In an attempt to accurately describe the extent of immunohistochemical staining of a tumor, the degree of IHC staining, if any, in each sub-cellular compartment in tumor cells is captured for each analyte. This algorithm includes capturing the percentage of tumor cells stained at each intensity level. A semi-quantitative intensity scale ranging from 0 for no staining to 3+ for the most intense staining is used. All of this information can be analyzed separately or used to calculate a variable, more continuous than simply Positive versus Negative, called the H-Score. This score is more representative of the staining of the entire tumor on the section. Although given sections may share the same simple intensity score, there clearly is a difference between a 3+ case with only 10% of the cells staining as compared to a 3+ case where greater than 90% of the cells are staining. This difference is easily picked up using H-Score method. An H-Score is typically calculated for staining of each sub-cellular compartment for both normal and tumor cells using the following formula; H-Score = (% at 0) * 0 + (% at 1+) * 1 + (% at 2+) * 2 + (% at 3+) * 3. Thus, this score produces a continuous variable that ranges from 0 to 300.
For semi-quantitative assays, controls periodically include paraffin section of cell blocks prepared from admixtures of target positive cell lines and peripheral blood leukocytes. These cell blocks contain mixtures of target cells at the 10%, 30%, 50%, 80%, and 90% levels.