Alternative tissue fixation for combined histopathological and molecular analysis in a clinically representative setting

AbstractFormalin is the principal tissue fixative used worldwide for clinical and research purposes. Despite optimal preservation of morphology, its preservation of DNA and RNA is poor. As clinical diagnostics increasingly incorporates molecular-based analysis, the requirement for maintaining nucleic acid quality is of increasing importance. Here we assess an alternative non-formalin-based tissue fixation method, PAXgene Tissue system, with the aim of better preserving nucleic acids, while maintaining the quality of the tissue to be used for vital existing diagnostic techniques. In this study, these criteria are assessed in a clinically representative setting. In total, 203 paired PAXgene Tissue and formalin-fixed samples were obtained. Blind-scored haematoxylin and eosin (H&E) sections showed comparable and acceptable staining. Immunohistochemistry (IHC) staining was suboptimal using existing protocols but improved with minor method adjustment and optimisation. Quality of DNA and RNA was significantly improved by PAXgene tissue fixation [RIN 2.8 versus 3.8 (p < 0.01), DIN 5.68 versus 6.77 (p < 0.001)], which translated into improved performance on qPCR assay. These results demonstrate the potential of PAXgene Tissue to be used routinely in place of formalin, maintaining adequate histological staining and significantly improving the preservation of biological molecules in the genomic era.

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Comprehensive Evaluation of PAXgene Fixation on Oral Cancer Tissues Using Routine Histology, Immunohistochemistry, and FTIR Microspectroscopy

Biomolecules ◽

10.3390/biom11060889 ◽

2021 ◽

Vol 11 (6) ◽

pp. 889

Author(s):

Pooja Lahiri ◽

Suranjana Mukherjee ◽

Biswajoy Ghosh ◽

Debnath Das ◽

Basudev Lahiri ◽

...

Keyword(s):

Comprehensive Evaluation ◽

Antigen Retrieval ◽

Tissue Fixation ◽

Ftir Microspectroscopy ◽

Protein Secondary Structures ◽

Associated Proteins ◽

Cancer Tissues ◽

Formalin Fixed ◽

Fixation System

The choice of tissue fixation is critical for preserving the morphology and biochemical information of tissues. Fragile oral tissues with lower tensile strength are challenging to process for histological applications as they are prone to processing damage, such as tissue tear, wrinkling, and tissue fall-off from slides. This leads to loss of morphological information and unnecessary delay in experimentation. In this study, we have characterized the new PAXgene tissue fixation system on oral buccal mucosal tissue of cancerous and normal pathology for routine histological and immunohistochemical applications. We aimed to minimize the processing damage of tissues and improve the quality of histological experiments. We also examined the preservation of biomolecules by PAXgene fixation using FTIR microspectroscopy. Our results demonstrate that the PAXgene-fixed tissues showed significantly less tissue fall-off from slides. Hematoxylin and Eosin staining showed comparable morphology between formalin-fixed and PAXgene-fixed tissues. Good quality and slightly superior immunostaining for cancer-associated proteins p53 and CK5/6 were observed in PAXgene-fixed tissues without antigen retrieval than formalin-fixed tissues. Further, FTIR measurements revealed superior preservation of glycogen, fatty acids, and amide III protein secondary structures in PAXgene-fixed tissues. Overall, we present the first comprehensive evaluation of the PAXgene tissue fixation system in oral tissues. This study concludes that the PAXgene tissue fixation system can be applied to oral tissues to perform diagnostic molecular pathology experiments without compromising the quality of the morphology or biochemistry of biomolecules.

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AI-based localization and classification of skin disease with erythema

Scientific Reports ◽

10.1038/s41598-021-84593-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ha Min Son ◽

Wooho Jeon ◽

Jinhyun Kim ◽

Chan Yeong Heo ◽

Hye Jin Yoon ◽

...

