List Mode Data Analysis Research Articles

Demonstration of INCC6 for advanced list-mode data acquisition and analysis using ALMM

The International Neutron Coincidence Counting (INCC) software plays an important role in nuclear safeguards and nuclear material control and accounting (NMC&A) measurements. While the current version, INCC5, represents the traditional standard utilized by inspectorates as well as facilities and practitioners alongside shift register hardware, LANL has recently developed an upgraded version of the software, INCC6. INCC6 offers the same analysis tools as INCC5 while adding new capabilities including acquisition and analysis of list-mode data, recording and storage of list-mode data files, and an expanded set of advanced analysis tools that make use of the additional information available from list-mode data. This paper presents the first demonstration of data acquisition and analysis using INCC6 and the Advanced List Mode Multiplicity Module (ALMM). The capability of INCC6 to perform live list-mode data acquisition and analysis is demonstrated for a variety of neutron sources with a range of neutron emission rates relevant for practical applications and validated against the results obtained with INCC5 and traditional shift register hardware. Advanced list-mode analysis tools introduced by INCC6, such as time-interval and coincidence matrix analysis, are also demonstrated. Finally, the capability of INCC6 to analyze list-mode data files is tested and validated against traditional shift register results using INCC5.

Novel Compton suppression equipment in [formula omitted]-ray spectrometry with list mode data acquisition

A novel Compton suppression device has been developed at the Radiation and Nuclear Safety Authority of Finland to improve the sensitivity of measurements in the Gamma Laboratory. It utilizes γ–γ anticoincidence, but operates as a full coincidence system. Specific software has been developed for the sorting, visualization and analysis of list mode data produced by the multi-detector list-mode devices in the Gamma Laboratory. By utilizing the software, the coincidence data can be accessed and the true-coincidence losses of photo-peaks of multiple gamma-ray-emitting nuclides restored in the analysis. This simplifies data analysis and further increases the sensitivity of the device in low count-rate gamma spectrometry.A detailed Geant4 simulation model of the device was developed and used to optimize the device as well as to support calibrations and complex analysis tasks.The setup has been integrated to current laboratory information management system used in the Gamma Laboratory. Compton suppression reduces the continuous background seen by the high-purity germanium detector by a factor of 3–10, in addition to comparable reduction in the Compton continuum of any peaks in the spectrum. A comparison with the results of conventional gamma-ray spectrometry is presented.

Using the JCC-71 neutron coincidence collar as a benchmark for detector characterization with PTR-32 list mode data acquisition

Augmenting or interchanging current international safeguards shift register−based data acquisition systems with list mode data acquisition systems gives nondestructive assay (NDA) systems greater versatility. Neutron list mode data analysis offers comparable analytical results to the more widely used shift register analysis in nuclear material quantification applications and offers several diagnostic tools that are specifically beneficial to in-laboratory characterization and calibration measurements. These benefits include identification of non-ideal behavior, optimization of operational parameters from a single measurement, and an improved understanding of the physics-based behavior of NDA systems for a more precise system representation and more confident assay results. In this work the advantages of using list mode data acquisition for detector characterization are demonstrated experimentally. Two commercial-off-the-shelf International Atomic Energy Agency-supported technologies are used for a typical detector characterization procedure and their performance is compared. Specifically, a 3He-based Canberra Industries JCC-71 Neutron Coincidence Collar is characterized using the Hungarian Institute of Isotopes’ Pulse Train Recorder-32 (PTR-32) list mode data acquisition system, and the results are compared to those obtained using the standard Canberra Industries JSR-15 model shift register. The quantitative results from the two systems are in agreement, which demonstrates that the PTR-32 is a technically viable alternative to conventional shift register electronics for this task. A suitable procedure for full instrument characterization is described, and the added benefits of list mode for characterization and data collection are discussed. This is an important step toward establishing a procedure for the routine use of list mode data acquisition and analysis for neutron NDA system characterization in safeguards field applications.

