ACVP-14 A-E: Next-Generation Multiplex vRNA and vDNA Lineage Specific In Situ Hybridization Detection With Immunohisto-Fluorescence or Chromogen in the Same Tissue Section with Quantitative Image Analysis in Fixed Tissues from Virally Infected Specimens

ACVP-14A: Next-Generation Multiplex vRNA and vDNA Lineage Specific in Situ Hybridization Combined with Immunohisto-Fluorescence for Cell Marker Detection in the Same Tissue Section with Quantitative Image Analysis in Fixed Tissues from Virally Infected Specimens

The Tissue Analysis Core within the AIDS and Cancer Virus Program will process, embed, and perform microtomy on fixed tissue samples presented in ethanol. HIV/SIV in situ hybridization for detection of vRNA and vDNA will be performed using the next-generation RNAscope technology platform using lineage specific in situ hybridization probes and kits designed and generated by Advanced Cell Diagnostic, Inc. These in situ hybridization probes and kits have been designed to increase both sensitivity and specificity for the viral lineage of interest. RNAscope and DNAscope will be perform simultaneously on the same tissue section, and then immunofluorescence will be performed to target cellular marker(s). This multiplex approach that combines RNAscope and DNAscope to identify and localize vDNA+ cells expressing, or not expressing, vRNA in the same section of tissue coupled with the ability to discriminate cell populations based on protein expression could provide a better understanding of the predominate location and phenotype of viral reservoirs within tissue microenvironments.

A high-resolution digital image of each sample will be generated using the Keyence BZ-X700 fluorescence digital microscope system or Fluoview FV10i confocal laser scanning system. Finally, under the guidance of a subject matter expert, the TAC will provide comprehensive representative pictures and quantitative image analysis using Cell Profiler and Keyence algorithm to determine the number and type of vRNA+ and or vDNA+ cells in each high-resolution image. The number of nuclei for each region of interest will be determined by segmentation using Cell Profiler to be able to report quantification by millions of cells. Relevant data will be provided electronically, and a report with interpretation/conclusions will be supplied. Sufficient basic consultation with a subject matter expert is included with the service.

ACVP-14B: Next-Generation Multiplex vDNA Lineage Specific in Situ Hybridization Detection in the Same Tissue Section with Quantitative Image Analysis in Fixed Tissues from Virally Infected Specimens

The Tissue Analysis Core (TAC) within the AIDS and Cancer Virus Program will process, embed, and perform microtomy on fixed tissue samples presented in ethanol. HIV/SIV in situhybridization for detection of vDNA will be performed using the next-generation DNAscope technology platform using lineage specific in situ hybridization probes and kits designed and generated by Advanced Cell Diagnostic, Inc. These in situ hybridization probes and kits have been designed to increase both sensitivity and specificity for the viral lineage of interest. A high-resolution digital image of each in situ hybridization sample will be generated using the Aperio ScanScope AT2 System. Regions of interest (ROI; 20–30) of a defined size will be highlighted on each digital image using the Aperio ImageScope software regions tool, and high-resolution images of ROIs will be generated using a custom-written software program created by the Imaging and Visualization Group that automatically extracts ROIs from the digital slide annotation layer. Finally, under the guidance of a subject matter expert, the TAC will provide comprehensive quantitative image analysis using a custom series of unique actions in Cell Profiler 3.0 to determine the number of vDNA+ cells per surface area in each high-resolution image. The number of nuclei for each ROI will be determine by segmentation using Cell Profiler to be able to report quantification by millions of cells.Relevant data will be provided electronically, and a report with interpretation/conclusions will be supplied. Sufficient basic consultation with a subject matter expert is included with the service.

