Protein profiling of arterial thrombosis
in acute myocardial infarction
[16-18] Applications of magnetic resonance
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[24] ase study: blood film review
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Editor’s letter
– Issue N°5 – October 2009
A society for all ages?
The first of October marked
the 10th anniversary of the
UN’s International Day
of Older Persons, and UN
Secretary-General Ban Kimoon’s message emphasised the need for persons
over 60 (currently twenty
percent of Europeans fall into this category) to be ‘both agents and beneficiaries
of development’. On the same day a robust study led by danish professor Kaare
Christensen was published in The Lancet, which concluded that if the increase
in life expectancy in developed countries
over the last two centuries is sustained in
this century, over half of the babies born
since the year 2000 will reach the age of
a hundred.
The ageing of the Western population has
a serious impact on the old-age dependency ratio (the number of retired people
divided by the number of working age
people). This ratio is expected to double
in forty years, greatly adding to the burden of the working population if action
is not taken. A frequently suggested but
facile solution to this complex problem
is to raise the legal age of retirement from
work. However, given that the average age
of European men’s exit from the workforce ranges from 58.6 years in Belgium
to 64.2 in the UK (in women it ranges
from 58.2 in Austria to 62.9 in Romania)
and that the official retirement age is 65
in most European countries, it must first
be ensured by healthcare providers, employers and indeed society as a whole that
the older workforce is healthy enough to
continue in employment, at least until the
legal retirement age. The most important
condition for older employees to work
longer (and be agents of development)
is good health.
Because the capacity for bearing weights
decreases as workers’ age increases, musculoskeletal disorders due to carrying
hefty loads or using heavy tools are the
most frequent causes of work-related disability in older workers. Older persons are
also more prone to work-related upper
limb problems caused by awkward posture, repetitive movements and too few
rest periods. In addition, work-related
stress, predominantly caused by the demands being made exceeding the worker’s
ability to cope without excessive working
hours, is more frequent in older workers. Perceptive employers can do much to
alleviate these problems, but commuting
older workers face an additional hazard:
they are less able to balance when standing in crowded commuter trains and
buses. Today’s older workers gave up their
seats for elderly users of public transport
when they themselves were young; if today’s young people cannot be persuaded
to emulate them, the provision of special
seats for older commuters is necessary.
People in the West are not only living
longer, but have fewer disabilities and
functional limitations than in the past,
largely because they are the beneficiaries
of development via healthcare systems offering effective diagnosis and treatment.
However, if health services are not to be
overwhelmed by an ageing population, it
is vital that both workers and those who
have retired from the workforce know
how to safeguard their own health. An
active life-style with regular exercise, a
healthy diet, no smoking, limited use of
alcohol and maintaining an appropriate
blood pressure will hopefully allow many
of today’s infants to celebrate a happy and
healthy 100th birthday.
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Disea se foc us
– Issue N°5 – October 2009
Rapid and accurate a-thalassaemia-screening
using quantitative real-time PCR technology
Alpha-thalassaemia, a common genetic disorder leading in its most moderate form to a hypochromic microcytic
anaemia, is mainly the result of deletions on chromosome 16. Current testing for a-thalassaemia is based on an
algorithm of exclusion-testing, which necessitates a wide range of dianostic procedures. Such procedures are labourintensive, time-consuming and not 100% specific. As a result, there is a need for a general, rapid and efficient screening
method. This article discusses the use of real-time quantitative PCR detection of deletions as one possible solution.
by Dr S. Brunner-Agten and Dr A. R. Huber
a-thalassaemia is a common genetic disorder,
which leads in its most moderate form to an
asymptotic anaemia with persistent hypochromia microcytosis. The clinical outcome of
more severe cases leads to very severe transfusion-dependent anaemia or hydrops fetalis.
The condition is mainly the result of deletions on chromosome 16, which contains, at
its telomeric region, two highly homologous
and closely linked genes (al- and a2-gene)
encoding the a globin chains. During meiosis, misalignment of chromosome homologues followed by reciprocal recombination
at three highly homologous segments (named
X, Y, and Z and separated by non-homologous segments [Figure 1]), results in various
deletion-duplication events.
Figure 2. Selected genotypes and corresponding ratio patterns.
The outcomes of the genetic disorder are
diverse and the severity is correlated with the
number of affected a globin loci and the exact
nature of the gene deletion [Figure 2].
is the result of the deletion or dysfunction of
three of the four a-globin alleles (--/-a). It
is characterised by microcytic hypochromic
haemolytic anaemia, hepatosplenomegaly,
mild jaundice and sometimes thalassaemialike bone changes.
The phenotypes of a-thalassaemia represent
two clinically significant forms, which are
Hb Bart hydrops fetalis (Hb Bart) syndrome
and haemoglobin H (HbH) disease. In HB
Bart, all four a-globin alleles are deleted or
inactivate (--/--) and death in the prenatal or
neonatal period is inevitable. HbH, however,
The phenotypes of a+-thalassaemia and
a0-thalassaemia, where just one or two
a-globin-genes are affected, are more common. a+-thalassaemia results from deletion or
dysfunction of one a-globin allele (a a/-a),
e.g. by reciprocal recombination between
the Z region, 3.7 kb apart, or between the X
Figure 1. Diagram of the a-globin gene cluster. Black boxes: highly homologous regions, separated by
non-homologous segments; white boxes: exons encoding the a-globin chains.
region, 4.2 kb apart, giving rise to the -a3.7kb
and -a4.2kb deletion, respectively [Figures
1 and 2]. Carriers of a+-thalassaemia, also
known as a-thalassaemia silent carrier, may
have a silent haematological phenotype or
present a moderate thalassaemia-like haematological picture. aº-thalassaemia however,
may be caused by extended deletions varying
from 100 kb to more than 250 kb resulting in
deletion or dysfunction of two a-globin alleles (homozygotes (-a/-a) or heterozygotes (a
a/--), e.g. -a SEA, -a TAI, -a FIL, -a MED, -(a)20.5kb.
Carriers of aº-thalassaemia, also known as
a-thalassaemia trait, show microcytosis (low
MCV), hypochromia (low MCH) and normal
percentages of HbΑ2 and HbF [1].
It is estimated that there are at least 200 million people worldwide affected by thalassaemia. In Switzerland, after iron deficiency,
thalassaemia is the most prevalent cause of
hypochromic anaemia [2, 3]. To offer genetic
counselling for couples who wish to start a
family, or to avoid unnecessary iron substitution, it is important to also identify the heterozygote carriers of a-thalassaemia who have
mild or even no symptoms.
Available methods for
alpha-thalassaemia screening
Current testing for a-thalassaemia is based
on an algorithm of exclusion-testing (i.e. to
exclude iron deficiency and β-thalassaemia
and other haemoglobinopathies), which
requires a wide range of procedures such as
hematological testing of red blood cell indices, peripheral blood smears, supravital staining to detect RBC inclusion bodies, qualitative and quantitative haemoglobin analysis,
bone marrow examination, and the in vitro
synthesis of radioactively-labelled globin
chains in affected individuals. However, the
final proof of the presence of an a-thalassaemia is only obtained using biomolecular
diagnostics [2]. This includes polymerase
chain reaction (gap-PCR) amplification of
the normal or aberrant a-globin gene [4,5],
ELISA for the detection of a–globin chains
in circulation [6] and hybridisation assays
with a-strips.
Current technologies are, however, labourintensive and time-consuming, and may still
not provide an accurate analysis of all variants of the diseases. There is a great need for a
general, rapid and efficient screening method,
which is completely standardised and suitable
for the routine laboratory.
Quantitative real-time PCR as an
a-thalassaemia screening method
Real-time Quantitative PCR technique (RTPCR) has been applied in different investigations including pathogen detection, allelic
discrimination, gene expression and gene regulation [7-9], as well as for the detection of duplications and deletions, e.g. in Duchenne and
Becker muscular dystrophies, cystic fibrosis
and neuroblastomas [10-12]. However, while
RT-PCR has also been applied for the detection
of a-thalassaemia [13], current methods only
allow for detection of several restricted mutations such as the southeast Asian type deletion,
or a group of three different deletions (-a 3.7kb,
-a SEA and -a MED).
We are now evaluating a new screening assay
(patent pending) for the detection of a-thalassaemia-causing deletions using multiple primer
sets, which enables classification of the genotype of an individual by performing only one
(or maximally two) single RT-PCR run.
In order to carry out the assay, genomic DNA
from human blood is extracted using a manual
or automated DNA purification method. Photometric quantification of genomic DNA is
performed on the NanoDrop liquid handling
device (Thermo Fisher Scientific, Inc.) and
– Issue N°5 – October 2009
only samples within a defined range of DNA
purity (260nm: 280nm ratio) are selected for
the experiments. A Light Cycler System is used
for RT-PCR. The specificity of the obtained
amplicons is analysed through melting curves,
gel electrophoresis and/or sequencing. Further quantification in reference to endogenous
controls (reference genes) allows identification
of the relative quantity of the amplified gene.
Through analysis of the obtained amplification
pattern we are able to define the genotype of the
individual. In the case of an aberrant genotype
(positive screening result), subsequent analysis,
e.g. sequencing, allows further characterisation
of the exact nature and location of the mutation
(if this is relevant information for the clinic).
In the case of negative screening results, no
additional work-up in the alpha-gene-cluster
is necessary.
With this quantitative RT-PCR assay we have
developed a new, completely standardised
method for routine laboratory alpha-thalassaemia-screening, which enables the genotype of
each patient to be classified by performing one
single RT-PCR run.
The implementation of this new method in a
diagnostic laboratory requires the assessment & search 24609
– Issue N°5 – October 2009
of a new algorithm for testing.
The two first steps normally carried out, namely the determination of the haemogram and the
iron metabolism parameters,
would be retained. However, after
iron deficiency and b-thalassaemia and haemoglobinopathies
are excluded, the new a-thalassaemias screening method can
be used instead of the common
molecular biological tests used
to date.
The quantification of the a-globin
gene will allow the determination
of the real prevalence of a-thalassaemia, with the detection of all
carriers who may otherwise be
subject to mis- or nondiagnosis.
This helps to provide genetic
advice and (prenatal) diagnostics.
Beyond this, the method can help
to minimise unnecessary, potentially toxic and expensive iron
substitution in patients who have
a-thalassaemia, rather than iron
deficiency anaemia.
1. Herklotz R, Risch L and Huber
AR. Hemoglobinopathies--clinical
symptoms and diagnosis of thalassemia and abnormal hemoglobins. Ther Umsch 2006; 63(1): 35-46.
2. Huber AR et al. Anomales Hämoglobin: Erscheinungsbilder und Abklärung. Swiss Medical Forum, 2004.
3. Huber AR et al. Thalassämie-Syndome: Klinik und Diagnose. Swiss
Medical Forum, 2004.
4. Chang JG et al. Rapid diagnosis of
alpha-thalassemia-1 of southeast
Asia type and hydrops fetalis by
polymerase chain reaction. Blood
1991; 78(3): 853-4.
5. K
o TM et al. Carrier detection and
prenatal diagnosis of alpha-thalassemia of Southeast Asian deletion by polymerase chain reaction.
Hum Genet 1992; 88(3): 245-8.
6. Ausavarungnirun R. et al. Detection of zeta-globin chains in the
cord blood by ELISA (enzymelinked immunosorbent assay):
rapid screening for alpha-thalassemia 1 (Southeast Asian type).
Am J Hematol 1998; 57(4): 283-6.
7. B
owie LJ et al. Detection of
alpha-thalassemias by multiplex
polymerase chain reaction. Clin
Chem 1994; 40(12):2260-6.
8. D
as H et al. Quantitation of Fas
and Fas ligand gene expression
in human ovarian, cervical and
endometrial carcinomas using
real-time RT_PCR. Br J Caner
2000; 82(10): 1682-8.
9. F
ujii K et al. Mutation detection
by TaqMan-allele specific amplification: application to molecular
diagnosis of glycogen storage disease type Ia and medium-chain
acyl-CoA dehydrogenase deficiency. Hum Mutat 2000; 15(2):
10. J ancourt F et al. Rapid identification of female carriers of DMD/
BMD by quantitative real-time
PCR. Hum Mutat 2004;. 23:
11. S chneider M et al. Detection of
exon deletions within an entire
gene (CFTR) by relative quantification on the LightCycler. Clin
Chem 2006; 52(11): 2005-12.
12. DePeter K et al. Quantification
of MYCN, DDX1, and NAG gene
copy number in neuroblastoma
using a real-time quantitative PCR
assay. Mod Pathol 2002; 15(2):
13. Armour JA et al. The detection of
large deletions or duplications in
genomic DNA. Hum Mutat 2002;
20(5): 325-37.
The authors
Dr Saskia Brunner-Agten and Prof.
Andreas R. Huber
Cantonal Hospital Aarau (KSA)
Address for correspondence:
Prof. Dr. med. Andreas R. Huber
Zentrum für Labormedizin
Kantonsspital Aarau AG
CH-5001 Aarau
Tel :+41 62 838 53 01
E-mail: [email protected]
Comments on
this article?
Feel free to post them at
The coinheritance of beta- and alpha- thalassaemia:
a review of one patient and her family
The diagnosis and management of alpha-thalassaemia may be complicated by the variability of the phenotype, which is due to the interaction of coinherited alpha-thalassaemia and the variable severity
of beta-thalassaemia mutations. A well-documented case of complex beta- and alpha-thalassaemia coinheritance is described in this
paper. Laboratory and clinical data for the patient and her family
were reviewed. The patient was an asymptomatic girl, one of identical twins. She presented at one month of age for follow-up of an
abnormal newborn-screening result (haemoglobin F only), which
initially suggested homozygosity for beta-thalassaemia. Extensive
studies on the patient and family revealed that she had coinherited
alpha-thalassaemia traits and homozygous beta-thalassaemia. This
case demonstrates the interaction of coinherited alpha- and betathalassaemia with the resultant amelioration of the clinical phenotype. It also highlights the importance of family studies and close
follow-up in diagnosing complex haemoglobinopathies.
Mast KJ, Hammond S, Qualman SJ, Kahwash SB. Lab Hematol.
2009;15(3):30-3. & search 24830
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– Issue N°5 – October 2009
Cardiovascular disease
Protein profiling of arterial thrombosis
in acute myocardial infarction
Atherosclerotic plaque rupture with subsequent mural thrombus formation is
considered the main event compromising epicardial flow in acute myocardial
infarction (AMI). In the past, only a few selected biomarkers have been
targeted to characterise the complex and poorly understood mechanisms
underlying acute coronary occlusion. Recent proteomic techniques now enable
a comprehensive analysis of protein alterations during atherothrombosis. As
the main pathophysiological determinants, proteins condition the propensity
and progression of atherothrombosis and cardiovascular disease. In particular,
profiling of cellular and soluble proteins from the arterial thrombus site may
identify local effectors that amplify the vascular occlusion process in AMI.
by Dr M. Kubicek, Dr K. Distelmaier and Dr I. M. Lang
Thrombus formation in acute
myocardial infarction
Thrombotic occlusion of an epicardial coronary artery upon atherosclerotic plaque rupture is considered the ultimate and key step
in acute myocardial infarction (AMI) [1].
