Nucleic acid quantity and quality from paraffin blocks: defining optimal fixation, processing and DNA/RNA extraction techniques.

Although the extraction and analysis of nucleic acids from formalin-fixed paraffin-embedded tissues is a routine and growing part of pathology practice, no generally accepted recommendations exist to guide laboratories in their selection of tissue fixation, processing and DNA/RNA extraction techniques. The aim of this study was to determine how fixation method and length, paraffin embedding, processing conditions and nucleic acid extraction methods affect quality and quantity of DNA and RNA, and their performance in downstream applications. Nine tissue samples were subjected to freezing, fixation in formalin for <24 h and 7 days followed by conventional processing, and fixation in molecular fixative for <24 h and 7 days followed by rapid processing. DNA and RNA were isolated using in-house extraction and commercial kits, and assessed by PCR reactions for amplicons with varying sizes ranging from 268 to 1327 bp and one-step RT-PCR for 621 bp and 816 bp amplicons of housekeeping genes. Molecular fixative (MF) appeared to perform well under nearly all circumstances (extraction methods, fixation lengths and longer amplicons), often performing as well as frozen samples. Formalin fixation generally performed well only for shorter length amplicons and short fixation (<24 h). WaxFree kit showed consistently higher success rates for DNA and poorer rates for RNA. RecoverAll kit generally performed suboptimally in combination with prolonged formalin fixation. In conclusion, the Molecular Fixative regardless of fixation length, and the rapid tissue processing system were able to preserve large DNA and RNA fragments in paraffin blocks, making these techniques preferable for use in downstream molecular diagnostic assays.

Inter-observer reproducibility of HER2 immunohistochemical assessment and concordance with fluorescent in situ hybridization (FISH): pathologist assessment compared to quantitative image analysis.

BACKGROUND: In breast cancer patients, HER2 overexpression is routinely assessed by immunohistochemistry (IHC) and equivocal cases are subject to fluorescent in situ hybridization (FISH). Our study compares HER2 scoring by histopathologists with automated quantitation of staining, and determines the concordance of IHC scores with FISH results. METHODS: A tissue microarray was constructed from 1,212 invasive breast carcinoma cases with linked treatment and outcome information. IHC slides were semi-quantitatively scored by two independent pathologists on a range of 0 to 3+, and also analyzed with an Ariol automated system by two operators. 616 cases were scorable by both IHC and FISH. RESULTS: Using data from unequivocal positive (3+) or negative (0, 1+) results, both visual and automated scores were highly consistent: there was excellent concordance between two pathologists (kappa = 1.000, 95% CI: 1-1), between two machines (kappa = 1.000, 95% CI: 1-1), and between both visual and both machine scores (kappa = 0.898, 95% CI: 0.775-0.979). Two pathologists successfully distinguished negative, positive and equivocal cases (kappa = 0.929, 95% CI: 0.909-0.946), with excellent agreement with machine 1 scores (kappa = 0.835, 95% CI: 0.806-0.862; kappa = 0.837, 95% CI: 0.81-0.862), and good agreement with machine 2 scores (kappa = 0.698, 95% CI: 0.6723-0.723; kappa = 0.709, 95% CI: 0.684-0.732), whereas the two machines showed good agreement (kappa = 0.806, 95% CI: 0.785-0.826). When comparing categorized IHC scores and FISH results, the agreement was excellent for visual 1 (kappa = 0.814, 95% CI: 0.768-0.856), good for visual 2 (kappa = 0.763, 95% CI: 0.712-0.81) and machine 1 (kappa = 0.665, 95% CI: 0.609-0.718), and moderate for machine 2 (kappa = 0.535, 95% CI: 0.485-0.584). CONCLUSION: A fully automated image analysis system run by an experienced operator can provide results consistent with visual HER2 scoring. Further development of such systems will likely improve the accuracy of detection and categorization of membranous staining, making this technique suitable for use in quality assurance programs and eventually in clinical practice.