Tomáš Kalina

EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes

Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2–7 sequential design–evaluation–redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.

EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.

Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients

BCR–ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR–ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR–ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR–ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR–ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR–ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR–ABL immunobead assay detects all types of BCR–ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within ∼4 h, and can be run in parallel to routine immunophenotyping.

Prognosis of children with mixed phenotype acute leukemia treated on the basis of consistent immunophenotypic criteria

Background Mixed phenotype acute leukemia (MPAL) represents a diagnostic and therapeutic dilemma. The European Group for the Immunological Classification of Leukemias (EGIL) scoring system unambiguously defines MPAL expressing aberrant lineage markers. Discussions surrounding it have focused on scoring details, and information is limited regarding its biological, clinical and prognostic significance. The recent World Health Organization classification is simpler and could replace the EGIL scoring system after transformation into unambiguous guidelines. Design and Methods Simple immunophenotypic criteria were used to classify all cases of childhood acute leukemia in order to provide therapy directed against acute lymphoblastic leukemia or acute myeloid leukemia. Prognosis, genotype and immunoglobulin/T-cell receptor gene rearrangement status were analyzed. Results The incidences of MPAL were 28/582 and 4/107 for children treated with acute lymphoblastic leukemia and acute myeloid leukemia regimens, respectively. In immunophenotypic principal component analysis, MPAL treated as T-cell acute lymphoblastic leukemia clustered between cases of non-mixed T-cell acute lymphoblastic leukemia and acute myeloid leukemia, while other MPAL cases were included in the respective non-mixed B-cell progenitor acute lymphoblastic leukemia or acute myeloid leukemia clusters. Analogously, immunoglobulin/T-cell receptor gene rearrangements followed the expected pattern in patients treated as having acute myeloid leukemia (non-rearranged, 4/4) or as having B-cell progenitor acute lymphoblastic leukemia (rearranged, 20/20), but were missing in 3/5 analyzed cases of MPAL treated as having T-cell acute lymphobastic leukemia. In patients who received acute lymphoblastic leukemia treatment, the 5-year event-free survival of the MPAL cases was worse than that of the non-mixed cases (53±10% and 76±2% at 5 years, respectively, P=0.0075), with a more pronounced difference among B lineage cases. The small numbers of MPAL cases treated as T-cell acute lymphoblastic leukemia or as acute myeloid leukemia hampered separate statistics. We compared prognosis of all subsets with the prognosis of previously published cohorts. Conclusions Simple immunophenotypic criteria are useful for therapy decisions in MPAL. In B lineage leukemia, MPAL confers poorer prognosis. However, our data do not justify a preferential use of current acute myeloid leukemia-based therapy in MPAL.

The role of nutrition and genetics as key determinants of the positive height trend

The aim of this study was to identify the most important variables determining current differences in physical stature in Europe and some of its overseas offshoots such as Australia, New Zealand and USA. We collected data on the height of young men from 45 countries and compared them with long-term averages of food consumption from the FAOSTAT database, various development indicators compiled by the World Bank and the CIA World Factbook, and frequencies of several genetic markers. Our analysis demonstrates that the most important factor explaining current differences in stature among nations of European origin is the level of nutrition, especially the ratio between the intake of high-quality proteins from milk products, pork meat and fish, and low-quality proteins from wheat. Possible genetic factors such as the distribution of Y haplogroup I-M170, combined frequencies of Y haplogroups I-M170 and R1b-U106, or the phenotypic distribution of lactose tolerance emerge as comparably important, but the available data are more limited. Moderately significant positive correlations were also found with GDP per capita, health expenditure and partly with the level of urbanization that influences male stature in Western Europe. In contrast, male height correlated inversely with childrens mortality and social inequality (Gini index). These results could inspire social and nutritional guidelines that would lead to the optimization of physical growth in children and maximization of the genetic potential, both at the individual and national level.

Transfer of genomics information to flow cytometry: expression of CD27 and CD44 discriminates subtypes of acute lymphoblastic leukemia.