ACVP-14C: Next-Generation Multiplex vDNA Lineage Specific in Situ Hybridization Combined with Immunohisto-Fluorescence for Cell Marker Detection in the Same Tissue Section with Quantitative Image Analysis in Fixed Tissues from Virally Infected Specimens

The Tissue Analysis Core (TAC) within the AIDS and Cancer Virus Program will process, embed, and perform microtomy on fixed tissue samples presented in ethanol. HIV/SIV in situ hybridization for detection of vDNA will be performed using the next-generation DNAscope technology platform using lineage specific in situ hybridization probes and kits designed and generated by Advanced Cell Diagnostic, Inc. These in situ hybridization probes and kits have been designed to increase both sensitivity and specificity for the viral lineage of interest. Immunohisto-fluorescence and DNAscope will be perform simultaneously on the same tissue section. A high-resolution digital image of each sample will be generated using the Keyence BZ-X700 fluorescence digital microscope system or Fluoview FV10i confocal laser scanning system. Finally, under the guidance of subject matter expert, the TAC will provide comprehensive representative pictures and quantitative image analysis using Cell Profiler and Keyence algorithm to determine the number and type of vDNA+ cells in each high-resolution image. The number of nuclei for each region of interest will be determine by segmentationusing Cell Profiler to be able to report quantification by millions of cells. Relevant data will be provided electronically, and a report with interpretation/conclusions will be supplied. Sufficient basic consultation with a subject matter expert is included with the service.

ACVP-14D: Next-Generation Multiplex vRNA Lineage Specific in Situ Hybridization Detection in the Same Tissue Section with Quantitative Image Analysis in Fixed Tissues from Virally Infected Specimens

The Tissue Analysis Core (TAC) within the AIDS and Cancer Virus Program will process, embed, and perform microtomy on fixed tissue samples presented in ethanol. HIV/SIV in situ hybridization for detection of vRNA will be performed using the next-generation RNAscope technology platform using lineage specific in situ hybridization probes and kits designed and generated by Advanced Cell Diagnostic, Inc. These in situ hybridization probes and kits have been designed to increase both sensitivity and specificity for the viral lineage of interest. RNAscope will be performed on the fully automated system BOND-RX from Leica. Using the BOND-RX to run RNAscope provides a more standardized approach; it reduces potential human error, increases throughput, and provides more consistent results between samples. A high-resolution digital image of each in situ hybridization sample will be generated using the Aperio ScanScope AT2 System. Regions of interest (ROI; 20–30) of a defined size will be highlighted on each digital image using the Aperio ImageScope software regions tool and high-resolution images of ROIs generated using a custom-written software program created by the Imaging and Visualization Group that automatically extracts ROIs from the digital slide annotation layer. Finally, under the guidance of a subject matter expert, the TAC will provide comprehensive quantitative image analysis using Cell Profiler to determine the number of virion and viral RNA+ cells per surface area in eachhigh-resolution image. The number of nuclei for each ROI will be determine by segmentation using Cell Profiler to be able to report quantification by millions of cells. Relevant data will be provided electronically, and a report with interpretation/conclusions will be supplied. Sufficient basic consultation with a subject matter expert is included with the service.

ACVP-14E: Next-Generation Multiplex vRNA Lineage Specific in Situ Hybridization Combined with Immunohisto-Fluorescence for Cell Marker Detection in the Same Tissue Section with Quantitative Image Analysis in Fixed Tissues from Virally Infected Specimens

The Tissue Analysis Core (TAC) within the AIDS and Cancer Virus Program will process, embed, and perform microtomy on fixed tissue samples presented in ethanol. HIV/SIV in situ hybridization for detection of vRNA will be performed using the next-generation RNAscope technology platform using lineage specific in situ hybridization probes and kits designed and generated by Advanced Cell Diagnostic, Inc. These in situ hybridization probes and kits have been designed to increase both sensitivity and specificity for the viral lineage of interest. Immunohisto-fluorescence and RNAscope will be perform simultaneously on the same tissue section. A high-resolution digital image of each sample will be generated using the Keyence BZ-X700 fluorescence digital microscope system or Fluoview FV10i confocal laser scanning system. Finally, under the guidance of a subject matter expert, the TAC will provide comprehensive representative pictures and quantitative image analysis using Cell Profiler and Keyence algorithm to determine the number and type of vRNA+ cells in each high-resolution image. The number of nuclei for each region of interest will be determine by segmentationusing Cell Profiler to be able to report quantification by millions of cells. Relevant data will be provided electronically, and a report with interpretation/conclusions will be supplied. Sufficient basic consultation with a subject matter expert is included with the service.