This thesis describes the research that was performed to unravel the function and development of human lymphoid tissue-resident (lt)NK cells in relation to the circulating CD56bright and CD56dim NK... Show moreThis thesis describes the research that was performed to unravel the function and development of human lymphoid tissue-resident (lt)NK cells in relation to the circulating CD56bright and CD56dim NK cells. Two methods, RNA sequencing and flow cytometry, both commonly used in the field of immunology, were employed at the bulk and single-cell level. We highlighted the complexity of single-cell analysis that could potentially lead to misinterpretation, but also the additional value over bulk approaches by uncovering the cellular heterogeneity and developmental trajectories. Finally, our workflow for single-cell analysis was applied on a patient more than 50 years after hematopoietic stem cell transplantation to perform combined analysis of the T cell phenotype and T cell receptor repertoire. Show less
Fosse, N.A. du; Bronsgeest, K.; Arbous, M.S.; Zlei, M.; Myeni, S.K.; Kikkert, M.; ... ; Akker, T. van den 2021
A primigravid woman with Covid-19 related respiratory insufficiency was admitted into a tertiary Intensive Care Unit at 23 3/7 weeks' gestation. Highly sensitive flow cytometry of peripheral... Show moreA primigravid woman with Covid-19 related respiratory insufficiency was admitted into a tertiary Intensive Care Unit at 23 3/7 weeks' gestation. Highly sensitive flow cytometry of peripheral leukocytes indicated significantly suppressed naive T- and B-cell compartments. The suppressed immune cell responses led us keep the initially started administration of corticosteroids for fetal and maternal indication at a low dose. After three weeks her B-cell response peaked, SARS-CoV-2 was cleared and clinical improvement ensued a week later. At 28 weeks' gestation, a son of 1570 g was born by cesarean section. She was extubated two days postpartum and discharged from hospital 5.5 weeks postpartum. Show less
The two branches of our highly advanced immune system work closely together to detect and eliminate pathological threats. The first line of defense is provided by the innate immune system via... Show moreThe two branches of our highly advanced immune system work closely together to detect and eliminate pathological threats. The first line of defense is provided by the innate immune system via detection of pathogenic or tumor cell fragments. Adaptive immune cells, on the other hand, recognize pathogens or malignant cells more specifically by scanning peptides, small protein fragments, presented by MHC molecules on other cells in our body. Specialized white blood cells (cytotoxic or killer T cells) can distinguish self- from non-self-peptides and directly eliminate cells that display signs of infection or mutation.The work described in this dissertation highlights how adaptive immunity can be used to our advantage, either from a therapeutic or diagnostic perspective. Immunotherapies that induce or promote anti-tumor or anti-viral responses have proven efficacious against infection and cancer. One strategy described is the development of chemically-modified epitopes as peptide vaccines, but small-molecule chemical drugs are also playing an increasing role in the field of cancer immunotherapy.In addition, monitoring of immune status and response to treatment, as well as mapping of epitopes, can aid diagnosis and design of treatment plans. The second part describes a novel method and application to visualize and monitor cytotoxic T cells. Show less
Sedek, L.; Theunissen, P.; Costa, E.S. da; Sluijs-Gelling, A. van der; Mejstrikova, E.; Gaipa, G.; ... ; EuroFlow Consortium 2019
Background: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of... Show moreBackground: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of markers differentially expressed on normal BCP and leukemic blasts is required.Methods: Multicenter analysis of CD73, CD86 and CD304 expression levels was performed in 282 pediatric BCP-ALL patients vs. normal bone marrow BCP, using normalized median fluorescence intensity (nMFI) values.Results: CD73 was expressed at abnormally higher levels (vs. pooled normal BCP) at diagnosis in 71/108 BCPALL patients (66%), whereas CD304 and CD86 in 119/202 (59%) and 58/100 (58%) patients, respectively. Expression of CD304 was detected at similar percentages in common-ALL and pre-B-ALL, while found at significantly lower frequencies in pro-B-ALL. A significant association (p = 0.009) was found between CD304 expression and the presence of the ETV6-RUNX1 fusion gene. In contrast, CD304 showed an inverse association with MLL gene rearrangements (p = 0.01). The expression levels of CD73, CD86 and CD304 at day 15 after starting therapy (MRD15) were stable or higher than at diagnosis in 35/37 (95%), 40/56 (71%) and 19/41 (46%) cases investigated, respectively. This was also associated with an increased mean nMFI at MRD15 vs. diagnosis of + 24 and +3 nMFI units for CD73 and CD86, respectively. In addition, gain of expression of CD73 and CD86 at MRD15 for cases that were originally negative for these markers at diagnosis was observed in 16% and 18% of cases, respectively. Of note, CD304 remained aberrantly positive in 63% of patients, despite its levels of expression decreased at follow-up in 54% of cases.Conclusions: Here we show that CD73, CD86 and CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients and that their expression profile remains relatively stable early after starting therapy, supporting their potential contribution to improved MRD analysis by flow cytometry. Show less
