Publications by authors named "Tomas Kalina"

89 Publications

Dissection of the Pre-Germinal Center B-Cell Maturation Pathway in Common Variable Immunodeficiency Based on Standardized Flow Cytometric EuroFlow Tools.

Front Immunol 2020 17;11:603972. Epub 2021 Feb 17.

Clinical and Translation Research Program, Cancer Research Centre (IBMCC, USAL-CSIC), Department of Medicine, Cytometry Service (NUCLEUS), University of Salamanca (USAL), Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.

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 memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVID patients.

Methods: In this collaborative multicentric study the EuroFlow PID 8-color , standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD).

Results: The allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5 CD38 CD21 CD24 (2.7 vs 5.6 cells/µl, p=0.0004) and CD5 CD38 CD21 CD24 (6.5 vs 17 cells/µl, p<0.0001) immature B cells (below normal HD levels in 22% and 37% of CVID patients). This was associated with an expansion of CD21CD24 (6.1 vs 0.74 cells/µl, p<0.0001) and CD21CD24 (1.8 vs 0.4 cells/µl, p<0.0001) naïve B-cell counts above normal values in 73% and 94% cases, respectively. Additionally, reduced IgMD (21 vs 32 cells/µl, p=0.03) and IgMD (4 vs 35 cells/µl, p<0.0001) MBC counts were found to be below normal values in 25% and 77% of CVID patients, respectively, always together with severely reduced/undetectable circulating blood pb. Comparison of the maturation pathway profile of pre-GC B cells in blood of CVID patients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%).

Conclusion: Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.603972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925888PMC
February 2021

A distinct CD38+CD45RA+ population of CD4+, CD8+, and double-negative T cells is controlled by FAS.

J Exp Med 2021 Feb;218(2)

Institute for Immunodeficiency, Center for Chronic Immunodeficiency, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

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 defined by a CD38+CD45RA+T-BET- expression pattern. These unconventional T cells are present in healthy individuals, are generated before birth, are enriched in lymphoid tissue, and do not expand during acute viral infection. They are characterized by a unique molecular signature that is unambiguously different from other known T cell differentiation subsets and independent of CD4 or CD8 expression. Functionally, FAS-controlled T cells represent highly proliferative, noncytotoxic T cells with an IL-10 cytokine bias. Mechanistically, regulation of this physiological population is mediated by FAS and CTLA4 signaling, and its survival is enhanced by mTOR and STAT3 signals. Genetic alterations in these pathways result in expansion of FAS-controlled T cells, which can cause significant lymphoproliferative disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20192191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658692PMC
February 2021

Analysis of Magnetic Anisotropy and Non-Homogeneity of S235 Ship Structure Steel after Plastic Straining by the Use of Barkhausen Noise.

Materials (Basel) 2020 Oct 15;13(20). Epub 2020 Oct 15.

Faculty of Civil Engineering, University of Žilina, Univerzitná 1, 010 26 Žilina, Slovakia.

This study investigates the microstructure, residual stress state, and the corresponding magnetic anisotropy of the ship structure samples made of S235 steel after uniaxial tensile deformation. A non-destructive magnetic technique based on Barkhausen noise is employed for fast and reliable monitoring of samples exposed to the variable degrees of plastic straining. It was found that the progressively developed plastic straining of the matrix results in an alteration of the easy axis of magnetization, stress anisotropy (expressed in residual stresses state) as well as the corresponding Barkhausen noise emission. Moreover, remarkable non-homogeneity can be found within the plastically strained region, especially when the localized plastic straining takes place.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ma13204588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602674PMC
October 2020

Danon disease is an underdiagnosed cause of advanced heart failure in young female patients: a LAMP2 flow cytometric study.

ESC Heart Fail 2020 10 13;7(5):2534-2543. Epub 2020 Jul 13.

Department of Cardiology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.

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, their early identification is critical to improve patient survival and facilitate genetic counselling. In this study, we evaluated the prevalence of DD among female patients with non-ischemic cardiomyopathy, who reached AHF and were younger than 40 years.

