[Self-medication with local anaesthetics by glaucoma patients using teletonometry]. / Selbstmedikation mit Lokalanästhetika durch Glaukompatienten bei der Teletonometrie.

BACKGROUND: Self-tonometry, a supplementary measurement of the intraocular pressure in ophthalmology by glaucoma patients using an automatic tonometer, will become more and more important in the future. As long as the self-tonometry has to work in the contact modus with the ocular surface, home application of a topical anaesthetic by the glaucoma patient will be a requirement for a successful measurement. So far no severe problems within this controlled self-medication have been seen. Nevertheless, public health authorities believe patient health is put at high risk by the application of local anaesthetics during self-tonometry. As there are no clinical studies of the health care, we evaluated the local tolerability of a topical anaesthetic in line with self-tonometry employing a modified tonometer OCUTON S. MATERIAL AND METHODS: A total of 100 glaucoma patients participated in a prospective clinical study of the routine clinical service in which each was monitored for 1 year. The telemonitoring involved self-tonometry for at least 6 months in every case and Ocuton S Proparakain-POS 0.5% eyedrops (proxymetacaine-HCl) were applied by the probands before every measurement of the intraocular pressure with a modified self-tonometer. Information regarding the local tolerability of the topical anaesthetic was analysed using a standardised questionnaire. The intensity of the following subjective symptoms was listed in separate visual analogue scales for: lacrimation, burning, foreign body sensation, mucus aggregation, pruritus and pain. RESULTS: Information from 83 glaucoma patients on local tolerability of proparacaine eyedrops could be analysed. For several reasons no data could be gathered from 17 probands, which were refusal to complete the questionnaire, cancelled participation and, in two test persons, there emerged an allergic reaction (local eyelid redness and swelling) which necessitated a change to a different topical anaesthetic. In all other participants the application was carried out without any significant local or systemic symptoms or side-effects. Immediately after application of the eyedrops 36.1% of the test persons suffered a minor conjunctival hyperaemia which eased off within 1 h in 20.4% of these patients. Of the interviewed glaucoma patients 91.5% judged the single symptoms on the visual analogue scale between zero and medium intensity. The severest effects, according to the subjective evaluation, were felt in symptoms of burning with a score of 94 and lacrimation graded 96. The least intensity was established in the symptom of mucus aggregation where 72.3% rated this symptom in the visual analogue scale between 0 and 10. The other symptoms pruritus, feeling of pressure and foreign body sensation hardly differed in subjective ratings. CONCLUSION: A self-medication with topical anaesthetics on undamaged ocular surfaces for self-tonometry purposes can be performed by glaucoma patients without a high risk potential. However, the application presupposes that routine ophthalmological examinations are carried out according to the ophthalmological associations' recommendations. Therefore, medical care concepts which integrate self-tonometry into routine ophthalmological services and comply with the complex requirements of a modern glaucoma management should be applied more often.

Validation of in vitro potency assays for tetanus immunoglobulin.

The European Pharmacopoeia (Ph. Eur.) monograph Human tetanus immunoglobulin (0398) gives a clear outline of the in vivo assay to be performed to determine the potency of human tetanus immunoglobulins during their development. Furthermore, it states that an in vitro method shall be validated for the potency estimation. Since no further guidance is given on the in vitro assay, every control laboratory concerned is free to design and validate an in-house method. At the moment there is no agreed method available. The aim of this study was to validate and compare 2 alternative in vitro assays, i.e. an enzyme-linked immunoassay (EIA) and a toxoid inhibition assay (TIA). The potency of 2 tetanus immunoglobulin preparations (Product 1, Product 2) was estimated against the WHO International Standard for tetanus immunoglobulin, using the tetanus EIA and TIA. The coefficient of variation (CV) to characterise the assay precision was 3.2% (EIA) and 3.6% (TIA), and the corresponding CV for intra-assay variation was 4.7% (EIA) and 5.5% (TIA). Using a spiking procedure, the 2nd part of the experiment investigated recovery of a known anti-tetanus potency. The recovery of samples spiked with defined amounts of reference preparation ranged from 104 112% (EIA) and 114 125% (TIA) respectively, resulting in a mean bias of 2.2 IU/ml (95% confidence interval (CI): -1.1-5.4 IU/ml, EIA) and 5.8 IU/ml (95% CI: 1.4 10.2 IU/ml, TIA). Good agreement was observed between the in vivo and in vitro assay results: the relative potency results of the EIA and TIA as compared to those of the in vivo assay performed by the manufacturers of the 2 tetanus immunoglobulins were for the EIA in the range of 104+/-10% for Product 1 and 100+/-6% for Product 2, and for the TIA in the range of 107+/-6% for Product 1 and 100+/-7% for Product 2. Tetanus EIA and TIA are suitable quality control methods for polyclonal tetanus immunoglobulin, which can be standardised in a quality control laboratory using a quality assurance system. In a collaborative study it will now be evaluated whether the validated methods can be proposed as common in vitro batch potency assays for replacement of the in vivo mouse assay.

