Immune monitoring technology primer: Single Cell Network Profiling (SCNP).

摘要: Description of the technology Understanding a patients’ immune status not only from cell phenotyping, but also through analysis functional signaling capacity, enables generation more comprehensive understanding complex mechanisms responsible for immunological tolerance in cancer, and generates data that is complementary to other non-functional phenotypic sets such as immunohistochemical profiling genomic analyses. Single network (SCNP) quantifies capacity connectivity at systems biology level. The based on multiparametric flow cytometry simultaneously multiple rare subsets, without need physical separation, both extracellular surface markers changes intracellular proteins response modulators. Quantifying modulated across panel modulators (e.g., IFNα, IFNγ, IL-4, IL-10, IL-27, antiCD3 etc.) pathways identifies which cannot be assessed by measuring basal (unmodulated) alone. A node defined combination modulator with readout. For example TLR4 - > p-Erk defines one modulation quantified increase levels compared unmodulated reference. Typically 3 nodes are captured per well subsets interest p-Erk, p-S6, IkB). application SCNP clinical decision-making requires high-content assays robust, accurate, quantifiable reproducible results time, operators instruments. Each procedural steps associated an assay, including pre-analytical sample handling, assay execution reagents, acquisition metrics, have been validated [1] (Figs. 1 2). Experimental setup performed using proprietary software experimental design/96well plate layouts capture contiguously linked, ensuring each correctly assigned. laboratory can many 30 samples assayed up 40 wells (approximately 200–500 dimensions comprising modulator/ inhibitor/intracellular readout/cell subset combinations) 2 days depending kinetic time points. statistical plan (SAP) drafted all studies beyond exploratory phase, upon clearly stated objectives. identification clinically classifiers frame development validation comparable technologies genomics, IHC) due requirements powering verification independent sets.