The Immunohistochemical Detection of Mismatch Repair Gene Proteins (MLH1, MSH2, MSH6, and PMS2): Practical Aspects in Antigen Retrieval and Biotin Blocking Protocols

The immunohistochemical detection of the mismatch repair (MMR) proteins is used as a screening test with microsatellite instability for the detection of hereditary nonpolyposis colon cancer (HNPCC). The authors describe a simple and cost-effective method using a pressure cooker and microwave oven for antigen retrieval and a modified method for applying a commercial biotin blocking kit. Colorectal tumors of 20 patients of the HNPCC spectrum were included in this study. Eighty paraffin sections were cut and submitted for immunohistochemical analysis using a routine protocol and a pressure cooker protocol. Parallel sections for biotin blocking were also run, including the modified biotin block for each protocol. The sections were incubated with the following antibodies: MLH1, MSH2, MSH6, and PMS2. All cases examined exhibited a normal expression of the MMR proteins in the nucleus and adjacent nonneoplastic tissue elements and consequently defined as having a normal expression of these proteins. Cases with tumor that exhibited a loss of the nuclear staining with the MMR proteins with a concurrent staining of the adjacent nonneoplastic cells were classified as abnormal MMR expression. The series of 20 cases using pressure cooker antigen retrieval produced superior results to the routine immunohistochemical protocol used previously in our laboratory. The modified biotin block also gave consistent results. The reproducibility and consistency of this procedure has resulted it in being used routinely for suspected HNPCC cases, both current and archival.