Methods And Results: The study cohort comprised 60 female patients: 47 (78%) heart transplant recipients, 2 (3%) patients treated with ventricular assist device, and 11 (18%) patients undergoing pre-transplant assessment. Aetiology of the cardiomyopathy was known in 15 patients (including two DD patients). LAMP2 expression in peripheral white blood cells (WBC) was tested by flow cytometry (FC) in the remaining 45 female patients. Whole exome sequencing was used as an alternative independent testing method to FC. Five additional female DD patients (two with different novel LAMP2 mutations) were identified by FC. The total prevalence of DD in this cohort was 12%. HCM phenotype (57% vs. 9%, P = 0.022) and delta waves identified by electrocardiography (43% vs. 0%, P = 0.002) were significantly more frequent in DD female patients.

Conclusions: Danon disease is an underdiagnosed cause of AHF in young female patients. LAMP2 expression testing in peripheral WBCs by FC can be used as an effective screening/diagnostic tool to identify DD in this patient population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ehf2.12823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524080PMC
October 2020

DUX4r, ZNF384r and PAX5-P80R mutated B-cell precursor acute lymphoblastic leukemia frequently undergo monocytic switch.

Haematologica 2020 07 9. Epub 2020 Jul 9.

CLIP-Dpt.of Paediatric Haematology/Oncology, Charles University, Prague, Czech Republic;

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 by flow cytometry (FC). Using exome and RNA sequencing, switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (5 cases with switch out of 5), rearranged DUX4 (DUX4r; 30 cases of 41) and rearranged ZNF384 (ZNF384r; 4 cases of 10). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype they could not identify cases who subsequently switched. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and PCR-determined MRD was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2020.250423DOI Listing
July 2020

Pigmentary retinopathy can indicate the presence of pathogenic LAMP2 variants even in somatic mosaic carriers with no additional signs of Danon disease.

Acta Ophthalmol 2021 Feb 13;99(1):61-68. Epub 2020 Jun 13.

Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.

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 patients with DD (3 males, 7 females) and a 45-year-old asymptomatic female somatic mosaic carrier of a LAMP2 disease-causing variant.

Results: All patients with manifest cardiomyopathy had pigmentary retinopathy with altered autofluorescence and diffuse visual field loss. Best corrected visual acuity (BCVA) was decreased (<0.63) in 8 (40%) out of 20 eyes. The severity of retinal pathology increased with age, resulting in marked cone-rod involvement overtime. Spectral-domain optical coherence tomography in younger patients revealed focal loss of photoreceptors, disruption and deposition at the retinal pigment epithelium/Bruch's membrane layer (corresponding to areas of marked increased autofluorescence), and hyperreflective foci in the outer nuclear layer. Cystoid macular oedema was seen in one eye. In the asymptomatic female with somatic mosaicism, the BCVA was 1.0 bilaterally. An abnormal autofluorescence pattern in the left eye was present; while full-field electroretinography was normal.

Conclusions: Detailed ocular examination may represent a sensitive and quick screening tool for the identification of carriers of LAMP2 pathogenic variants, even in somatic mosaicism. Hence, further investigation should be undertaken in all patients with pigmentary retinal dystrophy as it may be a sign of a life-threatening disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/aos.14478DOI Listing
February 2021

Flash survey on severe acute respiratory syndrome coronavirus-2 infections in paediatric patients on anticancer treatment.

Eur J Cancer 2020 06 7;132:11-16. Epub 2020 Apr 7.

Childrens Hospital Medical Center Schleswig-Holstein, Kiel, Germany.

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 intensive treatment and a dismal outcome. A weaker immunity of the elderly has been proposed as a possible explanation of this uneven age distribution. Thus, there is concern that children treated for cancer may allso be at risk for an unfavourable course of infection. Along the same line, anecdotal information from Wuhan, China, mentioned a severe course of COVID-19 in a child treated for leukaemia.

Aim And Methods: We made a flash survey on COVID-19 incidence and severity among children on anticancer treatment. Respondents were asked by email to fill in a short Web-based survey.

Results: We received reports from 25 countries, where approximately 10,000 patients at risk are followed up. At the time of the survey, more than 200 of these children were tested, nine of whom were positive for COVID-19. Eight of the nine cases had asymptomatic to mild disease, and one was just diagnosed with COVID-19. We also discuss preventive measures that are in place or should be taken and treatment options in immunocompromised children with COVID-19.

Conclusion: Thus, even children receiving anticancer chemotherapy may have a mild or asymptomatic course of COVID-19. While we should not underestimate the risk of developing a more severe course of COVID-19 than that observed here, the intensity of preventive measures should not cause delays or obstructions in oncological treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejca.2020.03.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141482PMC
June 2020

Editorial: Application of Cytometry in Primary Immunodeficiencies.

