Figure 11

Illustrating example of the impact of different sample preparation protocols on the immunophenotypic and light scatter features of lymphocytes from a normal peripheral blood (PB) sample (a) and blast cells from B-cell precursor acute lymphoblastic leukemia (BCP-ALL) (n=9; c) and how the harmonization process reduces such impact (b and d, respectively). In a and b, FSC versus SSC representation of duplicates of a sample stained with two different protocols (permeabilized versus non-permeabilized lymphocytes) is shown without (a) and with (b) data harmonization applied, respectively; in both a and b, green and violet populations correspond to non-permeabilized and permeabilized aliquots, respectively. In c and d, different BCP-ALL blast cell populations from nine different BCP-ALL patients each stained in five different aliquots with the BCP-ALL EuroFlow panel are displayed. Each population is represented as median values in a principal component (PC) 1 versus PC2 analysis diagram (automatic population separator (APS)1 view based on the discrimination obtained for the following parameters: FSC, SSC, CD19, CD34 and CD45), where paired duplicated samples are colored identically. In c samples contain both permeabilized and non-permeabilized aliquots within the panel and the harmonization process was applied for five patient samples (duplicates colored dark yellow, light green, dark violet, red and cyan) for which duplicates show a very close position in the APS1 view; conversely for the other pairs of duplicates (light yellow, dark green, violet, dark blue show greater differences between paired samples). In d, one group of duplicates was processed by permeabilizing all aliquots within the panel, while in the other group each sample contained permeabilized and non-permeabilized sample aliquots, with data harmonization being applied to the latter group; note that now all pairs of sample duplicates overlap, confirming that with data harmonization blast cell populations processed differently (permeabilized versus non-permeabilized) are highly comparable to those who underwent a uniform sample preparation protocol.