The propensity of vulnerable plaque rupture and subsequent thrombus formation
depends on a complex cascade of events
involving endothelium, inflammatory cells,
cytokines, endothelin, complement system,
apoptosis, T-lymphocytes, metalloproteinases, phospholipase A2, cholesterol, and
platelets [2]. However, the molecular and
cellular mechanisms underlying plaque rupture and thrombus formation in AMI are not
fully elucidated.
Timely recanalisation of the occluded artery
by percutaneous coronary intervention (PCI)
[Figure 1] is critical to limit cardiac mortality
and morbidity [3]. Implantation of drug eluting stents during PCI is carried out to reduce
re-stenosis and re-occlusion of the culprit
artery, which harbours the potential for late
thrombosis, thus compromising long-term
prognosis [4]. Impaired re-endothelialisation and local inflammatory reactions may
contribute to stent-thrombosis.
In the past, the basic knowledge of local processes underlying plaque rupture and thrombus formation has mainly been derived from
post-mortem material obtained hours after
the event, or from animal model systems [5].
Figure 1. Schematic diagram of sample collection (A) and processing (B) during PCI of acute myocardial
infarction patients. In C-E the culprit lesion is made visible (arrows indicate beginning of the thrombotic
occlusion site and the distal position of the aspiration catheter).
With the advent of modern aspiration catheters, fresh plaque, particulate thrombus material and also whole blood can be harvested
from the site of coronary occlusion during
primary PCI and compared to material aspirated at systemic sites of the same patients
[Figure 1]. Several studies have investigated
local concentrations of selected molecules at
the culprit lesion site [6-8].
Proteomic approaches to analyse
thrombosis in AMI
Changes in the abundance or structure of
plasma or cellular proteins at the thrombus site
can directly alter clot morphology and function and induce downstream events relative
to patient outcome. Recent technical achievements in liquid chromatography/mass spectrometry (LC-MS/MS) facilitate the global profiling of proteins and selected markers involved
in arterial thrombosis. In the following article,
we provide an overview of current proteomic
studies on relevant samples.
Proteome profiling of blood plasma
Direct proteomic analysis of whole plasma
from healthy individuals, unstable angina or
AMI patients has revealed differences in relatively highly abundant plasma proteins such
as alpha-1 antitrypsin, apolipoprotein A-1
and fibrinogen γ-chain [9], as well as AMI
specific proteolysis patterns (i.e. complement
C3f and fibrinopeptide A) [10]. Affinity
depletion of high abundant proteins enables
access to medium to low abundant plasma
proteins, but may also co-deplete disease
relevant proteins from the sample [11]. A
comprehensive analysis of immunodepleted
plasma with differential LC-MS/MS identified
that 95 out of 731 proteins differed between
individuals with or without angiographic
coronary artery disease (CAD) [12].
One approach to identifying less abundant
thrombosis-related proteins is to assess
plasma derived directly from the site of
plaque rupture rather than from peripheral
blood. We recently reported two-dimensional gel electrophoresis (2d-GE) of whole
plasma generated from the culprit or the
systemic site of AMI patients undergoing
PCI [13]. We showed a local accumulation
of complement effector proteins that are
involved in neutrophil recruitment to the culprit lesion site. Multiple immunodepletion
of the same samples followed
by both 2d-GE and LC-MS/MS
identified local accumulation of
additional complement effectors
and regulators [13] but also of
a novel factor (personal unpublished data). This low abundant
protein was shown to be specifically expressed and processed at
the thrombus site in vitro and in
vivo, and was proteomically not
detected in peripheral blood.
Proteome profiling of particulate thrombus material
Particulate thrombus is mainly
constituted by cross-linked fibrin,
but also contains a number of proteins and cells that may alter clot
structure and function. Howes et
al analysed clot-associated proteins by 2d-GE and LC-MS/MS
[14]. Our own group has identified 695 different protein species
in particulate thrombus material
harvested during PCI from AMI
patients (personal unpublished
data). Screening of a self-designed
proteomic database (
[15] for cell type-specific and functional signatures revealed neutrophil-specific effector functions
in particulate thrombus material. Interestingly, the proteome
of late STENT-induced clots differed markedly from that of native
thrombi when compared by spectral counting of shotgun LC-MS/
MS data (personal unpublished
data). We hope that these proteomic data will help to elucidate
the specific mechanisms of STENTinduced late thrombosis and help
to prevent these life-threatening
complications in the future.
Proteome profiling of
circulating cells
Circulating blood cells like
platelets or leukocytes can
interact with the vasculature
and the thrombus at the culprit
lesion site and, upon activation,
express specific adhesion molecules, secrete chemokines and
cytokines, liberate the contents
of alpha granules, generate reactive oxygen species (ROS) and
release microparticles (MP).
Changes in protein expression
and phosphorylation of platelets
have been studied upon in vitro
activation with thrombin [16],
thrombin receptor activating
peptide (TRAP) [17], or collagen-related peptide [18]. More
than 300 different proteins were
found to be released by platelets
following thrombin activation by
a combination of 2d-GE-based
and LC-MS/MS [19]. Several of
the identified proteins were not
previously assigned to platelets, including calumenin and
secretogranin 3, and were found
expressed in atherosclerotic but
not healthy arteries. Thrombogenic proteins have been identified, including platelet factor
4 (PF-4), associated with MP
released from ADP activated
platelets [20].
Barderas et al have analysed circulating peripheral blood monocytes isolated from patients with
acute coronary syndrome (ACS)
or stable CAD by 2d-GE and
LC-MS/MS [21]. The authors
– Issue N°5 – October 2009
identified several intracellular proteins that were altered in
the acute phase of ACS, changes
which were reversed to the level
of stable patients after a six
month period post-admission.
To date no proteome profile of
monocyte secretomes or monocytes from the culprit lesion site
have been reported.
We have recently demonstrated
that polymorphonuclear neutrophils (PMNs) accumulate at
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– Issue N°5 – October 2009
the site of plaque rupture in AMI, and that
the number of accumulated PMNs correlates with ST-segment resolution [22] and
enzymatic infarct size [13]. Secreted proteins
of activated PMNs have been mapped by
2d-GE- and 2D-LC-based approaches identifying thrombomodulatory factors such as
cathepsin G or calreticulin [23-24]. We have
assessed the proteome of PMNs harvested
from the culprit lesion site in AMI by 2d-GE
and LC-MS/MS (personal unpublished data).
In addition, we have employed metabolic in
vitro labelling with 35S methionine/cysteine
prior to autoradiography of 2d-gels generated
from PMN cytosols and supernatants [Figure
2]. This approach allows the identification
of low abundant cytokines, chemokines and
growth factors from cell supernatants [25]
while contaminating proteins from plasma,
platelets or erythrocytes are not labelled [26].
We have identified reproducible proteomic
alterations in PMNs incubated with plasma
generated from the culprit lesion site versus the systemic site [personal unpublished
data; see Figure 3]. These low abundant
proteins can in turn be assayed in clinical
specimens by high sensitive methods.
Atherothrombosis is one of the major causes of
morbidity and mortality. Identification of the
underlying molecular mechanisms is needed
to improve cardiovascular risk and prognosis. Modern proteome profiling approaches
from material harvested at the thrombus
site in AMI have a high potential to identify
important effector proteins implicated in
atherothrombosis. In parallel, in vitro stimulation of relevant primary cell types allows the
capture of low abundant proteomic changes.
Extended application of proteomic profiling
to different determinants of plaque rupture
and thrombosis will enable a comprehensive
assessment of the complex molecular processes relevant to atherothrombosis and acute
vascular syndromes.
1. Naghavi M et al. Circulation 2003;108(14).
2. Libby P. J Intern Med 2008;263:517-27.
3. D
e Luca G et al. J Am Coll Cardiol 2003;42:991-7.
Cardiovascular disease
Figure 2. Schematic diagram of 2d-GE prepared from in vitro labelled primary circulating blood cells.
4. Inoue T et al. Circ J 2009;73:615-21.
5. Cullen P et al. Arterioscler Thromb Vasc Biol
6. Maeda N et al. Fundam Clin Pharmacol
7. Maier W et al. Circulation 2005;111(11).
8. Suzuki M et al. Angiology 2006;57:459-63.
9. Mateos-Caceres PJ et al. J Am Coll Cardiol
10. Marshall J et al. J Proteome Res 2003;2(4)
11. Stempfer R et al. Electrophoresis 2008;29(21)
12. D
onahue MP et al. Am Heart J 2006;
13. Distelmaier K et al. Thromb Haemost
14. Howes JM et al. Diab Vasc Dis Res 2008;
15. Wimmer H et al. Electrophoresis 2009;
16. Maguire PB et al. Proteomics 2002;6:42-8.
17. Garcia A et al. Blood 2004 15;103:2088-95.
18. Garcia A et al. Proteomics 2006;6:5332-43.
19. Coppinger JA et al. Blood 2004;103:2096-104.
20. G
arcia BA et al. J Proteome Res 2005;
Figure 3. Pseudocolour-overlay (fluorographs magenta; autoradiographs green) of cell supernatants generated
from PMNs incubated with plasma obtained from the culprit lesion site (A, B) and the systemic site (C, D),
respectively. Arrows indicate altered protein spots that were affected by a specific neutrophil inhibitor.
21. B
arderas MG et al. Methods Mol Biol 20
22. Adlbrecht C et al. Thromb Haemost
23. B
oussac M et al. Electrophoresis 2000;
24. L
ominadze G et al. Mol Cell Proteomics
25. G
undacker N et al. Electrophoresis 2006;
26. H
audek VJ et al. J Proteome Res 2009;8:3834-43.
The authors
Markus Kubicek*, Klaus Distelmaier§, and Irene
M. Lang§
epartment of Medical and Chemical
Laboratory Diagnostics, Medical University of
Vienna and
Department of Internal Medicine II, Division of
Cardiology, Medical University of Vienna
Corresponding author:
Markus Kubicek
Department of Medical and Chemical Laboratory Diagnostics
Medical University of Vienna
Waehringer Guertel 18-20
A-1090 Vienna,
Tel: +43 1 40400 6436
Fax: +43 1 40400 6437
E-mail: [email protected]
Comments on this article?
Feel free to post them at
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– Issue N°5 – October 2009
Applications of magnetic resonance
spectroscopy in infectious diseases
Nuclear magnetic resonance spectroscopy (NMR) is an analytical
tool, widely used in physics, chemistry, material sciences, biochemistry,
biomedical sciences and medicine. Applications in microbiology and for
the diagnosis of infectious diseases are summarised in this article.
by Dr Uwe Himmelreich
Nuclear magnetic resonance spectroscopy
(NMR) is a powerful tool for the identification of chemicals, mainly in solutions. By this
means, magnetic properties of nuclei are used to
gain information on the chemical structure of a
molecule. The hydrogen atom (1H) is the most
sensitive and widely used isotope in NMR spectroscopy. It can also be used to gain information
on the distribution of molecules such as in magnetic resonance imaging (MRI). The application
of NMR spectroscopy to biological samples such
as cells, body fluids or biopsy samples provides
information on a large range of metabolites. Due
to the non-invasive nature of the technique, one is
not restricted to tissue extracts but can also study
aspects of cellular biochemistry in living systems.
In vivo NMR spectroscopy is routinely used in
the clinic, for example to diagnose and assess
tumours, stroke, metabolic diseases and other
pathologies [1]. Its attractiveness results from its
capability of simultaneous recognition of up to
hundreds of metabolites in living cells maintained
under physiological conditions. NMR spectroscopy requires homogeneous magnets operating
at high field strength (NMR spectrometer or
MRI scanner). Figure 1 illustrates the relationship
between magnetic field strength and the potential
of NMR in clinical diagnosis. Depending on the
nature of the sample (extracts, biopsies, small animals or humans), stronger or weaker magnets can
be used. The value of such a non-invasive diagnostic test performed on a patient is higher than
tests that require invasive methods such as the
collection of tissue samples.
The application of NMR spectroscopy in clinical microbiology and infectious diseases is still in
its infancy. Conventional diagnosis of infectious
diseases relies on microbiological tests based on
microbial culture and/or a limited array of biochemical tests. Disadvantages include the timeconsuming nature of the tests, and occasional
poor sensitivity and specificity. Newly emerged
approaches include molecular methods, which
are rapid and discriminatory but currently limited
in scope (ie target specific pathogens), miniaturised high throughput biochemical techniques and
immunological approaches. Technologies such as
NMR, but also infrared and Raman spectroscopy,
chromatography and mass spectrometry, generate complex data based on chemical composition
of microorganisms, which can detect phenotypic
differences in closely related species. The potential for adaptation of such platforms for diagnostic use has been facilitated by automation of
sample delivery and analysis, and development of
computer-based methods of data interpretation.
Characterisation of metabolites
isolated from microbial cultures
NMR spectra from secondary metabolites and
other compounds, such as proteins, lipids or
carbohydrates isolated from respective groups
of microorganisms, have been utilised since the
1970s for the classification and identification of
bacteria, fungi and lichenised fungi [2]. Compounds isolated from the cell wall or capsule material were most frequently utilised, but analyses of
relatively small compounds secreted from cell suspensions also reveal differences between groups
of microorganisms. Those approaches were, however, time consuming and lacked the sensitivity to
distinguish between closely related species.
Identification of microorganisms based
on NMR spectra of cell cultures
Figure 1. Comparison of the performance of NMR
spectroscopy on different samples
By using cell suspensions instead of extracts,
NMR spectroscopy can become a rapid
Figure 2. Comparison of NMR spectra from pus
of an abdominal abscess (D) and cell suspensions
of bacteria isolated from the pus ((A) Bacteroides
fragilis, (B) Streptococcus milleri, and (C)
Escherichia coli).
screening method. Although the spectral quality suffers substantially by using suspensions of
microorganisms, it is still sufficient to distinguish between different pathogenic species. For
example, linear discriminant analysis of NMR
data obtained from cell suspensions of various
taxa of bacteria is able to identify them rapidly
with an accuracy greater than 95% [3]. More
sophisticated analysis methods of NMR spectra are able to distinguished between two most
common pathogenic yeast species [4]. The identification accuracy for five different Candida species was comparable to molecular identification
methods, and in some cases superior to sophisticated traditional identification systems used
in clinical microbiology laboratories. Even the
identification to sub-species level was possible
and robust for the pathogenic yeast Cryptococcus
neoformans [5]. The ease of sample preparation,
rapid automated identification, and the robust
nature of combining NMR spectroscopy with
computerised data analysis methods are attractive for clinical and
industrial applications. In practice,
NMR-based identification may
be of most value for bacterial and
fungal species, which are relatively
slow growing or difficult to identify
by conventional methods.
susceptibility testing
Due to an increasing number of
microbial strains resistant to antibiotics, rapid drug susceptibility testing is critical for choosing
the right therapy for infectious
diseases. Since the exposure of a
microorganism to an antimicrobial drug most likely also influences its metabolism, it can be
expected that antimicrobial drug
pressure would similarly exert
measurable effects on the metabolism. This has been demonstrated
by measuring Metabolic End
Points (MEP) for several Candida
strains exposed to various antifungal drugs (caspofungin, AMB
and voriconazole) [6]. NMR can
simultaneously monitor utilisation
of substrates (glucose etc.) and
metabolites (acetate, ethanol etc.)
that were affected by the exposure
to the drug. Clear-cut and reproducible MEPs, defined as a >50%
change in metabolite or nutrient
concentrations were correlated
with minimal inhibitory concentrations determined by conventional tests. It was confirmed that
metabolic changes were evident
much earlier than drug-induced
inhibition of growth, the standard
endpoint currently used to determine susceptibility to drugs. This
illustrates the potential of NMR
spectroscopy to provide a more
rapid reading and thus to allow
selection of the most appropriate therapy earlier than by more
labour intense conventional tests.
