Validation and verification of examination procedures in medical laboratories: opinion of the EFLM Working Group Accreditation and ISO/CEN standards (WG-A/ISO) on dealing with ISO 15189:2012 demands for method verification and validation.

This paper reflects the opinion of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Accreditation and ISO/CEN standards (WG-A/ISO). It aims to provide guidance for drawing up local/national documents about validation and verification of laboratory methods. We demonstrate how risk evaluation can be used to optimize laboratory policies to meet intended use requirements as well as requirements of standards. This is translated in a number of recommendations on how to introduce risk evaluation in various stages of the implementation of new methods ultimately covering the whole process cycle.

Documenting metrological traceability as intended by ISO 15189:2012: A consensus statement about the practice of the implementation and auditing of this norm element.

ISO15189:2012 requires medical laboratories to document metrological traceability of their results. While the ISO17511:2003 standard on metrological traceability in laboratory medicine requires the use of the highest available level in the traceability chain, it recognizes that for many measurands there is no reference above the manufacturer's selected measurement procedure and the manufacturer's working calibrator. Some immunoassays, although they intend to measure the same quantity and may even refer to the same reference material, unfortunately produce different results because of differences in analytical selectivity as manufacturers select different epitopes and antibodies for the same analyte. In other cases, the cause is the use of reference materials, which are not commutable. The uncertainty associated with the result is another important aspect in metrological traceability implementation. As the measurement uncertainty on the clinical samples is influenced by the uncertainty of all steps higher in the traceability chain, laboratories should be provided with adequate and appropriate information on the uncertainty of the value assignment to the commercial calibrators that they use. Although the between-lot variation in value assignment will manifest itself as part of the long-term imprecision as estimated by the end-user, information on worst-case to be expected lot-lot variation has to be communicated to the end-user by the IVD provider. When laboratories use ancillary equipment that potentially could have a critical contribution to the reported results, such equipment needs verification of its proper calibration and criticality to the result uncertainty could be assessed by an approach based on risk analysis, which is a key element of ISO15189:2012 anyway. This paper discusses how the requirement for metrological traceability as stated in ISO15189 should be met by the medical laboratory and how this should be assessed by accreditation bodies.

Recommendation for the review of biological reference intervals in medical laboratories.

This document is based on the original recommendation of the Expert Panel on the Theory of Reference Values of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), updated guidelines were recently published under the auspices of the IFCC and the Clinical and Laboratory Standards Institute (CLSI). This document summarizes proposals for recommendations on: (i) The terminology, which is often confusing, noticeably concerning the terms of reference limits and decision limits. (ii) The method for the determination of reference limits according to the original procedure and the conditions, which should be used. (iii) A simple procedure allowing the medical laboratories to fulfill the requirements of the regulation and standards. The updated document proposes to verify that published reference limits are applicable to the laboratory involved. Finally, the strengths and limits of the revised recommendations (especially the selection of the reference population, the maintenance of the analytical quality, the choice of the statistical method used…) will be briefly discussed.

Accreditation process in European countries - an EFLM survey.

BACKGROUND: Accreditation is a valuable resource for medical laboratories. The development of quality systems based on ISO 15189 has taken place in many laboratories in the European countries but data about accreditation remain scarce. The EFLM Working Group "Accreditation and ISO/CEN standards" conducted a survey that reviews the current state of the accreditation process in European countries. METHODS: An on-line questionnaire was addressed to delegates of 39 EFLM scientific societies in March 2014. One answer by country was taken into account. The survey was dealing with mandatory status, number of accredited medical laboratories in each country, possibility of flexible scope and concerned medical fields. The status of point-of-care testing (POCT) in each country was also studied. RESULTS: Twenty-nine responses (74%) were registered. All the assessed countries (100%) have begun an accreditation process in various ways. All the national accreditation bodies (NAB) offer or are working to offer an ISO 15189 accreditation. The accreditation process most often concerns all phases of the examination and various medical fields. Medical laboratories are responsible for POCT in 20 (69%) countries. The accreditation process for POCT, according to ISO 15189 and ISO 22870, is also developing. CONCLUSIONS: While there are several variations in the approaches to accreditation of medical laboratories in the European countries, the ISO 15189 accreditation project has been widely accepted. The use of a unique standard and the cooperation among countries due to scientific societies, EFLM, accreditation bodies and EA enable laboratory professionals to move toward uniform implementation of the accreditation concept.

