John M. S. Bartlett.pdf - Bio-Nica.info

PCR Detection of Tumor Cells 195
4. The ASO primer does not generate specific PCR product or is not specific or sensitive
enough: Optimize PCR conditions and test primer for specificity or sensitivity. Devise
new primers. Change primer strategy (CDR3 or CDR1/ plus CDR3). If no specific product
can be generated at all, it is most probable that the sequence of the CDR-regions is not
that of the malignant clone.
5. qPCR results are implausible: This is most probably caused by a faulty dilution series.
Prepare dilution series anew and reanalyze the sample by qPCR.
5. Discussion
The described qPCR assay with ASO primers complementary to CDR regions of
the malignant clone can be used to quantitate the proportion of malignant cells in
peripheral blood (PB) and BM, aliquots of leukapheresis products, sorted cell fractions,
or in cultured cells. It is possible to use this method for quantitative follow up in the
course of therapy (16), to determine the tumor load of different stem cell sources (17),
to determine the effect of different mobilization regimens and the duration of collection
on the number of malignant cells in leukapheresis products (18,19) or to assess the
extent of involvement in the malignant process of different cell fractions, for example,
in the CD19+ or CD20+ cells of PB (20,21). The qPCR assay has evolved into an
accurate and very sensitive tool for the detection of malignant cells in MM.
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