The Joint Committee for Traceability in Laboratory Medicine (JCTLM): a global approach to promote the standardisation of clinical laboratory test results

Clinical laboratories are moving towards global standardisation to produce equivalent test results across space and time. Standardisation allows use of evidence-based medicine, eliminates the need of method-specific reference intervals, decision levels and cut-offs, and can be achieved by application of metrological principles. For example, in vitro diagnostics (IVD) manufacturers can make kit calibrators traceable to internationally recognised reference materials and reference methods.The first step towards standardisation is to identify appropriate reference materials and methods. This has been undertaken by a new international consortium, the Joint Committee for Traceability in Laboratory Medicine (JCTLM), formed in 2002. It brings together experts representing the clinical laboratory profession, government agencies, and manufacturers, to promote international comparability, reliability, and equivalence of measurement results in clinical laboratories for the purpose of improving healthcare. Through the efforts of the JCTLM, manufacturers are able to assign values to kit calibrators with consistency using appropriate higher order reference materials and methods, and traceability flowcharts, according to ISO Standards to ensure accuracy of test results and to promote assay performance harmonisation. Users of assay kits can assess suitability of calibrators on the basis of acceptable reference materials and/or methods identified by the JCTLM. The JCTLM exemplifies the dynamic nature of clinical laboratory medicine, the inherent spirit of cooperation among professionals in this scientific field, and the international desire to strive for the highest level of clinical laboratory practice for the benefit of patients.     

WHO informal consultation on regulatory evaluation of therapeutic biological medicinal products held at WHO Headquarters, Geneva, 19-20 April 2007.

This report reflects the discussion and conclusions of an informal consultation held on 19-20 April 2007 at the World Health Organization concerning the regulatory evaluation of therapeutic biological medicinal products. The objectives of this meeting were to discuss the current status of so-called "similar" biological medicinal products (biosimilars) and to review regulatory pathways and challenges in evaluating the quality, safety and efficacy of these products. Biosimilars are products that are subject to licensing with a reduced data package due to a proven 'similarity' to the licensed reference product. The meeting was attended by experts in biotherapeutics from regulatory agencies, industry and academia representing 16 countries worldwide. Dr. Elwyn Griffiths (Canada) acted as Chairman and Dr. James Robertson (UK) was the Rapporteur. The meeting strongly focused on the usage of biosimilars and the current regulatory situation in many different countries. The application of International Nonproprietary Names (INN) to biosimilars, their potential immunogenicity, and WHO international standards and reference materials were also discussed, alongside presentations from the innovator and generic manufacturing industries. The consultation recognized the importance of the terminology as well as a definition of biosimilars for future considerations of these products. However, achieving a global consensus on the terminology for these new challenging products was not attempted at the Consultation, and it was decided that a future WHO working group should act on this issue as a next step. For purposes of this meeting report only, the term 'biosimilars' is temporarily used to refer to this category of products. It became clear that biotherapeutics authorized on the basis of a reduced data package are available and being used in some countries, with more appearing on the market. The existence of divergent approaches to the regulatory oversight of biosimilars in different countries revealed a need for defining regulatory expectations for these products at the global level. While many countries are following the guideline developed within the EU for quality aspects, discrepancies remain regarding the non-clinical and clinical studies of these products. The Consultation recommended that the WHO should develop a guideline in this area in order to provide a framework for the development of regulatory pathways for these products worldwide. For this purpose, agreement on the scope, definition and terminology of these products was deemed necessary. The interchangeability and substitution of products were also flagged as areas in need of harmonization. A WHO working group should be established to develop a guideline that would promote global consensus on the regulation of biosimilars, assist in their registration and enhance the availability of safe and effective biosimilar products worldwide.     

International reference standards in coagulation

Measurement of coagulation factor activity using absolute physico-chemical techniques is not possible and estimation therefore relies on comparative bioassay relative to a reference standard with a known or assigned potency. However the inherent variability of locally prepared and calibrated reference standards can give rise to poor agreement between laboratories and methods. Harmonisation of measurement between laboratories at the international level relies on the availability of a common source of calibration for local reference standards and this is provided by the World Health Organization (WHO) International Standards which define the International Unit for the analyte. This article describes the principles, practices and problems of biological standardisation and the development and use of reference standards for assays of coagulation factors, with particular emphasis on WHO International Standards for both concentrates and plasma.     

