Flow cytometry data processing
From MIBBI
4. Data Analysis Details If data analysis has been performed the following details shall be specified: 4.1. List-mode Data File The list-mode data files (e.g., FCS files [18]) shall be provided directly or details on how they may be requested shall be stated. 4.2. Compensation Details 4.2.1. Compensation Description A description of the type of compensation used shall be included (e.g., no compensation, hardware compensation, computed compensation) and the spillover or compensation matrix shall be provided when possible (i.e., it may not be available for old data but shall be provided when available). While the spillover matrix is preferred, the compensation matrix is also acceptable. The type of the matrix (i.e., spillover vs. compensation) shall be explicitly stated. 4.2.2. Other Relevant Compensation Information Additional relevant compensation details shall be provided and may include information such as the FMO control. ISAC Recommendation MIFlowCyt - Minimum Information about a Flow Cytometry Experiment Version 1.0 / Feb 21st, 2008 14 4.3. Data Transformation Details The following shall be described for each data/parameter transformation performed during analysis when the transformation does not qualify as compensation (4.2): 4.3.1. Purpose of Data Transformation The purpose of each performed transformation shall be specified (e.g., data visualization, background correction, statistical analysis, quantitative flow cytometry, etc.). 4.3.2. Data Transformation Description Either the exact mathematical formulas/algorithms of each data transformation shall be supplied using an open and freely available specification, or a description of each transformation shall be provided. 4.3.3. Other Relevant Data Transformation Details Other relevant information about data transformation should be provided and may include specification of software (e.g., name, version, operating system), analysis date, and graphical visualization of the transformation process, which is especially essential for stepwise transformations such as used in quantitative cytometry, i.e., transformation from measured voltage to count of photons, to count of reporter molecules, to count of detector molecules, to count of analytes. 4.4. Gating (Data Filtering) Details Gating, or data filtering, is a process in flow cytometry in which a subset (subpopulation) of a larger set (population) is defined phenotypically. Gating significantly impacts all statistical and analytical results and thus it is crucial that all the gates be exactly mathematically described (e.g., using Gating-ML [9]). In case the exact gating/filtering description cannot be produced (e.g., software is incapable of exporting an exact description, unknown gate boundaries, probabilistic filtering algorithms, clustering analysis, etc.) detailed membership information should be provided for each gate/subpopulation. This should consist of a complete list of events within each particular subpopulation. The following information about gating shall be provided, or it shall be specified that no gate was applied: 4.4.1. Gate Description The subpopulation identified by the gate shall be briefly described (e.g., “IL-4 producing helper T cells of the CD3+CD4+ phenotype”). The gating strategy or a reference to where it is described in detail (e.g., a manuscript) should be provided. 4.4.2. Gate Statistics Percentage of events within the gate shall be provided specifically stating the denominator. The denominator shall be either the total population of ISAC Recommendation MIFlowCyt - Minimum Information about a Flow Cytometry Experiment Version 1.0 / Feb 21st, 2008 15 events (e.g., percentage of lymphocytes based on the total number of events) or another gate (e.g., percentage of CD4+ lymphocytes based on all lymphocytes). When the denominator is another gate, this shall also be exactly specified in recursive fashion so that the gating strategy can be followed up to the original data set. The denominator may not necessarily be a containing (superset) population for some statistics (e.g., the donor / host blood cell ratio). 4.4.3. Gate Boundaries Either the exact mathematical descriptions of each gate boundary shall be provided using an open and freely available specification, or this information shall be provided in the form of images. 4.4.4. Other relevant gate information Other descriptive statistics may be provided including the count of events, arithmetic mean, mode(s), median, coefficient of variation (CV), minimum value, maximum value, standard deviation, etc. A description of the relative intensity of staining of the markers defining the subpopulation identified by the gate may also be provided (e.g., CD3 negative, dim, moderate, or bright) [20]. A qualitative description of the subpopulation (e.g., between first and second log decade) and a reference (e.g., publication, individual, or other) for the definition of the qualitative descriptor may also provide useful information.
Notes
- No notes
Sources
- MIFlowCyt
