Biomedical subjects

B Rafferty

Publications and source records attributed to B Rafferty.

At least 19 recordsLinked to original sources

PubMed2003

Biological evaluation of recombinant human erythropoietin in pharmaceutical products.

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4% for the brilliant cresyl blue method, 14.1-30.8% for the selective hemolysis method and 8.5-19.7% for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2%), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products.

Animals↗

PubMed2002

In vitro identification of metabolic pathways and cytochrome P450 enzymes involved in the metabolism of etoperidone.

1. In vitro studies have been carried out to investigate the metabolic pathways and identify the hepatic cytochrome P450 (CYP) enzymes involved in etoperidone (Et) metabolism. 2. Ten in vitro metabolites were profiled, quantified and tentatively identified after incubation with human hepatic S9 fractions. Et was metabolized via three metabolic pathways: (A) alkyl hydroxylation to form OH-ethyl-Et (M1); (B) phenyl hydroxylation to form OH-phenyl-Et (M2); and (C) N-dealkylation to form 1-m-chlorophenylpiperazine (mCPP, M8) and triazole propyl aldehyde (M6). Six additional metabolites were formed by further metabolism of M1, M2, M6 and M8. 3. Kinetic studies revealed that all metabolic pathways were monophasic, and the pathway leading to the formation of OH-ethyl-Et was the most efficient at eliminating the drug. On incubation with microsomes expressing individual recombinant CYPs, formation rates of M1-3 and M8 were 10-100-fold greater for CYP3A4 than that for other CYP forms. The formation of these metabolites was markedly inhibited by the CYP3A4-specific inhibitor ketoconazole, whereas other CYP-specific inhibitors did not show significant effects. In addition, the production of M1-3 and M8 was strongly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activities in 13 different human liver microsome samples. 4. Dealkylation of the major metabolite M1 to form mCPP (M8) was also investigated using microsomes containing recombinant CYP enzymes. The rate of conversion of M1 to mCPP by CYP3A4 was 503.0 +/- 3.1 pmole nmole(-1) min(-1). Metabolism of M1 to M8 by other CYP enzymes was insignificant. In addition, this metabolism in human liver microsomes was extensively inhibited by the CYP3A4 inhibitor ketoconazole, but not by other CYP-specific inhibitors. In addition, conversion of M1 to M8 was highly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activity. 5. The results strongly suggest that CYP3A4 is the predominant enzyme-metabolizing Et in humans.

Chromatography, Liquid↗

PubMed2001

International Standards for hepatocyte growth factor/scatter factor: initial assessment of candidate materials and their evaluation by multicentre collaborative study.

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent paracrine growth factor with motogenic, mitogenic and morphogenic activities, and a potential therapeutic role in hepatic and renal disease, as well as diagnostic and prognostic applications. It is synthesised as an inactive, single-chain precursor that is cleaved by serine proteases to give a biologically active, heterodimeric form. To develop World Health Organization (WHO) International Standards (IS) for HGF/SF, candidate preparations of the two forms were assessed in a multicentre study in which they were compared with local standards by bioassay and immunoassay. Among laboratories, there was a wide variation in the estimates of potencies of the candidate standards in terms of in-house reference preparations, but between-assay and within-assay variabilities were low within laboratories. In some assay systems, the precursor and heterodimer showed different responses. Since both molecular forms are widely used in current assay systems, this suggested that a reference preparation was required for each form of the HGF/SF molecule. Accordingly, the Expert Committee on Biological Standardization of WHO established the heterodimeric material (96/564) as the first IS for HGF/SF, human, recombinant, with an assigned unitage of 4000 IU/ampoule and, for the purpose of immunoassay calibration, a nominal HGF/SF content of 4 microg/ampoule. The precursor preparation (96/556) was established as the first IS for HGF/SF (precursor) with an assigned unitage of 2000 IU/ampoule and, for the purpose of immunoassay calibration, a nominal HGF/SF (precursor) content of 4 microg/ampoule. The preparations can be obtained upon written request to the National Institute for Biological Standards and Control (NIBSC, PO Box 1193), by e-mail (standards@nibsc.ac.uk) or ordered at http://www.nibsc.ac.uk.

