Highly Efficient Assay of Circulating Tumor Cells by Selective Sedimentation with a Density Gradient Medium and Microfiltration from Whole Blood

TLDR

Size‑based isolation of circulating tumor cells often yields low purity and recovery because CTC sizes overlap with leukocytes, making filtration unreliable. The study introduces a highly sensitive, selective, and rapid method for isolating and detecting circulating tumor cells. The assay captures CTCs with anti‑EpCAM microbeads, then selectively sediments them in a density‑gradient medium while removing leukocytes and erythrocytes, and finally collects the purified cells by microfiltration, with optimization performed using MCF‑7 and DMS‑79 cell models to maximize sedimentation differences. The method achieved >99 % leukocyte removal, near‑perfect (~99 %) recovery of MCF‑7 cells, and high (~89 %) recovery of DMS‑79 cells, producing a highly pure CTC sample.

Abstract

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 μm) and DMS-79 small cell lung cancer cells (average diameter, 10 μm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (~99%) for MCF-7 cells and very high recovery rate (~89%) for DMS-79 cells, with minimal amounts of leukocytes present.