Front Immunol 2020 19;11:463. Epub 2020 Mar 19.

Department of Pediatrics, Leiden University Medical Center, Leiden, Netherlands.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.00463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096470PMC
March 2021

EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children.

Front Immunol 2020 19;11:371. Epub 2020 Mar 19.

Laboratory for Immunology, Department of Pediatrics, Leiden University Medical Center (LUMC), Leiden, Netherlands.

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 discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in ( = 4, two of them presented with Omenn syndrome), ( = 4, one of them with confirmed maternal engraftment), ( = 1), ( = 1), ( = 1), ( = 3, two of them with maternal engraftment) and ( = 1) and 11 other PID patients had diverse molecular defects [ ( = 1), ( = 2), ( = 1), ( = 1), del22q11.2 (DiGeorge = 4), ( = 1) and ( = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.00371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096355PMC
March 2021

The relationship between body composition and foot posture index in Special Olympics athletes.

Acta Bioeng Biomech 2019 ;21(4):47-52

Faculty of Sports Studies, Masaryk University, Brno, Czech Republic.

Purpose: In Special Olympics, athletes' foot deformities, abnormal postural patterns and overweight and obesity were observed to be more common than in the general population. Therefore, the purpose of this study was to analyze the relationship between body composition and foot posture index. Additionally, the effect of gender and other factors on the body composition and foot posture index in athletes with intellectual disability (ID) were analyzed.

Methods: 131 athletes with ID participated in this study: 36 table tennis players, 66 cross country skiers and 29 downhill skiers. Their body composition was assessed using the bioimpedance analysis InBody 230 (Biospace, Korea). The foot screening included the assessment of foot posture index, which evaluates the degree of pronated, supinated and neutral position of the foot, gait speed and the last part of the procedure consisted of a short questionnaire, providing the information about the type of housing, age, and the usage of orthotics.

Results: A low correlation between body composition and some of the foot posture index criteria was observed. The mean foot posture index score in athletes with ID was observed to be slightly pronated, as also described in the general population, with no statistically significant difference between female and male athletes. The gender differences were observed only in body composition.

Conclusions: The high body weight and body fat percentage observed in this study highlight the need for healthy lifestyle education in athletes with ID.
View Article and Find Full Text PDF

Download full-text PDF

Source
July 2020

Delineating Human B Cell Precursor Development With Genetically Identified PID Cases as a Model.

Front Immunol 2019 26;10:2680. Epub 2019 Nov 26.

Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands.

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 surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901940PMC
November 2020

Heterologous Cytomegalovirus and Allo-Reactivity by Shared T Cell Receptor Repertoire in Kidney Transplantation.

Front Immunol 2019 31;10:2549. Epub 2019 Oct 31.

Transplant Laboratory, Institute for Clinical and Experimental Medicine, Prague, Czechia.

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 peptide pools and alloantigen in 78 living donor/recipient pairs using the interferon-gamma Enzyme-Linked ImmunoSpot (ELISPOT) assay. To prove the hypothesis of cross-reactivity, we analyzed by applying next-generation sequencing the T cell receptor ß (TCR- ß) repertoire of CMV- and alloantigen-reactive T cells enriched from peripheral pre-transplant blood of 11 CMV-seropositive and HLA class I mismatched patients. Moreover, the TCR-repertoire was also analyzed in the allograft biopsies of those patients. There was a significant association between the presence of pre-transplant CMV immediate-early protein 1 (IE-1)-specific effector/memory T cells and acute renal allograft rejection and function ( = 0.01). Most importantly, we revealed shared TCR-ß sequences between CMV-IE1 and donor alloantigen-reactive T cells in all pre-transplant peripheral blood samples analyzed in CMV-seropositive patients who received HLA class I mismatched grafts. Identical TCR sequences were also found in particular in post-transplant allograft biopsies of patients with concomitant CMV infection and rejection. Our data show the presence of functional, cross-reactive T cells and their clonotypes in peripheral blood and in kidney allograft tissue. It is therefore likely that CMV-donor cross-reactivity as well as CMV specific T cell elicited inflammation is involved in the processes that affect allograft outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834532PMC
October 2020

Alu-mediated Xq24 deletion encompassing CUL4B, LAMP2, ATP1B4, TMEM255A, and ZBTB33 genes causes Danon disease in a female patient.

