scholarly journals Micro-region transcriptomics of fixed human tissue using Pick-Seq

2021 ◽  
Author(s):  
Zoltan Maliga ◽  
Ajit J. Nirmal ◽  
Nolan G. Ericson ◽  
Sarah A. Boswell ◽  
Lance U’Ren ◽  
...  
Keyword(s):  
Tumor Invasion ◽  
Parallel Imaging ◽  
Transcriptional Profiling ◽  
Human Melanoma ◽  
Fixed Tissue ◽  
Archival Tissue ◽  
Transcriptomic Data ◽  
Tissue Biology ◽  
Multiplexed Imaging ◽  
Fresh Frozen

ABSTRACTSpatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology and disease. Recently developed spatial transcriptomic methods use fresh-frozen specimens but most diagnostic specimens, clinical trials, and tissue archives rely on formaldehyde-fixed tissue. Here we describe the Pick-Seq method for deep spatial transcriptional profiling of fixed tissue. Pick-Seq is a form of micro-region sequencing in which small regions of tissue, containing 5-20 cells, are mechanically isolated on a microscope and then sequenced. We demonstrate the use of Pick-Seq with several different fixed and frozen human specimens. Application of Pick-Seq to a human melanoma with complex histology reveals significant differences in transcriptional programs associated with tumor invasion, proliferation, and immuno-editing. Parallel imaging confirms changes in immuno-phenotypes and cancer cell states. This work demonstrates the ability of Pick-Seq to generate deep spatial transcriptomic data from fixed and archival tissue with multiplexed imaging in parallel.

Molecular Diagnostics in Melanoma: Current Status and Perspectives

2011 ◽  
Vol 135 (7) ◽  
pp. 860-869 ◽  
Author(s):  
Soheil S. Dadras
Keyword(s):  
Molecular Diagnostics ◽  
Ribonucleic Acid ◽  
New Technologies ◽  
Mutational Analysis ◽  
Transcriptional Profiling ◽  
Clinical Information ◽  
Current Status ◽  
Comparative Genomic ◽  
Molecular Approaches ◽  
Fresh Frozen

Abstract Context.—In the current “molecular” era, the advent of technology, such as array-based platforms, systems biology, and genome-wide approaches, has made it possible to examine human cancers, including melanoma, for genetic mutations, deletions, amplification, differentially regulated genes, and epigenetic changes. Advancement in current technologies is such that one can now examine ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein directly from the patient's own tumor. Objective.—To apply these new technologies in advancing molecular diagnostics in melanoma has historically suffered from a major obstacle, namely, the scarcity of fresh frozen, morphologically defined tumor banks, annotated with clinical information. Recently, some of the new platforms have advanced to permit utilization of formalin-fixed, paraffin-embedded (FFPE) tumor specimens as starting material. Data Sources.—This article reviews the latest technologies applied to FFPE melanoma sections, narrowing its focus on the utility of transcriptional profiling, especially for melastatin; comparative genomic hybridization; BRAF and NRAS mutational analysis; and micro ribonucleic acid profiling. Conclusion.—New molecular approaches are emerging and are likely to improve the classification of melanocytic neoplasms.

scholarly journals FORMALIN FIXATION IN THE CYTOCHEMICAL DEMONSTRATION OF SUCCINIC DEHYDROGENASE OF MITOCHONDRIA

1961 ◽  
Vol 9 (2) ◽  
pp. 415-427 ◽  
Author(s):  
Donald G. Walker ◽  
Arnold M. Seligman
Keyword(s):  
Divalent Cations ◽  
Succinic Dehydrogenase ◽  
Staining Procedure ◽  
Frozen Sections ◽  
Tissue Slices ◽  
Fixed Tissue ◽  
Cytochemical Demonstration ◽  
Hematoxylin Staining ◽  
Fresh Frozen ◽  
Duodenal Epithelium

A variety of established methods for protecting mitochondria were tested on rat duodenal epithelium during the histochemical assay for succinic dehydrogenase. The use of sucrose at isotonic or hypertonic concentrations, 7.5 per cent polyvinylpyrrolidone, divalent cations, physiological salt solutions, phenazine methosulfate, coenzyme Q10, and menadione failed to improve the quality of the histochemical preparation once fresh frozen sections were prepared. However, preservation of mitochondrial integrity with little diminution in succinic dehydrogenase activity was obtained by fixing tissue slices (less than 1 mm. in thickness) in 8 per cent unneutralized, aqueous formaldehyde from 8 to 16 minutes at from 5° to 10°C. prior to freezing. To offset the inhibition of enzymatic activity it was necessary to extend the incubation period by 10 to 15 minutes. Two-micron-thick sections were easily obtained from the frozen blocks of such fixed tissue and incubated in the unmodified Nitro—BT-succinate medium. Once the optimum conditions for fixation of intestinal epithelium were determined, many other tissues were subjected to the same procedure. From the morphological standpoint the appearance of the mitochondria in these histochemical preparations compares favorably with the results obtained using the classical Regaud iron-hematoxylin staining procedure. With most tissues, the results are superior to those with fresh frozen sections. However, results with muscle, sperm, and kidney tubular epithelium are not as strikingly improved as with gut and liver.

Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons.

1989 ◽  
Vol 37 (6) ◽  
pp. 899-901 ◽  
Author(s):  
G R Chalmers ◽  
V R Edgerton
Keyword(s):  
Image Processing ◽  
Enzyme Activity ◽  
Succinate Dehydrogenase ◽  
Cytochrome Oxidase ◽  
Processing System ◽  
Tissue Fixation ◽  
Chemical Fixation ◽  
Fixed Tissue ◽  
Computer Image Processing ◽  
Fresh Frozen

The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.

