Articles
Open Access
Peer Reviewed
Effect of Repeatedly Used Fixative Solution on DNA Quality and PCR Amplification Success in Formalin-Fixed Paraffin-Embedded
Abstract
Formalin-fixed, paraffin-embedded (FFPE) tissues are widely used for histological and molecular analyses. Although fixative reuse can reduce costs and waste, its effects on DNA quality and downstream molecular applications remain a concern. This study investigated the effects of reusing 10% neutral buffered formalin (NBF) on H&E staining quality, DNA quality, and polymerase chain reaction (PCR) amplification. Four fixation conditions were evaluated: fresh NBF (0x; control) and NBF reused once (1x), twice (2x), or three times (3x), with six FFPE tissue sections assessed per condition. H&E staining quality was scored by a board-certified anatomic pathologist under blinded conditions. DNA concentration and purity were measured by spectrophotometry, DNA integrity was assessed by agarose gel electrophoresis, and PCR amplification of a 565-bp albumin gene fragment was evaluated. After three cycles of fixative reuse, 83% of tissue sections retained good H&E staining quality, with no statistically significant difference from the control (p>0.05). Good DNA integrity, however, was observed only in samples fixed with NBF reused up to twice. These findings suggest that NBF reuse should be limited to two cycles when FFPE tissues are intended for DNA analysis.
References
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3. Atanesyan L, Steenkamer MJ, Horstman A, Moelans CB, Schouten JP, Savola SP. Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA. Am J Clin Pathol. 2017;147: 60–68. https://doi.org/10.1093/AJCP/AQW205
4. Ottestad AL, Emdal EF, Grønberg BH, Halvorsen TO, Dai HY. Fragmentation assessment of FFPE DNA helps in evaluating NGS library complexity and interpretation of NGS results. Experimental and Molecular Pathology. 2022;126(April). https://doi.org/10.1016/j.yexmp.2022.104771
5. Chung J yong, Song JS, Ylaya K, Sears JD, Choi L, Cho H, et al. Histomorphological and Molecular Assessments of the Fixation Times Comparing Formalin and Ethanol-Based Fixatives. Journal of Histochemistry & Cytochemistry. 2018;66(2): 121–135. https://doi.org/10.1369/0022155417741467
6. Berrino E, Erika S, Chesta A, Detillo P, Bragoni A, Gagliardi A, et al. Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling. Lab Invest. 2023;104. https://doi.org/10.1016/j.labinv.2023.100280
7. Buesa RJ. Histology without formalin? Annals of Diagnostic Pathology. 2008;12: 387–396. https://doi.org/10.1016/j.anndiagpath.2008.07.004
8. Hardi Z, Wiryanti W, Durachim A, Rahmat M. efek penggunaan ulang larutan fiksatif formalin pada kualitas preparat histopatologi dan jumlah limbah yang dihasilkan. Current Biomedicine. 2024;2(2): 71–83. https://doi.org/10.29244/currbiomed.2.2.71-83
9. Rullier A, Amintas S, Marques M, Le Bail B, Belleannée G, Dubus P, et al. Recycled formalin: a new tool to mitigate the environmental impact of surgical pathology in routine practice. Pathology. 2025;57(4): 437–442. https://doi.org/10.1016/j.pathol.2024.12.639
10. Ariyadi T, Suryono H. Kualitas sediaan jaringan kulit metode microwave dan conventional histoprocessing pewarnaan hematoxylin eosin. Jurnal Labora Medika. 2017;1(1): 7–11. https://doi.org/10.26714/jlabmed.1.1.2017.07-11
11. Musyarifah Z, Agus S. proses fiksasi pada pemeriksaan histopatologik. Jurnal Kesehatan Andalas. 2018;7(3): 443–453. https://doi.org/10.25077/jka.v7i3.900
12. Allimi HS, Sari RVS, Apriliyani T, Nuriliani A, Retnoaji B, Saragih H, et al. Larutan Fiksatif Makromolekul pada Sediaan Histologis. Berkala Ilmiah Biologi. 2023;14(3): 41–49. https://doi.org/10.22146/bib.v14i3.6531
13. Li Y, Pillar N, Li J, Liu T, Wu D, Sun S, et al. Virtual histological staining of unlabeled autopsy tissue. Nature Communications. 2024;15(1684): 1–17. https://doi.org/10.1038/s41467-024-46077-2
14. Michaud K, Basso C, Boer HH De, Fracasso T, Gaspari M De. Ischemic and non-ischemic myocardial injuries at autopsy- an overview for forensic pathologists. International Journal of Legal Medicine. 2025;139: 1579–1596. https://doi.org/10.1007/s00414-025-03479-1
15. Kongtueng P, Charoenphon N, Senarat S, Thaochan N. Artifacts in microscopic slides and tem images of aquatic animals and insects: a 10-year retrospective observation. Malaysian Journal of Microscopy. 2024;20(2): 1–13. https://malaysianjournalofmicroscopy.org/ojs/index.php/mjm/article/view/805/466
16. Damayanti M, Ariyadi T, Tyas RA. Proses deparafinisasi sediaan jaringan ginjal dengan dan tanpa pemanasan menggunakan mineral oil pada pewarnaan hematoksilin-eosin. Jurnal Kesehatan Rajawali. 2021;11(2): 1–6. https://doi.org/10.54350/jkr.v11i2.104
17. Howat WJ, Wilson BA. Tissue fixation and the effect of molecular fixatives on downstream staining procedures. Methods. 2023;70(1): 12–19. https://doi.org/10.1016/j.ymeth.2014.01.022
18. Setiaputri AA, Barokah GR, Arbajayanti RD, Fabella N, Pertiwi RM, Nurilmala M, et al. Perbandingan metode isolasi dna pada produk perikanan segar dan olahan. JPHPI. 2020;23(3): 447–458. https://doi.org/10.17844/jphpi.v23i3.32314
19. Blouin T, Saini N. Aldehyde-induced DNA-protein crosslinks- DNA damage , repair and mutagenesis. Frontiers in Oncology. 2024;14(1478373): 1–10. https://doi.org/10.3389/fonc.2024.1478373
20. Paul P, Rajput S, Joshi P, Naithani M, Chowdhury N, Rao S, et al. Comparison of Fluorometric and UV Spectrophotometric Findings for DNA Isolated From Formalin-Fixed Paraffin-Embedded Blocks , Fine Needle Aspiration Cytology Smears , and Blood. Cureus. 2021;13(11): 1–6. https://doi.org/10.7759/cureus.19583
21. Matlock B. Assessment of Nucleic Acid Purity. Wilmington (MA), USA: Thermo Fisher Scientific; 2015