Table 2.
Main advantages and disadvantages of techniques used for circulating tumour cell (CTC) enrichment and analysis [Alunni-Fabbroni and Sandri, 2010; Paterlini-Brechot and Benali, 2007].
| Advantages | Disadvantages | |
|---|---|---|
| Enrichment | ||
| Immunomagnetic separation (manual) | Cells selected on basis of antigen expression using antibody coated magnetic beads. Versatility for positive or negative cell selection. | Dependent on epithelial marker expression which tumour cells may or may not express; cells may be lost in sample preparation |
| Immunomagnetic separation (CellSearch) | Semi-automated magnetic separation – easy to use. Blood samples stable for up to 96 hours. Automation permits reproducible results between users, laboratories and samples thus robust for multisite trials. | Dependent on epithelial marker (EpCam) expression. Challenging to extract pure CTC for further characterisation but enrichment good compared with other techniques. Expensive. |
| Centrifugation | Simple method. Isolates mononuclear cells on the basis of differences in density gradient. Nonexpensive. | Poor enrichment for CTCs - high contamination with WBCs. Large number CTCs potentially lost in processing |
| Filtration (ISET) | Isolates cells on basis of size differences (CTC large; WBC small). Effective method. Tumour cells amenable for molecular characterization. | Small tumour cells may not be detected by this method. Lower sensitivity compared with other techniques. |
| Analysis | ||
| Cytometric | Cells directly visualized by IHC or IF - permits evaluation of morphology and CTC enumeration. Cells can be extracted for molecular characterization, e.g. FISH, RT-PCR. In combination this allows greater specificity than nucleic acid methods. | Detection depends on epithelial or tumour marker expression. No pure CTC marker exists. False-positive cells may be enriched. Current techniques probably less sensitive than nucleic acid methods. |
| Nucleic Acid Based (RT-PCR) | Highly sensitive in particular with multimarker assay. Can be performed without enrichment step but at expense of sensitivity. Quantitative RT-PCR permits relative quantification. | Cells cannot be visualized directly for morphology or enumeration. Clinical correlations reported but difficult to standardize techniques across laboratories. No transcripts purely specific for tumour cells thus high false-positive rate. |
FISH, fluorescent in situ hybridization; IF, immunofluorescence; IHC, immunohistochemistry; ISET, isolation by size of epithelial tumour cells; RT-PCR, reverse transcription polymerase chain reaction; WBC, white blood cell.