Tomas Kalina

Charles University in Prague | CUNI · Department of Paediatric Haematology and Oncology (2. LF)

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 r...

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 dia...

Depletion of cellular immunity as a consequence of conditioning before allogeneic hematopoietic SCT (HSCT) frequently results in CMV reactivation, which may in turn lead to life-threatening infections and require timely antiviral treatment. We have investigated the functional signatures of CMV-specific CD4+ and CD8+ T-cells in 191 samples from 118...

Background B cells are central in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides (AAV). The efficacy of remission induction and maintenance by B cell depleting therapy strongly underlines the importance of B cells in the disease and the use of rituximab (RTX) has successfully been implemented in the treatmen...

Objectives B-cell depletion time after rituximab (RTX) treatment is prolonged in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) compared with other autoimmune diseases. We investigated central and peripheral B-cell development to identify the causes for the defect in B-cell reconstitution after RTX therapy. Methods We recru...

Understanding complex, organ-level single-cell datasets represents a formidable interdisciplinary challenge. This study aims to describe developmental trajectories of thymocytes and mature T cells. We developed tviblindi , a trajectory inference algorithm that integrates several autonomous modules - pseudotime inference, random walk simulations, re...

Understanding complex, organ-level single-cell datasets represents a formidable interdisciplinary challenge. This study aims to describe developmental trajectories of thymocytes and mature T cells. We developed tviblindi , a trajectory inference algorithm that integrates several autonomous modules - pseudotime inference, random walk simulations, re...

Detailed knowledge of the human B-cell development is crucial for proper interpretation of inborn errors of immunity and for malignant diseases. It is of interest to understand the kinetics of protein expression changes during the B cell development, but also to properly interpret the major and possibly alternative developmental trajectories. We ha...

Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain...

CONCLUSIONS Several works showed that patients with chronic myeloid leukemia (CML) diagnosed at the age of 18 to 39 years (adolescent and young adults, AYAs), had a worse outcome of tyrosine kinase inhibitors (TKI) therapy compared to elderly patients. Only limited data on the molecular background differing AYA from the other age groups are availab...

Herein, an advanced bioconjugation technique to synthesize hybrid polymer‐antibody nanoprobes tailored for fluorescent cell barcoding in flow cytometry‐based immunophenotyping of leukocytes is applied. A novel approach of attachment combining two fluorescent dyes on the copolymer precursor and its conjugation to antibody is employed to synthesize b...

Purpose:Anti-SARS-CoV-2 mRNA vaccines may elicit humoral responses in patients with primary antibody deficiency. However, this response is limited by its rapid waning and low levels of virus-neutralizing antibodies. There is limited evidence for the efficacy of the booster vaccination in patients with primary antibody deficiencies. Methods:This pro...

Understanding complex, organ-level single-cell datasets represents a formidable interdisciplinary challenge. This study aims to describe developmental trajectories of thymocytes and mature T cells. We developed tviblindi , a trajectory inference algorithm that integrates several autonomous modules - pseudotime inference, random walk simulations, re...

The distribution of fluorescence signals measured with flow cytometry can be influenced by several factors, including qualitative and quantitative properties of the used fluorochromes, optical properties of the detection system, as well as the variability within the analyzed cell population itself. Most of the single cell samples prepared from in v...

Triple‐negative breast cancer (TNBC) is an aggressive and complex subtype of breast cancer that lacks targeted therapy. TNBC manifests characteristic, extensive intratumoral heterogeneity that promotes disease progression and influence drug response. Single‐cell techniques in combination with next‐generation computation provide an unprecedented opp...

Background Common variable immunodeficiency (CVID) is characterized by an impaired post-vaccination response, high susceptibility to respiratory tract infections, and a broad spectrum of non-infectious complications. Thus, patients with CVID may be at high risk of coronavirus disease (COVID-19), and vaccination’s role in prevention is questionable....

Background : Common variable immunodeficiency (CVID) is characterized by an impaired post-vaccination response, high susceptibility to respiratory tract infections, and a broad spectrum of non-infectious complications. Thus, patients with CVID are at high risk of coronavirus disease 2019 (COVID-19), and vaccination’s role in prevention is questiona...

Background The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets an...

Our study presents a novel germline c.1715G>T (p.G572V) mutation in the gene encoding Toll‐like receptor 8 (TLR8) causing an autoimmune and autoinflammatory disorder in a family with monozygotic male twins, who suffer from severe autoimmune hemolytic anemia worsening with infections, and autoinflammation presenting as fevers, enteritis, arthritis,...

Reproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these lymphomas by flow cytometry and compared it to the WHO gold standard diagnosis. Overall, 662 samples from 662 patients representing nine disease categories wer...

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from lymphoblasts of T-cell origin. While T-ALL accounts for only 15% of childhood and 25% of adult ALL, 30% of patients relapse with a poor outcome. Targeted therapy of resistant and high-risk pediatric T-ALLs is therefore urgently needed, together with precision medicine...

