Quantitative immunohistochemistry and the use of cellular calibrators for HER2 receptor number determination.

It is suggested that high error rates in clinical IHC compared to other laboratory disciplines are due to a lack of traceable analytic standards with quantitative controls. Quantitative controls can be achieved by implementing calibrators into IHC workflows, but recently published approaches offer little discussion about what the ideal calibrator should look like. Here, we detail an IHC method that utilizes cellular calibrators to determine the average number of HER2 receptors in tumor cells of breast cancer tissue. Electrochemiluminescent immunoassay and flow cytometry were used to establish nominal HER2 amounts in the cellular calibrators. The quantitated number of HER2 receptors and associated 4B5 scores from commercially sourced breast cancer tumors were compared. The results were discordant, indicating differences in analytical performance. Cells are ideal calibrators for IHC because the antigens they express are structurally and functionally representative of the endogenous analyte being measured. They can also be stained and analyzed identically to tissues, and are easily cultured, expanded, and manipulated. However, novel methods for labeling cells with fixed amounts of proteins are needed. In addition, standards for validation of quantitative IHC methods should be developed and their clinical utility must be demonstrated.