Keyword(s):

Neural Network ◽

Skin Diseases ◽

Classification Model ◽

Screening Tests ◽

Sensitivity Score ◽

Common Skin ◽

Novel Method ◽

Improved Performance ◽

High Level

AbstractAlthough computer-aided diagnosis (CAD) is used to improve the quality of diagnosis in various medical fields such as mammography and colonography, it is not used in dermatology, where noninvasive screening tests are performed only with the naked eye, and avoidable inaccuracies may exist. This study shows that CAD may also be a viable option in dermatology by presenting a novel method to sequentially combine accurate segmentation and classification models. Given an image of the skin, we decompose the image to normalize and extract high-level features. Using a neural network-based segmentation model to create a segmented map of the image, we then cluster sections of abnormal skin and pass this information to a classification model. We classify each cluster into different common skin diseases using another neural network model. Our segmentation model achieves better performance compared to previous studies, and also achieves a near-perfect sensitivity score in unfavorable conditions. Our classification model is more accurate than a baseline model trained without segmentation, while also being able to classify multiple diseases within a single image. This improved performance may be sufficient to use CAD in the field of dermatology.

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Higher EU-TIRADS-Score Correlated with BRAF V600E Positivity in the Early Stage of Papillary Thyroid Carcinoma

Journal of Clinical Medicine ◽

10.3390/jcm10112304 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2304

Author(s):

Karolina Skubisz ◽

Joanna Januszkiewicz-Caulier ◽

Patrycja Cybula ◽

Elwira Bakuła-Zalewska ◽

Krzysztof Goryca ◽

...

Keyword(s):

Early Stage ◽

Braf V600e ◽

P Value ◽

Dna And Rna ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Ion Proton ◽

Indolent Disease ◽

Next Generation Sequencing Ngs ◽

Formalin Fixed

The data demonstrating a correlation between sonographic markers of malignancy of thyroid cancer (TC) and its genetic status are scarce. This study aimed to assess whether the addition of genetic analysis at the preoperative step of TC patients’ stratification could aid their clinical management. The material consisted of formalin-fixed paraffin-embedded tumor fragments of 49 patients who underwent thyroidectomy during the early stages of papillary TC (PTC). Tumor DNA and RNA were subjected to next-generation sequencing (NGS) on Ion Proton using the Oncomine™ Comprehensive Assay panel. We observed a significant correlation between BRAF V600E and a higher EU-TIRADS score (p-value = 0.02) with a correlation between hypoechogenicity and taller-than-wide tumor shape in analysed patients. There were no other significant associations between the identified genetic variants and other clinicopathological features. For TC patient’s stratification, a strong suspicion of BRAF V600E negativity in preoperative management of TC patients could limit the over-treatment of asymptomatic, very low-risk, indolent disease and leave room for active surveillance.

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The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽

10.1186/s12885-019-6363-0 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 4

Author(s):

Michal Marczyk ◽

Chunxiao Fu ◽

Rosanna Lau ◽

Lili Du ◽

Alexander J. Trevarton ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Rna Sequencing ◽

Rna Extraction ◽

Formalin Fixed Paraffin ◽

Ffpe Samples ◽

Formalin Fixed Paraffin Embedded ◽

The Impact ◽

Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

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ON THE ROLE OF HISTOMORPHOMETRIC (STEREOLOGICAL) MICROSTRUCTURE PARAMETERS IN THE PREDICTION OF VERTEBRAE COMPRESSION STRENGTH

Image Analysis & Stereology ◽

10.5566/ias.2028 ◽

2019 ◽

Vol 38 (1) ◽

pp. 63 ◽

Cited By ~ 2

Author(s):

Leszek Karol Wojnar ◽

Aneta Gądek-Moszczak ◽

Jacek Pietraszek

Keyword(s):

