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 EpCAMbased 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
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 ,
142 HHE 2018 | hospitalhealthcare . com