Journal: PLoS ONE

Article Title: Clinical Validation of an Ultra High-Throughput Spiral Microfluidics for the Detection and Enrichment of Viable Circulating Tumor Cells

doi: 10.1371/journal.pone.0099409

Figure Lengend Snippet: (A) CTC viability demonstrated by attachment to 2D Geltrex (Invitrogen)-coated substrate (72 hr after seeding). Isolated CTC were enriched for CD44. No cells were stained for CD45, indicating the absence of WBCs which did not adhere to substrate and were removed after washing with 1X PBS. Some CD44+cells were not stained for Hoechst (white arrows). Scale bar: 20 µm (B) Comparison of CTC isolation and recovery with CellSearch system. (C) Molecular FISH analysis on enriched CTCs of a patient with NSCLC. Cells were stained using Vysis ALK Break Apart FISH probe and counterstained with DAPI. The red and green signals demonstrated a distinct separation of the original fusion signal (arrows), indicating a rearrangement in the 2p23 ALK-gene locus. Scale bar: 16 µm. (D) MassArray spectra for a patient with NSCLC harboring EGFR L747_P753>S. Trace from FFPE, plasma and pooled CTCs illustrated. Percentage indicates calculated proportion of mutant allele against wild type allele (UEP: Unextended primer). ( i ) iPlex bi-allelic spectra on FFPE sample (33% mutant frequency), ( ii ) iPlex bi-allelic spectrum on plasma sample (32% mutant frequency), ( iii ) SABER mutant specific spectrum on plasma sample (Positive – high frequency), ( iv ) SABER mutant specific spectrum on CTCs (Positive – low frequency (n = 3/94), estimated mutant frequency of 1.4%) and ( v ) Representative iPlex & SABER (shown) spectrum on no-template control sample (Negative).

Article Snippet: Cleaned PCR product was spotted onto MassArray SpectroCHIPS II (Sequenom) using the MassARRAY RS1000 Nanodispenser.

Techniques: Isolation, Staining, Comparison, Clinical Proteomics, Mutagenesis, Control