Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still... Show moreExtracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact EV-based analysis and biomarker profiling, we investigated the presence and formation of EV-LPP complexes in biological samples by using label-free atomic force microscopy, cryo-electron tomography and synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high sensitivity single particle flow cytometry. Furthermore, we evaluated the impact on flow cytometric analysis in the presence of LPPs using in vitro spike-in experiments of purified tumour cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrate that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations, can influence quantitative and qualitative EV analysis. Lastly, we demonstrate that the colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down-stream EV analysis and EV biomarker profiling. Show less
Moekotte, L.; Kuiper, J.J.W.; Hiddingh, S.; Nguyen, X.T.A.; Boon, C.J.F.; Born, L.I. van den; ... ; Genderen, M.M. van 2023
PURPOSE. Eye inflammation may occur in patients with inherited retinal dystrophies (IRDs) and is seen frequently in IRDs associated with mutations in the CRB1 gene. The purpose of this study was to... Show morePURPOSE. Eye inflammation may occur in patients with inherited retinal dystrophies (IRDs) and is seen frequently in IRDs associated with mutations in the CRB1 gene. The purpose of this study was to determine the types of inflammatory cells involved in IRDs, by deep profiling the composition of peripheral blood mononuclear cells of patients with a CRB1-associated IRD. METHODS. This study included 33 patients with an IRD with confirmed CRB1 mutations and 32 healthy controls. A 43-parameter flow cytometry analysis was performed on peripheral blood mononuclear cells isolated from venous blood. FlowSOM and manual Boolean combination gating were used to identify and quantify immune cell subsets. RESULTS. Comparing patients with controls revealed a significant increase in patients in the abundance of circulating CD4+ T cells and CD8+ T cells that express sialyl Lewis X antigen. Furthermore, we detected a decrease in plasmacytoid dendritic cells and an IgA+CD24+CD38+ transitional B-cell subset in patients with an IRD. CONCLUSIONS. Patients with a CRB1-associated IRD show marked changes in blood leukocyte composition, affecting lymphocyte and dendritic cell populations. These results implicate inflammatory pathways in the disease manifestations of IRDs. Show less
Moekotte, L.; Kuiper, J.J.W.; Hiddingh, S.; Nguyen, X.T.A.; Boon, C.J.F.; Born, L.I. van den; ... ; Genderen, M.M. van 2023
PURPOSE. Eye inflammation may occur in patients with inherited retinal dystrophies (IRDs) and is seen frequently in IRDs associated with mutations in the CRB1 gene. The purpose of this study was to... Show morePURPOSE. Eye inflammation may occur in patients with inherited retinal dystrophies (IRDs) and is seen frequently in IRDs associated with mutations in the CRB1 gene. The purpose of this study was to determine the types of inflammatory cells involved in IRDs, by deep profiling the composition of peripheral blood mononuclear cells of patients with a CRB1-associated IRD.METHODS. This study included 33 patients with an IRD with confirmed CRB1 mutations and 32 healthy controls. A 43-parameter flow cytometry analysis was performed on peripheral blood mononuclear cells isolated from venous blood. FlowSOM and manual Boolean combination gating were used to identify and quantify immune cell subsets.RESULTS. Comparing patients with controls revealed a significant increase in patients in the abundance of circulating CD4+ T cells and CD8+ T cells that express sialyl Lewis X antigen. Furthermore, we detected a decrease in plasmacytoid dendritic cells and an IgA+CD24+CD38+ transitional B-cell subset in patients with an IRD. CONCLUSIONS. Patients with a CRB1-associated IRD show marked changes in blood leukocyte composition, affecting lymphocyte and dendritic cell populations. These results implicate inflammatory pathways in the disease manifestations of IRDs. Show less
