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
• algoritmy MeSH
• B-lymfocyty * patologie   MeSH
• imunofenotypizace * metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfoproliferativní nemoci * diagnóza   klasifikace   MeSH
• průtoková cytometrie * metody   MeSH
• senioři MeSH
• support vector machine MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie 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
• akutní myeloidní leukemie diagnóza   MeSH
• algoritmy * MeSH
• imunofenotypizace metody   MeSH
• leukocyty patologie   MeSH
• lidé MeSH
• průtoková cytometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We evaluated the immunophenotypic profile of 72 AMKL and 114 non-AMKL AML patients using the EuroFlow AML panel. Univariate and multivariate/multidimensional analyses were performed to identify most relevant markers contributing to the diagnosis of AMKL. AMKL patients were subdivided into transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), AML-not otherwise specified with megakaryocytic differentiation (NOS-AMKL), and AMKL-other patients (AML patients with other WHO classification but with flowcytometric features of megakaryocytic differentiation). Flowcytometric analysis showed good discrimination between AMKL and non-AMKL patients based on differential expression of, in particular, CD42a.CD61, CD41, CD42b, HLADR, CD15 and CD13. Combining CD42a.CD61 (positive) and CD13 (negative) resulted in a sensitivity of 71% and a specificity of 99%. Within AMKL patients, TAM and ML-DS patients showed higher frequencies of immature CD34+/CD117+ leukemic cells as compared to NOS-AMKL and AMKL-Other patients. In addition, ML-DS patients showed a significantly higher expression of CD33, CD11b, CD38 and CD7 as compared to the other three subgroups, allowing for good distinction of these patients. Overall, our data show that the EuroFlow AML panel allows for straightforward diagnosis of AMKL and that ML-DS is associated with a unique immunophenotypic profile.

• Publikační typ
• časopisecké články MeSH

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 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.

• MeSH
• laboratoře normy   MeSH
• lidé MeSH
• průtoková cytometrie normy   MeSH
• referenční standardy MeSH
• testování odbornosti laboratoří metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.

• MeSH
• hematologické nádory diagnóza   imunologie   MeSH
• imunofenotypizace normy   MeSH
• laboratoře normy   MeSH
• lidé MeSH
• monoklonální protilátky diagnostické užití   MeSH
• nádorové biomarkery imunologie   MeSH
• prognóza MeSH
• průtoková cytometrie přístrojové vybavení   metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Geografické názvy
• Evropa MeSH

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 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 BCP-ALL 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.

• MeSH
• 5'-nukleotidasa analýza   biosyntéza   MeSH
• antigeny CD86 analýza   biosyntéza   MeSH
• dítě MeSH
• GPI-vázané proteiny analýza   biosyntéza   MeSH
• lidé MeSH
• nádorové biomarkery analýza   MeSH
• neuropilin-1 analýza   biosyntéza   MeSH
• pre-B-buněčná leukemie patologie   MeSH
• předškolní dítě MeSH
• prekurzorové B-lymfoidní buňky patologie   MeSH
• reziduální nádor MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 (n = 4, two of them presented with Omenn syndrome), IL2RG (n = 4, one of them with confirmed maternal engraftment), NHEJ1 (n = 1), CD3E (n = 1), ADA (n = 1), JAK3 (n = 3, two of them with maternal engraftment) and DCLRE1C (n = 1) and 11 other PID patients had diverse molecular defects [ZAP70 (n = 1), WAS (n = 2), PNP (n = 1), FOXP3 (n = 1), del22q11.2 (DiGeorge n = 4), CDC42 (n = 1) and FAS (n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs).

• MeSH
• HLA-DR antigeny analýza   MeSH
• imunofenotypizace metody   MeSH
• kojenec MeSH
• lidé MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• primární imunodeficience diagnóza   imunologie   MeSH
• průtoková cytometrie metody   MeSH
• T-lymfocyty imunologie   MeSH
• těžká kombinovaná imunodeficience imunologie   MeSH
• thymus imunologie   MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.

• MeSH
• B-lymfocyty imunologie   MeSH
• dítě MeSH
• dospělí MeSH
• imunologická paměť imunologie   MeSH
• kojenec MeSH
• leukocyty imunologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfocyty imunologie   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• novorozenec MeSH
• novorozenecký screening metody   MeSH
• plazmatické buňky imunologie   MeSH
• předškolní dítě MeSH
• primární imunodeficience diagnóza   imunologie   MeSH
• průtoková cytometrie metody   MeSH
• senioři MeSH
• T-lymfocyty imunologie   MeSH
• těžká kombinovaná imunodeficience diagnóza   imunologie   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Projekt si klade za cíl zavést 8-mi barevnou standardizovanou cytometrickou diagnostiku hematologických malignit v návaznosti na evropský projekt Euroflow. Jednotně provedená diagnostika navíc umožní komputační zpracování dat a hledání prognostických vztahů v multicentrických souborech. Důležitou součástí projektu je vývoj jednotného postupu detekce odpovědi na léčbu (stanovení minimální reziduální nemoci), a zavedení metodiky externí kontroly kvality stanovení. Projekt je navržen jako projekt multicentrický se zastoupením 4 významných diagnostických center tak, aby bylo zajištěno dostatečné zastoupení všech klinických skupin hematologických malignit.; We expect end up with standardized 8-color flow cytometry for diagnosing and disease monitoring of various hematological malignancies as currently is within the Euroflow project under the development. Uniformly performed diagnostics across the centers inCzechia will enable searching new prognostic correlations and computer assisted diagnostic evaluation. Important part of the project is the development of minimal residual disease detection on common basis which will also enable inter-laboratory standardization and quality data control. Four centers focused on different patients group agreed in participation in the project and all disease categories are covered within these center.

• MeSH
• hematologické nádory diagnóza   prevence a kontrola   MeSH
• imunofenotypizace MeSH
• metody pro podporu rozhodování MeSH
• plošný screening MeSH
• průtoková cytometrie metody   normy   přístrojové vybavení   trendy   MeSH
• referenční standardy MeSH
• reziduální nádor MeSH
• Geografické názvy
• Česká republika MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• hematologie a transfuzní lékařství
• onkologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of therapy emerged as an independent unfavorable prognostic factor for DFS in childhood BCP-ALL in the discovery cohort-hazard ratio (95% confidence interval) of 2.50 (1-9.66); p = 0.05-together with the BM MRD status (p = 0.031). Further investigation of the predictive value of the combination of these two variables (%EC within stromal cells and MRD status at day +78) allowed classification of BCP-ALL into three risk groups with median DFS of: 3.9, 3.1 and 1.1 years, respectively (p = 0.001). These results were confirmed in two validation cohorts of childhood BCP-ALL (n = 74) (p = 0.001) and adult BCP-ALL (n = 40) (p = 0.004) treated at different centers. In summary, our findings suggest that an imbalanced EC/MSC ratio in BM at day +78 of therapy is associated with a shorter DFS of BCP-ALL patients, independently of their MRD status. Further prospective studies are needed to better understand the pathogenic mechanisms involved.

• Publikační typ
• časopisecké články MeSH