OBJECTIVES: The development of External Quality Assessment Schemes (EQAS) for clinical flow cytometry (FCM) is challenging in the context of rare (immunological) diseases. Here, we introduce a novel EQAS monitoring the primary immunodeficiency Orientation Tube (PIDOT), developed by EuroFlow, in both a 'wet' and 'dry' format. This EQAS provides feedback on the quality of individual laboratories (i.e., accuracy, reproducibility and result interpretation), while eliminating the need for sample distribution. METHODS: In the wet format, marker staining intensities (MedFIs) within landmark cell populations in PIDOT analysis performed on locally collected healthy control (HC) samples, were compared to EQAS targets. In the dry format, participants analyzed centrally distributed PIDOT flow cytometry data (n=10). RESULTS: We report the results of six EQAS rounds across 20 laboratories in 11 countries. The wet format (212 HC samples) demonstrated consistent technical performance among laboratories (median %rCV on MedFIs=34.5 %; average failure rate 17.3 %) and showed improvement upon repeated participation. The dry format demonstrated effective proficiency of participants in cell count enumeration (range %rCVs 3.1-7.1 % for the major lymphoid subsets), and in identifying lymphoid abnormalities (79.3 % alignment with reference). CONCLUSIONS: The PIDOT-EQAS allows laboratories, adhering to the standardized EuroFlow approach, to monitor interlaboratory variations without the need for sample distribution, and provides them educational support to recognize rare clinically relevant immunophenotypic patterns of primary immunodeficiencies (PID). This EQAS contributes to quality improvement of PID diagnostics and can serve as an example for future flow cytometry EQAS in the context of rare diseases.

• MeSH
• Immunophenotyping * standards   methods   MeSH
• Humans MeSH
• Flow Cytometry * standards   methods   MeSH
• Quality Control MeSH
• Immunologic Deficiency Syndromes diagnosis   immunology   MeSH
• Quality Assurance, Health Care MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Flow cytometry immunophenotyping is critical for the diagnostic classification of mature/peripheral B-cell neoplasms/B-cell chronic lymphoproliferative disorders (B-CLPD). Quantitative driven classification approaches applied to multiparameter flow cytometry immunophenotypic data can be used to extract maximum information from a multidimensional space created by individual parameters (e.g., immunophenotypic markers), for highly accurate and automated classification of individual patient (sample) data. Here, we developed and compared five diagnostic classification algorithms, based on a large set of EuroFlow multicentric flow cytometry data files from a cohort 659 B-CLPD patients. These included automatic population separators based on Principal Component Analysis (PCA), Canonical Variate Analysis (CVA), Neighbourhood Component Analysis (NCA), Support Vector Machine algorithms (SVM) and a variant of the CA(Canonical Analysis) algorithm, in which the number of SDs (Standard Deviations) varied for each of the comparisons of different pairs of diseases (CA-vSD). All five classification approaches are based on direct prospective interrogation of individual B-CLPD patients against the EuroFlow flow cytometry B-CLPD database composed of tumor B-cells of 659 individual patients stained in an identical way and classified a priori by the World Health Organization (WHO) criteria into nine diagnostic categories. Each classification approach was evaluated in parallel in terms of accuracy (% properly classified cases), precision (multiple or single diagnosis/case) and coverage (% cases with a proposed diagnosis). Overall, average rates of correct diagnosis (for the nine B-CLPD diagnostic entities) of between 58.9 % and 90.6 % were obtained with the five algorithms, with variable percentages of cases being either misclassified (4.1 %-14.0 %) or unclassifiable (0.3 %-37.0 %). Automatic population separators based on CA, SVM and PCA showed a high average level of correctness (90.6 %, 86.8 %, and 86.0 %, respectively). Nevertheless, this was at the expense of proposing a considerable number of multiple diagnoses for a significant proportion of the test cases (54.5 %, 53.5 %, and 49.6 %, respectively). The CA-vSD algorithm generated the smaller average misclassification rate (4.1 %), but with 37.0 % of cases for which no diagnosis was proposed. In contrast, the NCA algorithm left only 2.7 % of cases without an associated diagnosis but misclassified 14.0 %. Among correctly classified cases (83.3 % of total), 91.2 % had a single proposed diagnosis, 8.6 % had two possible diagnoses, and 0.2 % had three. We demonstrate that the proposed AI algorithms provide an acceptable level of accuracy for the diagnostic classification of B-CLPD patients and, in general, surpass other algorithms reported in the literature.

• MeSH
• Algorithms MeSH
• B-Lymphocytes * pathology   MeSH
• Immunophenotyping * methods   MeSH
• Middle Aged MeSH
• Humans MeSH
• Lymphoproliferative Disorders * diagnosis   classification   MeSH
• Flow Cytometry * methods   MeSH
• Aged MeSH
• Support Vector Machine MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.

