Verification and validation of diagnostic laboratory tests in clinical virology

doi:10.1016/j.jcv.2007.07.009

Journal of Clinical Virology

Volume 40, Issue 2, October 2007, Pages 93-98

https://doi.org/10.1016/j.jcv.2007.07.009 Get rights and content

Abstract

This review summarizes major issues of verification and validation procedures and describes minimum requirements for verification and validation of diagnostic assays in clinical virology including instructions for CE/IVD-labeled as well as for self-developed (“home-brewed”) tests or test systems. It covers techniques useful for detection of virus specific antibodies, for detection of viral antigens, for detection of viral nucleic acids, and for isolation of viruses on cell cultures in the routine virology laboratory.

Introduction

Routine viral diagnostics includes techniques for indirect and those for direct detection of viruses. Indirect detection of viruses is performed by serological studies. Techniques for direct detection of viruses include detection of viral antigens, detection of viruses or viral components by isolation of viruses on cell cultures (or through animal experiments) and detection of viral nucleic acids also referred to as nucleic acid testing (NAT). Furthermore, viral morphologic structures can be investigated by means of transmission electron microscopy.

Today, NAT has a major impact on viral diagnostics. Molecular assays are currently used routinely in almost all routine virological laboratories. Technological improvements, from automated sample preparation to real time amplification technology, provide the possibility to develop and introduce assays for most viruses of clinical interest. The risk of contamination has been reduced significantly and the turn-around time to generate results shortened. Both the sensitivity and the dynamic range have been improved. In contrast, standardization and quality assurance/quality control issues have often remained underdeveloped requiring urgent improvement.

Moreover, it must be taken into consideration that reliable viral diagnostics depends on pre-analytical issues either such as choice of the correct specimen, optimal sampling time with regard to the course of disease, and both time and conditions of the specimen transport to the laboratory.

Section snippets

Quality assurance/quality control

The European Union's Directive on In Vitro Diagnostic Medical Devices (98/79/EC) demands for data demonstrating that an IVD achieves the stated performance and will continue to perform properly after it has been shipped, stored, and put to use at its final destination (Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices, 1998). Furthermore, in the international standard DIN EN ISO 15189, special requirements for medical

Minimum requirements for verification and validation procedures for virological tests or test systems

Suitability of a technique does not necessarily mean that it is performed correctly and provides valid results. The IVD Directive 98/79/EC and the DIN EN ISO 15189 demand for verification or validation of each investigational procedure in order to prove both the correct application and the correct performance of a diagnostic test. The complexity and the extent of the verification or validation procedure depend on the fact whether an IVD-CE labeled test or a self-developed (“home-brewed”) test

Verification of IVD/CE-labeled tests or test systems for detection of virus specific antibodies, detection of viral antigens or NAT

If a new IVD/CE-labeled test or test system for detection of virus specific antibodies, detection of viral antigens or NAT is introduced in the routine diagnostic laboratory, verification experiments are performed to verify precision and, in case of a quantitative NAT test or test system, linearity (Table 2). In case of a qualitative test or test system, one positive and one low positive specimen are used for determination of intra-assay precision. Each sample is tested three times within a

General considerations when establishing a home-brewed NAT assay

When establishing a home-brewed NAT assay, primer and probe sequences must be checked carefully by use of a genome sequence databank. It is advisable to verify the amplification product by means of sequencing and to use a primer pair that has already been published in a highly recognized journal. The latter helps to avoid extended specificity testing. However, the published sequences should always be subjected to an alignment analysis with a genome sequence databank to ensure that the correct

Validation of home-brewed tests or test systems for detection of virus specific antibodies, detection of viral antigens or NAT

If a home-brewed test or test system for detection of virus specific antibodies, detection of viral antigens or NAT is introduced in the routine diagnostic laboratory, validation experiments are performed to validate sensitivity, specificity, precision and, in case of a quantitative test or test system, linearity (Table 3). The sensitivity is determined by testing 10 positive and 10 low positive specimens while the specificity is determined by analyzing 20 negative but potentially

Validation of isolation of viruses on cell cultures

Virus isolation on cell cultures is a technique which is difficult to standardize, thus validation becoming particularly demanding. First of all, the suitability of the cells for the detection of a certain virus has to be examined. During the implementation of new cell lines as an indicator system, the cell line should be tested for its susceptibility with two concentrations of both a reference virus strain and a wild type isolate. After titration of the virus stock, the inoculums should

Conclusion

Implementation of a new test or test system in the routine diagnostic virological laboratory demands for verification or validation procedures in compliance with a quality management system and according to DIN EN ISO 15189. While a CE/IVD-labeled test or test system requires verification, a home-brewed test or test system must be validated.

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ReviewVerification and validation of diagnostic laboratory tests in clinical virology