Skip to main content
Log in

Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections

  • Short communication
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

Antigen decay in archival formalin-fixed paraffin-embedded (FFPE) tissue sections for immunohistochemistry is a well-known phenomenon which may have repercussions on translational and research studies and length of storage time appears fundamental. The aim of this study was to evaluate all possible factors which may lead to antigen decay on a prospective standardized collection of human tissues with a panel of 14 routinely used antibodies. Serial slide sections from FFPE control tissues were stored using different methods (routine storage at room temperature, Parafilm® protected, paraffin coated and cold stored at 4 °C) and for different time periods: 1, 6, 9, 12, 24 and 36 months. Immunohistochemistry was performed at each time cutoff simultaneously on stored sections and on freshly cut sections using a panel of 14 antibodies. Immunoreactivity was compared with immunoreactions performed at time zero. Reduction in immunostaining was observed for a subset of antibodies (CD3, CD 31, CD117, estrogen and progesterone receptors, Ki67, p53, TTF-1, vimentin) while for others (smooth muscle actin, keratins 7, 20, AE1/AE3, 34βE12), no antigen decay was observed. Loss of antigenicity was proportional to tissue section age and was dependent on mode of storage with cold storage slides being the least affected. All antigens with reductions in immunosignal were nuclear or membranous, and they all required heat pre-treatment for antigen retrieval. In contrast to results from other studies, when pre-analytical factors are strictly controlled and standardized, antigen decay seems to be restricted to nuclear or membrane antigens which require heat antigen retrieval.

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

Access this article

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

References

  • Bertheau P, Cazals-Hatem D, Meignin V, de Roquancourt A, Vérola O, Lesourd A, Séné C, Brocheriou C, Janin A (1998) Variability of immunohistochemical reactivity on stored paraffin slides. J Clin Pathol 51:370–374

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Blind C, Koepenik A, Pacyna-Gengelbach M, Fernahl G, Deutschmann N, Dietel M, Krenn V, Petersen I (2008) Antigenicity testing by immunohistochemistry after tissue oxidation. J Clin Pathol 61:79–83

    Article  CAS  PubMed  Google Scholar 

  • Brandtzaeg P (1981) Prolonged incubation time in immunohistochemistry: effect on fluorescence staining of immunoglobulins and epithelial components in ethanol and formaldehyde-fixed paraffin-embedded tissues. J Histochem Cytochem 29:1302–1315

    Article  CAS  PubMed  Google Scholar 

  • Camp RL, Charette LA, Rimm DL (2000) Validation of tissue microarray technology in breast carcinoma. Lab Investig 80:1943–1949

    Article  CAS  PubMed  Google Scholar 

  • DiVito KA, Charette LA, Rimm DL, Camp RL (2004) Long-term preservation of antigenicity on tissue microarrays. Lab Investig 84:1071–1078

    Article  CAS  PubMed  Google Scholar 

  • Economou M, Schöni L, Hammer C, Galvàn JA, Mueller DE, Zlobec I (2014) Proper paraffin slide storage is crucial for translational research projects involving immunohistochemistry stains. Clin Transl Med 3:4. doi:10.1186/2001-1326-3-4

    Article  PubMed Central  PubMed  Google Scholar 

  • Jacobs TW, Prioleau JE, Stillman IE, Schnitt SJ (1996) Loss of tumor marker-immunostaining intensity on stored paraffin slides of breast cancer. J Natl Cancer Inst 88:1054–1059

    Article  CAS  PubMed  Google Scholar 

  • Karlsson C, Karlsson MG (2011) Effects of long-term storage on the detection of proteins, DNA, and mRNA in tissue microarray slides. J Histochem Cytochem 59:1113–1121. doi:10.1369/0022155411423779

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Katoh AK, Stemmler N, Specht S, D’Amico F (1997) Immunoperoxidase staining for estrogen and progesterone receptors in archival formalin fixed, paraffin embedded breast carcinomas after microwave antigen retrieval. Biotech Histochem 72:291–298

    Article  CAS  PubMed  Google Scholar 

  • Lisowski AR, English ML, Opsahl AC, Bunch RT, Blomme EA (2001) Effect of the storage period of paraffin sections on the detection of mRNAs by in situ hybridization. J Histochem Cytochem 49:927–928