• MeSH
• Child MeSH
• Adult MeSH
• Immunophenotyping methods   MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma * pathology   diagnosis   MeSH
• Child, Preschool MeSH
• Flow Cytometry * methods   MeSH
• Reproducibility of Results MeSH
• Neoplasm, Residual * pathology   diagnosis   MeSH
• Aged MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

• MeSH
• Single-Cell Analysis methods   MeSH
• Tumor Necrosis Factor Receptor Superfamily, Member 7 metabolism   immunology   MeSH
• Leukocyte Common Antigens * metabolism   immunology   MeSH
• CD8-Positive T-Lymphocytes * immunology   MeSH
• Adult MeSH
• Fetal Blood * immunology   cytology   MeSH
• Immunophenotyping methods   MeSH
• NK Cell Lectin-Like Receptor Subfamily B immunology   metabolism   MeSH
• Humans MeSH
• Flow Cytometry * methods   MeSH
• Software MeSH
• T-Lymphocyte Subsets immunology   metabolism   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH

Around half of people with severe COVID-19 requiring intensive care unit (ICU) treatment will survive, but it is unclear how the immune response to SARS-CoV-2 differs between ICU patients that recover and those that do not. We conducted whole-blood immunophenotyping of COVID-19 patients upon admission to ICU and during their treatment and uncovered marked differences in their circulating immune cell subsets. At admission, patients who later succumbed to COVID-19 had significantly lower frequencies of all memory CD8+ T cell subsets, resulting in increased CD4-to-CD8 T cell and neutrophil-to-CD8 T cell ratios. ROC and Kaplan-Meier analyses demonstrated that both CD4-to-CD8 and neutrophil-to-CD8 ratios at admission were strong predictors of in-ICU mortality. Therefore, we propose the use of the CD4-to-CD8 T cell ratio as a marker for the early identification of those individuals likely to require enhanced monitoring and/or pro-active intervention in ICU.

• MeSH
• CD4-Positive T-Lymphocytes immunology   MeSH
• CD8-Positive T-Lymphocytes immunology   MeSH
• COVID-19 immunology   MeSH
• Immunophenotyping methods   MeSH
• Intensive Care Units MeSH
• Middle Aged MeSH
• Humans MeSH
• Lymphocyte Count methods   MeSH
• CD4-CD8 Ratio methods   MeSH
• Prospective Studies MeSH
• SARS-CoV-2 immunology   MeSH
• Aged MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Observational Study MeSH

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r2 > 0.95 for all cell types in PB and r2 > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.

• MeSH
• Leukemia, Myeloid, Acute diagnosis   MeSH
• Algorithms * MeSH
• Immunophenotyping methods   MeSH
• Leukocytes pathology   MeSH
• Humans MeSH
• Flow Cytometry MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

OBJECTIVE:  Blood monocyte subsets are emerging as biomarkers of cardiovascular inflammation. However, our understanding of human monocyte heterogeneity and their immunophenotypic features under healthy and inflammatory conditions is still evolving. RATIONALE:  In this study, we sought to investigate the immunophenome of circulating human monocyte subsets. METHODS:  Multiplexed, high-throughput flow cytometry screening arrays and computational data analysis were used to analyze the expression and hierarchical relationships of 242 specific surface markers on circulating classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes in healthy adults. RESULTS:  Using generalized linear models and hierarchical cluster analysis, we selected and clustered epitopes that most reliably differentiate between monocyte subsets. We validated existing transcriptional profiling data and revealed potential new surface markers that uniquely define the classical (e.g., BLTR1, CD35, CD38, CD49e, CD89, CD96), intermediate (e.g., CD39, CD275, CD305, CDw328), and nonclassical (e.g., CD29, CD132) subsets. In addition, our analysis revealed phenotypic cell clusters, identified by dendritic markers CMRF-44 and CMRF-56, independent of the traditional monocyte classification. CONCLUSION:  These results reveal an advancement of the clinically applicable multiplexed screening arrays that may facilitate monocyte subset characterization and cytometry-based biomarker selection in various inflammatory disorders.

• MeSH
• Atherosclerosis diagnosis   immunology   MeSH
• Biodiversity MeSH
• Biomarkers metabolism   MeSH
• Phenotype MeSH
• Immunophenotyping methods   MeSH
• Blood Circulation MeSH
• Humans MeSH
• Lipopolysaccharide Receptors metabolism   MeSH
• Monocytes physiology   MeSH
• Flow Cytometry MeSH
• Receptors, IgG metabolism   MeSH
• High-Throughput Screening Assays MeSH
• Cell Separation MeSH
• Cluster Analysis MeSH
• Inflammation diagnosis   immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. METHODS: We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). RESULTS: We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. CONCLUSIONS: Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

• MeSH
• CD59 Antigens metabolism   MeSH
• Cell Differentiation MeSH
• Antigens, CD blood   physiology   MeSH
• Diagnosis, Differential MeSH
• Erythrocytes metabolism   pathology   MeSH
• Glycophorins metabolism   MeSH
• Immunophenotyping instrumentation   methods   standards   MeSH
• Cohort Studies MeSH
• Leukocytes pathology   MeSH
• Humans MeSH
• Monocytes metabolism   pathology   MeSH
• Neutrophils metabolism   pathology   MeSH
• Hemoglobinuria, Paroxysmal blood   classification   diagnosis   pathology   MeSH
• Leukocyte Count instrumentation   methods   MeSH
• Flow Cytometry instrumentation   methods   standards   MeSH
• Receptors, Transferrin blood   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

• MeSH
• Lymphoma, Large B-Cell, Diffuse * diagnosis   MeSH
• Adult MeSH
• Immunophenotyping methods   MeSH
• Clinical Laboratory Techniques methods   MeSH
• Comorbidity MeSH
• Palatine Tonsil pathology   MeSH
• Humans MeSH
• Parkinson Disease MeSH
• Treatment Outcome MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Male MeSH
• Publication type
• Case Reports MeSH

• Keywords
• Ibrutinib,
• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * diagnosis   therapy   MeSH
• Diagnosis, Differential MeSH
• Adult MeSH
• Fever etiology   MeSH
• Immunophenotyping methods   MeSH
• Blood Cell Count methods   MeSH
• Humans MeSH
• Antineoplastic Agents therapeutic use   MeSH
• Recurrence MeSH
• Bone Marrow Transplantation methods   MeSH
• Bone Marrow Examination methods   MeSH
• Treatment Outcome MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Case Reports MeSH