A new line density tracking algorithm for PEPT and its application to multiple tracers

A new algorithm for the analysis of list mode data from positron emission particle tracking experiments is described. The method is based upon the premise that the location of the tracer is within a region of space through which the majority of lines of response pass. The algorithm scans through the data set only once and makes use of the entire set of lines of response, thus efficiently achieving the maximum possible spatial resolution. The application of the approach to the tracking of both single and multiple tracers is explored.

Clinical cytometry in Europe, 2006

This year saw the transformation of the European Working Group on Clinical Cell Analysis (EWGCCA) into the European Society for Clinical Cell Analysis (ESCCA; www.escca.org). EWGCCA was established in 1996 in order to establish collaboration between large European centers, using (flow) cytometric cell analysis for clinical purposes. Being supported by a “concerted action” grant of the European Union 4th Framework Programme (1996–2000), EWGCCA has performed multicenter standardization trials and produced consensus documents in order to advance and standardize novel techniques for clinical cell analysis (1-11). With time, the activities of the group shifted from experimental to educational, and as such, EWGCCA has organized five annual Euroconferences (2001–2005) and eight workshops (1998–2005). Since 2005, the workshops have been organized as interactive sessions within the Euroconferences. Here, controversial issues in clinical cytometry were introduced by speakers with opposing points of view, followed by a debate in which the audience participated via an “interactive voting system.” Furthermore, EWGCCA has facilitated in 2004 the organization of the MASIR conferences: small European meetings held every 18 months and highly focused on “measuring antigen-specific immune responses” (www.masir.org; Refs.12 and13). Inspired by the Clinical Cytometry Society (CCS), EWGCCA took on the organization of an annual European Course in Clinical Cytometry preceding the Euroconference from 2005 onwards. In 2002, EWGCCA was legally established as a foundation under Dutch law. As the workshops and Euroconferences consistently attracted some 200 paying participants per year, the EWGCCA board decided to transform the foundation into a society with a council that is democratically elected by its members: ESCCA. The closed structure of the EWGCCA foundation did not fit with the need of a more widespread membership and did not allow the periodic renewal of its board by election. The goals of ESCCA are similar to those of EWGCCA. In addition, a close collaboration with CCS and the existing national European societies in the field of (clinical) cytometry as well as similar groups elsewhere in the world are considered of key importance. The 6th Euroconference on Clinical Cell Analysis was held in Stresa (Italy) from 14–16 September 2006, and preceded by the 2nd European Course in Clinical Cytometry (12–13 September). The three major areas addressed by the Course (and also by the Conference) were: (i) functional analyses of the immune system, with particular attention to HIV infection; (ii) diagnosis of clonal hematopoietic disorders; and (iii) minimal residual disease of hematological malignancies. While introductory lectures addressed instrument set-up and calibration, as well as technical aspects of multicolor flow cytometry, the students were offered theoretical lectures on the above three areas, and practiced list mode data analysis of various cases using computers equipped with software of the major instrument manufacturers.1 As for the Euroconference, the table gives an overview, by subject, of the abstracts by the invited speakers and the proffered peer-reviewed abstracts by the participants. Like most cytometry conferences, this Euroconference covers a fairly wide range of topics as technology is its common theme. However, the lack of focus is a potential weakness, and future Euroconferences will therefore center on only 3–4 main areas per congress. As the scientific organizing committee, we are very pleased with the financial support of our industrial partners, and with the opportunity offered by CCS and Clinical Cytometry to publish the abstracts that are shown in the following section. Jan W. Gratama*, Maria Arroz , Bruno Brando , Ingmar A. Heijnen?, Claudio Ortolani?, Stefano Papa**, * Erasmus MC Rotterdam, The Netherlands, Hospital de Egaz Moniz Lisbon, Portugal, Ospedale di Legnano Legnano, Italy, ? Kantonsspital Aarau, Switzerland, ? Ospedale SS. Giovanni e Paolo Venice, Italy, ** Università di Urbino Urbino, Italy.

