how efficient and automated can be serology and stool testing?

HOW EF FICIENT AND AUTOMATEDCAN BE SEROLOGY AND STOOL TESTING?

DIASORIN SCIENTIFIC CONTRIBUTIONEUROMEDLAB PARIS, JUNE 23, 2015

CONFERENCE INSIGHT BOOKLET

CONFERENCE INSIGHT

How efficient and automated can be Serology and Stool Testing?

DiaSorin scientific contribution

Euromedlab Paris, June 23, 2015

Moderator:V. Sambri

Faculty:P. HuynenB. Burde

Year XIII, N. 9, October 2015


DiaSorin scientific contribution Euromedlab Paris, June 23, 2015

ISBN 978 88 6756 1834ISSN 2038 8667

Editorial BoardElena BernacchiMassimo ChiesaMaddalena CastelliClaudio Oliveri

ProductionAnnalisa Pietrasanta

CONFERENCE INSIGHTYear XIII, N. 9, October 2015

© 2015 Springer Healthcare Italia S.r.l.Conference Insight. Registered in Milan - Registration n. 712 - 12/18/2002Publishing Director: Giuliana GerardoPrinted in Italy in October 2015 by Lazzati Industria Grafica S.r.l., Casorate Sempione (VA)Publication not for resale aimed at medical practitioners.All rights reserved throughout the world and in all languages. No part of this publication may be repro-duced, transmitted or stored in any form or by any means either mechanical or electronic, including photocopying, recording, or through an information storage and retrieval system, without the written permission of the copyright holder. The publishers have made every effort to trace the copyright holders for borrowed material. If they have inadvertently overlooked any, they will be pleased to make the neces-sary arrangements at the first opportunity. Although great care has been taken in compiling the content of this publication, the publisher and its servants are not responsible or in any way liable for the currency of the information, for any errors, omissions or inaccuracies, or for any consequences arising therefrom.Please be informed that the contents of this material may be used only if compliant with local laws and regulations.This publication is not a peer-reviewed publication. All opinions expressed in this publication reflect those of the authors and not necessarily those of Springer Healthcare Italy. The possible use of the trade names has the mere purpose of identifying the products and does not imply any suggestion of use. Each product must be used in accordance with the instructions for use (IFU) and/or summary of product characteristics (SPC) supplied by the relative manufacturing company.Publication made possible by an educational grant from DiaSorin

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DiaSorin scientific contributionEuromedlab Paris, June 23, 2015

Moderator:V. Sambri, MD PhDUnità di Microbiologia, Laboratorio Unico di Area Vasta Romagna, Cesena - Italy

Contents

Quality and Innovation, key factors for laboratory evolution 2P. Huynen, MDCentre Hospitalier Universitaire de Liège, Liège - Belgium

Efficiency improvement in stool testing: an automation approach 5 B. Burde, MDBioscientia Institut für Medizinische Diagnostik GmbH, Berlin - Germany

Questions and Answers 8


2

A laboratory has to face several challenges every day: a constant increase in the number of tests to be

performed, the need to provide results quickly and the need to achieve a lower turn-around-time (TAT),

while maintaining quality assurance and complete traceability of the results. In this context, in order to

meet all the clinical needs efficiently without any compromise on quality, one solution could be to look

towards innovation and optimize the laboratory with innovative systems (Figure 1).

Automated techniques are currently adopted for the most commonly used serological methods.

The first step towards innovation is to move away from the ELISA method to embrace a new technology,

chemiluminescence (CLIA), and the new fully automated analysers: LIAISON® and LIAISON® XL.

CLIA systems represent a technical improvement over the ELISA automated system. They reduce TAT,

while maintaining high quality and full traceability, with the added advantage of a random access system.

Ready-to-use reagents on board, a touch-screen monitor, auto-dilution, re-run and reflex testing auto-

matically performed by the system and a STAT position for emergency results are some of the technical

characteristics of the new systems. Moreover, with the LIAISON® systems it is possible to test several

types of samples (e.g., serum, cerebrospinal fluid and stools) at the same time, in the absence of

cross-contamination.

With LIAISON® XL, efficiency can be improved with several advantages: new infectious disease markers

(e.g., HIV, hepatitis C); a larger number of reagent integrals on board (from 15 to 25); no more daily

maintenance, and disposable tips. Moreover, most of the reagents are the same for the LIAISON® and

LIAISON® XL systems, which means easy validation files; the system also provides a backup, if needed.

Another challenge in a microbiology laboratory is performance. Implementation of highly automated

instrumentation fulfils the needs of the laboratory – i.e., TAT and logistic improvement without any

compromise on quality - and enables a good response to clinical needs, including confidence in the

results and flexibility (Figure 2).

