This study introduces an in-depth flow cytometric method for the analysis of nucleated erythroid progenitors during bone marrow regeneration. Initial immunophenotypic analysis with the conventional erythroid-associated markers CD36, CD71 and CD235a was supplemented with the analysis of additional markers, including CD105, CD117, CD45, CD38 and cell-scattered light characteristics. Our data show that the expression of CD105 and CD117 is critical for the distinction between four phenotypically different developmental stages of nucleated erythroid progenitors: pro-erythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts and orthochromatophilic erythroblasts. CD105 antigen expression was specifically associated with pro-erythroblasts and basophilic erythroblasts, whereas CD117 was expressed at the earliest pro-erythroblast stage. Both antigens were progressively lost throughout the course of differentiation. These data allow for the identification of aberrant erythroid development in acute erythroid leukemia or myelodysplastic syndrome.

The aim of this review is to evaluate recent immunophenotype knowledge of hematopoietic stem cells and their restricted progenies committed to myeloid lineage -common myeloid progenitors, myelo-monocytic progenitors, megakaryo-erythroid progenitors and granulocyte progenitors up to mature neutrophil granulocyte. This study evaluates also recent knowledge of immunophenotype of leukemic stem cells and their more differentiated progeny committed to myeloid lineage -acute myeloid leukemia blast cells with regard to their phenotypic similarity to normal stem and granulocyte committed progenitor cells. Improved knowledge of normal stem and progenitor cells phenotypes, identifying new leukemia-specific markers, searching for aberrant marker expression and evaluation of aberrant intensity or combination of various marker expressions is important for distinguishing normal cells from their malignant counterpart in view of the diagnostics of leukemias or follow-up of minimal residual disease.

The main aim of this study represents the extension of our studies using multiparametric flow cytometry analysis for exact definition of membrane and intracellular (cytoplasmic and nuclear) markers of acute leukemia cells of T-phenotype. The study of blasts of each patient with all available monoclonal antibodies targeted to T-cell differential antigens and against possible marker coexistence from different lineages has been performed. The main aim was concerned to more proper T-ALL diagnosis and stage definition and identification of the prognostic factors and the useful markers for the follow-up of T-ALL in remission. New knowledge of the T-cell maturation stages of hematopoietic cells in bone marrow and thymus has been applied, as each T-acute leukemia clone is representative of one blocked stage through maturation. We evaluated 44 patients with T-ALL by multiparameter flow cytometry. Patients with more favorable prognosis (i. e. those of cortical stage) could have been already differentiated at diagnosis from those, allocated to pro-T stage, with very immature phenotypes and of an unfavorable clinical course. These patients had very distinctive immunophenotes, CD1a and CD8 markers completely negative, CD7 and cCD3 positive; CD5 was weakly expressed and myeloid markers CD33 and CD13 were coexpressed, or immature markers CD34, HLA-DR were coexpressed, together with myeloid markers CD13 and CD33 of weak positivity. The patients were either completely unresponsive to therapy or because of persistent MRD during continuation therapy, indicated for allogeneic hematopoietic stem-cell transplant. The results have been discussed with similar the most relevant immunophenotypic results of others and mainly with gene-expressing profiling associated with a significantly worse clinical outcome.

In this review, we discuss methodological principles and clinical applications of minimal residual disease (MRD) assays based on multiparameter flow cytometry (MFC). The introduction of methods for MRD detection has revolutionized monitoring of treatment response in acute leukemia. Great progress has been made in the development of wide array of flow cytometric techniques for rare event detection. This advance was accompanied by increasingly greater understanding of the immunophenotypic features of leukemic and normal lymphoid cells, and of the antigenic differences that make MRD studies possible. Immunologic testing of MRD relies on "leukemia-associated" immunophenotypes which can be identified by MFC in the most of acute leukemia cases. The recent technical innovations in routine MFC (3 lasers and≥8 colors) and the new developments in software for data analysis make this technology the most attractive for MRD diagnostics. The importance of MFC methodology will be further strengthened by the ongoing international standardization efforts. Results of MRD testing provide unique and clinically important information. The systematic application of immunologic techniques to study MRD in clinical samples has demonstrated the prognostic significance of MRD in patients, leading to the use of MRD to regulate treatment intensity in many contemporary protocols. The identification of new markers of MRD should increase the sensitivity of MRD testing by MFC and is required to widen the applicability of MRD studies.

Reciprocal communication between hematopoietic cells and their surrounding bone marrow stroma is crucial for normal progression of hematopoiesis. This complex network of cell-to-cell signals in the microenvironment involves both cell contactmediated and paracrine cues. In hematological malignancies the intricate balance is, however, disrupted to support cancer progression. In order to detect altered microenvironmental reactivity of a hematopoietic cell sample, cellular functional assays can be designed to measure the cells' capacity to modulate stromal stress reactions, such as inflammation.Recently, we showed that human leukemic cell lines of monocytic origin can actively participate in modulation of stromal inflammation. In order to further functionally evaluate the hematopoietic cells' capacity to modulate stromal inflammation, we utilized an in vitro model of nemosis-induced inflammation of fibroblasts in a three-dimensional culture setting. This process of stromal inflammation in fibroblast aggregates is consistent, requires both cell-contact and paracrine signals, and can be produced on a large scale to support dose-dependent analyses. To extend our previous observations, we evaluated the effect of a wide panel of leukemia cell lines on cyclooxygenase-2 induction in fibroblast aggregates in co-culture. We also assessed the feasibility of the model to support clinical functional testing by utilizing the hematopoietic fraction of leukemia patients' bone marrow aspirates after immunophenotyping. Our results suggest that the stromal inflammation-modulating activity of these samples is differently modulated in cancer and in normal bone marrow. Moreover, differences in the samples' anti-inflammatory activity may reflect disease state.