NMR of biofluids and
biopsy material
Using isolated microorganisms for
the diagnosis of infectious diseases
is still very time consuming. Rapid
identification of microorganisms
without cultivation and isolation of
the organisms is therefore a longstanding aim for microbiologists.
The analysis of biofluids like urine,
cerebrospinal fluid (CSF) or blood
by NMR spectroscopy has a long database of NMR spectra from clintradition, in particular for toxico- ical specimens that would cover all
logical studies (for example [7]). In possible infective microorganisms.
contrast to applications in oncology
or toxicology, where only two or a The feasibility of identification of
few potential diagnoses are possi- infection-causing microorganisms
ble (for example malignant versus directly from biofluids by using
benign), applications of NMR spec- NMR spectroscopy has been demtroscopy for the identification of onstrated in proof-of-principle
infection-causing microorganisms studies based on pus from abscesses
from biofluids (CSF, blood, pus) is [8-10]. Those studies demonstrated
more difficult due to the diversity that: (1) the differences in specof pathogens and the associated troscopic pattern depend on the
pub LCI jib :Mise en page 1 05/10/09 17:45 Page1
difficulties of building up a large causative organisms rather than the
– Issue N°5 – October 2009
site of sample collection, (2) main
metabolites produced by bacteria
in liquid culture are also dominant
in pus samples, and (3) distinguishing between major groups of pathogens is feasible. Figure 2 shows the
example of 1H NMR spectra from a
human pus sample and bacterial cell
suspensions of organisms isolated
from the pus. Key bacterial metabolites such as organic acids (acetate,
butyrate or succinate) and amino
acid residues are also present in the
NMR spectrum of pus.
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– Issue N°5 – October 2009
The feasibility of NMR spectroscopy for rapid,
aetiological diagnosis of meningitis was demonstrated using as little as 10 µL CSF in animal
models for meningitis [11]. Accurate distinction between sterile CSF, and meningitis caused
by Streptococcus pneumoniae and Cryptococcus
neoformans was possible. An agreement of 98%
between the NMRbased classification and the
microbiological diagnosis was achieved.
The application of NMR for the diagnosis of infectious diseases based on biofluids is also promising
due to the ease of automated, high-throughput
data acquisition and analysis, and hence automated diagnosis of infections. The potential of
any high throughput diagnostic platform, which
will achieve maximum efficiency (cost/benefit)
when operating continuously, is dependent on
the number of samples of a given type and the
versatility of the platform. Different types of sample (cultured cells, cell supernatants, biofluids,
tissue biopsies) and different types of pathology
(microbiology, clinical chemistry, tissue pathology) are readily analysed in an NMR laboratory,
which makes such platforms potentially attractive
for large hospitals.
In vivo Magnetic Resonance
Spectroscopy of brain infections
Magnetic Resonance Imaging has increased the
ability to localise cerebral lesions but is not able
to provide a definite diagnosis. In patients with
focal lesions, stereotactic biopsy or open surgical
procedures are required to obtain tissue for histopathological and microbiological examination.
In contrast, in vivo NMR spectroscopy may provide a non-invasive alternative. Figure 3 shows
NMR images and spectra of an infective abscess
compared to a brain tumour. An NMR spectrum obtained from the location of the lesion
(indicated by the square box in the MRI) clearly
distinguishes between the two pathologies.
Identification of marker metabolites like acetate,
succinate and lactate can be used as an indication to distinguish between infective abscesses
and brain tumours non-invasively. Attempts
were already made to classify infection-causing
microorganisms based on in vivo NMR spectra
[10, 12, 13]. However, aetiological identification
of infective brain lesions using NMR spectra is
not yet available due to the low number of reference cases. As clinical management of both
diseases is different, in vivo NMR spectroscopy
can optimise therapy and avoid unnecessary
diagnostic procedures and surgery.
Microbiology laboratories in clinics, industry
and agriculture must be able to identify a wide
number of pathogens accurately and rapidly.
Microbiological tests should be inexpensive,
fully automated, not depend on specialist knowledge about the technology, be applicable to a
Figure 3. In vivo MR images and NMR spectra from a brain abscess and tumour. Distinction between tumour
(increased choline to creatine ratio) and abscess (presence of acetate and succinate) is possible based on
marker metabolite signals.
broad range of microorganisms and be as noninvasive as possible. Fully automated acquisition
of NMR spectra is available in many industrial
and pharmaceutical laboratories for screening of
large quantities of samples. On the other hand,
automated incubation of microbial cultures is
a reality in many microbiological laboratories.
Combining both approaches with automated
data analysis and currently available robotic
technology will result in a fully automated identification of microorganisms with high sample throughput, high accuracy and minimal
manual handling and processing. However, the
ultimate aim of future developments should be
the non-invasive diagnosis of certain infections
without the need of taking tissue samples.
1. Danielsen ER & Ross BR. Magnetic Resonance
Spectroscopy. Diagnosis of Neurological Diseases.
Marcel Dekker Inc, New York, 1998.
2. Grivet JP, Delort AM, Portais JC. Biochimie 2003;
85: 823–840.
3. Bourne R, Himmelreich U, Sharma A et al. J. Clin.
Microbiol 2001; 39: 2916-2923.
4. Himmelreich U, Somorjai RL, Dolenko B et al.
Appl. Environ Microbiol 2003; 69: 4566-4674.
5. Sorrell TC, Wright LC, Malik R & Himmelreich U.
FEMS Yeast Res 2006; 6: 558-566.
6. Coen M, Bodkin J, Power D et al. Antimicrob
Agents Chemother 2006; 50: 4018-4026.
7. Lindon JC, Holmes, E & Nicholson JK. Prog NMR
Spect 2004; 45: 109-143.
8. Garg M, Gupta RK, Husain M et al. Radiology 2004;
330: 519-527.
9. Himmelreich U, Dzendrowskyj T, Allen C et al.
Radiology 2001; 220: 122-128.
10. Himmelreich U, Accursso R, Malik R et al. Radiology 2005; 236: 261-270.
11. Himmelreich U, Malik R, Kühn T et al. PLoS One
2009; 4(4): e5328.
12. Agarwal M, Chawla S, Husain N et al. Neuroradiol
2004; 46: 211-215.
13. H
immelreich U & Gupta RK. Application of magnetic resonance for the diagnosis of infective brain
lesions. In: Webb GA(Ed.) Modern Magnetic Resonance 2006; 2: 991-999.
The author
Dr Uwe Himmelreich
Biomedical NMR Unit
Department of Medical Diagnostic Sciences
Katholieke Universiteit Leuven, Belgium
Jonas Berggren©Folio
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– Issue N°5 – October 2009
Rapid screening for
antibiotic-resistant bacteria
As bacterial resistance to antibacterial agents continues to spread globally,
the role of infection control is increasingly important in the battle against
multidrug-resistant hospital acquired infections. New developments in
chromogenic culture media help clinical laboratories to screen patient
samples quickly and easily for the presence of meticillin-resistant
Staphylococcus aureus, Extended Spectrum Beta-Lactamase-producing
organisms and Vancomycin Resistant Enterococci, allowing infections
caused by these multidrug-resistant bacteria to be identified rapidly so that
infection control procedures can be initiated at the earliest opportunity.
by J. E. C. Beaves
Microbiologists and infection control professionals worldwide are increasingly concerned
about the growing number of clinically significant microorganisms that have developed resistance to commonly prescribed antibiotics. Such
strains are a common cause of hospital-acquired
infections (HAI). More and more frequently,
they limit the choice of therapy available (often
resulting in the empiric use of more expensive
antimicrobial agents), cause increased morbidity
and mortality in patients, and result in prolonged
hospital stays. Measures to control such infections and to provide better outcomes are being
introduced throughout the developed world.
Meticillin-resistant Staphylococcus aureus infection was, for many years, the most notorious of
these HAIs, but now other, equally serious threats
are emerging. Health professionals worldwide are
showing particular concern about the increasing
prevalence of Extended Spectrum ß-Lactamase
(ESBL)-producing organisms and Vancomycin
Resistant Enterococci (VRE). Anxiety about
these emerging problems is now spreading to
the general public as new “super bugs” increasingly feature in the news headlines.
A joint working group from the European
Centre for Disease Prevention and Control
(ECDC) and the European Medicines Agency
(EMEA) recently produced a technical report
in response to the widening gap between the
increasing prevalence of multidrug-resistant
bacteria and the decline in development of
antibacterial agents aimed at treating infections with these organisms. The report
observed that “emerging and increasing resistance has become a threat to public health in
Europe and globally, so that we are now facing the possibility of a future without effective
antibiotics for several types of bacteria that
cause infection in humans.”
Some of the most common multidrug-resistant bacteria isolated from blood cultures (and
therefore with the potential to cause serious
Table 1. Bacteria responsible for bloodstream infections and resistances used as markers for resistance to
multiple antibiotics.
infections) in Europe, as identified in the ECDC/
EMEA report, are shown in Table 1.
The ECDC/EMEA working group reported
that very few antibacterial agents with new
mechanisms of action are under development
to meet the challenge of multidrug-resistant
bacteria and there is a particular lack of new
agents to treat infections due to multidrug
resistant Gram-negative bacteria, such as
Consequently, rapid detection of these resistant
organisms is increasingly important in order
to initiate the most appropriate treatment and
the necessary infection control procedures at
the earliest opportunity. Recent advances in the
development of chromogenic culture media
allow MRSA, VRE and ESBL-producing microorganisms to be detected directly from clinical
specimens in just 18-24 hours, providing an
important rapid screening tool for use in the
fight against multidrug-resistant bacteria.
MRSA is the most common cause of antibiotic-resistant HAI in many parts of the world,
including Europe, the Americas, North Africa,
the Middle East and the Far East. In Europe,
31 countries participating in the European
Antimicrobial Resistance Surveillance System (EARSS) reported 31,591 invasive S.
aureus isolates in 2007, 22% of which were
MRSA. The proportion of MRSA isolates varied across Europe, from 3% or less in northern countries to greater than 50% in some
southern countries.
However, the most recent figures show that
more European countries are reporting
decreasing trends in MRSA rates compared
with previous years, indicating that improved
control efforts can have a positive effect on
MRSA levels in hospitals, even in high endemic
countries. In England, for example, there has
been mandatory reporting of MRSA bacteraemias since 2005. Since that time, intense
scrutiny of the practices of individual hospitals, improved antibiotic prescribing policies
and infection control practices have assisted
in reducing the number of cases reported to
the Health Protection Agency (HPA) to more
than half - with 2932 cases reported in the
financial year 2008/2009 compared to 7096 in
2005/2006 [Figure 1].
– Issue N°5 – October 2009
Figure 1. MRSA bacteraemias reported in English NHS acute trusts.
The importance of screening as part of an effective infection control programme to limit the
spread of MRSA is well recognised and compulsory MRSA screening in elective admissions
was introduced in England in April of this year.
The effectiveness of a screening programme
relies on the speed and accuracy of results so
that colonised patients can be quickly and reliably targeted for isolation, decolonisation and
appropriate treatment.
Improved MRSA detection
Chromogenic MRSA culture media allow the
presumptive identification of MRSA to be
achieved from patient swabs or clinical isolates. Oxoid Brilliance MRSA Agar has been
shown to be one of the most selective MRSA
chromogenic media available and can provide
reliable results in as little as 18 hours. This
medium detects the phosphatase activity of
MRSA using a novel chromogenic compound.
When cleaved, the chromogen produces distinctive denim blue colonies. Antibacterial
agents within the medium inhibit the growth of
competing organisms, including MSSA, while
additional compounds suppress the expression
of phosphatase activity in other staphylococci.
These properties result in a highly selective
medium that demonstrates excellent sensitivity and specificity and allows results to be
obtained up to six hours earlier than alternative
chromogenic media.
The medium demonstrates exceptional positive and negative predictive values (98.1% and
99.2% respectively). These are higher than those
claimed by many PCR-based systems and yet
are achieved at a fraction of the cost. In circumstances where patient isolation facilities are in
short supply, leading to the cohorting of MRSApositive patients, good positive and negative
predictive values are essential. False-positive
results could lead to MRSA-negative patients
being put at increased risk of infection by prolonged stays in MRSA cohorted wards, in addition to the unnecessary and improper use of ‘last
resort’ antibiotics. False-negative results could
prevent an MRSA-positive patient from being
isolated and receiving appropriate treatment. In
an additional comparative trial, it was concluded
that the excellent selectivity of Oxoid Brilliance
MRSA Agar required fewer confirmatory tests,
concluding that it is a reliable and economical
option for clinical laboratories.
ESBL-producing micro-organisms
Extended Spectrum ß-Lactamase (ESBL)producing microorganisms are another class
of resistant bacteria that have been on the rise
since they were first characterised in Europe
in 1983. The relevant enzymes are found in a
family of organisms known as the Enterobacteriaceae, which includes common commensal flora, such as Klebsiella pneumoniae and
Escherichia coli.
Enterobacteriaceae are a significant cause of
nosocomial and community-acquired infections. Due to consistent reporting over the
last 26 years, it has been possible to identify
a number of factors that have contributed to
the increase of these infections, including the
overuse of antibiotics in humans and animals,
hospital cross-infection, human migration and
changes in the food chain [1].
The main therapeutic options for the treatment of
Enterobacteriaceae infections are ß-lactam antibiotics (broad spectrum penicillins and cephalosporins). However, ESBLs confer resistance to
these compounds. Furthermore, ESBL resistance
genes are encoded on freely transmissible genetic
elements, greatly increasing the risk of spread
of resistance to other organisms. The European Antibiotic Resistance Surveillance System
has been monitoring trends in the numbers of
bacteria producing these enzymes since 2000.
Previously, ESBLs were mostly found in Klebsiella species, with infections restricted to
certain vulnerable patient groups, often in
intensive care. However, a new class of ESBL
(referred to as CTX-M enzymes) has emerged,
which is widely detected among E. coli and
is most frequently found in hospital- and
community-acquired urinary tract infections. & search 24778
– Issue N°5 – October 2009
are confined to Enterococcus faecium, although
a few cases have been confirmed as originating
in Enterococcus faecalis. Typing has confirmed
that there are distinct differences between the
subpopulations that are found in hospitals,
and human commensal and animal strains.
The hospital-acquired strains have additional
genomic content with associated virulent factors (and ampicillin resistance in European
strains). Resistance to ampicillin tends to precede an increased VRE rate, which emerges
within several years [3].
The latest EARSS report (2006) showed that
there has been a continuous increase in the
number of invasive E. coli and K. pneumoniae isolates that are resistant to third generation cephalosporins and contain the ESBL
enzymes. The report included data from 800
laboratories in 31 countries and showed a
higher than 10% occurrence in half of the
enrolled countries [1].
