Effect of Fixatives and Duration of Fixation on Expression of MSI Markers.
Patrick A Adegboyega. LSU Health Sciences Center, Shreveport, LA
Background: Tumors with abnormal DNA mismatch repair (MMR) genes are described as having microsatellite instability (MSI) and have also been shown to have distinctive clinical and pathologic features including tumor response to chemotherapeutic agents and patients' survival. Hence, immunohistochemical expression of MMR gene products is used to screen for MSI status of tumors for therapeutic decisions and determination of prognosis. This study investigates the optimal tissue processing and fixative conditions for immunohistochemical assay of MMR proteins.
Design: Benign samples of 10 routine tonsillectomy specimens and 5 colectomy specimens were fixed separately in 10% neutral buffered formalin (NBF) solution and dissect aid (DA). Matched samples from each fixative were processed for routine paraffin embedding after fixation for 1, 7, 14, 21, 42, 84, 96, and 112 days. Section of each block was immunostained for MLH1, MSH2 and MSH6. Immunoreactivity was scored in a blinded fashion using a semi quantitative score of 0, 1, 2, 3, and 4. The fixatives' code was then broken.
Results: In tonsils, MLH1 expression in germinal centers was comparable in both fixatives for the first 4 weeks; and thereafter begins to fade out in fixative A samples – with even lower scores in the inter-follicular areas and in the overlying squamous mucosa. For fixative B, MLH1 and MSH6 expression were 4+ in all samples and in all compartments of the tonsil throughout the 16 weeks of tissue fixation; MSH2 expression was 3- 4+ but begins to fade out after 4 weeks. Fixative A samples expressed 1-2+ MSH6 only in the germinal centers and MSH2 was undetectable in most cases (8/10) – even with as little as 24 hours of fixation in fixative A.
In the colon, all fixative B samples stained strongly positive for MLH1, MSH2 and MSH6 and the strong staining reaction was maintained throughout the study. In comparison, fixative A samples' MLH1 scores were consistently at least 1+ lower than corresponding scores for fixative B samples. MSH2 and MSH6 had negative staining reaction for all colon samples processed in fixative A – with as early as 24 hours fixation.
Conclusions: The effect of tissue fixatives on levels of MSI markers is tissue dependent and also varies with the gene product examined. In the tonsil, the overlying squamous mucosa and interfollicular lymphoid cells are more negatively affected than the germinal center cells fixed in fixative A (DA). Compared with MLH1, MSH2 and MSH6 are more susceptible to the adverse effects of fixative A in all the tissue types studied. Fixative B (NBF) is the preferred fixative for MSI immunohistochemical assays.
Category: Quality Assurance
Monday, February 28, 2011 1:00 PM
Poster Session II # 228, Monday Afternoon