EuroFlow Lymphoid Screening Tube (LST) data base for automated identification of blood lymphocyte subsets

Flores‐Montero, Juan; Grigore, Georgiana; Fluxá, Rafael; Hernandez, Jessica; Fernández, Paula; Almeida, Júlia; Muñoz, Noemí Batista; Böttcher, Sebastian; Sędek, Łukasz; Velden, Vincent van der; Bárrena, Susana; Hernández, Alejando; Paiva, Bruno; Lécrevisse, Quentin; Lima, Margarida; Santos, Ana Filipa L.O.M.; Dongen, Jacques J.M. van; Órfão, Alberto

doi:10.1016/j.jim.2019.112662

Cited by 37 publications

(44 citation statements)

References 42 publications

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“…In parallel with the MG strategy, all FC standard data files were also analyzed with the AG&I module of Infinicyt ™ . The AG&I module compares each FC standard data file with a reference database of healthy controls using the automated gating and classification algorithms, as previously described in detail (18,19). This automated analysis included a first clustering step of all individual events in the data file, followed in a second step by classification of the resulting clusters of events into the cell populations identified a priori in the database, according to their characteristics in the multidimensional space generated by all the parameters evaluated.…”

Section: Discussionmentioning

confidence: 99%

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Improved Standardization of Flow Cytometry Diagnostic Screening of Primary Immunodeficiency by Software-Based Automated Gating

et al. 2020

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Background: Multiparameter 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. Methods: FC 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 ™ software (Cytognos). For comparison, percentage differences in absolute cell counts/µL were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on agematched 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. Results: The 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/µL) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on

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Section: Discussionmentioning

confidence: 99%

“…Due to major technological advances in multiparameter FC, data analysis has become increasingly complex and time-consuming (13,18,(21)(22)(23). This also implies higher variability and more subjectivity, as it is influenced by the observer's expertise.…”

Section: Discussionmentioning

confidence: 99%

Improved Standardization of Flow Cytometry Diagnostic Screening of Primary Immunodeficiency by Software-Based Automated Gating

et al. 2020

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“…The AGI tool is included in the Infinicyt software [7,21,22]. To validate the ALOT databases the Infinicyt software-version 2.0 release candidate (RC) 33 (Infinicyt 2.0.0c RC33) was used.…”

Section: Automated Gating and Identification Toolmentioning

confidence: 99%

“…Thus, automated gating strategies emerge as very attractive to include the gating procedure within the standardization process, speed up the analysis and improve the selection of abnormal cells to be further classified [14][15][16][17][18][19][20]. Previously, an automated gating and identification (AGI) tool was designed within the consortium and successfully validated with, e.g., the lymphoid screening tube for which a dedicated database was built [21]. In the current study, we built a reference database of normal peripheral blood (PB) and bone marrow (BM) samples stained with the previously validated ALOT antibody combination.…”

Section: Introductionmentioning

confidence: 99%

“…In the current study, we built a reference database of normal peripheral blood (PB) and bone marrow (BM) samples stained with the previously validated ALOT antibody combination. This database served as a reference for accurate, reproducible and AGI of distinct normal and different from normal cell subsets contained in independent ALOT data files [21]. We developed a pipeline combining this AGI tool with the database-guided algorithm to minimize human intervention and improve standardization.…”

Section: Introductionmentioning

confidence: 99%

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Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

et al. 2021

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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.

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Immune cell kinetics and antibody response in COVID‐19 patients with low‐count monoclonal B‐cell lymphocytosis

Oliva‐Ariza,

Fuentes‐Herrero,

Lecrevisse

et al. 2023

American J Hematol

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Low‐count monoclonal B‐cell lymphocytosis (MBLlo) has been associated with an underlying immunodeficiency and has recently emerged as a new risk factor for severe COVID‐19. Here, we investigated the kinetics of immune cell and antibody responses in blood during COVID‐19 of MBLlo versus non‐MBL patients. For this study, we analyzed the kinetics of immune cells in blood of 336 COVID‐19 patients (74 MBLlo and 262 non‐MBL), who had not been vaccinated against SARS‐CoV‐2, over a period of 43 weeks since the onset of infection, using high‐sensitivity flow cytometry. Plasma levels of anti‐SARS‐CoV‐2 antibodies were measured in parallel by ELISA. Overall, early after the onset of symptoms, MBLlo COVID‐19 patients showed increased neutrophil, monocyte, and particularly, plasma cell (PC) counts, whereas eosinophil, dendritic cell, basophil, and lymphocyte counts were markedly decreased in blood of a variable percentage of samples, and with a tendency toward normal levels from week +5 of infection onward. Compared with non‐MBL patients, MBLlo COVID‐19 patients presented higher neutrophil counts, together with decreased pre‐GC B‐cell, dendritic cell, and innate‐like T‐cell counts. Higher PC levels, together with a delayed PC peak and greater plasma levels of anti‐SARS‐CoV‐2‐specific antibodies (at week +2 to week +4) were also observed in MBLlo patients. In summary, MBLlo COVID‐19 patients share immune profiles previously described for patients with severe SARS‐CoV‐2 infection, associated with a delayed but more pronounced PC and antibody humoral response once compared with non‐MBL patients.

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EuroFlow Lymphoid Screening Tube (LST) data base for automated identification of blood lymphocyte subsets

Cited by 37 publications

References 42 publications

Improved Standardization of Flow Cytometry Diagnostic Screening of Primary Immunodeficiency by Software-Based Automated Gating

Improved Standardization of Flow Cytometry Diagnostic Screening of Primary Immunodeficiency by Software-Based Automated Gating

Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

Immune cell kinetics and antibody response in COVID‐19 patients with low‐count monoclonal B‐cell lymphocytosis

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