As with MRSA, there is a definite split between
the northern and southern European countries. The occurrence of ESBL isolates is still
considered low in the most northern European
countries compared to southern and eastern
countries. The lowest occurrence of ESBL clinical isolates is in the Nordic countries. However,
even there, awareness has heightened recently
as an outbreak of ESBL-producing Klebsiella
has been reported amongst newborns and children in a Swedish hospital. Although the outbreak has been relatively small (to date; four
children infected and an additional four identified as carriers), the effects have been devastating, claiming the lives of three newborn babies.
In southern and eastern Europe, there is an
increasingly high occurrence of ESBLs in both
nosocomial and community settings [1].
ESBL-producing isolates are normally resistant to a number of different antibiotic families
(including beta-lactams, fluoroquinolones,
aminoglycosides and trimetoprim-sulfametoxazole), which severely limits treatment options
and increases the multidrug-resistant ESBL
strains in both medical and social settings.
Rapid detection of ESBL production is, therefore, extremely important, so that an effective
antibiotic can be prescribed.
Rapid detection of ESBL-production
A new chromogenic medium, Oxoid Brilliance ESBL Agar, has been developed for the
presumptive identification of ESBL-producing
micro-organisms direct from clinical samples,
in just 24 hours. This carefully formulated
medium distinguishes between ESBL-producing E. coli and ESBL production in the KESC
group of bacteria (Klebsiella, Enterobacter, Serratia and Citrobacter).
Differentiation is achieved by using two chromogenic compounds which target galactosidase
and glucuronidase activity. The KESC group of
organisms produce galactosidase, resulting in
green colonies on the agar, while E. coli express
galactosidase and glucuronidase, producing
easily distinguished blue colonies [Figure 2].
Occasionally, E. coli will be galactosidase negative, but these organisms produce pink colonies. Proteus, Morganella and Providencia do
not utilise either chromogen, but produce tancoloured colonies with a brown halo due to the
Figure 2. ESBL-producing E. coli (blue colonies)
and KESC organisms (green colonies) growing on
Brilliance ESBL Agar.
deamination of tryptophan. The semiopaque
background of the medium contrasts with the
brightly coloured colonies and allows clear and
easy identification of target organisms.
The medium contains cefpodoxime and
additional antibacterial agents to inhibit
and to suppress the growth of less resistant
AmpC organisms and other non-ESBL flora,
including Stenotrophomonas maltophilia.
Inhibition of these organisms reduces the
incidence of false-positive results compared
to traditional culture media, minimising the
need for confirmatory testing.
An evaluation of the medium was performed
using a variety of isolates, including CTX-M,
TEM, SHV and K1-hyper-producing strains,
from clinical* and other sources. Results indicated that K1-hyper-producing (non-ESBL)
strains were inhibited while all representative
ESBL strains grew. The agar was selected by
MOSAR (Mastering Hospital Antimicrobial
Resistance in Europe) for use in a pioneering
European ESBL prevalence study (for further
information visit
Tracking VRE rates has become even more
important now that a link between ampicillin
resistance and VRE rates in hospital isolates has
become apparent. Within Europe, there is much
variability between surveillance systems that
have been set up to collect data on vancomycin
resistance in enterococci, and in some countries no data is collected. Due to these missing
and variable data, it is difficult to perform reliable statistical analysis. However, the EARSS
report for 2007 suggests that some trends have
emerged. There have been increasing VRE rates
in some countries, including Ireland, Germany
and Greece, whereas in the Nordic countries
and the Netherlands there is low VRE prevalence. Austria, Portugal and Italy have seen
decreases, but there is insufficient evidence to
link this to measures that these countries took
to contain outbreaks [3].
VRE-related infections are usually seen in hospital patients who are already very ill, in particular those who are immunocompromised,
those who have had previous treatment with
certain antibiotics (particularly cephalosporins
and glycopeptides), those on a prolonged hospital stay, or those in specialist units, such as
intensive care, oncology or renal units. Prompt
identification of infection in these vulnerable
patient groups is extremely important in order
to improve outcomes and to prevent the spread
of infection to other patients.
Vancomycin Resistant
Enterococci (VRE)
Glycopeptide resistance in enterococcal species
is also increasing and, in particular, Vancomycin Resistant Enterococci (VRE) have emerged
as significant nosocomial pathogens. This is
thought to be due to the increased use of vancomycin for treatment of MRSA, particularly
in the United States, and the use of a vancomycin-like glycopeptide (avoparcin) as a growth
promoter in animal husbandry in Europe [2].
There are a number of vancomycin resistance
mechanisms, of which the most genetically
mobile and widespread are VanA and the less
common VanB. The first report of VRE occurred
in the early 1980s in Europe. Most VRE strains
Figure 3. E. faecium (purple) and E. faecalis (light
blue) colonies growing on Brilliance VRE Agar.
Enhanced detection of VRE
Recently, Oxoid Brilliance VRE
agar, a new chromogenic screening plate for the detection of
VRE, has become available. This
medium provides presumptive
identification of vancomycin
resistant Enterococcus faecium and
Enterococcus faecalis, from a faecal
sample, swab, isolate or suspension, within just 24 hours. Differentiation between the two species
is achieved using two chromogenic compounds - one that targets phosphatase activity (present
in both species) and another that
targets α-galactosidase activity
(present in E. faecium). This results
in light blue E. faecalis colonies,
which are easily distinguished
from the indigo/purple colonies of
E. faecium [Figure 3].
help clinicians to provide the most
effective care, but can contribute to
more widespread epidemiological
studies and to our understanding
of these important pathogens.
1. Coque TM, Baquero F, Canton R.
Increasing Prevalence of ESBL-producing Enterobacteriaceae in Europe.
Eurosurveillance 2008; 13:47.
2. Bell JM, Paton JC, Turnidge J.
Emergence of Vancomycin Resistant Enteroccocci in Australia:
Phenotypic and Genotypic Characteristic of Isolates. J. Clin. Microbiol
1998; 36, 2187-2190.
3. Werner G, Coque TM et al. Emergence
and spread of Vancomycin Resistance
Among Enterococci in Europe. Eurosurveillance 2008; 13:47.
A full list of references is availble from
the author.
* Clinical isolates were provided by Dr.
Maurine A. Leverstein-van-Hall, Clinical Microbiologist, University Medical Centre Utrecht (UMCU)/National
– Issue N°5 – October 2009
Institute for Public Health and Environment (RIVM), Netherlands, and
Professor Youri Glupczynski, University
Clinic of the Catholic University of Louvain (UCL) Mont-Godinne, Belgium.
The author
James E. C. Beaves BSc.(Hons)
Clinical Applications Manager
Thermo Fisher Scientific
Basingstoke, Hants, UK.
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The growth of competing flora,
including E. gallinarum and E.
casseliflavus (both of which are
intrinsically resistant to vancomycin) is suppressed by the inclusion of vancomycin and additional
antibiotics in the medium.
The medium was evaluated in a
clinical trial, using a panel of 120
clinical isolates, and demonstrated
a sensitivity of 94.7% and 100% at
24 and 48 hours respectively.
Routine screening
Unlike some other rapid methods
for the identification of resistant
organisms, which require expensive or specialised equipment,
chromogenic culture media can be
adopted easily by clinical laboratories of any size. Brilliance MRSA,
VRE and ESBL media are supplied
in pre-poured agar plates that are
ready to inoculate, and give easyto-read results within 24 hours or
less, directly from clinical samples.
This speed, convenience and ease
of use make chromogenic media a
valuable option for routine screening for significant resistant microorganisms by clinical laboratories.
As resistance mechanisms, such as
those described above, continue
to challenge health professionals
around the world, it is important
to have such reliable and easily
accessible tools for local screening programmes, which not only
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– Issue N°5 – October 2009
Case study: how a general
hospital lab improved its blood
film review procedures
The haematology lab of Middlemore Hospital, South Auckland, New
Zealand, had been using the traditional light microscope to review blood
films. The laboratory is now using an automatic morphology system
to review the majority of its films. We spoke to John Peters, Scientist in
Charge of the haematology laboratory at Middlemore hospital, to see
how the system performs in practice compared with the manual system.
Q: Could you give us some background regarding the hospital at Middlemore and the main
activities of the haematology lab?
A: The hospital has approximately 900 beds at
its main facility but also has two smaller satellite facilities a short distance away. The hospital
is a general hospital and provides mental health
services, general surgery, paediatrics, womens’
health and geriatics. It also houses the largest
maternity unit and the largest accident and
emergency unit in New Zealand, as well as the
national burns unit and the largest renal inpatient and outpatient unit in the country. The satellite sites provide extensive outpatient services.
The laboratory serves all these areas and also
supports a haematology ward and clinic that
deal with chronic haematological patients, but
not with acute leukaemia or oncology patients.
The laboratory has four consultant haematologists covering both the lab, haematology ward
and clinics. There is one laboratory registrar
and approximately 18 full time equivalent staff,
12 of whom are scientists and the others of
whom are technicians.
Q: What patient population does your hospital/
lab serve and how many blood films does the lab
receive per year?
A: Middlemore hospital is one of three large
general hospitals serving the City of Auckland,
which has a population of 1.4 million people.
Middlemore hospital serves the southern section of the greater Auckland area with a population that is currently approximately 480,000
people but which is growing faster than that
in any other area of New Zealand. The haematology lab processes about 180,000 full blood
counts per year and of these, reviews approximately 46,000 films. The population served is
the poorest in the country and there are many
abnormal results.
Q: Since when has the lab been using automated
image analysis?
A: We have been using the CellaVision DM96
since May 2009. The lab also changed its cell
counters at that time and currently uses Sysmex
XE5000 Alpha systems.
Q: How do you find the DM96 system compared
with the manual morphology system?
A: There are two main advantages that we
have identified so far. These are speed and
the fact that the work is physically easier on
the staff. We have found the review rate to
be almost twice as fast as manual reviewing
when experience staff are using the system.
This is a big advantage for us, as all our staff
are experienced with the exception of students
undergoing training, and interns.
In the past we have had a few problems with
staff suffering from over-use of a combination
of microscopes and computers. The DM96 has
reduced these problems substantially since
there is less use of the manual microscope
and the actions necessary to focus and move
the stage. Approximately 4% of films are still
reviewed manually and the majority of these
are paediatric samples.
Having to look at a fixed number of cells
defines an end point to the review, and therefore an end to the process. The excellent WBC
categorisation and excellent morphological appearance of the cells makes the review
much easier and more accurate. We are much
more aware of abnormal cells and they cannot
be overlooked. If the DM96 identifies a blast
cell, for example, the staff member must look
at this and reclassify it if necessary. We have
the remote viewing stations set up with two
flat screen monitors running off a single PC,
John Peters, Scientist In Charge
of Middlemore Hospital
Haematology Laboratory.
making the film reviews a lot less labour intensive and physically less demanding than manual microscopic reviews. One monitor displays
the DM96 images and the other displays the
lab computer results and analyser scatter plots.
Images that we require for presentation work
and for our library of abnormal images are
kept either on CD or flash drive. The DM96 is
extremely good for auditing purposes as the scientist/technician who has reviewed the images
can easily be identified from the log in on the
computer. If a user is mis-categorising cells on
a regular basis then that user can be identified.
The DM96 is an excellent instrument for teaching new staff members RBC morphology, WBC
morphology and platelets morphology, and it
has an excellent library of images on board
that can be augmented. We don’t use the RBC
morphology recognition system but categorise the morphology ourselves and this is both
fast and accurate.
Q: Are there any improvements that you would
like to see in the system?
A: We would welcome a means of looking at the
tail of the film for platelet clumps, microfilaria
and clumps of abnormal cells. We would like to
see a modification of the DM96 main analyser
so that it could detect the last film in a cassette
and then reject it quickly and move on to the
next one. This would speed up the processing
of samples a lot when only partially loaded
cassettes are introduced into the instrument.
Q: Finally, could you summarise how the installation of the DM96 in your lab has helped both
clinicians and their patients?
A: The clinicians don’t use the DM96 but
the instrument allows the lab to analyse and
report on the samples in a more timely fashion and in a more standardised and accurate
manner. Education of the clinical staff is also a
lot easier with easy access to a large variety of
cells from the comprehensive library and from
patient samples.
CellaVision’s new analyser,
the DM1200, is now available,
see page 35.
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30 years of success: our company has matured!
To be honest on this, our 30th anniversary, we are rather
proud of what we have achieved during the past three
Thirty years in which we grew together as a team, but also
thirty years in which, in a sometimes difficult and always
highly competitive market, we asserted ourselves due to our
knowledge, our high-quality products and – last, but not least
– due to a trusted cooperation with our customers, to whom
we would here like to express our thanks.
We have also utilised our strengths in the development of a
new instrument, the Chloridmeter CM20: our ability to
produce high quality products and our knowledge of what
will satisfy the demands of our customers.
These are strengths that years ago resulted in our company’s
“We are the osmometer people.“
And today we are glad to be able to add:
”Always were, always will be.”
We are not an aloof large organisation: we know our
customers personally, are familiar with their work and their
problems, and thus know what they require from us and the
quality they expect from our products. We have remained a
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Ana l yt e of t he m ont h
– Issue N°5
N°6 – October 2009
Focus on Finland
Novel biochemical markers of
bone remodelling in the management
of osteoporosis
Currently available bone turnover markers are extensively used in research
applications and have also shown clinically interesting associations. Recent
advances in bone cell biology have led to the development of potential new
markers. Novel candidates include osteoclastic enzymes, non-collagenous
proteins, markers of bone matrix properties and regulators of bone cell activity.
by Dr Kaisa K. Ivaska
Bone is a metabolically active organ that is
continuously subjected to resorption and formation on the surface of the trabecular bone
area (typically in the interior of the bone) and
in the Haversian canals. During bone resorption, multinucleated osteoclasts degrade existing bone matrix. Resorption is followed by
bone formation, during which osteoblasts
synthesise new bone matrix which is gradually
mineralised. Most of the bone turnover takes
place in trabecular bone due to its higher surface area. Quantitative changes in bone metabolism can be assessed easily and non-invasively
by measuring biochemical markers of bone
remodelling in serum or urine. These markers, also known as bone turnover markers, are
skeletal proteins or their fragments, or enzymes
released from bone cells during bone turnover.
Formation and resorption are usually tightly
coupled in time and space. Therefore, in disease states where both events are coupled and
change in the same direction, such as in osteoporosis, any marker reflects the overall rate of
bone turnover.
Biomarkers for osteoporosis
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The most widely used currently available markers include bone specific alkaline phosphatase
(boneALP), osteocalcin (OC) and the N-terminal propeptide of type I collagen (PINP)
for bone formation, and crosslinked C and N
telopeptides (CTX and NTX) and deoxypyridinoline (DPD) for bone resorption. None of
the current markers are used to diagnose osteoporosis. Instead, bone mineral density (BMD)
measured by dual energy X-ray absorptiometry (DXA) is the single best method available
for confirming the diagnosis of osteoporosis,
defined as BMD value 2.5 standard deviations
or more below the mean of the young female
adult population. However, bone markers provide more dynamic and rapid measures of
skeletal status. They have been investigated in
osteoporosis for use in three main areas: (i)
monitoring anabolic or anti-resorptive therapy, (ii) predicting bone loss and the risk of
developing osteoporosis, and (iii) identifying
individuals with high risk of fracture.
