MIFlowCyt 1.0 http://sourceforge.net/projects/flowcyt/files/MIFlowCyt/ Taken direct from the web-available version 1.0 (PDF) on 2011-06-05 by Chris Taylor (chrisftaylor@gmail.com). A MIFlowCyt-compliant experimental description shall include information specified below. The list states the content of the provided information only; it does not imply the format of the information or whether an item should be directly provided or referenced. 1. Experiment overview The experiment overview shall contain the following information 1.1. Purpose A brief description of the goal of the experiment. This should include the rationale and hypothesis. 1.2. Keywords The keywords should include terms from an appropriate vocabulary (e.g., MeSH) to describe the experiment. 1.3. Experiment Variables Variables are attribute(s) that differ between samples within an experiment due to pre-existing differences in sample states or due to experimental manipulation of the samples. If applicable, a brief description of the conditional and/or manipulated variables in the experiment shall be provided (e.g., smoker vs. nonsmoker, IL-2 treatment vs. no treatment, knockout versus wild-type, varying number of transplanted cells, varying treatment dosage, etc.). The number of instances per experimental group should also be stated. 1.4. Organization The following shall be specified for the organization performing the experiment 1.4.1. Name 1.4.2. Address 1.5. Primary contact The following shall be specified for the experimental primary contact. The description may include information for additional individuals involved in the experiment, including their contact details and their role. 1.5.1. Name 1.5.2. Email address 1.6. Date The date or time period during which the investigation was performed (i.e., from collecting and treating samples to performing data analysis) shall be stated. 1.7. Conclusions A brief summary of the interpretation of the results or outcome of the experiment shall be provided if available. 1.8. Quality control measures A description of the quality control measures used such as replicates, calibrations, control assays, etc. shall be provided. If another experiment was performed for the purposes of establishing quality control standards, that experiment may be referenced. 1.9. Other Relevant Experiment Information Additional information about the experiment should be provided if relevant. funding announcements, related publications (which should be referenced by PMID), URIs, databases 2. Flow Sample/Specimen Details The flow sample details shall include a description of each sample material used in the experiment (2.1), (2.2), how they were treated (2.3) and what reagents were used (2.4) to fluorescently label the material. Relations between samples, aliquots, different treatments, and replicates shall be unambiguously described. 2.1. Sample/Specimen Material Description Sample materials shall be described according to 2.1.1, 2.1.2, or 2.1.3, based on the type of the sample material. Each sample shall be distinguished from other samples within the same experiment. 2.1.1. Biological Samples The following information about Biological Samples shall be provided 2.1.1.1. Biological sample description A description of the biological sample shall be provided, including the sample type, if relevant. C57BL/6 spleen, purified CD4+ lymphocytes, BALB/c thymocyte DNA, control patient PBMCs, protein lysate from lung cancer biopsy, peripheral blood from patient with Type I diabetes, liver biopsy 2.1.1.2. Biological Sample Source Description The source of the biological sample shall be described. If the source is a cell line the description shall include its name, ATCC [http://www.atcc.org/] (or equivalent) number, and cell type. wildtype mouse, C57BL/6 spleen, C57BL/6 splenocytes 2.1.1.3. Biological Sample Source Organism Description 2.1.1.3.1. Taxonomy The source organism shall be specified by genus and species. The terms should come from an appropriate standard such as the NCBI taxonomy database. Taxonomy information should also contain the type of subspecies and organism strain, if applicable. A standard taxonomy may be extended or a proprietary taxonomy may be used, especially if detailed identification is relevant and beyond the scope of standard taxonomies. Mus musculus if applicable should http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Root 2.1.1.3.2. Age The age shall be provided if applicable for the particular organism. It may also include the developmental state. 6 weeks, Theiler stage 23 if applicable shall 2.1.1.3.3. Gender The gender shall be provided if applicable for the particular organism. 