Sodium Alginate versus Plasma Thrombin Cell Blocks in Diagnostic Cytopathology: A Comparative Analysis

2021 ◽  
pp. 1-7
Author(s):  
Shruti Gupta ◽  
Upasana Gautam ◽  
Shaily Susheilia ◽  
Baneet Bansal ◽  
Radha Uppal ◽  
...  
Keyword(s):  
Sodium Alginate ◽  
Molecular Analysis ◽  
Low Cost ◽  
Nuclear Marker ◽  
Fine Needle Aspirates ◽  
Dna Yield ◽  
Morphological Evaluation ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

<b><i>Background:</i></b> Cell blocks (CBs) are an essential adjunct in cytopathology practice. The aim of this study was to compare 2 techniques of CB preparation – plasma thrombin (PT) method with sodium alginate (SA) method for overall cellularity, morphological preservation, obscuring artefacts, immunocytochemistry (ICC), suitability for molecular analysis, and cost of preparation. <b><i>Design:</i></b> A total of 80 fine-needle aspirates from various sites and serous effusion samples were included. Of these cases, by random selection, 40 each were prepared by PT method and SA methods, respectively. The haematoxylin-eosin-stained sections from the formalin-fixed, paraffin-embedded CBs from both methods were evaluated in a blinded fashion by 2 cytopathologists and scored for cellularity, artefacts, and morphological preservation and analysed by χ<sup>2</sup> test with Yates correction. We evaluated 6 cases from each method by ICC for a range of membrane, cytoplasmic and nuclear marker expression. DNA was extracted from four cases to evaluate their utility for molecular analysis. <b><i>Results:</i></b> CB sections from PT and SA techniques showed comparable cellularity and excellent cytomorphological preservation. Blue gel-like artefacts were common in the SA technique but did not interfere with morphological evaluation. ICC staining results were also similar. DNA yield and utility for PCR were also comparable. The SA-CB cost half that of PT-CB (USD 0.4 vs. USD 1). <b><i>Conclusion:</i></b> SA technique of CB preparation is an excellent low-cost alternative to PT method for CB preparation.

scholarly journals A protocol for good quality genomic DNA isolation from formalin-fixed paraffin-embedded tissues without using commercial kits

2021 ◽  
Author(s):  
Fazlur Rahman Talukdar ◽  
Irena Abramovic ◽  
Cyrille Cuenin ◽  
Christine Carreira ◽  
Nitin Gangane ◽  
...  
Keyword(s):  
Dna Isolation ◽  
Middle Income ◽  
Dna Yield ◽  
Genomic Dna Isolation ◽  
Novel Approach ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Commercial Kits ◽  
Formalin Fixed

DNA isolation from formalin fixed paraffin embedded (FFPE) tissues for molecular analysis has become a frequent procedure in cancer research. However, the yield or quality of the isolated DNA is often compromised, and commercial kits are used to overcome this to some extent. We developed a new protocol (IARCp) to improve better quality and yield of DNA from FFPE tissues without using any commercial kit. To evaluate the IARCp performance, we compared the quality and yield of DNA with two commercial kits, namely NucleoSpin DNA FFPE XS (MN) and QIAamp DNA Micro (QG) isolation kit. Total DNA yield for QG ranged from 120.0 to 282.0 ng (mean 216.5 ng), for MN: 213.6 to 394.2 ng (mean 319.1 ng), and with IARCp the yield was much higher ranging from 775.5 to 1896.9 ng (mean 1517.8 ng). Moreover, IARCp has also performed well in qualitative assessments. Overall, IARCp represents a novel approach to DNA isolation from FFPE which results in good quality and significant amounts of DNA suitable for many downstream genome-wide and targeted molecular analyses. Our proposed protocol does not require the use of any commercial kits for isolating DNA from FFPE tissues, making it suitable to implement in low-resource settings such as low and middle-income countries (LMICs).

scholarly journals A rapid technique for analysis of formalin-fixed, paraffin-embedded tissues by fluorescent in situ hybridization with alpha-satellite probes

