Table 1 Approaches for measuring peripheral biomarkers
Approach | Sample | Strengths | Manufacturers and/or examples of technologies |
|---|---|---|---|
Whole transcriptome profiling, RNA- seq, single-cell RNA-seq | RNA from PBMCs | RNA-seq • Fast and high efficiency • Broad, dynamic range • Detects differentially expressed genes • Measures average expression level • Uses millions of short reads (sequence strings), so all RNA in a sample can be investigated | • Illumina |
|  |  | Single-cell (scRNA-seq) • Measures the distribution of expression levels for each gene • Expression patterns of individual cells can be defined in complex tissues • High resolution of cell-to-cell variation | • Bio-Rad® single-cell RNA-sequencing solution • 10X Genomics |
Epigenetic differentiation-based immune-cell quantification | Genomic DNA from fresh or frozen whole blood, PBMCs | • Broad range of acceptable sample conditions (e.g. samples can be frozen and shipped without other steps) • Standardized measurements and circulating and tissue-infiltrating immune cells can be compared as an alternative to flow cytometry for peripheral blood samples and IHC for solid tissues | • Quantitative real-time PCR-assisted cell counting |
Chromosomal confirmation signatures | Blood | • CCSs can provide a stable framework from which changes in the regulation of a genome can be analyzed | • EpiSwitch™ |
Protein microarray | Fresh or frozen serum and plasma | • Versatile and robust platform • Miniaturized features, high throughput, and sensitive detections • Reduction in sample volume used • Variety of biological samples can be analyzed | • ProtoArray® (Life Technologies) can analyze serologic response of 9000 proteins simultaneously • SOMAscan® Assay • Olink Proteomics |
Mass spectrometry | Blood | • Mass spectrometry-based protein measurements in blood | • Biodesix |
Flow cytometry | Blood, fresh or frozen PBMCs, circulating tumor cells | • Multiparameter measurements at single-cell level • Rapid, high throughput manner • Cytometers available at reasonable cost • Recent advances in lasers/fluorochrome technology allows multiparameter analysis of rare cells (e.g. tumor antigen-specific T lymphocytes) | • BD LSRFortessa™ X-20 • BD FACSymphony™ |
Mass cytometry | Blood, fresh or frozen PBMCs | • Multiparameter single-cell analysis • Heavy metal ions as antibody labels overcome limitations of fluorescence-based flow cytometry • Little overlap between channels and no background (up to 40 labels per sample) • Increased number of phenotypic and functional markers can be probed | • Comprehensive analysis of profile and function of immune populations (e.g. time-of-flight cytometry by Helios™) |
T- and B-cell receptor deep sequencing | PBMCs formalin-fixed paraffin-embedded | • Identify changes in T- & B-cell populations, both in circulation and within tumors • Millions of T- & B-cell receptor sequences can be read from a single sample • Identify clonal expansion (measure of adaptive immune response) • Has been used to show clinical response to cancer immunotherapy | • ImmunoSEQ® immune profiling system at the deep level (Adaptive Biotechnologies) • Illumina HiSeq system |
ELISA and multiplex assays | Blood | • Widely used • Multiple biomarkers measured at once • Small sample volume • Measures soluble mediators (e.g. cytokines, chemokines, autoantibodies) | • ELISA • Multianalyte immunoassays: Simple Plex™ MesoScaleDiscovery Luminex, Quanterix™ |