2.1.1.3.4. Phenotype Appropriate phenotype characteristics such as disease state shall be described. increased tumorigenesis 2.1.1.3.5. Genotype Appropriate genotype characteristics shall be described. p53-/- 2.1.1.3.6. Treatment All source organism treatments shall be described. treatment with cyclooxygenase-2 (COX-2) inhibitor 2.1.1.3.7. Other Relevant Biological Sample Source Organism Information Additional information about the source organism should be provided if relevant. This may include anatomic location of the source, visit time/date, or additional details and comments. Other Relevant Biological Sample Information Additional information about the biological sample should be provided if relevant. This may include relevant information such as in vivo or in vitro treatment, stimulation, preparation / enrichment protocol, culture/growth protocol, collection time/date, whether the sample was frozen/banked/fixed, etc. 2.1.2. Environmental samples The following information about environmental samples (e.g., soil, sea water) shall be provided 2.1.2.1. Environmental Sample Description A description about the environmental sample shall be provided. The description should include relevant details. collection time/date, collection protocol, type of sample (e.g., seawater) Environmental sample location The location of the sample origin shall be specified. This should include longitude and latitude if relevant. 2.1.3. Other samples The following information about other samples (i.e., samples not qualifying as Biological Samples or Environmental Samples, such as calibrator samples) shall be provided. 2.1.3.1. Other Sample Description A description of the sample shall be provided. The description shall include information about the nature of the material in the sample. dyed plastic beads 2.2. Sample characteristics Key information about the possible and expected sample characteristics should be noted as they provide the rationale for the experimental design, including the choice of appropriate reagents. Sample characteristics description should include the expected and possible types of cells or other particles in the sample material. 2.3. Sample treatment(s) description The description shall include details about treatment agents, which play the role of experiment variables, or shall specify that samples were untreated. For example, a sample treated with an agent might be compared to an untreated sample; when reproducing and interpreting such an experiment, having access to details on treatment agents and conditions is essential in order to query and retrieve samples for further analysis. The treatment description should also contain other relevant treatment details such as, but not limited to, time, temperature, and concentration. 2.4. Fluorescence Reagent(s) Description The expected and possible types of cells or other particles in the sample material, and their expected and possible measurable characteristics should be noted as these represent the key information for choosing appropriate reagents with respect to the experimental goal. The following information shall be provided about each fluorescence reagent used. Information about the characteristic(s) being measured, as well as details about what plays the role of analyte, analyte detector, and analyte reporter should be descriptive enough to allow for accurate interpretation of the experiment data. 2.4.1. Characteristic(s) Being Measured The relative amount of molecules, properties, or processes being evaluated (e.g., CD25, apoptosis, membrane permeability, cell viability, oxidative burst). The characteristic(s) being measured shall be provided whenever there is ambiguity about the analyte being measured, such as when sample processing affects detection of the analyte (e.g., propidium iodide example in Table 1). The optical detector (e.g., FL1) or parameter (e.g., FL1-H, see 3.3.6) used primarily for this measurement shall be indicated. 2.4.2. Analyte What plays the role of the analyte? Which substance or chemical constituent is the subject of interest of the analytical procedure, what target is specifically bound by the analyte detector (e.g., CD25)? 2.4.3. Analyte Detector What plays the role of analyte detector? Which component of the fluorescence reagent specifically binds to the analyte to make it detectable (e.g., anti-CD25 antibody)? 2.4.4. Analyte Reporter (Fluorochrome) What plays the role of analyte reporter? Which component of the fluorescence reagent reports the presents of the analyte to the flow cytometer? What substance (label) is used to generate the measured signal (e.g., FITC)? 2.4.5. Clone Name or Number If the probe is a monoclonal antibody, the clone name or number shall be provided. 2.4.6. Reagent Manufacturer Name The reagent manufacturer shall be specified. MIFlowCyt Reagent Inc. 2.4.7. Reagent Catalogue Number The reagent catalogue number shall be specified. 