Am J Med Genet A 2020 01 15;182(1):219-223. Epub 2019 Nov 15.

Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, 1st Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic.

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 mutations cause Danon disease (DD). Cardiomyopathy is a dominant feature of DD present in both males and heterozygous females. No monogenic phenotypes have been associated with mutations in ATP1B4, TMEM255A, and ZBTB33 genes. To facilitate diagnostics and counseling in CS and DD families, we present a female DD patient with a de novo Alu-mediated Xq24 rearrangement causing a deletion encompassing CUL4B, LAMP2, and also the other three neighboring genes. Typical to females heterozygous for CUL4B mutations, the patient was CS asymptomatic, however, presented with extremely skewed X-chromosome inactivation (XCI) ratios in peripheral white blood cells. As a result of the likely selection against CUL4B deficient clones, only minimal populations (~3%) of LAMP2 deficient leukocytes were identified by flow cytometry. On the contrary, myocardial LAMP2 protein expression suggested random XCI. We demonstrate that contiguous CUL4B and LAMP2 loss-of-function copy number variations occur and speculate that male patients carrying similar defects could present with features of both CS and DD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ajmg.a.61416DOI Listing
January 2020

CD Maps-Dynamic Profiling of CD1-CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets.

Front Immunol 2019 23;10:2434. Epub 2019 Oct 23.

Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, Netherlands.

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, mapping the expression of CD1-CD100 ( = 110) on 47 immune cell subsets from blood, thymus, and tonsil using an eight-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABCs) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte, and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6820661PMC
October 2020

Comments on EuroFlow standard operating procedures for instrument setup and compensation for BD FACS Canto II, Navios and BD FACS Lyric instruments.

J Immunol Methods 2019 12 23;475:112680. Epub 2019 Oct 23.

CLIP, Department of Paediatric Hematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic. Electronic address:

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 an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jim.2019.112680DOI Listing
December 2019

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

Authors:
Andrea Cossarizza Hyun-Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam-Klages William W Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T Baldari Sudipto Bari Vincenzo Barnaba Joana Barros-Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A Boardman Christian Bogdan Jessica G Borger Giovanna Borsellino Philip E Boulais Jolene A Bradford Dirk Brenner Ryan R Brinkman Anna E S Brooks Dirk H Busch Martin Büscher Timothy P Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L Cardell Stefano Casola Marco A Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K Chattopadhyay Sue Chow Eleni Christakou Luka Čičin-Šain Mario Clerici Federico S Colombo Laura Cook Anne Cooke Andrea M Cooper Alexandra J Corbett Antonio Cosma Lorenzo Cosmi Pierre G Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang-Heine Martin S Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J Dress Diana Dudziak Michael Dustin Charles-Antoine Dutertre Friederike Ebner Sidonia B G Eckle Matthias Edinger Pascale Eede Götz R A Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S Falk Todd A Fehniger Mar Felipo-Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A Foulds Britta Frehse Paul S Frenette Stefan Frischbutter Wolfgang Fritzsche David W Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I Godfrey Christoph Goettlinger Jose M González-Navajas Carl S Goodyear Andrea Gori Jane L Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M Harpur Susanne Hartmann Andrea Hauser Anja E Hauser David L Haviland David Hedley Daniela C Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P Houston Bimba F Hoyer Bo Huang Fang-Ping Huang Johanna E Huber Jochen Huehn Michael Hundemer Christopher A Hunter William Y K Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans-Martin Jäck Peter K Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H E Kaufmann Baerbel Keller Steven L C Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui-Fern Koay Katja Kobow Jay K Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze-Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut-Landmann Michael D Leipold Leslie Y T Leung Megan K Levings Andreia C Lino Francesco Liotta Virginia Litwin Yanling Liu Hans-Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López-Botet Amy E Lovett-Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H Mair Alberto Mantovani Rudolf A Manz Aaron J Marshall Alicia Martínez-Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M McGuire Iain B McInnes Henrik E Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D Miller Kingston H G Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José-Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski-Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F Pickl Silvia Piconese Marcello Pinti A Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E M Pucillo Sally A Quataert Linda Quatrini Kylie M Quinn Helena Radbruch Tim R D J Radstake Susann Rahmig Hans-Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B M Remmerswaal Lisa Richter Laura G Rico Andy Riddell Aja M Rieger J Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala-de-Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes-Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U Scherer Matthias Schiemann Frank A Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R Schulz Cristiano Scottá Daniel Scott-Algara David P Sester T Vincent Shankey Bruno Silva-Santos Anna Katharina Simon Katarzyna M Sitnik Silvano Sozzani Daniel E Speiser Josef Spidlen Anders Stahlberg Alan M Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I M Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A W van Lier Marc Veldhoen Salvador Vento-Asturias Paulo Vieira David Voehringer Hans-Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V Walker Paul K Wallace Sa A Wang Xin M Wang Michael D Ward Kirsten A Ward-Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B Wing Rieke Winkelmann Thomas H Winkler Oliver F Wirz Alicia Wong Peter Wurst Jennie H M Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky

