Instructions for CPCTR TMA Block Production


Dr. Milton Datta, October, 2003

Logging in and Marking Specimens

  • Blocks are received from other facilities or from MCW. Blocks from other facilities were already be marked on both slides and tissue blocks to identify areas of tumor. Marking were performed by board certified surgical pathologists. Upon arrival at MCW all cases were re-reviewed by an additional board certified surgical pathologist and the markings checked before TMA production was initiated. If there are areas of benign prostate glands within the cancer, these areas were marked out so no punches were taken from those areas. If the cancerous area was small, a little arrow was placed to direct the punch.
  • The block edges are cleaned with a knife or razor blade.
  • Each block is labeled with a barcode containing the IMS number and the originating facility name and scanned into the block log. We make two copies of the block labels, one is placed on the block and one for on the working log sheet that is used when making the TMA block.
  • We write a TMA production number on the blocks starting with "1" through the final case number. These numbers are logged into the working log sheet and correlated with the CPCTR IMS number.
  • The corresponding IMS and production numbers are used to create the blockmap in Excel (see example #6) for visualizing the TMA.

    TMA Block Production

  • TMA production is accomplished using Beecher Tissue Microarrayer with the associated simple automated changes from the following reference:

    Matysiak BE, Brodzeller T, Buck S, French A, Counts C, Boorsma B, Datta MW,Kajdacsy-Balla AA. Simple, inexpensive method for automating tissue microarray production provides enhanced microarray reproducibility. Appl Immunohistochem Mol Morphol. 2003 Sep;11(3):269-73.

  • Detailed instructions are provided below as example protocol guidelines used in the TMA production:

    1. Setup the computer, and activate the motor controls (Motor 1 = X motor; Motor 2 = Y motor).
    2. Initialize the motors and tighten the set screws on the micrometers:
    3. Change the motor speed to 300 and Motor 1 to 4. Plug in machine. Hit run command. After it runs for about 3 seconds, hit run command and then stop command. Motor 1 should stop and motor 2 should continue running. Change motor 1 to 0 and change motor 2 to 4. Hit run command, then stop command and motor 2 should stop. Hit run command until you are able to see the set screw and tighten it comfortably. After motor 2 is set properly, change motor 2 to 0 and motor 1 to 4 and keep hitting run command until the motor 1 set screw is visible (preferably on the top).
    4. Manually wind the punch back to the beginning of the block (the lower left hand corner, array grid 1,1). Do not place the punch to close to the edge of the block or it will be hard to cut. Always align your punches using the RED punch; never the blue one. The recipient red one makes the holes and therefore is the aligning punch. After you have the punch position, press the "zero" key on the counter until it reads 0.00000 mm.
    5. Make your first punch with the recipient red punch. After that is done, empty the red punch by pushing down on the needle. Rotate the turning device to the donor blue punch and place the donor block table over the recipient block. Place donor block on the donor block table. Use the blue punch to take specimen punch from the marked areas.
    6. After the blue punch is complete, gently and slowly push the tissue down so a little bit of paraffin is showing from the donor punch. Then take a razor blade and cut away excess paraffin. This is done so that the donor tissue does not break while pushing it into the recipient block.
    7. After you have cut the excess paraffin, push the donor tissue core out all the way towards the recipient hole in the recipient block. Place the tissue about half way in the hole and lift up punch plate. Using the forceps handle, gently push the donor core the remaining way into the recipient core hole such that the top is flat and even with the recipient block. Remember to push the tissue down as far as you can without pushing too far into the block, or it will not get cut and will not be represented on the first few sections/slides produced from the TMA.
    8. Making and placement of the next TMA core. When this is complete, turn the value of motor 1 to 84 and press run command to take you to the coordinates of the next array space along the row (array 1,2). You can leave the value of motor 1 at 84 until you are done with the row (array 1,30).
    9. Completing the TMA cores for the row. Follow the above steps for remaining punches in the row, then you must manually return the row micrometer to the beginning of the row for the start of the next column (array 2,1-2,30). When you finish your row - change motor 1 to 4 and hit run command until you can see the setscrew to loosen it. Manually turn motor 1 back to the starting place on that row (array 2,1). The counter should be at "0" or around "0"when you are at the starting place in the row. You can be off by a little (+/- 2) with the wind back; it will not make a difference. After you are wound back the row micrometer, tighten the motor 2 set screw and set motor 2 to 180 and motor 1 to 0 and hit run command. Once you have the punch re-aligned, tighten the setscrew again and change the settings back to: Motor 1 = 0 and Motor 2 = 180 and press run command.
    10. Creation of the next row of cores in the TMA. Once you have the punch in position for the next row, change motor 1 to 84 and motor 2 to 0. Tighten the setscrew to motor 1 and leave motor 2 set screw tightened. The proceed as above - place a core hole in the recipient block with the recipient red punch, and using the donor blue punch cut a donor core, cut off excess paraffin, fully push donor tissue into the core hole.
    11. Repeat the punch steps until you are finished with the block

    Post-block processing
    1. Place block paraffin side down on a glass slide with the cores touching the slide and incubate at 37C for 15-20 minutes. Take block and slide out and gently push block against slide to flatten the cores for an even cutting surface. If you feel the block is not soft enough, put it back in the incubator for an additional few minutes. First try 3 minutes and continue in 3-minute intervals until you feel it is soft enough to mold a flat surface. Do not leave the block in for too long or you may melt the block and ruin the cores.
    2. Then place the block on an ice bath for about 20 minutes to separate the block from the slide. DO NOT pull the block off the slide before placing them in the ice bath, or the cores will pull out or tissue will be lost.
    3. After you remove the block from the slide you are ready to cut the block.