Immunohistochemistry basics

Author: Nat Pernick, M.D. (see Authors page)

Revised: 28 August 2015, last major update August 2013

Copyright: (c) 2003-2015,, Inc.

PubMed Search: immunohistochemistry+basic[TITLE]
Table of Contents
General | Common Errors
  • Immunohistochemistry (IHC) is a tool for surgical pathology and research
  • Diagnosis should be based on H&E morphology, with confirmation by immunohistochemistry or molecular testing; it is dangerous to use immunohistochemistry alone to make the diagnosis
  • A stain / result is not just positive or negative; focus on the types of cells that are immunoreactive and determine if they are tumor cells, inflammatory cells, normal cells or stromal cells; comparing the results to an H&E stained section or a negative control of the same block may be helpful (Am J Surg Pathol 2007;31:1627, J Clin Pathol 2011;64:466)
  • After you identify the type of cell staining, it is helpful to note the percentage of these cells staining, the intensity of staining (weak, 1+, 2+, 3+, 4+) and the pattern of staining (membranous, cytoplasmic, nuclear, dot-like)
  • The pattern of immunoreactivity should follow the anatomic distribution of the antigen before it is called positive / immunoreactive
  • Reference: CAP Laboratory Improvement Programs: Principles of Analytic Validation of Immunohistochemical Assays
  • Antibodies are often useful beyond their recommended expiration dates
  • Combining results from different studies may be hazardous, as studies may use different antibodies and different standards of interpretation
  • Recommended to interpret immunohistochemical stains in small needle core biopsy specimens based on the area with the greatest immunoreactivity (Am J Clin Pathol 2007;127:273)
Common Errors
  • Not using a positive or negative control; they are helpful in interpreting the staining pattern, particularly if it is heavy or weak
  • Other sources of error are ectopic antigen expression (may be due to abundant endogenous biotin, Hum Pathol 2011;42:369), cross reactions (Mod Pathol 2012;25:231), less specificity than thought (Int J Clin Exp Pathol 2012;5:137), use of the wrong secondary antibody (EJN Blog) or rarely the wrong primary antibody