[Histonet] Re: Negative IHC controls
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.
Hadi Yaziji, M.D.
Ancillary Pathways, LLC.
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