For immunophenotypic studies, all samples were systematically processed in parallel with the EuroFlow protocol versus the local routine procedures. Accordingly, the EuroFlow standard operating procedures (SOP) for instrument setup, instrument calibration, sample preparation, immunostaining and data acquisition16 were used at individual centers in parallel to the corresponding local protocols and techniques used for routine diagnosis and classification of hematological malignancies according to the WHO criteria. For data analysis, the Infinicyt software (Cytognos SL, Salamanca, Spain) was used in parallel to the local data analysis software programs and procedures.
For multivariate analysis of samples measured with the EuroFlow SOP and antibody panels, the Infinicyt software was used. For this purpose, the merge and calculation functions were applied for multi-tube panels prior to the analysis, as described elsewhere.31 (link), 32 (link) Briefly, prior to multivariate analyses, the populations of interest were selected and stored each in a distinct data file. Data files corresponding to the same cell population from an individual sample but stained with a different antibody tube of a multi-tube panel were merged into a single data file containing all information measured for that specific cell population. In a second step, ‘missing' data in one tube about markers only stained in the other tubes were calculated using previously described algorithms and tools implemented in the Infinicyt software.32 (link) Consequently, the generated final data file contained data about each parameter measured in the multi-tube panel for each of the events composing the cell population in that data file (Figure 2 ). This data file was further merged with the data files of other samples either to create a reference pool of a population of normal, reactive or malignant cells or to compare it with one or more of such reference pool data files, through multivariate analysis, for example, principal component analysis (PCA).31 (link)
For multivariate analysis of samples measured with the EuroFlow SOP and antibody panels, the Infinicyt software was used. For this purpose, the merge and calculation functions were applied for multi-tube panels prior to the analysis, as described elsewhere.31 (link), 32 (link) Briefly, prior to multivariate analyses, the populations of interest were selected and stored each in a distinct data file. Data files corresponding to the same cell population from an individual sample but stained with a different antibody tube of a multi-tube panel were merged into a single data file containing all information measured for that specific cell population. In a second step, ‘missing' data in one tube about markers only stained in the other tubes were calculated using previously described algorithms and tools implemented in the Infinicyt software.32 (link) Consequently, the generated final data file contained data about each parameter measured in the multi-tube panel for each of the events composing the cell population in that data file (
