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

In recent years the volume and complexity of flow cytometry data has increased substantially. This has led to a greater number of identifiable cell populations in a single measurement. Consequently, new gating strategies and new approaches for cell population definition are required. Here we describe how the EuroFlow Lymphoid Screening Tube (LST) reference data base for peripheral blood (PB) samples was designed, constructed and validated for automated gating of the distinct lymphoid (and myeloid) subsets in PB of patients with chronic lymphoproliferative disorders (CLPD). A total of 46 healthy/reactive PB samples which fulfilled predefined technical requirements, were used to construct the LST-PB reference data base. In addition, another set of 92 PB samples (corresponding to 10 healthy subjects, 51 B-cell CLPD and 31 T/NK-cell CLPD patients), were used to validate the automated gating and cell-population labeling tools with the Infinicyt software. An overall high performance of the LST-PB data base was observed with a median percentage of alarmed cellular events of 0.8% in 10 healthy donor samples and of 44.4% in CLPD data files containing 49.8% (range: 1.3–96%) tumor cells. The higher percent of alarmed cellular events in every CLPD sample was due to aberrant phenotypes (75.6% cases) and/or to abnormally increased cell counts (86.6% samples). All 18 (22%) data files that only displayed numerical alterations, corresponded to T/NK-cell CLPD cases which showed a lower incidence of aberrant phenotypes (41%) vs B-cell CLPD cases (100%). Comparison between automated vs expert-bases manual classification of normal (r2 = 0.96) and tumor cell populations (rho = 0.99) showed a high degree of correlation. In summary, our results show that automated gating of cell populations based on the EuroFlow LST-PB data base provides an innovative, reliable and reproducible tool for fast and simplified identification of normal vs pathological B and T/NK lymphocytes in PB of CLPD patients

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies — a EuroFlow study

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.The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 programme of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA). TS and LS were supported by a Scientific Grant from the Medical University of Silesia Nr. PCN-1-050/K/0/K

Bone Marrow Stromal Cell Regeneration Profile in Treated B-Cell Precursor Acute Lymphoblastic Leukemia Patients:Association with MRD Status and Patient Outcome

SIMPLE SUMMARY: For the last 20 years, measurable residual disease (MRD) has proven to be a strong prognostic factor in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effects of therapy on the bone marrow (BM) microenvironment and their potential relationship with MRD and patient outcome still remain to be evaluated. Here, we show that mesenchymal stem cells (MSC) and endothelial cells (EC) are constantly present at relatively low frequencies in normal BM and in most follow-up BM samples from treated BCP-ALL patients. Of note, their levels are independent of the MRD status. From the prognostic point of view, an increased percentage of EC among stromal cells (EC plus MSC) at day +78 of therapy was associated with shorter disease free survival (DFS), independently of the MRD status both in childhood and in adult BCP-ALL. Thus, an abnormally high EC/MSC distribution at day +78 of therapy emerges as an adverse prognostic factor, independent of MRD in BCP-ALL. ABSTRACT: 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

Generation of flow cytometry data files with a potentially infinite number of dimensions

Immunophenotypic characterization of B-cell chronic lymphoproliferative disorders (B-CLPD) is associated with the use of increasingly larger panels of multiple combinations of 3 to ≥6 monoclonal antibodies (Mab), data analysis being separately performed for each of the different stained sample aliquots. Here, we describe and validate an automated method for calculation of flow cytometric data from several multicolor stainings of the same cell sample - i.e., the merging of data from different aliquots stained with partially overlapping combinations of Mab reagents (focusing on ≥1 cell populations) - into one data file as if it concerned a single >super> multicolor staining. Evaluation of the performance of the method described was done in a group of 60 B-CLPD studied at diagnosis with 18 different reagents in a panel containing six different 3- and 4-color stainings, which systematically contained CD19 for the identification of B-cells. Our results show a high degree of correlation and agreement between originally measured and calculated data about cell surface stainings, providing a basis for the use of this approach for the generation of flow cytometric data files containing information about a virtually infinite number of stainings for each individual cellular event measured in a sample, using a limited number of fluorochrome stainings. © 2008 International Society for Advancement of Cytometry.Grant sponsor: European Commission (EuroFlow); Grant number: LSHB-CT-2006- 018708; Grant sponsor: Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Madrid, Spain; Grant number: ISCIII-RTICC RD06/0020/0035-FEDER; Grant sponsor: Ministerio de Educación y Ciencia, Madrid, Spain (Programa Hispano- Brasileño de Cooperación Universitaria); Grant number: Ref. PHB 2004-0800-PC.Peer Reviewe

