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Other issues
Preservation and storage
One issue is
preservation after sectioning. While the production of large
batches of microarray sections is most efficient, it raises
a separate problem of antigenic loss due, presumably, to tissue
oxidation. While this may not be the case if you use ZN-Formalin,
regular buffered formalin is more commonly used and does not
prevent oxidation after sectioning. Others and we have found
loss of antigenicity if sections are stored for as little as
a week prior to immunostaining. Work is underway to quantify
this loss. It appears that loss is an oxidative process since
the loss appears to be insensitive to storage temperature or
retrieval conditions. For tissue microarrays, we have found
that this loss can be prevented by sectioning without water
(using the tape transfer system), removal of the degreasing
agent after tape release by a short incubation in xylene, and
finally re-coating the slides in paraffin prior to storage
(Figure 1) and storage in a nitrogen safe. We have also begun
experimenting with preservatives (BHA or BHT) in the paraffin.
Figure 1
Are the small histo-spots representative of a whole section?
The major potential limitation of this technique is tissue
volume. Skeptics claim that the amount of material analyzed
is too small and potentially not representative of the entire
tumor. In a reproducibility study, the Sauter group found identical
Kaplan Meier curves were generated by analysis of 4 unique
sets of spots from the same patients (Torhorst et al, AJP 2001).
Thus although any given histo-spot may be negative on a given
array, the statistical power of analysis of hundreds or thousands
of cases eliminates the affect of variability of a single data
point in the ultimate conclusions. Our own study to assess
the number of histo-spots required to obtain an equivalent
result to a tissue section using the standard breast cancer
prognostic markers (Estrogen and progesterone receptors and
HER2 oncogene) shows that analysis of only 2 histo-spots results
in >95% accuracy (see figure in Camp et al, Lab Invest, 2000).
More recently, numerous other studies have found similar results.
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