Implementation of fully automated specimen processing and data acquisition for flow cytometric immunophenotyping
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Immunophenotyping
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Abstract Analysis of the immense complexity of the immune system is increasingly hampered by technical limitations of current methodologies, especially for multiparameter‐ and functional analysis of samples containing small numbers of cells. We here present a method, which is based on the stepwise functional manipulation and analysis of living immune cells that are self‐immobilized within microfluidic chips using automated epifluorescence microscopy overcoming current limitations for comprehensive immunophenotyping. Crossvalidation with flow cytometry revealed a 10‐fold increased sensitivity and a comparable specificity. By using small sample volumes and cell numbers (2–10 μl, down to 20,000 cells), we were able to analyze a virtually unlimited number of intracellular and surface markers even on living immune cells. We exemplify the scientific and diagnostic potential of this method by (1) identification and phenotyping of rare cells, (2) comprehensive analysis of very limited sample volume, and (3) deep immunophenotyping of human B‐cells after in vitro differentiation. Finally, we propose an informatic model for annotation and comparison of cytometric data by using an ontology‐based approach. The chip‐based cytometry introduced here turned out to be a very useful tool to enable a stepwise exploration of precious, small cell‐containing samples with an virtually unlimited number of surface‐ and intracellular markers. © 2008 International Society for Advancement of Cytometry
Immunophenotyping
Cytometry
Mass cytometry
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Sample (material)
Sample Preparation
Cytometry
Control sample
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Abstract Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.
Immunophenotyping
Identification
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Hematopathology
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Three- and four-color immunophenotyping is routine in traditional flow cytometry, as is measurement of cell proliferation, but there are drawbacks. The techniques cannot analyze cell morphology or permit restaining of cells of interest. This unit describes a slide-based method of immunophenotyping using a laser scanning cytometer. In general, many assays originally developed for flow can be adapted to LSC. Although the speed of analysis is comparatively slow, LSC has the advantage that cells are not lost. Considerable additional information can be obtained by morphological examination and/or by further staining because specimens can be repeatedly analyzed and archived. The method has potential to become a powerful tool in clinical diagnosis.
Immunophenotyping
Laser Scanning
Cytometry
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