[Scientific advice by the nationally competent authority and by the EMEA on the conduct of clinical trials]. / Beratung im Vorfeld klinischer Prüfungen durch die Bundesoberbehörde und Scientific Advice bei der EMEA.

Scientific advice for potential applicants for marketing authorization for medicinal products has been part of the tasks of the European Medicines Agency (EMEA) ever since its establishment in 1995, and has been of increasing significance. Based on Article 56(3) of Regulation (EC) No. 726/2004, this task is now the responsibility of an independent working group of the EMEA, the Scientific Advice Working Party (SAWP). National scientific and regulatory advice has also been part of the work of two national authorities in Germany, the Federal Agency for Medicinal Products and Medical Devices (BfArM) and the Paul Ehrlich Institute (PEI) for several years, but has gained in significance especially over the past 3 years. The basis for advice at a national level is Article 71c of the Law on Administrative Procedures (Verwaltungs-Verfahrensgesetz), the Drug Law, the Guidelines for the Evaluation of Medicinal Products (Arzneimittelprufrichtlinien), and relevant guidelines defining the scientific state of the art. A company developing medicinal products may consult the EMEA or the national authority at any time in order to obtain opinions on the investigations and studies on the pharmaceutical, preclinical and clinical development required for a particular stage of development. In this context, the focus is exclusively on the data required for the planned authorization and the assessment of the evaluation strategy suggested by the company itself. The agreement between the company and the regulatory authority prior to marketing approval is designed to achieve a more effective development of the product and to obtain a more rapid decision on a future authorization procedure. An important component of the scientific advice procedure is the discussion between the company and the agencies. The results of this oral exchange is always summarised in writing. Advice is available for all medicinal products, including "orphan drugs". At the European level, the authorization procedure is prepared with the EMEA which provides administrative and legal support, whereas the scientific opinion itself is elaborated by the members of SAWP, and is adopted by CHMP after a maximum of 3 months following the application. In addition to the written opinions elaborated by two members of SAWP, acting as coordinators, and the internal discussion, the exchange of positions within a "discussion meeting" is also an important part of the European procedure. Scientific advice is a fee-paying procedure, with certain exceptions. Experience from previous advice from the EMEA and the BfArM has shown a certain acceleration in subsequent procedures. This experience, however, also shows very clearly that it is the data actually established in the investigation, especially on efficacy and safety, which are important for a successful and fast approval procedure.

Collaborative study for establishment of a global standard for the potency assay of human anti-D immunoglobulin.

An international collaborative study aimed at establishing a global standard for the potency assay of anti-D immunoglobulin was started in 2002. 25 laboratories participated in this study run under the common aegis of the World Health Organization, the United States Food and Drug Administration (US-FDA) and the European Directorate for the Quality of Medicines (EDQM). The potencies of three candidate materials and the US-FDA standard (lot 3) included for comparison were evaluated using AutoAnalyzer, competitive enzyme-linked immunoassay (competitive EIA), flow cytometric methods or own "in-house" methods. Critical reagent, standardised procedures and standardised assay design were provided for either method, where appropriate. Central statistical evaluation of the potency data submitted by the participants was performed using a parallel line model. Agreement between laboratories and assay methods for all samples was observed. Intra-laboratory variability was lowest for laboratories performing flow cytometry and highest for laboratories that performed their in-house methods. Inter-laboratory variability was acceptable for all samples when assayed by AutoAnalyzer, competitive (EIA) and flow cytometric methods. It was concluded that sample A is most suitable to serve as a global standard and that sample C could serve as a reserve European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch provided that suitable stability is demonstrated. Sample A was adopted by the Ph. Eur. Commission at its 115th session (March 2003) as the first Ph. Eur. BRP (available from the EDQM: catalog number Y0000219) with the assigned potency of 285 IU/ampoule.