Animals↗

PubMed2001

Assessing potential secondary effects of countermeasures in agricultural systems: a review.

Secondary effects are defined as any positive or negative impacts resulting from the application of countermeasures other than radiological benefits or direct costs. They are categorised into environmental, radioecological, economic and social effects. Impacts on the environment may include changes in water, air and soil pollution or in the conservation and amenity value of an area. Radioecological effects occur when the countermeasure unintentionally alters the behaviour of the target radionuclide or any other radionuclide present. Economic effects may range from changes in agricultural income to environmental costs (e.g. impact of soil erosion on fisheries). Social effects relate to the acceptability of countermeasures, for example in terms of consumer confidence and animal welfare. Recent research into the identification and assessment of secondary effects is summarised. Non-quantitative and quantitative approaches are explained and formal evaluation procedures involving decision matrices and decision support systems are introduced. Examples of recent experimental and modelling work focusing on radiocaesium are given for the following countermeasures: soil application of potassium, administration of AFCF to livestock and ploughing techniques.

Agriculture↗

PubMed2001

FORIA: Forest Impact Analysis. An interactive decision support software providing information on the secondary effects of radiological countermeasures applied in forests.

FORIA (Forest Impact Analysis) is a decision support software that provides a synthesis of published information pertaining to the application of radiological countermeasures in forests and the secondary impacts that may result from their application. By linking relevant key parameters describing the forest environment and its social and economic roles, with information on the effects of countermeasures on a range of forest features, the potential secondary impacts of the countermeasures are indicated. User inputs refine this generic system to reveal the potential secondary effects of selected countermeasures for specific forest scenarios. FORIA has an Eastern European focus but is applicable to all forest scenarios.

Decision Support Techniques↗

PubMed2001

Calculation of the electronic structure of carbon films using electron energy loss spectroscopy.

The detailed understanding of the electronic properties of carbon-based materials requires the determination of their electronic structure and more precisely the calculation of their joint density of states (JDOS) and dielectric constant. Low electron energy loss spectroscopy (EELS) provides a continuous spectrum which represents all the excitations of the electrons within the material with energies ranging between zero and about 100 eV. Therefore, EELS is potentially more powerful than conventional optical spectroscopy which has an intrinsic upper information limit of about 6 eV due to absorption of light from the optical components of the system or the ambient. However, when analysing EELS data, the extraction of the single scattered data needed for Kramers Kronig calculations is subject to the deconvolution of the zero loss peak from the raw data. This procedure is particularly critical when attempting to study the near-bandgap region of materials with a bandgap below 1.5 eV. In this paper, we have calculated the electronic properties of three widely studied carbon materials; namely amorphous carbon (a-C), tetrahedral amorphous carbon (ta-C) and C60 fullerite crystal. The JDOS curve starts from zero for energy values below the bandgap and then starts to rise with a rate depending on whether the material has a direct or an indirect bandgap. Extrapolating a fit to the data immediately above the bandgap in the stronger energy loss region was used to get an accurate value for the bandgap energy and to determine whether the bandgap is direct or indirect in character. Particular problems relating to the extraction of the single scattered data for these materials are also addressed. The ta-C and C60 fullerite materials are found to be direct bandgap-like semiconductors having a bandgaps of 2.63 and 1.59eV, respectively. On the other hand, the electronic structure of a-C was unobtainable because it had such a small bandgap that most of the information is contained in the first 1.2 eV of the spectrum, which is a region removed during the zero loss deconvolution.

Journal Article↗

PubMed2001

Multicenter collaborative study to calibrate salmon calcitonin by bioassay and high-performance liquid chromatography: establishment of the third international standard.

