Detection of Circulating Tumor Cells in Blood Using an Optimized Density Gradient Centrifugation

Abstract

The aim of the study was to compare the new density gradient centrifugation system OncoQuick with the standard density gradient centrifugation system Ficoll for improved tumor cell enrichment in blood of tumor patients. Evaluation of OncoQuick and Ficoll density gradient centrifugation was performed by flow-cytometry and immunocytochemistry using 10 ml unspiked and tumor cell-spiked blood samples of tumor-free probands. From 10 ml blood, OncoQuick density gradient centrifugation separated a cell fraction which consisted of a mean cell number of 9.5X10 4 mononuclear cells compared to 1.8x10 7 cells by Ficoll. Density gradient centrifugation of tumor cell-spiked blood samples with OncoQuick and Ficoll led to similar tumor cell recovery rates, between 70% and 90% for both methods. The improved depletion of mononuclear blood cells by OncoQuick simplified further immunocytochemical evaluation of the enriched cell fraction, which could be spun onto 1-2 glass slides by cytocentrifugation. In comparison, the mononuclear cells separated by Ficoll had to be spun onto more than 50 glass slides for complete immunocytochemical evaluation. Consequently, tumor cell density on each cyto spin was higher after OncoQuick preparation compared to Ficoll. Density gradient centrifugation with OncoQuick results in higher relative tumor cell enrichment than Ficoll density gradient centrifugation. This simplifies further immunocytochemical tumor cell detection and is a promising tool for the detection of circulating tumor cells in blood of tumor patients. Introd,uctionTumor recurrence in cancer patients after curative tumor resection can be explained by the presence of minimal residual disease (MRD).

Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: Proof of concept

“…Simple micro-filtration gives the highest depletion of leukocytes, but the capture of CTCs is dictated by the membrane pore size, which may lead to decreased sensitivity (16). OncoQuick gradient centrifugation is able to enrich CTCs 100-fold greater than elutriation, but within a mixed leukocyte population (Greiner Bio-One) (14,51). Physical separation methods are sufficient to serve as a pre-enrichment step to significantly deplete leukocytes, but due to their low specificity CTCs are obtained FIG.…”

“…CTCs have generally been enriched based upon their physical character by their density using gradient centrifugation (9,14,15) and by their large size using micro-filtration (16)(17)(18)(19), or immunologically using antibodies targeting such as the epithelial cell adhesion molecule (EpCAM) that are immobilized on substrates including microfluidic chambers and magnetic beads for CTC adsorption (10,12,20). The enrichment of CTCs using gradient centrifugation and/ or immunomagnetic bead adsorption revealed a CTC incidence rate of 20% in ovarian cancer patients at primary diagnosis.…”

“…In an attempt to detect and enumerate CTCs following enrichment, fluorescent microscopy is commonly used with antibodies specific for epithelial cells including cytokeratin and EpCAM with or without the combination of a nuclear stain and CD45 for negative staining of leukocytes. Although enrichment by these techniques facilitates the detection of CTCs using subsequent fluorescent microscopy, they are not sufficient to isolate them due to the large background of leukocytes that persist reducing the purity of CTCs to 1% or less (14,20). The use of a functional assay to actively bind and separate CTCs from peripheral blood mononuclear cell (PBMNC) enriched samples via a cell adhesion matrix resulted in an increased median CTC count of 31.5 CTCs per mL of blood.…”

The application of circulating tumor cells detecting methods in veterinary oncology

“…The example of the commercial product suitable the enrichment of CTCs is OncoQuick ® (Greiner Bio One,Germany), which in test cell recovery gives 69% -91% reproduciblility and 1.08 -1.83 cell per 20 ml of blood sensitivity detection limit (Gertler et al 2003).…”

The clinical significance of circulating tumor cells in gastrointestinal cancer

“…OnkoQuick was developed to avoid the cross-contamination of different layers, resulting in higher recovery rate of CTCs. [10,11] Recently, RosetteSep TM (Stem Cell Technologies, Vancouver, British Columbia, Canada) developed a novel method based negative selection to improve the specifi city of standard gradient separation. [12,13] …”