Dako Autostainer - Manual (Page 125)

Dako Autostainer | User Guide 125
Section 22 | General Limitations
Immunohistochemistry is a multi-step diagnostic process that requires specialized training in the
selection of the appropriate reagents, tissue selection, fixation, and processing, preparation of the IHC
slide, and interpretation of the staining results.
Tissue staining is dependent on the proper handling and processing of tissues prior to staining.
Improper fixation, freezing, thawing, washing, drying, heating, sectioning or contamination with other
tissues or fluids may produce artifacts, antibody trapping, or false-negative results. Inconsistent
results may be due to variations in fixation and embedding methods, or to inherent irregularities within
the tissue.
Excessive or incomplete counterstaining may compromise proper results.
Use of old or unbuffered fixatives, or exposure of tissues to excessive heat (greater than 60
°C) during
processing may result in decreased staining sensitivity.
Normal/non-immune sera from the same animal source as the secondary antisera used in blocking
steps may cause false-negative or false-positive results due to auto-antibodies or natural antibodies.
False-positive results may be seen due to non-immunologic binding of reagents to tissue sections. In
some case the application of an alternate blocking reagent prior to incubation with the primary
antibody may be useful for reducing background. A recommended blocking reagent is Dako
® Protein
Block Serum-Free (Code No. X0909).
Unexpected negative reactions in poorly differentiated neoplasms may be due to loss or marked
decrease of antigen expression or nonsense mutation in the gene(s) coding for the antigen.
Unexpected positive staining in tumors may be from expression of an antigen not usually expressed in
morphologically similar normal cells, or from persistence or acquisition of an antigen in a neoplasm
that develops morphologic and immunohistochemical features associated with another cell lineage
(divergent differentiation). Histopathologic classification of tumors is not an exact science and some
literature reports of unexpected staining may be controversial.
The clinical interpretation of any positive staining or its absence should be complemented by
morphological and histological studies with proper controls. Evaluations should be made within the
context of the patient’s clinical history and other diagnostic tests. It is the responsibility of a qualified
pathologist who is familiar with the antibodies, reagents and methods used to interpret the stained
preparation. Staining must be performed in a certified licensed laboratory under the supervision of a
pathologist who is responsible for reviewing the stained slides and assuring the adequacy of positive
and negative controls.
Reagents may demonstrate unexpected reactions in previously untested tissues. The possibility of
unexpected reactions in tested tissue groups cannot be completely eliminated due to biological
variability of antigen expression in neoplasms, or other pathological tissues. Contact your local Dako
representative with documented unexpected reactions.
Tissues from persons infected with hepatitis B virus and containing hepatitis B surface antigen
(HBsAg) may exhibit nonspecific staining with horseradish peroxidase.