Immunophenotypic characterization of leukemic cells with the use of flow cytometry (FC) is a fundamental tool in acute lymphoblastic leukemia (ALL) diagnostics. A variety of genetic aberrations underlie specific B-cell precursor ALL (BCP-ALL) subtypes and their identification is of great importance for risk group stratification. These aberrations include: ETV6::RUNX1 fusion gene, Philadelphia chromosome (BCR::ABL1 fusion gene), rearrangements of the KMT2A, TCF3::PBX1 fusion gene and changes in chromosome number (hyperdiploidy and hypodiploidy). Diagnostic panels for BCP-ALL usually include B-cell lineage specific antigens: CD19, CD10, CD20, maturation stage markers: CD34, CD10, CD38, TdT, IgM and other markers useful for possible genetic subtype indication. Some genetic features of leukemic cells (blasts) are associated with expression of certain antigens. This review comprehensively summarizes all known research data on genotype-immunophenotype correlations in BCP-ALL. In some cases, single molecules are predictive of particular genetic subtypes, i.e., NG2 with KMT2A gene rearrangements or CD123 with hyperdiploidy. However, much more information on possible genotype or prognosis can be obtained with wider (≥8-color) panels. In several studies, a quantitative antigen expression scale and advanced statistical analyses were used to further increase the specificity and sensitivity of genotype/immunophenotype correlation detection. Fast detection of possible genotype/immunophenotype correlations makes multicolor flow cytometry an essential tool for initial leukemia diagnostics and stratification.

Flow cytometry technique (FC) is a standard diagnostic tool for diagnostics of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) assessing the immunophenotype of blast cells. BCP-ALL is often associated with underlying genetic aberrations, that have evidenced prognostic significance and can impact the disease outcome. Since the determination of patient prognosis is already important at the initial phase of BCP-ALL diagnostics, we aimed to reveal specific genetic aberrations by finding specific multiple antigen expression patterns with FC immunophenotyping. The FC immunophenotype data were analysed using machine learning methods (gradient boosting, decision trees, classification rules). The obtained results were verified with the use of repeated cross-validation. The t(12;21)/ETV6-RUNX1 aberration occurs more often when blasts present high expression of CD10, CD38, low CD34, CD45 and specific low expression of CD81. The t(v;11q23)/KMT2A is associated with positive NG2 expression and low CD10, CD34, TdT and CD24. Hyperdiploidy is associated with CD123, CD66c and CD34 expression on blast cells. In turn, high expression of CD81, low expression of CD45, CD22 and lack of CD123 and NG2 indicates that none of the studied aberrations is present. Detecting aberrations in pediatric BCP-ALL, based on the expression of multiple markers, can be done with decent efficiency.

Objective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients’ samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.