One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides

Abstract

Background The Fluorescence In Situ Hybridization (FISH) technique is a very useful tool for diagnostic and prognostic purposes in molecular pathology. However, clinical testing on patient tissue is challenging due to variables of tissue processing that can influence the quality of the results. This emphasizes the necessity of a standardized FISH protocol with a high hybridization efficiency. We present a pretreatment protocol that is easy, reproducible, cost-effective, and facilitates FISH on all types of patient material simultaneously with good quality results. During validation, FISH analysis was performed simultaneously on formalin-fixed paraffin-embedded, fresh frozen and cytological patient material in combination with commercial probes using our optimized one-fits-all pretreatment protocol. An optimally processed sample is characterized by strong specific signals, intact nuclear membranes, non-disturbing autofluorescence and a homogeneous DAPI staining. Results In our retrospective cohort of 3881 patient samples, overall 93% of the FISH samples displayed good quality results leading to a patient diagnosis. All FISH were assessed on quality aspects such as adequacy and consistency of signal strength (brightness), lack of background and / or cross-hybridization signals, and additionally the presence of appropriate control signals were evaluated to assure probe accuracy. In our analysis 38 different FISH probes from 3 commercial manufacturers were used (Cytocell, Vysis and ZytoLight). The majority of the patients in this cohort displayed good signal quality and barely non-specific background fluorescence on all tissue types independent of which commercial probe was used. Conclusion The optimized one-fits-all FISH method is robust, reliable and reproducible to deliver an accurate result for patient diagnostics in a lean workflow and cost-effective manner. This protocol can be used for widespread application in cancer and non-cancer diagnostics and research.

Fluorescent In Situ Hybridisation (FISH) as a Follow-up Test for Postnatal Microarray Results

“…In this study, the hybridisation time was reduced, and the work ow was simpli ed compared to that of the standard overnight hybridisation procedure. Signal intensity and speci city were comparable to standard hybridisation procedures as demonstrated by [10,12,13]. The Vysis IntelliFISH Hybridization Buffer (Abbott Molecular Inc., Des Plaines, IL) therefore provides an e cient work ow for peripheral blood cultures and a reduced the turnaround time for results as seen in similar studies by [10,12,13].…”

“…Signal intensity and speci city were comparable to standard hybridisation procedures as demonstrated by [10,12,13]. The Vysis IntelliFISH Hybridization Buffer (Abbott Molecular Inc., Des Plaines, IL) therefore provides an e cient work ow for peripheral blood cultures and a reduced the turnaround time for results as seen in similar studies by [10,12,13]. In all, FISH is a useful follow-up test for genomic imbalances and may further detect aberrations that are not detected by other molecular laboratory techniques.…”

ALK-positive histiocytosis: a new clinicopathologic spectrum highlighting neurologic involvement and responses to ALK inhibition

“…Molecular analysis was performed as part of initial diagnosis at referring institutions or done in the process of central review, when sufficient tumor tissue was available. For analyses performed during central review, fluorescence in situ hybridization (FISH) analysis was performed using CytoCell ® dual color ALK break-apart probes (Oxford Gene Technology, UK), as previously described 54 . Targeted RNA sequencing was performed using customized Archer ® FusionPlex ® next-generation sequencing (NGS) panels, which allow the detection of both fusions with known or unknown fusion partners.…”

Identification of Pneumocystis jirovecii with Fluorescence In-Situ Hybridization (FISH) in Patient Samples—A Proof-of-Principle

“…Autofluorescence, which is a considerable challenge for FISH with more complex sample matrices as summarized recently [14], may be an alternative explanation for the recorded poor specificity. Admittedly, tissue specimens are pretty tough materials for FISH regarding a variety of interfering factors affecting the diagnostic accuracy as detailed elsewhere [62]. To the authors' experience, visualization of staining with the FITC/FAM (fluorescein isothiocyanate/fluorescein amidite) dyes is particularly prone to autofluorescence, while this phenomenon is less pronounced e.g., with cyanine dyes like the fluorophores Cy3 and Cy5 which were applied with the paraffin-embedded tissues.…”