Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Improved method increases sensitivity for circulating hepatocellular carcinoma cells.

Author(s): Liu HY, Qian HH, Zhang XF, Li J, Yang X, Sun B, Ma JY, Chen L, Yin ZF

Publication: World J Gastroenterol, 2015, Vol. 21, Page 2918-25

PubMed ID: 25780289 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   Sensitivity of CTC detection in peripheral blood was compared using the ASGPR+ cell selection and the CD45+ leukocyte depletion methods. Five mL of venous blood was collected into EDTA tubes from 32 patients with hepatocellular carcinoma, 17 patients with other types of cancer (3 breast, 2 lung, 3 esophageal, 5 gastric, and 4 colorectal), 40 patients with other liver diseases (12 benign intrahepatic space-occupying lesions, 3 acute hepatitis A, 6 chronic hepatitis B, 4 chronic hepatitis C, and 15 cirrhosis), and 20 healthy volunteers. For the CD45-depletion method, red blood cells were removed from whole blood specimens by Ficoll density gradient, CD45+ cells were removed using antibody-coated magnetic beads, and the remaining cells were cytocentrifuged on polylysine-coated slides, and stored at 4°C until analysis by immunofluorescence staining. The ASGPR+ selection method entailed identification of CTCs by immunofluorescent staining for cytokeratin and CPS1. Blood from a healthy volunteer was spiked with 10, 50, 250, or 1000 HepG2 cells (a human liver cancer cell line) and recovery was measured after enrichment by both methods, and then in CD45+ depleted samples subjected to immunofluorescence staining with a ASGPR and CPS1 antibody cocktail.

   Summary of Findings:

   A significantly higher number of HepG2 cells were recovered by CD45+ depletion than by ASGPR+ selection in a blood specimen from a healthy volunteer that had been spiked with 10, 50, 250, or 1000 cells (P<0.05, P<0.01, P<0.01, and P<0.01, respectively). CTCs were detected in 29/32 (91%) patients with hepatocellular carcinoma using the CD45-depletion method and an ASGPR and CPS1 antibody cocktail, while CTCs were not detectable in blood from healthy volunteers or patients diagnosed with any other form of cancer or liver disease. The CD45-depletion method in conjunction with an ASGPR and CPS1 antibody cocktail resulted in a significantly higher CTC count than the ASGPR+ selection method (P=0.001), and increased sensitivity by 12%-21% in hepatocellular carcinoma patients with more than 40 CTCs.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Fluorescent microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Fluorescent microscopy Specific	Targeted peptide/protein	CD45 DAPI cytokeratin asialoglycoprotein receptor (ASGPR) carbamoyl phosphate synthetase 1 (CPS1)

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