Flexible scope for ISO 15189 accreditation: a guidance prepared by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Accreditation and ISO/CEN standards (WG-A/ISO).

The recent revision of ISO15189 has further strengthened its position as the standard for accreditation for medical laboratories. Both for laboratories and their customers it is important that the scope of such accreditation is clear. Therefore the European co-operation for accreditation (EA) demands that the national bodies responsible for accreditation describe the scope of every laboratory accreditation in a way that leaves no room for doubt about the range of competence of the particular laboratories. According to EA recommendations scopes may be fixed, mentioning every single test that is part of the accreditation, or flexible, mentioning all combinations of medical field, examination type and materials for which the laboratory is competent. Up to now national accreditation bodies perpetuate use of fixed scopes, partly by inertia, partly out of fear that a too flexible scope may lead to over-valuation of the competence of laboratories, most countries only use fixed scopes. The EA however promotes use of flexible scopes, since this allows for more readily innovation, which contributes to quality in laboratory medicine. In this position paper, the Working Group Accreditation and ISO/CEN Standards belonging to the Quality and Regulation Committee of the EFLM recommends using an approach that has led to successful introduction of the flexible scope for ISO15189 accreditation as intended in EA-4/17 in The Netherlands. The approach is risk-based, discipline and competence-based, and focuses on defining a uniform terminology transferable across the borders of scientific disciplines, laboratories and countries.

EFLM Working Group Accreditation and ISO/CEN standards on dealing with ISO 15189 demands for retention of documents and examination objects.

Many aspects of the activity of a medical laboratory have to be documented so as to facilitate the maintenance of the ongoing quality of service. As a consequence, many documents, forms and reports are generated. The retention time for each of these has to be specified. In addition to medical laboratory reports as part of the patient's medical record, the medical laboratory has to retain many documents and specimens according to national legislation or guidance from professional organizations, if these exist. If not, the laboratory management needs to define a retention schedule, which shall define the storage conditions and period of storage, according to ISO 15189:2022 requirements for retention of general quality management documents and records. The EFLM Working Group on Accreditation and ISO/CEN standards provides here a proposal on retention periods of documentation and specimens based on a failure-mode-effects-analysis (FMEA) risk-based approach, a concept of risk reduction that has become an integral part of modern standards.

An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting.

BACKGROUND: Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable. METHODS: Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described. RESULTS: The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed. CONCLUSIONS: The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale) vaccine clinical trials.

Intrahost evolution of envelope glycoprotein and OrfA sequences after experimental infection of cats with a molecular clone and a biological isolate of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a member of the genus Lentivirus and causes AIDS-like disease in its natural host, the cat. Like other lentiviruses, FIV displays a high degree of nucleotide sequence variability that is reflected in both the geographic distribution of the viruses and the different cat species that are infected. Although a lot of data on sequence variation at the population level is available, relatively little is known about the intrahost variation of FIV sequences. In the present study, cats were infected with either a biological isolate of FIV or a molecular clone that was derived from the same isolate, AM19. After infection, the cats were monitored for up to 3 years and at various time points sequences were obtained of virus circulating in the plasma. Regions of the env gene and the orfA gene were amplified, cloned and their nucleotide sequence analyzed. Furthermore, the extent of sequence variation in the original inocula was also determined. It was found that FIV is displaying relative little sequence variation during infection of its host, both in the env and the orfA gene, especially after infection with molecular clone 19k1. Although the extent of variation was higher after infection with biological isolate AM19, a large portion of these variant sequences was already present in the inoculum.

Evaluation of vaccination strategies against infection with feline immunodeficiency virus (FIV) based on recombinant viral vectors expressing FIV Rev and OrfA.

In recent years it has become clear that cell-mediated immunity is playing a role in the control of lentivirus infections. In particular, cytotoxic T lymphocyte responses have been associated with improved outcome of infection, especially those directed against the regulatory proteins like Rev and Tat, which are expressed early after infection. Therefore, there is considerable interest in lentiviral vaccine candidates that can induce these types of immune responses. In the present study, we describe the construction and characterisation of expression vectors based on recombinant Semliki Forest virus system and modified vaccinia virus Ankara for the expression of feline immunodeficiency virus (FIV) accessory proteins Rev and OrfA. These recombinant viral vectors were used to immunize cats using a prime-boost regimen and the protective efficacy of this vaccination strategy was assessed after challenge infection of immunized cats with FIV.

