124
and in situ hybridization assays. The American Society of
Clinical Oncology/College of American Pathologists (ASCO/
CAP) guidelines recommend that cold ischemic times be lim-
ited to less than 1 h for breast samples undergoing HER2 test-
ing [ 32 , 33 ]. For endoscopic biopsies, however, an expert
panel has recommended that they should be fixed as soon as
they are taken, avoiding drying of the tissue, whereas the cold
ischemic time for surgical samples should be less than 20 min
[ 34 ].
(b) Fixation Procedure
The fixation procedure is the most critical pre-analytical fac-
tor in determining the success or failure of any immunohisto-
chemical or in situ hybridization assay. The ASCO/CAP
guidelines recommend tissue for HER2 testing be fixed in 10%
neutral buffered formalin, and the duration of fixation prior to
processing be between 8 and 48 h [ 33 ]. Therefore, the type of
fixative and fixation duration should be documented and where
there is any doubt, the clinician submitting the samples should
be contacted by the laboratory as soon as possible. In our expe-
rience, fixation times of less than 6 h duration have an adverse
effect on nuclear integrity when performing the routine
HER2-C17 Dual ISH assay. Over-fixation may also lead to
weak or absent HER2 and C17 signals. In this case, increasing
the pre-treatment component times may also recover the loss
of the HER2 and C17 signals [ 35 ].
(c) Tissue Sectioning
Too thick sections with inadequate paraffin wax removal as
well as nuclear bubbling can hamper interpretation. It is rec-
ommended that 4 μm sections be the standard thickness for
sections used in ISH assays [ 35 ]. In addition, that the type of
slide used to pick up the tissue section and perform the HER2
assay on can also affect assay results [ 36 ].
(d) Drying (Baking)
Longer duration of drying sections at higher temperatures
has been shown to decrease both the staining intensity and
percentage of tumor staining in HER2 IHC assays [ 37 ].
(e) Storage and Aging
For optimal results, paraffin tissue sections for HER2 testing
should be cut just prior to testing. It has been shown that the
aging of stored tissue sections on slides adversely effects the
accuracy of the HER2 assay, as immunoreactivity is seen to
reduce significantly over time as well as the environment in
which archival paraffin tissue blocks are stored [ 38 ]. Older
archival blocks may also require increased pre-treatment to
achieve adequate signal intensity for enumeration of HERr2
signals using the Dual in situ hybridization assay [ 39 ].Duminda Subasinghe et al.