Radar plots facilitate differential diagnosis of acute promyelocytic leukemia and NPM1+ acute myeloid leukemia by flow cytometry.

BACKGROUND: Acute promyelocytic leukemia (APL) is one of the most life-threatening hematological emergencies and requires a prompt correct diagnosis by cytomorphology and flow cytometry (FCM) with later confirmation by cytogenetics/molecular genetics. However, nucleophosmin 1 muted acute myeloid leukemia (NPM1+ AML) can mimic APL, especially the hypogranular variant of APL. Our study aimed to develop a novel, Radar plot-based FCM strategy to distinguish APLs and NPM1+ AMLs quickly and accurately. METHOD: Diagnostic samples from 52 APL and 32 NPM1+ AMLs patients were analyzed by a 3-tube panel of 10-color FCM. Radar plots combining all markers were constructed for each tube. Percentages of positive leukemic cells and mean fluorescence intensity were calculated for all the markers. RESULTS: APL showed significantly higher expression of CD64, CD2, and CD13, whereas more leukemic cells were positive for CD11b, CD11c, CD15, CD36, and HLA-DR in NPM1+ AMLs. Radar plots featured CD2 expression, a lack of a monocytic component, lack of expression of HLA-DR and CD15, and a lack of a prominent CD11c+ population as recurring characteristics of APL. The presence of blasts with low SSC, presence of at least some monocytes, some expression of HLA-DR and/or CD15, and a prominent CD11c population were recurrent characteristics of NPM1+ AMLs. Radar plot analysis could confidently separate all hypergranular APL cases from any NPM1+ AML and in 90% of cases between variant APL and blastic NPM1+ AML. CONCLUSION: Radar plots can potentially add to differential diagnostics as they exhibit characteristic patterns distinguishing APL and different types of NPM1+ AMLs.

Characteristics of Lymphoproliferative Disorders with More Than One Aberrant Cell Population as Detected by 10-Color Flow Cytometry.

BACKGROUND: We have evaluated the frequency of lymphoproliferative disorders with more than one aberrant population of monotypic B-cells detected during routine hematopathological diagnostics. MATERIALS AND METHODS: 2600 samples peripheral (blood, bone marrow, fine-needle aspirate, lymph node, and pleural fluid cell suspensions) were analyzed using a 10-color B-cell panel and a 10-color T-cell panel. A 10-color plasma cell/lymphoplasmacytic panel was performed when appropriate. RESULTS: 790/2600 samples (30%) showed at least one aberrant B-cell population and 27(1%) showed an aberrant T-cell population. 41/790 samples (5.1%) showed two aberrant B-cell populations. Thirteen patients had two B-cell populations with different surface immunoglobulin restriction (one kappa+ and one lambda+), most with B-cell chronic lymphocytic leukemia-related phenotype. Five cases showed two B-cell populations with the same light chain restriction but distinctly different immunophenotypes. In 23 cases, two populations had the same light chain restriction and differed by expression of one or 2 markers, thus, a possibility of intraclonal differentiation could not be excluded. Cases with possible intraclonal differentiation had a significantly higher proportion of aberrant B-cells than those with two coexisting aberrant B-cell populations (49.9% vs. 27.7%, p = 0.008). In only one sample one population of clonal B-cell and one clonal T-cell population with large granular lymphocyte related phenotype were found. CONCLUSION: Using our panels 5.1% of cases with lymphoproliferative disorder-associated aberrant findings show two aberrant (clonal) lymphoid and/or plasma cell populations. © 2016 International Clinical Cytometry Society.

Visualization of Cell Composition and Maturation in the Bone Marrow Using 10-Color Flow Cytometry and Radar Plots.

