Skip to main content
SpringerLink
Log in

The quality of DNA isolated from autopsy formalin-fixed and formalin-fixed paraffin-embedded tissues: study of 1662 samples

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

There are enormous formalin-fixed paraffin-embedded tissue archives and a constantly growing number of methods for molecular analyses but, the isolation of DNA from this tissue is still challenging due to the damaging effect of formalin on DNA. To determine the extent to which DNA purity, yield and integrity depend on the process of fixation in formalin, and to what extent on the process of tissue paraffin embedding, we compared the quality of DNA isolated from fixed tissues and DNA isolated from tissues embedded in paraffin blocks after fixation.

Methods and results

Heart, liver and brain tissues obtained from healthy people who suddenly died a violent death were fixed in 10% buffered formalin as well as in 4% unbuffered formalin for 6 h, 1–7 days (every 24 h), 10, 14, 28 days and 2 months. Additionally, the same tissues were fixed in 4% unbuffered formalin embedded in a paraffin block and stored from a few months to 30 years. The yield and purity of the DNA samples isolated from these tissues were measured using spectrophotometry. PCR amplification of the hTERT gene was performed to evaluate the degree of DNA fragmentation. Although the purity of the DNA isolated from almost all tissue samples was satisfactory, the DNA yields changed significantly. There was a decrease in successful PCR amplification of the hTERT gene in DNA samples isolated from tissue fixed in buffered and unbuffered formalin for up to 2 months from 100% to 8.3%. Archiving the tissue in paraffin blocks for up to 30 years also impacts the integrity of DNA, so there was a decrease in PCR amplification of the hTERT gene from 91% success to 3%.

Conclusion

The largest decrease in DNA yield was observed after tissue formalin fixation after 14 days of fixation in buffered and unbuffered formalin. DNA integrity depends on the time of tissue formalin fixation, especially after 6 days for tissue fixed in unbuffered formalin, while for tissue fixed in buffered formalin the time is prolonged up to 28 days. The age of paraffin blocks also impacted DNA integrity, after 1 year and 16 years of archiving the paraffin blocks of tissues, there was a decrease in the success of PCR amplification.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Begum S, Prabhu V, Mohanty V, Upadhyaya KK, Abdulla R (2022) Unveiling the arcanum of formalin-fixed paraffin-embedded archival tissue blocks: a valuable resource for genomic DNA extraction. J Oral Maxillofac Pathol 26(2):289. https://doi.org/10.4103/jomfp.jomfp_424_20

    Article  PubMed  PubMed Central  Google Scholar 

  2. Viljoen R, Reid KM, Mole CG, Rangwaga M, Heathfield LJ (2022) Towards molecular autopsies: development of a FFPE tissue DNA extraction workflow. Sci Justice 62(2):137–144. https://doi.org/10.1016/j.scijus.2021.12.005

    Article  PubMed  Google Scholar 

  3. Gaffney EF, Riegman PH, Grizzle WE, Watson PH (2018) Factors that drive the increasing use of FFPE tissue in basic and translational cancer research. Biotech Histochem 93(5):373–386. https://doi.org/10.1080/10520295.2018.1446101

    Article  CAS  PubMed  Google Scholar 

  4. Gouveia GR, Ferreira SC, Ferreira JE, Siqueira SA, Pereira J (2014) Comparison of two methods of RNA extraction from formalin-fixed paraffin-embedded tissue specimens. Biomed Res Int. https://doi.org/10.1155/2014/151724

    Article  PubMed  PubMed Central  Google Scholar 

  5. Potluri K, Mahas A, Kent MN, Naik S, Markey M (2015) Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue. Anal Biochem 486:17–23. https://doi.org/10.1016/j.ab.2015.06.029

    Article  CAS  PubMed  Google Scholar 

  6. Rabelo-Gonçalves E, Roesler B, Guardia AC, Milan A, Hara N, Escanhoela C, Almeida J, Boin I, Zeitune JM (2014) Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma. Pathol Res Pract 210(3):142–146. https://doi.org/10.1016/j.prp.2013.11.003

    Article  CAS  PubMed  Google Scholar 

  7. Lucena-Aguilar G, Sánchez-López AM, Barberán-Aceituno C, Carrillo-Ávila JA, López-Guerrero JA, Aguilar-Quesada R (2016) DNA source selection for downstream applications based on DNA quality indicators analysis. Biopreserv Biobank 14(4):264–270. https://doi.org/10.1089/bio.2015.0064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cantagalli VD, Leite KRM, Srougi M, Gattas GJF (2011) Quality control of DNA from formalin fixed and paraffin embedded prostate biopsy for forensic analysis. Forensic Sci Int 3:e39–e40

