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Figurę 7. Effect of time between completion of staining and data acquisition in the flow cytometer (0 h, 1 h, 3 h and 24 h) and the sample preparation protocol on the mean fluorescence intensity (MFI) of CD19-phycoerythrin cyanin 7 (PECy7), CD4-peridinin chlorophyll protein cyaninS.5 (PerCPCy5.5) and CD8-allophycocyanin hilite7 (APCH7) on peripheral blood (PB) B-cells, CD4 T-cells and CD8^hi T-cells, using ammonium chloride (a) or FACS Lysing Solution (b) as lysing reagents. Three different sample preparation protocols were evaluated: SLNW; SLW and SLWF. Results are shown as mean values (open circles) and 95% confidence intervals (vertical lines). FACS Łyse, FACS Lysing Solution; NH„CI, ammonium chloride. SLW, stain-lyse-wash; SLWF, stain-lyse-wash-fix; SLNW, stain-lyse-no wash.

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number of parameters simultaneously assessed in greater numbers of individual cells, and the expanded number of variables that might have an impact on the quality of the results.⁴⁴"⁴⁷ Moreover, these technical improvements have not been paralleled (or followed) by innovations of data analysis and interpretation tools in the software packages routinely used in hematology laboratories. This lack of innovation has further contributed to the increased complexity of immunophenotyping of hematological malignancies.^44,45 In recent years, the EuroFlow Consortium has proposed several new data analysis tools⁴⁸"⁵⁰ aimed at decreasing such complexity through the development of new and morę objective data analysis and interpretation strategies.⁴⁸"⁵¹ These novel tools have been progressively incorporated into the Infinicyt software (Cytognos SL) developed by the EuroFlow Consortium.

In this section we describe the new data analysis strategy proposed by the EuroFlow Consortium to be used in combination with the EuroFlow antibody panels and the EuroFlow SOPs for multiparameter immunophenotypic diagnosis and dassification of hematological disorders.

Merge of flow cytometry data files and calculation of 'missing values'

The EuroFlow antibody panels are composed of multiple 8-color combinations of antibodies that contain three or four fluoro-chrome-conjugated antibodies as common backbone markers, essential for gating the cells of interest in every aliquot of a sample stained with a specific EuroFlow antibody panel.²⁹ The Merge function (first step in Figurę 8) was used to fuse different data files corresponding to distinct aliquots of the same sample, each stained with a unique combination of reagents from the

Figurę 8. Flow chart diagram illustrating the sequential steps used during data analysis for the evaluation of the performance of the EuroFlow antibody panels.

EuroFlow antibody panels. This results in a new single merged data file that contains all information measured in the same sample.^52,53 Such data file consists of a data matrix (Figurę 9) in which the information (the measured parameters) for each different cellular event evaluated is aligned in one column per