HUPO Biological Reference Material Initiative Workshop 26 September 2009, Toronto,Canada

The Biological Reference Material Initiative Workshop held at the Toronto HUPO congress on 26 September 2009, focused on the development of new biological reference materials and tools for the assessment of reproducibility, the solutions to many of the technical challenges in proteomics and protein‐based molecular diagnostics. This half‐day meeting included presentations from leading scientists from the worldwide proteomic community, who shared a common interest in standardization and increased accuracy of proteomic data. The conclusion was that proteomics is highly sensitive to both biological and technical variability. It is this biological and technical variance, when not accounted for by experiment design, that invalidates proteomic experiments, but both of these issues can be dealt with by tackling reproducibility.     

WHO Study Group on cell substrates for production of biologicals, Geneva, Switzerland, 11–12 June 2007

For many years, the World Health Organization (WHO) has provided global leadership in defining technical specifications for quality assurance and safety of biological medicines produced in cell substrates. Current WHO requirements for the use of animal cells as substrates for production of vaccines and other biologicals were adopted by the WHO Expert Committee on Biological Standardization in 1996 (WHO TRS 878). Since then, significant progress especially in the development of vaccines in novel continuous cell lines of mammalian origin as well as in insect cells has been made and consequently there is an increasing need for the re-evaluation of existing criteria for the acceptability of such cell lines. In addition there is also a need to consider new issues in cell substrate safety arising from these new cell types and developments in technology and scientific knowledge. In response to these demands, the WHO Study Group on Cell Substrates was formed in 2006 to initiate revision of WHO requirements and to address the need for further research in this area. At its second meeting on 11-12 June 2007, the Study Group reviewed scientific data that would form the basis for new recommendations and made a number of proposals for further investigations. The Study Group is working on the preparation of a revised WHO document, and a broad consultation with regulators, manufacturers, and other relevant parties is planned for 2008.     

The standardization of infectivity titrations of poliovaccines--a WHO collaborative study

The reproducibility of a method for infectivity titrations of live poliovaccines using microtitre plates was investigated in a WHO collaborative study involving eight laboratories. The large variation (up to 100-fold) in estimates of infectivity observed between laboratories using their local methods of assay was reduced to no more than fourfold when a common method was used. However, expressing infectivities relative to those of the monovalent reference viruses improved the agreement between the laboratories irrespective of the titration method employed. On the basis of these results, WHO adopted the common method used in the study as its recommended method for poliovirus titrations and established the preparations studied as international reference materials for the infectivity titrations of live poliovaccines.     

Optimization of the formulation for a candidate lyophilised tetanus toxoid reference preparation

Tetanus toxoid is a vital primary reference material used for standardization of assays required to establish the antigenic purity of tetanus toxoid for vaccine production. Several formulations were assessed and ampouled fills of each formulation lyophilised. The relative Lf content determined by Ramon flocculation, SRD, and ELISA assays was measured. The stability of the tetanus toxoid activity in each formulation was assessed by accelerated degradation studies. Formulations containing glycine were not suitable in flocculation tests but both sorbitol and trehalose formulations were. The trehalose/sodium chloride formulation had a good appearance, showed good activity in all assays and maintained its activity best under stress conditions. This formulation has been applied to a large scale batch of ampoules prepared as a WHO candidate replacement standard, evaluated in a collaborative study and accepted as a replacement WHO IS for use in flocculation test (WHO ECBS, October 2007, ref no BS/07.2061). The stability of this formulation was also excellent for the large scale batch. The benefits of using thermal analysis and freeze drying microscopy coupled with small scale lyophilisation trials in order to screen formulations for the preparation of batches of biological reference materials are demonstrated.     

Diagnostic kits in parasitology: which controls?