Compression Strength ◽

Correlation Coefficients ◽

Diagnostic Techniques ◽

Micro Ct ◽

Compression Tests ◽

Mineral Density ◽

3D Images ◽

Microstructure Parameters

The well-documented relation between bone mineral density (BMD) and bone compression strength constitutes the basis for osteoporosis diagnostics and the assessment of fracture risk. Simultaneously, this relation demonstrates a considerable scatter of results as bones of identical mineral density may have significantly different properties. The experimentally confirmed theorem that two materials or tissues of identical microstructure have identical properties leads to the evaluation of various quantitative stereological parameters (also referred to in biomedicine as histomorphology). These parameters, obtained from analysis of 2D or 3D images, have been used in numerous attempts to explain changes in bone strength. Although numerous correlation dependencies, often with high correlation coefficients, were evaluated, we do not know which parameters are worth evaluating, and there is no physical interpretation of these relations. An extended statistical analysis was accomplished on the basis of analysis of 3D images from 23 lumbar (L3) vertebrae scanned with micro-CT and the results of subsequent compression tests. A new parameter called SDF (structure destruction factor) was proposed in order to characterise the quality of 3D trabecular structures, and its significance was demonstrated. The final correlation function, which uses only three stereological parameters, made it possible to predict compression strength with considerable precision. The estimated values correlated very well with the apparent values (correlation coefficient r=0.96). Finally, the stereological parameters most suitable for characterisation of bone compression strength were chosen and a mechanism responsible for the changes in mechanical properties was proposed. The results obtained defined the necessary improvements in diagnostic techniques that would allow for more efficient quantitative microstructure evaluation and guidelines on how to improve treatment of patients with weakened bones.

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KOMITE AUDIT UNTUK MENINGKATKAN TATA KELOLA PEMERINTAHAN DAN PENYAJIAN LAPORAN KEUANGAN DALAM RANGKA PENINGKATAN KINERJA PEMERINTAH DAERAH (STUDI KASUS PEMERINTAH DAERAH KABUPATEN BELITUNG)

Jurnal Magister Akuntansi Trisakti ◽

10.25105/jmat.v1i1.4903 ◽

2014 ◽

Vol 1 (1) ◽

pp. 1

Author(s):

Alexander Ikhsan ◽

Antar MT Sianturi

Keyword(s):

Local Government ◽

Audit Committee ◽

Good Governance ◽

Financial Statements ◽

Financial Statement ◽

Audit Committees ◽

Government Performance ◽

Qualitative Descriptive ◽

Improved Performance

This research is a qualitative descriptive study entitled “Audit Committee to Improve Governance and Financial Statement in order to Improved Performance of Local Government (Case Study of District Government Belitung)”. The purpose of this study was to determine how much influence of the audit committee in the local government in order to improve governance and the quality of local government financial statement, and its associations with local government performance. Measuring the importance of the audit committee, which was measured using interviews with 15 respondents who have an influence on governance in the Belitung District Based on these results, we can conclude that audit committees in local government will improve the governance and the quality of local government financial statements presentation. And from the results of this study also found that the good governance and quality of local government financial statements presentation will improve the overall local government performance

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STUDIES ON NUCLEIC ACID METACHROMASY

The Journal of Cell Biology ◽

10.1083/jcb.27.2.327 ◽

1965 ◽

Vol 27 (2) ◽

pp. 327-336 ◽

Cited By ~ 41

Author(s):

Ned Feder ◽

Merrill K. Wolf

Keyword(s):

Nucleic Acid ◽

Ribosomal Rna ◽

Toluidine Blue ◽

Color Contrast ◽

Tissue Sections ◽

Dna And Rna ◽

Excellent Preservation ◽

Structural Preservation ◽

Intense Color

Acrolein-fixed, polyester wax-embedded tissue sections showed excellent preservation of light microscopic architecture and, when stained with toluidine blue, intense color contrast between DNA, which stained orthochromatically, and RNA, which stained metachromatically. This method has practical value for differentiating DNA from RNA in the same section. The color contrast was impaired by substituting formaldehyde for acrolein or paraffin for polyester wax, and was negligible in tissues fixed in formaldehyde or Carnoy's fluid and embedded in paraffin. Quality of structural preservation paralleled degree of color contrast. Metachromatic staining can be analysed, by the quantitative parameters of Bradley and colleagues, to provide inferences regarding the conformation of biopolymers in tissue sections. Comparison of the nucleic acid color contrasts in toluidine blue-stained sections with titrations of fixative-treated nucleic acids against toluidine blue in solution indicated a greater difference in conformation between DNA- and RNA-protein in acrolein-polyester sections than between acrolein-treated free DNA and RNA in solution. This is supported by recent evidence that the conformation of ribosomal RNA is quite different in whole ribosomes from that assumed by the same RNA free in solution. The acrolein-polyester method may enhance color contrast by providing superior preservation of ordered nucleoprotein conformations.