Novakova, M.; Glier, H.; Brdickova, N.; Vlkova, M.; Santos, A.H.; Lima, M.; ... ; Kalina, T. 2019
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... Show moreA 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 instruments from 9 manufacturers capable of 3-laser excitation and >= 8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of >= 8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles. Show less
Flow cytometry immunophenotyping is essential for diagnosis, classification and monitoring of clonal hematopoietic diseases, particularly of hematological malignancies and primary... Show moreFlow cytometry immunophenotyping is essential for diagnosis, classification and monitoring of clonal hematopoietic diseases, particularly of hematological malignancies and primary immunodeficiencies. Optimal use of immunophenotyping for these purposes requires detailed knowledge about the phenotypic patterns of normal hematopoietic cells.In the past few decades, flow cytometry has benefited from technological developments allowing simultaneous analysis of multiple antigen stainings with >= 3-35 distinct fluorochrome-conjugated antibodies for increasingly higher numbers of cells. These advances have contributed to expand our knowledge about the phenotypic differentiation profiles of normal hematopoietic cells, from uncommitted CD34(+) precursors in the bone marrow (BM) and peripheral blood (PB), to the several hundreds of populations of circulating myeloid and (B and T) lymphoid cells identified so far. Detailed dissection of the normal phenotypic profiles of hematopoietic cells has settled the basis for identification of aberrant phenotypes on leukemia and lymphoma cells. Thus, it has contributed to: i) more sensitive identification of leukemia/lymphoma cells (especially when represented at low frequencies in a sample), and ii) more accurate classification of hematological malignancies. In this manuscript, we review the major phenotypic features of hematopoietic cells, from the more immature BM CD34(+) precursors committed to the myeloid and lymphoid lineages toward mature hematopoietic cells circulating in PB (e.g. neutrophils, monocytes, basophils, eosinophils, dendritic cells, erythroid cells, and B- and T-cells) and those homing to other tissues (e.g. plasma cells, mast cells). Show less
Kalina, T.; Brdickova, N.; Glier, H.; Fernandez, P.; Bitter, M.; Flores-Montero, J.; ... ; Orfao, A. 2019
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... Show moreEuroFlow 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 participation in the EuroFlow QA is to evaluate whether the technical quality of the data generated by the laboratory is comparable to the data of the EuroFlow members and thus if a non-EuroFlow member participant can use the EuroFlow reference sample database for his own patient evaluation. Also it assesses whether data are sufficiently standardized for automated population gating and alarm notification. By spring 2018, a total 87 laboratories from 32 countries on five continents have registered for the EuroFlow QA program. We evaluated 163 results of 2015-2016 QA rounds, where we noted clear improvement in the score of first-time participants (median score of 91% correct) when they participated second time or later (median score of 94% correct, p = 0,017), which was comparable to EuroFlow member scores (median score of 97% correct). Among frequent mistakes, we found non-adherence to the EuroFlow protocols (improper reagent used), improper gating and some compensation issues. In summary, we show that EuroFlow QA has a positive impact on improvement of standardized data quality of non-member laboratories adhering to the EuroFlow standard operating procedures and reagent panels. Show less
Bottcher, S.; Velden, V.H.J. van der; Villamor, N.; Ritgen, M.; Flores-Montero, J.; Escobar, H.M.; ... ; Orfao, A. 2019
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... Show moreThe 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 for mouse immunoglobulin light chains was utilized to compare the mean fluorescence intensity of 1323 consecutive antibody lots to the currently used lot of the same monoclonal antibody. In total, 157 different monoclonal antibodies were assessed over seven years. Median relative difference between consecutive lots was 3.8% (range: 0.01% to 164.7%, interquartile range: 1.3% to 10.1%). The relative difference exceeded 20% in 8.8% of all comparisons. F1TC labelled monoclonal antibodies (median relative difference: 2.1%) showed a significantly smaller variation between lots than antibodies conjugated to PE (3.5%), PECy7 (3.9%), PerCPCy5.5 (5.8%), APC (5.8%),APCH7 ( 7.4%), and APCC750 (14.5%). Reagents labelled with Pacific Blue (1.4%), Pacific Orange (2.4%), HV450 (0.7%), and HV500 (1.7%) demonstrated more consistent results compared to conjugates of BV421 (4.1%) and BV510 (16.2%). Additionally, significant differences in lot-to-lot fluorescence stability amongst antibodies labelled with the same fluorochrome were observed between manufacturers.These observations might guide future quality recommendations for the production and application of fluorescence-labelled monoclonal antibodies in multicolor flow cytometry. (C) 2017 Published by Elsevier B.V. Show less
