Determination of leucocyte antibody binding capacity (ABC): the need for standardization.
The flow cytometric determination of antigen density, or cellular antibody binding capacity, is now an accepted technique for the characterization of cells in health and disease. In HIV infection, for example, antigen density changes in CD38 expression may be an important indicator of disease progression. Our experience of using one such method, Quantum Simply Cellular, which measures antibody binding capacity (ABC), has highlighted several technical factors which can affect the results. We report the influence of pH, incubation temperature and time, antibody fluorochrome and titre, as well as lysing reagent (FACS Lysing Solution v. Ortho-mune Lysing Reagent) on the ABC of anti-CD3, CD4 and CD8 of normal lymphocytes. In addition, the effect of single, double or triple-staining was assessed. The results indicate that the ABC values are influenced by all the variables studied. The pH range tested (6.0-9.0) demonstrated that pH 7.4 gave maximal binding. Furthermore, temperature also influenced the pH of the two lysing solutions, and thus potentially the ABC. Antibody concentration, fluorochrome and staining technique are also important factors with an observed difference of up to 458,855 ABC between the various fluorochromes. In addition a maximal difference of 130,119 ABC was observed between single and triple staining techniques. In conclusion, if antigen quantification is to be used in the clinical setting, an internationally standardized method is required to ensure the reproducibility of results from centre to centre. Our data suggests that single staining, using fluorescein isothiocynate (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 system.
UK NEQAS Leucocyte Immunophenotyping Schemes, Department of Haematology, Royal Hallamshire Hospital, Sheffield, UK.
D
D
Barnett
I
I
Storie
G A
GA
Wilson
V
V
Granger
J T
JT
Reilly
D000937:Antigen-Antibody Reactions; D017252:CD3 Complex / Q000276:immunology*; D015704:CD4 Antigens / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D016827:CD8 Antigens / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002458:Cell Fractionation; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006405:Hematology / Q000592:standards*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016130:Immunophenotyping / Q000592:standards*; D012015:Reference Standards; D012996:Solutions / Q000494:pharmacology; D013696:Temperature
D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D005456:Fluorescent Dyes; D012996:Solutions; D016650:Fluorescein-5-isothiocyanate