Bioingeniøren (Nov 2019)
Optimizing of protocols regarding immunohistochemic detection of mismatch repair-proteins
Abstract
Background: Immunohistochemical staining can be used to detect mismatch repair proteins such as MLH1, PMS2, MSH2 and MSH6. These proteins are normally expressed in the cell nucleus. Loss of one or several of these proteins can indicate Lynch syndrome. The purpose of the study was to determine the most optimal testing variables for detection of MMR proteins by using the primary antibodies anti-MLH1, anti-PMS2, anti-MSH2 and anti-MSH6. Material and methods: Formalin fixed, paraffin-embedded tissue samples, mainly from colon, was used. In addition, tissue from uterus, small intestine, rectum and ventricle were also tested. The samples were previously evaluated by a pathologist, and approximately half of the samples had normal protein expression for MLH1, PMS2, MSH2 and MSH6, while the remaining samples had absence of MLH1 and PMS2 or MSH2 and MSH6. The method used was immunohistochemical staining performed on BenchMark Ultra (Ventana/Roche). Results: The four primary antibodies detected the MMR proteins. Four optimized protocols were prepared for the primary antibodies. The most optimal protocol for anti-MLH1 had antigen retrieval time of 64 minutes, incubation time with antibody of 32 minutes and amplification of 4 minutes. For anti-PMS2, antigen retrieval time of 92 minutes, incubation time with antibody of 40 minutes and amplification of 8 minutes were the most optimal protocol. Furthermore, antigen retrieval time of 64 minutes and incubation time with antibody of 32 minutes were most optimal for anti-MSH2. For anti-MSH6, a protocol with antigen retrieval time of 64 minutes and incubation time with antibody of 12 minutes were most optimal. Conclusion: Four optimal protocols were prepared for each of the four primary antibodies in consultation with a pathologist. These analyses were verified and implemented in the routine diagnostics of Lynch Syndrome at the Unit for Immunohistochemistry, Department of Pathology, St. Olav's Hospital.