Eur J Immunol 2019 Oct;49(10):1457-1973

Max Planck Institute for Infection Biology, Berlin, Germany.

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 types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.201970107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350392PMC
October 2019

Novel SAMD9 Mutation in a Patient With Immunodeficiency, Neutropenia, Impaired Anti-CMV Response, and Severe Gastrointestinal Involvement.

Front Immunol 2019 18;10:2194. Epub 2019 Sep 18.

Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czechia.

Mutations in the Sterile alpha motif domain containing 9 () 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 unclear. Here, we present a patient with a novel mutation in (c.2471 G>A, p.R824Q), manifesting with prominent gastrointestinal tract involvement and immunodeficiency, but without any sign of adrenal insufficiency typical for MIRAGE syndrome. He suffered from severe CMV (cytomegalovirus) infection at 3 months of age, with a delayed development of T lymphocyte functional response against CMV, profound T cell activation, significantly reduced B lymphocyte counts and impaired lymphocyte proliferative response. Cultured T cells displayed slightly lower calcium flux and decreased survival. At the age of 6 months, he developed severe neutropenia requiring G-CSF administration, and despite only mild morphological and immunophenotypical disturbances in the BM, 78% of the BM cells showed monosomy 7 at the age of 18 months. Surprisingly, T cell proliferation after CD3 stimulation and apoptosis of the cells normalized during the follow-up, possibly reflecting the gradual development of monosomy 7. Among other prominent symptoms, he had difficulty swallowing, requiring percutaneous endoscopic gastrostomy (PEG), frequent gastrointestinal infections, and perianal erosions. He suffered from repeated infections and periodic recurring fevers with the elevation of inflammatory markers. At 26 months of age, he underwent HSCT that significantly improved hematological and immunological laboratory parameters. Nevertheless, he continued to suffer from other conditions, and subsequently, he died at day 440 post-transplant due to sepsis. Pathogenicity of this novel mutation was confirmed experimentally. Expression of mutant caused a significant decrease in proliferation and increase in cell death of the transfected cells. We describe a novel mutation in a patient with prominent gastrointestinal and immunological symptoms but without adrenal hypoplasia. Thus, SAMD9 mutations should be considered as cause of enteropathy in pediatric patients. The insufficient therapeutic outcome of transplantation further questions the role of HSCT in the management of patients with mutations and multisystem involvement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759462PMC
November 2020

Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges.

Authors:
Tomas Kalina

Cytometry A 2020 02 8;97(2):137-147. Epub 2019 Oct 8.

CLIP-Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Medical School, Charles University and University Hospital Motol, Prague, Czech Republic.

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 on a complete set of methods for sample preparation. The term "standardization" is used to describe the complete set of methodical steps, while "harmonization" is used for partial agreement on the method. Standardization can provide a platform for improved reproducibility of cytometry results over prolonged periods of time, across different sites and across different instruments. For the purpose of structured discussion, several desired aims are described: common interpretation of the immunophenotype definition of a target subset, accurate quantification, reproducible pattern of a multicolor immunophenotype, and reproducible intensity of all measured parameters. An overview of how standardization was approached by several large consortia is provided: EuroFlow, The ONE Study, Human Immunology Project Consortium (HIPC), and several other groups. Their particular aims and the tools adopted to reach those aims are noted. How those standardization efforts were adopted in the field and how the resulting outcome was evaluated is reviewed. Multiple challenges in the instrument hardware design, instrument setup tools, reagent design, and quality features need to be addressed to achieve optimal standardization. Furthermore, the aims of different studies vary, and thus, the reasonable requirements for standardization differ. A framework of reference for the reasonable outcomes of different approaches is offered. Finally, it is argued that complete standardization is important not only for the reproducibility of measurements but also for education, for quality assessment and for algorithmic data analysis. The different standardized approaches can and in fact should serve as benchmarking reference tools for the development of future flow cytometry studies. © 2019 International Society for Advancement of Cytometry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.23901DOI Listing
February 2020

Relevance of Antibody Validation for Flow Cytometry.