Background and Aims: Coronary atherosclerosis is a chronic non-resolving inflammatory process wherein the interaction of innate immune cells and platelets plays a major role. Circulating... Show moreBackground and Aims: Coronary atherosclerosis is a chronic non-resolving inflammatory process wherein the interaction of innate immune cells and platelets plays a major role. Circulating neutrophils, in particular, adhere to the activated endothelium and migrate into the vascular wall, promoting monocyte recruitment and influencing plaque phenotype and stability at all stages of its evolution. We aimed to evaluate, by flow cytometry, if blood neutrophil number and phenotype—including their phenotypic relationships with platelets, monocytes and lymphocytes—have an association with lipid-rich necrotic core volume (LRNCV), a generic index of coronary plaque vulnerability, in a group of stable patients with chronic coronary syndrome (CCS). Methods: In 55 patients, (68.53 ± 1.07 years of age, mean ± SEM; 71% male), the total LRNCV in each subject was assessed by a quantitative analysis of all coronary plaques detected by computed tomography coronary angiography (CTCA) and was normalized to the total plaque volume. The expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1, CXCR4 and CD41a cell surface markers was quantified by flow cytometry. Adhesion molecules, cytokines and chemokines, as well as MMP9 plasma levels, were measured by ELISA. Results: On a per-patient basis, LRNCV values were positively associated, by a multiple regression analysis, with the neutrophil count (n°/µL) (p = 0.02), neutrophil/lymphocyte ratio (p = 0.007), neutrophil/platelet ratio (p = 0.01), neutrophil RFI CD11b expression (p = 0.02) and neutrophil–platelet adhesion index (p = 0.01). Significantly positive multiple regression associations of LRNCV values with phenotypic ratios between neutrophil RFI CD11b expression and several lymphocyte and monocyte surface markers were also observed. In the bivariate correlation analysis, a significantly positive association was found between RFI values of neutrophil–CD41a+ complexes and neutrophil RFI CD11b expression (p < 0.0001). Conclusions: These preliminary findings suggest that a sustained increase in circulating neutrophils, together with the up-regulation of the integrin/activation membrane neutrophil marker CD11b may contribute, through the progressive intra-plaque accumulation of necrotic/apoptotic cells exceeding the efferocytosis/anti-inflammatory capacity of infiltrating macrophages and lymphocytes, to the relative enlargement of the lipid-rich necrotic core volume of coronary plaques in stable CAD patients, thus increasing their individual risk of acute complication. Show less
Gruijs, M.; Zeelen, C.; Hellingman, T.; Smit, J.; Borm, F.J.; Kazemier, G.; ... ; Egmond, M. van 2023
Circulating tumor cells (CTCs) have been detected in many patients with different solid malignancies. It has been reported that presence of CTCs correlates with worse survival in patients with... Show moreCirculating tumor cells (CTCs) have been detected in many patients with different solid malignancies. It has been reported that presence of CTCs correlates with worse survival in patients with multiple types of cancer. Several techniques have been developed to detect CTCs in liquid biopsies. Currently, the only method for CTC detection that is approved by the Food and Drug Administration is CellSearch. Due to low abundance of CTCs in certain cancer types and in early stages of disease, its clinical application is currently limited to metastatic colorectal cancer, breast cancer and prostate cancer. Therefore, we aimed to develop a new method for the detection of CTCs using the Attune NxT-a flow cytometry-based application that was specifically developed to detect rare events in biological samples without the need for enrichment. When healthy donor blood samples were spiked with variable amounts of different EpCAM+EGFR+ tumor cell lines, recovery yield was on average 75%. The detection range was between 1000 and 10 cells per sample. Cell morphology was confirmed with the Attune CytPix. Analysis of blood samples from metastatic colorectal cancer patients, as well as lung cancer patients, demonstrated that increased EpCAM+EGFR+ events were detected in more than half of the patient samples. However, most of these cells showed no (tumor) cell-like morphology. Notably, CellSearch analysis of blood samples from a subset of colorectal cancer patients did not detect CTCs either, suggesting that these blood samples were negative for CTCs. Therefore, we anticipate that the Attune NxT is not superior to CellSearch in detection of low amounts of CTCs, although handling and analysis of samples is easier. Moreover, morphological confirmation is essential to distinguish between CTCs and false positive events. Show less
Pan, K. van der; Bruin-Versteeg, S. de; Damasceno, D.; Hernandez-Delgado, A.; Sluijs-Gelling, A.J. van der; Bossche, W.B.L. van den; ... ; EuroFlow Consortium 2022
Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease... Show moreInnate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual "expert-based") gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings. Show less
Neirinck, J.; Emmaneel, A.; Buysse, M.; Philippe, J.; Gassen, S. van; Saeys, Y.; ... ; Bonroy, C. 2022
IntroductionMultiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium... Show moreIntroductionMultiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium recently developed the PID Orientation Tube (PIDOT) as a universal screening tool to identify lymphoid-PID in suspicious patients. Although PIDOT can identify different lymphoid-PIDs with high sensitivity, clinical validation in a broad spectrum of patients with suspicion of PID is missing. In this study, we investigated the diagnostic performance of PIDOT, as part of the EuroFlow diagnostic screening algorithm for lymphoid-PID, in a daily practice at a tertiary reference center for PID. MethodsPIDOT was tested in 887 consecutive patients suspicious of PID at the Ghent University Hospital, Belgium. Patients were classified into distinct subgroups of lymphoid-PID vs. non-PID disease controls (non-PID DCs), according to the IUIS and ESID criteria. For the clinical validation of PIDOT, comprehensive characterization of the lymphoid defects was performed, together with the identification of the most discriminative cell subsets to distinguish lymphoid-PID from non-PID DCs. Next, a decision-tree algorithm was designed to guide subsequent FCM analyses. ResultsThe mean number of lymphoid defects detected by PIDOT in blood was 2.87 times higher in lymphoid-PID patients vs. non-PID DCs (p < 0.001), resulting in an overall sensitivity and specificity of 87% and 62% to detect severe combined immunodeficiency (SCID), combined immunodeficiency with associated or syndromic features (CID), immune dysregulation disorder (ID), and common variable immunodeficiency (CVID). The most discriminative populations were total memory and switched memory B cells, total T cells, TCD4+cells, and naive TCD4+cells, together with serum immunoglobulin levels. Based on these findings, a decision-tree algorithm was designed to guide further FCM analyses, which resulted in an overall sensitivity and specificity for all lymphoid-PIDs of 86% and 82%, respectively. ConclusionAltogether, our findings confirm that PIDOT is a powerful tool for the diagnostic screening of lymphoid-PID, particularly to discriminate (S)CID, ID, and CVID patients from other patients suspicious of PID. The combination of PIDOT and serum immunoglobulin levels provides an efficient guide for further immunophenotypic FCM analyses, complementary to functional and genetic assays, for accurate PID diagnostics. Show less
Neirinck, J.; Emmaneel, A.; Buysse, M.; Philippé, J.; Gassen, S. van; Saeys, Y.; ... ; Bonroy, C. 2022
IntroductionMultiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium... Show moreIntroductionMultiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium recently developed the PID Orientation Tube (PIDOT) as a universal screening tool to identify lymphoid-PID in suspicious patients. Although PIDOT can identify different lymphoid-PIDs with high sensitivity, clinical validation in a broad spectrum of patients with suspicion of PID is missing. In this study, we investigated the diagnostic performance of PIDOT, as part of the EuroFlow diagnostic screening algorithm for lymphoid-PID, in a daily practice at a tertiary reference center for PID. MethodsPIDOT was tested in 887 consecutive patients suspicious of PID at the Ghent University Hospital, Belgium. Patients were classified into distinct subgroups of lymphoid-PID vs. non-PID disease controls (non-PID DCs), according to the IUIS and ESID criteria. For the clinical validation of PIDOT, comprehensive characterization of the lymphoid defects was performed, together with the identification of the most discriminative cell subsets to distinguish lymphoid-PID from non-PID DCs. Next, a decision-tree algorithm was designed to guide subsequent FCM analyses. ResultsThe mean number of lymphoid defects detected by PIDOT in blood was 2.87 times higher in lymphoid-PID patients vs. non-PID DCs (p < 0.001), resulting in an overall sensitivity and specificity of 87% and 62% to detect severe combined immunodeficiency (SCID), combined immunodeficiency with associated or syndromic features (CID), immune dysregulation disorder (ID), and common variable immunodeficiency (CVID). The most discriminative populations were total memory and switched memory B cells, total T