Abstract B48: Immunohistochemistry and RT-PCR evaluation of fixative effects in a model tissue system

The standard practice in hospital pathology laboratories is to preserve patient clinical tissue specimens as fixed, paraffin-embedded (FPE) tissue. FPE specimens are used for routine pathologic examination, immunohistochemistry (IHC) studies, and a variety of molecular diagnostic assays. The results of these studies assist in determining the patient9s clinical status and in therapeutic decision making. However, the methodology for tissue fixation is not standardized across laboratories and a number of different tissue fixatives are currently commercially available. The use of different tissue fixatives may significantly affect the performance of IHC and molecular diagnostic assays. The results of nine common tissue fixatives and their effects on both IHC- and RNA-based molecular assays are reported. Using human placenta as a model tissue system, nine common fixatives (B5, Bouin9s, ethyl alcohol 70%, formalin, Hollandes, Penfix, Prefer, Zenker9s, and zinc formalin) were compared for effects on six IHC assays and a panel of 42 gene targets by RT-PCR assays, as well as performance relative to fresh (RNAlater®) or frozen (OCT) unfixed tissue. The 42-gene panel assessed by RT-PCR included the six genes assessed by IHC. The IHC assays were scored using a semiquantitative method. For RT-PCR, raw assay scores were derived and subsequently normalized. Different fixatives resulted in varying effects on IHC and molecular assay performance. Per gene across each fixative, mRNA expression levels assessed by RT-PCR assays demonstrated wide variation, which could be largely corrected for by normalization. Variation in immunoreactivity as a function of tissue fixative was also observed with IHC assays. Compared to IHC, RT-PCR assays demonstrated greater sensitivity and were able to detect lower levels of gene expression, when the IHC assay gave negative results. Interestingly, fixative related effects were not always similar between IHC and RT-PCR assays. Therefore, it is recommended that the effects of tissue fixation be taken into consideration when performing data analysis and making comparisons between IHC and molecular diagnostic assays. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B48.