The Human Immune Monitoring CoRE (HIMC) provides comprehensive immune monitoring services for clinical and translational studies.
The HIMC provides consultation regarding optimal project-specific immunologic assessment and innovative approaches to evaluate immune responses in clinical studies. Our staff of researchers will offer expert advice to assist the investigator in designing and conducting the proper experiment and preparing the appropriate samples for analysis.
We will help define the endpoints of the immune assay, assay parameters, sample requirements (collection, labeling, storage, handling, and shipping) and processing, reagents and controls, data analysis, data management expectations, and estimated costs. We perform immune assays assuring consistent performance, allowing for data mining across the studies and in a cost-effective manner due to our bulk and batch purchasing power.
Blood and Biospecimen Processing, Cell Isolation, Banking and Nucleic Acid Isolation
The HIMC has three separate rooms, including one for comprehensive biospecimen processing from sample acquisition processing in real time; a room for management and tracking in the LIMS, ILab, and a room dedicated to Freezerwork. Two rooms are equipped with tissue culture hoods, tissue culture incubators, centrifuges, and computer with a bar coding label printer. Our tissue culture room also has the clinical grade Coulter Ac-T 5diff Hematology Analyzer, which reads 18 parameters for the CBC/differential of the whole blood. HIMC is currently operating as GLP to help us ensure the uniformity, consistency, reliability, reproducibility, quality, and integrity of laboratory procedures. All procedures have written SOPs that we strictly adhere to, properly documenting any deviations and corrective actions. Through our procedure worksheets we capture all critical information including signature or initials of the person performing the procedure. Quality Assurance reviews all worksheets and archives them in the appropriate binders.
HIMC has implemented a HIPAA compliant and secure bar-coded sample management and tracking system using the software Freezerwork (v6.1) supported by the School’s secure server. Through this system we ensure crucial sample data management in a proper and reliable manner, maintaining sample quality for biomedical research, as well as getting the most valuable samples to the researchers in a timely and efficient way. As a result, we ship samples to other sites in a streamlined manner, use the checkout/check-in feature to manage sample usage and availability and to meet all regulatory requirements with an automated audit trail. We bar code all the samples in a unique identification number using the bar code printer and place them in storage locations. We track and manage the complete life cycle of biospecimens from acquisition to usage in audit trail tracking.
The following services are provided for the investigators at MSSM and through collaborative relationships with other performance sites such as Cancer Immunotherapy Trials Network (CITN), Eastern Cooperative Oncology Group (ECOG) and Hoosier Oncology Group (HOG).
- Serum and plasma processing and storage
- PBMC processing and storage
- PBMC cryopreservation
- PAXgene collection and storage
- PAXgene DNA and RNA extraction
- Tempus tube collection and storage
- Tempus tube RNA extraction
- Tissue DNA/RNA isolation
- Shipment of cryovials in dry-shipper with temperature sensor and monitor
Cell Sorting and enrichment
The HIMC is equipped with a Robosep robotic magnetic cell sorting system and has optimized protocols for isolation of major immune subsets for downstream analyses, such as isolation of CD4 and CD8 T cells for antigen specific stimulation assays. We can perform more advanced cell sorting applications in collaboration with the Flow Cytometry CoRE using a 5-laser BD™ FACSAria-II cell sorter installed in a Baker Bioprotect® III – Biosafety Cabine, through robotic magnetic cell sorting of RoboSep.
Nucleic acid isolation
The HIMC offers optimized nucleic acid isolation and QC from biospecimens using either a QiaCube or QiaSymphony platform, which allows us to perform high throughput automated extraction of 96 samples in a single run. We can also perform downstream transcriptional profiling experiments in collaboration with the Genomics CoRE.
Cytometric Immune Monitoring Assays
The HIMC offers deep expertise in high dimensional cytometric analyses of the immune system.
The HIMC is equipped with a 5 laser BD LSRFortessa dedicated to supporting multiparametric flow cytometry analyses in immune monitoring studies. This instrument includes a 96-well autosampler unit to facilitate high thorough putsample acquisition. Only trained technicians operate the HIMC’s LSRFortessa, following strict SOPs to optimize and track instrument performance and ensure data quality and reproducibility.