Cytometry A 2020 02 2;97(2):126-136. Epub 2019 Oct 2.

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain.

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 with the proper performance, how to validate antibody reagents for flow cytometry, and how to interpret the validation results. The achievements of cytometry moved the field to a higher number of measured parameters, large data sets, and computational data analysis approaches. These advancements pose an increased demand for antibody reagent performance quality. This review summarizes the codevelopment of cytometry, antibody development, and validation strategies. It discusses the diverse issues of the specificity, cross-reactivity, epitope, titration, and reproducibility features of antibody reagents, and this review discusses the validation principles and methods that are currently available and those that are emerging. We argue that significant efforts should be invested by antibody users, developers, manufacturers, and publishers to increase the quality and reproducibility of published studies. More validation data should be presented by all stakeholders; however, the data should be presented in sufficient experimental detail to foster reproducibility, and community effort shall lead to the public availability of large data sets that can serve as a benchmark for antibody performance. © 2019 International Society for Advancement of Cytometry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.23895DOI Listing
February 2020

EuroFlow-Based Flowcytometric Diagnostic Screening and Classification of Primary Immunodeficiencies of the Lymphoid System.

Front Immunol 2019 13;10:1271. Epub 2019 Jun 13.

Department of Medicine, Cancer Research Centre (IBMCC, USAL-CSIC), Cytometry Service (NUCLEUS), University of Salamanca (USAL), Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.

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 innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.01271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585843PMC
October 2020

Fluorochrome choices for multi-color flow cytometry.

J Immunol Methods 2019 12 7;475:112618. Epub 2019 Jun 7.

Cancer Research Center (IBMCC-CSIC/USAL-IBSAL), Cytometry Service (NUCLEUS) and Department of Medicine, University of Salamanca, Salamanca, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC) CB16/12/00400, Instituto de Salud Carlos III, Madrid, Spain.. Electronic address:

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 fluorescence profiles (excitation and emission), ii) relative brightness, iii) fluorescence overlap, iv) fluorochrome stability, and v) reproducible conjugation to antibodies. Here we used 75 samples (45 bone marrow and 30 blood) to illustrate EuroFlow strategies for evaluation of compatible fluorochromes, and how the results obtained guide fluorochrome selection as a critical step in the antibody-panel building process. Our results allowed identification of optimal fluorescence profiles (e.g. higher fluorescence intensity and/or resolution with limited fluorescence overlap into neighbor channels) for brilliant violet (BV)421 and BV510 in the violet laser and allophycocyanin (APC) hilite 7 (H7) or APC C750 in the red laser vs. other candidate fluorochromes generally applied for the same detectors and here evaluated. Moreover, evaluation of the same characteristics for another group of fluorochromes (e.g. BV605, BV650, PE CF594, AF700 or APC AF700) guided selection of the most appropriate fluorochrome conjugates to be combined in a multi-color antibody panel. Albeit this is a demanding approach, it could be successfully applied for selection of fluorochrome combinations for the EuroFlow antibody panels for diagnosis, classification and monitoring of hematological malignancies and primary immunodeficiencies. Consequently, sets of 8-, 10- and 12-color fluorochrome combinations are proposed as frame of reference for initial antibody panel design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jim.2019.06.009DOI Listing
December 2019

The coast of giants: an anthropometric survey of high schoolers on the Adriatic coast of Croatia.

PeerJ 2019 17;7:e6598. Epub 2019 Apr 17.

Faculty of Sports Studies, Masaryk University, Brno, Czech Republic.

The aim of this anthropometric survey was to map regional differences in height and body proportions in eight counties adjacent to the Adriatic coast of Croatia. Body height was measured in 1,803 males and 782 females aged 17-20 years at 66 schools in 23 towns. When corrected for population size in regions, mean male height is 182.6 cm in all eight counties, 182.8 cm in seven counties of Adriatic Croatia, and 183.7 cm in four counties of Dalmatia proper. Regional variation is considerable: from 180.6 cm in the county of Karlovac to 184.1 cm in the county of Split-Dalmacija. The mean height of females is based on more limited data (168.0 cm in seven counties). These results show that young men from Dalmatia are currently the tallest in the world in the age category of 18 years, and the north-to-south gradient of increasing stature on the Adriatic coast largely mirrors that in neighbouring Bosnia and Herzegovina (BiH). The extraordinary values of height in Croatia and BiH can most likely be explained by unique genetic predispositions that are shared by the local populations of the Dinaric Alps.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7717/peerj.6598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475134PMC
April 2019

Implementation of Mass Cytometry for Immunoprofiling of Patients with Solid Tumors.