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Optimisation of DNA and RNA extraction from archival formalin-fixed tissue

Nucleic Acids Research ◽

10.1093/nar/27.16.e12-i ◽

1999 ◽

Vol 27 (16) ◽

pp. i-iii ◽

Cited By ~ 19

Author(s):

N. J. Coombs ◽

A. C. Gough ◽

J. N. Primrose

Keyword(s):

Rna Extraction ◽

Fixed Tissue ◽

Formalin Fixed Tissue ◽

Dna And Rna ◽

Formalin Fixed

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A Study of Bifurcation Design Used in CD-ELISA Micro-Fluidic Platform

ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology ◽

10.1115/nemb2010-13079 ◽

2010 ◽

Author(s):

Samuel I. En Lin

Keyword(s):

Coriolis Force ◽

Clinical Diagnostics ◽

Biological Molecules ◽

Navier Stokes ◽

Navier Stokes Equation ◽

Safety Testing ◽

Micro Channels ◽

Simulation Results ◽

Incompressible Navier Stokes Equation ◽

Detection And Quantification

Enzyme-linked immunosorbent assays (ELISA), one of the most common immunoassays, is widely used for detection and quantification of chemical and biological molecules and is becoming more and more important in clinical diagnostics, food safety testing, and environmental monitoring. A major challenge in developing the CD-ELISA is to split the flow (e.g., bio-reagents) evenly on the micro-channels. The Coriolis force resultant from CD rotation can disturb the flow in the splitter region and thus cause the failure mode in delivering the solution from each reservoir in a pre-specified manner. In this study, we investigate on the effects of inlet pressure and Coriolis force on the splitting ratio under two splitter structures. The analysis is based on the incompressible Navier-Stokes equation and the simulation results agree well with our experimental work.

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White matter imaging of ethanol-fixed rat brain by phase-contrast X-ray computed tomography

Acta Radiologica ◽

10.1177/02841851211030776 ◽

2021 ◽

pp. 028418512110307

Author(s):

Thet-Thet- Lwin ◽

Akio Yoneyama ◽

Shogo Kokubo ◽

Hiroko Maruyama ◽

Kazuyuki Hyodo ◽

...

Keyword(s):

White Matter ◽

Phase Contrast ◽

Three Dimensional ◽

Image Contrast ◽

Cerebral White Matter ◽

Tissue Fixation ◽

X Ray ◽

Density Values ◽

X Ray Computed ◽

Formalin Fixed

Background Phase-contrast X-ray computed tomography imaging (PCI) based on crystal X-ray interferometry can detect minute density differences within biological soft tissues without contrast agents. Ethanol fixation yields increased tissue-background density differences due to the dehydrating and delipidifying effects of ethanol. Purpose To obtain high image contrast of cerebral white matter structures in PCI, tissue fixation using ethanol and routinely used formalin have been examined. Material and Methods Ethanol-fixed (EF) (n = 4) and formalin-fixed (FF) (n = 4) rat brains were imaged by crystal X-ray interferometry-based PCI. Tissue staining/microscopy was also performed for histological comparison and myelin density evaluation. Three-dimensional white matter tract images were reconstructed. Results Superior image contrast was obtained in the images of EF brains (EF images) compared to those of formalin-fixed brains (FF images), particularly for white matter structures. Significant density differences between the white matter structures and hippocampus ( P < 0.01)/thalamus ( P < 0.001) were observed in the EF, but not FF, images. Ethanol fixation enhanced the image contrast of white matter tracts by approximately sixfold compared to formalin fixation, and close agreement (r2 = 0.97; P < 0.05) between the density values on the CT images and the myelin density values in histological images was observed for the EF brains. Three-dimensional reconstruction of the white matter tracts was possible from the EF images, but not FF images. Conclusion Ethanol fixation resulted in marked contrast enhancement of cerebral white matter structures in PCI. Thus, high-resolution PCI using ethanol for tissue fixation could be valuable for experimental neurological studies and postmortem neuropathology evaluation.

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