    Google Scholar 

  9. Panchal NK, Bhale A, Chowdary R, Verma VK, Beevi SS (2020) PCR amplifiable DNA from breast disease FFPE section for mutational analysis. J Biomol Tech 31(1):1–6. https://doi.org/10.7171/jbt.20-3101-001

    Article  PubMed  PubMed Central  Google Scholar 

  10. Seiler C, Sharpe A, Barrett JC, Harrington EA, Jones EV, Marshall GB (2016) Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality? BMC Clin Pathol 16:17. https://doi.org/10.1186/s12907-016-0039-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sengüven B, Baris E, Oygur T, Berktas M (2014) Comparison of methods for the extraction of DNA from formalin-fixed, paraffin-embedded archival tissues. Int J Med Sci 11(5):494–499. https://doi.org/10.7150/ijms.8842

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gouvegia GR, Ferreira SC, Siqueira SAC, Pereira J (2016) Nucleic acids extraction from formalin-fixed and paraffin-embedded tissues. In: Larramendy ML, Soloneski S (eds) Nucleic acids-from basic aspects to laboratory tools. IntechOpen, UK, pp 27–36

    Google Scholar 

  13. Carlsson J, Davidsson S, Fridfeldt J, Giunchi F, Fiano V, Grasso C, Zelic R, Richiardi L, Andrén O, Pettersson A, Fiorentino M, Akre O (2018) Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies. BMC Med Res Methodol 18(1):161. https://doi.org/10.1186/s12874-018-0628-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Santos S, Sá D, Bastos E, Guedes-Pinto H, Gut I, Gärtner F, Chaves R (2009) An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues. Res Vet Sci 86(3):421–426. https://doi.org/10.1016/j.rvsc.2008.08.007

    Article  CAS  PubMed  Google Scholar 

  15. Ohmomo H, Komaki S, Ono K, Sutoh Y, Hachiya T, Arai E, Fujimoto H, Yoshida T, Kanai Y, Sasaki M, Shimizu A (2021) Evaluation of clinical formalin-fixed paraffin-embedded tissue quality for targeted-bisulfite sequencing. Pathol Int 71(2):135–140. https://doi.org/10.1111/pin.13054

    Article  CAS  PubMed  Google Scholar 

  16. Bonin S, Stanta G (2013) Nucleic acid extraction methods from fixed and paraffin-embedded tissues in cancer diagnostics. Expert Rev Mol Diagn 13(3):271–282. https://doi.org/10.1586/erm.13.14

    Article  CAS  PubMed  Google Scholar 

  17. Lv Y, Li S, Li Z, Tao R, Shao Y, Chen Y (2020) Quantitative analysis of noncoding RNA from paired fresh and formalin-fixed paraffin-embedded brain tissues. Int J Legal Med 134(3):873–884. https://doi.org/10.1007/s00414-019-02210-1

    Article  PubMed  Google Scholar 

  18. Reid KM, Maistry S, Ramesar R, Heathfield LJ (2017) A review of the optimisation of the use of formalin fixed paraffin embedded tissue for molecular analysis in a forensic post-mortem setting. Forensic Sci Int 280:181–187. https://doi.org/10.1016/j.forsciint.2017.09.020

    Article  CAS  PubMed  Google Scholar 

  19. Yi QQ, Yang R, Shi JF, Zeng NY, Liang DY, Sha S, Chang Q (2020) Effect of preservation time of formalin-fixed paraffin-embedded tissues on extractable DNA and RNA quantity. J Int Med Res 48(6):300060520931259. https://doi.org/10.1177/0300060520931259

    Article  CAS  PubMed  Google Scholar 

  20. Hewitt SM, Lewis FA, Cao Y, Conrad RC, Cronin M, Danenberg KD, Goralski TJ, Langmore JP, Raja RG, Williams PM, Palma JF, Warrington JA (2008) Tissue handling and specimen preparation in surgical pathology: issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med 132(12):1929–1935. https://doi.org/10.1043/1543-2165-132.12.1929

    Article  PubMed  Google Scholar 

  21. Ferruelo A, El-Assar M, Lorente JA, Nin N, Peñuelas O, Fernández-Segoviano P, Gonzalez C, Esteban A (2011) Transcriptional profiling and genotyping of degraded nucleic acids from autopsy tissue samples after prolonged formalin fixation times. Int J Clin Exp Pathol 4(2):156–161

