DETERMINATION OF LEUKOCYTE ANTIBODY-BINDING CAPACITY (ABC) - THE NEEDFOR STANDARDIZATION

The flow cytometric determination of antigen density, or cellular anti body binding capacity, is now an accepted technique for the characteri zation of cells in health and disease, In HIV infection, for example, antigen density changes in CD38 expression may be an important indicat or of disease progression, Our experience of using one such method, Qu antum Simply Cellular, which measures antibody binding capacity (ABC), has highlighted several technical factors which can affect the result s. We report the influence of pH, incubation temperature and time, ant ibody fluorochrome and titre, as well as lysing reagent (FACS Lysing S olution v. Ortho-mune Lysing Reagent) on the ABC of anti-CD3, CD4 and CD8 of normal lymphocytes. In addition, the effect of single, double o r triple-staining was assessed. The results indicate that the ABC valu es are influenced by all the variables studied. The pH range tested (6 .0-9.0) demonstrated that pH 7.4 gave maximal binding. Furthermore, te mperature also influenced the pH of the two lysing solutions, and thus potentially the ABC. Antibody concentration, fluorochrome and stainin g technique are also important factors with an observed difference of up to 458 855 ABC between the various fluorochromes. In addition a max imal difference of 130119 ABC was observed between single and triple s taining techniques, In conclusion, if antigen quantification is to be used in the clinical setting, an internationally standardized method i s required to ensure the reproducibility of results from centre to cen tre, Our data suggests that single staining, using fluorescein isothio cynate (FITC) conjugated antibodies with all reagents at pH 7.4 + 0.1, with incubation and lysing carried out at 20 + 1 degrees C, could be used as a 'benchmark' method for ABC determination using the QSC syste m.