2009 Vienna - European Society of Human Genetics

Laboratory and quality management
system using more than 7,800,000* independently called bases with
quality value (QV) 20 or better data (*data estimate excludes the calls
< QV20 which are also used).
P15.07
system Validation of a New Real time PcR and High Resolution
melting instrument: the Lightscanner32 (Ls32)
C. Gundry, D. David, R. Weigel, R. Lems, M. Poulson, R. Lundstrom, B. Wade,
D. Hawks, L. Caldwell, S. Moore, M. Ferguson, B. Dorcheus, D. Kane, R. Abbott,
D. Nielsen, J. T. McKinney;
Idaho Technology, Inc., Salt Lake City, UT, United States.
Rapid air-thermocycling was introduced by Idaho Technology in 1991,
resulting in the development of the LightCycler in 1996. Licensed to
Roche (1998), the LightCycler is now commonplace throughout the
world with an install base >7000. High-resolution melting was introduced
by Idaho Technology in 2003 with the HR-1 instrument and is
now a validated technology for mutation scanning and genotyping.
The LightScanner32 (LS32), capable of both rapid real-time PCR and
high-resolution melting, has been developed by Idaho Technology and
launched in February 2009. LS32 combines features of the LightCycler
and HR-1 instruments (see Table 1). System validation tested instrument
and software functionality independently and as integrated components.
Instrument control and analysis modules were tested with
independent 16-rotor experiments as part of the formal system validation
plan. Integrated system testing included: multiplex qPCR with
color-compensation; qPCR dynamic range, precision, and accuracy;
high-resolution melting; multi-users and database’s; customizable data
reports. Over 100 experiments representing specific test cases were
performed for each design input and product specification. All design
inputs and product specifications related to system performance and
experimental applications were successfully verified with the planned
test cases. System validation resulted in final modifications to software
and the operator manual, with any potential software modifications
validated by re-running the appropriate test cases. LS32 system performance
was documented via Idaho Technology’s quality system and
approved for launch in February 2009.
Instrument Comparison
LightCycler(Roche)
HR-
1(IdahoTech)
LS32(IdahoTech)
Amplification Yes No Yes
Analysis Mode qPCR, Melting Curves
Hi-Res
Melting
qPCR, Melting
Curves,Hi-Res Melting
Acquisition
Mode
Single/Step/
Continuous
Continuous
Single/Step/
Continuous
Data Resolution 4.1 pts/Co 400 pts/Co 400 pts/Co Sample
Capacity
32 1 32
P15.08
the mLPA-dHPLc procedure to analyse the NF1 gene
S. Pinson;
Hospices Civils de Lyon, Lyon, France.
The identification of mutations in the NF1 gene causing type 1 neurofibromatosis
(OMIM-162200) is still presenting a considerable amount
of work mainly because of the large size of the gene and the restricted
number of recurent mutations. The high frequency of NF1 which affect
1 in 3500 individuals lead us to choose two complementary methods
for NF1 gene analysis:
- the multiplex ligation-dependant probe amplification (MLPA) for the
large deletion and duplication detection (P081 and P082 - P122C1)
- and the automated denaturing high performance liquid (dHPLC)
screening method.
The MLPA method was validated by the detection of 19 known large
NF1 gene deletions. We also tested 39 different mutations that would
interfere with the MLPA results.
The dHPLC was optimised for a rapid screening of the 60 exons and
the splice junctions of the NF1 gene. The dHPLC conditions were validated
by the detection of 260 known variants located in two thirds of
the NF1 exons. We also evaluated the sensitivity of dHPLC for somatic
mosaicism mutation.
The sensitivity was evaluated in a MLPA/dHPLC analysis of a panel of
150 unrelated french NF1 patients with at least two consensus diagnostic
criterias.
Seven large deletions were first detected by P081/082/P122C1 MLPA
(6 total and 1 partial).
