Division of Haematology Laboratory & Blood Bank, Department of Anatomical & Cellular Pathology, Prince of Wales Hospital

There have been 130 groups of 'cluster of differentiation' (CD) of leucocyte monoclonal antibodies debuted since its introduction in 1982. The categorization of monoclonal antibodies by this CD system enables their evaluation and comparison in a consistent way. For diagnostic work, only a small number of these evaluated reagents are of importance in clinical laboratories; unevaluated research antibodies are best avoided. Antibodies without CD designations, but are generally used, include antibodies to FMC7, terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase, etc. Newer antibodies, such as anti-Cyclin D1 and ALK, help to pinpoint the diagnosis of mantle cell lymphomas and anaplastic large cell lymphomas, especially those occur in childhood, respectively. Monoclonal antibodies, as reagents, may be of different class, subclass or isotypes. They may be unconjugated or conjugated to fluorochromes. And they may be applied in different techniques, such as immunofluorescence microscopy, flow cytometry or immunocytochemistry, in different specimens e.g. whole blood, isolated cells, smears, imprints, fine needle aspirates and tissue sections. Immunophenotypic studies, when first developed, were performed by microscopic evaluation of antibody staining; at present, flow cytometry is the preferred method on account of its higher precision and objectivity.

There are several technical factors that require considerations in the application of flow cytometry. (1) Antibody labeling: Phycoerythrin (PE), and other newer and more sensitive fluorochromes release higher quantum of light than traditional fluorescein isothiocyanate (FITC), and they should be used for single-colour analysis. In case of two-colour analysis, the brighter antibody should be used to detect the weaker antigen. (2) Isotypic (negative) controls: They are used to determine background fluorescence of the sample (which includes autofluorescence and non-specifically bound antibody). They are also employed to set photomultification tube voltages and for setting the position of the analysis regions. However, they are not without problems: they may not have the same characteristics as the test monoclonal antibodies; their fluorochrome to protein ratio may be different; they may bind differently to different cell populations, and isotypic controls from different manufacturers are different. (3) Antibody concentration: The optimal working dilution, also known as titre value, defined as the amount of antibody that is required to saturate the maximum number of antigen-binding sites on a selected cell population, should be established for every batch of test monoclonal antibody. (4) Cell concentration: Final cell counts should be standardized in the order of 10⁶ cells/ml. Dead cells, which give rise to nonspecific binding and data misinterpretation, can be a problem. However, they can be easily "gated" out using appropriate DNA staining dye (e.g. propidium iodide) employing multicolour analysis. (5) Fc blocking: The Fc fragments of immunoglobulins have high and saturable binding affinity to Fc receptors present on the cell surface of monocytes or histiocytes of the reticulo-endothelial system. Blocking Fc receptors, by preincubation with rabbit or human serum, would make a difference in data collection and analysis, particularly when studying leukaemias with strong monocytic components. Another method to nullify this phenomenon is the use of fluorescent (Fab)² fragments (i.e. Fc-cleaved) in indirect immunofluorescence analysis. (6) Quality control: Running daily minimal positive and negative control and participation in an external proficiency programme should be undertaken in every clinical flow cytometry laboratory.