Feasibility of Multicentric Assessment of MRD in All by Flow-Cytometry: Standardization and Experience of the I-BFM-ALL FCM-MRD Study Group.

Detection of minimal residual disease (MRD) in ALL using flow cytometry (FC) is feasible in nearly 100% of patients (pts.) and gives superior prognostic information. This has been evidenced by several single-laboratory studies. It is, however, not known to date whether FC MRD detection can reliably be standardized, so that multiple laboratories could collaborate to provide MRD information of similar quality in large and international treatment trials. On the basis of the current AIEOP-BFM-ALL 2000 trial, which comprehensively includes children with ALL from Austria, Germany, Italy, and Switzerland, a multicenter observational study of FC-MRD was initiated in 2000 in 4 collaborating laboratories. Until 2004, n=395 pts. have been prospectively recruited to this investigation (consecutive per center). Samples for evaluation were blood (PB) and bone marrow (BM) from early treatment time-points (diagnosis, days 8, 15, 22, 33, 52, 78). Lineage-specific, standardized 3-(5)-tube staining panels were based on 4-color FC. To foster inter-laboratory standardization, an extensive quality program was launched. This included full-staff meetings twice yearly (including list-mode data review), personnel exchange, assessment of concordance in list-mode data analysis, and sample exchange. We report now on results of the latter two issues. List-mode data files from 93 samples (from 15 pts., i.e. (3)-4 consecutive pts. per center selected by a certain time-point) were shared between all 4 centers for parallel analysis. By peer-review, 48/93 samples (51,5%) were finally regarded as MRD-positive. Concordance of all 4 centers in positive-negative estimates was reached in 71% of samples (66/93), and of at least 3 centers in 95,7% (89/93). In MRD-positive samples, concordance in quantitative estimates (<5x- difference) from all 4 centers was evident in 70,8% (34/48), and from at least 3 centers in 81% (39/48). Lowest concordance was reached in samples with very low amounts of MRD (<10−4). Surveying the available 369 single MRD-results (data from 93 samples x 4 centers), 341 (92,4%) were correct regarding positive-negative estimates (range by centers: 88,0 – 97,8%). From 174 positive MRD-measures, correct log-levels were documented in 167 (96%) overall (range by centers: 93,2 – 97,8%). Sample exchange (n=38 follow-up samples from 10 consecutive pts.) by express mail yielded a 2-center concordance of 92% regarding positive-negative MRD-estimates. Quantitative results in MRD-positive samples (n=21 paired data sets) were highly concordant (in 86% <5x-difference) despite transportation, and differences in FC instrumentation and software usage. In conclusion, multicenter MRD-evaluation by FC in large treatment trials for ALL is feasible, but extensive quality standardization is necessary. Performance should be monitored throughout the trial period to warrant comparable results when assessed in multiple centers (Figure 1. Concordance: MRD-positive samples per series). [Display omitted]

Flow cytometric enumeration of antigen-specific T lymphocytes.

Until the second half of the 1990s, quantification of MHC-restricted, peptide-specific T lymphocytes required cell culture– based techniques of mononuclear cell suspensions that were often laborious, cumbersome, and prone to bias towards cellular subsets with growth advantage. Initially, limiting dilution assays were used. These assays include multiple days of culturing T lymphocytes in the presence of antigen and antigen-presenting cells, and the functional read-outs produced were T-cell proliferation (1) or cytolytic activity (2). However, such assays may not accurately reflect the function and frequency of T cells in vivo, due to selective proliferation and apoptosis that occur over time in culture. Alternatively, assays for T-cell activation have been developed based on the upregulation of the early activation marker CD69 (3) and/or the production of cytokines upon specific stimulation. The cytokines thus being secreted by the T cells can be measured

Lymphoma discrimination by computerized triple matrix analysis of list mode data from three‐color flow cytometric immunophenotypes of bone marrow aspirates

