High-Content Flow Cytometry and Temporal Data Analysis for Defining a Cellular Signature of Graft-Versus-Host Disease.
Brinkman R.R et al., Biology of Blood and Marrow Transplantation
Acute graft-versus-host disease (GVHD) is diagnosed by clinical and histologic criteria that are often nonspecific and typically apparent only after the disease is well established. Because GvHD is mediated by donor T cells and other immune effector cells, we sought to determine whether changes within a wide array of peripheral blood lymphocyte populations could predict the development of GvHD. Peripheral blood samples from 31 patients undergoing allogeneic blood and marrow transplant were analyzed for the proportion of 121 different subpopulations defined by 4-color combinations of lymphocyte phenotypic and activation markers at progressive time points posttransplant. Samples were processed using a newly developed high content flow cytometry technique and subjected to a spline- and functional linear discriminant analysis (FLDA)-based temporal analysis technique. This strategy identified a consistent posttransplant increase in the proportion and extent of fluctuation of CD3+CD4+CD8[beta]+ cells in patients who developed GVHD compared to those that did not. Although larger prospective clinical studies will be necessary to validate these results, this study demonstrates that high-content flow cytometry coupled with temporal analysis is a powerful approach for developing new diagnostic tools, and may be useful for developing a sensitive and specific predictive test for GvHD.
Patient identification has been removed.
Identification of compounds that enhance the anti-lymphoma activity of rituximab using flow cytometric high-content screening. Gasparetto, M., et al., J Immunol Methods, 2004. 292(1-2): p. 59-71.
In this report, we describe a new flow cytometry technique termed flow cytometric high-content screening (FC-HCS) which involves semi-automated processing and analysis of multiparameter flow cytometry samples. As a first test of the FC-HCS technique, we used it to screen a 2000-compound library, called the National Cancer Institute (NCI) Diversity Set, to identify agents that would enhance the anti-lymphoma activity of the therapeutic monoclonal antibody rituximab. FC-HCS identified 15 compounds from the Diversity Set that significantly enhanced the ability of rituximab to inhibit cell cycle progression and induce apoptosis in lymphoma cells. The validity of the screening results was confirmed for several compounds using additional assays of cell proliferation, apoptosis and cell growth. The FC-HCS technique was relatively simple and reliable and could process up to 1000 samples/day on a single flow cytometer. The FC-HCS technique may be useful for a variety of applications including drug discovery, immunologic monitoring of patients, functional genomics studies and tissue engineering efforts.
Demonstration of how to use flowCore to analyze data from a 96 well plate experiment
The file contains 5 plates, staining description, and R scripts.
Large testing data sets from FACSAria and FACSVantage SE, created by BD FACSDiva (v5.0.1)
Large data sets for testing purposes; list mode data with up to a million of events in over 20 parameters.
Normal donor study
Over 8GB of data from a study of immune responses to infectious agents and cancer antigens among 40 healthy young adults
Fundamental intention is to measure immune responses to infectious agents and cancer antigens among healthy young adults. The antigenic stimuli include chronic and episodic in vivo stimuli (cytomegalovirus and flu, respectively), and clinically relevant stimuli for which we expect the donor pool to be unexposed (HIV and a set of cancer related antigens).
The tactic of the experiment was to add fresh peripheral blood mononuclear cells (lymphocytes and monocytes, PBMCs) to an array of antigenic stimuli and a set of controls, allow the cells to respond to the stimuli for 6 hours, then measure the responses and the cell types of the responders by staining the samples with a set of fluorescent antibodies and acquiring data on a four color flow cytometer. In detail, each PBMC sample was distributed in 96 stimulation wells
in a microtiter tray then stained with one of two 4 color antibody cocktails. 40 such PBMC samples each generated 96 flow cytometry list files. Detailed description is included in the zip file.