Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood

Frank A W Coumans,Markus Beck,Leon W M M Terstappen,Guus Van Dalum,Timothy W Secomb

doi:10.1371/journal.pone.0061770

Abstract

A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system.

Highlights

Circulating tumor cells (CTC) predict survival in patients with various metastatic cancers [1,2,3,4,5,6,7,8]
A large part of the TEM grid is covered with a red blood cell layer, the image in panel C of figure 2 shows the only part of the filter that was not covered with red blood cells, comprising 17% of the total filter area
Good agreement on the median size was found, the % CV on the size was larger by the Coulter principle than by the imaging approach, up to double for the cells from the tumor cell lines and almost five times higher for leukocytes

Summary

Introduction

Circulating tumor cells (CTC) predict survival in patients with various metastatic cancers [1,2,3,4,5,6,7,8] Enumeration of these CTC is a great technological challenge [9]. CTC are extremely rare cells typically 1–10 CTC among ,66106 leukocytes, ,26108 platelets and ,46109 erythrocytes per ml of blood [10]. This implies that any assay for their enumeration must be able to handle a large number of cells. The epithelial cell adhesion molecule (EpCAM) is frequently used for CTC enrichment as it has little or no expression on leukocytes, and is expressed by the CTC in most patients [12,32,33]

Methods

Results

Conclusion