Mass Cytometery (CyTOF)
The CyTOF2 is cutting edge instrument that merges the high-throughput single cell analysis of flow cytometry with the specificity and resolution of mass spectrometry to allow highly multi-parametric single cell analysis of heterogeneous cell samples. Mass cytometry is conceptually similar to conventional flow cytometry, but instead of fluorescently labeled antibodies, mass cytometry utilizes antibodies conjugated to isotopically-enriched metals tags to label samples. The stained single cell suspensions are nebulized and passed through argon plasma that atomizes and ionizes the cells into individual ion clouds, which then pass through a series of mass optics and are analyzed by time-of-flight spectrometry. The multi-atomic signals associated with the labeled antibodies are integrated to derive single cell data much like those generated by conventional flow cytometers, though overcoming the limitations of spectral overlap and allowing the simultaneous detection of over 40 antibodies. The HIMC has integrated the CyTOF2 as part of a full service mass cytometry program and has established robust SOPs for instrument operation, optimization and QC to ensure the generation of high-quality, highly-reproducible data.
High dimensional immunophenotyping
The HIMC has established standard panels for flow and mass cytometric immunophenotyping of human peripheral blood, bone marrow and tissue-derived cell samples. Our mass cytometry staining panels allow the identification of all major immune cell subsets including neutrophils, eosinophils, basophils, monocytes, dendritic cells, plasmacytoid dendritic cells, B lymphocytes, natural killer (NK) cells, NKT cells, and effector and regulatory T lymphocyte subsets. These panels can be expanded and customized to further characterize cellular heterogeneity within these subsets, or incorporated into functional profiling experiments. The HIMC also has expertise in developing and optimizing customized cytometric profiling panels for specific applications, such as minimal residual disease (MRD) monitoring.
Functional cytometric profiling
The HIMC has extensive expertise with functional immune profiling by flow and mass cytometry. We have optimized mass cytometry panels to simultaneous evaluate the production of multiple cytokines on a single cell level using intracellular cytokine staining (ICS). We have also optimized panels to measure dynamic changes in single cell protein phosphorylation. These functional panels can be applied to profile responses to a range of stimuli and can also be combined with surface immunophenotyping panels to characterize the phenotype of responding populations.
Reagent bank and custom CyTOF antibody conjugation
In addition to offering a large repository of validated metal-conjugated antibodies and cell labeling reagents, the HIMC offers all the necessary reagents and expertise to perform custom isotopic conjugation and validation of new antibodies for specific CyTOF projects.
Cytometric data management and analysis
All mass cytometry data generated at the HIMC undergo initial QC analysis and post-processing including bead-based normalization and barcode deconvolution. Processed files are uploaded and shared with investigators through our institutional Cytobank server (link: https://mtsinai.cytobank.org/). In addition, the HIMC offers both assisted and comprehensive analysis of flow and mass cytometry data. Experiments can be analyzed and visualized using a variety of software platforms including Cytobank, which offers dimensionality reducing visualizations such as SPADE and viSNE. The HIMC is also actively developing novel data processing and analysis pipelines using Mt. Sinai’s Minerva supercomputer to facilitate the analysis of complex multidimensional datasets.
Ex vivo cell culture, stimulation and antigen specific assays
The HIMC has a range of protocols for ex vivo stimulation of immune cells to evaluate both general and antigen-specific immune functional responses.
In vitro sensitization with antigen and expansion of T lymphocytes
For many antigens, frequency of antigen-specific T cells ex vivo may be too low to reliably measure immune responses. This is true of many tumor antigens but also for some weaker viral antigens. One way to overcome this limitation is to expand antigen specific T cells in vitro using overlapping peptide libraries, preferably independently for CD8 and CD4 T cells that tend to peak at different times after culture. This in vitro sensitization step is designed to only expand in vivo generated T cell responses while minimizing de novo generation of responses from in vitro stimulation, with minimal skewing of repertoires. Although the assay is no longer directly quantitative following presensitization, comparison of eventual frequencies across samples is still possible when assays are performed simultaneously. Besides providing more robust results, presensitization also allows for testing more specificity controls.
