RESUMEN
The present study was aimed to test the hypothesis that paracetamol (PCM) can precipitate autistic like features when used to counteract vaccine-induced fever using experimental rat pups. The pups were treated with measles mumps rubella (MMR) vaccine, diphtheria tetanus and pertussis (DPT) vaccines and lipopolysaccharide (LPS) with subsequent PCM treatment. The pups were evaluated for postnatal growth (weight gain, eye opening) and behavior alterations (swimming performance, olfactory discrimination, negative geotaxis, nociception, and locomotor activity) by performing battery of neurobehavioral test. Significant correlation was observed between social behavioral domains (nociception, anxiety and motor coordination) and pro-inflammatory load in the pups when treated with MMR/LPS along with PCM. A significant change in pro and anti-inflammatory (IL-4, IL-6, IL-10) markers were observed in rats treated with PCM, MMR, LPS, DPS alone or in combination with MMR, LPS and DPT (5128.6 ± 0.000, 15,488 ± 0.000***, 9661.1 ± 157.29***a, 15,312 ± 249.29***, 10,471 ± 0.00***a, 16,789 ± 273.34*** and 12,882 ± 0.00***a). Pups were also scrutinized for the markers of oxidative stress, inflammation and histopathologically. All the treatment groups showed significant alteration in the behavioral changes, oxidative markers (TBARS-in control-4.33 ± 0.02, PCM-9.42 ± 0.18***, MMR-5.27 ± 0.15***, MMR + PCM-8.57 ± 0.18*** a, LPS-6.84 ± 0.10***, LPS + PCM-4.51 ± 0.30***a, DPT-5.68 ± 0.12***, DPT + PCM-7.26 ± 0.18***a) and inflammatory markers without following any specific treatment. These observation could be accorded to variable phenotypes of autistic spectrum disorders (ASDs).
Asunto(s)
Acetaminofén/toxicidad , Antipiréticos/toxicidad , Trastorno Autístico/etiología , Inflamación/etiología , Acetaminofén/administración & dosificación , Acetaminofén/farmacología , Animales , Animales Recién Nacidos , Antipiréticos/administración & dosificación , Antipiréticos/farmacología , Conducta Animal/efectos de los fármacos , Vacuna contra Difteria, Tétanos y Tos Ferina/administración & dosificación , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Endotoxinas/toxicidad , Exotoxinas/toxicidad , Femenino , Fiebre/tratamiento farmacológico , Vacuna contra el Sarampión-Parotiditis-Rubéola/administración & dosificación , Vacuna contra el Sarampión-Parotiditis-Rubéola/toxicidad , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
This paper describes the results of a study of the effects of modified housing conditions, conditioning and habituation on humans using a rabbit model for monitoring whole-cell pertussis vaccine (pWCV)-induced adverse effects. The study has been performed with reference to previous vaccine safety studies of pWCV in rabbits in which results were difficult to interpret due to the large variation in experimental outcome, especially in the key parameter deep-body temperature (T(b)). Certain stressful laboratory conditions, as well as procedures involving humans, e.g. blood sampling, inoculation and cage-cleaning, were hypothesized to cause this large variation. The results of this study show that under modified housing conditions rabbits have normal circadian body temperatures. This allowed discrimination of pWCV-induced adverse effects in which handled rabbits tended to show a dose-related increase in temperature after inoculation with little variance, whereas non-handled rabbits did not. Effects of experimental and routine procedures on body temperature were significantly reduced under modified conditions and were within the normal T(b) range. Handled animals reacted less strongly and with less variance to experimental procedures, such as blood sampling, injection and cage-cleaning, than non-handled rabbits. Overall, handling had a positive effect on the behaviour of the animals. Data show that the housing modifications have provided a more robust model for monitoring pWCV adverse effects. Furthermore, conditioning and habituation of rabbits to humans reduce the variation in experimental outcome, which might allow for a reduction in the number of animals used. In addition, this also reduces distress and thus contributes to refining this animal model.