A Variation of 3D Printer Machinery for Spectroscopic Tissue Analysis

2019 ◽  
Author(s):  
Laura Connolly
Keyword(s):  
Raman Spectroscopy ◽  
Learning Algorithm ◽  
Imaging Techniques ◽  
Optical Data ◽  
Chicken Breast ◽  
Fixed Tissue ◽  
Tissue Samples ◽  
Target Registration Error ◽  
The Future ◽  
Fresh Frozen

PURPOSE: Raman Spectroscopy is amongst several optical imaging techniques that have the ability to characterize tissue non-invasively. To use these technologies for intraoperative tissue classification, fast and efficient analysis of optical data is required with minimal operator intervention. Additionally, there is a need for a reliable database of optical signatures to account for variable conditions. We developed a software system with an inexpensive, flexible mechanical framework to facilitate automated scanning of tissue and validate spectroscopic scans with histologic ground truths. This system will be used, in the future, to train a machine learning algorithm to distinguish between different tissue types using Raman Spectroscopy.  METHODS: A sample of chicken breast tissue is mounted to a microscope slide following a biopsy of fresh frozen tissue. Landmarks for registration and evaluation are marked on the specimen using a material that is recognizable in both spectroscopic and histologic analysis. The slides are optically analyzed using our software. The landmark locations are extracted from the spectroscopic scan of the specimen using our software. This information is then compared to the landmark locations extracted from images of the slide using the software, ImageJ. RESULTS: Target registration error of our system in comparison to ImageJ was found to be within 1.1 mm in both x and y directions. CONCLUSION: We demonstrated a system that can employ accurate spectroscopic scans of fixed tissue samples. This system can be used to spectroscopically scan tissue and validate the results with histology images in the future

Contrast Enhancement of Fresh Frozen-Dried Tissue for Light and Electron Microscopy

1977 ◽  
Vol 35 ◽  
pp. 548-549
Author(s):  
Patrick W. Bankston ◽  
Louis Terracio
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Biological Tissues ◽  
Ice Crystal ◽  
Fixed Tissue ◽  
X Ray ◽  
Fresh Frozen ◽  
Electron Microscopes ◽  
Specific Reactions ◽  
Cellular Components

Recent improvements in the technique of freeze-drying now routinely provide biological tissues with morphological preservation ultrastructurally comparable to conventionally-fixed tissue. Using this technique, tissue is frozen on a metal surface, dried in vacuo, vapor fixed with OSO4 and directly embedded in polymers suitable for sectioning for light or electron microscopy. Tissue processed in this manner can be used for such specialized applications as x-ray microanalysis and radioautography of soluble substances. Further, it is possible to eliminate OSO4 before embedding making frozen-dried tissue useful for investigation of cellular components by cytochemical reactions performed on sectioned material. Many of these procedures require that the tissue be free of OSO4 initially but that OSO4 be present for final viewing. In addition, frozen-dried tissue requires preliminary viewing in the light and electron microscopes to evaluate the extent of ice crystal damage before use for specific reactions. However, it is difficult to stain unfixed plastic embedded tissue for light microscopy and equally difficult to provide adequate contrast to these for electron microscopy.

scholarly journals Transcriptional profiling of degraded RNA in cryopreserved and fixed tissue samples obtained at autopsy

2006 ◽  
Vol 6 (1) ◽  
Author(s):  
Andrew C Haller ◽  
Deepa Kanakapalli ◽  
Rosemarie Walter ◽  
Samir Alhasan ◽  
James F Eliason ◽  
...  
Keyword(s):  
Transcriptional Profiling ◽  
Fixed Tissue ◽  
Tissue Samples

Retrospective Analysis of Ploidy in Primary Osseous and Extraosseous Ewing Family Tumors in Children

1998 ◽  
Vol 84 (4) ◽  
pp. 493-498 ◽  
Author(s):  
Daniela Perotti ◽  
Valentina Corletto ◽  
Roberto Giardini ◽  
Antonina Parafioriti ◽  
Franca Fossati-Bellani ◽  
...  
Keyword(s):  
Ewing’S Sarcoma ◽  
Ewing's Sarcoma ◽  
Image Cytometry ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Frozen Tissue ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Ewing Family Tumors ◽  
Formalin Fixed

Aims To restrospectively study the DNA content in a series of childhood Ewing Family Tumors (EFT), and to investigate its prognostic value. Methods The study was performed on a series of 27 EFTs (osseous Ewing's sarcoma, 18 cases; extraos-sous Ewing's sarcoma, 2; peripheral neuroepithelioma, 4; Askin Rosai tumors, 3). Ploidy was investigated using both flow cytometry (FCM) and image cytometry (ICM) on tumor cell suspensions from formalin-fixed paraffin-embedded specimens or fresh frozen tissue obtained from the primary tumor at diagnosis. Results Ploidy was evaluable by FCM in all cases, and by ICM in 23/27. When fresh frozen tissue and paraffin-embedded samples from the same tumor were available for analysis, they yielded equal results. The rate of agreement between FCM and ICM was 82%. The majority of cases were diploid, and in the present series aneuploidy seemed to be associated with a poor outcome. Conclusions These results suggest that aneuploidy could be an indicator of a bad prognosis in EFT; however, the small number of cases precludes any conclusion of statistical value. Larger restrospective studies on ploidy using archival material could be performed and their reliability is supported by the concordance of results from fresh and formalin-fixed tissue.

Sign in / Sign up

Export Citation Format

Share Document