Domain- and nucleotide-specific Rev response element regulation of feline immunodeficiency virus production.

Computational analysis of feline immunodeficiency virus (FIV) RNA sequences indicated that common FIV strains contain a rev response element (RRE) defined by a long unbranched hairpin with 6 stem-loop sub-domains, termed stem-loop A (SLA). To examine the role of the RNA secondary structure of the RRE, mutational analyses were performed in both an infectious FIV molecular clone and a FIV CAT-RRE reporter system. These studies disclosed that the stems within SLA (SA1, 2, 3, 4, and 5) of the RRE were critical but SA6 was not essential for FIV replication and CAT expression. These studies also revealed that the secondary structure rather than an antisense protein (ASP) mediates virus expression and replication in vitro. In addition, a single synonymous mutation within the FIV-RRE, SA3/45, reduced viral reverse transcriptase activity and p24 expression after transfection but in addition also showed a marked reduction in viral expression and production following infection.

Sensory nerve conduction studies in neuralgic amyotrophy.

Neuralgic amyotrophy is a painful, episodic peripheral nerve disorder localized to the brachial plexus. Sensory symptoms occur in 80% of the patients. We assessed the frequency of abnormalities in sensory nerve conduction studies of the lateral and medial antebrachial cutaneous, radial sensory, median sensory, and ulnar sensory nerves in 112 patients. Sensory nerve conduction studies showed abnormalities in <20% of nerves, even when the nerve was clinically affected. The lateral and medial antebrachial cutaneous nerves were most often abnormal, in 15% and 17% of nerves. No correlation with the presence or localization of clinical deficits was found. Brachial plexus sensory nerve conduction studies seem to be of little diagnostic value in neuralgic amyotrophy. Our findings also indicate that some sensory lesions may be in the nerve roots instead of the plexus. An examination of normal sensory nerve conduction studies does not preclude neuralgic amyotrophy as a diagnosis.

Evaluation of ISCOM-adjuvanted subunit vaccines containing recombinant feline immunodeficiency virus Rev, OrfA and envelope protein in cats.

For the development of feline immunodeficiency virus (FIV) vaccines mostly structural proteins have been evaluated for their capacity to induce protective immunity. In the present study, subunit vaccines containing recombinant FIV accessory proteins Rev and OrfA were evaluated in cats. Cats were vaccinated repeatedly with these proteins, adjuvanted with immune stimulating complexes (ISCOMs). In addition, cats were vaccinated with bacterially expressed fragments spanning the entire FIV envelope protein, either alone or in combination with the regulatory proteins. Subsequently, the cats were challenged with a homologous FIV strain to assess the level of protective immunity achieved with the respective vaccination regimens. Although the vaccines proved to be immunogenic, vaccinated cats were not protected from infection with FIV.

Antibodies specific for hypervariable regions 3 to 5 of the feline immunodeficiency virus envelope glycoprotein are not solely responsible for vaccine-induced acceleration of challenge infection in cats.

In a previous vaccination study in cats, the authors reported on accelerated feline immunodeficiency virus (FIV) replication upon challenge in animals vaccinated with a candidate envelope subunit vaccine. Plasma transfer studies as well as antibody profiles in vaccinated cats indicated a causative role for antibodies directed against the hypervariable regions HV3, HV4 and HV5 (HV3-5) of the envelope glycoprotein. The present study was designed to investigate further the contribution of antibodies in envelope vaccine-induced acceleration of FIV infection. To this end, regions HV3-5 of the envelope glycoprotein were deleted from the original vaccine, thus addressing the contributing role of antibodies directed against these hypervariable regions. Interestingly, this approach did not prevent acceleration of challenge infection. Analysis of the antibody responses in the respective groups suggested that removal of HV3-5 redirected the humoral immune response towards other regions of the envelope glycoprotein, indicating that these regions can also induce antibodies that accelerate virus replication.