Calibration of the WHO 5th IS for Blood Coagulation Factor IX, Concentrate and Ph. Eur. Human Coagulation Factor IX Concentrate Biological Reference Preparation Batch 3 and investigation of the suitability of an IS as potency standard for purified full-length recombinant FIX.

A joint World Health Organization (WHO) - European Directorate for the Quality of Medicines & HealthCare (EDQM) study was run to calibrate the WHO 5th International Standard (IS) for Blood Coagulation Factor IX (FIX), Concentrate, and European Pharmacopoeia (Ph. Eur.) Human Coagulation Factor IX concentrate Biological Reference Preparation (BRP) Batch 3. The suitability of the 4th IS as a potency standard for purified full-length recombinant FIX (rFIX) was also investigated. Forty-nine laboratories contributed data for the calibration of 2 plasma-derived FIX candidates, relative to the 4th IS, from clotting and chromogenic assays. The intra-laboratory variability was reasonably low; the inter-laboratory variation was lower for sample B (14/148) than for sample C (14/162). Although there were no discrepancies between clotting and chromogenic assays, a significantly lower potency was obtained for sample C with clotting assays when buffer rather than FIX-deficient plasma was used as pre-diluent. A significant assay discrepancy was observed with estimates for the 4th IS for Blood Coagulation Factors FII, VII, IX, X, Plasma against the 4th IS, resulting in a clotting to chromogenic activity ratio of 1.11. The study also investigated the comparability of the plasma-derived concentrate standard with the rFIX products and considered the establishment of an IS for rFIX. The 3 rFIX products currently licensed were represented in this study. Data from 49 laboratories for 2 rFIX candidates were received, with additional results for another full-length rFIX test sample returned by 6 laboratories. The intra-laboratory variability when the rFIX samples were assayed against the 4th IS was acceptably low. Although the full-length rFIX could be assayed against the plasma-derived 4th IS and provided statistically valid results, there were large discrepancies among the clotting assays using different APTT reagents. The inter-laboratory variability of the chromogenic assays was similarly high. There were also significant clotting and chromogenic assay discrepancies. The data from the present study indicate that a recombinant standard for rFIX products will minimise assay discrepancies and improve inter-laboratory agreement. However, they also underline that the value assignment of the 1st rFIX IS needs careful consideration. The Expert Committee on Biological Standardization (ECBS) of WHO was therefore not requested to consider the establishment of an IS for rFIX. In order to ensure continued harmonised standards, sample B (14/148) was established as the WHO 5th IS for Blood Coagulation Factor IX, Concentrate, and as Ph. Eur. Human Coagulation Factor IX, concentrate BRP Batch 3 with the functional activity of 10.5 IU/ampoule.

Establishment of an erythropoietin CRS with stable measurable dimer content for SEC system suitability qualification

The European Pharmacopoeia (Ph. Eur.) monograph 1316 'Erythropoietin concentrated solution' prescribes that the dimer content of therapeutic erythropoietin (EPO) preparations must not exceed 2% as determined by Size-Exclusion Chromatography (SEC). This report describes the evaluation of a candidate Chemical Reference Substance (cCRS) to serve as system suitability reference material for the qualification of SEC systems used to assess dimer and oligomer content in EPO solutions. The study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) was performed with the participation of six European laboratories which tested the candidate material and the EPO for physicochemical tests CRS batch 1. The candidate material was shown to be a suitable reference material for the determination of the resolving capability of the SEC system for separation of dimer and higher oligomers from monomeric EPO. The cCRS was adopted by the Ph. Eur. Commission as Erythropoietin for SEC system suitability CRS batch 1 following consideration of the report. The importance of the resolving capability of the SEC system, as defined by the peak ratios or the peak-to-valley resolution, together with the asymmetry of the peaks eluted, and the linear response of the UV detector were all seen as critical parameters. Therefore, the monograph Erythropoietin concentrated solution (1316) was revised concomitantly to take account of the CRS and to set acceptance criteria for these critical parameters..

International reference reagents to standardise blood group genotyping: evaluation of candidate preparations in an international collaborative study.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate, in an international collaboration, four lyophilised genomic DNA preparations, selected from genotyped and phenotyped donors by the study organisers, for their suitability to standardise and control blood group genotyping procedures for common ancestral Caucasian and Black African alleles. MATERIALS AND METHODS: Twenty-nine laboratories performed 'blind' testing of replicated ampoules of the candidate reference reagents, RBC1 (10/232), RBC4 (10/236), RBC5 (10/238) and RBC12 (10/234), using a range of genotyping procedures, most commonly classical PCR using allele or sequence specific primers. RESULTS: The majority of laboratories reported blood group genotypes in accordance with those determined by the study organisers and the serological phenotypes. Despite an overall high level of accuracy in genotyping, the identified errors and inconsistencies, and the limited genotyping capabilities of many laboratories, confirmed the need for validated reference materials to control test procedures. CONCLUSIONS: The establishment of RBC1, RBC4, RBC5 and RBC12 as World Health Organization Reference Reagents will facilitate international standardisation of blood group genotyping and ensure that such tests are sufficiently sensitive and specific.

Genetic reference materials and their application to haematology.

Genetic investigations are becoming increasingly useful and widespread in many areas of human health. However, there is a worldwide lack of certified reference materials for use in genetic testing, meaning that tests are being run without well validated controls and new assays are more difficult to develop and validate. We have responded to this challenge by starting a programme of developing genetic reference materials (GRMs) for international accreditation and worldwide distribution. Our approach has been to make materials for disorders where testing is commonplace and genotyping errors have been demonstrated. To ensure a continuing supply of DNA, cell lines are established from consenting, phenotypically well-characterised patients and are then grown up in bulk for genomic DNA extraction to yield up to 100 milligrams of DNA. In most cases the DNA is then formulated, freeze-dried and sealed in glass ampoules to ensure greater stability over time and obviate the need for chilled transportation. In this paper we explore the options and routes available to the production of DNA reference materials and describe the establishment of the first internationally recognised reference materials for human genomic DNA, with particular reference to some genetic tests carried out frequently within haematological and cardiovascular laboratories.

