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In-Situ Hybridaization Quantification

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An Integrated Optical Density Based Measurement to Evaluate CISH Staining in Tissue Sections

Introduction:

  • For many years, chromogenic in-situhybridization (CISH), or its fluorescent counterpart (FISH) have been successfully utilized to evaluate gene alterations in various cell and tissue types.
  • Recently, CISH has emerged as a powerful alternative to immunohistochemistry (IHC) to quantify a gene product, particularly when specific antibody reagents are not available or when the expression patterns of highly homologous proteins need to be evaluated.
  • There are many established IHC scoring paradigms to evaluate the expression levels of specific proteins; however there is no well-defined scoring protocol for RNA CISH.
  • Dot-counting is one proposed approach, but counting frequently becomes challenging due to variability in dot staining intensity, dot size, and dot frequency.
  • This variability can impact both subjective interpretation and development of automated scoring algorithms.
  • We developed a tissue image analysis approach that can accurately distinguish and classify cells according to a summary cell-based measurement of Integrated Optical Density (IOD).
  • The algorithm incorporates dot intensity, dot size, and dot frequency for RNA CISH applications.
  • We first demonstrated that IOD assessment of target genes in various tissues performs similar to the conventional method of counting the number of dots in an ideal setting where dot intensity, size, and frequency is relatively uniform.
  • We then extended the IOD classification to the more common RNA CISH situation where the variability in dot size, intensity, and frequency compromises a reliable dot number assessment.
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