The development of new diagnostic tools particularly for some parasitic "neglected diseases", is slowed or even hindered by limited resources assigned for basic and applied research in public institution and private sector. Even if the time-line and costs needed for developing a new In Vitro Diagnostic (IVD) test are generally lower compared to vaccines or new drugs, industry is poorly engaged in investing resources due to the perception of limited markets. To accelerate the development of diagnostics for the world's most deadly diseases, the World Health Organization's (WHO) Special Programme for Research and Training in Tropical Diseases (TDR), the United Nations Development Programme, the World Bank and the Gates Foundation, last year launched a new initiative, FIND (Foundation for Innovative New Diagnostics, www.finddiagnostics.org). The aim is to "apply the latest biotechnology innovations to develop and validate affordable diagnostic tests for diseases of the developing world". Ideally, a new diagnostic test should be accurately evaluated prior to use in medical practice. The first step would be a pre-clinical evaluation, an analytic study to determine its laboratory performance. A crucial point in this phase is the calibration of reagents (antigens, antibodies, DNA probes, etc.) against a standard reference preparation. WHO, through the WHO International Laboratories for Biological Standards, "provides International Biological Reference Preparations which serve as reference sources of defined biological activity expressed in an internationally agreed unit" (www.who.int/biologicals/IBRP/index.htm). Standardization allows "comparison of biological measurements worldwide" and ensures the reliability of diagnostic procedures. These preparations are generally intended for use in the characterization of the activity of secondary reference preparations (regional, national or in-house working standards). Unfortunately, international reference standards for parasitic diseases are not available at present, except for Toxoplasma antibodies. The first international standard reagent for Anti-Toxoplasma Serum was established in 1968 and at present, an international standard reference serum, Anti-toxoplasma serum, human TOXM is available at the National Institute for Biological Standards and Control (NIBSC) in UK. Several collaborative, multicenter studies were carried out to assess the performance of different methods and commercial tests for the diagnosis of toxoplasmosis, by providing to participating laboratories a panel of well-defined sera to be tested. A four-phase process following well-accepted methodological standards for the development of diagnostics, analogous to those internationally accepted for drugs and vaccines was recently proposed. The pre-clinical evaluation, the analytic study to assess sensitivity, specificity, predictive values in laboratory (phase I), should be followed by a proof of principle study to distinguish diseased from healthy persons in easily accessible populations (phase II). The evaluation of test performance in populations of intended use (phase III), and finally the delineation of cost-effectiveness and societal impact of new tests in comparison with existing tools (phase IV) should complete the validation procedure. In this context, national regulatory agencies play a major role in pre-market approval and post-market surveillance of IVDs. The European Community in 1998 approved a directive (Directive 98/79/EC) which rules the marketing of IVD medical devices, in order to harmonise the performance levels and standards in European countries. But, among IVDs for parasitic diseases, only those to detect congenital toxoplasmosis are submitted to defined procedures to provide the verification of products before their placing on the market and the surveillance after their marketing by a notified body, which perform appropriate examinations, tests and inspections to production facilities to verify if the device meets the requirements of the directive. In U.S.A., the Food and Drug Administration (FDA), through the Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD), provides a comprehensive and regulatory activity for IVDs through pre-market evaluation and post-market surveillance. In developing countries, the scarcity of resources limits the procedures through which the national control authority can assure safety, quality and efficacy of products marketed, both imported and locally manufactured.     

Comparative analysis of stability and toxicity profile of three differently capped gold nanoparticles for biomedical usage

Nowadays gold nanoparticle (GNP) is increasingly being used in drug delivery and diagnostics. Here we have reported a comparative analysis of detailed stability and toxicity (in vitro and in vivo) profile of three water soluble spherical GNPs, having nearly similar size, but the surfaces of which were modified with three different capping materials aspartic acid (GNPA), trisodium citrate dihydrate (GNPC) or bovine serum albumin (GNPB). Spectral analyses on the stability of these GNPs revealed that depending on the nature of capping agents, GNPs behave differently at different environmental modalities like wide range of pH, high salt concentrations, or in solutions and buffers of biological usage. GNPB was found to be extremely stable, where capped protein molecule successfully maintained its secondary structure and helicity on the nanoparticle, whereas colloidal stability of GNPA was most susceptible to altered conditions. In vitro cytotoxicity of these nanoparticle formulations in vitro were determined by water soluble tetrazolium and lactate dehydrogenase assay in human fibroblast cell line (MRC-5) and acute oral toxicity was performed in murine model system. All the GNPs were non-toxic to MRC-5 cells. GNPC had slight hepatotoxic and nephrotoxic responses. Hepatotoxicity was also evident for GNPA treatment. Present study established that there is a correlation between capping material and stability together with toxicity of nanoparticles. GNPB was found to be most biocompatible among the three GNPs tested.     

The Second International Standard for somatropin (recombinant DNA-derived human growth hormone): preparation and calibration in an international collaborative study.

