RESUMEN
The aim of this study was to assess the incidence of DNA aneuploidy in Polish children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and the relationship between aneuploidy and immunological phenotype, age, leukocyte count, S-phase fraction (SPF) and early response to induction chemotherapy assessed by the percentage of residual blast cells in bone marrow aspirates. The study group consisted of 267 patients. DNA content and immunophenotype were assessed in the bone marrow before treatment using multicolor flow cytometry (FC). DNA aneuploidy was detected in 50/267 (19%) patients. High hyperdiploidy was found to be associated with lower leukocyte count (p = 0.006) and common ALL immunophenotype. Flow cytometry analysis revealed that high hyperdiploid BCP-ALL patients showed significantly higher expression of CD9, CD20, CD22, CD58, CD66c, CD86 and CD123 antigens as compared to other groups of ploidy. In contrast, CD45 showed decreased expression. The percentage of leukemic blasts at diagnosis was lower in high hyperdiploid BCP-ALL cases than in diploid (79% vs. 85.7%, p = 0.001). The difference in minimal residual disease (MRD) levels on day 15 and 33 of induction therapy between analyzed groups was not significant. This study showed that high hyperdiploidy is associated with lower WBC count and specific immunological phenotype. Flow cytometric evaluation of expression of selected antigens can be used for fast identification of markers of aneuploidy in pediatric BCP-ALL, before genetic tests results are available. Understanding the biological significance of aneuploidy in leukemia can potentially be exploited therapeutically using targeted therapies against specific blast cell subclones.
RESUMEN
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.