Diks, A.M.; Bonroy, C.; Teodosio, C.; Groenland, R.J.; Mooij, B. de; Maertelaere, E. de; ... ; Berkowska, M.A. 2019
Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in... Show moreObtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (approximate to transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies.EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 degrees C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for < 24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes.Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population. Show less
Velden, V.H.J. van der; Flores-Montero, J.; Perez-Andres, M.; Martin-Ayuso, M.; Crespo, O.; Blanco, E.; ... ; Orfao, A. 2019
Within EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and... Show moreWithin EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and prone to operational mistakes. We therefore evaluated dried formats of the lymphocytosis screening tube (LST) and of the primary immune deficiency orientation tube (PIDOT). Both tubes were evaluated on normal and/or on patient samples, comparing the mean fluorescence intensity of specific lymphocyte populations. Our data show that the dried tubes and liquid counterparts give highly comparable staining results, particularly when analyzed in multidimensional plots. In addition, the use of dried tubes may result in a reduced staining variability between different samples and thereby contributes to the generation of more robust data. Therefore, by using ready-touse reagents in a dried single test tube format, the laboratory efficiency and quality will be improved. (C) 2017 Erasmus MC, University Medical Center Rotterdam. Published by Elsevier B.V. Show less
Glier, H.; Novakova, M.; Marvelde, J. te; Bijkerk, A.; Morf, D.; Thurner, D.; ... ; Kalina, T. 2019
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... Show moreThis 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 infra-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. Show less
Blanco, E.; Perez-Andres, M.; Sanoja-Flores, L.; Wentink, M.; Pelak, O.; Martin-Ayuso, M.; ... ; Orfao, A. 2019
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. > 20 years ago, the International Union of... Show moreThe clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. > 20 years 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 and IgA subclasses for a broad range of laboratory assays, except for flow cytometry. Here we analyzed the performance of commercially available Ab clones to detect IgG and IgA subclasses in memory B-cells and plasma cells (PCs) by flow cytometry. In a first step, 28 Ab clones were evaluated in peripheral blood from healthy donors. Only 17/28 clones showed reactivity against IgG and IgA subclasses expressed on the B-cell and PC surface membrane, including Ab clones for IgG(1) (SAG1, HP6188, HP6001 and HP6186), IgG(2) (SAG2, HP6014 and HP6002), IgG(3) (SAG3, HP6095 and HP6050), IgG(4) (SAG4), IgA(1) (SAA1, H69-11.4 and B3506B4) and IgA(2) (SAA2, 2E2, and A9604D2). In a second step, for each Ig subclass a single clone was selected according to its specificity and fluorescence intensity (resolution power), for further more detailed validation (SAG1, SAG2, SAG3, SAG4, SAA1 and SAA2). This validation process was carried out in 4 different laboratories by testing the selected Ab clones in human peripheral blood, bone marrow and tonsil samples, using different staining protocols (e.g. surface membrane and/or cytoplasmic staining). All selected Ab clones displayed strong positivity, high specificity and optimal resolution between negative and positive cells. Alternative Ab clones were also validated. Thus, our results show the feasibility of using the validated Ig subclass Ab clones in combination with other B cell-associated markers for detailed dissection of the memory B-cell and PC compartments that express distinct Ig subclasses in different human tissues. Show less
The presence of mast cells in human atherosclerotic plaques has been associated with adverse cardiovascular events. Mast cell activation, through the classical antigen sensitized-IgE binding to... Show moreThe presence of mast cells in human atherosclerotic plaques has been associated with adverse cardiovascular events. Mast cell activation, through the classical antigen sensitized-IgE binding to their characteristic Fcε-receptor, causes the release of their cytoplasmic granules. These granules are filled with neutral proteases such as tryptase, but also with histamine and pro-inflammatory mediators. Mast cells accumulate in high numbers within human atherosclerotic tissue, particularly in the shoulder region of the plaque. These findings are largely based on immunohistochemistry, which does not allow for the extensive