Receiver Operating Characteristic Analysis for MIF.

<p>ROC analysis was performed using the R Package software version 1.0-4 as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041052#pone.0041052-Team1" target="_blank">[53]</a>.</p

RNA recoveries in the “Enriched CTC” fractions in melanoma patients vs. healthy controls.

<p>There is a statistically significant difference between RNA yield levels of melanoma patients and the healthy controls (p = 0.005) based on the nonparametric Wilcoxon rank-sum test. There was no statistically significant correlation between melanoma stage and RNA yield. This was true for scale 1 (stage 1,2,3, or 4; p = 0.739) and scale 2 (where the scale separates 0, IA, IB, IIA, IIB, IIC, etc.; p = 0.948).</p

Marker RNA levels for MLANA and MIF in melanoma patients vs. healthy controls.

<p>Blood was fractionated as described using OncoQuick columns, RNA was purified from the enriched CTC fractions, and MLANA and MIF RNA levels were quantified by QPCR. Panel A shows results with blood drawn on the same day as excision of melanomas, or the corresponding healthy controls. Panel B shows results with blood drawn one week after excision. Melanoma patients and healthy controls are as noted on the X-axis, and Staging information for melanoma patients is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041052#pone-0041052-t002" target="_blank">Table 2</a>. The Y-axis depicts RNA copy numbers per ml of blood. Shown to the far right of Panel A are calculated transcript numbers for 2 patients with squamous cell carcinoma of skin and for a patient with neurofibromatosis (left to right, respectively).</p

Melanoma and Pancreatic CTCs Captured on Filters.

<p>CTCs were enriched from blood samples from another melanoma patient and from a patient with pancreatic cancer using OncoQuick, CTCs from the enriched fraction were captured on filters, and cells were cultured as described. Upper row shows a brightfield shot of pancreatic CTCs growing on the filter, and immunofluorescent staining of CTCs for pan-KRT and CD-45 (as indicated). Lower row shows CTCs from another melanoma patient. Inset in the brightfield panel shows DAPI staining for 2 of the cells on the filter (the 3^rd cell also stained positively, not shown), which was difficult to photograph due to inherent fluorescence from the filters.</p

Marker RNA levels for TYR and MITF in melanoma patients vs. healthy controls.

<p>Blood was fractionated as described using OncoQuick columns, RNA was purified from the enriched CTC fractions, and TYR and MITF RNA levels were quantified by QPCR. Panel A shows results with blood drawn on the same day as excision of melanomas, or the corresponding healthy controls. Panel B shows results with blood drawn one week after excision. Staging information for melanoma patients is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041052#pone-0041052-t002" target="_blank">Table 2</a>. The Y-axis depicts RNA copy numbers per ml of blood. Shown to the far right of Panel A are calculated transcript numbers for 2 patients with squamous cell carcinoma of skin and for a patient with neurofibromatosis (left to right, respectively).</p

AJCC Stage of Melanoma Patients.

<p>AJCC Stage of Melanoma Patients.</p

Representative Melanoma CTCs Captured on a “Splitable Gap” Filter Device.

<p>CTCs were enriched from blood from melanoma patients using OncoQuick and the enriched CTC fraction was then passed through the filter device. Filters containing captured cells were then placed in culture for various times up to ∼ 42 days, and cells were subsequently stained with DAPI, and stained for immunofluorescence using pan-KRT and CD-45 antibodies as described. Left panels show brightfield pictures of cells within wells of the filter; large wells are 50 µm in diameter, whereas the surrounding smaller pores are 7.5 µm in diameter. Center panels show immunofluorescent staining of cells with for pan-KRT marker, and right panels show staining for the common leukocyte antigen CD-45. All cells shown stained positively with DAPI.</p

OncoQuick Enrichment and Recovery of Melanoma CTCs.

<p>Left Panel shows a schematic of OncoQuick enrichment. Right Panel shows recovery of SK-MEL-28 human melanoma cells from blood. Known numbers of SK-MEL-28 cells were added to 7.5 ml of blood, and processed using OncoQuick spin columns as described. Recovery of cells was assessed using QPCR amplification of KRT8 and 18 RNAs as described.</p