Antigen Specific Intracellular staining of cytokines (ICS)
We can measure antigen specific responses using multiparametric intracellular cytokine staining detected by flow or mass cytometry. This sophisticated approach allows us to quantify and characterize antigen-reactive T cells at the single cell level, allowing an evaluation of polyfunctional cytokine production in conjunction with additional surface or intracellular markers to further define their cellular phenotype. We can perform ICS directly ex vivo or following presensitization (see above), using whole blood, PBMC or from purified CD4 and/or CD8 compartments.
ELISPOT (Enzyme Linked Immunosorbent SPOT) assay is a robust and standardized test to quantify functional antigen-reactive T cells at the single cell level for capacity to produce cytokines such as IFN-γ. PBMCs or purified and/or expanded CD4 or CD8 T cells are stimulated with antigen and cultured in a 96 well plate with a nitrocellulose membrane coated with antibody specific for IFN-γ. Results are provided as a number of spots (equivalent to reactive cells) out of cells tested. The test is very sensitive and allows for multiple specificity controls.
Tetramer/pentamer staining is a cytometric assay for precise quantification and phenotypic analysis of antigen-specific CD8 T cells using HLA-peptide complexes as multimers to bind to T cell receptors specific for defined combinations of epitopes and alleles. The method is available for antigens for which a peptide epitope has been defined in the context of common alleles such as HLA-A1, A2, B35, Cw3, or others. Tetramer staining can be combined with antibodies against multiple surface markers expressed on antigen-specific T cells, to determine memory status or homing receptor molecules for example. Exploratory HLA class II tetramers for CD4 T cells may also be available, with additional validation required. The HIMC is also working to develop highly multiplexed mass cytometry based tetramer assays to allow simultaneous detection of several antigen specific responses in a single sample.
Frequency of antigen-specific CD4+ T cells using CD154 assay
Additional assays are under development, such as assessing T cell specificity with CD154 surface expression, which allows for sorting and further expansion of even rare antigen-specific T cell populations. Consultation with the immune CoRE is encouraged to help investigators design the best assays for their interests.
Antigen-specific antibody responses
We offer a grand serology (ELISA) assay to test of the presence of antibodies against defined antigens in serum or plasma from peripheral blood. Currently, a tumor panel of defined tumor antigens is available (includes NY-ESO-1, SSX2, Melan-A, TP53…), though the test is easily customizable to include additional antigens of choice, or to test for other subclasses and isotypes such as IgE or IgG4. The assay is an ELISA (enzyme-linked immunosorbent assay) with full-length proteins or overlapping peptides, to determine primarily linear epitopes. The sensitivity of the assay is very high, requiring only microliters of serum or plasma, reproducible, controlled, and results are reported following titration to allow for quantitative comparisons between samples. This flexible assay can also be adapted for ascites, urine, or supernatants of cell cultures where immunoglobulin may be present.
Seromics: protein array antibody profiling
Seromics is an exploratory, hypothesis-generating platform that tests thousands of human proteins simultaneously as potential targets of autoantibodies from patient serum or plasma. The method only requires few microliters of material, and may be customized for applications other than IgG detection. It is ideally suited to comprehensively look for serum antibody changes at the antigen-specific level following treatment, for example immunotherapy, vaccine, chemotherapy, or radiotherapy. Results are normalized and expressed as fold change from pre- to post-treatment. Alternatively, the seromic platform can be used to define biomarkers or sets of antigens present at baseline in specific patient populations, and that could be used as prognostic or predictive markers. This approach requires a minimal number of samples (20) to be tested in order to establish cutoffs of specific responses. Data analysis is provided, and results are expressed as a list of antigens scored for their likelihood of being immunogenic, spontaneously or as a change related to treatment.
Measure of Soluble Factors in Biospecimens
Following are details on our analyses types.
