Global Journal of Biology, Agriculture & Health Sciences
Open Access

ISSN: 2319–5584



Design, optimization and validation of a flow cytometry assay for quantitation of Igg-anti-D (rho) in the industrial process control of the anti rho-gamma globulin production

Sergio A Oviedo, Susana Vitali, Jorge Zarzur

One of the main causes of the hemolytic disease of the newborn is due to the generation of anti-D antibodies, in response to a continuous antigenic challenge in pregnant mothers who do not possess the D antigen in their red blood cells. In order to prevent complications, the monitoring of anti-D antibody levels in pregnant women is carried out and the sensitization is prevented by prophylaxis with anti-D immunoglobulin (IgG anti-D). To date, the monitoring and titration of anti-D in plasma and finished products (IgG anti-D) is carried out continuous flow analysis with a Technicon Autoanalyzer, Radio Immuno Assay (RIA) and Enzymo Immuno Assay (EIA). In our laboratory, for the quantification of anti-D antibodies of the Gamma-Rho UNC and the products of the elaboration process, a "house made" technique of flow cytometry was developed as an alternative test to the reference method of the European Pharmacopoeia. Polyclonal anti-Rho antibodies and an anti FITC (fluorescein isothiocyanate) -labeled human IgG were used to measure the concentration of anti-D antibodies in a commercial preparation of gammaglobulin, semi-processed and human plasma. The method was developed using the 1st international WHO standard for anti-D IgG (69/419). At the concentration of red cells used in the method (5 x 104 cells/ul) there is no interference by auto agglutination. The obtained dose response curve (MFI vs. Log C) was linear in the adopted work range, presenting a correlation coefficient of 0.99. The specificity and recovery tests were satisfactory, the negative controls showed insignificant levels of fluorescence and did not present a serious interference in the determination. Knowing that the levels of anti-D sites antigen by red blood cell, vary according to the phenotype, our results show that the globules with phenotypes R1R1 present the best performance for this test, and can be replaced if necessary by R1R2 cells. The method was validated following the criteria of the group of Experts N°6B of the NIBSC and the European Pharmacopoeia who have standardized a similar procedure to be applied in the assessment of anti-D. Flow Cytometry allowed to obtain accurate, precise, sensitive and specific determinations at different concentrations of anti-D (100-0.9 ug/ ml) and in different biological matrices such as finished product, pre-filled concentrate product and fraction II of Cohn's method. The advantages found in the application of this method, in addition to the relatively low cost, include a strong fluorescence signal that does not require amplification, the use of highly diluted samples that minimize the risk of agglutination by IgM-Anti D or polymers. It is a simple, fast and reliable method to determine the serum levels of anti-D or quantify the concentration in an immunoglobulin solution.