Asunto(s)
Temperatura Corporal/efectos de los fármacos , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Vivienda para Animales , Conejos/fisiología , Pruebas de Toxicidad/métodos , Animales , Anticuerpos Antibacterianos/sangre , Recuento de Células Sanguíneas/veterinaria , Temperatura Corporal/fisiología , Peso Corporal/efectos de los fármacos , Femenino , Manejo Psicológico , Hematócrito/veterinaria , Hemoglobinas/análisis , Humanos , Masculino , Modelos Animales , Distribución Aleatoria , Telemetría/veterinariaRESUMEN
This report reflects the discussion and conclusions of a WHO group of experts from National Regulatory Authorities (NRAs), National Control Laboratories (NCLs), vaccine industries and other relevant institutions involved in standardization and control of diphtheria, tetanus and pertussis vaccines (DTP), held on 20-21 July 2006 and 28-30 March 2007, in Geneva Switzerland for the revision of WHO Manual for quality control of DTP vaccines. Taking into account recent developments and standardization in quality control methods and the revision of WHO recommendations for D, T, P vaccines, and a need for updating the manual has been recognized. In these two meetings the current situation of quality control methods in terms of potency, safety and identity tests for DTP vaccines and statistical analysis of data were reviewed. Based on the WHO recommendations and recent validation of testing methods, the content of current manual were reviewed and discussed. The group agreed that the principles to be observed in selecting methods included identifying those critical for assuring safety, efficacy and quality and which were consistent with WHO recommendations/requirements. Methods that were well recognized but not yet included in current Recommendations should be taken into account. These would include in vivo and/or in vitro methods for determining potency, safety testing and identity. The statistical analysis of the data should be revised and updated. It was noted that the mouse based assays for toxoid potency were still quite widely used and it was desirable to establish appropriate standards for these to enable the results to be related to the standard guinea pig assays. The working group was met again to review the first drafts and to input further suggestions or amendments to the contributions of the drafting groups. The revised manual was to be finalized and published by WHO.
Asunto(s)
Vacuna contra Difteria, Tétanos y Tos Ferina/normas , Difteria/prevención & control , Tétanos/prevención & control , Tos Ferina/prevención & control , Animales , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Humanos , Ratones , Control de Calidad , Estándares de Referencia , Suiza , Vacunas Combinadas/normas , Vacunas Combinadas/toxicidad , Organización Mundial de la SaludRESUMEN
While evaluating vaccine efficacy against clinical Bordetella pertussis isolates in mice, after challenge vaccinated mice showed increased lung pathology with eosinophilia, compared to challenged, non-vaccinated animals. This led us to study bacterial clearance, lung pathology, lung TNF-alpha expression, and parameters of immediate hypersensitivity (IH), being serum IgE levels, eosinophil numbers in the bronchoalveolar lavage fluid, and ex vivo IL-4, IL-5, IL-10, IL-13, and IFN-gamma production by the bronchial lymph node cells. BALB/c mice received a combined Diphtheria (D), Tetanus (T), Poliomyelitis, and whole-cell Pertussis vaccine (WCV), a combined D, T, and three-component acellular Pertussis vaccine (ACV), aluminium hydroxide adjuvant, or PBS, 28 and 14 days before B. pertussis infection. Similarly treated non-infected mice were taken as a control. Infection induced pathology; this induction was stronger after (especially WCV) vaccination. WCV but not ACV vaccination induced TNF-alpha expression after challenge. After challenge, IH parameters were strongly increased by (especially ACV) vaccination. Vaccinated IL-4 KO mice showed similar clearance and pathology, in the absence of IgE and with reduced numbers of eosinophils. Vaccinated (Th1-deficient) T-bet KO mice showed reduced clearance and similar pathology. In summary, after challenge vaccination increased lung pathology, TNF-alpha expression (only WCV), and IH parameters. Th1 cells were critical for clearance.
Asunto(s)
Bordetella pertussis/inmunología , Hipersensibilidad Inmediata/inducido químicamente , Pulmón/patología , Vacuna contra la Tos Ferina/inmunología , Tos Ferina/inmunología , Administración Intranasal , Animales , Bordetella pertussis/crecimiento & desarrollo , Vacuna contra Difteria, Tétanos y Tos Ferina/administración & dosificación , Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Femenino , Hipersensibilidad Inmediata/metabolismo , Inmunoglobulina E/sangre , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Vacuna contra la Tos Ferina/administración & dosificación , Vacuna contra la Tos Ferina/toxicidad , Factor de Necrosis Tumoral alfa/metabolismo , Vacunas Acelulares/administración & dosificación , Vacunas Acelulares/inmunología , Vacunas Acelulares/toxicidad , Tos Ferina/prevención & controlRESUMEN
GlaxoSmithKline plc has developed and launched Boostrix, a subunit vaccine for use in adolescents and adults as a booster immunization against diphtheria, tetanus and pertussis (DTPa) infections.
