H.Schimmel, Beijing 16/10/2007 Institute for Reference Materials and Measurements (IRMM) Geel, Belgium .

H.Schimmel, Beijing 16/10/2007 1

Institute for Reference Materials and Measurements (IRMM)Geel, Belgium

http://www.irmm.jrc.be http://www.jrc.cec.eu.int

Challenges for International Standardisation and TraceabilityGenetic Testing

H. Schimmel

H. Schimmel, Beijing 16/10/2007

Implications of Genetic Testing

• Can be highly predictive for the future health

• Can be carried out at any stage of life and be applied for pre-natal or pre-implantation diagnostics.

• Relevant to healthy people as well as to those showing symptoms of an unhealthy condition.

• May have also important implications for the relatives of the person tested.

• The genotype established by a single laboratory test is usually not repeated and forms a permanent part of the medical record of the patient.


Laboratory performance (I)• PT in collaboration with DGKL, Instand e.V. and

QualiCont kht. on the prothrombin gene G20210A mutation (Factor II Leiden)

• 198 laboratory results:

– False negative: 5.8 % (20210A mutation present)

– False positive: 3.2% (G20210 wildtype)

• Typical figures in the genetic testing area.

• Error rate when additional mutations are present (about half of methods influenced):

– False negative: 9.5 % (G20210 + 20210A heterozygous + T20175G + 20179-80 del AC)

– False positive: 5.3 % (G20210 wildtype + C20209T)


Laboratory Performance (II)

• False genotypes (18 in total) concentrate on 13 laboratories only. Reasons:

– Insufficiently robust home-brew LightCycler assays

– Mixing up of the results post-analytically

– Genotypes assigned incorrectly although the raw data showed the expected patterns.

• Increase in risk to obtain false results in the presence of additional mutations is method dependent.


Laboratory performance (III)

• In the absence of additional mutations the majority of the false genotyping results arose from inadvertence and not from technical failure of the method as such.

– Apply stringent QC in laboratory procedures

• Alertness for the presence of additional mutations

• Discourage use of insufficiently robust methods


In vitro Diagnostics Medical Devices Directive 98/79/EC

NOT COVERED

• CRMs

• Materials used for EQA schemes

• Devices manufactured and used within the same Institution

COVERED need of CE Mark

• Calibrators, control materials needed to establish or verify performance of devices (commercial or not)


Annex I.A.3 : Essential requirement

The traceability of values assigned to calibrators and/or control materials must be assured through :

• available reference measurement procedures

and/or

• available reference materials of higher order.


Need for Quality Assurance

• Importance of genetic testing steadily increases.

• Crucial for quality assurance and to establish the required reliability:

– Appropriate reference materials

– Thorough assessment of the performance of methods and stringent quality assurance in accordance with ISO 15189 and ISO 17025.

• Traceability is a tool to achieve comparability of quantity values.


International Standardisation

• Settled for quantitative measurements

• Space for interpretation of existing standards related to qualitative measurements and eventually need for new standards. Reference to this area is already made in various standards:

– E.g. ISO Guide 35 (2006): Under definition of a CRM, Note 1) The concept of values includes qualitative attributes such as identity or sequence. Uncertainties for such attributes may be expressed as probabilities.


Same language ?


International Standardisation

Various standardization bodies (ISO REMCO, ISO TC 212, etc)

Professional organizations (CLSI, AOAC, IUPAC, Eurachem, etc)

Networks (EUROGENTEST, JCTLM, etc)

Are considering additional guidelines on qualitative analysis (identity checks)

IRMM is involved in the process and gives input in the various committees favouring a common approach


Categories of genetic Testing (I)• Identification of variants or abnormalities in a nucleic

acid sequence such as mutation(s), translocation(s), duplication, amplification and deletion(s).

• Determination of the number of nucleotide repeats or the length of nucleic acid sequence in terms of base pairs or number of nucleotide repeats.

• Measurement procedure producing a signal within a dynamic range and which is turned into a qualitative format by defining a cut off value or by setting up the measurement procedure in a way that it gives at a certain concentration level of the analyte a detectable signal.

• Measurement procedures of which results are expressed quantitatively e.g. gene expression.


RM requirements• The type of reference material which is required for

QC or calibration depends on the analytical problem.

• In all cases the production is an integrated process of (GUM, ISO Guide 35, ISO 15194):

– correct selection, preparation,

– homogeneity and stability (short-term and long-term) demonstration,

– accurate and traceable characterisation (for CRMs),

– Commutability / validation of applicability


Applicability of a RM• Assessment of Commutability / validation of

applicability

– Limited availability of human material

– Reference materials are usually synthetic or derived form cell lines and are highly processed DNA/RNA

– RMs/CRMs are either pure or added to a matrix

– RMs/CRMs may have been designed to work with a dedicated platform.

• Therefore the reference materials may not perform identically to a patient sample.


