The rise in the analytical speed of mutiparameter flow cytometers made possible by the introduction of digital instruments, has brought up the possibility to manage progressively higher number of ...parameters simultaneously on significantly greater numbers of individual cells. This has led to an exponential increase in the complexity and volume of flow cytometry data generated about cells present in individual samples evaluated in a single measurement. This increase demands for new developments in flow cytometry data analysis, graphical representation, and visualization and interpretation tools to address the new big data challenges, i.e. processing data files of ≥10–25 parameters per cell in samples with >5–10 million cells (= up to 250 million data points per cell sample) obtained in a few minutes.
Here, we present a comprehensive review of some of the tools developed by the EuroFlow consortium for processing flow cytometric big data files in diagnostic laboratories, particularly focused on automated EuroFlow approaches for: i) identification of all cell populations coexisting in a sample (automated gating); ii) smart classification of aberrant cell populations in routine diagnostics; iii) automated reporting; together with iv) new tools developed to visualize n-dimensional data in 2-dimensional plots to support expert-guided automated data analysis. The concept of using reference data bases implemented into software programs, in combination with multivariate statistical analysis pioneered by EuroFlow, provides an innovative, highly efficient and fast approach for diagnostic screening, classification and monitoring of patients with distinct hematological and immune disorders, as well as other diseases.
Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel ...(T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.
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.
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 ...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/numyogenin/CD4-EpCAM/CD56/GD2/smCD3-CD19/cyCD3-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. In addition, STOT allowed accurate discrimination among the four most common subtypes of malignant CD45− CD56++ non-hematopoietic solid tumors: 13/13 (GD2++ numyogenin− CD271−/+ nuMyoD1− CD99− EpCAM−) neuroblastoma samples, 5/5 (GD2− numyogenin++ CD271++ nuMyoD1++ CD99−/+ EpCAM−) rhabdomyosarcomas, 2/2 (GD2−/+ numyogenin− CD271+ nuMyoD1− CD99+ EpCAM−) Ewing sarcoma family of tumors, and 7/7 (GD2− numyogenin− CD271+ nuMyoD1− 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.
Multiple myeloma remains largely incurable. However, a few patients experience more than 10 years of relapse-free survival and can be considered as operationally cured. Interestingly, long-term ...disease control in multiple myeloma is not restricted to patients with a complete response, since some patients revert to having a profile of monoclonal gammopathy of undetermined significance. We compared the distribution of multiple compartments of lymphocytes and dendritic cells in the bone marrow and peripheral blood of multiple myeloma patients with long-term disease control (n=28), patients with newly diagnosed monoclonal gammopathy of undetermined significance (n=23), patients with symptomatic multiple myeloma (n=23), and age-matched healthy adults (n=10). Similarly to the patients with monoclonal gammopathy of undetermined significance and symptomatic multiple myeloma, patients with long-term disease control showed an expansion of cytotoxic CD8(+) T cells and natural killer cells. However, the numbers of bone marrow T-regulatory cells were lower in patients with long-term disease control than in those with symptomatic multiple myeloma. It is noteworthy that B cells were depleted in patients with monoclonal gammopathy of undetermined significance and in those with symptomatic multiple myeloma, but recovered in both the bone marrow and peripheral blood of patients with long-term disease control, due to an increase in normal bone marrow B-cell precursors and plasma cells, as well as pre-germinal center peripheral blood B cells. The number of bone marrow dendritic cells and tissue macrophages differed significantly between patients with long-term disease control and those with symptomatic multiple myeloma, with a trend to cell count recovering in the former group of patients towards levels similar to those found in healthy adults. In summary, our results indicate that multiple myeloma patients with long-term disease control have a constellation of unique immune changes favoring both immune cytotoxicity and recovery of B-cell production and homing, suggesting improved immune surveillance.
Summary
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 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.
About 25% of the patients with the translocation t(11;19)(q23;p13.3)/KMT2A-MLLT1 present three-way or more complex fusions, associated with a worse prognosis, suggesting that a particular mechanism ...creates functional KMT2A fusions for this condition. In this work, we show a cryptic three-way translocation t(9;11;19). Interestingly, long-distance inverse polymerase chain reaction sequencing revealed a KMT2A-MLLT1 and the yet unreported out-of-frame SEC16A-KMT2A fusion, associated with low SEC16A expression and KMT2A overexpression, in an infant with B-acute lymphoblastic leukemia presenting a poor prognosis. Our case illustrates the importance of molecular cytogenetic tests in selecting cases for further investigations, which could open perspectives regarding novel therapeutic approaches for poor prognosis childhood leukemias.
A
bstract
The production of
W
and
Z
bosons in association with jets is studied in the forward region of proton-proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, ...corresponding to an integrated luminosity of 1.98 ± 0.02 fb
−1
. The
W
boson is identified using its decay to a muon and a neutrino, while the
Z
boson is identified through its decay to a muon pair. Total cross-sections are measured and combined into charge ratios, asymmetries, and ratios of
W
+jet and
Z
+jet production cross-sections. Differential measurements are also performed as a function of both boson and jet kinematic variables. All results are in agreement with Standard Model predictions.
A search for $CP$ violation in the decay $Λ^0_b$ → $pK$- $μ$+$μ$- is presented. This decay is mediated by flavour-changing neutral-current transitions in the Standard Model and is potentially ...sensitive to new sources of CP violation. The study is based on a data sample of proton-proton collisions recorded with the LHCb experiment, corresponding to an integrated luminosity of 3 fb-1. The $Λ^0_b$ → $pK$- $μ$+$μ$- decay is observed for the first time, and two observables that are sensitive to different manifestations of $CP$ violation are measured, $ΔΑ$CP $\equiv$ $ΔΑ$CP ($Λ^0_b$ → $pK$- $μ$+$μ$-) - $ΔΑ$CP($Λ^0_b$ → $pK$- $J/Ψ$) and a C P T ^ - o d d , where the latter is based on asymmetries in the angle between the $μ$+$μ$- and $pK$- decay planes. These are measured to be Δ A C P = ( − 3.5 ± 5.0 ( s t a t ) ± 0.2 ( s y s t ) ) × 10 − 2 , a C P T ^ - o d d = ( 1.2 ± 5.0 ( s t a t ) ± 0.7 ( s y s t ) ) × 10 − 2 , and no evidence for $CP$ violation is found
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