A preparation of somatropin (recombinant DNA-derived human growth hormone) was prepared as lyophilised ampoules according to WHO procedures for international biological standards. The candidate preparation (98/574) was evaluated in an international collaborative study (16 laboratories, nine countries), with the following aims: (i) to determine the suitability of the preparation to serve as the International Standard for somatropin by studying its performance in the current range of physico-chemical and biological assay methods employed for somatropin; (ii) to assign a content in terms of the existing (first) International Standard for somatropin, using the currently recognised assay procedure (Size Exclusion High Performance Liquid Chromatography, SE HPLC); (iii) to confirm the specific biological activity of the candidate preparation; (iv) to confirm the stability of the candidate preparation. On the basis of the collaborative study WHO agreed that: the preparation in ampoules coded 98/574 is suitable to serve as the next WHO International Standard for somatropin; the preparation in ampoules coded 98/574 should be established as the second International Standard for somatropin, with a defined ampoule content of 1.95 mg total somatropin plus somatropin-related proteins per ampoule; the specific activity of the preparation should be defined as 3.0 IU/mg somatropin.     

WHO international reference reagents for bovine and porcine proinsulins

Candidate preparations for International Reference Reagents (IRR) for immunoassays of bovine and porcine proinsulin were evaluated in an international collaborative study. With the authorization of the Expert Committee on Biological Standardization of WHO, the following preparations were established as IRRs: bovine proinsulin (code 84/514, defined ampoule content 25 micrograms) and porcine proinsulin (84/528, 20 micrograms). The content of ampoules of these materials is defined in terms of mass rather than international units of activity, therefore they are IRR rather than International Standards. Both preparations are intended as primary reference reagents for the calibration of immunoassays.     

Technical note: comparison of the Maresh reference data with the who international standard for normal growth in healthy children

The Maresh reference data on stature and long bone lengths in a sample of healthy middle-class children from Denver, Colorado [Maresh: Am J Dis Child 66 (1943) 227-257; Maresh: Am J Dis Child 89 (1955) 725-742; Maresh: Human growth and development (1970) p 155-200], have been used extensively by biological anthropologists to estimate juvenile age and body size using skeletal elements and to assess growth in skeletal series from different ethnic populations or archaeological cultural groups. How well these data reflect the potentially diverse growth patterns of healthy human populations from different geographic areas is unknown. Similarly, the efficacy of using the Maresh reference data to estimate stunting prevalence in prehistoric populations is unknown. This report presents the results from a comparison of the Maresh data on supine length and standing height to the World Health Organization (WHO) international child growth standard. The WHO growth standard is meant to depict typical human growth under optimal conditions and can be used to assess children worldwide, regardless of ethnicity and socioeconomic status. The results from this comparison indicate that although the Maresh reference data generally conform to the WHO standard, reflecting a normal human growth pattern, and therefore serve as a suitable reference for comparative studies of growth patterns, these reference data are not suitable for estimating stunting prevalence.     

World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA

Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the “1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection” (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.     

Calibration of replacement international standards of diphtheria and tetanus toxoids for use in flocculation test

The 1st International Reference Reagents (IRR) of Diphtheria and Tetanus Toxoids for Flocculation Test (DIFT and TEFT) were established by the WHO in 1988. These reagents are essential for the standardization of assays used to calculate Lf units of toxoids. Candidate replacement materials were provided by several European vaccine manufacturers and were formulated and freeze-dried at NIBSC. This paper provides a summary of the results of an international collaborative study including 18 laboratories from 16 countries, which examined the candidate replacement materials in a variety of methods. Materials 02/176 and 04/150 were proposed and adopted by the Expert Committee on Biological Standardization of WHO in October 2007 as 2nd WHO International Standards of Diphtheria and Tetanus Toxoid for use in Flocculation Test. The replacement standards were assigned the value of 1100 and 690Lf/ampoule, respectively, based on results of flocculation tests carried out using provided reagents. Material coded 02/176 fully complied with the WHO specifications for stability, residual moisture content, precision of fill and sterility. Stability of material coded 04/150 was slightly lower than expected but predictions were based only on 2-year data and were to be further monitored, post-adoption.     

