scholarly journals 11 A multi-physics approach enabling rare cell isolation with high recovery and high purity

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A11-A11
Author(s):  
Liping Yu ◽  
Silin Sa ◽  
Alice Wang
Keyword(s):  
Flow Cytometry ◽  
Sample Preparation ◽  
Single Cell ◽  
Tumor Cells ◽  
Whole Blood ◽  
Cell Isolation ◽  
High Recovery ◽  
Color Flow ◽  
Single Cell Genomics ◽  
Cell Processing

BackgroundAdvancements in fields of multi-omics analysis and cell-based therapies depend upon efficient cell processing tools to isolate rare cancer and immune cells from complex biologic samples as an initial step in sample preparation. Conventional technologies are limited in automation, recovery and purity. We present an integrated system based on multiple physics principles with built-in novel technologies to achieve cell purification, concentration and target cell isolation, with high recovery at an unprecedented flow rate. This platform, the Multi-physics Automated Reconfigurable Separation (MARS), combines tunable, acoustic cell processing and in-flow immuno-magnetic separation technologies, enabling automation of the entire cell sample preparation workflow for proteomics and genomics analysis.MethodsCirculating tumor cells (CTC) are present in extreme low frequency in blood stream (1–100 in billions of blood cells) thus it has been a challenge to isolate CTCs with high recovery. We have developed protocols on MARS to isolate CTCs from whole blood for multi-color flow cytometry analysis. To demonstrate the extent of enrichment of tumor cells in whole blood, PC3 cells were used for spike recovery. RBC lysed blood sample was then loaded on MARS and automatically processed through cell washing, concentration, and magnetic depletion. Enriched tumor cells were collected and analyzed by flow cytometry.ResultsResults show > 4 log enrichment of tumor cells and average recovery of spiked CTC > 85% in the clinical relative range <100 cells per ml of whole blood (R2=0.929) with a throughput of 60 ml/hr. Isolated cells were confirmed to be cancer cells with imaging analysis and single cell genomic sequencing. The protocol was also validated with other cell line cells such as A549. The purity of the cells prepared by MARS are ideal for single cell genomics platforms.ConclusionsThe fluidics of MARS is also replaceable and can be sterilized to minimize sample to sample contamination. The high molecular debris removal achieved by MARS is ideal for single cell genomics platforms, as is the first-to-market automated and integrated sample preparation and cell separation system designed to be a versatile tool for downstream cell analysis.

DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY

1987 ◽  
Author(s):  
S J Shattil ◽  
J A Hoxie ◽  
M Cunningham ◽  
C S Abrahms ◽  
J O’Brien ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Platelet Activation ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Membrane Surface ◽  
White Blood Cells ◽  
Color Flow ◽  
Platelet Surface ◽  
Activated Platelets

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.

Abstract 3065: Single cell isolation and DNA analysis from circulating tumor cells using a self sorting nanowell plate

2014 ◽  
Author(s):  
Joost F. Swennenhuis ◽  
Arjan G.J. Tibbe ◽  
Michiel Stevens ◽  
Hien Duy Tong ◽  
Cees J.M. van Rijn ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Dna Analysis ◽  
Cell Isolation ◽  
Single Cell Isolation

Abstract 1606: Single cell isolation and DNA analysis from circulating tumor cells using self-sorting nanowell plates

2015 ◽  
Author(s):  
Joost F. Swennenhuis ◽  
Arjan GJ Tibbe ◽  
Michiel Stevens ◽  
Nikolas H. Stoecklein ◽  
Rui Neves ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Dna Analysis ◽  
Cell Isolation ◽  
Single Cell Isolation
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