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Funabashi KS, Barcelos D, Visoná I, e Silva MS, e Sousa ML, de Franco MF, Iwamura ES, (2012) DNA extraction and molecular analysis of non-tumoral liver, spleen, and brain from autopsy samples: the effect of formalin fixation and paraffin embedding. Pathol Res Pract 208(10):584–591. https://doi.org/10.1016/j.prp.2012.07.001

    Article  CAS  PubMed  Google Scholar 

  23. Vitošević K, Todorović M, Varljen T, Slović Ž, Matić S, Todorović D (2018) Effect of formalin fixation on PCR amplification of DNA isolated from healthy autopsy tissues. Acta Histochem 120(8):780–788. https://doi.org/10.1016/j.acthis.2018.09.005

    Article  CAS  PubMed  Google Scholar 

  24. Bass BP, Engel KB, Greytak SR, Moore HM (2014) A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen? Arch Pathol Lab Med 138(11):1520–1530. https://doi.org/10.5858/arpa.2013-0691-RA

    Article  PubMed  Google Scholar 

  25. Skage M, Schander C (2007) DNA from formalin-fixed tissue: extraction or repair? That is the question. Marine Biol Res 3(5):289–295. https://doi.org/10.1080/17451000701473942

    Article  Google Scholar 

  26. Ludyga N, Grünwald B, Azimzadeh O, Englert S, Höfler H, Tapio S, Aubele M (2012) Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses. Virchows Arch 460(2):131–140. https://doi.org/10.1007/s00428-011-1184-9

    Article  CAS  PubMed  Google Scholar 

  27. Sarnecka AK, Nawrat D, Piwowar M, Ligęza J, Swadźba J, Wójcik P (2019) DNA extraction from FFPE tissue samples—a comparison of three procedures. Contemp Oncol (Pozn) 23(1):52–58. https://doi.org/10.5114/wo.2019.83875

    Article  CAS  PubMed  Google Scholar 

  28. Bonnet E, Moutet ML, Baulard C, Bacq-Daian D, Sandron F, Mesrob L, Fin B, Delépine M, Palomares MA, Jubin C, Blanché H, Meyer V, Boland A, Olaso R, Deleuze JF (2018) Performance comparison of three DNA extraction kits on human whole-exome data from formalin-fixed paraffin-embedded normal and tumor samples. PLoS ONE 13(4):e0195471. https://doi.org/10.1371/journal.pone.0195471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Turashvili G, Yang W, McKinney S, Kalloger S, Gale N, Ng Y, Chow K, Bell L, Lorette J, Carrier M, Luk M, Aparicio S, Huntsman D, Yip S (2012) Nucleic acid quantity and quality from paraffin blocks: defining optimal fixation, processing and DNA/RNA extraction techniques. Exp Mol Pathol 92(1):33–43. https://doi.org/10.1016/j.yexmp.2011.09.013

    Article  CAS  PubMed  Google Scholar 

  30. Farrugia A, Keyser C, Ludes B (2010) Efficiency evaluation of a DNA extraction and purification protocol on archival formalin-fixed and paraffin-embedded tissue. Forensic Sci Int 194(1–3):e25–e28. https://doi.org/10.1016/j.forsciint.2009.09.004

    Article  CAS  PubMed  Google Scholar 

  31. Rich J, Dean DE, Powers RH (2005) Forensic medicine of the lower extremity. Humana Press, Totowa

    Book  Google Scholar 

  32. Ferrer I, Armstrong J, Capellari S, Parchi P, Arzberger T, Bell J, Budka H, Ströbel T, Giaccone G, Rossi G, Bogdanovic N, Fakai P, Schmitt A, Riederers P, Al-Sarraj S, Ravid R, Kretzschmar H (2007) Effects of formalin fixation, paraffin embedding, and time of storage on DNA preservation in brain tissue: a BrainNet Europe study. Brain Pathol 17(3):297–303. https://doi.org/10.1111/j.1750-3639.2007.00073.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Nam SK, Im J, Kwak Y, Han N, Nam KH, Seo AN, Lee HS (2014) Effects of fixation and storage of human tissue samples on nucleic Acid preservation. Korean J Pathol 48(1):36–42. https://doi.org/10.4132/KoreanJPathol.2014.48.1.36

    Article  PubMed  PubMed Central  Google Scholar 

  34. Legrand B, Mazancourt Pd, Durigon M, Khalifat V, Crainic K (2002) DNA genotyping of unbuffered formalin fixed paraffin embedded tissues. Forensic Sci Int 125(2–3):205–211. https://doi.org/10.1016/s0379-0738(01)00641-7