2.4.8. Other Relevant Reagent Information When secondary antibodies are used, the primary analyte (the analyte bound by the primary antibody) and the reporter component of the secondary antibody (the reporter that generates the measured signal) shall be clearly specified as described in 2.4.2, 2.4.3, and 2.4.4. If relevant, the primary and secondary antibodies shall be described individually (e.g., in the case that the use of a secondary antibody significantly impacts the interpretation of results because of nonspecific fluorescence, etc.). The description should also contain other relevant details which may include lot number, concentration, label incorporation method, separation technique, antibody staining procedures and wash steps. 3. Instrument details The following details shall be specified about instruments used to analyze samples. It is expected that this information will be automatically provided by flow cytometry instruments in instrumentation description files along with list-mode data files, such as FCS files [Seamer, L.C., et al., Proposed new data file standard for flow cytometry, version FCS 3.0. Cytometry, 1997. 28(2): p. 118-22]. For instrument components that are not user configurable and that are part of a standard model, the instrument manufacturer and model number shall be sufficient if this uniquely specifies the required information. For all other cases where the make and model number is not sufficient to specify the required information, the individual component description shall be specified as described below. 3.1. Instrument Manufacturer The instrument manufacturer shall be specified by stating its name. A URL pointing to manufacturer web pages may also be provided. MIFlowCyt Cytometry Systems, Inc. 3.2. Instrument Model The instrument model shall be specified. It may include the instrument serial number. CytMeter Elite-2000 3.3. Instrument Configuration and Settings The system and its configurations have significant impact on experimental results and as such the following shall be provided. 3.3.1. Flow cell and fluidics The flow cell of the instrument shall be described as follows. 3.3.1.1. Flow Cell Type The flow cell type including the material shall be provided. stream-in-air, cuvette 3.3.1.2. Other Relevant Flow Cell and Fluidics Information Other relevant flow cell details should be provided and may include flow cell manufacturer name (e.g., MIFlowCyt Optics, Inc.) and model number (e.g., PRO-CFC-75), flow cell outside shape (e.g., rectangular with an attached sphere, spherical, square) and dimensions, flow channel shape (e.g., rectangular, circular, triangular) and dimensions, sheath flow velocity (e.g., 10 l/s), sheath flow pump type (e.g., gas pressure, vacuum, gear), sample flow velocity (e.g., 7.5 l/s) and sample flow pump (e.g., gas pressure, syringe). Light source(s) Each light source (e.g., laser) shall be described stating the following. 3.3.2.1. Light Source Type The type of the light source shall be provided. laser, diode laser, xenon lamp 3.3.2.2. Light Source Excitatory Wavelength The excitatory wavelength shall be provided. 488 nm, 633 nm 3.3.2.3. Light Source Power at the Excitatory Wavelength The power of the light source shall be provided. If possible, this should be measured at the intersection of the light source beam with particles; see also 3.3.3. 180 mW, 50 mW 3.3.2.4. Light Source Polarization Polarization shall be specified. If possible, this should be specified at the intersection of the light source beam and particles; see also 3.3.3. linear, >100:1, in the direction of the sample flow 3.3.2.5. Light Source Beam Light source beam dimensions and geometry shall be specified at the flow cell or at the intersection of the beam with particles. elliptical 22 x 66 µm 3.3.2.6. Other Relevant Light Source Information Additional relevant light source details should be provided and may include light source manufacturer name (e.g., MIFlowCyt Lasers, Inc.), light source model name (e.g., 302C Krypton-Ion Laser), light source part number (e.g., I-326), noise (e.g., less than 1%), pointing and power stability (e.g., less than 1% over 1 hour), etc. 3.3.3. Excitation Optics Configuration If possible, the light source power (3.3.2.3), polarization (3.3.2.4), and beam profile (3.3.2.5) should be specified at the intersection of light source beam with particles. If this is not feasible for a particular researcher then details about the excitation optics shall be included to allow for an approximation of the light characteristics at the beam/sample intersection point. This description shall detail all components along the excitation optical path (i.e., from light sources to the flow cell). Each component shall be described stating its type (e.g., beam expander), manufacturer (e.g., MIFlowCyt Optics, Inc.), and model number (e.g., BE03M). 