J Immunol Res 2019 11;2019:6705949. Epub 2019 Feb 11.

Department of Genetics and Microbiology, Faculty of Science, Charles University, BIOCEV, Průmyslová 595, 25250 Vestec, Czech Republic.

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 method is necessary for obtaining reliable results. Also, with respect to procedure length and costs, sample preparation, staining, and storage should be optimized. Here, we designed a panel of 31 antibodies which allows for identification of several subpopulations of lymphoid and myeloid cells in a solid tumor and peripheral blood simultaneously. For sample preparation, disaggregation of tumor tissue with two different collagenases combined with DNase I was compared, and removal of dead or tumor cells by magnetic separation was evaluated. Two possible procedures of barcoding for single-tube staining of several samples were examined. While the palladium-based barcoding affected the stability of several antigens, the staining with two differently labeled CD45 antibodies was suitable for cells isolated from a patient's blood and tumor. The storage of samples in the intercalation solution for up to two weeks did not influence results of the analysis, which allowed the measurement of samples collected within this interval on the same day. This procedure optimized on samples from patients with head and neck squamous cell carcinoma enabled identification of various immune cells including rare subpopulations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2019/6705949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388349PMC
June 2019

The EuroFlow PID Orientation Tube for Flow Cytometric Diagnostic Screening of Primary Immunodeficiencies of the Lymphoid System.

Front Immunol 2019 4;10:246. Epub 2019 Mar 4.

Department of Immunology, Erasmus MC, Rotterdam, Netherlands.

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 all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID (without a need for specificity to diagnose particular PID) and to guide and prioritize further diagnostic modalities and clinical management. The tube composition has been defined in a stepwise manner through several cycles of design-testing-evaluation-redesign in a multicenter setting. Equally important appeared to be the standardized pre-analytical procedures (sample preparation and instrument setup), analytical procedures (immunostaining and data acquisition), the software analysis (a multidimensional view based on a reference database in Infinicyt software), and data interpretation. This standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID patients with defined genetic defects. In addition, an application of new EuroFlow software tools with multidimensional pattern recognition was designed with inclusion of maturation pathways in multidimensional patterns (APS plots). The major advantage of the EuroFlow approach is that data can be fully exchanged between different laboratories in any country of the world, which is especially of interest for the PID field, with generally low numbers of cases per center.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.00246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410673PMC
May 2020

Cytometric analysis of cell suspension generated by cavitron ultrasonic surgical aspirator in pediatric brain tumors.

J Neurooncol 2019 May 2;143(1):15-25. Epub 2019 Mar 2.

CLIP - Childhood Leukaemia Investigation Prague, Prague, Czech Republic.

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 analyze the composition of material collected using the CUSA. Cell suspensions were prepared from CUSA aspirates. Then sample viability was assessed by conventional flow cytometry and subsequently stained with a panel of 31 metal-labeled antibodies.

Results: Viability assessment was performed using conventional flow cytometry. Viability of cells in the acquired samples was below 50% in 16 of 19 cases. A mass cytometry investigation and subsequent analysis enabled us to discriminate brain tumor cells from contaminating leukocytes, whose proportions varied across the specimens. The addition of the viability marker cisplatin directly into the mass cytometry panel gave the means to selecting viable cells only for subsequent analyses. The proportion of non-viable cells was higher among tumor cells compared leukocytes.

Conclusions: When the analysis of the tumor cell immunophenotype is performed with markers for determining viability, the expression of the investigated markers can be evaluated. Suitable markers can be selected by high-throughput methods, such as mass cytometry, and those that are diagnostically relevant can be investigated using flow cytometry, which is more flexible in terms of time.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11060-019-03135-wDOI Listing
May 2019

Defects in memory B-cell and plasma cell subsets expressing different immunoglobulin-subclasses in patients with CVID and immunoglobulin subclass deficiencies.