Somatic mutations are a common molecular mechanism through which chronic myeloid leukemia (CML) cells acquire resistance to tyrosine kinase inhibitors (TKIs) therapy. While most of the mutations in the kinase domain of BCR-ABL1 can be successfully managed, the recurrent somatic mutations in other genes may be therapeutically challenging. Despite th...

Background: Inborn errors of immunity (IEI) are genetic disorders characterized by various degrees of immune dysregulation that can manifest as immune deficiency, autoimmunity or autoinflammation. The routine use of next-generation sequencing in the clinic has facilitated the identification of an ever-increasing number of IEI, revealing the roles...

The immunogenicity of the novel mRNA COVID-19 vaccine in immunocompromised lung transplant recipients is still unknown. We compared the antibody response after the first and second doses of the BNT162b2 mRNA COVID-19 vaccine (Pfizer/BioNTech) with the response after natural SARS-CoV-2 infection in lung transplant recipients. None of the vaccinees t...

Introduction Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memo...

The identification and characterization of rare immune cell populations in humans can be facilitated by their growth advantage in the context of specific genetic diseases. Here, we use autoimmune lymphoproliferative syndrome to identify a population of FAS-controlled TCRαβ+ T cells. They include CD4+, CD8+, and double-negative T cells and can be de...

Background Acute leukemia is the most common malignancy of childhood. Most of pediatric acute leukemias are derived from B-cell precursor, and about 15% are from T-cell origin and lead to T-cell acute lymphoblastic leukemia (T-ALL). Despite recent progress in treatment, the prognosis of T-ALL patients is worse, and ~30% of cases relapse (Pui CH, Se...

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell s...

Aims Danon disease (DD) is a rare X‐linked disorder caused by mutations in the lysosomal‐associated membrane protein type 2 gene (LAMP2 ). DD is difficult to distinguish from other causes of dilated or hypertrophic cardiomyopathy (HCM) in female patients. As DD female patients regularly progress into advanced heart failure (AHF) aged 20–40 years, t...

Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with early switch to the monocytic lineage and loss of the B-cell immunophenotype, including CD19 expression. Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced switch detectable...

Purpose Danon disease (DD) is a rare X‐linked disorder caused by pathogenic variants in LAMP2 . DD primarily manifests as a severe cardiomyopathy. An early diagnosis is crucial for patient survival. The aim of the study was to determine the usefulness of ocular examination for identification of DD. Methods Detailed ocular examination in 10 patient...

Background ALPS patients show impaired generation of humoral memory for T independent antigens whereas they generate memory for self-antigens due to impaired FAS-dependent removal of autoreactive germinal center B cells. It is known that FAS signaling via caspase activation results in cell apoptosis. However, FAS ligation may also initiate or modul...

INTRODUCTION Since the beginning of COVID-19 pandemic, it is known that the severe course of the disease occurs mostly among the elderly, whereas it is rare among children and young adults. Comorbidities, in particular, diabetes and hypertension, clearly associated with age, besides obesity and smoke, are strongly associated with the need for inten...

Introduction Since the beginning of COVID-19 pandemics, it is known that the severe course of the disease occurs mostly among elderly, whereas it is rare among children and young adults. Comorbidities, in particular diabetes and hypertension, clearly associated with age, besides obesity and smoke are strongly associated with the need of intensive t...

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discus...

B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with sur...

Cullin 4B (CUL4B), lysosomal‐associated membrane protein Type 2 (LAMP2), ATP1B4, TMEM255A, and ZBTB33 are neighboring genes on Xq24. Mutations in CUL4B result in Cabezas syndrome (CS). Male CS patients present with dysmorphic, neuropsychiatric, genitourinary, and endocrine abnormalities. Heterozygous CS females are clinically asymptomatic. LAMP2 mu...

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets and their malignant counterparts. Here, we present the results of the first...

Cytomegalovirus (CMV) infection is associated with allograft rejection but the mechanisms behind are poorly defined yet. Although cross-reactivity of T cells to alloantigen and CMV has been hypothesized, direct evidence in patients is lacking. In this observational cohort study, we tested the pre-transplant effector/memory T cell response to CMV pe...

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapp...

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell ty...

There is an agreement in the field that interlaboratory reproducibility of flow cytometry measurements as well as the whole studies might be improved by a consensual use of methodological approach. Typically, a consensus is made on a crucial markers needed in the immunostaining panel, sometimes on the particular fluorochrome conjugates and rarely o...

Antibody reagents are the key components of multiparametric flow cytometry analysis. Their quality performance is an absolute requirement for reproducible flow cytometry experiments. While there is an enormous body of antibody reagents available, there is still a lack of consensus about which criteria should be evaluated to select antibody reagents...

This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is...

Mutations in the Sterile alpha motif domain containing 9 (SAMD9) gene have been described in patients with severe multisystem disorder, MIRAGE syndrome, but also in patients with bone marrow (BM) failure in the absence of other systemic symptoms. The role of hematopoietic stem cell transplantation (HSCT) in the management of the disease is still un...

Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to...