3.3.4. Optical Filters Each optical filter of the instrument shall be described stating the following. 3.3.4.1. Optical Filter Type The optical filter type shall be specified. band pass filter, long pass filter, dichroic long pass filters, short pass filter, dichroic short pass filter, polarizer excitation filter, grating, prism 3.3.4.2. Transmitted Wavelengths Light wavelengths transmitted by the filter shall be specified 488/25 nm, greater than 670 nm, less than 620 nm 3.3.4.3. Optical Filter Installation Date As optical filters of all types are subject to degradation, filter performance needs to be monitored at intervals to verify continued performance at an acceptable level [Kelley, K.A. and J.L. McDowell, Practical considerations for the selection and use of optical filters in flow cytometry. Cytometry, 1988. 9(4): p. 277-80]. The optical filter installation date shall be specified. January 15, 2007 3.3.4.4. Optical Filter Manufacturer The optical filter manufacturer shall be specified. MIFlowCyt Filter Inc. 3.3.4.5. Optical Filter Model Number The optical filter model number shall be specified model #1234 3.3.4.6. Other Relevant Optical Filter Information Other relevant optical filter details should be provided. 3.3.5. Optical Detectors Each optical detector (e.g., photomultiplier tube) shall be described stating the following. 3.3.5.1. Optical Detector Name The optical detector name shall be specified. FSC, SSC, FL1, FL2, FL3, FL4 3.3.5.2. Optical Detector Type The type of the optical detector shall be provide photodiode, photomultiplier tube 3.3.5.3. Optical Detector Voltage The optical detector voltage shall be specified. 300V, 700V 3.3.5.4. Optical Detector Amplification Type The optical detector amplification type shall be specified (e.g., linear, log). For log amplification, the number of decades shall be provided (e.g., 4 decades). For a linear amplification, the linear gain shall be provided (e.g., linear gain of 2.0). Additional details including the actual amplifier profile may be provided. 3.3.5.5. Other Relevant Optical Detector Information Additional relevant optical detector details should be provided and may include the optical detector manufacturer name and model number (e.g., MIFlowCyt Photonics Inc., PMT #R9220), minimal, maximal, and peak wavelength sensitivity (e.g., 185 nm, 900 nm, and 650 nm), the internal gain (e.g., 10^7), or the rise time (e.g., 2.2ns). 3.3.6. Optical Paths The full optical path shall be given for each measured parameter where applicable (i.e., excluding time). The optical path shall start with specification of the light source(s), which shall be followed by enumeration of all optical components (e.g., optical filters, beam splitters, mirrors, spectrometer, etc.) that contribute to the particular parameter. If nonimaging components are used, this shall be explicitly noted. The components shall be enumerated corresponding to their order along the light path in the instrument. The description shall include how components are used if relevant (e.g., passed vs. reflected light for a dichroic filter). The optical path description shall specify the optical detector used to measure the particular parameter. It shall also state whether the height, width, or area of the detected signal is used, and specify the threshold value if set. In addition, the collection angle shall be specified for the forward scatter detector. See Table 2 for an example of optical path components and Table 3 for an example of optical path details. As for other sections, we are not specifying the format for providing this information. 3.4. Other Relevant Instrument Details Additional relevant instrument details should be provided and may include machine-specific information such as information about automated agitation, temperature control, controlled volume dispensing, sampling from microtiter plates, auto-boost, auto-flush, etc. Other custom settings shall be provided if relevant and may include setting name, description, and value. 4. Data Analysis Details If data analysis has been performed the following details shall be specified. if applicable shall 4.1. List-mode Data File The list-mode data files (e.g., FCS files [ Seamer, L.C., et al., Proposed new data file standard for flow cytometry, version FCS 3.0. Cytometry, 1997. 28(2): p. 118-22]) 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. 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. data visualization, background correction, statistical analysis, quantitative flow cytometry 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 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) [ Borowitz, M.J., et al., U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: data analysis and interpretation. Cytometry, 1997. 30(5): p. 236-44]. 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.