Improved methods using the reverse transcriptase polymerase chain reactionto detect tumour cells

Reverse transcriptase polymerase chain reaction (RT-PGR) is increasingly us ed to detect small numbers of circulating tumour cells, though the clinical benefit remains controversial. The largest single contributing factor to t he controversy of its value is the different approaches to sample processin g. The aim of this study was to compare the sensitivity and reproducibility of RT-PCR for the detection of tumour cells after four commonly used diffe rent methods of sample processing. Using RT-PCR, one tumour cell spiked in 2 mi of whole blood was detected after analysis of separated mononuclear ce ll RNA, whole blood total or poly-A(+) RNA. No false positives were identif ied with any method. However, the reproducibility of tumour cell detection was reduced after isolation of the mononuclear cell fraction. Only analysis of poly-A(+) RNA had a sensitivity of 100% in all the cell spiking experim ents. In patient blood samples, analysis of poly-A(+) RNA increased the num ber of blood samples positive for tyrosine hydroxylase (TH) mRNA compared w ith those positive after analysis of total RNA. This may reflect high level s of cDNA reducing the efficiency of the PCR. Isolation of poly-A(+) RNA in creases the sensitivity and reproducibility of tumour cell detection in per ipheral blood.