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As the target cell population may not be known in advance or might have been defined previously, a different strategy is required in each situation.In the former situation, a rapid screening step based on a limited number of antibodies (preferably in a single tube) directed at differential identification of all relevant cell subsets in the sample is generally most efficient (Figure 1).Such a diagnostic algorithm is proposed here by the Euro Flow Consortium (Figure 1) as a critical prerequisite in the design of optimal antibody panels for diagnostic immunophenotyping.
Consequently, it has acquired a prominent position in the current World Health Organization (WHO) classification of hematological malignancies.
Preferably, the immunophenotypic profiles of suspected cells should be compared with those of normal hematopoietic cells.
The evaluation of the performance of a given antibody conjugate may be based on absolute measures (for example, fluorescence intensity and stain index (SI) obtained for a given control cell population), but these criteria may not apply once the marker is combined with other reagents in a single-tube combination or a multi-tube antibody panel.
Therefore, each reagent needs to be evaluated for its unique staining pattern obtained for the distinct cell populations in a multidimensional space defined by all parameters of the newly designed multicolor tube.
Consequently, such careful selection of reagents is essential for the design of standardized multicolor antibody combinations that provide unique staining patterns for each normal or aberrant cell population in a given sample.