[Histonet] Re: Negative IHC controls


Dear colleagues,

The CAP checklist has a good discussion of negative IHC controls. One  
cant expand by reading the quoted references in order to understand  
the background behind negative controls. Many of the opinions  
mentioned in this discussion are also excellent and make scientific  

Briefly, a negative control is used to ensure that any signal on the  
patient's tissue that *contains* the primary antibody is not a false- 
positive signal. That false-positive signal could be secondary many  
factors including internal tissue properties (i.e., endogenous biotin  
in the case of ABC detection system, inadequate fixation, etc.),  
unusually high antibody concentration, excessive pretreatment,  
prolonged incubation with the primary antibody, inadequate wash  
steps, lifting of tissue section, etc.

If a case has 5 antibodies with 2 different detection systems and 3  
different pretreatment methods, in order to fully address the issue  
of negative control, one should include negative control sections  
from the same patient by eliminating each one of the above-mentioned  
parameters, one at a time, which means running multiple negative  
controls for the same tissue.

In real life, however, that doesn't seem to be necessary.  Replacing  
the primary antibody with a non-specific IgG would suffice. And that  
is in compliance with the CAP requirements.

For the sake of standardization, a few manufacturers are in the  
process of developing multi-pellet controls for all the different  
steps in the process, which has the potential of achieving a very  
high level of specificity in terms of eliminating any possibility of  
false-positive signal. In our laboratory, we have been running a  
single patient's section with non-specific IgG serum per tissue block.

Best regards,

Hadi Yaziji, M.D.
Ancillary Pathways, LLC.

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