Fluorescence-based in situ hybridization (FISH) is used to visualize DNA or localized RNA in cells, tissues and tumors. Providing a reliable means for studying the genetic composition of cells in mitosis as well as interphase, with high detection sensitivity and capacity for multiplicity, FISH is also frequently used to demonstrate the presence of cancer gene rearrangements characteristic of certain hematologic malignancies.
Two main components are required in the preparation of a FISH assay: the DNA probe and the target DNA to which the probe will be hybridized. To date, a wide range of commercially available probes have been developed for a number of common targets, however their reliable use requires stringent validation and routine monitoring of assay performance. Consistent and well-validated reference materials are therefore essential to ensure accuracy.
Variability within a standard FISH based workflow
Horizon Diagnostics (HDx) has worked closely with the key European and US pathology proficiency schemes, FISH laboratories and biotech companies to develop FISH Reference Standard Slides. The reference materials are provided in cell culture spread or formalin-fixed, paraffin embedded (FFPE) sections as appropriate for the tumor type being evaluated.
Tumor Successful execution of the analytical phase is completely dependent on correct performance during the pre-analytical phase
Tumor profiles and genotype can often be specific to individual samples and depending on the sample the proportion of normal versus tumor cells can be highly variable. Genoytping a sample from a 10% tumor cell population may be more challenging to score and genotype compared to a sample from a 100% tumor cell population.
Sample fixation processes are highly variable and will impact on the quality of the extractable DNA. High-grade formalin for a short time (i.e. less than 24 hours) will preserve the quality DNA within the samples and provide a greater DNA yield compared to Low-grade formalin for a longer time (i.e. more than 24 hours).
Hybridisation conditions need to be optimised for each probe and chromosomal abnormality.
Increased pepsin treatment step can lead to the over-digestion of cells and as a consequence a lower quality signal for sample analysis
The choice of microscope will impact on the requirement for post-image analysis and adjustment to reduce background staining prior to scoring.
Genotyping and nuclei scoring is probe and mutation specific. It is critical to understand the expected staining for single-probes, dual-probes and breakapart-probes prior to analysis. Reference slides can assist with the interpretation of daily results and help with the initial setup of a FISH based assay.