A global standard for anti-D immunoglobulin: international collaborative study to evaluate a candidate preparation.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate a lyophilized anti-D immunoglobulin preparation to serve as a global standard for potency assays of anti-D immunoglobulin products. MATERIALS AND METHODS: The candidate global standard, 01/572, was calibrated against the World Health Organization (WHO) International Reference Preparation (IRP) for anti-D immunoglobulin, human (68/419), along with two reserve candidate reference preparations, in an international collaborative study involving 25 laboratories in 15 countries. The United States Food and Drug Administration (US-FDA) Center for Biologics Evaluation and Research (CBER) Standard for anti-D immunoglobulin, Lot 3, was included for comparison. Most laboratories (20/25) performed AutoAnalyser methodology, competitive enzyme-linked immunoassay (EIA) and/or flow cytometry. RESULTS: The overall mean potency of the candidate global standard, 01/572, was 284.5 international units (IU)/ampoule, with an interlaboratory variability, expressed as a percentage geometric coefficient of variation (% gcv), of 9.7. The mean potency of the US Standard was 859.4 IU/ml with an interlaboratory variability of 9.5% gcv, excluding an outlier. The mean potencies of the reserve preparations per ampoule/vial were 110.6 IU and 106.7 IU when calibrated against the IRP, and 112.2 IU and 106.6 IU when calibrated against 01/572, respectively, with interlaboratory % gcv values of 9.6-18.3 (excluding outliers). CONCLUSIONS: Preparation 01/572 proved more suitable for use as a global standard than the reserve candidate preparations and was established, with an assigned potency of 285 IU/ampoule, by the WHO as the 2nd International Standard for anti-D immunoglobulin; by FDA-CBER as the Standard for anti-D immunoglobulin, Lot 4; and by the European Directorate for the Quality of Medicines (EDQM) as the 1st Biological Reference Preparation for anti-D immunoglobulin.

N-ras mutation in a canine lymphoma: short communication.

Lymphomas of dogs were investigated by molecular genetic methods. Regions of exon 1 and 2 of the N-ras gene, which harbours the mutation hot spots (codons 12, 13 and 61) were screened. A GGT [symbol: see text] GAT (glycine [symbol: see text] aspartic acid) mutation in codon 13 was present in a multicentric-type lymphoma of a 1-year-old male dog.

Validation of flow cytometry to quantify the potency of anti-D immunoglobulin preparations.

BACKGROUND AND OBJECTIVES: Flow cytometry has been recommended as an alternative to that of autoanalyser methodology for estimation of anti-D potency. This investigation was performed to validate the flow cytometry method based on a quality assurance system. MATERIALS AND METHODS: A flow cytometry method based on indirect labelling of Rh(D)-positive red blood cells was validated using the parameters precision and accuracy and was compared to the autoanalyser data of manufacturers of anti-D immunoglobulin preparations. RESULTS: The experiment first investigated the possible differences between assays from single donors compared with a pooled assay. Red blood cells of four individual donors and their pooled red blood cells were interchangeable. There was no significant difference between donors, on one hand, and between the use of a single donation and the pooled red blood cells, on the other hand (P = 0.695). The two-sided 95% confidence intervals (CIs) of the difference between single donors and the pool ranged from -4.6% to 4.7%. The intermediate variability was determined by standard deviation (SD) = 48.4 IU/ml [coefficient of variation (CV) = 3.8%]; the repeatability was SD = 34.3 IU/ml (CV = 2.7%). Using a spiking experiment, the second part of the experiment investigated recovery of a known anti-D potency. The recovery of samples spiked with defined amounts of reference preparation was 97.7-101%, with a mean bias of -1.3 (95% CI: -4.1 to 1.6). The results of the flow cytometry assay, as compared to those of the autoanalyser performed by the manufacturers of anti-D immunoglobulin preparations for those manufacturers who have their method validated in-house, ranged from 87 to 129%, indicating good correlation. CONCLUSIONS: Flow cytometry is a suitable quality control method for polyclonal anti-D immunoglobulins, which can be standardized in a quality control laboratory using a quality assurance system.

International collaborative study to evaluate methods for quantification of anti-D in immunoglobulin preparations.

BACKGROUND AND OBJECTIVES: The disadvantages of autoanalyser methodology for anti-D potency estimation have prompted the search for an alternative reference method. The aim of this study was to carry out a direct comparison of autoanalyser methodology, competitive enzyme-linked immunoassay (EIA) and flow cytometry. MATERIALS AND METHODS: The anti-D potencies of nine immunoglobulin preparations were estimated against the World Health Organization (WHO) International Reference Preparation for anti-D immunoglobulin, using the three different assay methods described above, in an international collaborative study involving 18 laboratories. RESULTS: No significant differences in potency estimates for five of nine samples were identified using the three different methods. For six of nine samples the mean potency estimates obtained using competitive EIA lay between those of the autoanalyser and flow cytometry. Flow cytometry gave the lowest estimates for seven of nine samples. The range of intralaboratory variability, expressed as percentage geometric coefficients of variation (% gcv) and excluding extreme values, was as follows: autoanalyser (eight laboratories), 0.4-17.0; competitive EIA (12 laboratories), 2.6-34.8; and flow cytometry (eight laboratories), 2.9-30.0. The range of interlaboratory variability (expressed as % gcv) was: autoanalyser (eight laboratories, eight samples), 10.8-17.6; competitive EIA (12 laboratories, nine samples), 10.3-17.3; flow cytometry (nine laboratories, eight samples), 6.2-16.1. CONCLUSIONS: The study did not show clear superiority of one method over the others. Both competitive EIA and flow cytometry are acceptable alternatives to autoanalyser methodology for polyclonal anti-D potency estimation. For monoclonal anti-D, further work is necessary to determine the most appropriate method for potency testing.