The HIMC offers Luminex-based assays to measure biomarkers including cytokines, chemokines, hormones, nitric oxide and growth factor, cell signaling and phospho-protein detection in pathway specific for immune call activation. The HIMC is equipped with a Luminex 200 system that is capable of multiplexing up to 41analytes simultaneously in a single well of microtiter plate with Data analysis via X-ponent 3.1. The HIMC is actively involved in several research and development efforts to improve the robustness and reliability of multiplexed protein measurement.
SOMAlogic soluble factor profiling analysis (under development)
SomaLogic's SOMAscan assay is a cutting edge platform that offers a comprehensive profile of the proteomic and soluble factors in various biospecimens using high throughput protein analysis. The assay detects and measures 1,310 analytes across a wide dynamic range in small volumes of multiple biological sample types.
TCRbeta repertoire High Throughput Sequencing with Adaptive Biotechnology
The adaptive immune system has the potential to recognize a vast number of antigens through the combinatorial diversity of αβ TCRs. Estimates based on direct sequencing of TCRβ chains indicate that at any one time, an individual carries over 10 million unique TCRβ CDR3 chains. We are developing a platform for in-depth analysis of the T-cell receptor repertoire, a specific and important part of the immune system for the selection, function, and diversity of T cells in collaboration with Adaptive Biotechnology.
Microbiome of the human gut
The microbiome of the human gut differs to a large degree from person to person and has an important influence on health and disease due to its interaction with the human immune system. Composition of the microbial residents in the gut has been implicated in many autoimmune diseases such as inflammatory bowel disease. Functional profiling is a critical new target discovery for diagnostics and therapeutics in complex inflammatory conditions. HIMC processes stool samples, isolates microbial DNA and constructs the bar-coded sample library for the sequencing. Sequencing and data analysis pipelines are currently under the development.
We employ the following data sharing resources.
Mount Sinai Sharefile
MountSinai ShareFile system, our online secure database, integrates the results of all standard assays across projects. As an investigator you are able to access your own projects.
Please contact firstname.lastname@example.org if you have HIMC data and need help accessing MountSinaiShareFile system.
Cytobank is a cloud-based platform that facilitates storage, analysis, visualization and sharing of cytometric data sets. The HIMC administers Mount Sinai’s institutional Cytobank Platform, which includes a secure, dedicated hosted server to enable rapid data sharing and processing.
Minerva HPC integration
The HIMC is currently developing novel data processing pipelines to leverage the capabilities Mt. Sinai’s Minerva Supercomputer for advanced data analyses.
We offer the most advanced equipment in the field for your research.
- Mass Cytometer (CyTOF2), a state of the art mass cytometer that allows highly multi-parametric single cell analysis
- LSRFortessa BD special order analytical flow cytometer (5 lasers) with high throughput capability (96 well format) to support large clinical study analysis
- Luminex 200 System capable of multiplexing up to 41 analytes simultaneously in a single micro titer plate well
- Tissue culture room dedicated for patient sample processing and to perform immune function assays, 2 laminar hoods, 4 tissue culture incubators, centrifuges (two Eppendorf 5425R and 5810R), microscope, water bath
- Infrastructure for sample collection, processing and storage at dedicated -20oC, -80oC and two mobile Liquid nitrogen tanks with temperature monitors
- RoboSep for magnetic bead based cell separation/isolation
- QiaSymphony machine for automated nucleic acid isolation in high throughput manner
- Cryovials shipper in liquid N2 with inner temperature sensor/monitor
- Sample bar coding facility and management/tracking via Freezerwork server maintained by MSSM
- Nanodrop 2000 Spectrophotometer (ABI), shared equipment
- ELISPOT reader, shared equipment
- Coulter Ac-T 5diff Hematology Analyzer, shared equipment
- BioTek Synergy Neo HTS Multi-Mode Reader with top dual PMT filter optics with integrated BioStack automated stacker, shared equipment
- BioTek 405™ Touch Microplate Washer with 96-tube Dual-Action™ manifold and integrated BioStack automated stacker, shared equipment
Equipment - Request Forms, Human Immune Monitoring CoRE
To request Nucleic Acid Isolation please fill out the Nucelic Acid Isolation Request Form
You can also make an iLab Reservation