Fluorochrome selection is a key step in designing multi-color antibody panels. The list of available fluorochromes is continuously growing, fitting current needs in clinical flow cytometry to simultaneously use more markers to better define multiple leukocyte subpopulations in a single tube. Several criteria guide fluorochrome selection: i) the flu...

Background In 2014, we used flow‐cytometry (FC) to describe a subtype of pediatric B cell precursor acute lymphoblastic leukemia (BCP ALL) with a lineage switch of leukemic blasts into monocytoid cells (swALL) during early treatment (Slamova et al., 2014). Although part of the swALL cases harbored deletion of ERG (ERGdel) and/or IKZF1 alterations,...

Purpose The aim of this study was to test the possibility of using specimens obtained by a cavitron ultrasonic surgical aspirator (CUSA) in flow and mass cytometry investigations of pediatric brain tumors. Methods CUSA specimens obtained from 19 pediatric patients with brain tumors were investigated. Flow and mass cytometry methods were applied to...

In the rapidly evolving field of primary immunodeficiencies (PID), the EuroFlow consortium decided to develop a PID orientation and screening tube that facilitates fast, standardized, and validated immunophenotypic diagnosis of lymphoid PID, and allows full exchange of data between centers. Our aim was to develop a tool that would be universal for...

Monitoring immune responses to solid cancers may be a better prognostic tool than conventional staging criteria, and it can also serve as an important criterion for the selection of individualized therapy. Multiparametric phenotyping by mass cytometry extended possibilities for immunoprofiling. However, careful optimization of each step of such met...

Background: Predominantly antibody deficiencies (PADs) are the most prevalent primary immunodeficiencies, but their B-cell defects and underlying genetic alterations remain largely unknown. Objective: We investigated patients with PADs for the distribution of 41 blood B-cell and plasma cell (PC) subsets, including subsets defined by expression o...

Background: Common variable immunodeficiency disorder (CVID) is one of the most frequent inborn errors of immunity, increased occurrence of malignancies, particularly lymphomas, and gastric cancers, has long been noted among CVID patients. Multifactorial etiology, including immune dysregulation, infections, chronic inflammation, or genetic backgrou...

Introduction: Recently we described a subgroup of pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP ALL) with switching from B to monocytic lineage in early phase of the therapy (Slamova et al., 2014). In a limited cohort of patients with switching ALL (swALL), we observed inferior response to treatment with discrepancy of...

Next generation sequencing has enabled rapid diagnosis of patients with monogenic diseases and discovery of novel disease-causing variants. However, the functional characterization of their role in disease pathogenesis still remains challenging. Reduced folate carrier (RFC) encoded by the SLC19A1 gene is one of the main folate transporters in mamma...

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation...

Danon disease (DD) is an X‐linked disorder caused by mutations in the lysosomal‐associated membrane protein 2 (LAMP2) gene (Xq24). DD is characterized by cognitive deficit, myopathy, and cardiomyopathy in male patients. The phenotype is variable and mitigated in females. The timely identification of de‐novo LAMP2 mutated family members, many of who...

EuroFlow Quality Assessment was designed to provide a feedback on the quality of the standardization effort in executing the EuroFlow protocols for sample preparation and instrument setup. It was first beta-tested by the members of the EuroFlow consortium internally (2010-2013) and opened to the external participants from 2015 onwards. The goal of...

PurposeSignal transducer and activator of transcription 1 gain-of-function (STAT1 GOF) mutations are the most common cause of chronic mucocutaneous candidiasis (CMC). We aim to report the effect of oral ruxolitinib, the Janus kinase (JAK) family tyrosine kinase inhibitor, on clinical and immune status of a 12-year-old boy with severe CMC due to a n...

Since publication of this article, the authors identified an error in the acknowledgments section. The original sentence in acknowledgments reads as follows.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL, and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a referenc...

Background: Humoral immunocompetence develops stepwise throughout life and contributes to individual susceptibility to infection, immunodeficiency, autoimmunity and neoplasia. Immunoglobulin heavy chain (IgH)-isotype serum levels can partly explain such age-related differences, but their relationship with the IgH-isotype distribution within memory...

A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instrumen...

The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. >20years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG a...

Background: Lately, mounting evidence has shown that B cells play an important role in the pathogenesis of type 1 diabetes (T1D). Here, we present alterations in B cell subsets including BAFF receptor (BAFFR) expression in cohorts of patients with type 1 diabetes (T1D) and their relatives. Patients and methods: B cells were studied in 438 patien...

The fluorescence detected using fluorochrome-labelled monoclonal antibodies depends not only on the abundance of the target antigen, but amongst many other factors also on the effective fluorochrome-to-antibody ratio. The diagnostic approach of the EuroFlow consortium relies on reproducible fluorescence intensities over time. A capture bead system...

The development of acute lymphoblastic leukemia (ALL) typically involves the acquisition of at least two genetic events. The initial mutation leads to the formation of a persistent, but clinically covert, preleukemic clone. Additional genetic changes then lead, in a stepwise manner, to complete