Salmon calcitonin (sCT) is widely used therapeutically in the treatment of patients with postmenopausal osteoporosis, Paget's disease, and some forms of hypercalcemia. Preparations of synthetic calcitonin peptides of high purity and reproducibility are now routinely produced and physicochemical methods, particularly reverse-phase high-performance liquid chromatography (RP-HPLC), are replacing the in vivo biological assay for monitoring and calibration. Although the bioassay is no longer required for routine batch control in Europe, calcitonin bioassays are still required in some countries and in the development of new products. Stocks of the Second International Standard (IS) for salmon calcitonin are now depleted and, to replace it with a new calibrant, an international collaborative study was organized in which the aims were to: determine the activity of the candidate sCT by in vivo bioassay in terms of the second IS; assess the stability of the preparation after accelerated thermal degradation; estimate the purity of the ampouled candidate preparation; and determine the sCT content in gravimetric units by HPLC. The HPLC data in terms of ampoule content were in good agreement giving an estimate of 23.1 (coefficient of variation [CV] 3.8%) microg per ampoule. The HPLC chromatograms revealed a small, but detectable, degree of heterogeneity, which possibly occurred during the formulating or ampouling procedures, resulting in a reduction in monocomponent content (purity) from 96% to 92%. The biological activity of the ampoule contents in international units (IU) was calculated from the mass value and the internationally agreed-upon figure of 6000 IU/per mg for the specific activity of salmon calcitonin. This gave a value of 138 IU per ampoule, which was in good agreement with the biological assay estimate (140 IU per ampoule). The preparation of sCT was subsequently adopted as the Third International Standard by the World Health Organization with an assigned content of 138 IU per ampoule.

Animals↗

PubMed2001

Multicentre collaborative study to calibrate IGF-II by bioassay and immunoassay: establishment of the First WHO Reference Reagent.

A preparation of recombinant insulin-like growth factor-II (IGF-II) (NIBSC code 96/538) was compared with local standards in bioassays and immunoassays by eight laboratories in four countries to assess its suitability for use as a World Health Organisation (WHO) reference reagent. Estimates of relative potencies for the bioassays gave a geometric mean of 1.04 (0.94--1.16) microg of local standard per microg of 96/538. Estimates of relative immunological activities by immunoassay gave a geometric mean of 1.15 (0.94--1.38) microg of local standard per microg of 96/538. The study provided evidence that a common standard for rhIGF-II would be helpful and that 96/538 was sufficiently stable to serve as a reference reagent. Accordingly 96/538 was established as the First WHO Reference Reagent for IGF-II, human, recombinant, and assigned a unitage of 5000 units per ampoule and on the basis of the immunoassay results a nominal mass content of 5 microg per ampoule.

Biochemistry↗

PubMed2001

On the origin of transverse incoherence in Z-contrast STEM.

We use a Bloch wave approach to further investigate the origins of the incoherent nature of Z-contrast imaging using an ADF detector in a STEM. We discuss how, although at high angles the collected electrons will be mostly thermally scattered in addition to the elastic scattering, it is not the thermal scattering that destroys the coherence, rather the combination of the large detector with the high-angle elastic scattering. This incoherent nature of the elastic scattering arises through the filtering of the 1s-type Bloch states by the detector geometry. We show that it is this filtering that renders an atomic column an independent scatterer insensitive to the configuration of neighbouring columns. It also makes the image contrast insensitive to the effects of beam spreading onto neighbouring columns as the probe propagates through the crystal. We also discuss the implications of this for previous calculations of the intensity of Z-contrast images.

Journal Article↗

PubMed2000

Z-contrast Imaging in an Aberration-corrected Scanning Transmission Electron Microscope.

We show that in the limit of a large objective (probe-forming) aperture, relevant to a spherical aberration corrected microscope, the Z-contrast image of a zone-axis crystal becomes an image of the 1s Bloch states. The limiting resolution is therefore the width of the Bloch states, which may be greater than that of the free probe. Nevertheless, enormous gains in image quality are expected from the improved contrast and signal-to-noise ratio. We present an analytical channeling model for the thickness dependence of the Z-contrast image in a zone-axis crystal, and show that, at large thicknesses, columnar intensities become proportional to the mean square atomic number, Z(2).