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.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

A direct comparison of the antigen-specific antibody profiles of intravenous immunoglobulins derived from US and UK donor plasma.

BACKGROUND AND OBJECTIVES: Intravenous immunoglobulin (IVIG) is used in a range of immunodeficiency states that require a broad spectrum of protective antibodies to a range of common pathogens. A comparison of the antigen-specific antibody profile of preparations of an IVIG (Vigam) derived from US and UK sourced plasma was performed, and these preparations were also compared with three other IVIG products from different manufacturers. MATERIALS AND METHODS: Antibodies against a range of bacterial and viral pathogens were measured by immunoassay. RESULTS: Similar profiles were found for Vigam made from UK and US source plasma and also for the other three IVIGs tested, but some specific differences were observed. CONCLUSIONS: IVIG preparations have a similar therapeutic spectrum of antibodies when prepared from plasma sourced either from the UK or the US.

Solvent/detergent treatment does not alter the tolerance or uptake of human normal immunoglobulin for intramuscular injection.

BACKGROUND AND OBJECTIVES: The tolerability and pharmacokinetics of a solvent/detergent-treated intramuscular immunoglobulin were compared with those of the standard product. MATERIALS AND METHODS: Single, 750-mg intramuscular (i.m.) injections were administered to a total of 36 healthy individuals: 23 in a double-blind trial and 13 in an open trial. Changes in specific serum hepatitis A and hepatitis B antibodies were monitored for a period of up to 3 months postinjection. RESULTS: No serious adverse reactions were reported, and the bioavailability of the solvent/detergent-treated preparation was equivalent to that of the standard i.m. immunoglobulin. CONCLUSION: There is no evidence that solvent/detergent treatment alters the pharmacokinetics or tolerance of human normal immunoglobulin, but it offers additional assurance against potential virus transmission.

Clinical experience with a new solvent detergent-treated intravenous immunoglobulin free of hypotensive effects.

OBJECTIVE: To see if modifications to the processing of intravenous immunoglobulin to include a virus inactivation stage alter immunoglobulin G (IgG) resulting in hypotension in patients. METHODS: Clinical trials were done involving extensive patient monitoring during infusion: in vitro - testing for markers of hypotension, and in vivo - an animal model which closely simulates clinical use. RESULTS: No hypotensive response was seen in the animal model or clinical trial. CONCLUSIONS: The production process used does not damage IgG or create vaso-active kinins as the preparation was free of hypotensive effects.

Fingerprinting of glycans as their 2-aminoacridone derivatives by capillary electrophoresis and laser-induced fluorescence.

Capillary electrophoresis and laser-induced fluorescence detection have been used to fingerprint the 2-aminoacridone derivatives of complex glycans released from bovine fetuin and human IgG monoclonal antibodies. The utility of this method in distinguishing between N- and O-linked oligosaccharides and in determining the presence of sialic acid residues in glycan mixtures at an early stage of analysis has been demonstrated.

Red-cell IgM-antibody capture assay for the detection of Mycoplasma pneumoniae-specific IgM.

A red-cell IgM-antibody capture assay has been developed for detecting Mycoplasma pneumoniae-specific IgM, which is based on the adsorption or 'capture' of IgM from patients' sera onto so-called 'inagglutinable' bovine red cells, chemically linked with anti-human mu. When M. pneumoniae antigen is added to the system, the red cells agglutinate in the presence of M. pneumoniae-specific IgM. The test was compared with the mu-capture ELISA described by Wreghitt & Sillis (1985), and was found to give comparable results. The two tests had similar sensitivity and specificity and could detect M. pneumoniae-specific IgM for a similar time (up to 6 months) after proven M. pneumoniae infection. However, the red-cell antibody capture assay is a much more simple and rapid test, taking only 1 h to perform (compared to 24 h for mu-capture ELISA). The red-cell IgM-antibody capture assay is therefore amenable to rapid diagnosis of M. pneumoniae infection and the institution of early appropriate antibiotic therapy.

Experimental studies on red cell-based assays for total IgE and allergen-specific IgE.

The potential of red cell-based assays for IgE and allergen-specific IgE has been examined using mouse monoclonal anti-human IgE antibodies and chimaeric human IgE anti-4-hydroxy-3-iodo-5-nitrophenacetyl. Experiments concerned with developing a red cell IgE antibody-capture assay for allergen-specific IgE have pointed to the advantages of presenting the allergen on a second agglutinable red cell.

Isolation of low-frequency class-switch variants from rat hybrid myelomas.

Class-switch variants have been isolated from rat-rat hybrid myelomas by sib selection using a simple assay based on red cell-labelled antiglobulins. The variants detected are consistent with the gene order deduced from molecular cloning. They appear to arise spontaneously at a rate approximately ten-fold lower than for mouse cell lines but the rate of switching back to the parental isotype is substantial in comparison. An IgG2b variant antibody having the same specificity as CAMPATH-1 for human lymphocytes and monocytes is active in antibody-dependent cell-mediated killing (unlike the parental IgG2a) and may prove to be a valuable therapeutic antibody for immunosuppression and treatment of leukaemia and lymphoma.

The N-terminal sequence of albumin Redhill, a variant of human serum albumin.

Albumin Redhill, a variant human albumin, has been isolated by fast protein liquid chromatofocusing. The N-terminal sequence of this protein corresponded to that of albumin A except that one additional arginine residue was attached to the N-terminus.