Comparison of Fluorometric and UV Spectrophotometric Findings for DNA Isolated From Formalin-Fixed Paraffin-Embedded Blocks, Fine Needle Aspiration Cytology Smears, and Blood

Pranoy Paul,Nilotpal Chowdhury,Swati Rajput,Shalinee Rao,Manju O Pai,Prashant Joshi,Manisha Naithani

doi:10.7759/cureus.19583

Abstract

Introduction: Fine needle aspiration cytology (FNAC) smear may serve as a convenient sample for DNA extraction for molecular pathology in addition to more commonly used formalin-fixed paraffin-embedded (FFPE) sections. DNA quantification done by fluorometer is more accurate than UV vis spectrophotometer regardless of the source. This study was conducted to compare DNA yield and quality from cytology smears, FFPE sections and peripheral blood using both fluorometer and spectrophotometer. Further, introspection was made to check for the adequacy of DNA extracted from cytology smears with respect to DNA extracted from core biopsies.Method: DNA was extracted from 10 fresh peripheral blood samples, core biopsies and FNAC smears. The DNA was quantified using a fluorimeter and UV vis spectrophotometer in all cases.Results: Statistically significant difference was seen between the data obtained from UV vis spectrophotometry and flourometry. The quantity of DNA extracted from FNAC smears was higher than that of core biopsy as per fluorometry data (mean DNA of core biopsy = 1.9ng/µl, of FNAC = 3.3ng/µl).Conclusion: DNA estimation by fluorometry is more accurate and precise than spectrophotometry in FFPE, FNAC and whole blood samples. DNA yield from FNAC slides is comparable to that from core biopsies.

Highlights

Fine needle aspiration cytology (FNAC) smear may serve as a convenient sample for DNA extraction for molecular pathology in addition to more commonly used formalin-fixed paraffin-embedded (FFPE) sections
The quantity of DNA extracted from FNAC smears was higher than that of core biopsy as per fluorometry data
Mean DNA yield in blood, FFPE and FNA cytological smear measured by fluorometer and spectrophotometer was found to be 10.99, 1.9, 3.3 and 29.76, 69.9, 119.9 respectively (Tables 1, 2, 3)

Summary

Introduction

Fine needle aspiration cytology (FNAC) smear may serve as a convenient sample for DNA extraction for molecular pathology in addition to more commonly used formalin-fixed paraffin-embedded (FFPE) sections. DNA quantification done by fluorometer is more accurate than UV vis spectrophotometer regardless of the source. This study was conducted to compare DNA yield and quality from cytology smears, FFPE sections and peripheral blood using both fluorometer and spectrophotometer. Archival cytology smears obtained from fine needle aspiration cytology technique (FNAC) can be another appropriate source for isolating nucleic acids for downstream processes [24]. A simplified and portable but less commonly used method of estimating nucleic acid concentration is by fluorometer. According to numerous independent studies, fluorometer is shown to be a much more accurate and reproducible mode of nucleic acid estimation [9,10]. Since most mutation analysis kits available in the market do not explicitly mention mode of DNA estimation in their methodology for preparation of amplification mix, using UV vis spectrophotometer readings to set up a reaction may severely decrease the sensitivity of the assay

Methods

Results

Discussion

Conclusion