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Decreasing formalin concentration improves quality of DNA extracted from formalin-fixed paraffin-embedded tissue specimens without compromising tissue morphology or immunohistochemical staining
  1. Michele Cummings1,
  2. Henry King1,
  3. James Hurst1,
  4. Georgette Tanner1,
  5. Leah Khazin1,
  6. Phillip Thompson2,
  7. Allan Gray2,
  8. Narinder Gahir1,
  9. Caroline Cartlidge1,
  10. Zara Farooq1,
  11. Keyura Raveendran1,
  12. Katie Allen1,2,
  13. Olorunda Rotimi2,
  14. Nicolas M Orsi1
  1. 1 Women's Health Research Group, Pathology & Data Analytics, Leeds Institute of Medical Research at St. James's, University of Leeds, Wellcome Trust Brenner Building, Leeds, UK
  2. 2 Department of Histopathology, St. James's University Hospital, Leeds, UK
  1. Correspondence to Dr Nicolas M Orsi, Pathology and Data Analytics, Leeds Institute of Medical Research at St. James's, University of Leeds, Wellcome Trust Brenner Building, Beckett St., Leeds LS9 7TF, UK; n.m.orsi{at}


Genomic technologies are increasingly used clinically for both diagnosis and guiding cancer therapy. However, formalin fixation can compromise DNA quality. This study aimed to optimise tissue fixation using normal colon, liver and uterus (n=8 each) by varying neutral buffered formalin (NBF) concentration (1%–5% w/v) and fixation time (24–48 hours). Fixation using 4% NBF improved DNA quality (assessed by qPCR) compared with routine (4% unbuffered formal saline-fixed) specimens (p<0.01). Further improvements were achieved by reducing NBF concentration (p<0.00001), whereas fixation time had no effect (p=0.110). No adverse effects were detected by histopathological or QuPath morphometric analysis. Immunohistochemistry for multicytokeratin and α-smooth muscle actin revealed no changes in staining specificity or intensity in any tissue other than on liver multicytokeratin staining intensity, where the effect of fixation time was more significant (p=0.0004) than NBF concentration (p=0.048). Thus, reducing NBF concentration can maximise DNA quality without compromising tissue morphology or standard histopathological analyses.

  • PCR
  • morphology
  • morphometry
  • molecular pathology
  • immunohistochemistry

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  • Handling editor Runjan Chetty.

  • MC and HK contributed equally.

  • Contributors NMO and OR: conceived and designed the study. HK, LK, PT, AG, CC, ZF and KR: conducted laboratory analyses. MC, HK, JH GT and NG: performed data analysis. KA and NMO: carried out histopathological assessment. MC and NMO: drafted the manuscript. All authors approved the final version of the manuscript.

  • Funding Funding for this study was provided by Leeds Cares.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; internally peer reviewed.