Journal Article↗

PubMed2000

The development of a sialic acid specific lectin-immunoassay for the measurement of human chorionic gonadotrophin glycoforms in serum and its application in normal and Down's syndrome pregnancies.

OBJECTIVE: We have developed and validated a lectin-immunoassay for the recognition of sialic acid residues on hCG glycoforms in serum. DESIGN: This assay employs a hCG specific capture antibody and a sialic acid specific lectin (Wheat Germ Agglutinin) labelled with horse radish peroxidase. RESULTS: The standard curve covered hCG concentrations of 0-4000 IU/l (3rd IS for hCG, 75/537) with an analytical sensitivity of 1.0 IU/l. The within and between batch coefficient of variation was < 7% for all doses. Cross-reactivity of < 1% with TSH, LH, FSH, hCGalpha, hCGbeta and desialylated hCG confirmed assay specificity. Dilutions of serum of < 10% final concentration were parallel to the standard curve (within and between batch CV, < 6%). The assay working range was 100 - > 500 000 IU/l and the recovery of hCG from serum was in the range of 94.5% to 115.4%, with a mean value of 102.1%. The assay detected a time dependent change in hCG sialylation during normal pregnancy with the relative abundance of sialylated hCG declining after week 9 and increasing after week 15 of gestation. In addition preliminary studies showed that maternal serum hCG concentrations measured with the lectin-immunoassay were elevated in high risk Down's pregnancies (as defined by conventional screening tests between weeks 16-18 gestation, median multiple of median, 3.14; range 1.81-19.12, P < 0. 001) and low risk (1.57, 0.49-6.14, P = 0.034) compared to normal (1. 00, 0.32-3.20) pregnancies. Furthermore, the lectin immunoassay had greater discriminatory power compared to conventional immunoassay of hCG and hCGbeta between normal and both low and high risk Down's pregnancies. CONCLUSION: This assay will allow analysis of serum samples for the investigation of sialylated variants of hCG glycoforms in various pathological and physiological situations.

Analysis of Variance↗

PubMed2000

Zero loss peak deconvolution for bandgap EEL spectra.

This paper shows how bandgap EEL spectra are commonly processed either by deconvolution or subtraction methods in an attempt to remove effects arising from the finite width and long tail of the zero loss peak. This paper will compare the two main methods, and show that the deconvolution method is significantly more reliable and free of user-interpretation or artefacts. We first consider how the zero loss peak is produced and what effects experimental conditions have upon it, and then show that only by the deconvolution method can accurate bandgap energies and bandgap densities of states be determined.

Journal Article↗

PubMed2000

Long-term trends of radiocesium activity concentrations in vegetation in Irish semi-natural ecosystems.

137Cs activity concentrations were determined in vegetation from four Irish blanket bog ecosystems during the summer months of 1989-1997. The dominant and abundant vegetation species were sampled at each site and the data were used to estimate long-term trends of 137Cs activity concentrations. A general decline in the 137Cs activity concentrations in vegetation was observed from all sites sampled. The fastest and most consistent long-term rates of 137Cs decline were observed in the bryophyte and lichen vegetation group with 137Cs effective half-life (T ef) values of 2.2 to 10.7 y. The ericoid group with the exception of one case also showed a significant long-term decline in 137Cs activity concentrations with T ef values of 3.5 to 12.4 y as did the rush species Juncus squarrosus (T ef range 9.3 to 12.8 y). The sedge and grass groups showed the slowest and the least consistent rates of 137Cs decline, with T ef values of 2.9 to 59.8 y. Preliminary evaluation of 137Cs activity concentrations in sheep from some of the studied sites suggests that the decline found in vegetation is reflected in sheep.