BACKGROUND: The enormous potential of complex data files generated by 10-color flow cytometry (FC) is hindered by the requirement for exhaustive manual gating and the complexity of multidimensional data visualization. We propose a model using radar plots (RPs), to improve FC data visualization by capturing multidimensionality and integration of FC findings. METHOD: We analysed 12 normal/reactive bone marrow (N/R BM) samples and 12 BM samples from patients with myelodysplasia (MDS) with 10-color FC. All identifiable cell clusters were individually marked, grouped, and visualized on radar plots. RPs were optimized to de-clutter the cell clusters and map BM cell composition and maturation. RESULTS: A total of 27 immature and mature cell clusters were identified and visualized on 8 multidimensional radar plots. The RPs displayed flow cytometry findings of normal BM in an integrated fashion to maximize overall insight into the data set. The constructed map of bone marrow cell composition was reproducible in all normal BM samples analyzed. Analysis of the pilot cohort of patient samples confirmed the presence of MDS-related changes. These changes are readily identifiable on RPs. CONCLUSION: We demonstrated that the cell clusters of normal BM can be mapped on multidimensional radar plots, which provide an inclusive insight into BM cell composition and maturation. These reproducible RPs present a comprehensive and comprehensible visual display of differentiation and maturation of haematopoietic cells in normal BM, and can be used as a reference map to assess abnormal haematopoiesis in MDS. © 2017 International Clinical Cytometry Society.

A standardized immune phenotyping and automated data analysis platform for multicenter biomarker studies.

The analysis and validation of flow cytometry-based biomarkers in clinical studies are limited by the lack of standardized protocols that are reproducible across multiple centers and suitable for use with either unfractionated blood or cryopreserved PBMCs. Here we report the development of a platform that standardizes a set of flow cytometry panels across multiple centers, with high reproducibility in blood or PBMCs from either healthy subjects or patients 100 days after hematopoietic stem cell transplantation. Inter-center comparisons of replicate samples showed low variation, with interindividual variation exceeding inter-center variation for most populations (coefficients of variability <20% and interclass correlation coefficients >0.75). Exceptions included low-abundance populations defined by markers with indistinct expression boundaries (e.g., plasmablasts, monocyte subsets) or populations defined by markers sensitive to cryopreservation, such as CD62L and CD45RA. Automated gating pipelines were developed and validated on an independent data set, revealing high Spearman's correlations (rs >0.9) with manual analyses. This workflow, which includes pre-formatted antibody cocktails, standardized protocols for acquisition, and validated automated analysis pipelines, can be readily implemented in multicenter clinical trials. This approach facilitates the collection of robust immune phenotyping data and comparison of data from independent studies.

Screening bone marrow samples for abnormal lymphoid populations and myelodysplasia-related features with one 10-color 14-antibody screening tube.

BACKGROUND: We have designed one-tube 14-antibody 10-color flow cytometry (FCM) panel that would provide maximum information on lymphoid and myeloid cell subsets in bone marrow aspirates (BMA) from patients with cytopenia(s). SAMPLES AND METHODS: BMA from 8 normal donors, from 286 non-malignant hospital controls, 92 myelodysplastic syndromes (MDS), 47 myeloproliferative neoplasms (MPN), and from 14 MDS/MPN patients were investigated. One tube 14-monoclonal antibody (MAb) 10-fluorochrome panel included: kappa+CD4 FITC, Lambda+CD8 PE, CD3 + CD14 ECD, CD34 APC, CD20+CD56 PC7, CD10 APC-A750, CD19 APC-A700, CD33 PC5.5, CD5 PB, and CD45 KO. Kappa/lambda expression was evaluated separately in CD19+, CD10+ and CD5+ B-cells. CD4+CD3+, CD8+CD3+, CD5+CD3+ T-lymphocyte subsets were enumerated. Blasts were evaluated using CD45/SSC and CD34 gating. The FCM score for MDS (sc. Ogata score) included CD34+ myeloblast and B-progenitor cluster size, myeloblast/lymphocyte CD45 expression, and granulocyte/lymphocyte SSC ratio. RESULTS: Abnormal lymphoid populations or increased plasma cells were found in 18 patients (4%). A 43/92 BMA from MDS and 7/14 from MDS/MPN patients had score >2. Score >2 had 92.5% positive predictive value for MDS/MDS-MPN diagnosis. Negative predictive value for MDS/MDS-MPN was 83% for scores under 3 and 88% for scores under 2. All but two of normal/hospital control samples had FCM score <3 (99%). Differences in scores between MDS & MDS/MPN and the control groups were statistically significant (P < 0.0001). CONCLUSIONS: Our one-tube FCM panel can be easily applied to screening for aberrant lymphoid populations and myelodysplasia-related features. MDS scores >2 are highly indicative of MDS or MDS-MPN.