Bone markers are useful tools in assessment of
response to anti-resorptive or anabolic therapy.
Significant changes in markers occur within
months whereas it usually takes a few years to
detect a significant change in BMD. Monitoring the efficacy of bone-active drugs is currently the most promising and widely known
clinical application. Another area where markers may be of use is bone loss. Bone loss can
be assessed by DXA if serial measurements
are performed but it takes years to detect significant changes due to measurement imprecision and the relatively slow rate of change in
BMD. The assessment of bone markers could
be of use in identifying individuals who have
increased bone turnover and who may be at
the greatest risk for bone loss and of developing osteoporosis We have found that women
who had constantly high bone turnover in
serial measurements lost significantly more
bone over five years than women with constantly low turnover. They also progressed to
the level of osteoporosis more frequently [1].
Serial assessment of bone turnover may thus
assist in decision-making and targeting preventive measures. One of the most important
challenges in the management of osteoporosis
is, however, to identify individuals who are at
high risk for fragility fractures. Low BMD is
a strong risk factor for fractures but there is a
great overlap in BMD of individuals with and
without fracture. Apparently, BMD is only one
of a number of risk factors and can capture only
one aspect of the likelihood of fracture. Due to
the low sensitivity of BMD testing, there is a
need for additional measures to identify individuals who are at high risk for fractures and
who might need treatment, despite not reaching the diagnostic threshold for osteoporosis.
During recent years risk-based assessment has
been applied and the Fracture Risk Assessment
Tool (FRAX) integrating clinical risk factors
has been developed to evaluate fracture risk
of patients. High levels of bone markers have
been shown to be associated with fractures
independently of BMD in postmenopausal
women in several prospective studies [2]. The
results have been most consistent with resorption markers. Markers could add information,
particularly for individuals with normal or
moderately low bone mass (osteopenia), who
Overview on existing bone turnover markers (black) and novel candidate markers (orange). Abbreviations:
PINP, N-terminal propeptide of type I collagen; Dkk1, Dickkopf 1; boneALP, bone-specific alkaline phosphatase;
OPG, osteoprotegerin; sRANKL, soluble receptor activator of nuclear factor kappa B; OC, osteocalcin; CTX and
NTX, C- and N-terminal crosslinked telopeptide of type I collagen; DPD, deoxypyridoline; TRACP5b, tartrateresistant acid phosphatase 5b.
are asymptomatic but have increased bone
metabolism and may thus be at increased risk
for developing osteoporosis and osteoporotic
fractures in the future [3].
Novel candidate markers of bone
Currently available bone turnover markers are
extensively used in research applications and
have shown clinically interesting associations,
but they also have some limitations. Markers
reflect quantitative changes but do not provide
information on structural abnormalities of
bone. Furthermore the relative contribution of
trabecular, cortical and periosteal bone may vary
depending on disease, age or therapy. Some analytes also have high variability or are not bonespecific. Recent progress in the identification of
important pathways in bone physiology has led
to the development of potential new biochemical markers. Candidates include osteoclastic
enzymes, non-collagenous proteins and markers of bone matrix properties. In addition, regulators of bone cell activity have been studied.
fractures, in particular for vertebral fractures
[5]. Cathepsin K is a member of the cysteine
protease family and is secreted into the resorption lacuna, where it contributes to the cleavage
of collagen molecules and leads to degradation
of the organic matrix. Cathepsin K is endocytosed with the degradation products, transported through the cell and released into the
circulation where it can be detected by immunoassay. It is a potential marker of osteoclast
number but because the circulating concentrations are low, the accurate determination with
current assays remains challenging.
Non-collagenous matrix proteins and
their fragments
Although circulating osteocalcin is widely used
as an index of bone formation, some of the
fragments are derived from the bone resorption process when osteocalcin is released from
the bone matrix. These fragments are rapidly
cleared through glomerular filtration and accumulate in the urine. Urinary OC fragments
– Issue N°5 – October 2009
consist of the mid-molecule portion, which
appears to be protected from degradation in
the kidney. Immunoassays for urinary osteocalcin fragments have been developed and
theoretically, urinary osteocalcin fragments
may constitute a more specific bone resorption
marker than type I collagen-related fragments
[6]. Other potential non-collagenous markers
include bone sialoprotein, for example. It has
been shown to be a product of active osteoblast, but is strongly upregulated in a variety of
human primary cancers, including breast, prostate and lung cancer. The limited clinical data
suggest that BSP may be interesting for use in
patients with malignant bone disease.
Markers of bone matrix properties
Pentosidine is formed by non-enzymatic glycation of type I collagen when sugar is present in
extracellular matrix. It is a crosslinking molecule although the precise location in collagen
helix is not known. High pentosidine content in bone matrix has been associated with
decreased resistance to fracture. High serum
pentosidine has recently been shown to be
moderately associated with vertebral fracture
in postmenopausal women with type 2 diabetes [7]. C-terminal crosslinked telopeptide of
type I collagen, CTX, is a collagen fragment
of eight amino acids, which exists in a nonisomerised and an isomerised form referred
to as α-CTX and β-CTX, respectively. β-CTX
contains a β-aspartyl peptide bond in L-enantiomeric form, which is considered to result
from the ageing of extracellular proteins. The
isomerisation process reaches a maximum
of approximately three years after the bone is
mineralised, and therefore the released β-CTX
reflects the degradation of relatively old bone.
On the contrary, the non-isomerised α-CTX
presumably results from the degradation of relatively young bone. The urinary ratio of these
Osteoclastic enzymes
Tartrate-resistant acid phosphatase 5b
(TRACP5b) belongs to the family of five isoenzymes of acid phosphatases. Two isoforms of
TRACP exist in circulation. TRACP5a originates from macrophages and dendritic cells,
whereas TRACP5b is derived from the osteoclasts. TRACP5b is believed to destroy the
endocytosed bone matrix degradation products during transcytosis through the osteoclast.
TRACP5b is highly expressed and secreted by
both resorbing and non-resorbing osteoclasts
into the circulation. Recent data show that it
reflects the number of osteoclasts rather than
osteoclastic activity. S-TRACP has very little
circadian variation and is not influenced by
renal function or by food intake [4]. Using a
population-based design, serum TRACP5b
was found to be predictive for forthcoming
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– Issue N°5
N°6 – October 2009
two forms can be measured by immunoassays
and it gives information on collagen maturation which could contribute to bone matrix
quality. This is supported by studies which have
demonstrated that the urinary α/β CTX ratio
is predictive for fractures independent of BMD
and bone turnover, suggesting that the ratio
provides additional information beyond bone
mineral density and turnover rate [8].
Regulators of bone cell activity
Osteoprotegerin (OPG) and receptor activator for nuclear factor kappa B ligand (membrane-bound RANKL, secreted sRANKL) are
expressed by osteoblasts and are important
local regulators of bone turnover. The interaction of RANKL with the receptor on osteoclast precursors induces the differentiation
and maturation of osteoclasts, while OPG acts
as a decoy receptor and prevents osteoclastogenesis. At present it remains unclear what
proportion of circulating OPG is monomeric,
dimeric or bound to RANKL and which form
is the most biologically relevant to measure.
Due to these limitations, conflicting data
exist. It is also unclear how systemic concentrations reflect local cytokine concentrations
in bone. Wnt signalling, on the other hand,
plays a key role in the regulation of osteoblastic activity. Different secreted proteins
inhibit Wnt signalling in osteoblasts, such as
soluble frizzled-related proteins (sFRPs), Wnt
inhibitory factor-1 (WIF1), Dickkopfs 1-4
(Dkk1-4) and sclerostin. An assay for circulating Dkk-1 has been developed and increased
levels have been detected e.g. in patients with
breast cancer bone metastases [9]. The role in
postmenopausal osteoporosis remains to be
determined. With the advances in bone cell
biology, other regulatory molecules are also
emerging. Recent findings suggest that bone
is an important endocrine organ producing
Focus on Finland
hormones, such as FGF-23 regulating phosphate metabolism in the kidneys and uncarboxylated osteocalcin affecting adipose tissue
and pancreatic beta cells [10].
Progress in the characterisation of important
biological pathways in bone cell biology and
the components of bone matrix has led to the
development of new candidate markers. Of
these, TRACP5b, the α/β CTX ratio, serum
pentosidine and urinary osteocalcin fragments
have been shown to be associated with fracture
risk in preliminary clinical studies. Post-translational modifications of bone matrix proteins
are interesting candidates since they may have
a role in determining the mechanical competence of bone. Thus they would complement the
existing markers by providing information on
qualitative changes in bone matrix properties.
Further studies are required to elucidate the
clinical value of novel markers in osteoporosis,
alone and in combination with existing markers. The list of available markers should further
expand in the near future.
1. Ivaska KK, Lenora J, Gerdhem P, Akesson K,
Vaananen HK, Obrant KJ. Serial assessment
of serum bone metabolism markers identifies
women with the highest rate of bone loss and
osteoporosis risk. J Clin Endocrinol Metab 2008;
2. Garnero P. Markers of bone turnover for the prediction of fracture risk. Osteoporos Int 2000; 11
Suppl 6:S55-65.
3. Sornay-Rendu E, Munoz F, Garnero P, Duboeuf
F, Delmas PD. Identification of osteopenic women
at high risk of fracture: the OFELY study. J Bone
Miner Res 2005; 20(10):1813-9.
4. Halleen JM, Tiitinen SL, Ylipahkala H, Fagerlund KM, Vaananen HK. Tartrate-resistant acid
phosphatase 5b (TRACP 5b) as a marker of bone
resorption. Clin Lab 2006; 52(9-10):499-509.
5. Gerdhem P, Ivaska KK, Alatalo SL, Halleen JM,
Hellman J, Isaksson A, Pettersson K, Vaananen
HK, Akesson K, Obrant KJ. Biochemical markers
of bone metabolism and prediction of fracture in
elderly women. J Bone Miner Res 2004; 19(3):38693.
6. I vaska KK, Kakonen SM, Gerdhem P, Obrant KJ,
Pettersson K, Vaananen HK. Urinary osteocalcin
as a marker of bone metabolism. Clin Chem 2005;
7. Yamamoto M, Yamaguchi T, Yamauchi M, Yano
S, Sugimoto T. Serum pentosidine levels are
positively associated with the presence of vertebral fractures in postmenopausal women with
type 2 diabetes. J Clin Endocrinol Metab 2008;
8. G
arnero P, Cloos P, Sornay-Rendu E, Qvist P, Delmas PD. Type I collagen racemization and isomerization and the risk of fracture in postmenopausal women: the OFELY prospective study. J Bone
Miner Res 2002; 17(5):826-33.
9. Voorzanger-Rousselot N, Goehrig D, Journe
F, Doriath V, Body JJ, Clezardin P, Garnero P.
Increased Dickkopf-1 expression in breast cancer
bone metastases. Br J Cancer 2007; 97(7):964-70.
10. Fukumoto S, Martin TJ. Bone as an endocrine organ. Trends Endocrinol Metab 2009;
The author
Dr Kaisa K. Ivaska
University of Turku, Institute of Biomedicine
Department of Cell Biology and Anatomy
FI-20520 Turku, Finland
Clinical and Molecular Osteoporosis Research Unit
Lund University
Department of Orthopaedics
Malmö University Hospital
SE-20502 Malmö , Sweden
Decreased bone mineral density in adults born
with very low birth weight
In a study published in the open-access medical journal, PLoS
Medicine, Petteri Hovi and colleagues from the National Institute for Health and Welfare Helsinki, Finland evaluated skeletal health in 144 adults (ages ranging from 18 to 27 years) who
were born preterm with very low birth weight. They showed
that as adults these individuals have significantly lower bone
mineral density than do their term-born peers and suggest
that this finding translates into increased risk for osteoporosis
in adulthood for these individuals.
info%3Adoi%2F10.1371%2Fjournal.pmed.1000135 & search 24791
The Finnish diagnostics industry
The Finnish diagnostics industry is rich with companies and research groups, which
provide technologies and products to global markets. The diagnostics industry can
be categorised into several speciality areas. These include companies that supply
reagents and services, those supplying complete diagnostic kits, as well as companies
that produce either instrumentation or electronic and mechanical components for
incorporation into instrumentation in many different areas. The activities of some
of these companies, as well as some of their new products, are outlined.
Ani Labsystems Ltd
This established Finnish diagnostics company is
dedicated to the production and sales of immunoassays. The core business is high quality EIAs and
FEIAs for infectious diseases and neonatal screening. For 25 years, and through changing ownership (formerly Labsystems), Ani Labsystems has
been a key pioneer in the development of the first
microplate arrays, screening of metabolic disorders
for neonates and avidity methods. The company
strongly invests in research and development, and
state of the art production. The quality system complies with ISO 13485:2003 and IVD directive 98/79/
EC. The assays to diagnose infectious diseases
include kits for antibodies to Mycoplasma pneumoniae, Chlamydia pneumoniae, Bordetella pertussis
and Chlamydia trachomatis, as well as a full Toxo
panel. The company offers PKU, TSH, Toxo-IgM,
G6PD, 17-OHP, Galactosaemia and IRT for neonatal screening, and HIV and hepatitis B for blood
screening. A unique assay to detect coeliac disease
and a kit for quantitative IgD EIA are also available.
HyTest Ltd
HyTest Ltd is a biotechnology-oriented company
based in Turku, that manufactures and markets
high-quality immunological reagents for industrial
and research applications. The product portfolio
includes cardiac markers, hormones and toxins,
human proteins, neuroscience-related reagents,
infectious and autoimmune disease reagents. The
company was established in 1994 and has experienced rapid growth over the subsequent years. The
solid scientific background of its staff and continuous investments in R&D together with dedication
to customer service and focus on global operations
from day one have combined to ensure its success.
HyTest is a leading supplier of several reagents
worldwide, such as antibodies and antigens of troponin I and troponin complex. The inherent quality of its reagents and extensive customer support
has helped the company to become a recognised
name in the industry. The diagnostic industry is in
a state of constant development. In response to this
challenge, the company is continuously working
to bring new products to the market as well as to
improve the existing ones. Its R&D specialists maintain close ties with the scientific community and
engage in joint research with Finnish and international academic diagnostic research organisations.
HyTest is an ISO 9001:2000 certified company for
the manufacture and distribution of immunological reagents. It strives to completely understand
and satisfy the needs of its customers in both the
industrial and research communities.
Based in Helsinki, Labquality is an independent
and impartial organisation founded in 1971 and
owned by the Finnish Society of Clinical Chemistry, the Association of Finnish Local and Regional
Authorities, 19 local hospital districts, the Finnish
Medical Association, the Finnish Red Cross, the
Association of Medical Service Providers and the
Finnish Union of Experts in Science. Labquality
provides External Quality Assessment (EQA) programmes for clinical laboratories and point-of-care
tests performed in social and healthcare units. Its
mission is to support, promote and improve quality
in clinical testing, analytics and diagnostics. Labquality has 23 employees, and more than 100
experts are involved in
the schemes. In 2009,
Labquality had 4174 client laboratories based in
42 different countries.