    Article  CAS  PubMed  Google Scholar 

  35. Niland EE, McGuire A, Cox MH, Sandusky GE (2012) High quality DNA obtained with an automated DNA extraction method with 70+ year old formalin-fixed celloidin-embedded (FFCE) blocks from the indiana medical history museum. Am J Transl Res 4(2):198–205

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Lin J, Kennedy SH, Svarovsky T, Rogers J, Kemnitz JW, Xu A, Zondervan KT (2009) High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil. Anal Biochem 395(2):265–267. https://doi.org/10.1016/j.ab.2009.08.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Okello JB, Zurek J, Devault AM, Kuch M, Okwi AL, Sewankambo NK, Bimenya GS, Poinar D, Poinar HN (2010) Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues. Anal Biochem 400(1):110–117. https://doi.org/10.1016/j.ab.2010.01.014

    Article  CAS  PubMed  Google Scholar 

  38. Greytak SR, Engel KB, Zmuda E, Casas-Silva E, Guan P, Hoadley KA, Mungall AJ, Wheeler DA, Doddapaneni HV, Moore HM (2018) National Cancer Institute Biospecimen Evidence-Based Practices: harmonizing procedures for nucleic acid extraction from formalin-fixed, paraffin-embedded tissue. Biopreserv Biobank 16(4):247–250. https://doi.org/10.1089/bio.2018.0046

    Article  PubMed  PubMed Central  Google Scholar 

  39. Robbe P, Popitsch N, Knight SJL, Antoniou P, Becq J, He M, Kanapin A, Samsonova A, Vavoulis DV, Ross MT, Kingsbury Z, Cabes M, Ramos SDC, Page S, Dreau H, Ridout K, Jones LJ, Tuff-Lacey A, Henderson S, Mason J, Buffa FM, Verrill C, Maldonado-Perez D, Roxanis I, Collantes E, Browning L, Dhar S, Damato S, Davies S, Caulfield M, Bentley DR, Taylor JC, Turnbull C, Schuh A (2018) Clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the 100000 Genomes Project. Genet Med 20(10):1196–1205. https://doi.org/10.1038/gim.2017.241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors would like to thank the Faculty of Medical Sciences, University of Kragujevac and the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (No 451-03-47/2023-01/200111).

Funding

This study was supported by grant (project JP: 05/13) from the Faculty of Medical Sciences, University of Kragujevac, Serbia.

Author information

Authors and Affiliations

  1. Faculty of Medical Sciences, Department of Forensic Medicine, University of Kragujevac, Serbia, Kragujevac, Serbia

    Katarina Vitošević, Živana Slović, Ivana Radaković & Miloš Todorović

  2. Department of Forensic Medicine and Toxicology, University Clinical Center Kragujevac, Kragujevac, Serbia

    Katarina Vitošević, Živana Slović & Miloš Todorović

  3. Faculty of Medical Sciences, Department of Genetics, University of Kragujevac, Serbia, Kragujevac, Serbia

    Danijela Todorović & Dušan Radojević

  4. Institute of Legal Medicine, Faculty of Medicine, University of Belgrade, Belgrade, Serbia

    Tatjana Varljen

  5. Faculty of Medical Sciences, University of Kragujevac, Serbia, Kragujevac, Serbia

    Vanja Čanović

Authors
  1. Katarina Vitošević
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Danijela Todorović
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Živana Slović
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tatjana Varljen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ivana Radaković
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dušan Radojević
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Vanja Čanović
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Miloš Todorović
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by KV, MT and DT. The first draft was written by KV and MT and all authors commented on previous version of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Katarina Vitošević.

Ethics declarations

Competing interests

All authors declare that they have no conflict of interest.

Ethics approval

Institutional ethical approval was obtained in compliance with the Declaration of Helsinki. Authorization to use human biological samples for research was obtained. The Ethics Committee of the University of Kragujevac, Faculty of Medical sciences, the Ethic Committee of University Clinical Centre of Kragujevac (No. 01–2798), Appeal Public Prosecutor's Office from Kragujevac (A No. 79/13) and Higher Court in Kragujevac (SU-VIII-110/13) agreed to these investigations. Informed consent was obtained from the relatives of the subjects when they arte identified and available.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vitošević, K., Todorović, D., Slović, Ž. et al. The quality of DNA isolated from autopsy formalin-fixed and formalin-fixed paraffin-embedded tissues: study of 1662 samples. Mol Biol Rep 50, 6323–6336 (2023). https://doi.org/10.1007/s11033-023-08491-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-023-08491-5

Keywords

Search

Navigation