1998 ◽  
Vol 21 (4) ◽  
pp. 447-448 ◽  
Author(s):  
Nilce Barril ◽  
Andréa Borduchi Carvalho-Salles ◽  
Eloiza Helena Tajara
Keyword(s):  
Fluorescent In Situ Hybridization ◽  
Low Cost ◽  
Fish Analysis ◽  
Alpha Satellite ◽  
Chromatin Decondensation ◽  
Rapid Procedure ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

We describe a rapid procedure for preparing archival tissues for interphase FISH analysis. The present protocol differs from others previously described because it allows the obtention of nuclei in satisfactory number and quality without using special equipments, adhesive-treated slides or solutions for chromatin decondensation. The method is of low cost and useful for retrospective analyses of formalin-fixed, paraffin-embedded samples.

scholarly journals Effect of Different Proteinase K Digest Protocols and Deparaffinization Methods on Yield and Integrity of DNA Extracted From Formalin-fixed, Paraffin-embedded Tissue

2020 ◽  
Vol 68 (3) ◽  
pp. 171-184 ◽  
Author(s):  
Zoe Frazer ◽  
Changyoung Yoo ◽  
Manveer Sroya ◽  
Camille Bellora ◽  
Brian L. DeWitt ◽  
...  
Keyword(s):  
Cell Number ◽  
Proteinase K ◽  
Dna Integrity ◽  
Epithelial Tumor ◽  
Dna Yield ◽  
Tissue Sections ◽  
Epithelial Tumor Cell ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

DNA extracted from formalin-fixed, paraffin-embedded tissue sections is often inadequate for sequencing, due to poor yield or degradation. We optimized the proteinase K digest by testing increased volume of enzyme and increased digest length from the manufacturer’s protocol using 54 biospecimens, performing the digest in centrifuge tubes. Doubling the quantity of proteinase K resulted in a median increase in yield of 96%. Applying the optimized proteinase K protocol to sections deparaffinized on microscope slides generated a further increase in yield of 41%, but only at >50,000 epithelial tumor cells/section. DNA yield now correlated with (χ2 = 0.84) and could be predicted from the epithelial tumor cell number. DNA integrity was assayed using end point multiplex PCR (amplicons of 100–400 bp visualized on a gel), quantitative PCR (qPCR; Illumina FFPE QC Assay), and nanoelectrophoresis (DNA Integrity Numbers [DINs]). Generally, increases in yield were accompanied by increases in integrity, but sometimes qPCR and DIN results were conflicting. Amplicons of 400 bp were almost universally obtained. The process of optimization enabled us to reduce the percentage of samples that failed published quality control thresholds for determining amenability to whole genome sequencing from 33% to 7%.

scholarly journals DNA Yield From Tissue Samples in Surgical Pathology and Minimum Tissue Requirements for Molecular Testing

2015 ◽  
Vol 140 (2) ◽  
pp. 130-133 ◽  
Author(s):  
Melissa C. Austin ◽  
Christina Smith ◽  
Colin C. Pritchard ◽  
Jonathan F. Tait
Keyword(s):  
Molecular Testing ◽  
Prognostic Information ◽  
Tissue Samples ◽  
Institutional Practice ◽  
Tissue Volume ◽  
Dna Yield ◽  
Molecular Assays ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Context Complex molecular assays are increasingly used to direct therapy and provide diagnostic and prognostic information but can require relatively large amounts of DNA. Objectives To provide data to pathologists to help them assess tissue adequacy and provide prospective guidance on the amount of tissue that should be procured. Design We used slide-based measurements to establish a relationship between processed tissue volume and DNA yield by A260 from 366 formalin-fixed, paraffin-embedded tissue samples submitted for the 3 most common molecular assays performed in our laboratory (EGFR, KRAS, and BRAF). We determined the average DNA yield per unit of tissue volume, and we used the distribution of DNA yields to calculate the minimum volume of tissue that should yield sufficient DNA 99% of the time. Results All samples with a volume greater than 8 mm3 yielded at least 1 μg of DNA, and more than 80% of samples producing less than 1 μg were extracted from less than 4 mm3 of tissue. Nine square millimeters of tissue should produce more than 1 μg of DNA 99% of the time. Conclusions We conclude that 2 tissue cores, each 1 cm long and obtained with an 18-gauge needle, will almost always provide enough DNA for complex multigene assays, and our methodology may be readily extrapolated to individual institutional practice.

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