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4.4.5.3. Scan the Control TMA

  1. Take references for the microscope
  2. When a batch is finished staining we first check that the slides are designated in the Specimen_Table ([4.3.1](#431-specimen_table “Title”)).
    • If the slide is designated in the Specimen_Table_N; check that the ‘M’ numbers are written on the slides and that they are the correct numbers
    • If the slide is not designated in the Specimen_Table_N insert the required information and give the slides SampleNames corresponding to the next available number (again values should not be repeated even if a batch fails)
  3. Set up the scanning exposures (the ‘Protocol’) for the TMA in the proper <spath> folder
    • Name the protocol and the task list-> Control_TMA_XXXX_YYY
      • XXXX -> the TMA ID (ie. 1372)
      • YYY -> the TMA cut number (ie. 126)
      • Old scanning protocols will be located in any Clinical_Specimen folder under Protocols
  4. Name the control TMA slide -> Control_TMA_XXXX_ZZ_MM.DD.YYYY
    • XXXX is the TMA ID
    • ZZ is the TMA cut number
    • MM.DD.YYYY is the month, day, and year of when staining finished
  5. Scan the whole slide overview on the microscope
  6. Create an annotation scanning plan in Phenochart for the Vectra3
    • Open the TMA in Phenochart
    • Click Login at the upper left-hand corner
      • Type your name or initials
    • Click TMA in the upper middle of the screen
    • Change the plan so that the bottom right core is labeled [1,1]
    • Change the Imaging Size to 3x3
    • Draw the rectangle around all cores for Phenochart to locate the cores
    • To do this click and hold near one of the corners and drag across to the opposite corner (a search box should be displayed by Phenochart)
      • If the cores are mislabled, you need to delete the region and draw the rectangle again
      • You may need to draw several different rectangular regions to get the cores to be labeled properly
    • If a core is missing right-click the location of the core and select Add missing core and select the appropriate number
      • If any cores are completely missing from the slide, do not image them, but do image partial cores
    • Adjust the 3x3 images so that each box is centered over the TMA spot
    • Once you are satisfied click Accept Grid and close Phenochart
  7. Create an annotation scanning plan in Phenochart for the Vectra Polaris
    • Open the TMA in Phenochart
    • Click Login at the upper left-hand corner
      • Type your name or initials
    • Click TMA in the upper middle of the screen
    • Change the plan so that the bottom left core is labeled [1,1]
      • For TMA 1372 the rows should go from 6 tp 1 and the columns from 1 to 4
      • For TMA 1544 the rows should go from 11 to 1 and the columns from 1 to 9
      • For TMA 1642 the rows should go from 5 to 1 and the columns from 1 to 5
    • Change the Core size: to 2.0 mm
    • Draw a rectangle around all cores for Phenochart to locate the cores
    • To do this click and hold near one of the corners and drag across to the opposite corner (a search box should be displayed by Phenochart)
      • If the cores are mislabled, you need to delete the region and draw the rectangle again
      • You may need to draw several different rectangular regions to get the cores to be labeled properly
    • If a core is missing right-click the location of the core and select Add missing core and select the appropriate number
      • Add all cores regardless of if any cores are completely missing from the slide
    • Adjust the core images so that each circle is centered over the TMA spot
      • Adjust core location by holding down Ctrl and dragging the image
    • Once you are satisfied click Accept Grid and close Phenochart
  8. Select the Acquire MSI fields task for the TMA on the microscope and scan the slide
  9. Do the quality control of the TMA
    • Open images from three tonsil cores from a previous Control TMA in inForm
      • Be sure that the TMA cores came from the same cohort and from a Batch that worked
    • Open the fields that most closely match the three cores from the current TMA in inForm
    • Open the Algorithm with a current library, created for each Clinical Specimen Project
    • Prepare all images with the library
    • Compare the counts and the stain quality between the old and the new TMA
      • Click on the icon with the box next to a mouse pointer to compare the counts between the old and new TMAs
      • It is easiest to write down the Opal and its respective counts range on a sheet of paper to refer to when switching between old and new versions
      • The colors are always as follows
        • DAPI - Blue
        • Opal480 - Pink
        • Opal520 – Green
        • Opal540 – Yellow
        • Opal570 – Red
        • Opal620 – Orange
        • Opal650 – Cyan
        • Opal690 – Magenta
        • Opal780 - White
      • Note: Opal480 and Opal 780 are not used on the Vectra3 and should be omitted
      • Look to see if general patterns are the same and if intensities vary
      • It is usually easy to see differences visually and use counts as a sanity check for your eyes
      • Because the of the similarities in the cores and stains within a cohort the variation between slides should be minimal
  10. Give the TMA a BatchID.txt file and give it the next corresponding BatchID
  11. Fill in the BatchID on the Specimen_Table.xlsx if it is not already present