Michael Liew, Leslie Rowe, Kristina Moore, Emily Aston, Kathryn O'Brien, Maria Longhurst, Jason Kenney, Marshall Priest, Wenhua Zhou, Diane Wilcock, Anton Rets, Rodney Miles
{"title":"MYC和BCL6快速分离数字荧光原位杂交技术的临床应用验证。","authors":"Michael Liew, Leslie Rowe, Kristina Moore, Emily Aston, Kathryn O'Brien, Maria Longhurst, Jason Kenney, Marshall Priest, Wenhua Zhou, Diane Wilcock, Anton Rets, Rodney Miles","doi":"","DOIUrl":null,"url":null,"abstract":"<p><strong>Objective: </strong>Detection of gene rearrangements in <i>MYC</i> (a family of regulator genes and proto-oncogenes) and human B-cell lymphoma 6 (<i>BCL6</i>) using fluorescence <i>in situ</i> hybridization (FISH) are important in the evaluation of lymphomas, in particular diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma. Our current clinical MYC and BCL6 FISH workflow involves an overnight hybridization of probes with digital analysis using the GenASIs Scan and Analysis instrument (Applied Spectral Imaging). In order to improve assay turnaround time SureFISH probes were validated to reduce the hybridization time from 16 hours down to 1.5 hours.</p><p><strong>Methods: </strong>Validation was a four-phase process involving initial development of the assays by testing new probes in a manual protocol, and cytogenetic studies to confirm the probe specificity, sensitivity, and localization. In the next phase, the assays were validated as a manual assay. The third phase involved development of the digital FISH assays by testing and optimizing the GenASIs Scan and Analysis instrument. In the final phase, the digital FISH assays were validated.</p><p><strong>Results: </strong>Cytogenetic studies confirmed 100% probe sensitivity/specificity, and localization patterns. Negative reference range cutoffs calculated from 20 normal lymph nodes using the inverse of the beta cumulative probability density function (Excel BETAINV calculation) were 11% inclusive for both manual and digital MYC and BCL6 assays. There was 100% concordance between the manual and digital methods. The shortened hybridization time decreased the overall workflow time by 14.5 hours.</p><p><strong>Conclusions: </strong>This study validates the use of the SureFISH MYC and BCL6 probes on formalin fixed paraffin embedded (FFPE) tissue sections using a hybridization time of 1.5 hours that shortened the overall workflow by 14.5 hours. The process described also provides a standardized framework for validating digital FISH assays in the future.</p>","PeriodicalId":13943,"journal":{"name":"International journal of clinical and experimental pathology","volume":"16 4","pages":"76-85"},"PeriodicalIF":1.1000,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10165167/pdf/ijcep0016-0076.pdf","citationCount":"0","resultStr":"{\"title\":\"Validation of MYC and BCL6 rapid break apart digital fluorescence in situ hybridization assays for clinical use.\",\"authors\":\"Michael Liew, Leslie Rowe, Kristina Moore, Emily Aston, Kathryn O'Brien, Maria Longhurst, Jason Kenney, Marshall Priest, Wenhua Zhou, Diane Wilcock, Anton Rets, Rodney Miles\",\"doi\":\"\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Objective: </strong>Detection of gene rearrangements in <i>MYC</i> (a family of regulator genes and proto-oncogenes) and human B-cell lymphoma 6 (<i>BCL6</i>) using fluorescence <i>in situ</i> hybridization (FISH) are important in the evaluation of lymphomas, in particular diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma. Our current clinical MYC and BCL6 FISH workflow involves an overnight hybridization of probes with digital analysis using the GenASIs Scan and Analysis instrument (Applied Spectral Imaging). In order to improve assay turnaround time SureFISH probes were validated to reduce the hybridization time from 16 hours down to 1.5 hours.</p><p><strong>Methods: </strong>Validation was a four-phase process involving initial development of the assays by testing new probes in a manual protocol, and cytogenetic studies to confirm the probe specificity, sensitivity, and localization. In the next phase, the assays were validated as a manual assay. The third phase involved development of the digital FISH assays by testing and optimizing the GenASIs Scan and Analysis instrument. In the final phase, the digital FISH assays were validated.