Quality and Innovation, key factors for laboratory evolution

P. Huynen

Figure 1. The challenge of our laboratory

75.000

100.000

125.000

150.000

2006 2008 2010 2012 2014

↑ N tests/year New clinical needs↓TAT

Quality assurance→ ↑ administrative

tasksAccreditation → traceability

Same human resources

Tota

l num

ber

of t

ests

To satisfy all the clinical needs efficiently and without any compromise on quality, one solution could be to look towards innovation.

CLIA systems reduce the TAT, with high quality, full traceability and with the advantage of a random access system.

CONFERENCE INSIGHT

3

The chronological evolution of the Liège laboratoryThe laboratory of Infectious Serology and Antigen Detection is part of the division of Clinical Microbiology

of the University Hospital of Liège and performs both infection serology and antigen detection for infec-

tious diseases including Clostridium difficile.

The collaboration with DiaSorin started in 2001. Since that time, DiaSorin has been able to support the

evolution of the Liège laboratory with an increasing number of infectious disease markers available on

the fully-automated analyser and with the ability to consolidate the serology platform.

After that, in addition to the ETI-Max 3000 (ELISA analyzer), between 2003 and 2006 three LIAISON®

systems were acquired and an increasing number of infectious disease markers were implemented over

the years. In 2011, with the advent of the LIAISON® XL system, the LIAISON® was gradually phased out.

Finally, C. difficile diagnosis on stool specimens was introduced in 2013, first on the LIAISON® and then

on the LIAISON® XL (Figure 3).

Experience with Clostridium difficile infection diagnosisA fully automated solution is necessary for the diagnosis of C. difficile infection especially in a laboratory

that performs a large number of tests per year.

The advantages of a random access system are flexibility and the ability to provide results quickly and

several times a day, as with the rapid tests but with the full traceability of an automated system. More-

over, the LIAISON® can run toxin tests on stools but also on bacterial colonies of C. difficile.

The picture shows the workflow in the Liège laboratory. Blood and cerebrospinal fluid samples are ali-

quoted (if needed) and then tested either manually or by the ELISA automated system or by CLIA. For C. difficile diagnosis, after a short extraction step, stools go directly to the LIAISON® system for antigen and

toxin detection. At the same time, they are also incubated for 24-48 hours in the bacteriology laboratory,

and the colonies obtained after incubation can be tested for toxins on the LIAISON®, if needed (Figure 4).

Future perspectivesA large panel of fully automated parameters has been implemented on the LIAISON® system over the

years, and new parameters will be available in the near future, e.g., for Chlamydia, Bordetella pertussis

toxins, and, for stool testing, for adenovirus and rotavirus antigens.

Figure 2. Interaction between laboratory and clinicians

↑ N tests

↑ N tests

Good responseto clinical needs

Same human resources

Full automationSerum, CSF - Stool

↓TAT

Quality of the results

Moving from ELISA to CLIA allows us to consolidate our serology platform and to successfully face all our challenges. Quality and innovation are key success factors for the evolution of the laboratory


4

To conclude, clinical needs have to be met efficiently and without any compromise on quality.

Moving from ELISA to CLIA allows us to consolidate a serology platform and successfully tackle

all the challenges faced by laboratories. Indeed quality and innovation are key success factors

for the evolution of the laboratory and contribute to improving performance with a good response

to clinical needs.

Figure 4. From sample to results

Blood, CSF

Infe

ctio

us S

erol

ogy

Bac

teri

olog

y

C. dif�cile

Manual serology

ELISA

CLIA

Aliquoting

Stool

24 - 48h

GDHToxins

C. dif�cile

12 min.

Figure 3. Innovation in our laboratory

ETI-max (ELISA)

2001 2003 2005 2007 2009 2011 2013 2015

LIAISON® (CLIA)

M. & C. pneumo, C. tracho,Parasites, Measles, Mumps,

Respi virus, VZV,HIV, HCV, HSV, Aspergillus

M.& C.pneumo, C. tracho,Parasites, Measles, Mumps,

Respi virus, HIV, HCV, PARVO

C. pneumo, C. tracho,Paras, Measles, Mumps,Respi virus, Aspergillus,

HTLV

C. pneumo, C. tracho,Respi virus, Asperg,

HTLV, ParasitesH. PYLORI

HBV, HAV,EBV, CMV,Toxo, Rub,

Borrelia

CSFBorrelia

STOOLC.dif�cile

HSV, VZV,Syphilis

HIV, HCV,M. pneumo

Parvo

MeaslesMumps

144.000 tests/y.

