1233 Methods
Several laboratory techniques are available to determine HER2 sta-
tus. Gene amplification, which is the fundamental driver, is assessed
by in situ hybridization (ISH) and resultant protein expression is
assessed by immunohistochemistry (IHC).Pre-analytical issuesThere are crucial issues affecting test tech-
niques that occur at pre-analytical phase. Pre-analytical phase of
testing involves procurement, fixation and processing of tissue, and
final slide preparation. The time lapse between procurement of tis-
sue sample with severance of blood supply to fixation (cold isch-
emic time), duration of fixation before processing, the age of the
tissue blocks, and exposure of unstained slides before staining
could have implications on the final results. All the above steps of
the pre- analytical phase can have great variability within the same
laboratory as well as from laboratory to laboratory and thereby can
greatly affect accuracy of the result. There is a major international
push to standardize these steps as much as practicably possible to
limit this variability and enhance reproducibility of immunohisto-
chemical and in situ hybridization assays [ 30 , 31 ].
(a) Cold ischemic time
The time from biopsy or resection to placing them in ade-
quate formalin fixative ensuring satisfactory tissue penetration
and fixation can affect the HER2 testing result. It is critical to
limit the cold ischemic time as degradation of the tissue struc-
tures increases conversely. This can lead to false-negative as
well as false-positive staining in both immunohistochemical3.1 Attention
to the Factors
Affecting the Accuracy
of the Tests
Fig. 2 Cell block of a peritoneal effusion sample with metastatic esophagogastric
junction adenocarcinoma featuring 3+ HER2 staining by immunohistochemistry
(×200)HER2