cells, TCD4+cells, and naive TCD4+cells, together with serum immunoglobulin levels. Based on these findings, a decision-tree algorithm was designed to guide further FCM analyses, which resulted in an overall sensitivity and specificity for all lymphoid-PIDs of 86% and 82%, respectively. ConclusionAltogether, our findings confirm that PIDOT is a powerful tool for the diagnostic screening of lymphoid-PID, particularly to discriminate (S)CID, ID, and CVID patients from other patients suspicious of PID. The combination of PIDOT and serum immunoglobulin levels provides an efficient guide for further immunophenotypic FCM analyses, complementary to functional and genetic assays, for accurate PID diagnostics. Show less
Simple Summary Flow cytometry allows detailed characterization of large numbers of cells and plays an important role in the diagnosis of acute myeloid leukemia. To facilitate analysis of... Show moreSimple Summary Flow cytometry allows detailed characterization of large numbers of cells and plays an important role in the diagnosis of acute myeloid leukemia. To facilitate analysis of flowcytometric data, reference databases of normal bone marrow samples and samples from acute myeloid leukemia patients, together with new software tools, are required. We here report on the building of a large database of acute myeloid leukemia patients (n = 1142) and 22 normal samples. We report on the quality assessment procedure used and its validation, discuss potential pitfalls, and provide possible solutions for avoiding such flaws in the construction of other databases. Our data show that obtaining and collecting reproducible flow cytometric data over time and across centers is feasible, but also that strict quality assessment remains crucial, even when standardized protocols for staining and instrument settings are being used in a multicenter setting. Flowcytometric analysis allows for detailed identification and characterization of large numbers of cells in blood, bone marrow, and other body fluids and tissue samples and therefore contributes to the diagnostics of hematological malignancies. Novel data analysis tools allow for multidimensional analysis and comparison of patient samples with reference databases of normal, reactive, and/or leukemia/lymphoma patient samples. Building such reference databases requires strict quality assessment (QA) procedures. Here, we compiled a dataset and developed a QA methodology of the EuroFlow Acute Myeloid Leukemia (AML) database, based on the eight-color EuroFlow AML panel consisting of six different antibody combinations, including four backbone markers. In total, 1142 AML cases and 42 normal bone marrow samples were included in this analysis. QA was performed on 803 AML cases using multidimensional analysis of backbone markers, as well as tube-specific markers, and data were compared using classical analysis employing median and peak expression values. Validation of the QA procedure was performed by re-analysis of >300 cases and by running an independent cohort of 339 AML cases. Initial evaluation of the final cohort confirmed specific immunophenotypic patterns in AML subgroups; the dataset therefore can reliably be used for more detailed exploration of the immunophenotypic variability of AML. Our data show the potential pitfalls and provide possible solutions for constructing large flowcytometric databases. In addition, the provided approach may facilitate the building of other databases and thereby support the development of novel tools for (semi)automated QA and subsequent data analysis. Show less
Sbrana, S.; Cecchettini, A.; Bastiani, L.; Giorgi, N. di; Mazzone, A.; Ceccherini, E.; ... ; Rocchiccioli, S. 2022
Background: Atherosclerosis is a chronic inflammatory disease. The balance between proand anti-inflammatory factors, acting on the arterial wall, promotes less or more coronary plaque macro... Show moreBackground: Atherosclerosis is a chronic inflammatory disease. The balance between proand anti-inflammatory factors, acting on the arterial wall, promotes less or more coronary plaque macro-calcification, respectively. We investigated the association between monocyte phenotypic polarization and CTCA-assessed plaque dense-calcium volume (DCV) in patients with stable coronary artery disease (CAD). Methods: In 55 patients, individual DCV component was assessed by quantitative CTCA and normalized to total plaque volume. Flow cytometry expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1 and CXCR4 was quantified. Adhesion molecules and cytokines were measured by ELISA. Results: DCV