|Elizabeth J. Cochran, MD|
I was recently contacted by the affable Elizabeth Cochran, MD asking if I would post a query regarding MIB-1 counts on brain tumors. Does anyone know if there is a standardized approach to this? Please tell us your approach to MIB-1 quantification in the comment section. Dr. Cochran takes an approach based on an article she had read in Human Pathology some time ago, summarized as follows:
1. Examine control section stained with MIB-1 antibody to confirm that the stain worked.
2. Survey the slide of the case to be counted to find the area(s) that have the most stained nuclei.
3. Put reticule in right eyepiece of microscope.
4. Place the area identified in #2 under the reticule at 40x power.
5. Using the hand held counter, count all the immunoreactive nuclei within the entire grid.
6. Count all nuclei that touch the right and bottom outer border of the grid, but NOT the nuclei that touch the upper and left borders.
7. Record this number in the lab book.
8. Count all nuclei present within one vertical column of the grid. This column should be randomly chosen.
9. Record this number in the lab book.
10. Count all nuclei present within one horizontal column of the grid. Again this column should be randomly chosen, and recorded in the lab book.
11. Again, be sure to count all nuclei that touch the right and bottom outer border of the grid, but NOT the nuclei that touch the upper and left borders
12. Repeat this procedure for at least two more high power fields on the section that contains the most numerous immunostained nuclei and record all values in the lab book.
13. Calculate the MIB-1 proliferative index as follows:
a. For each of the fields counted: Add all of the nuclei counted in each row and column from each field and multiple by five. Divide the number of the immunostained nuclei counted in that field by the calculated sum of all of the nuclei in that field.
b. The total number of all cells counted in all fields should be > 1000. If it is substantially less than 1000, count another field, as described above.
c. Calculate the average of the three fields and record the proliferative index in the lab book.