Abstract
RNA ISH (Ribonucleic acid in situ hybridization) assays are an ever-expanding application due to the ability to evaluate molecular targets with the added benefit of retaining tissue morphology. The rate limiting step in RNA ISH assays is the time consuming and error prone method of manually counting signal when reviewing under a microscope. The Aperio RNA ISH algorithm offers a reproducible, fast, and quantitative method of evaluating tissue samples which have been stained to detect RNA ISH signal. This single algorithm can be used on numerous tissue types for both single and dual-plex assays.
Within this paper, we describe a validation study, which was performed in order to verify the correlation between the Aperio RNA ISH algorithm and the current gold standard method of manual interpretation. A total of 30 digital slides ranging in tissue source and assays types were scored manually by a scientist and the resulting data were correlated with scores obtained from the Aperio RNA ISH algorithm. In both modalities, the number of cells, count of signal within the cell, and signal in all tissue were recorded. The high level of correlation between the two methods (>R2 0.99) confirm that automated image analysis can be used as a fast and reproducible alternative to the traditional methods of manual interpretation.
Introduction
RNA ISH is a rapidly growing method for the analysis of molecular targets within tissue samples. It enables identification of individual copies of targets, whilst maintaining tissue morphology, a feature which is lost in other methods such as PCR1. RNA ISH technology is being used in many areas of cancer research today2-5. In addition, there is the potential benefit of being able to combine RNA ISH assays with traditional IHC assays, thereby enabling users to be able to visualize both RNA and protein status on a single slide6. Manual interpretation of RNA ISH signal is time-consuming and typically reverts to the semi-qualitative approach of ordinal scores (0, 1+, 2+, 3+), which often mask discrete cohorts that are readily identifiable when quantitative analysis is performed. Moreover, manual reads are subject to inter- and intra-observer variability, resulting in a lack of reproducibility and standardization in formalin fixed paraffin embedded tissue staining interpretation7–10. The Aperio RNA ISH algorithm identifies and counts single or dual-plex chromogenic signals and will distinguish whether the expression is within the nuclei or cytoplasmic cellular compartments of FFPE tissue. It has been optimized for usage on brightfield digital slides from Aperio scanners or the Ariol System, at both 20x and 40x resolution. Individual signals or clusters of signals can be identified within the subcellular compartments, in uncategorized tissue (i.e. tissue not identified as a nuclei or cytoplasm) or across all tissue within the sample. The resulting data provides quantitative numerical counts of cells, signals and clusters. In addition, a semi-quantitative score is also generated by the algorithm, RNA ISH Score (0, 1+, 2+, 3+, 4+). Herein, we describe the validation study which was performed in order to ensure the Aperio RNA ISH algorithm correlates with the current gold standard of manual interpretation.Methods & Materials
A total of 30 FFPE whole tissue sections were gathered, from human and animal sources (kidney, liver, prostate, breast, small intestine, head and neck, ovarian cell lines, colon, placenta, skin, and rat kidney). Tissue sections were stained with single-plex RNA ISH probes (Red, Brown or Green chromogens) or dual-plex (Red/Brown, Brown/Green chromogens) either manually or on the BOND RX IHC/ISH stainer.>> Download the full Application Note as PDF
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