Immunomonitoring Core

Immunomonitoring covers a broad range of technologies used to characterize and monitor immune responses in health and disease. At Houston Methodist, the Immunomonitoring Core supports researchers and clinicians in studying interactions between the immune system, cancer cells and other disease-associated cells. These approaches are applicable across cancer, inflammatory and infectious diseases, neuroscience, respiratory diseases and other areas where multidimensional characterization of immune responses is important. Immunomonitoring can also be used to evaluate therapeutic responses, investigate mechanisms of action, identify biomarkers, and assess or predict treatment-related adverse events.

Biomarker discovery and translational research increasingly rely on high-plex approaches capable of profiling proteins at the single-cell level, within intact tissues, or in liquid biopsy samples. The Houston Methodist Immunomonitoring Core provides mass cytometry (CyTOF) using the Helios™ CyTOF system, Imaging Mass Cytometry (IMC) using the Hyperion™ Imaging System, and Olink proteomic assays using the Olink Signature Q100 system.

The Helios CyTOF system uses metal-isotope-tagged antibodies to measure more than 40 cellular markers simultaneously in single-cell suspension samples. By distinguishing signals based on mass rather than fluorescence wavelength, CyTOF minimizes spectral overlap and enables detailed characterization of immune-cell phenotypes, functional states, activation and signaling.

The Hyperion Imaging System applies IMC to intact tissue sections, enabling simultaneous visualization of more than 40 molecular markers while preserving tissue architecture and spatial relationships among individual cells. This supports characterization of cell populations, functional phenotypes, tissue organization, cell-cell interactions, and biomarker distribution within tumors and other disease microenvironments.

Olink Target and Reveal platforms enable high-plex protein biomarker analysis using proximity extension assay (PEA) technology in volume-limited samples, such as plasma, serum, cerebrospinal fluid, conditioned media, tissue and cell lysate, and bronchoalveolar lavage fluid. Olink Target panels provide focused measurement of predefined protein sets, while Olink Reveal enables NGS-based broader proteomic profiling for biomarker discovery and hypothesis generation.

Surface Plasmon Resonance (SPR) enables real-time, label-free characterization of molecular interactions, including binding affinity, kinetics and specificity.

The Core also supports complementary high-dimensional approaches, including spatial transcriptomics, to integrate molecular, cellular and spatial information across biological samples.

Biology

• Olink Target and Reveal high-plex proteomic biomarker profiling
• Mass Cytometry (CyTOF)
• Imaging Mass Cytometry (IMC)
• Surface plasmon resonance (SPR)
• Spatial Transcriptomics

Bioinformatics

• CyTOF and IMC data analysis
• Olink Target and Reveal data analysis
• Spatial Transcriptomics data analysis

CyTOF

For analysis using the Helios™ CyTOF system, tissues should be processed and digested into single cell suspension; approximately one to three million cells per sample are preferred. Red blood cells should be removed, and samples should have good cell viability with minimal debris and aggregation, as excessive dead cells or aggregates may compromise staining quality and clog the instrument. Samples should be delivered on ice before 1 p.m. on the scheduled day.

IMC

Tissues should be appropriately fixed in formalin or paraformaldehyde (PFA) according to the tissue type and established histology procedures. When arranging tissue in a cassette, samples should be positioned to facilitate sectioning and subsequent region-of-interest selection. Long or narrow tissues, such as bone marrow specimens, should be oriented consistently and arranged without unnecessary gaps.

Fixed tissues may be embedded in paraffin. After the antibody panel has been finalized and any required antibody conjugation and validation have been completed, tissue blocks can be submitted for sectioning. Freshly prepared sections are preferred to help preserve antigenicity. Tissue should generally be sectioned at approximately 5 µm, with one tissue section per slide. An adjacent section should be used for H&E staining. A low-magnification image of the entire H&E slide should also be submitted, with the regions of interest (ROIs) clearly marked for IMC acquisition, together with higher-magnification images of the selected ROIs.

Olink

For Olink Target and Reveal assays, EDTA plasma is the most commonly used sample type, although other matrices, including plasma collected with other anticoagulants, serum, cerebrospinal fluid (CSF), conditioned media, bronchoalveolar lavage fluid, and appropriately prepared tissue or cell lysates, may also be evaluated depending on the assay and study design. Olink assays generally require only 1–4 µL of sample, making them well suited for volume-limited specimens.

Samples should be collected and processed consistently across all study groups and stored frozen until analysis. Repeated freeze-thaw cycles should be minimized. Samples with substantial hemolysis, contamination, precipitation or other quality concerns may affect assay performance. Randomization of samples across rows, columns and assay plates is strongly recommended to minimize potential technical bias. For Olink Target 96 or Reveal studies involving samples analyzed across multiple assay runs or batches, especially longitudinal studies or pooled analyses, appropriate bridging samples should be included across batches to facilitate normalization and minimize batch effects.

Procedures

CyTOF

For the Helios™ CyTOF system, cells will first be labeled with a metal-conjugated viability reagent and appropriate blocking reagents. Surface-marker staining will be performed before fixation and permeabilization. If intracellular markers are included in the panel, cells will be fixed and permeabilized using an appropriate staining kit, such as a Foxp3/transcription-factor staining kit, followed by intracellular antibody staining. Cells will then be incubated with a metal-containing DNA intercalator for nuclear identification, typically overnight at 4°C. Before acquisition, samples will be washed and filtered through a 40 µm cell strainer to minimize aggregates.

IMC

Slides with tissue sections will be incubated at 60°C overnight to improve tissue adhesion and facilitate deparaffinization. Slides will then be deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval will typically be performed using Tris-EDTA buffer at 95°C for 20 minutes in a temperature-controlled microwave; alternative retrieval buffers or conditions may be used depending on tissue type and antibody requirements. After cooling, slides will be blocked with 3% BSA for one hour and incubated with metal-conjugated antibodies overnight at 4°C. The following day, slides will be washed and stained with a metal-containing DNA intercalator for nuclear identification. Slides will be stored at 4°C until IMC acquisition.

Olink

For Olink Target assays, samples are analyzed using proximity extension assay (PEA) technology on the Olink Signature Q100 platform. Pairs of oligonucleotide-labeled antibodies bind their respective target proteins, allowing complementary DNA sequences to hybridize and be extended. The resulting DNA reporters are subsequently quantified to provide multiplex protein measurements. Depending on the selected Target panel, results are reported as normalized protein expression (NPX) values or quantitative concentrations where supported by the assay.

For Olink Reveal, the same PEA principle is combined with next-generation sequencing (NGS) readout to enable substantially broader protein profiling. Following PEA and library preparation, DNA reporter sequences are quantified by NGS, followed by quality control, normalization, and bioinformatic analysis to generate protein-expression data for biomarker discovery and downstream statistical analysis.