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Immunocytochemistry and Immunohistochemistry

Immunocytochemistry and Immunohistochemistry

Immunocytochemistry (ICC) and immunohistochemistry (IHC) are techniques that use antibodies to check for specific proteins or antigens in a sample. The technique was first published by Albert Coons in 1941 to detect the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus. Immunocytochemistry helps localization of proteins or antigens in individual cells like blood cells, stem cells, etc., while immunohistochemistry does the same for tissue samples.
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Updated: Apr 18, 2026

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

Published on: July 26, 2019

A reference image-based method for optimization of clinical immunohistochemistry.

Marcel Kap¹, King H Lam¹, Patricia Ewing-Graham¹

¹Department of Pathology, Erasmus MC, Rotterdam, The Netherlands.

|February 3, 2015

Summary

This summary is machine-generated.

Optimizing immunohistochemistry (IHC) protocols by adjusting analytical factors, not pre-analytical ones like cold ischaemic and formalin fixation time (CIT and FFT), improved staining consistency. This method aids in reducing interlaboratory variation for reliable IHC results.

Keywords:

FFPE cold ischaemic time formalin fixation time immunohistochemistry interlaboratory variation optimization pre-analytical phase

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Area of Science:

Anatomical Pathology
Biochemistry
Immunology

Background:

Intralaboratory variation in immunohistochemistry (IHC) is often attributed to pre-analytical factors such as cold ischaemic time (CIT) and formalin fixation time (FFT).
Standardized control tissues with known pre-analytical histories are essential for method development and validation.

Purpose of the Study:

To develop and evaluate a novel method for optimizing IHC protocols.
To assess the impact of CIT and FFT on IHC staining using control tissue blocks.
To compare IHC outcomes with digitized reference slides for quality assessment.

Main Methods:

Control tissue specimens were divided and subjected to varying CIT and FFT.
Immunohistochemistry was performed using 34 antibodies and standard operating procedures.
Staining intensity was scored, and IHC protocols were adapted by modifying antibody dilution and antigen retrieval methods.

Main Results:

Seven antibodies were affected by CIT, 13 by FFT, and five by both.
Protocol optimization, primarily through analytical adjustments, improved staining consistency for most antibodies.
Nine protocols remained un-optimizable, but optimized results met adequate quality standards when compared to reference slides.

Conclusions:

Optimizing the analytical phase of IHC protocols is more effective than altering pre-analytical conditions for improving consistency.
This method, if supported by global references, has the potential to significantly reduce interlaboratory variation in IHC.
Further standardization of pre-analytical workflows is recommended to complement analytical optimization efforts.