Asunto(s)
Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Adolescente , Adulto , Niño , Vacuna contra Difteria, Tétanos y Tos Ferina/química , Vacuna contra Difteria, Tétanos y Tos Ferina/farmacocinética , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Humanos , Inmunización Secundaria , Relación Estructura-ActividadRESUMEN
The required in vivo assays for the release of Whole Cell Pertussis Vaccine are the Mouse Weight Gain test (MWGT) for assessment of specific toxicity and the Mouse Protection test (MPT) to estimate the potency. Despite the fact that both assays are criticised for the use of large number of animals, poor precision or insensitivity, more precise alternatives--such as the Leukocytosis Promotion test (LP-test) and Pertussis Serological Potency Test (PSPT)--are still not fully accepted. During the optimisation of the production process, the need for more accurate parameters to determine toxicity and potency became essential. To reduce substantially the number of animals, we have combined the MWGT with the LP-test and PSPT in one model, named the Mouse Toxicity and Immunogenicity test (MTI-test). Animals are inoculated with half a human dose and are weighed individually pre-immunisation and 16 hours (parameter for endotoxin), three and seven days post immunisation. Additionally, the number of leukocytes (parameter for PT) is determined on day 7 and after 28 days the animals are bled individually for immunogenicity testing (parameter for potency). A lyophilised reference vaccine is included as a positive control to standardise the assay and to determine its reproducibility. The advantage of this modified procedure is that the data on toxicity and immunogenicity are obtained from the individual mouse, which enables statistical analysis to be made. The leucocytosis data can be used to check whether mice were vaccinated correctly, allowing for the elimination of incorrectly vaccinated mice from the assay. Furthermore, this assay can be used to determine the consistency of production, the influence of adjuvant on toxicity, as well as the composition and stability of vaccines.
Asunto(s)
Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Modelos Animales , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Peso Corporal , Femenino , Humanos , Leucocitos/inmunología , Leucocitos/metabolismo , Masculino , Ratones , Tamaño de los Órganos , Toxina del Pertussis/inmunología , Toxina del Pertussis/metabolismo , Bazo/anatomía & histologíaRESUMEN
Vaccination of young children with diphtheria, tetanus, poliomyelitis and pertussis (DTPoP) vaccine is effective in preventing outbreaks of whooping cough but adverse events sometimes occur. This pilot study shows that in freely-moving rats, multiple treatment with DTPoP (at day 0 and day 5, 6 ml/kg i.v.) increased heart rate (HR) for 5 days after the first treatment and decreased diastolic blood pressure (DBP) for at least 26 days after the first treatment and inhibited the circadian rhythm of HR and DBP for at least 10 days. DTPo vaccine, containing no pertussis vaccine, was free of such effects. Thus, in rats, the pertussis component of DTPoP acts on the cardiovascular system and disturbs its circadian rhythm. The contribution of these findings to clinical adverse effects is as yet unknown and needs further research.
Asunto(s)
Sistema Cardiovascular/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Toxoide Diftérico/toxicidad , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Vacuna contra la Tos Ferina/toxicidad , Vacuna Antipolio de Virus Inactivados/toxicidad , Toxoide Tetánico/toxicidad , Vacunas Combinadas/toxicidad , Animales , Presión Sanguínea/efectos de los fármacos , Toxoide Diftérico/administración & dosificación , Vacuna contra Difteria, Tétanos y Tos Ferina/administración & dosificación , Esquema de Medicación , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Vacuna contra la Tos Ferina/administración & dosificación , Proyectos Piloto , Vacuna Antipolio de Virus Inactivados/administración & dosificación , Ratas , Ratas Wistar , Telemetría , Toxoide Tetánico/administración & dosificación , Vacunas Combinadas/administración & dosificaciónAsunto(s)
Vacuna contra Difteria, Tétanos y Tos Ferina/normas , Toxoide Tetánico/normas , Animales , Bioensayo/métodos , Vacuna contra Difteria, Tétanos y Tos Ferina/administración & dosificación , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Relación Dosis-Respuesta a Droga , Control de Calidad , Estándares de Referencia , Toxoide Tetánico/administración & dosificación , Toxoide Tetánico/toxicidadRESUMEN
The mouse weight gain test was evaluated for its value in toxicity testing for pertussis vaccines. When the reference whole cell pertussis vaccine was tested at dilutions of 1 in 1 and 1 in 16, the mice which received the 1 in 1 dilution achieved the greatest weight gain by the seventh day of injection, although they experienced a more significant weight loss during the first 24 hours than both the normal control mice or those that received the 1 in 16 dilution. A commercial diphtheria-tetanus-acellular pertussis vaccine with an increased level of pertussis toxin activity significantly accelerated the weight gain of mice. The effect was lost by heating the vaccine at 80 degrees C for 2 hours. A 1 micrograms dose of endotoxin induced a significant weight loss in mice during the first 24 hours of injection followed by weight gain at the same rate as that of the normal control. Pertussis toxin accelerated the weight gain of mice at a dose of 2 micrograms to a level exceeding that of the normal control mice throughout the observation period of 11 days. Pertussis toxin, when inoculated with endotoxin, showed a marked effect of helping mice to recover quickly from the endotoxin-induced initial weight loss to the level of those receiving only pertussis toxin. The effect of pertussis toxin on the acceleration of the weight gain of mice showed the possibility of inappropriate interpretation of the test results. It suggests, therefore, the necessity for separate quantitative tests for controlling the vaccine's toxicities.