Animals↗

PubMed2000

Reference reagents for prostate-specific antigen (PSA): establishment of the first international standards for free PSA and PSA (90:10)

BACKGROUND: Prostate-specific antigen (PSA) measurements in serum by immunoassay are widely used in the screening, diagnosis, and monitoring of patients with prostate cancer although the lack of common reference reagents has led in the past to wide differences in estimates. We report here the results of a WHO international collaborative study in which two preparations of PSA representative of the main immunoreactive components in serum, free PSA and PSA 90:10, and a preparation of recombinant DNA-derived PSA were assessed as potential standards for the calibration of diagnostic immunoassays for PSA. METHODS: Coded vials of the candidate materials and serum preparations containing PSA in the clinically important range were provided to the 10 laboratories in the study, and participants were asked to perform PSA assays currently in use in their laboratories. Data from 89 immunoassays by 26 different method-laboratory combinations were contributed to the study and analyzed centrally at the National Institute for Biological Standards and Control. RESULTS: Potency estimates of the preparations relative to the in-house calibrators were in good agreement with the target value of 1 microg of total PSA/vial, the preparation of free PSA giving 1.10 microg/vial (95% confidence interval, 0.99-1.21 microg/vial) and PSA 90:10, 1.11 microg/vial (95% confidence interval, 1.04-1.18 microg/vial). No immunoreactivity was detected in ampoules containing the recombinant material. Use of a common standard of PSA 90:10 significantly reduced the between-laboratory geometric coefficients of variation for serum samples included in the study and gave a much narrower range of potency estimates. CONCLUSIONS: The preparation of free PSA was established by WHO as the First International Standard for PSA (free) with an assigned content of 1 microg of total PSA per vial. In addition, the preparation of bound PSA was established as the First International Standard for PSA (90:10) with an assigned content of 1 microg of total PSA per vial.

Calibration↗

PubMed1999

Electronic structure analysis of (In, Ga, Al) N heterostructures on the nanometre scale using EELS.

In this work the local electronic structure of MOVPE-grown (In, Ga, Al) N heterostructures has been investigated by electron energy loss spectroscopy (EELS). The cold field-emission scanning transmission electron microscope (VG HB501) used was equipped with a dedicated parallel EELS system which provides high dispersions at an energy resolution of 0.35 eV with the use of subnanometre electron probes the spatial resolution of the measurements depends on the physical localization of the scattering process itself and thus is in the order of nanometres. The low-loss region of the energy spectra gives information on plasmon excitations and transitions across the bandgap. The main problem on looking at the bandgap region of EELS spectra is to separate the bandgap signal from the fading tail of the zero-loss peak. High energy resolution and application of suitable deconvolution routines for removal of the zero-loss peak extract improved information from this energy region. Thus the EEL spectra of different group III nitrides reveal the onset of the bandgap itself and the characteristic shape of the joint density of states. From these results the local optical properties can be deduced via a Kramers-Kronig transformation. The data obtained show detailed structure on the energy scale and are in excellent agreement with optical ellipsometric results. In comparison with these techniques EELS methods yield a superior spatial resolution of better than 10 nm which enables detailed investigation of the effect of local defects and boundaries on the optical properties.

Journal Article↗

PubMed1999

Comparison of pituitary and recombinant human thyroid-stimulating hormone (rhTSH) in a multicenter collaborative study: establishment of the first World Health Organization reference reagent for rhTSH.