CONFERENCE INSIGHT

5

To understand the possible application of fully automated methods in microbiology, it is first necessary to

focus on why, where and how these tests, and in particular immunological stool testing, should be used.

The main reasons to run a test are to identify the cause of symptoms, to treat the patient appropriately

and to avoid complications.

Sometimes there are no standardized reasons to request a test. A study from the Netherlands (Van Den Berg RJ et al) tested a number of stool samples for C. difficile; the stool samples were divided into two

groups, in one group C. difficile testing was requested and in the other it was not. The results showed

the same C. difficile positivity rate (8%) in the two groups. So, why was this test requested or why was

it not?

Immunological tests using blood are very well established in that they have good sensitivity and speci-

ficity. By contrast, stool testing is challenging as there could be different concentrations of the target in

each sample and many endogenous materials can interfere with the test. However, when an infection

(e.g., C. difficile) is local it must be tested on the spot.

Concerning methodology, there is a choice between classic culture microscopy and immunological

methods. Classic microbiology is tricky, slow and needs experienced personnel; on the other hand,

immunological tests are highly automated, quick to run and require no special experience of the staff.

Immunological methodsImmunoassays are all based on specific antigen-antibody reactions. They can be used to test for virus-

es, bacteria, toxins, drugs, etc. Typical examples of these methods are: EIA (enzyme-linked immunosor-

bent assay), ELISA (enzymatic colour reaction), RIA (radioimmune assay) and CLIA (chemiluminescence

assay).

In ELISA (and EIA), an antibody (or antigen on EIA) is marked with an enzyme. The reaction catalysed

by the enzyme is a representation of the presence of the antigen. The result can usually be detected by

a colour change, fluorescence or chemiluminescence.

Another typically used test is the Sandwich-ELISA. The Sandwich-ELISA uses two different antibodies

(Ab) binding to the specific antigen, but on different epitopes.

Chemiluminescence (CLIA) is a process in which a chemical reaction emits electromagnetic radiation

in the visible light region. The maximum light emission is up to 1 sec., which allows a very high sample

turnover that is important if rapid testing is needed.

Automation in microbiologyAutomated processes are fast, turn-around-time is short and staff costs per specimen are low. On the

other hand, the implementation of automated systems is complex and the cost is high.

The main opposition to automating microbiology is that microbiology is strictly separated in many labo-

ratories. As an example, in a typical workflow for stool testing the only connection between microbiology

and the laboratory is the administration (Figure 1).

Efficiency improvement in stool testing: an automation approach

B. Burde

“Automation” means not only running a test but also integrating the test into the laboratory

We have designed a new workflow to transfer microbiology tests into an automated laboratory, to overcome old boundaries and establish an interactive process


6

In order to automate microbiology, a new workflow has been designed to transfer microbiology tests into

an automated laboratory, to overcome old boundaries and establish an interactive process. This workflow

shifts the microbiology section into the automated laboratory. Entry of the specimen and registration of

the patient and testing are done in the laboratory; in the processing phase there is interaction between

the two sections, and validation and report printing is done in the laboratory. This way, there is dynamic

registration and interactive processing. (Figure 2)

Figure 1. Typical Workflow

Figure 2. New Workflow

➜ Microbiology takes much time ➜ Microbiology is a separate section

➜ Interaction only for registration and reporting

LABORATORY

Entry (Specimen)

Registration(Patient)

Print(Report)

MICROBIOLOGY

Registration(Test)

Processing

Validation

Stool testing runs in microbiology!

➜ Dynamic registration ➜ Interactive processing

LABORATORY

Entry (Specimen)

Registration(Patient)

Processing

Print(Report)

MICROBIOLOGY

Registration(Test)

Processing

Validation

Shift “Microbiology” into “Automated”-Laboratory

The new workflow is much faster and provides a better test quality

CONFERENCE INSIGHT

7

To test the differences between classic and immunological tests, and between classic and automated

workflow, a good example could be the diagnosis of C. difficile.

C. difficile is an anaerobe, Gram-positive, spore-forming bacteria. Pathogenic C. difficile strains produce

multiple toxins; complications are toxic megacolon, colon perforation and septic shock. “Classic” tests

to detect C. difficile in stools are: culture or PCR tests. These classic tests show a good correlation with

immunological tests, such as glutamate-dehydrogenase (GDH) or toxin tests.

