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.


OBJECTIVE
To establish a novel method of detecting circulating carcinoma cells.


METHODS
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.
Guo
Junming
J
Ningbo University School of Medicine, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. junmingguo@yahoo.com
Yao
Feng
F
Lou
Yanlu
Y
Xu
Changfeng
C
Xiao
Bingxiu
B
Zhou
Wenhong
W
Chen
Jian
J
Hu
Yaoren
Y
Liu
Zhong
Z
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000235:genetics / Q000473:pathology*; D005260:Female; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D008113:Liver Neoplasms / Q000097:blood / Q000235:genetics / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D012334:RNA, Neoplasm / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000235:genetics* / Q000378:metabolism
D014408:Biomarkers, Tumor; D012334:RNA, Neoplasm; D000509:alpha-Fetoproteins