Fluorescent in situ hybridization heating pretreatment: The key is temperature control

Tojo, Marta; Couso, Elena; Vázquez-Boquete, Ángel; Pérez-Becerra, Raquel; García‐Caballero, Tomás; Forteza, Jerónimo; Fraga, Máximo

doi:10.1111/j.1440-1827.2010.02600.x

Cited by 4 publications

(4 citation statements)

References 22 publications

(36 reference statements)

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“…Previous studies on FISH pretreatment methods described the commonly used pretreatment agent sodium thiocyanate as too harsh when applied to tissue, because tissue sections were found to detach from the slides and were susceptible to mechanical disaggregation [11, 12]. In addition some studies suggested that the optimal concentration, incubation time, and incubation temperatures, of these pretreatment agents must be titrated for the different tissue sections and tissue types [6, 13]. This would suggest the use of multiple protocols for FISH in one laboratory.…”

Section: Discussionmentioning

confidence: 99%

“…Additionally all reagents excluding probes are prepared in-house, making it very cost effective. Leers et al and Tojo et al suggested that the heating of cells in an acidic environment with the pretreatment agent, citric acid buffer followed by a proteolytic step improved the fluorescent signals in FFPE tissue significantly [6, 13].…”

Section: Discussionmentioning

confidence: 99%

“…The technical challenge of applying FISH lies within the pretreatment protocol. An efficient pretreatment protocol should expose the target genes and allow the penetration of the probes without significantly altering the integrity and morphology of the tissue [6]. In the current study we have developed a general pretreatment method in order to standardize our FISH procedure for many tissue types and probes produced by different manufacturers (Fig.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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“…We briefly mention some of the effects of these fixatives on nucleic acids. Formalin fixation induces DNA-protein and RNA-protein cross-links (5, 6), which may prevent efficient nucleic acid extraction (7) as well as in situ hybridization (8)(9)(10). Nucleic acid fragmentation (11) and loss may occur by prolonged fixation of the specimen or changes in the pH of the fixative (12)(13)(14)(15).…”

Section: Types Of Fixativesmentioning

confidence: 99%

Pre-analytical Practices in the Molecular Diagnostic Tests, A Concise Review

et al. 2021

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Molecular assays for detection of nucleic acids in biologic specimens are valuable diagnostic tools supporting clinical diagnoses and therapeutic decisions. Pre-analytical errors, which occur before or during processing of nucleic acid extraction, contribute a significant role in common errors that take place in molecular laboratories. Certain practices in specimen collection, transportation, and storage can affect the integrity of nucleic acids before analysis. Applying best practices in these steps, helps to minimize those errors and leads to better decisions in patient diagnosis and treatment. Widely acceptable recommendations, which are for optimal molecular assays associated with pre-analytic variables, are limited. In this article, we have reviewed most of the important issues in sample handling from bed to bench before starting molecular tests, which can be used in diagnostic as well as research laboratories. We have addressed the most important pre-analytical points in performing molecular analysis in fixed and unfixed solid tissues, whole blood, serum, plasma, as well as most of the body fluids including urine, fecal and bronchial samples, as well as prenatal diagnosis samples.

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A new method for real-time evaluation of pepsin digestion of paraffin-embedded tissue sections, prior to fluorescence in situ hybridisation

et al. 2017

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Fluorescence in situ hybridisation (FISH) is a molecular cytogenetic technique, which is regularly applied to formalin-fixed paraffin-embedded (FFPE) tissue sections of a variety of cancers to assess chromosomal aberrations. However, high-quality FISH requires optimal enzymatic digestion, and insufficient digestion is not noted until the hybridisation signals are evaluated in the fluorescence microscope. As a consequence, FISH results may be unreliable, and the experiment might have to be repeated. To solve this problem, we developed a new method for real-time evaluation of enzymatic tissue digestion. Termination of enzyme activity at the proper time facilitates successful hybridisation, and experiments do not have to be repeated. We first performed FISH on 20 FFPE samples, which had been pepsin digested for different times, and this revealed distinct morphological changes within the nucleus and perinuclear space that were detectable by light microscopy. These observations suggested that the presence of intact and clear bare nuclei, surrounded by a translucent perinuclear space, might serve as an indicator of adequate digestion. We developed a protocol for assessment of this indicator, based on morphological features, and applied this to a collection of 400 tissue samples, partly of breast cancer and partly of different types of lymphoma, prior to FISH. The FISH success rate was 99.5% (398/400), which was significantly higher than that of the conventional method. In all successful cases, morphological signs of adequate digestion were paralleled by easily interpretable FISH signals. This new method for the real-time assessment of digestion quality improved the success rate of FISH and in addition was simple and rapid.

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Fluorescent in situ hybridization heating pretreatment: The key is temperature control

Cited by 4 publications

References 22 publications

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

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

Pre-analytical Practices in the Molecular Diagnostic Tests, A Concise Review

A new method for real-time evaluation of pepsin digestion of paraffin-embedded tissue sections, prior to fluorescence in situ hybridisation

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