Detecting carcinoma cells in peripheral blood of patients with hepatocellular carcinoma by immunomagnetic beads and rt-PCR.

BACKGROUND

Increasing the sensitivity and specificity of detecting circulating carcinoma cells of patients with hepatocellular carcinoma (HCC) is very important for monitoring recurrence.

GOAL

To establish a novel method of detecting circulating carcinoma cells.

STUDY

For method development, 3 sets of controls using HCC cell line HepG2 cells were used. (A): Serial dilutions of HepG2 cells were directly used to extract total RNA for nested reverse transcription-polymerase chain reaction (RT-PCR). (B): Five milliliter of healthy blood was spiked with a serial dilution of HepG2 cells and was used for Ficoll density gradient centrifugation to recover cells. The cells were used to extract total RNA for RT-PCR. (C): After cell recovery with the same procedure as B, the cells were sorted sequentially by CD45 and Ber-EP4 immunomagnetic beads and used for RNA extraction and RT-PCR. For clinical samples, 44 patients with HCC and 7 healthy subjects were included. The alpha-fetoprotein mRNA was amplified using nested RT-PCR technique.

RESULTS

The spiking experiments using HepG2 cells showed that 10 cells in 5 mL blood could be detected by method C and an excellent dose-response to the number of spiked cells. Whereas, method B lacked any dose-response and would yield high false-positive rates. In clinical samples, the improved method led to a positive detection rate of 52.9%, 76.9%, and 92.9% in Child-Plug class A, B, and C, respectively. There was significant difference between class A and class C (P<0.05). The total positive detection rate was 72.7%.

CONCLUSIONS

Combining negative and positive immunomagnetic beads with RT-PCR technique may improve the sensitivity and specificity of detecting circulating HCC cells.