HHE Pathology and diagnostics supplement 2018 - Page 10

other solid tumours) and other leukocyte antigens, to achieve a CTC-enriched sample. The RosetteSep TM CTC Enrichment Cocktail (STEMCELL Technologie) offers a unique method for further depletion of unwanted cells by integrating immunoaffinity-based enrichment with density centrifugation. RosetteSep™ targets unwanted cells and forms a pellet with red blood cells (RBCs) through tetrameric antibody complexes that target an extensive mixture of specialised antigens. An immunomagnetic version is also available – the EasySep™ system – which contains magnetic nanoparticles and tetrameric antibody complexes targeting CD45, for example. Some technologies are flexible and can function either as positive or as negative enrichment systems by applying different antibodies (for example, replacing anti-EpCAM, specific for epithelial cells with anti-CD45, specific for leucocytes). For example, the CTC-iChip could use an immunomagnetic selection with functionalised beads against EpCAM or CD45 and CD66b. 17 Positive selection Mostly used, immunomagnetic systems target an antigen with an antibody, which is coupled to a magnetic bead, and the antigen–antibody complex is isolated via exposure to a magnetic field. Positive selection is usually carried out with antibodies against the epithelial cell adhesion molecule (EpCAM). Among the current EpCAM- based technologies, the FDA-approved CellSearch™ system has gained considerable attention over the past ten years and is frequently compared with all new CTC detection methods as the gold standard. However, capturing CTCs lacking EpCAM has involved the use of antibody cocktails against various other epithelial cell surface antigens (for example, EGFR, MUC1), or against tissue-specific antigens (for example, PSA, HER2) and against mesenchymal or stem cell antigens (Snail, ALDH1). 11 Many commercial platforms for CTC detection using positive magnetic enrichment are available, including: • Based on EpCAM: MagSweeper 12 or MACS technologies from Miltenyi 13 • Using different cocktails of antibodies: IsoFlux TM , 14 AdnaTest from Adnagen. 15 At present, there is a focus on the development of microfluidics devices (‘chips’) such as Ephesia Chip 16 or CTC-iChip. 17 Interestingly, a unique in vivo device, GILUPI CellCollector ® , was developed to capture CTCs directly in the vein of patients, with a structured medical Seldinger guidewire functionalised with an antibody targeting EpCAM to trap CTCs. This wire allows screening of a large volume of blood during the 30-min collection period. Positive selection of CTCs requires an assumption about the unknown nature of CTCs in an individual blood sample. This bias is avoided by negative selection in which the blood sample is depleted of unwanted cells. Negative selection Negative enrichment uses an indirect method to isolate CTCs: they target and remove background cells, such as leucocytes, using antibodies against CD45 (which is not expressed on carcinomas or 10 HHE 2018 | hospitalhealthcare.com Physical properties Numerous marker-independent techniques have been developed for CTC isolation and detection. Label-free enrichment processes based on physical properties, such as density, size, deformability, and electric charge, avoid molecular bias induced by variability of cell biomarker expression associated with tumour heterogeneity. Density selection Density gradient centrifugation was one of the first methods recorded for CTC isolation. 18 Although not originally developed for CTC isolation, Ficoll-Paque ® , a density gradient medium for the separation and isolation of mononuclear cells, has been used in research for some time. However, after such a non-specific pre-analytical process, CTCs are still present in a large number of leucocytes; indeed, only erythrocytes and polynuclear cells are depleted using Ficoll-Paque centrifugation. To improve this enrichment, a subsequent positive or negative step is usually required and performed. Designed for CTC isolation, OncoQuick (Greiner Bio-One) employs a liquid separation medium that has been optimised for the specific enrichment of CTCs only, based on their buoyant density under appropriate conditions and no additional step is required because even the leucocytes are eliminated from the cell monolayer between the plasma and the Ficoll. Size selection Microfiltration technologies, based on the precedent that CTCs generally exhibit a larger morphology than leukocytes, such as ScreenCell ® , 19 ISET ® , 20 CellSieve™, 21,22 or Parsortix™ 23 involve flowing blood through pores or microfluidic steps of calibrated size to trap larger cells (the CTCs) while smaller cells pass through. Some other size-based microfluidic devices use inertial focusing to separate CTCs from blood. Vortex technology relies on inertial microfluidics and laminar microscale vortices to position cells along channel walls upstream of micro-vortices designed to stably trap CTCs. 24 In the same way,