– Issue N°5 – October 2009
antibodies, as well as research and development,
are conducted from the head office. Actim rapid
tests are manufactured at the Joensuu site in Eastern Finland. The company employs around 100
highly trained professionals, and the product
range covers monoclonal antibodies for a variety
of markers. In order to meet the needs of different
assay technologies, there are multiple clones for
the most important analytes. MedixMAB monoclonal antibodies are manufactured in vitro using
the latest manufacturing technology and serum
or protein free culture media. All antibodies are
fully characterised and purified by affinity chromatography. Diagnostic tests are marketed under
the Actim range of rapid tests. Two of the most
successful products are Actim PROM and Actim
Partus. The Actim PROM rapid test reliably
detects premature rupture of foetal membranes
while Actim Partus is a fast and simple bedside
test to estimate the ripeness of the cervix during
pregnancy. In addition, the company has tests for
respiratory (Actim Influenza A&B) and infectious diseases (Actim CRP) and gastroenterology
(Actim Fecal Blood and Actim Pancreatitis).
Reagena Ltd
Based in Kauniainen, Reagena is an emerging diagnostics company in the Nordic region, focused on
developing novel POC immunoassay tests in the
areas of infectious disease testing and immunity.
Aimed at developing biomarker-based novel rapid
tests, Reagena’s unique ReaMobile Diagnostic Test
System commands respect in the POC testing market as it facilitates faster diagnosis and treatment of
patients. As a product developed to address the
needs of patients quickly and effectively, the wireless ReaMobile diagnostic test system has a unique
place in the market as it fulfills the need for faster
diagnosis in the immunoassay segment. With an
ability to provide both quantitative and qualitative
results, this test system utilising a mobile hand held
reader is a valuable product addition.
Medix Biochemica
owned by the Minerva
Foundation, is a nonprofit foundation in
medical research with
half a century of extensive experience. The
head office is located
in Kauniainen, near
Helsinki. Most activities, such as the production of monoclonal
External Quality
Assessment Services
Controls, Calibrators and
Reference materials for
Internal Quality Assessment & search 24833
– Issue N°5 – October 2009
Product News
Tests for Toxoplasma gondii
Ani Labsystems
has updated the
well-known Toxoplasma gondii IgG
Avidity EIA test,
which was the
first Toxoplasma
avidity test available globally. The
new T. gondii mIgM EIA and IgG EIA tests are being
developed to form a full “Toxo-package”. The T. gondii mIgM EIA is a microcapture test, that can be run
simultaneously with the T. gondii IgG EIA. The latter
test provides quantitative results, and automatically
generates the guidelines for the avidity assay (only
2 points dilution). Generally, the reliability of the
original avidity test is now combined within a full
package of tests that can be run fully automated as
well as manually. For neonatal screening, a fluorometric Neonatal Toxoplasma gondii IgM FEIA is
offered. The photometric version of the test, Neonatal Toxoplasma gondii IgM EIA, has recently been
clinically evaluated; using over 1000 patient samples
and almost 300 CDC samples, the test was found to
have excellent specificity and sensitivity.
useful for the development of high sensitivity
cTnI assays, and also validated pairs suitable for
lateral flow assays. A new generation of cTnI
antibodies is currently under development.
Work with brain natriuretic peptides is ongoing,
and BNP, proBNP and NT-proBNP antibodies
and antigens are available. In addition a new
type of BNP/proBNP immunoassay, the “single
epitope” sandwich assay, has been developed,
which differs from the conventional format of
sandwich immunoassays. In the single epitope
sandwich assay, the capture antibody recognises
the antigen (BNP or proBNP), whereas the detection antibody is specific to the complex of capture
antibody and antigen.
The single epitope
sandwich approach
has great advantages
compared with conventional
especially in the case
of unstable antigen
holds the intellectual
property rights for
this invention.
Ani Labsystems Ltd
Hytest Ltd
Vantaa, Finland
Turku, Finland
Medica stand Hall 03/C83
Medica stand Hall 03/F44
detects influenza type A and B from respiratory
samples. It has been proven that this test also
detects the currently circulating Influenza A
(H1N1) variant. Using this simple dipstick test,
influenza can be diagnosed in just 15–20 minutes, including the time for sample collection.
Similarly, the symptoms of respiratory syncytial virus (RSV), especially in infants, are very
similar to the common cold and influenza.
Diagnosing the disease early reduces the risk
of serious complications, such as pneumonia.
Actim RSV is a novel test that allows an instant
RSV diagnosis to be made. All original and
patented Actim tests are based on monoclonal
antibodies developed and produced exclusively
by Medix Biochemica.
Medix Biochemica
Kauniainen, Finland
Medica stand Hall 03/F44 & search 24798 & search 24819
Troponin I and Brain Natriuretic
Peptide cardiac markers
Rapid diagnostic system & search 24803
HyTest specialists have been involved in cardiac
troponin I (cTnI) studies for more than 15 years.
Currently many well characterised antibodies
directed to different regions of the cTnI molecule are available. Many of these antibodies are
used in commercial assays. The company has
determined antibody pairs and combinations
Tests to differentiate between common
cold, influenza and RSV
Diagnosing viral infections is more important
than ever. The influenza A (H1N1) pandemic
has magnified the need for a reliable and fast
method of testing symptomatic patients. Quick
diagnosis is vital in managing the disease effectively with antiviral treatment. Actim Influenza
A&B is a rapid qualitative in vitro assay that
Action needed in
pandemic scale.
As the influenza A (H1N1) infection is sweeping
the world, there is an urgent need for a reliable
rapid test to diagnose patients with symptoms. & search 24788
Copyright © 2009 Medix Biochemica.
We are also looking
for new distributors
for Actim tests!
The original Actim Influenza A&B rapid
test is the answer you are looking for.
The fast and reliable ReaMobile Rapid Diagnostic
System is based on a novel lateral-flow immunoassay technology, which consists of a handheld ReaScan test reader, ReaScan rapid tests and
ReaMobile software. The test procedure takes
10-15 minutes and requires minimal training.
Results can be transferred wirelessly via a ReaLink
Bluetooth device to a mobile phone, or via cable
to a computer for data processing. The rapid test
portfolio includes quantitative and qualitative
applications for CRP, hsCRP, Strep A, and Puumala IgM among others. The test menu is steadily increasing to cover the important diagnostic
markers analysed at the point-of-care. Licensing
or OEM options are available.
Reagena also manufactures a wide range of products for staining in microbiology, pathology and
haematology. One of the most attractive and
widely used products is REASTAIN Quick-Diff
KIT, an optimised colour and fixative composition for the differential staining of cellular elements in peripheral blood smears.
The staining procedure with readyto-use solutions
takes only three
Toivala, Finland
Medica stand Hall 03/F33 & search 24799
News in brief
Severe stress can cause stroke
Many patients urgently admitted to
hospital with cerebral infarction state
that they were under great stress over
a prolonged period prior to suffering
their stroke. This was shown in a unique
patient study conducted in cooperation
between the Sahlgrenska Academy at
the University of Gothenburg and Sahlgrenska University Hospital, Sweden. The study is published in the scientific journal BMC Medicine.
Nearly 600 patients were asked to complete a questionnaire in this study,
no later than ten days after being admitted to Sahlgrenska University
Hospital with acute cerebral infarction. In the questionnaire, the patients
were asked to choose between six different alternatives to indicate how
stressed they had felt before their stroke, from “never been stressed” to
“constantly stressed over the past five years”. The patients’ responses were
compared with subjects in a healthy control group who were asked the
same question.
The study shows that there is a link to stress in those cases where the stroke
is caused by atherosclerosis or to blood clots that have developed locally in
the smaller vessels of the brain. The link was also found for those patients
in whom it had not been possible to establish the cause of the stroke despite
an extensive evaluation. On the other hand, the researchers could not see
any independent correlation with stress for those patients who had had a
stroke due to a blood clot from the heart.
– Issue N°5 – October 2009
Jane Norman, of the University of
Edinburgh, and colleagues from
NHS National Services in Scotland,
researched a population-wide database of linked maternity records,
infant health and death records in
Scotland. They identified 1.49 million singleton births between 1980 and 2004, of which 90,000 were preterm
births. Both spontaneous preterm births and medically-induced preterm
births increased between 1980 and 2004; in absolute terms the rates of
increase in each type of preterm birth were similar. Examining the database,
the researchers found that maternal complications including pre-eclampsia and placenta previa played a decreasing role in preterm births over the
period, whilst gestational and pre-existing diabetes played an increasing
role. The researchers also found that there was an overall decline in stillbirth
and in neonatal and perinatal deaths amongst preterm babies in the period
covered – although at 28 weeks gestation and beyond, stillbirths and perinatal deaths were reduced amongst medically-induced preterm babies, but
were not reduced in babies who were spontaneously preterm.
Key role played by metalloprotease in survival
of Leishmania parasite
Leishmania is a deadly parasitic disease
that affects over 12 million people worldwide, with more than 2 million new cases
reported every year. Until recently, it was
not known exactly how the parasite survives inside human cells. However, a team
led by Dr Martin Olivier from the Research
Institute of the McGill University Health
Centre (RI-MUHC) and McGill University, Canada has elucidated the mechanism. It is hoped the new study,
published in Science Signaling, will lead to development of the first
prophylactic treatment for leishmania.
The parasites enter the bloodstream and are ingested by macrophages
where they block immune function and multiply, spreading to other tissues in the body. The researchers discovered that the metalloprotease
GP63, which is found on the surface of the parasite, plays a role in neutralising the macrophages’ defences. The GP63 protease directly activates
other key molecules that negatively regulate the function of the host cell.
The work is significant in that it is the first study that explains how the
leishmania parasite blocks the immune function of macrophages.
For more information
please visit our booth K88
Hall 3 at MEDICA
Spontaneous and medically induced preterm births
contribute equally to the rising rate of preterm births
Research published recently in the open access journal PLoS Medicine
shows that the rising rate of preterm birth in Scotland is as much a result
of an increase in spontaneous preterm birth as it is of preterm birth that
is medically-induced to avoid risking the lives of the mother and child.
The results emphasise that preterm birth, which remains the single biggest cause of infant death in many developed countries, continues to
be a major obstetric and neonatal problem despite the reductions that
there have been in stillbirths and perinatal deaths as a result of improved
maternal medical care.
BioPorto Diagnostics A/S
Grusbakken 8
DK-2820 Gentofte
(+45) 4529 0000
(+45) 4529 0001
[email protected] & search 24783
– Issue N°5 – October 2009
News in brief
Breakthrough test can prevent fatal kidney failure
Acute kidney injury has long been recognised as a
devastating disorder, which often arises as a complication of other serious illnesses. Despite great efforts to
understand and treat this condition the area has seen
little or no improvement in over 60 years. Paradoxically, the incidence of this potentially deadly disorder
has been rising during the last decades and is now
reaching epidemic proportions. Each year, more than
13 million patients suffer acute kidney injury, of whom more than 30%
have a fatal outcome. With a move that will prove to be a major advance
in the diagnosis of this growing number of affected patients, Denmarkbased BioPorto Diagnostics has announced the successful development of
a groundbreaking test called NGAL that can diagnose acute kidney injury.
A test like this has been on the wish list of doctors worldwide for decades.
Dr Lars Otto Uttenthal, CSO of BioPorto Diagnostics, said that the NGAL
test had the potential to revolutionise the way kidney injury was managed.
Existing methods of determining kidney injury do not provide useful
information about the state of the patient until one to three days after an
injury has occurred. In contrast, the NGAL test will reveal the occurrence
Pos for
of kidney injury within a few hours. Dr Uttenthal said that doctors now
had an opportunity to predict a patient’s outcome – they could take proactive steps at an early stage that would prevent kidney injury from turning
into the very dangerous state of established kidney failure. Thea Olesen,
CEO of BioPorto, said that the icing on the cake was that the NGAL test
was designed to be used on apparatus that already existed in laboratories
and hospitals. In this way the company has ensured that the test will be
affordable and readily available to doctors worldwide. In a market that is
estimated to be worth over a billion dollars, the NGAL test could grab
a considerable share, solidifying BioPorto’s position as the leading player
in the NGAL market. The new NGAL test is developed in collaboration
with one of the world’s leading manufacturers of diagnostic tests and is
currently being prepared for production. The test will be presented this
month at ASN Renal Week in San Diego, USA, the yearly meeting point
for kidney specialists from all over the world.
Promising results for rapid viral diagnosis
tests in emergency rooms
Rapid viral diagnosis tests for respiratory diseases in children who arrive
in emergency departments have
the potential to reduce pressures on
health systems by enabling doctors
to reach a quicker diagnosis, according to Dr Quynh Doan of the British
Columbia Children’s Hospital in Vancouver, Canada, lead researcher of a recent study.
Children who are admitted to emergency departments with cold and
flu symptoms and fever undergo various diagnostic tests and are often
prescribed antibiotics as a precautionary measure, even though viruses,
which are often the cause, do not respond to antibiotics. The burden on
health systems is huge, not only financially, but also in terms of the time
and staff required to reach a diagnosis. Rapid viral diagnostic methods
could help deliver fast, accurate diagnoses, and enable a much more
appropriate use of antibiotics.
The study included data from four trials, which together included 1,588 children. There was some evidence that rapid viral testing reduced use of other
blood or urine tests, chest X-rays and antibiotics, but the results were not
significant. However, the researchers suggest that further, sufficiently large
studies could reveal the true impact of faster tests.
High-sensitivity bone marrow aspiration technology
enhances leukaemia cell detection
3*-4-5-6 NOV. 2009 • CNIT Paris la Défense – France
*Scientific conferences only
Organised by
Superconducting Quantum Interference Device (SQUID) enhanced the
ability to rapidly quantify the amount of nanoparticle-bound tumour
cells in a sample at least 10 fold, and increased sensitivity of minimal
residual disease measurements. Results of this proof-of-concept study
are published in Cancer Research, a journal of the American Association for Cancer Research. These findings are a result of a collaborative
research effort between Senior Scientific, LLC, and the University of New
Mexico, USA. The study was funded by a small business innovation grant
awarded by the National Cancer Institute, USA. The scientists developed
the magnetic marrow biopsy needle in an effort to target tumour cells
with nanoparticles and then preferentially extract the tumour cells with a
magnetic needle. They used anti-CD34 antibody-loaded magnetic nanoparticles to detect CD34+ cells as an indicator of leukaemia. To quantify
the cells recovered, they coupled this nanoparticle-mediated fishing for
leukaemic cells with SQUID. The technique enhanced the sensitivity of
measuring minimal residual disease over standard pathology methods
for patients undergoing chemotherapy.
cli_gb.indd 1
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© 2009 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc.
and its subsidiaries.
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 IntrLogic™ 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
Moving science forward & search 24853
Thermo Scientific Revco refrigerators
and freezers are available for general and
specialized applications including pharmacy,
blood banking and chromatography.
