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544 Chapter 10: Graft Manipulation
power of a technique, since it can be relatively easy to achieve impressive
numbers of logs of depletion by increasing the initial level of infiltration. Under
these conditions, many depletion methods will perform efficiently. That may not
be true under clinically applicable conditions, where the best chances of affecting
outcome are when the tumor burden is low within the graft, and the aim is to
achieve complete removal. In that case, the limiting factors to the technique
become more apparent and the characteristics of the residual cells provide useful
information on how to optimize the purging system. For example, in antibodymediated
purging methods, an evaluation of target antigen density on the cells that
escape elimination is invaluable in developing a strategy to improve efficacy. In
general, low antigen density cells are usually more difficult to purge, particularly
when using antibodies and complement 12
; however, cells with excessively high
expression of an antigen may also be able to evade elimination. 13
The use of a
clinically appropriate model system in combination with multiple sensitive assays
for residual target cells is, therefore, central to the development of an effective
purging technology.
Detection technologies. Validation of any new purging technology is also
critically dependent on the use of a sensitive, well-characterized method for the
detection of the target cell population. Molecular techniques are capable of
impressive levels of sensitivity but may not provide quantitative information.
They will also detect material that has been released from dead or dying target
cells, thereby underestimating the purging efficiency. Nonetheless, these assays
can still be regarded as the gold standard for evaluating purging methods.
Purging to PCR negativity has become the synonym for complete purging in
many diseases. 4,8
In some studies, however, the sensitivity and reproducibility of
the detection technique is not reported, making it difficult to determine what a
negative finding truly represents. It becomes all the more important, under such
circumstances, to think in terms of absolute numbers of cells rather than levels of
depletion. Based on preliminary purging experiments, and knowing the
sensitivity of the detection technique, it should be possible to calculate the
probable number of target cells that may remain after purging. The volume and
number of samples of the postpurge product that would be required to detect
these cells with a high degree of probability can then be determined. Publications
still appear in which a single small volume sample of a purged graft is used to
demonstrate purging efficacy, under conditions where elementary mathematics
and the Poisson distribution would clearly indicate that a negative result would
essentially be guaranteed.
Tumor enrichment. Recently, there has been a renewed emphasis on using
tumor enrichment techniques to improve the sensitivity of tumor cell detection in
clinical samples. 14
In most cases, this involves using tumor-directed antibodies and