values were significantly associated, by multiple regression analysis, with the expression (RFI) of CCR5 (p = 0.04), CX3CR1 (p = 0.03), CCR2 (p = 0.02), CD163 (p = 0.005) on all monocytes, and with the phenotypic M2-like polarization ratio, RFI CCR5/CD11b (p = 0.01). A positive correlation with the increased expression of chemokines receptors CCR2, CCR5 and CX3CR1 on subsets Mont was also present. Among cytokines, the ratio between IL-10 and IL-6 was found to be strongly associated with DCV (p = 0.009). Conclusions: The association between DCV and M2-like phenotypic polarization of circulating monocytes indicates that plaque macro-calcification in stable CAD may be partly modulated by an anti-inflammatory monocyte functional state, as evidenced by cell membrane receptor patterns. Show less
Sedek, L.; Flores-Montero, J.; Sluijs, A. van der; Kulis, J.; Marvelde, J. te; Philippe, J.; ... ; Orfao, A. 2022
Simple Summary Objective: interpretation of flow cytometry may be hampered by a lack of standardized sample preparation procedures. The EuroFlow consortium conducted a series of experiments to... Show moreSimple Summary Objective: interpretation of flow cytometry may be hampered by a lack of standardized sample preparation procedures. The EuroFlow consortium conducted a series of experiments to determine the potential impact of different pre-analytical and analytical factors on the variability of results in terms of relative cell populations distribution and marker expression levels. The experiments were performed on healthy donors and patients with different hematological malignancies (e.g., acute leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome) to mimic real-world clinical settings. Overall, the results showed that sample storage conditions, anticoagulant use, and sample processing protocol might need to be tailored for sample and cell type(s), as well as to the specific markers evaluated. However, defining of well-balanced boundaries for storage time to 24 h, staining-acquisition delay to 3 h, and choosing a washing buffer of pH within the range of 7.2 to 7.8 would be a valid recommendation for most applications and circumstances described herein. Objective: interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients' samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein. Show less
Diks, A.M.; Versteegen, P.; Teodosio, C.; Groenland, R.J.; Mooij, B. de; Buisman, A.M.; ... ; Berkowska, M.A. 2022
Pertussis is a vaccine-preventable disease caused by the bacterium Bordetella pertussis. Over the past years, the incidence and mortality of pertussis increased significantly. A possible cause is... Show morePertussis is a vaccine-preventable disease caused by the bacterium Bordetella pertussis. Over the past years, the incidence and mortality of pertussis increased significantly. A possible cause is the switch from whole-cell to acellular pertussis vaccines, although other factors may also contribute. Here, we applied high-dimensional flow cytometry to investigate changes in B cells in individuals of different ages and distinct priming backgrounds upon administration of an acellular pertussis booster vaccine. Participants were divided over four age cohorts. We compared longitudinal kinetics within each cohort and between the different cohorts. Changes in the B-cell compartment were correlated to numbers of vaccine-specific B- and plasma cells and serum Ig levels. Expansion and maturation of plasma cells 7 days postvaccination was the most prominent cellular change in all age groups and was most pronounced for more mature IgG1+ plasma cells. Plasma cell responses were stronger in individuals primed with whole-cell vaccine than in individuals primed with acellular vaccine. Moreover, IgG1+ and IgA1+ plasma cell expansion correlated with FHA-, Prn-, or PT- specific serum IgG or IgA levels. Our study indicates plasma cells as a potential early cellular marker of an immune response and contributes to understanding differences in immune responses between age groups and primary vaccination backgrounds. Show less
Verbeek, M.W.C.; Buracchi, C.; Laqua, A.; Nierkens, S.; Sedek, L.; Flores-Montero, J.; ... ; Velden, V.H.J. van der 2021
The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic... Show moreThe standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct. Show less
