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4.4.8. MergeConfig Tables

Merge Configuration file is a csv spreadsheet which indicates how the markers will be analyzed and merged to a single coordinate system where only one phenotype call exists for each cell. Columns are defined below, column names are case senstitive. Mark fields that do not have a designation as NA in character columns and 0s in numeric columns. Additional information on the merge can be found in the MaSS documentation here.

Figure 2 Image

  • BatchID[int]: indicates which batch the slides were stained in. This variable is used a record keeping method and does not indicate settings in the merge code
  • Opal[int]: should be the name of the Fluor which targets a given antibody a. stains should be added in numeric order starting with DAPI
  • Target[string]: name if the antigen the applied antibody is targeting
    • name does not need to be very technical by should unique
    • the name should also be used for:
      • the opal labels in inForm
      • the positive phenotype for each marker
      • the folder for each of the separate inForm outputs
      • Exception: the tumor marker (also designated in ImageQA)
        • For this marker, use 'Tumor' to desgnate the output folder
        • Optionally: use 'Tumor' when desgniating that antibody in inForm.
        • Must use same name for phenotype and opal namings
  • Compartment[string]: The cell compartment from the inForm tables to use when loading in the database. Options include EntireCell, Membrane, Nucleus, and Cytoplasm
  • TargetType[string]: 'Lineage' or 'Expression'
    • 'Lineage'
      • use this to denote markers that define the cell type\ function (e.g. Macrophages, TCells, Tumor cells, Tregs)
    • 'Expression':
      • Markers that may be expressed at varying levels on many given cell types (e.g. PD1, PDL1) and will not affect the phenotype call of other cells
  • CoexpressionStatus[float]: different for Expression and Lineage phenotypes
    • Exrpression markers: input the opal dyes of the lineage marker(s) it predominately co-expresses with
      • e.g. PD1(650) may co-expresses predominantly with CD8 (540) and FoxP3 (570), add 540,570 to this input for PD1
    • Lineage markers: input the opal dyes of other Lineage marker(s) that will be allowed to co-express with the specified marker
      • e.g. if CD8+ (540) - FoxP3+ (570) cells are accepted; in the CD8 row add “570” for FoxP3 and in the FoxP3 row add “540” for CD8
  • SegmentationStatus[int]: This is a numeric value; 1-X for the different types of segmentation that may exist, each marker with the same number should be processed with the same cell segmentation algorithm in inform
    • e.g. for a specified panel all markers may have the same segmentation and would have (1)s except Tumor which would have (2)s
    • The primary segmentation (1) should be the more reliable algorithm and usually correspond to smaller cells
    • “Other” cells will be defined by the primary segmentation (1)
  • SegmentationHierarchy[int]: For Lineage markers only; create an order of phenotypes/ segmentation you believe will be most accurate – this is primarily based off of cell morphology
    • Use NA for expression markers
    • The code will remove cells according to this column
    • lower cells take precedence over higher number cells when they collide
    • e.g.: A cell given a positive phenotype for CD8 in the CD8 algorithm and positive for CD163 in the CD163 algorithm; if CD8 (1) is ranked higher than CD163 (2) then the CD163 designation will be removed
    • Only cells that are allowed co-expression should have the same number. If two cells co-express with the same cell but not each other (CD4-FoxP3 and CD8-FoxP3); use the higher number for one of the two cells (CD4 or CD8) and the double co-expressed cell (FoxP3). Use a lower number for the other cell (CD4 or CD8). In the example with CD8-CD4-FoxP3 we usually use the numbers 1-2-1. CD4-FoxP3 will still be found based off of co-expression status but CD8-FoxP3 cells will take precedence.
  • NumberofSegmentations[int]: this value indicates how many segmentations were used for each antibody
    • as of the current update this setting is only supported on markers designated as ‘expression’
    • lineage cells that are from separate segmentations but are allowed to coexpress will create unexpected results
  • ImageQA[string]: This column allows a user to set 2 different conditions of the batch
    • 'Immune': The user can set one cell type as the ‘Immune’ cell
    • Fields with the highest density of this cell type will be selected by the CreateImageQAQC algorithm to be assessed
    • there needs to be at least one and only one of these designations for the MaSS code to work
    • 'Tumor': The user can set one cell type as ‘Tumor’
    • This is an optional field and will tell the code to narrow down the QA to fields to only include those with more than 60 of this cell type
    • There can be only one cell of this type
    • Only set one marker as ‘Immune’ and only one marker as ‘Tumor’
  • Colors[string]: specify the colors for the create image qa qc protocol. Colors can be specified by the color name or abbreviations below.
    • Options: 'red','green','blue','cyan', 'magenta','yellow','white','black','orange','coral'
    • Abbreviations: 'r','g','b','c','m','y','w','k','o','l'