Visit us at Medica 2009
Dusseldorf/Germany – November 18-21
Hall 01 / Both E11
– Issue N°5 – October 2009
Medica Preview
NGAL assay for use on clinical chemistry analysers
Over the past few years NGAL has become
established as a new biomarker for diagnosing acute kidney injury (AKI). Using NGAL
to diagnose AKI enables a marked improvement in the management of
affected patients. Currently there is no preferable technology for the early
diagnosis of AKI. Existing methods of determining kidney damage, e.g.
the commonly used measurement of serum creatinine, only indicate renal
failure resulting from a prior kidney injury at a relatively late stage (24-72
hours) after the injury has occurred, making this worthless for early diagnosis. Moreover, numerous studies have shown that NGAL responds earlier than all other renal status markers and shows a proportionate response
to injury. NGAL, therefore, permits the early diagnosis and prognostic
stratification of acute kidney injury and is expected to enable new specific
therapies to be developed.
Until now only ELISA-based NGAL assays have been commercially
available, and the lack of a more suitable assay platform has impeded the
translation of this promising biomarker from research into routine diagnostics, but NGAL will soon be available for use on clinical chemistry
analysers. BioPorto Diagnostics is expanding its NGAL portfolio with
the forthcoming launch of a turbidimetic NGAL test that is suitable for
use with both blood and urine samples. The test is designed for routine
diagnostic use on a wide range of automated clinical chemistry analysers
that are already running in central laboratories in hospitals all over the
world. Like the majority of other high volume routine biomarkers, the new
NGAL test is based on the principle of turbidimetry, recognised to combine high diagnostic performance with fast turnaround time and therefore
implicitly addressing the acute aspect of NGAL evaluation.
BioPorto Diagnostics A/S
Gentofte, Denmark
Medica stand Hall 03/K88 & search 24817
Slide scanner for optimal histological examinations
With its unprecedented scanning speed and
top-quality on-screen imaging, the new Leica
SCN400 Slide Scanner offers an alternative to
microscopy for the examination of histological
samples in pathology, research and teaching. The
custom tailored lens for a digital sensor, specially
designed for high-res scans, ensures that the
resolution and colour fidelity of the image on
the screen are just as good as that of the microscope image. Thanks to the
dynamic focus principle which keeps the sample in focus for the full duration
of the scan, even difficult samples can be effortlessly digitised. The slide scanner is able to load and scan up to four specimens at a time. With a scanning
rate of 100 secs per 15 x 15 mm at 20x magnification, sample throughput is
substantially increased. The matching Autoloader Leica SL801 is capable of
scanning up to 384 samples at the same time, overnight if required, offering
completely new options for automated operation. The user can keep loading new samples or remove finished scans without interrupting the process.
Once a sample has been digitised, it can be easily retrieved, processed and
made available to a defined group of users.
Leica Microsystems GmbH
Wezlar, Germany
Medica Stand Hall 10/C32 & search 24818
Light resistant tubes
Light-sensitive substances such as certain vitamins and enzymes, metallic
salts, photosensitisers and antibodies coupled with fluorescent dyes must
be stored under special conditions in the laboratory to ensure they are not
degraded by UV radiation, leading to a loss of functionality. Existing brown
glass flasks and polypropylene tubes can effectively protect these solutions
and reagents from the harmful effects of light. However, they are not generally suitable for storing smaller sample sizes in the millilitre range. The
portfolio of Light Protection Tubes for storing light-sensitive materials and
light-sensitive reactions, which includes light-protection centrifuge tubes
in 15 and 50 mL sizes, has been augmented by a 1.5 mL light-protection
polypropylene reaction tube with attached cap. This is a major improvement
for scientists who, until now, had to wrap smaller vessels in aluminium foil.
Compared to transparent standard tubes, products made from pigmented
polypropylene display no light transmission in a broad wavelength range to
ensure optimal protection of the light-sensitive sample.
Greiner Bio-One GmbH
Frickenhausen, Germany
Medica stand Hall 03/B70 & search 24736 & search 24810
Medica Preview
Fully automated clinical chemistry analysers
The Analyticon Biolyzer 200 is a fully automated clinical chemistry benchtop analyser
with a throughput of up to 450 tests/hour. The
system requires minimal space, while offering a broad range of parameters and easy-tooperate software, and is therefore a convenient
solution for small to medium sized laboratories. A second clinical chemistry analyser will
be launched in the first quarter of 2010 with an even higher throughput.
The Biolyzer 600 will extend the company’s line of analysers, covering
medium sized laboratories or even the large laboratory as a specialised
chemistry system and backup solution. A broad panel of parameters
is available for both analysers as well as an optional direct ISE-unit for
measuring electrolytes. Both systems use the same reagent concept,
taking advantage of nearly 30 years of the company’s experience in
producing high-quality clinical chemistry reagents.
Lichtenfels, Germany
– Issue N°5 – October 2009
Analyser for blood samples
The CellaVision DM1200 automates the work
that is traditionally carried out by laboratory
personnel using microscopes. The analyser
performs a preliminary WBC differential
and RBC characterisation, and provides
functionality for platelet estimation. Using
technology for digital image analysis, cells in
blood can be classified automatically, reducing the time taken and providing more standardised analyses. Final verification can be performed by
medical technologists on their computer screens. The results are then sent
automatically to the LIS for archiving. The digital analysis allows results
to be shared and discussed with colleagues within or outside the hospital.
The analyser is compatible with all other products from the company, and
can easily become part of any haemotology workflow.
CellaVision AB
Lund, Sweden
Medica Stand Hall 01/A32 & search 24802
Medica Stand Hall 3/E67 & search 24842
ISE module for clinical chemistry analyser
Designed using automated 2-point calibration for
measuring sodium, potassium and chloride in whole
blood, serum and diluted urine, the ISE module can
be customised for the user’s clinical chemistry analyser easily and flexibly. Results are available within
30 seconds, and the newly developed high sensitive
electrodes and module allow calibration to be carried out only once a
day. Very accurate results are obtained with a C.V of less than 0.5%.
Jokoh Co Ltd
Tokyo, Japan
Medica Stand Hall 01/B26 & search 24800
Filter tips for safer pipetting and greater accuracy
The unique SafetySpace filter tips have more
space between the sample and the filter than
conventional filter tips usually have, thus the
user does not need to worry about the sample
touching the filter regardless of the pipetting
technique or type of liquid being handled. Accurate and precise results are obtained even when
pipetting foaming liquids such as buffers and
proteins. Unlike with other filter tips, these tips
are also suitable for reverse pipetting as well as
for multiple dispensing with electronic pipettes. Designed to meet high
quality and purity demands, these tips are ideal for molecular biology,
microbiology and cell culture applications, as well as radioactive work.
The tip range covers seven different sizes from 10 µL up to 1200 µL.
Packed in colour-coded single tray boxes, and certified DNase, RNase
and endotoxin free as well as pre-sterilised, the tips are compatible with
all Biohit pipettes and most other pipette brands.
Biohit Ltd
Helsinki, Finland
Medica Stand Hall 02/A49 & search 24812 & search 24792
Medica Preview
Cystatin C assay
The range of tests available on MODULAR Analytics and cobas platforms for the assessment
of glomerular filtration rate (GFR) has been
extended with the launch of a new assay for the
early assessment of glomerular filtration rate
(GFR) in patients with impaired renal function.
The Roche Cystatin C assay is more sensitive than
creatinine measurements, allowing diagnosis of
chronic kidney disease (CKD) at an early stage
when therapeutic intervention is possible.
Unlike creatinine, cystatin C levels are not
affected by muscle mass, age (below 50), gender
or inflammation and cystatin C is only eliminated
via filtration, making it a convenient and accurate
marker for the assessment of GFR. By indicating a
reduction in GFR sooner, it is ideal for the assessment of patients in the early stages of CKD and
reduces the need to perform invasive determinations of GFR. The Roche Cystatin C assay is promoted for use in the assessment of patients under
the age of 60, for early diagnosis of initially minor
kidney damage (for example in diabetes patients).
It can also be used in monitoring kidney disease
and post-transplantation patients, and for dose
adjustment of renally excreted drugs. The assay
is easy to use, requires minimal handling, and
produces highly precise and sensitive results from
low sample volumes (2µL).
Roche Diagnostics GMBH
Penzberg, Germany
it is easy to disassemble for cleaning and servicing.
Five models are available, with adjustable volumes
ranging from 0.25mL to 60mL. High precision and
accuracy is ensured through several stages of strict
quality checks during the manufacturing process. Each instrument is individually calibrated in
accordance with ISO 8655 standards and comes
with an individual calibration certificate.
Bottle top dispensor
Responding to current needs, the Microlit bottle
top dispensor is a unique combination of affordable pricing and high performance. The simple
design together with a variety of useful features
facilitates all routine dispensing needs. The plunger
movement is smooth and effortless. Each instrument comes with four adapters to fit most laboratory reagent bottles. The safety discharge system
reduces the risk of accidental dispensing and the
nozzle cap prevents any unwanted drops landing
on the work space. The instrument is fully autoclavable at 121 °C, 15psi, and whilst there is no need
for disassembling the instrument for autoclaving,
Medica Preview
Designer antigen-based ELISAs
A new milestone in the serological
diagnosis of coeliac disease
DNA sequence coding for
Gliadin analogous peptides
Medica Stand Hall 03/K36 & search 24843
Upright microscope with built-in
LED illumination
Offering consistent longterm performance for
biological and medical
applications, the costeffective CX21LED clinical microscope with builtin LED illumination can
illuminate samples with
a similar light intensity
to that provided by halogen bulbs. Furthermore, the LED light source
produces a more uniform, controllable and stable illumination for high-quality imaging. As an
environmentally-friendly and easy-to-use system,
this microscope is also ideal for educational use.
Hamburg, Germany
Medica Stand Hall 10/C20, 15/G12 & search 24809
Rapid test for TB
TB DNA rapid
test is an accurate,
speedy and crosscontamination
acid detection kit for the qualitative detection
of Mycobacterium tuberculosis (TB) DNA in
human samples, and a clinical diagnostic tool
for TB infection. The test kit includes sample
preparation, nucleic acid isothermal amplification & hybridisation, and finally detection
using an innovative, cross-contaminationproof, user-friendly device that easily fits into
the human hand. Superior sensitivity of has
been demonstrated compared to solid media
culture and smear microscopy.
MP Biomedicals
Santa Ana, CA, USA
Medica Stand Hall 03/H56 & search 24745
Peptide 1
Peptide 2
GAF(gliadin analogous
fusion peptide)
Lucknow, India
Medica Stand Hall 02/A07 & search 24742
– Issue N°5 – October 2009
Chemical synthesis
Sequence optimisation
and assembly
Expression in
E. coli
The Anti-Gliadin (GAF-3X) ELISA is based on
a state-of-the-art recombinant gliadin-analogue
fusion peptide, and provides significantly higher
sensitivity and specificity than conventional
anti-gliadin ELISAs. At 95% specificity the new
ELISA showed a sensitivity of 83%/94% (IgA/
IgG), compared to 54%/31% (IgA/IgG) for a
conventional ELISA. The Anti-Gliadin (GAF3X) ELISA (IgG) is especially suited for detection of patients with IgA deficiency, which is
frequently associated with coeliac disease.
A new standard in the diagnosis
of Wegener’s granulomatosis
The Anti-PR3-hn-hr ELISA employs an optimised
antigen combination of human native (hn) and a
designer, human expressed recombinant (hr) proteinase 3, which provides an unsurpassable antigen
spectrum and unrivalled sensitivity for the detection of anti-PR3 autoantibodies. In a clinical study,
94% of cANCA reactive sera (indirect immunofluorescence) showed a positive reaction in the AntiPR3-hn-hr ELISA, compared with 88% for capture
ELISA and 78% for conventional ELISA.
A novel ELISA to aid diagnosis of
primary biliary cirrhosis
PBC spec.
PBC spec.
138 aa
118 aa
PBC spec.
82 aa
The Anti-M2-3E ELISA is based on a designer
fusion protein, which contains all three relevant
enzyme complexes (3E) of the mitochondrial
antigen M2 (AMA M2): pyruvate dehydrogenase, branched-chain 2-oxoacid dehydrogenase
and oxoglutarate dehydrogenase. In a clinical
study the Anti-M2-3E ELISA showed a previously unattained sensitivity of 93%, compared
to 79% for a conventional anti-M2 ELISA based
solely on pyruvate dehydrogenase. The specificity of the new test system was 98%.
Luebeck, Germany
Medica Stand Hall 1, Booth A08 & search 24835
– Issue N°5 – October 2009
Medica Preview
Custom-made horseradish peroxidase and glucose oxidase
BBI Enzymes has
launched a new
custom specification service which
offers horseradish
glucose oxidase tailor-made exclusively for customers. The efficient service creates the enzymes
specifically to each customer’s own unique
requirements, and promises batch to batch consistency, optimised isoenzyme content, and high
purity and specificity. This service is the first
launch from the company’s new state of the art
facility following a significant investment into
BBI Enzymes. Promising a fast turnaround and
guaranteeing highly competitive pricing, the
service comes backed by BBI Enzymes’ 50 years
of expertise and is ideal for anyone looking for a
guaranteed enzyme supply.
BBI Enzymes
Blaenavon, Gwent, UK
susceptibility to HIV. Many physicians do not
routinely complete an examination unless a possible pregnancy is involved and yet this is the most
common form of vaginal disease. The standard
Gram stain test is, however, time consuming.
A 60 second cost effective diagnostic tool for a
quick, qualified, quality diagnosis of bacterial
vaginosis, the VGTest, is now available. This new
diagnostic tool will enable specialists, general
practitioners and laboratories dedicated to women’s healthcare, to quickly test, diagnose and so
provide appropriate on the spot treatment. Based
on ion mobility spectrometry technology, already
a proven technology for detecting drugs, chemicals and explosives, the VGTest shows excellent
laboratory results and provides a correct diagnosis within 60 seconds. The test detects elevated
levels of trimethylamine (TMA), which is present
in cases of infection or dysfunction.
Arad, Israel
Medica Stand Hall 03/E44 & search 24845
Medica Stand Hall 1/F24 & search 24797
Rapid diagnosis of bacterial
Between 10-64% of
the female population
worldwide is at risk of
the vaginal condition
bacterial vaginosis at
any given time. The disease very often remains
asymptomatic and is thus undetected. BV can
cause gynaecological and obstetric complications, including miscarriages, preterm births,
pelvic inflammatory disease (PID), chorioamnionitis, puerperal endometritis and increased
Anti-human adiponectin Mabs
Adiponectin is an important regulator of lipid
and glucose metabolism with anti-diabetic, antiinflammatory and anti-atherogenic properties.
The potential diagnostic use of adiponectin has
been a subject of increasing interest in recent
years. There are several oligomeric forms of native
adiponectin circulating in the blood, namely low-,
medium- and high-molecular weight forms. Biological activity of adiponectin is mediated by the
high-molecular weight form (HMW) and, not
surprisingly, it has been suggested recently that
concentration of the HMW form of adiponectin or the ratio HMW/total adiponectin (sum
of three types of oligomers) in serum correlates
more strongly with insulin resistance and other
measures of type 2 diabetes than total adiponectin. A new generation of anti-human adiponectin
monoclonal antibodies suitable both for research
purposes (Western blotting, direct ELISA) and
for the development of adiponectin-specific sandwich immunoassays are now available, as well as
native antigen purified from human plasma.