Background The goal of this study was to evaluate a self-learning algorithm for the computer classification of information extracted from flow cytometric immunophenotype list mode files from high-grade non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and multiple myeloma (MM). Materials and Methods Bone marrow aspirates (BMA) were obtained from untreated NHL (n = 51), HD (n = 9), or MM (n = 13) patients. Bone marrow aspirates were not infiltrated in NHL and HD patients as confirmed by thorough histologic and cytologic investigation; however, MM patients showed an infiltration rate >50% by malignant myeloma cells. Peripheral blood leukocyte (PBL) samples were taken from age-matched healthy volunteers (n = 44) as easily available control material. A second control group of 15 healthy volunteers, from whom BMA and PBL samples were available, allowed us to differentiate whether the observed classification results on malignant samples were due to the malignant process or simply to the inherent differences between BMA and PBL. Bone marrow aspirates and PBL were analyzed by the same immunophenotyping antibody panel (CD45/14/20, CD4/8/3, kappa/CD19/5, lambda/CD19/5). The acquired list mode data files were analyzed and classified by the self-learning triple matrix classification algorithms CLASSIF1 following a priori separation of the data into a learning set and unknown test set. After completion of the learning phase, known patient samples were reclassified and unknown samples prospectively classified by the algorithm. Results Highly discriminatory information was extracted for the various lymphoma entities. The most discriminating information was encountered in antibody binding, antibody binding ratios, and relative antibody surface density parameters of leukocytes rather than in percentage frequencies of discrete leukocyte subpopulations. Samples from healthy controls were classified as normal in 97.2% of the cases, whereas those of NHL, HD, and MM patients were on average correctly classified in 80.8% of the cases. Conclusions Although no detectable lymphoma cells were present in BMA of NHL and HD patients, the CLASSIF1 classification of the immunophenotypes of morphologically normal cells provided a surprisingly good disease discrimination equal or better than that obtained by examining pathological lymph nodes according to the respective literature. The results are suggestive for a lymphoma-related and disease-specific antigen expression shift on normal hematopoietic bone marrow cells that can be used to discriminate the underlying disease (specificity of unspecific changes), i.e., in this case NHL from HD. Multiple myeloma patients were discriminated by changes on malignant as well as on normal bone marrow cells. Cytometry 41:9–18, 2000 © 2000 Wiley-Liss, Inc.

Lymphoma discrimination by computerized triple matrix analysis of list mode data from three-color flow cytometric immunophenotypes of bone marrow aspirates

The goal of this study was to evaluate a self-learning algorithm for the computer classification of information extracted from flow cytometric immunophenotype list mode files from high-grade non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and multiple myeloma (MM). Materials and Methods Bone marrow aspirates (BMA) were obtained from untreated NHL (n = 51), HD (n = 9), or MM (n = 13) patients. Bone marrow aspirates were not infiltrated in NHL and HD patients as confirmed by thorough histologic and cytologic investigation; however, MM patients showed an infiltration rate >50% by malignant myeloma cells. Peripheral blood leukocyte (PBL) samples were taken from age-matched healthy volunteers (n = 44) as easily available control material. A second control group of 15 healthy volunteers, from whom BMA and PBL samples were available, allowed us to differentiate whether the observed classification results on malignant samples were due to the malignant process or simply to the inherent differences between BMA and PBL. Bone marrow aspirates and PBL were analyzed by the same immunophenotyping antibody panel (CD45/14/20, CD4/8/3, kappa/CD19/5, lambda/CD19/5). The acquired list mode data files were analyzed and classified by the self-learning triple matrix classification algorithms CLASSIF1 following a priori separation of the data into a learning set and unknown test set. After completion of the learning phase, known patient samples were reclassified and unknown samples prospectively classified by the algorithm. Highly discriminatory information was extracted for the various lymphoma entities. The most discriminating information was encountered in antibody binding, antibody binding ratios, and relative antibody surface density parameters of leukocytes rather than in percentage frequencies of discrete leukocyte subpopulations. Samples from healthy controls were classified as normal in 97.2% of the cases, whereas those of NHL, HD, and MM patients were on average correctly classified in 80. 8% of the cases. Although no detectable lymphoma cells were present in BMA of NHL and HD patients, the CLASSIF1 classification of the immunophenotypes of morphologically normal cells provided a surprisingly good disease discrimination equal or better than that obtained by examining pathological lymph nodes according to the respective literature. The results are suggestive for a lymphoma-related and disease-specific antigen expression shift on normal hematopoietic bone marrow cells that can be used to discriminate the underlying disease (specificity of unspecific changes), i.e., in this case NHL from HD. Multiple myeloma patients were discriminated by changes on malignant as well as on normal bone marrow cells.

