IHC quantification

Immunohistochemical staining was evaluated on an Olympus BX53 light microscope at 100×, 200× and 400× magnifications and images were captured using an Olympus DP25 camera and Olympus CellSens Dimension software. All the immunohistochemical slides were semi-quantitatively analyzed and interpreted without knowledge of the histologic diagnosis by one person in order to eliminate interobserver bias in interpretation of the immunohistochemical results. These slides were analyzed at 200× using two different methods as follows:

Score of 0 = no staining; 1 = 1 % to 25 % positive; 2 = 26 % to 50 % positive; 3 = 51 % to 75 % positive; 4 = 76 % to 100 % positive.

An immunoreactivity score (IRS) was utilized as previously described [24, 28]. IRS values are calculated as the staining intensity value (0 to 3) multiplied by the estimated value of the percentage of positively stained cells. Percentage of stained cells was scored as: 1 = 10 % to 25 % positive; 2 = 26 % to 50 % positive; 3 = 51 % to 75 % positive; 4 = 76 % to 100 % positive. Intensity was scored as: 0 = no staining; 1 = weak staining; 2 = moderate staining; 3 = strong staining. If less than 10 % of the cells were stained, a score of zero was given. Therefore, the total IRS ranged from 0 to 12.

While the quartile scoring method is currently the more standard method used by pathologists, the IRS scoring method is a standard research tool and its utility has been validated in multiple prior studies ^24,28. Therefore, both semi-quantitative methods were evaluated to determine the best method in grading and diagnosing dysplasia in BE.