Asunto(s)
Toxina del Pertussis , Vacuna contra la Tos Ferina/toxicidad , Factores de Virulencia de Bordetella/toxicidad , Aumento de Peso/efectos de los fármacos , Animales , Bioensayo , Glucemia/efectos de los fármacos , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Endotoxinas/toxicidad , Epinefrina/farmacología , Escherichia coli , Femenino , Hipoglucemia/etiología , Ratones , Ratones Endogámicos , Factores de TiempoRESUMEN
The mouse paw edema test and the tests for the determination of leukocytosis-stimulating and histamine-sensitizing activity are reproducible if standard samples are used and the properties under test are evaluated with reference to the characteristics of standard samples. The standard character of the weight gain test on mice is also enhanced if all conditions of the test are standardized. The most accurate information on the toxicity of the pertussis component of adsorbed DPT vaccine can be obtained if a single human dose (0.5 ml) is injected to mice and the toxicity of the vaccine is estimated from the weight gain registered in the test mice and the control animals. The use of standard samples in each test makes it possible to observe the conditions for controlling the toxicity of the tested preparations.
Asunto(s)
Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Vacuna contra la Tos Ferina/toxicidad , Adsorción , Animales , Bulgaria , Checoslovaquia , Vacuna contra Difteria, Tétanos y Tos Ferina/normas , Relación Dosis-Respuesta a Droga , Hungría , Métodos , Ratones , Vacuna contra la Tos Ferina/normas , Polonia , U.R.S.S.RESUMEN
The quality of 14 lots of acellular pertussis-diphtheria-tetanus (AC-PDT) vaccines manufactured by the Kitasato Institute during the period 1987-1990 were investigated. The geometric means of HSU, LPU, and BWDU were 0.078, 0.257, and 7.33 per ml respectively. The potency was higher than 14 IU per ml. These results indicated the consistency of the Kitasato AC-PDT vaccines. The antibody response to the AC-PDT vaccines was measured in primary and secondary vaccinated mice by ELISA. IgG antibody response to FHA and PT was obtained in all immunized mice (P less than 0.001) after the primary injection. In contrast, IgG antibody response to fimbriae 2 showed a significant titer rise (P less than 0.001) after the booster injection. The results indicated that the Kitasato AC-P vaccines consisted of protein, PT and FHA as the major antigens, and a little agglutinogen as the minor antigen.
Asunto(s)
Adhesinas Bacterianas , Anticuerpos Antibacterianos/biosíntesis , Vacuna contra la Tos Ferina/inmunología , Animales , Antígenos Bacterianos/inmunología , Peso Corporal , Bordetella pertussis/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Ensayo de Inmunoadsorción Enzimática , Femenino , Hemaglutininas/inmunología , Histamina/farmacología , Leucocitosis/etiología , Ratones , Vacuna contra la Tos Ferina/toxicidad , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/toxicidad , Factores de Virulencia de Bordetella/inmunologíaRESUMEN
The results of the weight gain test on mice have shown that acellular pertussis vaccine is less toxic than the pertussis component of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine due to a lower content of endotoxin in the acellular vaccine; but the leukocytosis-promoting and histamine-sensitizing activities of JNIH-6 and adsorbed DPT vaccines are indicative of incomplete inactivation of Bordetella pertussis toxin. The content of incompletely inactivated B. pertussis toxin is practically the same in both preparations, constituting 1/100-1/200 of the calculated initial activity. For this reason, the use of the new pertussis vaccine also involves a risk of development of serious postvaccinal reactions and/or complications caused by this toxin. Search for the optimum method of inactivation of B. pertussis main toxin should be continued. As shown by the enzyme immunoassay, acellular pertussis vaccine used in the same immunizing dose as adsorbed DPT vaccine induces a more intensive immune response to hemagglutinin and B. pertussis toxin. This is due to higher residual toxicity of the corpuscular component of adsorbed DPT vaccine. Induction of antibodies to B. pertussis toxin has been shown to decrease in response to injection of acellular pertussis vaccine containing a certain residual amount of incompletely inactivated B. pertussis toxin.
