autologous blood and marrow transplantation - Blog Science ...

Gee 545
immunomagnetic particles to identify and concentrate the tumor cells. While this
approach is certainly of value for diagnostic specimens, it should be used
cautiously in purging applications. The tendency has been to use the same
antibodies for purging and for concentrating the residual tumor cells in the purged
graft to aid in their detection. This can be self-defeating if the residual cells have
escaped purging by their inability to bind those particular monoclonal reagents. An
alternative strategy that can be used under these circumstances is to enrich the
residual tumor cells by depleting CD45-positive normal leukocytes. Obviously this
can only be used in diseases in which the tumor population does not express the
CD45 antigen.
Antibody-based detection techniques are generally about an order of magnitude
less sensitive than molecular techniques. Although methods are available to allow
flow cytometers to detect extremely small numbers of residual tumor cells, the
technique of choice has been to use immunocytochemistry. 1415
This offers the
combination of detection by positive staining and visual confirmation of the
appropriate morphology of the stained cell. Careful validation of the method may
allow limits of detection of about one positive event in 1 million cells; however, as
indicated previously, it then becomes crucial to examine a sufficiently large
number of cells to obtain accurate information. This endeavor may be facilitated by
computerized image analysis, in which many cells can be scanned by the system
and positive events recorded and retrieved subsequently for visual examination. 14
Detection methods that rely on molecular or surface markers do not give any
indication of the likely proliferative potential of the tumor cells, which can be
obtained from various in vitro colony-forming assays. 16
Again, these suffer from
limitations, in that failure to grow in culture may reflect the inadequacy of the
culture conditions, rather than the absence of tumor cells. Positive growth is the
only result that can be interpreted with any degree of certainty.
Detection technology is, therefore, inextricably linked to purging. Ideally, the
combination of molecular, antibody-based, and colony-forming assays should be
used, since each provides complementary, if incomplete, information. These assays
must also be used within their limits of detection—which seems intuitively obvious
but is not always the case in some studies. Purging that achieves a negative result
should then be reported as achieving depletion to the level of sensitivity of the
assay, with an indication of how many residual cells may still be present in the
purged graft. Purging performed on heavily infiltrated grafts is, as discussed above,
not representative of most clinical situations, but it may give at least some
indication of the tumor removal capacity of a purging technique. When the
technique is then used under more clinically appropriate conditions, and achieves
elimination to the level of sensitivity of the detection assays, this capacity
measurement can provide some idea of the degree of additional tumor removal that
may have been achieved.