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Molecular diagnostics in routine practice: Quality issues and application to complex disease

Posted on: Sunday, 3 August 2003, 06:00 CDT

Abstract

The public already has concerns about 'the new genetics', and it is clear that confidence can only be maintained by scrupulous attention to quality. Standards can be improved by harmonization of methods, discouraging poor practice and using appropriate internal and external quality controls. At present, despite the profound implications of genetic test results, few genetic tests are subject to sufficient scrutiny. The Human Genome Project will lead to the identification of numerous genetic variations contributing to multifactorial diseases, and high-throughput technologies will permit the generation of disease-susceptibility profiles. Clinical laboratories will need to develop the wherewithal to handle these data and present them in a format that is clinically useful.

Ann Clin Biochem 2003; 40: 309-312

Introduction

'I had,' said he, 'come to an entirely erroneous conclusion which shows, my dear Watson, how dangerous it always is to reason from insufficient data.' (Arthur Conan Doyle, The Adventure of the Speckled Band)

Molecular testing is becoming an increasingly important part of the repertoire of specialist biochemistry laboratories, particularly for the diagnosis and assessment of monogenic disorders.1,2 In the post-genomic era it is likely that the genes that contribute significantly to complex disorders such as cancer,3 cardiovascular disease4,5 and diabetes6 will be elucidated. It is also likely that the genetic determinants of individual responses to particular forms of therapeutic intervention (drug treatment, radiotherapy, etc.) will be identified,7 permitting better-targeted, individualized treatment3 that will be more effective, safer for the patient and cheaper for the National Health Service. It is also inevitable that high-throughput methodologies will be developed1,8,9 and applied more widely.

This is an exciting prospect, but it will inevitably impose a considerable burden of responsibility on hospital laboratories to provide an efficient, timely and accurate molecular diagnostic service. This will include the provision of a post-analytical interpretative service, which for complex disease may not be a simple matter.3,10 It may necessitate a parallel development of more sophisticated medical bioinformatics within pathology.

Quality in molecular biology testing: a lesson for clinical biochemistry from forensic science?

The analytical methods associated with molecular diagnostics also require robust quality-assurance procedures, including external quality assurance11-15 (Box 1). The importance of these procedures has become evident from the experience of colleagues in other disciplines, particularly forensic pathology.16-18

Molecular biology testing (genetic finger-printing) was first applied for legal purposes in an immigration case, in which it was used to ascertain maternity.19 At the request of the Midland's Police, genetic fingerprinting was first used in a criminal case in 1986(20) in an attempt to verify a suspect's confession to two rape- murders; the test showed the suspect to be innocent. The first convictions made on the basis of genetic finger-printing were made in 1987 in the UK (Regina v Milas) and USA (The People v Andrews).

Although DNA evidence appeared incontrovertible, its use was challenged soon after its introduction. In 1988, Jose Castro was accused of killing a neighbour Vilma Ponce and her 2-year-old daughter, in New York, on the basis of DNA analysis of a bloodstain on his watch. The laboratory reported that the DNA profile of Ponce matched that on the defendant's watch and that the frequency of that pattern in the general population was 1:189 200 000. However, the DNA patterns of Ponce and the blood found on the watch exhibited 'band shifting,' i.e. the DNA bands in the two samples did not line up exactly. The laboratory claimed that the DNA sample derived from the blood spot on the watch migrated faster on the gel because of degradation. However, the laboratory did not use any internal control for band shifting to substantiate its claim. And the probability calculation was not made against the relevant ethnic group (Hispanic). Hence, in this particular instance, the evidence was suspect on the basis of pre-analytical, analytical and post- analytical processing. The legal experts decided in an unprecedented pre-trial hearing that the laboratory procedures were so 'flawed' that the resulting evidence was unreliable. It was subsequently concluded that quality-assurance programmes in individual laboratories alone are insufficient to ensure high standards and that external mechanisms are needed to ensure adherence to the practices of quality assurance. The Court recommended '. . . that laboratories should adhere to high quality standards (such as those defined by the Technical Working Group on DNA Analysis Methods (TWGDAM) and the DNA Advisory Board), be accredited for DNA work (by such organizations as the American Society of Crime Laboratory Directors-Laboratory Accreditation Board (ASCLD-LAB)), and should participate regularly in proficiency tests.' Although forensic scientists are operating under particularly demanding constraints,21,22 should routine diagnostic laboratories be any less rigorous in their approach to DNA testing? The implications of an erroneous result may be greater than for other routine tests.

Experience of maintaining the quality of genetic testing

The European Molecular Genetics Quality Network (EMQN) has been organizing quality-assurance schemes in molecular diagnostics for several years. It has reported that '. . . overall technical performance showed a high diagnostic standard. Nevertheless, serious genotyping errors have occurred in some schemes . . . The error rates (for some tests) ranged between 18 and 24%'.23 This cannot be regarded as an acceptable standard of performance, particularly among laboratories with an expertise in this type of testing. UK NEQAS (http://www.ukneqas.org.uk/ Directory/GENET) and the EMQN (http://www.emqn. org/eqa) have established external quality assurance (EQA) schemes for a limited number of monogenic disorders. However, it should be a matter of concern that for many genetic tests there are currently no EQA schemes (Box 2).