Asunto(s)
Vacuna contra la Tos Ferina/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Peso Corporal/efectos de los fármacos , Bordetella pertussis/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Evaluación Preclínica de Medicamentos , Hemaglutininas/inmunología , Histamina/inmunología , Inmunidad Activa/efectos de los fármacos , Inmunidad Activa/inmunología , Inmunización , Leucocitosis/sangre , Leucocitosis/inducido químicamente , Ratones , Toxina del Pertussis , Vacuna contra la Tos Ferina/toxicidad , Factores de Tiempo , Factores de Virulencia de Bordetella/inmunologíaRESUMEN
The test for the evaluation of the toxicity of different types of pertussis preparations as manifested by their in vitro influence on mouse thymic cells (T test) has been finally worked out. The use of the T test has made it possible to reveal the nonstandard character of the production lots of adsorbed diphtheria-pertussis-tetanus vaccines, both whole-cell vaccine and Japanese acellular vaccine. The degree of the in vitro damaging action of pertussis preparations on mouse thymic cells greatly depends on the residual content of Bordetella pertussis nontoxoidized toxin which, in contrast to B. pertussis lipopolysaccharide and filamentous hemagglutinin, produces pronounced cytotoxic action on mouse thymic cells.
Asunto(s)
Vacuna contra la Tos Ferina/toxicidad , Timo/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Relación Dosis-Respuesta a Droga , Dosificación Letal Mediana , Ratones , Ratones Endogámicos CBA , Timo/citología , Factores de TiempoRESUMEN
The influence of pertussis preparations, introduced by oral and parenteral routes, on the detoxifying function of the liver and the state of the nervous system of the animals was studied by methods used in pharmacology and toxicology. The use of these methods made it possible to find out side effects produced by corpuscular pertussis vaccine, introduced parenterally, on the detoxifying function of the liver and the state of the nervous system of the animals. The negative influence on the nervous system was more pronounced after the injection of the commercial adsorbed diphtheria-pertussis-tetanus vaccine used in this investigation than after the injection of pertussis monovaccine. The oral administration of corpuscular pertussis vaccine exerted no negative influence on the above-mentioned body functions of the animals.
Asunto(s)
Vacuna contra la Tos Ferina/toxicidad , Administración Oral , Animales , Vacuna contra Difteria, Tétanos y Tos Ferina/administración & dosificación , Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Relación Dosis-Respuesta Inmunológica , Evaluación Preclínica de Medicamentos , Inyecciones Intraperitoneales , Hígado/efectos de los fármacos , Ratones , Sistema Nervioso/efectos de los fármacos , Vacuna contra la Tos Ferina/administración & dosificación , Factores de TiempoRESUMEN
A total of 112 samples of adsorbed DPT vaccine were tested in mice for freedom from abnormal toxicity by three procedures: (a) single human dose by intraperitoneal route; (2) single human dose by subcutaneous route; and (3) half the single human dose by intraperitoneal route. The percentage of samples passing by the three methods was 25, 90 and 54.5, respectively. Method 3, which is being recommended now by the World Health Organization, is supported by the results of this study to be neither very stringent nor very lenient to pass the adsorbed DPT vaccine for freedom from abnormal toxicity.
Asunto(s)
Vacuna contra Difteria, Tétanos y Tos Ferina/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Femenino , Cobayas , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Masculino , RatonesRESUMEN
Since the DPT vaccine is broadly used for the prevention of diphtheria, tetanus and pertussis' its preparations should meet special quality requirements. The present study was aimed at evaluating the reproducibility of laboratory methods utilized to assess the protective activity and toxicity of the pertussis component as well as at examining the feasibility of expert estimations of product quality to enhance the validity of findings. The results of interlaboratory comparative examination of quality parameters of the tested preparations revealed that the routine laboratory quality control methods were not sufficiently standardized as their application in different laboratories did not always produce identical results. The WHO criteria to evaluate the toxicity of pertussis vaccines are far from perfect since vaccines with pronounced toxicity can only be distinguished from those with moderate to mild toxicity after the administration to tested mice of vaccines at a single infant dose of 0.5 ml. To enhance the reproducibility of methods employed in the laboratory, appropriate standard specimens of the preparation should be used serving as a measure of different quality parameters; all test conditions should be also standardized as far as possible. To enable objective quality evaluation of medical biological preparations, the degree of experiment reproducibility should be regularly verified in interlaboratory tests on an international scale as well as inside those countries which have several manufacturers of a given preparation. It appears expedient to set up an international system of expert evaluation of quality of biological preparations by appointing several regional and national centres which meet the requirements for expert laboratories.