Hytest ltd
Turku, Finland
Medica Stand Hall 03/F44 & search 24738
Expanded range of quality controls
Acusera, Randox’ range of quality control sera, has
expanded to include more new controls. The controls now include: blood gas; coagulation; drugs
of abuse; liquid cardiac; liquid clinical chemistry;
maternal screening; tumour markers and urinalysis. These assayed quality controls are highly
accurate, and are supplied with instrument and
method-specific values. Bottles, caps and packaging are all colour-coded to help distinguish different analyte levels and minimise costly errors. The
company also offers customised quality control
materials tailored to the customer’s specifications
allowing uncompromised performance.
Randox Laboratories
Crumlin Co. Antrim, UK
Mecica Stand Hall 03/A08 & search 24743
Software for analysis of flow
cytometer data
A new version of the software for the analysis of
data acquired with FlowCytomix, a technology
for multiple analyte detection on a flow cytometer, is available. FlowCytomix Pro 2.3 is very
user-friendly, and incorporates many new features. Analysis is even more precise, since the new
software offers the option to assay single samples
and standards, duplicates or triplicates. File information is retained for further analysis, and the
loading of files is faster. Individual settings can be
saved at different stages, facilitating subsequent
analyses. All plots generated in the course of an
analysis can be saved and printed for improved
data preparation.
Bender MedSystems GmbH
Vienna, Austria
Medica Stand Hall 03/F56 & search 24793 & search 24741
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Clean room for autoimmunity tests, Biosystems
Biosafety laboratory for infectious immunology tests, BioSystems
Costa Brava 30, 08030 Barcelona (Spain) Tel. +34-93 311 00 00 Fax +34-93 346 77 99
[email protected] • & search 24529
– Issue N°5 – October 2009
Product News
Single and multi channel pipettes
Fully autoclavable and UV resistant, the Smart
range of Accumax pipettes includes both single
and multi channel models. An over-moulded
TPE grip, sensitive 4 digits counter, Teflon sealing and unique locking system facilitate use.
The ergonomic design and engineering ensure
light weight handling, smooth plunger action
and ease in operation.
This range of pipettes is subject to the same
stringent quality management system which
made Accumax the first and only micropipette
manufacturer in Asia-Pacific to have a calibration laboratory accredited with the highest
standard for calibration: ISO 17025. & search 24795
Gandhinagar, Gujarat, India
Medica Stand Hall 3/K66 & search 24846
Hepcidin ELISA
A test kit is now available that facilitates the
timely diagnosis of iron disorders, including
haemochromatosis and anaemia; this diagnostic kit measures the level of the hormone hepcidin. In recent years, scientists have discovered
that hepcidin helps regulate the amount of iron
in humans. An unbalanced iron level can lead
to many common medical conditions including anaemia and iron overload diseases, or it
can occur in chronic kidney disease, inflammation, or diabetes mellitus. The DRG Hepcidin 25
(C-Terminal) ELISA Kit will be available to medical professionals worldwide, providing the first
simple, fast and accurate method to test patient
hepcidin levels. The results offer more information to clinicians to facilitate the diagnosis and
treatment of medical conditions including iron
deficiency diseases, some of the most common
diseases worldwide. The kit provides accurate
information and is user-friendly.
DRG International
Mountainside, NJ, USA & search 24794 & search 24811
Lab systems for mid-volume
clinical laboratories
The Dimension Vista 500 Intelligent Lab System is the latest addition to Siemens’ family
of ultra-integrated chemistry and immunochemistry systems. The new system has a test
menu of more than 115 assays and offers test
panels for anaemia, cardiac disease, thyroid
disorders, therapeutic drug monitoring, protein testing, drugs-of-abuse testing and routine
and specialty chemistry testing—all on one
system and from a single patient sample. With
the option of two configurations, the system
meets the varying needs of mid-volume laboratories: throughput is up to 1,000 patient tests
per hour. The Dimension Vista 1000T Intelligent Lab System is also available by seamlessly adjoining two Dimension Vista 500 Systems, doubling the throughput capacity to up
to 2,000 tests per hour and providing mirror
back-up capabilities and increased walk-away
time for laboratory personnel. Identical to the
other Dimension Vista systems, four advanced
detection technologies including photometry,
nephelometry, V-LYTE electrolyte and LOCI
advanced chemiluminescence are used on the
systems, resulting in a broad test menu. The
LOCI technology allows access to fast, sensitive immunoassay testing and a fast analytical
turnaround time of 10 minutes for critical cardiac tests such as high-sensitivity Troponin I.
Siemens Healthcare Diagnostics
Newark, DE, USA & search 24748
Buprenorphine Enzyme
Beckman Coulter has expanded
its drugs of
abuse test menu
to include an
enzyme immunoassay for the
opioid, buprenorphine. The test provides fast
screening for the qualitative and semi-quantitative determination of norbuprenorphine
(buprenorphine metabolite) in human urine.
Reagents and calibrators for the assay are liquid and ready-to-use, eliminating the need
Product News
to mix, hydrate or pre-dilute reagents before
testing. The assay’s cutoff value is 10ng/mL
for norbuprenorphine, and it delivers analytical sensitivity of 3ng/mL for buprenorphine and norbuprenorphine (the active,
dealkylated metabolite of buprenorphine). It
cross-reacts at 94% with buprenorphine. Like
all the company’s Drugs of Abuse Test assays,
this test is formulated to deliver the critical
elements of effective DAT analysis, namely
speed, accuracy, ease-of-use and economy.
When used in conjunction with Synchron
UniCel Clinical Chemistry Systems and i
class Integrated Systems, this high quality
assay provides timely, reliable and efficient
DAT results.
Beckman Coulter, Inc
Nyon, Switerland & search 24844
HA LT for automated measurement
of hyaluronic acid
– Issue N°5 – October 2009
Semi-automatic analyser
Using state-ofthe-art technology, the BTS-350
new generation
based on LED
technology. It embraces concepts such as low
energy consumption, low toxicity and a practically unlimited lifetime. Currently this is the
only semi-automatic analyser that functions
on a complete range of LEDs, has a USB drive
to allow archiving and data transfer and a battery pack that not only functions as a back up
when electricity fails, but also allows the analyser to be used anywhere at anytime. Furthermore, with hard coated filters, advanced
ergonomics and improved optics, as well as low
energy operation, the analyser requires basically no maintenance. In addition to the new
and improved hardware technology, an intuitive, straightforward and user-friendly software encompasses all of the parameters necessary for routine assays with an excellent quality
control scheme.
An ideal solution to low-to-midsize laboratories worldwide, this analyser is flexible, reliable
and optimised for IVD and laboratory testing.
BioSystems SA
Barcelona, Spain & search 24814
Diatron’s Reagent
Division offers high
quality reagents for
Sysmex, Mindray,
Beckman Coulter,
Abbott, ABX, Erma
hematology analyzers
• Long stability
• Proven reliability
• Cyanide free
• 20 years of experience
• High quality standards:
A unique new test for the automated measurement of Hyaluronic Acid (HA) in serum
and plasma using standard biochemical
analysers is now available. Patient samples
are mixed with a recombinant HA binding
protein (HABP) in the HA LT reagent. Latex
particles coated by anti-HABP antibody are
added resulting in increasing turbidity. The
high molecular weight glycosaminoglycan
HA is the best single biomarker for hepatic
fibrosis. It shows a negative predictive value
(NPV) of up to 100% for cirrhosis of various aetiologies. Several biomarker combinations have been validated to enable the
accurate staging of different fibrosis stages
as non-invasive alternatives to liver biopsies.
Fibrometer and Hepascore are the most powerful scores that include HA in their panels.
HA LT by Wako can significantly improve
such scores in terms of automation, simplicity and analytical performance. The test
is highly precise, accurate and offers a high
linear range from 10 to 1000 ng/mL.
Whole genome amplification kit
Current single-cell
WGA kits using
molecular displacement amplification
(MDA) technology
do not offer reproducible amplification
of whole genomes,
causing sporadic allele and locus dropouts, which
seriously compromise results in Preimplantation
Genetic Diagnosis (PGD). Hence, microarray and
qPCR genetic analysis of single cells using MDAamplified DNA is noisy and produces unreliable
data. The PicoPlex Single Cell Whole Genome
Amplification (WGA) kit allows researchers to
get the same amount of genomic information
from one cell as they do from 10,000 cells. This
technology enables labs to begin qPCR, microarray or sequencing analysis less than three hours
after sample collection. The kit reproducibly
amplifies total DNA one million-fold from single cells to produce five micrograms of amplified
DNA in less than three hours.
Wako Chemicals GmbH
Rubicon Genomics Inc.
Neuss, Germany
Ann Arbour, MI, USA & search 24815 & search 24807
ISO 9001 and ISO 13485
• Produced in EU
• Reasonable price
For more information please contact us at
[email protected]
Visit us on Medica 2009
Hall 3, Stand A07 & search 24641
– Issue N°5 – October 2009
Surgical Pathology of Endocrine and
Neuroendocrine Tumors
Ed. by Ashraf Khan,
Pub. by Springer (Humana), 2009, 242pp, € 149
Surgical pathology is the
cornerstone in the management of neoplastic disorders. Written for the practicing surgical pathologist in
mind, this book, one of the
Current Clinical Pathology
series, provides an up-todate text on surgical pathology of endocrine
and neuroendocrine tumours. The text begins
with radiological imaging of tumours, followed by a section on fine needle aspiration
biopsy. The main section focuses on surgical
pathology of endocrine and neuroendocrine
tumours. The volume closes with applications
of molecular techniques and their potential for
the future. Written by a panel of internationally recognised pathologists who are experts in
their fields, the authors contribute their own
valuable insights into making this new book
an essential resource for practicing general and
specialist surgical pathologists and pathologists
in training.
Methods and Protocols
Ed. by Chi Wai Eric So
Pub. by Springer (Humana), 2009, 428pp, €74,95
The molecular basis and
pathogenesis of leukaemia
have been clarified, but better strategies are needed to
diagnose, classify and treat
this biologically and clinically diverse disease. Here
experts bring together a wide
range of state-of-the-art laboratory methods
and detailed protocols that are useful for both
clinical and basic research scientists working
on the disease. The volume provides techniques
for: prenatal backtracking of leukaemic clones;
molecular diagnosis; detection of genome-wide
genetic abnormalities and profiling; identification of unknown fusion genes; monitoring of
minimal residual diseases; disease modelling
using murine and human primary haematopoietic cells; studying of normal and malignant
haematopoiesis; identification of interacting
partners with leukaemia-associated oncoproteins; and global characterisation of genomewide epigenetic changes in leukaemic cells.
Clinical and Diagnostic Virology
by Goura Kudesia and Tim Wreghitt
Pub. by Cambridge University Press 2009,
276pp, £29.99
This basic but comprehensive
text is aimed at all healthcare
professionals who need a
clear understanding of medical virology. Written by two
highly experienced virologists, the book is divided into
five sections, namely individual viruses; other related agents; clinical
syndromes; diagnostic techniques; and patient
management. The individual virus chapters
provide information on incubation period,
infectivity, control of infection and management. The clinical syndrome chapters provide
information on the clinical presentation of disease, thus enabling the reader to search according to patient symptoms rather than referring
to several individual virus chapters. The standard chapter formats, simple language and liberal use of tables, figures and algorithms enable
quick access to key information, and the comprehensive coverage of all viral agents is unique
in a practical guide of this size.
Cambridge University Press
New York, NY, USA
New York, NY, USA
Cambridge, UK & search 24847 & search 24687 & search 24848
Calendar of events
November 4-6, 2009
Journées Internationales de
Biologie (JIB)
Paris, France
Syndicat des Biologistes & Reed
Expositions France
Tel. +33 1 47 56 50 79
Fax +33 1 47 56 52 58
e-mail : [email protected]
LABEX is a very experienced producer of
reagents for various types of blood cell
counters. All products are CE-marked and
marketed world-wide through exclusive
distributors. For further information about
products, your local supplier or distribution
partnership, please contact LABEX.
Welcome to our new stand location
Hall 1/F29
At MEDICA 2009
18 -21 NOV. 2009
P.O. Box 22159
SE-250 23 Helsingborg
Tel +46 42 32 40 00
Fax +46 42 20 27 71
[email protected] &&search
November 5-7, 2009
7th Annual World Congress on
Insulin Resistance
San Francisco, CA, USA
Tel. +1 818 342 1889
Fax +1 818 342 1538
e-mail: insulinresistance@
November 18-20, 2009
ESCMID Conference on Enterococci: from Animals to Man
Barcelona, Spain
European Society of Clinical
Microbiology and Infectious
Tel. +41 61 686 7799
Fax +41 61 686 7798
e-mail: [email protected]
November 18-21, 2009
Düsseldorf, Germany
e-mail: [email protected]
November 19-22, 2009
AMP 2009
Kissimmee, FL, USA
Association for Molecular
Tel. +1 301 634 7939
Fax +1 301 6347990
e-mail: [email protected]
December 11-13, 2009
Pragati Maidan, New Delhi,
Vantage Trade Fairs (P) Limited
Tel. +91 11 3058 0444 / 3058 0777
Fax +91 11 3058 1000
[email protected]
January 16-17, 2010
Melanoma 2010: 20th Annual
Cutaneous Malignancy Update
San Diego, CA, USA
Scripps Health
Tel. +1 858-652-5400
Fax +1 858-652-5565
[email protected]
January 25-28, 2010
MEDLAB at Arab Health 2010
Dubai, United Arab Emirates
IIR Middle East
Tel. +971 4 3365161
Fax +971 4 3364021
Microbiology & Infectious
Vienna, Austria
Congrex Switzerland Ltd
Tel. +41 61 686 77 11
Fax +41 61 686 77 88
e-mail: [email protected]
February 25-28, 2010
Early Disease Detection and
Prevention (EDDP) conference
Munich, Germany
Paragon Conventions
Tel. +41 22 5330 948
Fax +41 22 5802 953
e-mail: [email protected]
April 18-21, 2010
63rd CMEF Spring 2010
Shenzen, China
Reed Sinopharm Exhibitions
Tel. +86 10 6202 8899 ext
Fax +86 20 6235 9314
e-mail: [email protected]
February 27- March 2, 2010
The Role of Telomeres and
Telomerase in Cancer research
Fort Worth, TX, USA
American Association for
Cancer Research
Tel. +1 215 440 9300
Fax +1 215 440 9313
e-mail: [email protected]
April 10-13, 2010
ECCMID 2010 – 20th
European Congress of Clinical
May 9-13, 2010
Focus 2010
Glasgow, UK
Association for Clinical
For more events see:
Dates and descriptions of future
events have been obtained from
official industrial sources. CLi cannot
be held responsible for errors,
changes or cancellations.
Are we going in the
right direction?
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the right path with its broad menu and connectivity options.
With more than 20 years of proven immunoassay performance, the new IMMULITE® 2000 XPi* System maximizes
productivity and reliability. As with all IMMULITE® systems, it features a wide range of routine, allergy and
specialty assays, while easily connecting to automation and informatics platforms. Find out which IMMULITE
system will place your lab on the right path at
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A91DX-9054-A1-4A00 & search 24782
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© 2009 Siemens Healthcare Diagnostics Inc. All rights reserved.
IMMULITE is a trademark of Siemens Healthcare Diagnostics Inc.
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EIA Kit, April 2008 & search 24784