Services

Study Design:

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 end points 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. Immune assays are performed in assurance of consistent performance, allows data mining across the studies and in a cost-effective manner due to bulk and batch purchasing power.

Immune Monitoring Assays

1. Immune cell profiling with polychromatic flow cytometry and CyTOF

2. Antigen-specific cellular assays

  • Separation of CD4 and CD8 T cells
  • In vitro sensitization with antigen and expansion of T lymphocytes
  • Intracellular staining of cytokines (ICS)
  • ELISPOT assay
  • Tetramer assay
  • Frequency of antigen-specific CD4+ T cells using CD154 assay (in development)

3. Antigen-specific antibody responses

  • Grand serology (ELISA) for characterized antigens
  • Seromics: protein array antibody profiling

4. Measure of soluble factors in serum and plasma

  • Multiplex analysis

5. Blood Processing, Banking and Nucleic Acid Isolation

  • 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

6. Cell Sorting, enrichment and nucleic acid isolation

7. TCRbeta repertoire High Throughput Sequencing with Adaptive Biotechnology

8. Functional Assays for Leukocyte subsets

9. Assay results are available through secure MountSinai ShareFile systems

 


 Immune cell profiling with polychromatic flow cytometry and CyTOF

Flow Cytometry

Phenotypic analysis of immune cells are analyzed according to established cell staining panels for effector and regulatory T lymphocyte subsets, B lymphocytes, natural killer (NK) cells, NKT cells, monocytes, neutrophils, dendritic cells (DC), and plasmacytoid dendritic cells. BD LSRFortessa analytical flow cytometer (Becton Dickinson) with high throughput capability (96 well format) to support large clinical study analysis is dedicated to the HIMC. Price List

 CyTOF

CyTOF Mass Cytometer (DVS Sciences) is a multiparametric biological analyzer, similar to flow cytometry except it operates based on time-of-flight Mass cytometry. Using antibodies tagged with various heavy metal ions, over 30 parameters can be analyzed simultaneously in a single tube without the need for compensation at high acquisition rate. We are developing and standardizing immunophenotyping CyTOF panels in collaboration with Flow Cytometry CORE. Price List


 Antigen-specific cellular assays

Separation of CD4 and CD8 T cells

To allow accurate quantification of CD8 and CD4 T cells independently, we offer separation of lymphocytes using magnetic beads, so that responses can be compared using a common denominator of initial T cells tested. A minimum of 10-20 million PBMC is recommended, though starting from fewer numbers is feasible. Price List

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. Price List

Intracellular staining of cytokines (ICS)

Intracellular staining of cytokines is a sophisticated flow cytometry-based method to quantify antigen-reactive T cells at the single cell level for their capacity to produce multiple cytokines and to characterize surface or intracellular markers that may be coexpressed. It is a combination of functional and phenotypic analyses, and can be used to measure Th profiles, memory or homing receptors, or polyfunctionality of T cell responses. It can be used ex vivo or following presensitization (see above), from PBMC or from purified CD4 and/or CD8 compartments. Price List

ELISPOT assay

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. Price List

Tetramer assay

Tetramer/pentamer staining is a flow cytometry-based assay for precise quantification and phenotypic analysis of antigen-specific CD8 T cells. It uses 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. It allows to tests 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, though additional validation would be required. Price List

Frequency of antigen-specific CD4+ T cells using CD154 assay (in development)

Additional assays are under development, such as assessing T cell specificity with CD154 surface expression, that 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

Grand serology (ELISA) for characterized antigens

Serum or plasma from peripheral blood is used to test for presence of IgG responses to defined antigens. 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. Price List

Seromics: protein array antibody profiling

Seromics is an exploratory, hypothesis-generating platform that allows to test 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. Price List


 Measure of soluble factors in serum and plasma

Multiplex analysis

Luminex based assay is used to measure biomarkers, cytokines, chemokines, hormones, nitric oxide and growth factor, cell signaling and phospho-protein detection in pathway specific for immune call activation. A Luminex 200 system is capable of multiplexing up to 41analytes simultaneously in a single well of microtiter plate with Data analysis via X-ponent 3.1. Price List


 Blood Processing, Banking and Nucleic Acid Isolation

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

Price List


 Cell Sorting, enrichment and nucleic acid isolation

Purification of Leukocyte subsets and DNA/RNA isolation for genomic profiling and functional Assays of specific Leukocyte subsets (in collaboration with the Genomic Institute). Purification of immune cell subsets is performed either i) using a 5-laser BD™ FACSAria-II cell sorter installed in a Baker Bioprotect® III – Biosafety Cabinet in the Shared Research Facility of Flow Cytometry or, ii) through robotic magnetic cell sorting of RoboSep. Nucleic acid isolation service is provided from whole blood collected in application specific vacutainers, i.e., tempus, Pax tube and tissues such as biopsy and tumor samples using automated QiaCube. Transcriptional profiling is performed by the Genomics Core. Price List


 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.


 Functional Assays for Leukocyte subsets

Functional characterization of human leukocyte subsets by separation and co-culture assay ( i.e., immunogenicity, cytotoxicity and immunosuppression). Protocol specifics upon discussion with the PI.


 Assay results are available through secure MountSinai ShareFile systems

MountSinai ShareFile system our online secure database, integrates the results of all standard assays across projects. Each investigator can access his/her own projects.

Please contact seunghee.kim-schulze@mssm.edu if you have HIMC data and need help accessing MountSinai ShareFile system

Contact Us

Please contact us for consultation:

Human Immune Monitoring CORE (HIMC)
Hess Center for Science and Medicine
1470 Madison Avenue
5th Floor, Rooms 310/313
New York, New York 10029

Send e-mail
Tel: 212-824-9354