Special difficulties associated with DNA assessment of complex diseases

Several important multifactorial diseases have a strong genetic component,5,24 and hence the value of genetic testing in assessing disease susceptibility is potentially very great; however, at present the application of genetic testing to these conditions is severely hampered for a number of reasons (Box 3). There are insufficient prospective data to establish allele-specific disease risk, and even fewer in which gene-gene or gene-environment interactions have been modelled. In addition, none of these genetic tests have been fully evaluated for their clinical utility (Box 4). The complexity of the data is such that it is unrealistic to expect clinicians to find them clinically useful without providing interpretative guidelines. At present we are poorly equipped to provide this.

Conclusion

Although high-throughput molecular biology testing within hospital laboratories is an exciting prospect, a great deal needs to be done to ensure quality of analysis and interpretation. A first step would be to introduce EQA schemes for tests that are currently, or are becoming, widely used.

(C) 2003 The Association of Clinical Biochemists

Box 1. Assuring high-quality molecular testing requires detailed standard operating procedures

Pre-analytical handling

* Patient identification

* Patient sampling

* Specimen collection

* Specimen labelling

* Specimen preservation, transportation and storage before testing

Analysis

* Reagent quality checks (e.g. in date, etc.)

* Equipment validation to ensure volumetric and thermal accuracy and regular documented instrument maintenance

* PCR procedure

* Sample preparation including a check that DNA is present and is amplifiable by the PCR reaction (i.e. detection of inhibitors)

* Amplification controls, including an assessment of reagent (primers and enzymes) quality and analytical sensitivity and specificity

* Quality of detection system, including restriction enzymes, or reagents for colorimetric end-point

* Internal controls (known normal and abnormal samples; molecular weight standards; Mendelian inheritance? (racial/ethnicity study)

* Criteria to identify normal and abnormal results

* Record of failed nucleic acid isolation, or analysis and system to inform clinician

* Band-matching criteria

* Regular testing by all members of technical staff

Post-analytical quality

* System to detect clerical errors in a timely manner

* Appropriate turnaround time and system of monitoring this

* Participation in proficiency testing with:

* Survey samples integrated into routine analysis

* Regular review of proficiency testing reports

* Clinical validation by:

* Investigation of discrepancies

* Comparing statistics (allele frequencies, etc.) with other laboratories

* Report of appropriate format

* Regular review of current technology

Modified from reference 25. PCR, polymerase chain reaction.

Box 2. Current availability of external quality-assurance schemes for DNA testing

External quality assurance scheme available

UK NEQAS

* Factor V^sub Leiden^

* HCV RNA quantitation

* Hereditary haemochromatos\is

* HIV-1 RNA quantitation

* HLA DQ-B1 and-B2

* HLA DR B27

* Linkage marker analysis

* Prothrombin mutations

EMQN schemes

* Charcot-Marie-Tooth

* Cystic fibrosis

* Duchenne muscular dystrophy

* Familial breast/ovarian cancer

* Fragile X syndrome

* Friedreich's ataxia

* Hereditary haemochromatosis

* Huntington disease

* Prader-Willi/Angelman syndromes

* Retinoblastoma

* Y chromosome microdeletions

External quality assurance scheme not widely available

* [alpha]^sub 1^Antitrypsin genotype

* Achondroplasia

* Adrenoleucodystrophy

* Apolipoprotein E genotype

* Clonal rearrangements for lymphomas

* Human papilloma virus

* Incontinenta pigmenti

* Lesch-Nyhan syndrome

* Menke disease

* MEN type I and II

* Methylenetetrahydrofolate reductase mutation

* Porphyria

HCV, human cytomegalovirus; HIV, human immunodeficiency virus; MEN, multiple endocrine neoplasia.

Box 3. Additional challenges in the genetic analysis of complex diseases

* Multifactorial: genetic and environmental

* May be ecogenetic interactions

* Polygenic

* Limited number of loci identified

* Very limited quantitative data on risk

* Genetic heterogeneity

* Different genetic loci

* Variants at single locus

* Differences between ethnic groups

* Continuous versus dichotomous trait

* Biological variability of trait

* Arbitrary definition of category (e.g. definition of hypertension or dyslipidaemia)

Box 4. Testing the validity and utility of a marker for predictive

* Identify genetic variation

* Test its relationship to disease phenotype by segregation analysis or population studies

* Evaluate efficiency to discriminate cases from controls

* Assess benefits to patient of early diagnosis

* Formal economic analysis

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Accepted for publication 22 February 2003

Gordon AA Ferns1,2, David O'Dowd1, Gwen Wark1 and Nadine Collins1

Addresses

1The Clinical and Molecular Diagnostics Laboratories The Royal Surrey County Hospital Egerton Road, Guildford GU2 7XX, UK

2The Centre for Clinical Science & Measurement University of Surrey Guildford GU2 7XH, UK

Correspondence

Professor GAA Ferns

E-mail: g.ferns@surrey.ac.uk

Copyright Royal Society of Medicine Press Ltd. Jul 2003

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