Categories of genetic Testing (II)• Identification of variants or abnormalities in a nucleic

acid sequence such as mutation(s), translocation(s), duplication, amplification and deletion(s).

• RMs with known sequence used as a QC material, not as a calibrator

– Assigned property: sequence (matching backward and forward sequencing reduces the probability of stating a wrong sequence to a negligible level).

– Validation of the material with a range of relevant methods

• Challenge:

– To adjust copy numbers in a way that a method operated under sub-optimal conditions would fail as it would do with typical patient samples


Plasmid pIRMM-0001 (3310 bp)

NciI 47NciI 82

NdeI 183

PvuII 306

399HindIIISphIPstISalIXbaIBamHINciISmaIXmaINciISacIEcoRI450

HindIII 559

XbaI 622

NciI 762

BclI 828

EcoRI 1075

PvuII 1253

NciI 1810

NciI 2506

ScaI 2802NciI 2857

G 943

Factor II

AP(R)

Case StudyCRM production for the prothrombin G20210A mutation


Characterization

• Minimum sample intake

• Quantification of pDNA (instructions for use)

• Qualitative PCR (homogeneity)

• Quantitative real-time PCR (homogeneity, stability)

• Genotyping using the LightCycler technology (identity)

• DNA sequencing (identity)


Long-term stability of pIRMM-0001 (I)Long-term stability of pIRMM-0001 at -20 °C

y = 0.0004x + 0.9898

R2 = 0.0242

0.97

0.98

0.99

1.00

0.00 2.00 4.00 6.00

Time (months)N

orm

alis

ed

Ct

va

lue

Long-term stability of pIRMM-0001 at 4 °Cy = -0.0005x + 0.9878

R2 = 0.0268

0.97

0.98

0.99

1.00

0.00 2.00 4.00 6.00

Time (months)

Nor

mal

ised

Ct v

alue


Validation study of IRMM/IFCC-490, -491 and -492 materials application

Lab Method Type Correctness of genotyping

Homozygous wildtype

Homozygous mutant

Heterozygouswildtype/G20210A

mutantBlank

1 PCR LightCycler Y Y Y Y

2 MultiplexPCR

Home brewed Y Y Y Y

3 Multiplex PCR

Home brewed Y Y Y Y

4 PCR LightCycler Y Y Y Y

5 RFLP1 Home brewed Y Y Y Y

5 PCR LightCycler Y Y Y N

6 RFLP2 Home brewed Y Y Y Y

7 PCR Rotor-Gene Y Y Y Y


Categories of genetic Testing (III)

• Counting of the number of nucleotide repeats or the determination of the length of nucleic acid sequence in terms of base pairs.

• RMs with DNA fragments of known length or number of nucleotide repeats.

• Challenges:

– Difficulties in quantifying the target by rt-PCR methods due to lacking specificity of primers and probes.

– Difficulties in long range sequencing of repeat sequences.

– Production of RMs not simple (unstable insertions)

– Influence of sequence on the electrophoretic migration


Categories of genetic Testing (IV)

• Measurement procedure producing a signal within a dynamic range but turned into a qualitative format by applying a cut off value or by setting up the measurement procedure in a way that it gives at a certain concentration level of the analyte a detectable signal.

• For these procedures the ability to distinguish positive and negative sample populations can be best described by the limit of detection, which is a quantity value.

• Usually such procedures are applied for presence / absence testing, i.e. the focus is on the detection of the lowest possible copy number


Categories of genetic Testing (V)

• RMs below, at and above LOD are required to assess the stability of the LOD and thus of the performance in terms of efficiency.

• Challenges:

– Lack of PCR independent methods which would allow the determination of low copy numbers.

– All sorts of sample handling problems common to measurements at low concentration level (adsorption, instability, stochastic effects etc).

– The determination of the LOD may depend on the properties of the RM, i.e. the DNA/RNA surrounding the target sequence and the matrix, i.e. commutability issues arise.


Categories of genetic Testing (VI)

• Quantitative measurement procedures (e.g. gene expression).

– Currently not widely applied in the genetic testing area but of growing importance.

• Commutable RMs with defined copy numbers required for calibration.

• Challenges:

– The determination of the absolute copy number is related to large measurement uncertainties due to the exponential nature of the usually applied DNA amplification techniques.

– Commutability phenomena have to be expected.

– In particular RNA is unstable and armoured RNA may not behave similarly during sample preparation.


CONCLUSIONS

• In terms of written standards a coordinated approach is required to clarify aspects related to qualitative measurements.

• Quantitative measurements or the assessment of the LOD of methods used in the genetic testing area are constituting the more significant technical challenges.

H.Schimmel, Beijing 16/10/2007 Institute for Reference Materials and Measurements (IRMM) Geel, Belgium .

Documents

schimmel slide

c20209t slide

false results

laboratory performance

laboratory results

common approach slide

iso guide

false genotypes