The immunophenotype of different immature, myeloid and B-cell lineage-committed CD34+ hematopoietic cells allows discrimination between normal/reactive and myelodysplastic syndrome precursorsCD34+ cells phenotype in myelodysplastic syndromes

Occurrence of phenotypic abnormalities in CD34+ hematopoietic progenitor and precursor cells (HPC) and their major B-cell and nonlymphoid compartments has been frequently reported in myelodysplastic syndromes (MDS). Here, we analyze for the first time the numerical and phenotypic abnormalities of different maturation-associated subsets of bone marrow (BM) CD34+ HPC from 50 newly diagnosed MDS patients in comparison to normal/reactive BM (n=29). Our results confirm the existence of heterogeneously altered phenotypes among CD34+ HPC from MDS and indicate that such variability depends both on the relative distribution of the different subsets of CD34+ HPC committed into the different myeloid and B-lymphoid compartments, and their immunophenotype (for example, higher reactivity for CD117 and CD13 and lower expression of CyMPO, CD64 and CD65 on CD34+ immature and neutrophil precursors), a clear association existing between the accumulation of CD34+ HPC and that of immature CD34+ HPC. Interestingly, expansion of erythroid- and neutrophil-lineage CD34+ cells is detected in low-grade MDS at the expense of CD34+ plasmacytoid dendritic cell and B-cell precursors, while expansion of immature CD34+ precursors occurs in high-grade MDS. On the basis of the number and severity of the phenotypic abnormalities detected, a scoring system is proposed that efficiently discriminates between normal/reactive and MDS CD34+ HPC, the mean score significantly increasing from low- to high-grade MDS.Peer Reviewe

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

In recent years the volume and complexity of flow cytometry data has increased substantially. This has led to a greater number of identifiable cell populations in a single measurement. Consequently, new gating strategies and new approaches for cell population definition are required. Here we describe how the EuroFlow Lymphoid Screening Tube (LST) reference data base for peripheral blood (PB) samples was designed, constructed and validated for automated gating of the distinct lymphoid (and myeloid) subsets in PB of patients with chronic lymphoproliferative disorders (CLPD). A total of 46 healthy/reactive PB samples which fulfilled predefined technical requirements, were used to construct the LST-PB reference data base. In addition, another set of 92 PB samples (corresponding to 10 healthy subjects, 51 B-cell CLPD and 31 T/NK-cell CLPD patients), were used to validate the automated gating and cell-population labeling tools with the Infinicyt software. An overall high performance of the LST-PB data base was observed with a median percentage of alarmed cellular events of 0.8% in 10 healthy donor samples and of 44.4% in CLPD data files containing 49.8% (range: 1.3–96%) tumor cells. The higher percent of alarmed cellular events in every CLPD sample was due to aberrant phenotypes (75.6% cases) and/or to abnormally increased cell counts (86.6% samples). All 18 (22%) data files that only displayed numerical alterations, corresponded to T/NK-cell CLPD cases which showed a lower incidence of aberrant phenotypes (41%) vs B-cell CLPD cases (100%). Comparison between automated vs expert-bases manual classification of normal (r2 = 0.96) and tumor cell populations (rho = 0.99) showed a high degree of correlation. In summary, our results show that automated gating of cell populations based on the EuroFlow LST-PB data base provides an innovative, reliable and reproducible tool for fast and simplified identification of normal vs pathological B and T/NK lymphocytes in PB of CLPD patients

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

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 ( 0.95 for all cell types in PB andr(2) > 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)

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present