J Allergy Clin Immunol 2019 09 28;144(3):809-824. Epub 2019 Feb 28.

Department of Medicine, Cancer Research Centre (IBMCC, USAL-CSIC), Cytometry Service (NUCLEUS), University of Salamanca (USAL), Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain; Biomedical Research Networking Centre Consortium of Oncology (CIBERONC), number CB16/12/00400, Instituto de Salud Carlos III, Madrid, Spain. Electronic address:

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 of distinct immunoglobulin heavy chain subclasses.

Methods: Blood samples from 139 patients with PADs, 61 patients with common variable immunodeficiency (CVID), 68 patients with selective IgA deficiency (IgAdef), 10 patients with IgG subclass deficiency with IgA deficiency, and 223 age-matched control subjects were studied by using flow cytometry with EuroFlow immunoglobulin isotype staining. Patients were classified according to their B-cell and PC immune profile, and the obtained patient clusters were correlated with clinical manifestations of PADs.

Results: Decreased counts of blood PCs, memory B cells (MBCs), or both expressing distinct IgA and IgG subclasses were identified in all patients with PADs. In patients with IgAdef, B-cell defects were mainly restricted to surface membrane (sm)IgA PCs and MBCs, with 2 clear subgroups showing strongly decreased numbers of smIgA PCs with mild versus severe smIgA MBC defects and higher frequencies of nonrespiratory tract infections, autoimmunity, and affected family members. Patients with IgG subclass deficiency with IgA deficiency and those with CVID showed defects in both smIgA and smIgG MBCs and PCs. Reduced numbers of switched PCs were systematically found in patients with CVID (absent in 98%), with 6 different defective MBC (and clinical) profiles: (1) profound decrease in MBC numbers; (2) defective CD27 MBCs with almost normal IgG MBCs; (3) absence of switched MBCs; and (4) presence of both unswitched and switched MBCs without and; (5) with IgG MBCs; and (6) with IgA MBCs.

Conclusion: Distinct PAD defective B-cell patterns were identified that are associated with unique clinical profiles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2019.02.017DOI Listing
September 2019

CVID-Associated Tumors: Czech Nationwide Study Focused on Epidemiology, Immunology, and Genetic Background in a Cohort of Patients With CVID.

Front Immunol 2018 22;9:3135. Epub 2019 Jan 22.

Department of Immunology, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czechia.

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 background, is suggested to contribute to tumor development. Here, we present the results of the first Czech nationwide study focused on epidemiology, immunology and genetic background in a cohort of CVID patients who also developed tumors The cohort consisted of 295 CVID patients followed for 3,070 patient/years. Standardized incidence ratio (SIR) was calculated to determine the risk of cancer, and Risk ratio (RR) was established to evaluate the significance of comorbidities. Moreover, immunophenotyping, including immunoglobulin levels and lymphocyte populations, was assessed. Finally, Whole exome sequencing (WES) was performed in all patients with lymphoma to investigate the genetic background. Twenty-five malignancies were diagnosed in 22 patients in a cohort of 295 CVID patients. SIR was more than 6 times greater in comparison to the general population. The most common neoplasias were gastric cancers and lymphomas. History of Immune thrombocytopenic purpura (ITP) was established as a potential risk factor, with over 3 times higher risk of cancer development. The B cell count at diagnosis of lymphoma was reduced in the lymphoma group; moreover, post-treatment B and T cell lymphopenia, associated with poorer outcome, was found in a majority of the patients. Intriguingly, no NK cell depression was observed after the chemotherapy. WES revealed heterogeneous genetic background among CVID patients with tumors, identifying gene variants associated with primary immunodeficiencies (such as CTLA4, PIK3CD, PMS2) and/or increased cancer susceptibility (including BRCA1, RABEP1, EP300, KDM5A). The incidence of malignancy in our CVID cohort was found to be more than 6 times greater compared to the general population. Gastric cancers and lymphomas were the most frequently diagnosed tumors. ITP was identified as a risk factor for malignancy in CVID patients. WES analysis confirmed a wide genetic heterogeneity among CVID patients. The identified causative or modifying gene variants pointed to errors in mechanisms contributing to both immunodeficiency and malignancy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.03135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349737PMC
October 2019

A high-throughput pipeline for validation of antibodies.

Nat Methods 2018 11 30;15(11):909-912. Epub 2018 Oct 30.

Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway.

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 followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-018-0179-8DOI Listing
November 2018