High Definition Single Cell Assay (HD-SCA) workflow

The importance of the liquid biopsy in understanding of the metastatic process and the potential use as a noninvasive route for cancer detection, characterization, and monitoring warrants the development of robust, reproducible detection and characterization of analytes for use in the clinical settings. HDSCA is our toolset to explore different phases of a disease at the single cell level. HDSCA uses the โ€˜No-Cell-Left-Behindโ€™ approach to identify rare cells and characterizes them by single cell genomics and single cell proteomics. Towards that end, we have established critical academic and public-private partnerships. The ultimate goal is for our research to enable and motivate the necessary studies for clinical utility.

HDSCA has utility both as a clinical care tool and a research tool. The HDSCA workflow maintains a complete chain of custody from the patient data to the bulk sample to the individual analyte. As circulating tumor cell (CTC) assays are becoming more utilized, there is a need for development of standard operating procedures using the best pre-analytical conditions for blood collection, handling, and processing.  As a member of the Blood Profiling Atlas Commons (Blood PAC) we are making available standard operating procedures and data to the scientific community to advance the development of clinically useful tests by standardizing the methods for analysis of CTC morphology, genomics, and proteomics, with concurrent cell-free genomics (cfDNA).

The third generation HDSCA workflow provides the opportunity to identify epithelial, mesenchymal, endothelial, hematopoietic cells, as well as large extracellular events (LEVs), building a platform capable of providing a more comprehensive overview of the circulating rare events and capturing the heterogeneity of the liquid biopsy.

Click here for a video describing the approach used by HD-SCA.

Click image to view single CTCs

OPTICOLL: technical validation of hd-sca

Optimizing Parameters and Techniques in Circulating Tumor Cell Collection (OPTICOLL)

The high-definition single-cell assay (HD-SCA) is a circulating tumor cell (CTC) assay has utility both as a clinical care tool and a research tool. As CTC assays are becoming more utilized, there is a need for development of standard operating procedures (SOPs) using the best pre-analytical conditions for blood collection, handling and processing.  This project is supported by the NCI Center for Strategic Scientific Initiatives and is being managed by Leidos Biomedical Research by means of a subcontract with the University of Southern California.

Phase I Study design of sample collection.
Phase I Study design of sample collection.

Phase I of the study evaluated the effect of blood collection tube (BCT) type and time-to-assay (TTA) (i.e., time from blood collection to processing at the laboratory) in the performance of the assay in terms of cell enumeration and a key downstream analysis intrinsic to the HD-SCA platform: single cell genomics.

  • Comparison of 4 Blood Collection Tubes
  • Comparison of 2 Time To Assay timepoints
  • Readouts:
    • Enumeration of HD-CTCs
    • Enumeration of candidate cells
    • Enumeration of non-CTCs
    • Assessment of morphology
    • Assessment of genotype/phenotype
  • Comparison of HD-SCA platform to the CellSearch™ system

Outcomes: The results demonstrated that both BCT type and TTA significantly impact CTC detection. We found that the Cell-free DNA BCT (Streck tube) provided superior assay performance at 24 hour TTA when compared to other BCT types such as EDTA, Citrate, and Heparin. In addition, the Cell-free DNA BCT when processed at 24h, as opposed to 72 hours, provided higher detection rates.  The HD-CTC assay proved to be more sensitive as compared to the CellSeach™ methodology.

Phase II CTC/cfDNA genomic analysis workflow
Phase II CTC/cfDNA genomic analysis workflow.

Phase II of the study will characterize the effect of two TTAs (24h vs. 48h) in the enumeration of CTCs and specific high-content measurements including whole genome single-cell copy number variation (CNV) and targeted sequencing. This work will also characterize the effect of fresh versus frozen (FVF) plasma preparations in whole genome CNV and targeted sequencing of cell-free DNA (cfDNA). In addition, we will finalize current draft SOPs and experimental protocols for cfDNA isolation from plasma and library construction, single cell genomics, and Imaging Mass Cytometry (IMC). The IMC or scanning CYTOF is a new and novel instrument that will be validated and optimized to determine the protein expression of a panel of markers in CTC and related rare cells identified using the HD-SCA platform.

Phase II Imaging Mass Cytometer (IMC) Workflow.

Expected Outcomes:

  • Characterize Circulating Tumor Cells (CTCs) from liquid biopsies from breast cancer patients
    • Perform HD-SCA analysis to identify CTCs and provide a high content data set comprised of at least 6 parameters, in addition to CTC counts
    • Perform single cell genomic analysis of the CTCs identified
    • Perform targeted mutation sequencing of CTC genomic DNA isolated from single cells
    •  Perform single cell proteomic analysis of CTCs using mass cytometry
  • Characterize circulating cell-free DNA (cfDNA) from liquid biopsies 
    • Isolate cfDNA from liquid biopsies
    • Perform copy number variation profiling analysis on the isolated cfDNA
    • Perform targeted mutation sequencing of cfDNA