BACKGROUND: The increasing use of recombinant-DNA-derived materials in therapy and diagnosis poses a new challenge for biological standardization, that of developing reference preparations appropriate for both the native and recombinant products. Here we report the results of an international collaborative study that was carried out under the auspices of WHO to assess the suitability of a preparation of recombinant thyroid-stimulating hormone (rTSH; 94/674) to serve as a potential standard for the calibration of diagnostic immunoassays compared with the International Reference Preparation (IRP) for human TSH (80/558). METHODS: Coded samples were provided to the 33 laboratories in the study, and participants were asked to perform TSH assays currently in use in their laboratories. Twenty-eight laboratories contributed 93 immunoassays in 41 different method-laboratory combinations, and an additional 5 laboratories contributed bioassay data. All data were analyzed centrally at the National Institute for Biological Standards and Control. RESULTS: The results obtained in different laboratories and with different assay systems revealed significant variability between estimates of rTSH relative to the IRP. These ranged from 5. 51 mIU (95% limits, 3.95-7.67 mIU) per ampoule by RIA to 7.15 mIU (95% limits, 6.7-7.63 mIU) per ampoule by immunofluorometric assay. However, the results showed that the assignment of a value of 6.70 mIU per ampoule of 94/674 would give reasonable continuity with the IRP in many assay systems. CONCLUSIONS: The preparation was established as the First WHO Reference Reagent for TSH, human, recombinant, to provide a means of validating assay performance and to maintain continuity with the IRP without compromising clinical data.

Freeze Drying↗

PubMed1998

Recombinant human albumin as a stabilizer for biological materials and for the preparation of international reference reagents.

Recombinant human albumin expressed in Saccharomyces cerevisiae was compared with native human serum albumin in its physicochemical properties and in its use as a stabilizer in lyophilized preparations of thyroid-stimulating hormone (TSH), interleukin 15 (IL-15) and granulocyte colony-stimulating factor (G-CSF). Advantages of recombinant albumin include its lack of potential human contaminants and infectious agents. When used at concentrations of 0.1-0.2% (w/v), recombinant albumin was equivalent to native serum albumin in its capacity to protect immunological, biological and biochemical properties of TSH, IL-15 and G-CSF. Physicochemical characteristics of the two forms of albumin including their binding to fatty acids were also similar. The recombinant form of albumin used in this study should be considered as a suitable stabilizer in the preparation of lyophilized products and reference reagents.

Endotoxins↗

PubMed1998

Epoetin alfa and beta differ in their erythropoietin isoform compositions and biological properties.

Epoetin alfa and beta are the two forms of recombinant DNA-derived erythropoietin (rEPO), both synthesized in Chinese hamster ovary cells, which are used for the treatment of erythropoietin (EPO)-responsive anaemias. Several batches of each of these rEPOs were compared for differences in their EPO isoform compositions by isoelectric focusing (IEF) and in a range of lectin-binding assays, and for differences in their EPO activities by in-vivo and in-vitro mouse bioassays and by immunoassay. Epoetin beta was found to differ from epoetin alfa in containing: (a) a greater proportion of more basic isoforms, (b) a greater proportion of EPO binding to Erythrina cristagalli agglutinin (which binds N-glycans with nonsialylated outer Gal beta1-4GlcNAc moieties), and (c) isoforms with higher in-vivo:in-vitro bioactivity ratios. Epoetin beta also contained slightly more than epoetin alfa of EPO binding to Lycopersicon esculentum agglutinin (which binds N-glycans containing repeating Gal beta1-4GlcNAc sequences), to the leucoagglutinin of Phaseolus vulgaris (which binds tetraantennary and 2,6-branched triantennary N-glycans) and to Agaricus bisporus agglutinin (which binds Gal beta1-3GalNAc containing O-glycans). No differences were found between the two rEPOs in their binding to a further five lectins. The differences between the isoform composition of epoetin alfa and beta, and the smaller inter-batch differences appear to be due to differences in glycosylation. The higher murine in-vivo:in-vitro bioactivity ratio of epoetin beta compared to epoetin alfa could not be explained in terms of differences in their degrees of sialylation, but was consistent with differences in their pharmacokinetics and pharmacodynamics observed in human subjects. There have been no reports that epoetin alfa differs from epoetin beta in its clinical efficacy, but the differences between epoetin alfa and beta in some analytical systems suggest that there might be a need for separate international standards for these two types of rEPO.

Animals↗