    Article  CAS  PubMed  Google Scholar 

  • Litlekalsoy J, Vatne V, Hostmark JG, Laerum OD (2007) Immunohistochemical markers in urinary bladder carcinomas from paraffin-embedded archival tissue after storage for 5–70 years. BJU Int 99:1013–1019

    Article  PubMed  Google Scholar 

  • Mirlacher M, Kasper M, Storz M, Knecht Y, Dürmüller U, Simon R, Mihatsch MJ, Sauter G (2004) Influence of slide aging on results of translational research studies using immunohistochemistry. Mod Pathol 17:1414–1420

    Article  PubMed  Google Scholar 

  • Nuovo AJ, Garofalo M, Mikhail A, Nicol AF, Vianna-Andrade C, Nuovo GJ (2013) The effect of aging of formalin-fixed paraffin-embedded tissues on the in situ hybridization and immunohistochemistry signals in cervical lesions. Diagn Mol Pathol 22:164–173. doi:10.1097/PDM.0b013e3182823701

    Article  CAS  PubMed  Google Scholar 

  • Olapade-Olaopa EO, MacKay EH, Habib FK (1998) Variability of immunohistochemical reactivity on stored paraffin slides. J Clin Pathol 51:943

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Olapade-Olaopa EO, Ogunbiyi JO, MacKay EH, Muronda CA, Alonge TO, Danso AP, Moscatello DK, Sandhu DP, Shittu OB, Terry TR, Wong AJ, Habib FK (2001) Further characterization of storage-related alterations in immunoreactivity of archival tissue sections and its implications for collaborative multicenter immunohistochemical studies. Appl Immunohistochem Mol Morphol 9:261–266

    CAS  PubMed  Google Scholar 

  • Prioleau J, Schnitt SJ (1995) p53 antigen loss in stored paraffin slides. N Engl J Med 332:1521–1522

    Article  CAS  PubMed  Google Scholar 

  • Puchtler H, Meloan SN (1985) On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry 82:201–204

    Article  CAS  PubMed  Google Scholar 

  • Ramos-Vara JA, Webster JD, DuSold D, Miller MA (2014) Immunohistochemical evaluation of the effects of paraffin section storage on biomarker stability. Vet Pathol 51:102–109. doi:10.1177/0300985813476067

    Article  CAS  PubMed  Google Scholar 

  • Shin HJ, Kalapurakal SK, Lee JJ, Ro JY, Hong WK, Lee JS (1997) Comparison of p53 immunoreactivity in fresh-cut versus stored slides with and without microwave heating. Mod Pathol 10:224–230

    CAS  PubMed  Google Scholar 

  • True LD (2014) Methodological requirements for valid tissue-based biomarker studies that can be used in clinical practice. Virchows Arch 464:257–263. doi:10.1007/s00428-013-1531-0

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • van den Broek LJ, van de Vijver MJ (2000) Assessment of problems in diagnostic and research immunohistochemistry associated with epitope instability in stored paraffin sections. Appl Immunohistochem Mol Morphol 8:316–321

    PubMed  Google Scholar 

  • Werner M, Chott A, Fabiano A, Battifora H (2000) Effect of formalin tissue fixation and processing on immunohistochemistry. Am J Surg Pathol 24:1016–1019

    Article  CAS  PubMed  Google Scholar 

  • Wester K, Wahlund E, Sundström C, Ranefall P, Bengtsson E, Russell PJ, Ow KT, Malmström PU, Busch C (2000) Paraffin section storage and immunohistochemistry. Effects of time, temperature, fixation, and retrieval protocol with emphasis on p53 protein and MIB1 antigen. Appl Immunohistochem Mol Morphol 8:61–70

    CAS  PubMed  Google Scholar 

  • Xie R, Chung JY, Ylaya K, Williams RL, Guerreo N, Nakatsuka N, Badie C, Hewitt SM (2011) Factors influencing the degradation of archival formalin-fixed paraffin-embedded tissue section. J Histochem Cytochem 59:356–365. doi:10.1369/0022155411398488

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical standard

All procedure and studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Federica Grillo.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Grillo, F., Pigozzi, S., Ceriolo, P. et al. Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections. Histochem Cell Biol 144, 93–99 (2015). https://doi.org/10.1007/s00418-015-1316-4

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-015-1316-4

Keywords

Navigation