Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

The immunophenotypes of blast cells in B-cell precursor acute lymphoblastic leukemia: how different are they from their normal counterparts?

BACKGROUND: Currently, there are three major maturational stages of CD19 antigen expressing B-cell precursors (hematogones). In B-cell precursor acute lymphoblastic leukemia (BCP-ALL), the malignant counterpart of hematogones, the leukemic blasts share common phenotypic features. The aim of the study was to enumerate the actual differences between the leukemic blasts in the CD10+ and CD10- subgroups of BCP-ALL and hematogones by assessing the expression of the antigens: TdT, CD34, CD45, CD10, CD38, CD20 and CD22. METHODS: To enable quantitative assessment of antigen expression on the different cell types, an objective scale of antigen expression was developed, the basis of which was direct fluorescence measurement using multicolor flow cytometry. RESULTS: All cases of CD10+ BCP-ALL clustered with type 1 hematogones. Among CD10-- BCP-ALL subgroup, 54.5%, 27.3% and 18.2% of cases clustered with type 1, 2 and 3 hematogones, respectively. In contrast to the CD10- blasts, the CD10+ blasts exhibited significantly higher levels of TdT, CD22, CD34 and CD20 expression. Conversely, CD10- blasts showed significantly higher expression of CD45 than CD10+ blasts, and a higher rate of CD45 antigen overexpression than CD10+ blasts (54.5% vs. 14.9% of cases, respectively). CONCLUSIONS: Multiparameter flow cytometry combined with the use of absolute antigen expression scale based on direct fluorescence measurement, has enabled a clear distinction between blasts in BCP-ALL cases and their normal counterparts. This novel and previously undescribed method has allowed the comparative analysis of antigen expression between leukemic blasts and different types of their normal counterparts.