Reduction of interlaboratory variability in flow cytometric immunophenotyping by standardization of instrument set-up and calibration, and standard list mode data analysis

Two workshops addressed the question to which degree standardization of instrument set-up and calibration, and standard list mode data analysis would reduce interlaboratory variability of flow cytometric results on prestained peripheral blood mononuclear cells (PBMC). Standard instrument set-up included uniform positioning of the "windows of analysis" for the forward and sideward light scatter and fluorescence (FL) 1 (i.e., fluorescein isothiocyanate [FITC]) and 2 (i.e., phycoerythrin [PE]) parameters. Reference standards and PBMC, double-stained with FITC- and PE-conjugated monoclonal antibodies covering a wide range of FL intensities and coexpression patterns, were sent out to 25 laboratories in Workshop 1 and to 35 laboratories in Workshop 2 with the following requests: a) to set up instruments according to local and standard protocols, b) to acquire list mode data on the PBMC with both instrument settings, and c) to analyze both datasets according to local protocols. Standard analysis of the list mode data acquired with uniform instrument settings was performed centrally using so-called "latent class model" software (Van Putten et al., Cytometry 14:86-96, 1993). This software provides an automated, "no-gating" analytical method of lymphocyte immunophenotypes and employs fixed FL marker settings as defined prior to each analytical run. In Workshop 1, these markers were set in identical histogram channels for all instruments based on results obtained with a reference instrument. Standard analysis of list mode data acquired after uniform instrument set-up led only to a 13% reduction of interlaboratory variability of results as compared to data analysis using local protocols. The standard protocol for instrument set-up led to uniform positioning of relatively strong FL signals but variable positioning of unstained cells on the FL histogram scales. Hence, standard FL marker settings were inappropriate for some instruments. Therefore, instrument responses to FITC and PE signals in Workshop 2 were calibrated using microbeads labeled with FITC or PE in a range of predefined FL intensities expressed in MESF units (molecules of equivalent soluble fluorochrome). That approach allowed the positioning of the FL markers for the standard analysis on the basis of identical FL1 and FL2 intensities, expressed in MESF units, for all instruments. Standard analysis of list mode data acquired after uniform instrument set-up and calibrated FL marker settings led to a 43% reduction of interlaboratory variability as compared to data analysis to local protocols. We conclude that standard list mode data analysis using fixed FL marker settings reduces the interlaboratory variability of flow cytometric results on prestained PBMC, provided that the instruments have been set up in a uniform way and that FL markers have been standardized on the basis of calibration of each instrument's response to the corresponding FL signals.

Reduction of interlaboratory variability in flow cytometric immunophenotyping by standardization of instrument set‐up and calibration, and standard list mode data analysis