Transfer of genomics information to flow cytometry: expression of CD27 and CD44 discriminates subtypes of acute lymphoblastic leukemia

Detection of residual B precursor lymphoblastic leukemia by uniform gating flow cytometry

Residual disease (RD) is an important prognostic factor in acute lymphoblastic leukemia (ALL). Flow cytometry (FC)‐based RD detection is easy to perform, but interpretation requires expert analysis due to individual differences among patients.

Acute lymphoblastic leukemia incidence during socioeconomic transition: selective increase in children from 1 to 4 years

Pre-school acute lymphoblastic leukemia (ALL) peak is consistent in developed but not in developing countries and its magnitude apparently correlates with the socioeconomic status. A population-based study describing ALL incidence during socioeconomic transition has been lacking. Central European post-communist countries (with very low foreign migration and centralized statistics) offer reliable data for the period before and during major socioeconomic changes. Population-based data on Czech ALL patients younger than 18 years were taken from two independent Czech national registries partially overlapping in time (1980–1998, n = 1236 and 1991–1999, n = 570). During the 1980s and 1990s, ALL incidence among children 1–4 years increased 1.5 times (P = 0.01). This increase was more prominent in females than in males (slopes 0.13 and 0.09, P values 0.03 and >0.05, respectively). No significant change was observed in other age groups (0, 5–9, 10–14, 15–17 years or all others combined). We discuss possible underlying socioeconomic factors including infant care and breast-feeding, hygiene, birth order, industry and pollution. Moreover, we try to pinpoint the immunophenotypic/molecular-genetic subsets of ALL that might be socioeconomically affected. Selective increase of ALL in children 1–4 years old provides epidemiological evidence that etiology and/or trigger mechanisms are different for a considerable proportion of these children and that these mechanisms are exogenous.

CD2-positive B-cell precursor acute lymphoblastic leukemia with an early switch to the monocytic lineage

Switches from the lymphoid to myeloid lineage during B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment are considered rare and thus far have been detected in MLL-rearranged leukemia. Here, we describe a novel BCP-ALL subset, switching BCP-ALL or swALL, which demonstrated monocytosis early during treatment. Despite their monocytic phenotype, ‘monocytoids’ share immunoreceptor gene rearrangements with leukemic B lymphoblasts. All swALLs demonstrated BCP-ALL with CD2 positivity and no MLL alterations, and the proportion of swALLs cases among BCP-ALLs was unexpectedly high (4%). The upregulation of CEBPα and demethylation of the CEBPA gene were significant in blasts at diagnosis, prior to the time when most of the switching occurs. Intermediate stages between CD14negCD19posCD34pos B lymphoblasts and CD14posCD19negCD34neg ‘monocytoids’ were detected, and changes in the expression of PAX5, PU1, M-CSFR, GM-CSFR and other genes accompanied the switch. Alterations in the Ikaros and ERG genes were more frequent in swALL patients; however, both were altered in only a minority of swALLs. Moreover, switching could be recapitulated in vitro and in mouse xenografts. Although children with swALL respond slowly to initial therapy, risk-based ALL therapy appears the treatment of choice for swALL. SwALL shows that transdifferentiating into monocytic lineage is specifically associated with CEBPα changes and CD2 expression.

Kinetics of dendritic cells reconstitution and costimulatory molecules expression after myeloablative allogeneic haematopoetic stem cell transplantation: implications for the development of acute graft-versus host disease.

Allogeneic hematopoetic stem cell transplantation (HSCT) represents a unique opportunity to monitor the kinetics of reconstitution of dendritic cells (DCs) and their dynamics in distinct pathologies. We analyzed DCs reconstitution after myeloablative HSCT. We separately analyzed patients with acute GVHD. DCs were monitored from the earliest phase of hematopoetic reconstitution until day +365. Both myeloid DCs and plasmacytoid DCs appeared at earliest stages after engraftment and relative numbers within white blood cells compartment peaked between days 19-25 after HSCT. Their proportion then gradually declined and absolute numbers of both DC subsets remained lower than in controls during the whole follow-up. Patients with acute GVHD had significantly lower numbers of circulating DCs. Decrease in DC counts preceded onset of clinical symptoms by at least 24 h and was independent of corticosteroids administration. This study reveals quantification of plasmacytoid and myeloid DCs as a potential biomarker for the prediction of acute GVHD development.