Tumour suppressor gene p53 mutation in a case of haemangiosarcoma of a dog.

Haemangiosarcomas of dogs were analysed by molecular genetic techniques. Regions of the tumour suppressor gene p53, including the well-known tumour hot spots (codons 175, 245, 248, 249, 273 and 282) were screened. A 24 bp deletion was detected in exon 5 of the gene.

Genetic mapping with SNP markers in Drosophila.

Map-based positional cloning of Drosophila melanogaster genes is hampered by both the time-consuming, error-prone nature of traditional methods for genetic mapping and the difficulties in aligning the genetic and cytological maps with the genome sequence. The identification of sequence polymorphisms in the Drosophila genome will make it possible to map mutations directly to the genome sequence with high accuracy and resolution. Here we report the identification of 7,223 single-nucleotide polymorphisms (SNPs) and 1,392 insertions/deletions (InDels) in common laboratory strains of Drosophila. These sequence polymorphisms define a map of 787 autosomal marker loci with a resolution of 114 kb. We have established PCR product-length polymorphism (PLP) or restriction fragment-length polymorphism (RFLP) assays for 215 of these markers. We demonstrate the use of this map by delimiting two mutations to intervals of 169 kb and 307 kb, respectively. Using a local high-density SNP map, we also mapped a third mutation to a resolution of approximately 2 kb, sufficient to localize the mutation within a single gene. These methods should accelerate the rate of positional cloning in Drosophila.

Lack of sequence variation in sporadic bovine leucosis in regions of tumour suppressor genes p53 and p16.

Regions of the promoter and exons 5-8 of the tumour suppressor gene p53 were analysed in 25 cases of sporadic bovine leucosis. The study included 17 cases of juvenile leucosis, five cases of adult leucosis and three cases of skin leucosis. Exon 2 of tumour suppressor gene p16 was also investigated in the same samples. No sequence variations were present in the analysed areas of the genes. In p53, this fact represents a clear difference in comparison with enzootic bovine leucosis. In p16, no comparative data are available.

A screen for genes required for meiosis and spore formation based on whole-genome expression.

BACKGROUND: Meiosis is the process by which gametes are generated with half the ploidy of somatic cells. This reduction is achieved by three major differences in chromosome behavior during meiosis as compared to mitosis: the production of chiasmata by recombination, the protection of centromere-proximal sister chromatid cohesion, and the monoorientation of sister kinetochores during meiosis I. Mistakes in any of these processes lead to chromosome missegregation. RESULTS: To identify genes involved in meiotic chromosome behavior in Saccharomyces cerevisiae, we deleted 301 open reading frames (ORFs) which are preferentially expressed in meiotic cells according to microarray gene expression data. To facilitate the detection of chromosome missegregation mutants, chromosome V of the parental strain was marked by GFP. Thirty-three ORFs were required for the formation of wild-type asci, eight of which were needed for proper chromosome segregation. One of these (MAM1) is essential for the monoorientation of sister kinetochores during meiosis I. Two genes (MND1 and MND2) are implicated in the recombination process and another two (SMA1 and SMA2) in prospore membrane formation. CONCLUSIONS: Reverse genetics using gene expression data is an effective method for identifying new genes involved in specific cellular processes.

Presence of p53 mutations in feline neoplasms.

A region from exon 4 to 8 of the tumour suppressor gene p53 was analysed in 60 feline tumours (30 fibrosarcomas, seven malignant histiocytomas, three lymphosarcomas, five basal cell tumours, five squamous cell carcinomas, two adenocarcinomas of tubular skin glands, one undifferentiated carcinoma of the skin, seven mammary carcinomas). Missense mutations were detected in two fibrosarcomas, one malignant fibrous histiocytoma, the undifferentiated carcinoma of the skin and one mammary carcinoma. One nonsense mutation was detected in one fibrosarcoma and one deletion/frameshift-mutation was observed in one squamous cell carcinoma.

Comparative studies in the promoter and exon 1 regions of tumour suppressor p53 in several mammalian species: absence of mutations in a panel of spontaneous domestic animal tumours.

Tumour suppressor p53 is critical in a broad panel of tumour types in human, mouse and other mammals. Regions of the promoter and exon 1 play an important role in expression of p53. In the present study, the DNA sequences of promoter and exon 1 regions of four domestic animal species (dog, cat, horse and cattle) are determined and compared with experimental rodents (mouse, rat and hamster) and man. A broad panel of tumour types have been investigated for mutations in this regulatory area in 90 canine, 136 feline, 25 equine and 10 bovine patients. No mutation was detected in any of the tumours analysed.