Early diagnosis of pediatric cancer is key for adequate patient management and improved outcome. Although multiparameter flow cytometry (MFC) has proven of great utility in the diagnosis and... Show moreEarly diagnosis of pediatric cancer is key for adequate patient management and improved outcome. Although multiparameter flow cytometry (MFC) has proven of great utility in the diagnosis and classification of hematologic malignancies, its application to non-hematopoietic pediatric tumors remains limited. Here we designed and prospectively validated a new single eight-color antibody combination-solid tumor orientation tube, STOT-for diagnostic screening of pediatric cancer by MFC. A total of 476 samples (139 tumor mass, 138 bone marrow, 86 lymph node, 58 peripheral blood, and 55 other body fluid samples) from 296 patients with diagnostic suspicion of pediatric cancer were analyzed by MFC vs. conventional diagnostic procedures. STOT was designed after several design-test-evaluate-redesign cycles based on a large panel of monoclonal antibody combinations tested on 301 samples. In its final version, STOT consists of a single 8-color/12-marker antibody combination (CD99-CD8/(nu)myogenin/CD4-EpCAM/CD56/GD2/(sm)CD3-CD19/(cy)CD3-CD271/CD45). Prospective validation of STOT in 149 samples showed concordant results with the patient WHO/ICCC-3 diagnosis in 138/149 cases (92.6%). These included: 63/63 (100%) reactive/disease-free samples, 43/44 (98%) malignant and 4/4 (100%) benign non-hematopoietic tumors together with 28/38 (74%) leukemia/lymphoma cases; the only exception was Hodgkin lymphoma that required additional markers to be stained.& nbsp;In addition, STOT allowed accurate discrimination among the four most common subtypes of malignant CD45(-) CD56(++) non-hematopoietic solid tumors: 13/13 (GD2(++) (nu)myogenin(-) CD271(-/+) (nu)MyoD1(-) CD99(-) EpCAM(-)) neuroblastoma samples, 5/5 (GD2(-) (nu)myogenin(++) CD271(++) (nu)MyoD1(++) CD99(-/+) EpCAM(-)) rhabdomyosarcomas, 2/2 (GD2(-/+) (nu)myogenin(-) CD271(+) (nu)MyoD1(-) CD99(+) EpCAM(-)) Ewing sarcoma family of tumors, and 7/7 (GD2(-) (nu)myogenin(-) CD271(+) (nu)MyoD1(-) CD99(-) EpCAM(+)) Wilms tumors. In summary, here we designed and validated a new standardized antibody combination and MFC assay for diagnostic screening of pediatric solid tumors that might contribute to fast and accurate diagnostic orientation and classification of pediatric cancer in routine clinical practice. Show less
Diks, A.M.; Khatri, I.; Oosten, L.E.M.; Mooij, B. de; Groenland, R.J.; Teodosio, C.; ... ; Berkowska, M.A. 2021
Antigen-specific serum immunoglobulin (Ag-specific Ig) levels are broadly used as correlates of protection. However, in several disease and vaccination models these fail to predict immunity. In... Show moreAntigen-specific serum immunoglobulin (Ag-specific Ig) levels are broadly used as correlates of protection. However, in several disease and vaccination models these fail to predict immunity. In these models, in-depth knowledge of cellular processes associated with protective versus poor responses may bring added value. We applied high-throughput multicolor flow cytometry to track over-time changes in circulating immune cells in 10 individuals following pertussis booster vaccination (Tdap, Boostrix(R), GlaxoSmithKline). Next, we applied correlation network analysis to extensively investigate how changes in individual cell populations correlate with each other and with Ag-specific Ig levels. We further determined the most informative cell subsets and analysis time points for future studies. Expansion and maturation of total IgG1 plasma cells, which peaked at day 7 post-vaccination, was the most prominent cellular change. Although these cells preceded the increase in Ag-specific serum Ig levels, they did not correlate with the increase of Ig levels. In contrast, strong correlation was observed between Ag-specific IgGs and maximum expansion of total IgG1 and IgA1 memory B cells at days 7 to 28. Changes in circulating T cells were limited, implying the need for a more sensitive approach. Early changes in innate immune cells, i.e. expansion of neutrophils, and expansion and maturation of monocytes up to day 5, most likely reflected their responses to local damage and adjuvant. Here we show that simultaneous monitoring of multiple circulating immune subsets in blood by flow cytometry is feasible. B cells seem to be the best candidates for vaccine monitoring. Show less
