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.

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.

the most common applications of flow cytometry (Fc) include diagnostics of haemato-oncological disorders, based on analysis of bone marrow, peripheral blood (PB), or cerebrospinal fluid (csF) samples. a proper diagnostic process requires standardisation in setting the optimal time frame between material collection and the assay. unfortunately, this might be difficult to achieve in daily practice due to unintended shipment delays, which might compromise large-scale multicentre studies. thus, material fixation should be considered as a solution. the most widely used fixative agents are: paraformaldehyde, transFix ® , cyto-chex ® , and serum-containing media. in this review, we attempted to summarise the literature data on the influence of sample storage under different temperatures and times combined with different fixation conditions on the cell count and marker expression levels. Based on the findings of several extensive studies employing fixed PB samples, it can be concluded that the performance of particular fixative greatly depends on the analysed marker and specific PB cell population expressing a given antigen. Preservation of absolute cell count was usually better in cyto-chex ® -fixed PB samples, whereas transFix ® tended to better stabilise marker expression levels. csF-based studies reveal that both serum-containing media and transFix ® can prevent cellular loss and enhance Fc-based detection of leptomeningeal localisations of haematological malignancies, the latter being more available and having longer shelf-life. as both cell count and marker expression level are the main determinants of quality of biological samples dedicated to Fc analyses, it remains to be addressed by the investigators which is the fixative of choice for their specific research aims.

Flow cytometry (FcM) is a precise and well-established tool to assess the minimal residual disease (Mrd) level in childhood acute lymphoblastic leukemia (all). it is crucial to distinguish leukemic cells from their normal counterparts; thus new markers should be evaluated, to increase the accuracy of the analysis.the expression of cd73 on blast cells was measured and compared at the day of diagnosis and at days 15 and 33 of treatment. to determine antigen expression levels, a normalized scale based on median fluorescence intensity (nMFi) was used. the study group consisted of 188 patients from the Polish Pediatric leukemia and lymphoma study group.From 177 patients with positive Mrd at day 15 of treatment, in 147 (83.1%) cases an increase of cd73 expression was observed (mean increase of +17 nMFi units). in addition, an increase of cd73 expression was noted in 26 of 31 (83.9%) patients at day 33 of treatment. in turn, a decrease of cd73 expression was observed only in 13/177 (7.3%) and 1/31 (3.2%) cases at days 15 and 33 of treatment, respectively. in 17 (9.6%) patients no change in expression of cd73 between diagnosis and day 15 of treatment was observed.in the great majority of cases the expression of cd73 is not only stable but increases during the early stages of treatment, which makes it a very useful marker to be used for Mrd monitoring in childhood B-cell precursor (BcP)-all patients.