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Determination of leucocyte antibody binding capacity (ABC): the need for standardization
D Barnett1, I Storie, G A Wilson
1UK NEQAS Leucocyte Immunophenotyping Schemes, Department of Haematology, Royal Hallamshire Hospital, Sheffield, UK.
Insights
Optimizing antigen density measurement requires standardizing technical factors. Standardizing pH, temperature, and staining methods is crucial for reproducible cellular antibody binding capacity (ABC) results in clinical settings.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Flow cytometry is vital for characterizing cells in health and disease.
- Antigen density, measured as cellular antibody binding capacity (ABC), is important in conditions like HIV infection.
- Technical variations can significantly impact ABC measurements.
Purpose of the Study:
- To investigate the influence of various technical factors on antibody binding capacity (ABC) measurements.
- To identify optimal conditions for reproducible ABC determination using the Quantum Simply Cellular (QSC) system.
- To propose a standardized 'benchmark' method for ABC quantification.
Main Methods:
- Assessed the impact of pH, incubation temperature and time, antibody fluorochrome and titre, and lysing reagents on ABC.
- Evaluated the effect of single, double, and triple-staining techniques.
- Utilized anti-CD3, CD4, and CD8 antibodies on normal lymphocytes.
Main Results:
- ABC values were significantly affected by pH, temperature, antibody concentration, fluorochrome, and staining technique.
- Optimal binding occurred at pH 7.4.
- Differences in ABC values up to 458,855 were observed between fluorochromes, and 130,119 between single and triple staining.
Conclusions:
- Standardization of methods is essential for reliable clinical application of antigen quantification.
- A proposed benchmark method involves single staining with FITC-conjugated antibodies at pH 7.4 and 20°C for ABC determination using QSC.
- This standardized approach aims to improve inter-center reproducibility.
Abstract:
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.