</p><p><strong>Results: </strong>Cytogenetic studies confirmed 100% probe sensitivity/specificity, and localization patterns. Negative reference range cutoffs calculated from 20 normal lymph nodes using the inverse of the beta cumulative probability density function (Excel BETAINV calculation) were 11% inclusive for both manual and digital MYC and BCL6 assays. There was 100% concordance between the manual and digital methods. The shortened hybridization time decreased the overall workflow time by 14.5 hours.</p><p><strong>Conclusions: </strong>This study validates the use of the SureFISH MYC and BCL6 probes on formalin fixed paraffin embedded (FFPE) tissue sections using a hybridization time of 1.5 hours that shortened the overall workflow by 14.5 hours. The process described also provides a standardized framework for validating digital FISH assays in the future.</p>\",\"PeriodicalId\":13943,\"journal\":{\"name\":\"International journal of clinical and experimental pathology\",\"volume\":\"16 4\",\"pages\":\"76-85\"},\"PeriodicalIF\":1.1000,\"publicationDate\":\"2023-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10165167/pdf/ijcep0016-0076.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"International journal of clinical and experimental pathology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"ONCOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"International journal of clinical and experimental pathology","FirstCategoryId":"1085","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"ONCOLOGY","Score":null,"Total":0}
Validation of MYC and BCL6 rapid break apart digital fluorescence in situ hybridization assays for clinical use.
Objective: Detection of gene rearrangements in MYC (a family of regulator genes and proto-oncogenes) and human B-cell lymphoma 6 (BCL6) using fluorescence in situ hybridization (FISH) are important in the evaluation of lymphomas, in particular diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma. Our current clinical MYC and BCL6 FISH workflow involves an overnight hybridization of probes with digital analysis using the GenASIs Scan and Analysis instrument (Applied Spectral Imaging). In order to improve assay turnaround time SureFISH probes were validated to reduce the hybridization time from 16 hours down to 1.5 hours.
Methods: Validation was a four-phase process involving initial development of the assays by testing new probes in a manual protocol, and cytogenetic studies to confirm the probe specificity, sensitivity, and localization. In the next phase, the assays were validated as a manual assay. The third phase involved development of the digital FISH assays by testing and optimizing the GenASIs Scan and Analysis instrument. In the final phase, the digital FISH assays were validated.
Results: Cytogenetic studies confirmed 100% probe sensitivity/specificity, and localization patterns. Negative reference range cutoffs calculated from 20 normal lymph nodes using the inverse of the beta cumulative probability density function (Excel BETAINV calculation) were 11% inclusive for both manual and digital MYC and BCL6 assays. There was 100% concordance between the manual and digital methods. The shortened hybridization time decreased the overall workflow time by 14.5 hours.
Conclusions: This study validates the use of the SureFISH MYC and BCL6 probes on formalin fixed paraffin embedded (FFPE) tissue sections using a hybridization time of 1.5 hours that shortened the overall workflow by 14.5 hours. The process described also provides a standardized framework for validating digital FISH assays in the future.
期刊介绍：
The International Journal of Clinical and Experimental Pathology (IJCEP, ISSN 1936-2625) is a peer reviewed, open access online journal. It was founded in 2008 by an international group of academic pathologists and scientists who are devoted to the scientific exploration of human disease and the rapid dissemination of original data. Unlike most other open access online journals, IJCEP will keep all the traditional features of paper print that we are all familiar with, such as continuous volume and issue numbers, as well as continuous page numbers to keep our warm feelings towards an academic journal. Unlike most other open access online journals, IJCEP will keep all the traditional features of paper print that we are all familiar with, such as continuous volume and issue numbers, as well as continuous page numbers to keep our warm feelings towards an academic journal.
分享
京公网安备 11010802042870号
京ICP备2023020795号-1
求助内容：
应助结果提醒方式：
扫码关注我们