Landscape analysis of NTD diagnostics and considerations on the development of a strategy for regulatory pathways

Access to quality-assured, accurate diagnostics is critical to ensure that the 2021–2030 neglected tropical disease (NTD) road map targets can be achieved. Currently, however, there is limited regulatory oversight and few quality assurance mechanisms for NTD diagnostic tools. In attempting to address such challenges and the changing environment in regulatory requirements for diagnostics, a landscape analysis was conducted, to better understand the availability of NTD diagnostics and inform future regulatory frameworks. The list of commercially available diagnostics was compiled from various sources, including WHO guidance, national guidelines for case detection and management, diagnostic target product profiles and the published literature. The inventory was analyzed according to diagnostic type, intended use, regulatory status, and risk classification. To estimate the global need and size of the market for each type of diagnostic, annual procurement data were collected from WHO, procurement agencies, NGOs and international organizations, where available and global disease prevalence. Expert interviews were also conducted to ensure a better understanding of how diagnostics are procured and used. Of 125 diagnostic tools included in this analysis, rapid diagnostic tools accounted for 33% of diagnostics used for NTDs and very few diagnostics had been subjected to regulatory assessment. The number of tests needed for each disease was less than 1 million units per annum, except in the case of two diseases, suggesting limited commercial value. Despite the nature of the market, and presumed insufficient return on commercial investment, acceptable levels of assurance on performance, quality and safety of diagnostics are still required. Priority actions include setting up an agile, interim, stepwise risk assessment mechanism, in particular for diagnostics of lower risk, in order to support national NTD programmes and their partners with the selection and procurement of the diagnostics needed to control, eliminate and eradicate NTDs.     

Measurement of cytokines by bioassays: theory and application

A large number of cytokines have been characterized, of which several have proved successful in the clinic as biotherapeutic agents for malignant, infectious or autoimmune diseases. As biologically active proteins, they cannot be fully characterized by physicochemical methods alone. Thus, biological assays (bioassays) have become increasingly important for their biological characterization and potency determinations. Since cytokines exert various biological activities in vitro, cultured cell line-based bioassay methods have mainly been developed to quantify potency. Such bioassays, like all biological systems, are inherently variable. Thus, measurement of potency of a particular cytokine must be made relative to a common, stable, reference preparation of the same cytokine to permit valid inter-assay and inter-laboratory comparisons. The development and establishment of appropriate primary reference preparations as World Health Organization (WHO) International standards (IS) and reference reagents (RR) is essential for the standardization of bioassays. This review addresses the practical and statistical considerations for the development of valid bioassays, the preparation and use of WHO IS and RR and, in brief, the types of bioassay methods applicable to potency measurements of individual cytokines. More extensive details for the potency determinations of tumor necrosis factor-alpha (TNF-alpha), related cytokines, and biotherapeutic anti-TNF-alpha products are provided.     

WHO international reference reagents for human proinsulin and human insulin C-peptide

Candidate preparations for international reference reagents for immunoassays of human proinsulin and human insulin C-peptide were evaluated in an international collaborative study. With the authorization of the Expert Committee on Biological Standardization of WHO, the following preparations were established as international reference reagents: human proinsulin (84/611, nominal ampoule content 6 micrograms) and human insulin C-peptide (84/510, 10 micrograms). Each preparation is intended as a primary reference reagent for the calibration of immunoassays.     

Synthesis and in vitro biological activity of retinyl retinoate, a novel hybrid retinoid derivative

A new hybrid, retinyl retinoate 1, was synthesized with a condensing reaction between retinol and retinoic acid to improve the photo-stability, and the in vitro biological activity of the hybrid was analyzed. This retinol derivative had enhanced thermal stability and decreased photosensitivity, and exhibited decreased cell toxicity compared to that of retinol. In addition, RAR activity analysis showed that retinyl retinoate 1 had higher inhibitory activity against c-Jun than retinol and showed superior effects on collagen synthesis compared to retinol. Thus, retinyl retinoate 1 may have the potential to be conveniently used as an additive in cosmetics for prevention and improvement of skin aging and medicines for the treatment of skin troubles due to its excellent stability under severe and accelerated conditions.     

Updating the International Health Regulations

First adopted in 1951, the International Health Regulations (IHR) provide the international legal framework for efforts to prevent and control the cross-border spread of communicable diseases. In 1995, after outbreaks of emerging infections had rendered the IHR increasingly obsolete, the 192 member states of the World Health Organization (WHO) requested a major updating of the regulations to adapt them to the highly mobile, globalized world of the 21st century. After negotiations in 2004 and 2005, the revised IHR text was adopted unanimously by the World Health Assembly, WHO's highest policymaking body. This article reviews the 2005 regulations and discusses their implications for the international response to natural epidemics and to incidents involving the accidental or deliberate release of biological or chemical agents or radiological materials.