Phase 2 of Ki67 project: Instructions on how to standardize your scoring pattern for this study of Ki67 assessment on TMA cores.

Transcription

1 Phase 2 of Ki67 project: Instructions on how to standardize your scoring pattern for this study of Ki67 assessment on TMA cores. 1

2 Please use the nuclei counter web application when scoring (described on pg pages of these instructions). Please use the pattern & number of nuclei shown at lf left. Please sequentially score every definite invasive cancer nucleus in the search area. The website contains H&E images to assist you. Please do NOT score in clumps or clusters. This does not mean to avoid clumps or clusters of cells, but rather to score across them in a typewriter pattern. The layout of the TMA and its representation in the counter software will make it clear which core to score first. 2

3 start here (nucleus #1) X stop when 250 nuclei counted stop when 500 nuclei counted X start here (nucleus #251) 3

4 H&E images of each core are provided on the website (see below). You may find it helpful to keep the H&E image open while you score the core. 4

5 Note on the following slides: Slides 6-11 below show marked images from the calibration website. Although Phase 2 is on glass slides, these images are provided d here to help you standardize your assessment pattern and the intensity threshold you use while scoring on the glass slides in Phase 2. 5

6 Correct scoring pattern 6

7 Scoring in clumps and clustersleadsto leads to higher inter observer variability. Wrong scoring pattern 7

8 Do not score DCIS. WRONG Correct (DCIS not scored) 8

9 What counts as positive? Positive = Any definite brown staining in the nucleus of an invasive breast cancer cell, above the surrounding background in cytoplasm and extracellular matrix. Example images of positive ii and negative follow. 9

10 Examples of range of staining levels that should be considered POSITIVE (red squares) for Ki67 (unmarked & marked side by side) Positive nuclei are ordered from weakest to strongest brown stain. 10

11 Examples of range of staining levels that should be considered NEGATIVE (green circles) for Ki67 (unmarked & marked side by side) Negative nuclei are ordered from weakest to strongest blue stain. 11

12 Nuclei counter web application Please use the nuclei counter web application shown in the figure on the next slide. (The web link will be sent to you.) Before you begin, please practice on the mini TMA i of three breast cancer cores stained for Ki67 that will be provided in the package shipped to you. IMPORTANT: The web page will time out in THREE HOURS if there is no save activity. Therefore, please SAVE your progress periodically to avoid webpage time out and data loss. You can go back to edit your scoring on a previous core by clicking "Show previous image above Nuclei count commands. But remember to click SAVE nuclei counts to save your work before you do so. If you find the nuclei counter is not working, make sure you have the latest Java download, found by clicking the "Free Java Download" button at 12

13 Nuclei counter: Practice session interface shows the number of remaining TMA cores View H&E image Remember to save your work [1] test sound [2] shows the location of the TMA core to be scored [3] text box for cell counter commands. Cursor has to be placed in this box for counter to work (producing sounds and tracking counts). (See instructions on next page.) [5] click to save score and proceed to next TMA core [4] text box for optional comments 13

14 Nuclei counter web application instructions To enter nuclei counts data, please follow these steps: 1. There are 3 commands available for the cell counter, namely: d for positive nuclei count s for negative nuclei count z for undo a single nucleus count 2. Each command will produce a unique sound. Another unique sound will be produced when the total nuclei count reaches 250 and Please make sure your computer is able to produce these sounds by using the buttons, indicated by [1] in the picture, to test the sounds. 4. The location of the TMA core to be scored is indicated by the text description of the TMA core as well as the sector map image, both indicated by [2] in the picture. Please go to this core on the glass slide. 5. Type the cell counter commands (s, d, or z) in the text box indicated by [3] in the previous slide. Your cursor has to be placed in this box for the counter to work, producing sounds and tracking counts. You will be prompted when the count reaches 250 and If you would like to make any comments regarding a particular TMA core, please do so in the text box indicated by [4] in the picture. 7. Once you are satisfied with your score and (optional) comments, click on the button indicated by [5] in the picture, to proceed to the next core. 14

15 Nuclei counter: Interface for the phase 2 scoring Remember to save your work [3] text box for cell counter commands. Cursor has to be placed in this box for counter to work (producing sounds and tracking counts). (See instructions on previous page.) pg 15

16 IMPORTANT: Before you submit your scores and before you ship the slides to the next lab Once you complete the scoring & before you submit, please make sure all 50 cores are accounted for in the web application by doing the following: 1. Log back into your main page. 2. Click the "show report" link next to your nuclei count session. This report will show a summary of your scores. 3. Double-check to make sure that any cores listed as 0% have a comment noting why (for example, if they are unscorable) or, if it was accidentally skipped, that you go back to score it. 4. Samuel Leung at Samuel.Leung@vch.ca letting him know you are done. 16

17 Contact information If you have any questions or difficulties, please contact Sam Leung at: Phone: x62649 (Pacific Time) or Thank you. 17