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Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish "simple" from bilineal/"complex" mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter-laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: Flow cytometry is a valuable part in the routine diagnostics of acute leukemia (AL). Although internationally recognized definitions of main AL subsets are available, there is currently no consensus format for the short summary of clinical flow cytometry reports. Since clinical reports are too long for most database purposes, there is a need for a standardized format of their short summaries., Methods: The Associazione Italiana Ematologia Oncologia Pediatrica--Berlin Frankfurt Muenster (AIEOP-BFM) Flow Network that encompasses reference diagnostics laboratories in Australia, Austria, Czechia, Germany, Israel, Italy, and Switzerland have designed a pro-forma for the summary of flow cytometry results in the diagnosis of leukemia. The process involved several meetings and other communications, during which the group established a consensus on the essentials that lead to the diagnostic conclusions in childhood AL., Results: The "Flow Diagnostics Essential (FDE) Code" is a result from an agreement within the AIEOP-BFM Flow Network. In a standardized format, it reports the extent of the infiltration by a malignant clone, followed by description antigen expression as strong, weak or negative, and a diagnostic conclusion., Conclusions: A consensus brief format (the "FDE Code") has been designed as a brief summary of the diagnostic immunophenotype of childhood AL. It is also applicable for the diagnostic investigation of other malignancies by flow cytometry. The FDE code may be included in the final clinical report and/or used in the setting of a multicenter clinical trial database., (© 2013 Clinical Cytometry Society.)

Treatment response is a strong outcome predictor for childhood acute lymphoblastic leukemia (ALL). Here, we evaluated the predictive impact of flow cytometric blast quantification assays (absolute blast count, BC, and blast reduction rate, BRR) in peripheral blood (pB) and/or bone marrow (BM) at early time points of induction therapy (days 0, 8 and 15) on the remission status in the AIEOP-BFM-ALL 2000 protocol. At the single parameter level (905 patients), the strongest predictive parameter for the remission status as a dichotomous minimal residual disease (MRD) parameter (positive/negative) has been provided by the BC at day 15 in BM (cutoff: 17 blasts/microl; 50 vs 15%; odds ratio: 5.6; 95% confidence interval: 4.1-7.6, P<0.001), followed by the BRR at day 15 in BM and by the BC at day 8 in pB (odds ratios: 3.8 and 2.6, respectively). In the multiple regression analysis (440 patients), BC in pB (d0 and d8) and in BM (d15) as well as BRR at day 8 in pB provided significantly contributing variables with an overall correct prediction rate of 74.8%. These data show that the quantitative assessment of early response parameters, especially absolute BCs at day 15 in BM, has a predictive impact on the remission status after induction therapy.

Background: Identification of minor cell populations, e.g. leukemic blasts within blood samples, has become increasingly important in therapeutic disease monitoring. Modern flow cytometers enable researchers to reliably measure six and more variables, describing cellular size, granularity and expression of cell-surface and intracellular proteins, for thousands of cells per second. Currently, analysis of cytometry readouts relies on visual inspection and manual gating of one- or two-dimensional projections of the data. This procedure, however, is labor-intensive and misses potential characteristic patterns in higher dimensions., Results: Leukemic samples from patients with acute lymphoblastic leukemia at initial diagnosis and during induction therapy have been investigated by 4-color flow cytometry. We have utilized multivariate classification techniques, Support Vector Machines (SVM), to automate leukemic cell detection in cytometry. Classifiers were built on conventionally diagnosed training data. We assessed the detection accuracy on independent test data and analyzed marker expression of incongruently classified cells. SVM classification can recover manually gated leukemic cells with 99.78% sensitivity and 98.87% specificity., Conclusion: Multivariate classification techniques allow for automating cell population detection in cytometry readouts for diagnostic purposes. They potentially reduce time, costs and arbitrariness associated with these procedures. Due to their multivariate classification rules, they also allow for the reliable detection of small cell populations.

Background: Multiparametric flow cytometry has become an indispensable but complex tool for the diagnosis of acute leukemias. Interpretation of immunophenotypic data within a six-parameter analytical space relies on the standardization and validation of the instrument, the reagents, and the procedure. To address whether or not residual normal lymphocytes, usually present within leukemic samples, can serve as internal quality control for fluorescence intensity, 116 leukemic and 35 normal samples were analyzed., Methods: Eight laboratories participated in the study and recruited a total of 151 individuals including 29 patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), 77 with acute myeloid leukemia (AML), 10 with T-cell precursor acute lymphoblastic leukemia (T-ALL), and 35 normal bone marrow donors. Lymphocytes were gated according to the CD45hi/SSClo gating strategy, after which median fluorescence intensities (MFI) as well as percentages of positive cells (%positive) for CD19, CD22, CD7, and CD3 were recorded. Nonparametric statistics were used to compare variation within and between laboratories., Results: Normal lymphocytes within leukemic samples do not show substantial differences compared to lymphocytes from normal controls with respect to expression of CD19, CD22, CD7, and CD3. In particular, longitudinal control charts of MFI values for CD3 antigen provide useful information on analytical and instrument performance., Conclusion: Residual normal lymphocytes can serve as internal quality control for studies addressing fluorescence intensity in the setting of immunophenotyping of acute leukemias., (Copyright (c) 2005 Wiley-Liss, Inc.)