A Survey of Flow Cytometry Data Analysis Methods <s> Quality Assessment. <s> Highthroughput cell-based assays with flow cytometric readout provide a powerful technique for identifying components of biologic pathways and their interactors. Interpretation of these large datasets requires effective computational methods. We present a new approach that includes data pre-processing, visualization, quality assessment, and statistical inference. The software is freely available in the Bioconductor package prada. The method permits analysis of large screens to detect the effects of molecular interventions in cellular systems. <s> BIB001 </s> A Survey of Flow Cytometry Data Analysis Methods <s> Quality Assessment. <s> 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+CD8beta+ 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. <s> BIB002 </s> A Survey of Flow Cytometry Data Analysis Methods <s> Quality Assessment. <s> Heterogeneity in the differentiation behavior of hematopoietic stem cells is well documented but poorly understood. To investigate this question at a clonal level, we isolated a subpopulation of adult mouse bone marrow that is highly enriched for multilineage in vivo repopulating cells and transplanted these as single cells, or their short-term clonal progeny generated in vitro, into 352 recipients. Of the mice, 93 showed a donor-derived contribution to the circulating white blood cells for at least 4 months in one of four distinct patterns. Serial transplantation experiments indicated that two of the patterns were associated with extensive self-renewal of the original cell transplanted. However, within 4 days in vitro, the repopulation patterns subsequently obtained in vivo shifted in a clone-specific fashion to those with less myeloid contribution. Thus, primitive hematopoietic cells can maintain distinct repopulation properties upon serial transplantation in vivo, although these properties can also alter rapidly in vitro. <s> BIB003 </s> A Survey of Flow Cytometry Data Analysis Methods <s> Quality Assessment. <s> Suspensions of multipotent hematopoietic stem cells with long-term repopulating activity can now be routinely isolated from adult mouse bone marrow at purities of 30%. A robust method for obtaining these cells in a single step using multiparameter cell sorting to isolate the CD45(mid)lin(-)Rho(-)SP subset is described here, together with a detailed protocol for assessing their regenerative activity in mice transplanted with single cells. These procedures provide unprecedented power and precision for characterizing the molecular and biological properties of cells with hematopoietic stem cell activity at the single cell level. <s> BIB004
The basis of the quality assessment method proposed in  BIB002 is that, given a cell line, or a single sample, divided in several aliquots, the distribution of the same physical or chemical characteristics (e.g., side light scatter (SSC) or forward light scatter (FSC)) should be similar between aliquots. To test this hypothesis, five distinct visualization methods were implemented to explore the distributions and densities of ungated FCM data: Empirical Cumulative Distribution Function (ECDF) plots, histograms, boxplots, and two types of bivariate plots. Hahne et al. BIB001 also propose a set of visualization tools to inspect box plots of fluorescent values, number of cells, and a measure defined as "odds ratio" for similar samples within a plate. These different graphical methods provide investigators with different views of the data and can quickly flag the samples that are different from the rest. As the flagged samples may be anomalous for biological reasons, these samples are worth studying further, and some determination as to whether the sample presents data quality issues or rather presents real biological significance should be made  . Problems with the cell suspension, clogging of the needle, or similar issues can cause unusual patterns in the data. flowQ R package  addresses such problems by developing several approaches that detect disturbances in the flow of cells and also detect unusual patterns in the acquisition of fluorescence and light scatter measurements over time. These are detected dynamically by identifying trends in the signal intensity over time or local changes in the measurement intensities. The underlying hypothesis is that measurement values are acquired randomly; hence there should not be any correlation to time. Other quality assessment strategies may include investigating the number of events or the number of live cells within a sample. Furthermore, specific statistical tests addressing quality assurance requirements of an experiment can be developed. For example, in the FCM experiments to monitor clonal repopulation of engrafted single cell hematopoietic stem cells in mice BIB003 BIB004 , blood samples are taken and divided into three aliquots. Each aliquot is stained with cocktail specific for detecting granulocytes/monocytes, B cells, and T cells. The percentages of each cell type from the donor population should add to roughly 100%; otherwise possible problems with the staining or the gating have occurred. Using such criterion, automated quality assurance tools can be developed to identify possible problems in the experiments.