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Effect of fixation time on breast biomarker expression: a controlled study using cell line-derived xenografted (CDX) tumours
  1. K R Kao1,2,
  2. T Hasan1,
  3. A Baptista3,4,
  4. T Truong1,
  5. L Gai2,
  6. A C Smith3,5,
  7. S Li1,
  8. P Gonzales1,
  9. K Voisey2,
  10. P Eriwvo2,
  11. J Power1,2,
  12. N Denic2
  1. 1 Division of Biomedical Sciences, Faculty of Medicine, Memorial University, St. John's, Newfoundland and Labrador, Canada
  2. 2 Division of Anatomical Pathology, Laboratory Medicine Program, Eastern Health, St. John's, Newfoundland and Labrador, Canada
  3. 3 Department of Cancer Cytogenetics, Laboratory Medicine Program, University Health Network, Toronto General Hospital, Toronto, Ontario, Canada
  4. 4 Instituto de Pesquisa Pelé Pequeno Princípe, Curitiba, Paraná, Brazil
  5. 5 Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Canada
  1. Correspondence to Dr K R Kao, Division of Anatomical Pathology, Laboratory Medicine Program, Eastern Health, 300 Prince Philip Drive, St. John's, NL, Canada A1B 3V6; kkao{at}


Aims Altering the length of time specimens are placed in fixative without compromising analytical testing accuracy is a continuous challenge in the anatomical pathology lab. The aim of this study was to determine under controlled conditions the effects of variable fixation time on breast biomarker expression in human breast cancer cell line-derived xenografted (CDX) tumours.

Methods CDX tumours using strong oestrogen receptor (ER)-positive, Her2-negative (MCF7) and weak ER-positive, Her2 equivocal (T47D) breast cancer cell lines were fixed for various times ranging from 1 to 336 hours in 10% neutral buffered formalin. CDX tumours were processed according to routine biomarker testing protocols and stained for ER and Her2 immunohistochemistry (IHC) and processed for HER2 fluorescence in situ hybridisation (FISH). The tumours were evaluated using Allred scoring for ER and current ASCO/CAP guidelines for Her2, and by objective cell counting methodology.

Results No differences were found in expression of ER in either MCF7 or T47D CDX tumours under variable fixation. T47D tumours displayed equivocal Her2 staining when fixed for 24 hours, but fixation for ≤8 hours resulted in consistently negative staining while tumours fixed for >72 hours demonstrated consistent equivocal staining (p<0.01). Cell counting assays revealed only a significant increase in sensitivity in tumours fixed for >72 hours (p<0.01). As expected, FISH results were unaffected by variable fixation.

Conclusions Neither shortened nor prolonged fixation affects ER expression, consistent with previous findings. In equivocal Her2-expressing tumours, however, increasing fixation increased the sensitivity of Her2 IHC reporting while not affecting FISH.


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  • Handling editor Runjan Chetty

  • Contributors KRK designed experiments, collected and interpreted data, wrote the manuscript and supervised research. TH performed all xenograft experiments, collected data and assisted in manuscript preparation. AB performed FISH analysis, collected and interpreted data and contributed to writing of manuscript. TT performed initial fixation audit and collected data. LG interpreted data and contributed to manuscript preparation. ACS designed experiments and contributed to manuscript preparation. SL, PG and KV collected data. PE, JP and ND interpreted data.

  • Funding Newfoundland & Labrador Centre for Applied Health Research (Enhancing Health in Newfoundland and Labrador).

  • Competing interests None declared.

  • Ethics approval HREA Newfoundland and Labrador.

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

  • Data sharing statement All data generated by this study are presented either in the text or as online supplementary information.