Overview

immunohistochemistry, ihc controls

Avoid errors

IHConctrols® are on-slide controls developed to verify correct performance of the stain on each and every slide. Boston Cell Standards sets a new bar in Clinical IHC laboratory quality control with IHControls and IHCalibrators.  Because they are manufactured to have analytically traceable concentrations to an international standard, you know that your control is consistently at the correct concentration for your immunostain. IHCalibrators, the first and only calibrators on the market, allow you to compare your immunostain’s analytic sensitivity against your peer group.  These features far surpass homemade tissue controls and cell line controls in both performance and cost-effectiveness.

What are Quantitative IHControls®

The HER-2/ER/PR Reference IHControls® are on-slide immunohistochemistry (IHC) staining controls. Positive controls are important for testing to see if the immunohistochemical assay worked properly. HER-2/ER/PR formalin-fixed changes in stain intensity (beyond the normal variability) likely indicate a problem.  Corrective action may be required, at the discretion of the laboratory. Changes in stain intensity of the IHControls can be due to variability with antigen retrieval, reagents, protocols, or the instrument.

immunohistochemistry, tissue testing

How Quantitative IHControls are made

Our controls are made of 8-micron diameter glass microbeads coated with a portion of the antigen, such as HER-2, ER, or PR. The microbeads are dispersed within a liquid matrix that solidifies after dispensing onto a microscope slide. The solidified matrix is porous to IHC staining reagents. Because the matrix is porous, antibodies and other reagents that are applied during the IHC staining procedure will stain the microbeads in a similar fashion as they stain tissue sections.

 

Figure legend: IHControls are made up of a suspension of hundreds of thousands of small glass microbeads (per vial), each microbead being the approximate size of a cell. The microbeads are coated with antigen fragments (peptides) that are immunoreactive with one or more of the antibodies commonly used in immunohistochemistry.

immunohistochemistry calibrators, IHC calibrators

How Quantitative IHControls work

IHControls have been extensively characterized for their ability to test for adequate antigen retrieval (5). Like tissue sections, they are already formalin-fixed.  Like tissue sections, antigen retrieval usually increases stain intensity, sometimes dramatically.

 

In tissue sections, formalin fixation causes cross-links within a single protein or between adjacent proteins. If a protein cross-link brings another protein at or near the antibody epitope, then it can sterically interfere with antibody binding. Antigen retrieval reverses formaldehyde-induced crosslinks and dissociates proteins that are sterically interfering with antibody binding (6).

 

We use the same formaldehyde fixation chemical reaction on the IHControls as used in the fixation of the tissue. During manufacture, we incubate peptide-coated glass microbeads with formaldehyde and an irrelevant protein. This causes the irrelevant protein to be covalently bound to the peptide, blocking antibody binding to the peptide epitope. Antigen retrieval reverses the reaction and re-exposes the peptide epitope for binding to the antibody.

 

 

(6) SA Bogen, K Vani, & SR Sompuram. 2009. Molecular Mechanisms of Antigen Retrieval: Antigen Retrieval Reverses Steric Interference Caused By Formalin-Induced Crosslinks. Biotechnic & Histochem. 84(5):207-215.