The Ig superfamily protein glycoprotein A33 (GPA33) has been implicated in immune dysregulation, but little is known about its expression in the immune compartment. Here, we comprehensively... Show moreThe Ig superfamily protein glycoprotein A33 (GPA33) has been implicated in immune dysregulation, but little is known about its expression in the immune compartment. Here, we comprehensively determined GPA33 expression patterns on human blood leukocyte subsets, using mass and flow cytometry. We found that GPA33 was expressed on fractions of B, dendritic, natural killer and innate lymphoid cells. Most prominent expression was found in the CD4(+) T cell compartment. Naive and CXCR5(+) regulatory T cells were GPA33(high), and naive conventional CD4(+) T cells expressed intermediate GPA33 levels. The expression pattern of GPA33 identified functional heterogeneity within the CD4(+) central memory T cell (Tcm) population. GPA33(+) CD4(+) Tcm cells were fully undifferentiated, bona fide Tcm cells that lack immediate effector function, whereas GPA33(-) Tcm cells exhibited rapid effector functions and may represent an early stage of differentiation into effector/effector memory T cells before loss of CD62L. Expression of GPA33 in conventional CD4(+) T cells suggests a role in localization and/or preservation of an undifferentiated state. These results form a basis to study the function of GPA33 and show it to be a useful marker to discriminate between different cellular subsets, especially in the CD4(+) T cell lineage. Show less
Bras, A.E.; Osmani, Z.; Haas, V. de; Jongen-Lavrencic, M.; Marvelde, J.G. te; Zwaan, C.M.; ... ; Velden, V.H.J. van der 2020
Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a... Show moreGiven its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as >= 20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry. Show less
Linskens, E.; Diks, A.M.; Neirinck, J.; Perez-Andres, M.; Maertelaere, E. de; Berkowska, M.A.; ... ; Bonroy, C. 2020
BackgroundMultiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows... Show moreBackgroundMultiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results.MethodsFC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt (TM) software (Cytognos). For comparison, percentage differences in absolute cell counts/mu L were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples.ResultsThe AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/mu L) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I.ConclusionAltogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles. Show less
Background:FoxP3(+)follicular regulatory T cells (Tfr) have been identified as the cell population controlling T follicular helper (Tfh) cells and B cells which, are both involved in effector... Show moreBackground:FoxP3(+)follicular regulatory T cells (Tfr) have been identified as the cell population controlling T follicular helper (Tfh) cells and B cells which, are both involved in effector immune responses against transplanted tissue. Methods:To understand the biology of Tfr cells in kidney transplant patients treated with tacrolimus and mycophenolate mofetil (MMF) combination immunosuppression, we measured circulating (c)Tfh and cTfr cells in peripheral blood by flow cytometry inn= 211 kidney transplant recipients. At the time of measurement patients were 5-7 years after transplantation. Of this cohort of patients, 23.2% (49/211) had been previously treated for rejection. Median time after anti-rejection therapy was 4.9 years (range 0.4-7 years). Age and gender matched healthy individuals served as controls. Results:While the absolute numbers of cTfh cells were comparable between kidney transplant recipients and healthy controls, the numbers of cTfr cells were 46% lower in immunosuppressed recipients (p< 0.001). More importantly, in transplanted patients, the ratio of cTfr to cTfh was decreased (median; 0.10 vs. 0.06), indicating a disruption of the balance between cTfr and cTfh cells. This shifted balance was observed for both non-rejectors and rejectors. Previous pulse methylprednisolone or combined pulse methylprednisolone + intravenous immunoglobulin anti-rejection therapy led to a non-significant 30.6% (median) and 51.2% (median) drop in cTfr cells, respectively when compared to cTfr cell numbers in transplant patients who did not receive anti-rejection therapy. A history of alemtuzumab therapy did lead to a significant decrease in cTfr cells of 85.8% (median) compared with patients not treated with anti-rejection therapy (p< 0.0001). No association with tacrolimus or MMF pre-dose concentrations was found. Conclusion:This cross-sectional study reveals that anti-rejection therapy with alemtuzumab significantly lowers the number of cTfr cells in kidney transplant recipients. The observed profound effects by these agents might dysregulate cTfr functions. Show less
Kalina, T.; Abraham, R.S.; Rizzi, M.; Burg, M. van der 2020
