J Clin Pathol 61:1184-1192 doi:10.1136/jcp.2007.047720
  • Demystified

Technical pitfalls potentially affecting diagnoses in immunohistochemistry

  1. E Leonardo2
  1. 1Department of Biomedical Sciences and Human Oncology, University of Turin, Torino, Italy
  2. 2Department of Pathology, SCDU Pathological Anatomy, ASO S Luigi–Orbassano, Torino, Italy
  1. Professor G Bussolati, Department of Biomedical Sciences and Human Oncology, University of Turin, Via Santena 7, 10126 Torino, Italy; gianni.bussolati{at}
  • Accepted 15 January 2008
  • Published Online First 6 March 2008


Result of the immunohistochemical reactions routinely used in diagnostic surgical pathology should be properly interpreted, since false results, related to technical and interpretative pitfalls may lead to incorrect diagnosis. The main sources of such pitfalls are reviewed, analytically described and related to different steps (fixation, tissue processing and embedding, decalcification, antigen retrieval) which may affect the accuracy of immunohistochemistry. In addition, the presence of endogenous enzyme activity, improper binding of avidin to endogenous biotin, incorrect use of antibodies, chromogen and detection systems, as well as incorrect interpretation may produce unreliable data. The high frequency and extension of such pitfalls make mandatory the use of internal and external controls and adoption of cross-validation programmes. The present study, supported by an extensive review of the related literature, is intended as a guideline leading to proper interpretation of immunohistochemical data, an essential component of the diagnostic process. Experience on the antigen retrieval procedures for different antigens is also presented.


  • Funding: This paper was supported by grants from Piedmont Region (Ricerca Sanitaria Finalizzata 2006).

  • Competing interests: None.