In order to test the new workflow using LIAISON® XL, a highly automated laboratory and a classic micro-

biology section were used in parallel. The comparison shows that the new workflow is much faster than

the old one and the automated quality control ensures a better test quality. (Figure 3)

In conclusion, the aim of the new workflow is to achieve a fast and specific diagnosis. Immuno-

assays are as good in stools as in blood and allow for “on the spot” testing. Many methods are

available for bacteria, viruses and other pathogens. Finally, automation makes it possible to cross

laboratory boundaries and achieve goals together.

CONFIRMATION

SCREENING

RESULT

–+

+

(Re�ex)

Parallel

+

+–

––

C. Dif�cileinfection

NoC. Dif�cileinfection

Toxinnot present

Toxin A/B

GDH Culture

Toxin A/BPCR

MICROBIOLOGYAUTOMATED LAB

Automation makes it possible to cross laboratory boundaries and to achieve goals together

Figure 3. Example: Analytical pathway for C. difficile


8

Q: How are the reports of the cultures and toxicological tests provided? Are they integrated or supplied

separately?

A (Burde): We provide a single integrated report comprising all the results.

Q: How do you handle any discrepant results between a culture and an immunological test?

A (Huynen): The culture is used to dispel doubts in the event of discordant immunological tests (anti-

gens and toxins). If these are both positive, the culture is irrelevant.

Q: How do you tackle the issue of making the laboratory and microbiology staff feel responsible with

regard to the use of automatic testing?

A (Burde): We have had to make them gradually take on more responsibility.

My view is that we should not bring microbiology to automation but automation to microbiology. This way,

the responsibility for the preparation of specimens remains with the same people.

Q: What can you tell me about the risk of contamination? It is possible to test stool and blood at the

same time?

A (Huynen): Because we too initially had doubts about this, in the early phases we validated the use of

both matrices on the same system, and found that there were no problems.

Q: Dr. Huynen, how do you go about the diagnosis of H. pylori?A (Huynen): We use both direct methods (culture and antigen detection) and the search for antibodies,

as requested by the clinicians. The search for antibodies is especially useful for IgG.

Q: Glutamate-dehydrogenase (GDH) followed by toxin tests. Do you always do that or do you carry out

the toxin tests only when the GDH is positive?

A (Burde): Only when the GDH is positive. In the event of a positive GDH, the LIAISON® system automat-

ically runs the toxin test so the technologists do not even need to look at the GDH results.

Q: The recent literature suggests the possible use of PCR testing without GDH plus toxin testing. Is that

possible for you?

A (Burde): The PCR test is not reimbursed in Germany, so the patient has to pay for it. Another problem

is that it is too sensitive, and it detects toxins even after the infection has subsided.

Questions and Answers

CONFERENCE INSIGHT

9

Q: Given that it can take some time for the samples to reach the laboratory after collection and the

antigen may degrade in the meantime, would it not be better to run the PCR test, which is not affected

by this problem?

A (Huynen): We carry out the toxin tests on samples stored in a refrigerator for a maximum of three

days.

Q: How do you handle the transport and extraction of the sample. Do you do these steps yourselves or

send the samples to a satellite laboratory?

A (Huynen): When the stool sample arrives, one aliquot is kept for culture and another one goes to the

technologist who extracts it and feeds it to the LIAISON® systems.

Q: Do you have any advice for the accreditation for stool testing?

A (Huynen): Generally, to accredit such a test you need to have a reference method carried out in

parallel.

Bibliography• Van Den Berg RJ, Vaessen N, Endtz HP, et al. Evaluation of real-time PCR and conventional diagnostic methods

for the detection of Clostridium difficile-associated diarrhoea in a prospective multicentre study. J Med Microbiol. 2007 Jan;56(Pt 1):36-42

• Huynen P, Toussaint F, Gerard C, et al. A clinical lab experience with an automated HIV antigene/antibody (AG/AB) combined tests. 24th ECCMID, 2014 (Barcelona, Spain)

• Huynen P, Toussaint F, Melin P, et al. Validation of a new automated chemiluminescent assay for serodiagnosis of human parvovirus B19 infection. 30th RICAI, 2010 (Paris, France).

• Huynen P, Toussaint F, Melin P. Validation of a new automated chemiluminescent assay for serodiagnosis of human mycoplasma pneumonia infection. 22th ECCMID, 2012 (London, UK).

• Huynen P, Melin P, Mathieu J, et al. LIAISON VZV IgG and VZV IgM assay: a comparative study. 17th ECCMID/ 25th ECC, 2007 (Munich, Germany).

• Casprini P, Degl’Innocenti R, Brunelli T, et al. Diagnosi microbiologica di Infezione da Clostridium difficile: presentazione di un percorso a step automatizzato. XLIII AMCLI, 2014 (Rimini, Italy).

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how efficient and automated can be serology and stool testing?