characterization of these mast cells and of the local mast cell activation mechanisms. In this study, we thus aimed to develop a new flow-cytometry based methodology in order to analyze mast cells in human atherosclerosis. We enzymatically digested 22 human plaque samples, collected after femoral and carotid endarterectomy surgery, after which we prepared a single cell suspension for flow cytometry. We were able to identify a specific mast cell population expressing both CD117 and the FcεR, and observed that most of the intraplaque mast cells were activated based on their CD63 protein expression. Furthermore, most of the activated mast cells had IgE fragments bound on their surface, while another fraction showed IgE-independent activation. In conclusion, we are able to distinguish a clear mast cell population in human atherosclerotic plaques, and this study establishes a strong relationship between the presence of IgE and the activation of mast cells in advanced atherosclerosis. Our data pave the way for potential therapeutic intervention through targeting IgE-mediated actions in human atherosclerosis. Show less
Human blood monocytes are divided into populations based on the differential expression of CD14 and CD16 receptors: CD14 (+) CD16(classical), CD14 (+) CD16 (+) (intermediate), and CD14(-)CD16(+) ... Show moreHuman blood monocytes are divided into populations based on the differential expression of CD14 and CD16 receptors: CD14 (+) CD16(classical), CD14 (+) CD16 (+) (intermediate), and CD14(-)CD16(+) (non-classical). Given their functional differences and their role in pathogenesis of chronic obstructive pulmonary disease (COPD), monocyte profiling is of clinical interest. Here we investigated blood monocyte subsets in clinically stable COPD patients with alpha1-antitrypsin (AAT) deficiency (PiZZ, n = 7) and with normal AAT variant (PiMM, n = 7). Peripheral whole blood was collected in sodium heparin tubes and incubated with LPS (from E. coli; 1 mu g/ml) or placebo for 6 h at 37 degrees C, 5% CO2. To profile monocyte subsets we performed flow cytometry analysis based on HLA-DR and CD14/CD16 staining. HLA-DR + subsets of cells did not differ between PiZZ and PiMM COPD, and healthy controls (n = 7), used as a reference. Monocyte profiling, which express the CD14 and CD16, but not the HLA-DR (HLA-DR-) showed that intermediate monocytes subset was lowest in PiZZ group, and almost totally disappeared from blood treated with LPS. The non-classical subset was almost absent in PiZZ patients independently of LPS treatment. Recent studies demonstrate that non-classical monocytes exhibit a unique ability to protect the vascular endothelium under both homeostatic and inflammatory conditions whereas intermediate monocytes are recruited at a later stage of inflammation, and are associated with secretion of cytokines/chemokines and wound healing. Evident alterations in blood monocyte subsets together with a partial reduction of AAT levels, an important anti-inflammatory protein, can be key factors for the early manifestation of emphysema in some PiZZ AATD carriers. Show less
Slütter, B.A.; Depuydt, M.A.C.; Duijn, J. van; Bot, I.; Wezel, A.; Koppejan, H.; ... ; Kuiper, J. 2018
CD8+ T-cells are numerous in early and advanced atherosclerotic lesion, but their role in atherogenesis and plaque stability is still under debate. Recent work in murine models suggests the... Show moreCD8+ T-cells are numerous in early and advanced atherosclerotic lesion, but their role in atherogenesis and plaque stability is still under debate. Recent work in murine models suggests the existence multiple CD8+ T-cell subsets that may differently impact the progression of disease. Here we aim to characterize the CD8+ T-cell population in human atherosclerotic lesions using a combination of histology, flow cytometry and mass cytometry (Cytof).Fresh carotid or femoral endarterectomy and matching blood samples were collect from a local hospital. Part of the sample was fixed for histology and the remainder was digested to obtain single cell suspensions. Cells were labelled with fluorescently labelled antibodies for flowcytometry and isotope labelled antibody for mass cytometry. Results were analyzed with Flowjo (flowcytometry and mass cytometry) and Cytosplore (mass cytometry) software.CD8+ T-cells were detected in every lesion (n=42) analyzed and on average constituted 23% of the total leukocyte. The percentage of CD8+ T-cells inversely correlated with the percentage of macrophages in the lesion, suggesting a role for CD8+ T-cells in preventing macrophage accumulation in the lesion. Mass cytometry revealed at least 13 phenotypically distinct CD8+ T-cells in the lesions, terminally differentiated CD27-/CD28- representing the largest CD8+ section.Human atherosclerotic lesions contain various CD8+ T-cell populations that may differentially affect disease progression and lesion stability. Although the overall effect of CD8+ T-cell presence in the lesion appear to be beneficial, identifying the protective subsets and expanding them may open new avenues for treatment. Show less
Heiden, P.L.J. van der; Egmond, H.M. van; Veld, S.A.J.; Meent, M. van de; Eefting, M.; Wreede, L.C. de; ... ; Jedema, I. 2018