Two workshops addressed the question to which degree standardization of instrument set-up and calibration, and standard list mode data analysis would reduce interlaboratory variability of flow cytometric results on prestained peripheral blood mononuclear cells (PBMC). Standard instrument set-up included uniform positioning of the “windows of analysis” for the forward and sideward light scatter and fluorescence (FL) 1 (i.e., fluorescein isothiocyanate [FITC]) and 2 (i.e., phycoerythrin [PE]) parameters. Reference standards and PBMC, double-stained with FITC- and PE-conjugated monoclonal antibodies covering a wide range of FL intensities and coexpression patterns, were sent out to 25 laboratories in Workshop 1 and to 35 laboratories in Workshop 2 with the following requests: a) to set up instruments according to local and standard protocols, b) to acquire list mode data on the PBMC with both instrument settings, and c) to analyze both datasets according to local protocols. Standard analysis of the list mode data acquired with uniform instrument settings was performed centrally using so-called “latent class model” software (Van Putten et al., Cytometry 14:86–96, 1993). This software provides an automated, “no-gating” analytical method of lymphocyte immunophenotypes and employs fixed FL marker settings as defined prior to each analytical run. In Workshop 1, these markers were set in identical histogram channels for all instruments based on results obtained with a reference instrument. Standard analysis of list mode data acquired after uniform instrument set-up led only to a 13% reduction of interlaboratory variability of results as compared to data analysis using local protocols. The standard protocol for instrument set-up led to uniform positioning of relatively strong FL signals but variable positioning of unstained cells on the FL histogram scales. Hence, standard FL marker settings were inappropriate for some instruments. Therefore, instrument responses to FITC and PE signals in Workshop 2 were calibrated using microbeads labeled with FITC or PE in a range of predefined FL intensities expressed in MESF units (molecules of equivalent soluble fluorochrome). That approach allowed the positioning of the FL markers for the standard analysis on the basis of identical FL1 and FL2 intensities, expressed in MESF units, for all instruments. Standard analysis of list mode data acquired after uniform instrument set-up and calibrated FL marker settings led to a 43% reduction of interlaboratory variability as compared to data analysis to local protocols. We conclude that standard list mode data analysis using fixed FL marker settings reduces the interlaboratory variability of flow cytometric results on prestained PBMC, provided that the instruments have been set up in a uniform way and that FL markers have been standardized on the basis of calibration of each instrument's response to the corresponding FL signals. Cytometry 30:10–22, 1997. © 1997 Wiley-Liss, Inc.

Use of biocarrier beads and flow cytometry for single-cell studies of fibronectin gene regulation in dibutyrl cyclic AMP reverse transformed CHO-K1 cells

The protease sensitivity of a number of cell surface or cytoskeletal components and the relationship of these to the substratum in attached cells has prevented the quantitative measurement of their expression by flow cytometry. Using traditional cell sorting techniques, cells must be treated with a protease to detach them from a substrate in order to produce a single-cell suspension. Unfortunately, proteolytic treatment alters or destroys a number of cellular proteins. Fibronectin either on the cell surface or as part of the substratum laid down by the cell is particularly sensitive to proteases, preventing its quantitative study by flow cytometry. To circumvent these problems and produce a single cell suspension necessary for flow cytometric analysis, CHO-K1, a Chinese hamster ovary cell line, were grown in suspension on specially-treated 25 microns biocarrier beads. The CHO-K1 cell line is composed of transformed epithelial-like cells that have lost the fibronectin deposit around their cell membranes. To restore the typical fibroblastic deposit of fibronectin, the cells attached to beads were induced by dibutyryl cAMP to undergo a reverse transformation reaction to restore fibroblastic morphology and the typical fibroblastic deposite of fibronectin on the cell surface and substratum. The cells attached to beads were then immunofluorescently labeled for the protease-sensitive, extracellular matrix component, fibronectin, and examined on a flow cytometer. Cell surface fibronectin heterogeneity was then examined on a cell-by-cell basis as a function of cell cycle using Hoechst 33342 dye that binds to AT base pairs of cellular DNA. Dual laser measurement and multiparameter list mode data analysis were used to study the relationship between cell surface fibronectin of biocarrier bead attached cells and cell cycle.

The Application of a Small-Size Array Processor in List Mode Data Analysis

An ANALOGIC array processor AP400 has been connected to a PDP-11/44. To test the speeding-up characteristics of the AP400 used for list mode data analysis two monitoring routines of the HOOPSY-program system1 have been transferred to the array processor. Running these routines the AP400 was faster by a factor of 40 to 60 than the PDP-11/44.