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Title: The Effect of Fibrosis on DNA Yield from Formalin Fixed Paraffin Embedded Tissues
Authors: Muhammad Naveed, Hassan Tariq, Asma Gul, Natasha Arzo, Muhammad Hussain, Sumaira Buksh
Journal: Pakistan Armed Forces Medical Journal (PAFMJ)
Publisher: Army Medical College, Rawalpindi.
Country: Pakistan
Year: 2024
Volume: 74
Issue: 3
Language: English
Keywords: Deoxyribonucleic acidFormalin-fixed paraffin-embeddedFibrosis
Objective: To report the effect of fibrosis on yield of Deoxyribonucleic Acid extraction from Formalin-Fixed Paraffin-Embedded tissue.
Study Design: Cross sectional study.
Place and Duration of Study: Department of Molecular Pathology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, Jul to Dec 2021.
Methodology: Fifty-four Formalin-Fixed Paraffin-Embedded tissues having size <2cm containing fibrosis (Mild, Moderate, Severe) were included in the study. Formalin-Fixed Paraffin-Embedded tissues having tissue size > 2cm were excluded. Deoxyribonucleic Acid was extracted and quantified from Formalin-Fixed Paraffin-Embedded blocks.
Results: Nine (17%) Formalin-Fixed Paraffin-Embedded tissues had no fibrosis, 28(51%) had Mild fibrosis, 9(17%) and 8(15%) had Moderate and Severe fibrosis. Measurement scale in 5 different stages for evaluation of our quantified results were used. For specimens with no fibrosis the DNA quantity fell on scale 1, 0(0%); on scale 2, 1(11%); on scale 3, 0(0%); on scale 4, 1(11%); and on scale 5, 7(78%). Deoxyribonucleic Acid yield in moderate fibrosis specimens fell on scale 1, 0(0%)’ on scale 2, 7(78%); on scale 3, 0(0%); on scale 4, 1(11%); and on scale 5, 1(11%). Among severe fibrosis the Deoxyribonucleic Acid quantity fall on scale 1, 5(62%); on scale 2, 3(38%); on scale 3, 0(0%); on scale 4, 0(0%); and on scale 5, 0(0%).
Conclusion: Formalin-Fixed Paraffin-Embedded tissues having severe fibrosis resulted in significantly lower Deoxyribonucleic Acid yield as compared to Formalin-Fixed Paraffin-Embedded tissues having no fibrosis
To determine the effect of fibrosis on the yield of Deoxyribonucleic Acid (DNA) extraction from Formalin-Fixed Paraffin-Embedded (FFPE) tissues.
A cross-sectional study was conducted using 54 FFPE tissue samples (size <2cm) with varying degrees of fibrosis (Mild, Moderate, Severe). DNA was extracted and quantified using the QIAamp FFPE DNA tissue kit and a Qubit fluorometer. Data was analyzed using SPSS version 25.
graph TD;
A["Collect FFPE Tissue Samples"] --> B["Categorize by Fibrosis Level: Mild, Moderate, Severe, None"];
B --> C["Extract DNA using QIAamp FFPE DNA tissue kit"];
C --> D["Quantify DNA using Qubit fluorometer"];
D --> E["Analyze Data using SPSS v25"];
E --> F["Determine Effect of Fibrosis on DNA Yield"];
Fibrosis, characterized by an increased amount of collagen and fibronectin in the extracellular matrix, appears to hinder DNA extraction from FFPE tissues, leading to lower yields. This can impact downstream molecular analyses. The study controlled for technical factors like fixative type, time, and temperature to isolate the effect of fibrosis.
- Tissues with severe fibrosis yielded significantly lower DNA amounts compared to tissues with no or mild fibrosis.
- Among specimens with no fibrosis, 78% yielded DNA in scale 5 (>=3.00 ng/ul).
- Among specimens with moderate fibrosis, 78% yielded DNA in scale 2 (0.001 ng/ul to 0.99 ng/ul).
- Among specimens with severe fibrosis, 62% yielded DNA in scale 1 (0.00 ng/ul) and 38% in scale 2 (0.001 ng/ul to 0.99 ng/ul).
Severe fibrosis in FFPE tissues significantly reduces DNA yield, which has implications for molecular testing and research.
- Study duration: July to December 2021. (Confirmed in text)
- Total samples analyzed: 54 FFPE tissue samples. (Confirmed in text)
- Percentage of samples with mild fibrosis: 51% (28 out of 54). (Confirmed in text)
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