Assuntos
Animais , Masculino , Camundongos , Medula Óssea/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Floxuridina/toxicidade , Imunossupressores/toxicidade , Timo/patologia , Peso Corporal/efeitos dos fármacos , Corticosterona/sangue , Desoxicitidina/toxicidade , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Isomerismo , Tamanho do Órgão/efeitos dos fármacos , Contagem de Reticulócitos/efeitos dos fármacosRESUMO
PURPOSE: The implementation of an in vivo assay to determine the biological activity of human recombinant erythropoietin (Hu-r EPO) is essential. The purpose of this study was to perform and optimize the conditions of an easy in vivo bioassay suitable for routine testing of quality control of Hu-r EPO preparations. MATERIAL AND METHODS: Normocythemic 8 weeks female mice treated with different Hu-r EPO doses were employed. The reticulocyte response was measured by flow cytometry and by visual count in a Neubauer cell count chamber, after selective red blood cell haemolysis. A unique subcutaneous injection with blood extraction 96 hours later was the schedule employed. The reticulocyte count measured by both methods was plotted against the log dose of Hu-r EPO. RESULTS: The dose-response curve obtained was linear between 5 and 160 UI/mouse and the doses chosen for future assays were 10, 30 and 90 UI/mouse. The use of at least 6 animals per dose and not less than 3 assays to obtain reliable limits according to international regulations is convenient. Thirty assays were performed in four different samples and were analyzed by parallel lines (3 + 3) relating the response with the log dose. The coefficient of correlation between both methods was 0.989, so they are equivalent. CONCLUSIONS: This method is suitable because fewer animals and bioassays are necessary to obtain fiducial limits according to international requirements. It is in agreement with the tendency to reduce the number of animals used for bioassay because ethical and economic reasons.
Assuntos
Bioensaio/métodos , Contagem de Eritrócitos/efeitos dos fármacos , Eritropoetina/farmacologia , Contagem de Reticulócitos/efeitos dos fármacos , Animais , Bioensaio/economia , Separação Celular , Relação Dose-Resposta a Droga , Eritropoetina/administração & dosagem , Eritropoetina/normas , Estudos de Avaliação como Assunto , Feminino , Citometria de Fluxo , Humanos , Injeções Subcutâneas , Camundongos , Controle de Qualidade , Proteínas Recombinantes , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The diagnosis of megaloblastic anaemias caused by cobalamine or folate deficiency are still difficult. The dosage of these two substances help to differenciate between both carencies, but it is not determinant of any of them and is an expensive method. Homocisteinuria (HC), methylmalonuria (MMA) and formiminoglutamic acid (FIGLU) are cheap tests which could help in the differential diagnosis, if they are used properly. We report 62 patients to whom we made these test simultaneously. All of the patients received 10 micrograms of vit B12 and after 72 hours, 1 mg/day of folic acid (for 3 days). In both cases waiting for the increase of reticulocytyes up to 150 x 10(9)/L as a form of therapeutic test of diagnosis. By this simple way we have detected 97.9% of specificity for cobalamin deficiency of the MMA test, and only 4.2% for HC. This last test had increased its specificity up to 91.6% in association with the negative FIGLU test. We have also found a high specificity (92.3%) for FIGLU due to the detection of folate deficiency, in opposition with other authors who had described it as low as 50%. We have also compared the costs of the 3 tests with the dosage of cobalamine and folate, and we have found that the formers are 11 times less expensive than the last ones.