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One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides
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. Thi...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580589/ https://www.ncbi.nlm.nih.gov/pubmed/31236139 http://dx.doi.org/10.1186/s13039-019-0442-4 |
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author | Richardson, Shivanand O. Huibers, Manon M. H. de Weger, Roel A. de Leng, Wendy W. J. Hinrichs, John W. J. Meijers, Ruud W. J. Willems, Stefan M. Peeters, Ton L. M. G. |
author_facet | Richardson, Shivanand O. Huibers, Manon M. H. de Weger, Roel A. de Leng, Wendy W. J. Hinrichs, John W. J. Meijers, Ruud W. J. Willems, Stefan M. Peeters, Ton L. M. G. |
author_sort | Richardson, Shivanand O. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6580589 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-65805892019-06-24 One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides Richardson, Shivanand O. Huibers, Manon M. H. de Weger, Roel A. de Leng, Wendy W. J. Hinrichs, John W. J. Meijers, Ruud W. J. Willems, Stefan M. Peeters, Ton L. M. G. Mol Cytogenet Methodology 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. BioMed Central 2019-06-17 /pmc/articles/PMC6580589/ /pubmed/31236139 http://dx.doi.org/10.1186/s13039-019-0442-4 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Richardson, Shivanand O. Huibers, Manon M. H. de Weger, Roel A. de Leng, Wendy W. J. Hinrichs, John W. J. Meijers, Ruud W. J. Willems, Stefan M. Peeters, Ton L. M. G. One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides |
title | One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides |
title_full | One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides |
title_fullStr | One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides |
title_full_unstemmed | One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides |
title_short | One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides |
title_sort | one-fits-all pretreatment protocol facilitating fluorescence in situ hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580589/ https://www.ncbi.nlm.nih.gov/pubmed/31236139 http://dx.doi.org/10.1186/s13039-019-0442-4 |
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