Mutations were identified in 136 among the 143 remaining patients
with a global mutation detection rate of 96% [CI95%: 91-98].
Our results confirm that the association of the MLPA and dHPLC techniques
provides an accurate and fast method for the identification of
NF1 mutations.
P15.09
information on quality assurance in genetic testing in Europe:
Orphanet/ EuroGentest
L. Desmet 1 , N. Nagels 1 , M. A. Morris 2 , M. Jovanovic 3 , I. Caron 3 , M. Hanauer 3 ,
S. Aymé 3 , E. Dequeker 1 ;
1 Centre for Human Genetics, University of Leuven, Belgium, 2 Laboratoire de
Diagnostic moléculaire, Service de Médecine Génétique, University Hospital,
Geneva, Switzerland, 3 INSERM, SC11 / Orphanet, Paris, France.
Given that the outcome of genetic testing has a great impact on the
life of patients and their entourage, the quality of genetic testing is
of utmost importance. Since December 2008, the EuroGentest portal
of the Quality Assurance database moved to the Orphanet website.
Data about the quality management of laboratories offering medical
genetic testing are now linked to their tests and contact details. To
ensure the highest possible reliability of the quality assurance data,
replies of laboratories are validated by comparison with EQA providers
and accreditation bodies by EuroGentest, prior to dissemination via
Orphanet database. Currently, the database includes data from 1,308
laboratories. Quality data comprise laboratory accreditation status with
a link to the accreditation scope, participation in genetic external quality
assessment and the presence of a quality manager.
With the developing awareness of the central role of QAu, we believe
that the uptake of quality data in Orphanet will benefit laboratories by
encouraging and providing recognition of their investment in QAu and
by the possibility of a better informed choice for referral of tests, consumers
(patients, doctors, laboratories, etc.) by a greater transparency
and a possibility of a better-informed choice, and quality organizations
(EQA providers, accreditation bodies etc.) by greater visibility and recognition
of their roles.
Orphanet is continuously updated and participation in the database is
freely open to all European laboratories offering human medical genetic
testing (www.orpha.net). If you have any further questions concerning
quality data, please contact the EuroGentest team at QAuSurvey@
eurogentest.org.
P15.10
the role of EuroGentest and cF Network in measuring the
improvement of quality assurance in genetic testing laboratories
S. Berwouts 1 , M. A. Morris 2 , E. Dequeker 1 ;
1 Centre of Human Genetics, University of Leuven, Leuven, Belgium, 2 Laboratoire
de Diagnostic Moléculaire, Service de Médecine Génétique, Hôpitaux
Universitaires de Genève, Geneva, Switzerland.
Laboratories across Europe are increasingly evolving towards implementation
of quality assurance (QAu) and ISO 15189:2007 is being
widely implemented by genetic testing services. The EuroGentest
workshops on QAu and the CF Network cystic fibrosis (CF) external
quality assessment (EQA) scheme provide useful instruments to examine
the evolution of quality in laboratories.
35 institutions that participated in the workshops were surveyed about
their accreditation status and implementation of different “quality parameters”.
Accreditation changed from 37% of the laboratories in 2005
to 49% in 2007. The majority of the non-accredited laboratories initially
participated in EQA schemes (72% in 2005, 83% in 2007), whereas
technical aspects such as validation of methods (0% in 2005, 22% in
2007) tend to be addressed later in the move towards accreditation.
Overall, implementation of all the parameters surveyed increased with
time. A follow-up survey will be performed in 2009.
152 laboratories participated in the CF EQA scheme in each of 2005,
2006 and 2007. Improved error rates in genotyping (4% in 2005, 2% in
2007) and interpretation errors (14% in 2005, 7% in 2007) are encouraging.
Similar improvements are apparent for the inclusion in reports
of elements required by ISO 15189:2007, such as patient name (98%
in 2005 and 2007), sample number (86% in 2005, 89% in 2007) and
gender (53% in 2005, 63% in 2007). 2008 scheme data will be analysed.