[Recombinant human erythropoietin: method of in vivo evaluation using normocythemic mice].
OBJECTIVE
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.


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.
Albertengo
M E
ME
Departamento de Productos Biológicos, Instituto Nacional de Medicamentos (INAME), Ciudad de Buenos Aires, Argentina.
Valcarce
G A
GA
Oliva
L M
LM
Baiges
D L
DL
Alonso
B S
BS
Chiale
C A
CA
D000818:Animals; D001681:Biological Assay / Q000191:economics / Q000379:methods*; D002469:Cell Separation; D004305:Dose-Response Relationship, Drug; D004906:Erythrocyte Count / Q000187:drug effects*; D004921:Erythropoietin / Q000008:administration & dosage / Q000494:pharmacology* / Q000592:standards; D005069:Evaluation Studies as Topic; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007279:Injections, Subcutaneous; D051379:Mice; D011786:Quality Control; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D017701:Reticulocyte Count / Q000187:drug effects*; D012680:Sensitivity and Specificity
D011994:Recombinant Proteins; D004921:Erythropoietin