Cancer Institute A national cancer institute
designated cancer center

Human Immune Monitoring Center Shared Resource

http://himc.stanford.edu

Facility Director:  Holden Maecker, PhD [maecker]
Mark Davis, PhD [mdavis pmgm2.stanford.edu]

Contact

Holden Maecker, PhD
Office: Rm 0125A, CCSR Building
Stanford, CA 94305
(650) 723-1671
E-mail: maecker@stanford.edu [maecker]
Lab: Rm 0128, CCSR Building
Stanford, CA 94305
(650) 723-1671

Overview

Immune Monitoring Center (HIMC) Shared Resource provides state-of-the-art assays that measure biomarkers relevant to the immune system or specific pathogens. Examples of biomarkers that are specifically relevant to cancer include changes in lymphocyte signaling pathways, serum cytokines, and specific immune responses that target tumor cells. This resource offers Cancer Center members specialized assays that monitor the effects and efficacy of immunotherapies and vaccination strategies that are becoming increasingly important alternatives to conventional forms of cancer treatment. In addition, pathogens have been implicated in the etiology of many cancers, and the HIMC offers array methodologies for pathogen identification.

The HIMC initiated operations in early 2007, and has already attracted many faculty users in the past two years; about 74% of the current users are Cancer Center members. We expect an increasing demand for our services as new projects in the immune modulation of cancer are developed.

Major services provided by the HIMC are Luminex bead-based assays for measuring cytokines, FACS phenotyping and phosphoflow analysis for cell characterization, and the Firefly nanofluidics assay method for detecting phosphorylated proteins.

Future goals of the HIMC are to introduce for general use the multiplex peptide-MHC tetramer technology recently developed by the Davis lab to quantitate and characterize T cell responses to tumor associated antigens and the complementary ELISPOT analysis, which quantitates T and B cell responses. The shared resource will also offer full bioinformatics consulting and data management for its users.

Services

Central Sample Reception, Logging, Processing and Repository

The HIMC will inventory the samples on arrival.  The blood samples are identified only with a unique barcode.  No patient information is sent with the samples to the HIMC. We process blood into RNA, DNA, serum, plasma and viable buffy coats.  Quality of RNA is assessed by an Agilent bioanalyzer after preparation and before labeling for microarrays.    Serum is alloquated into 200 ul volumes and frozen at -80°C.  The freezers are on an emergency power system and are located in a secure room with keycard access.  Any patient related data (if any is provided) will be parsed to a SQL database on our HIPPA server.  The server is on it’s own subnet behind two firewalls on the Stanford Campus in a locked facility managed by Stanford’s Medical Information Technology (MEDIT) Group.  All data is backed up daily to another secure server.  Access is by secure user ID and all sessions are logged).  Samples and any data generated will be linked by the barcode information and stored on the three HIMC data servers.  The data files are only available to researchers directly connected to this study.    The servers are managed by the HIMC, housed in a secure data center managed by Stanford MEDIT and backed up daily.  All our servers used RAID drives that protects against data loss through individual hard drive failure.

Specialized Immune Monitoring and Pathogen Assays

The HIMC has implemented several multiplexed analyte assays on serum, plasma, ascites or other biological fluids to assess immune response of the patients at the time of blood draw or in frozen samples. We are also providing broad scale pathogen identification arrays such as “Greenchip” (Lipkin) and “Virochip” (deRisi) as many cancers have been linked to an infectious (usually viral) agent.

Luminex Bead-based Assays

The HIMC uses a Luminex LabMap200 System together with Panomics antibodies to currently measure the levels of 42 cytokines from sera or cell supernatant samples. Cytokines and chemokines play critical roles in regulating the development, trafficking and activation of immune cells.  There are currently over 100 cytokines and chemokines whose coordinate or discordant regulation is of clinical and research interest to cancer researchers.  The HIMC uses a Luminex LabMap 200 system which can simultaneously assay of up to 100 analytes in a single well of a microtiter plate, using very small sample volumes (40 microliters).  The Luminex system offers comparable sensitivity to traditional ELISA-based systems, but with additional advantages, including extended dynamic range and smaller sample size.

Phenotyping and Phosphoflow Analysis

The HIMC uses an Becton Dickinson LSRII flow cytometer to perform high throughput cell characterization. With the capability of 18 parameter analysis, the cytometer measures cell surface markers, intracellular phosphoproteins and intracellular cytokines. We use flow cytometry to determine the relative frequency of the different types of white blood cells in a given blood sample. This analysis can be used to both flag cellular anomalies and to normalize gene expression data, should that be needed. Other markers can be added to sub-panels for additional phenotype information. These sub-panels can also include intracellular cytokines such as IFN-gamma, IL-2, MIP1-beta and TNF-alpha, after the appropriate stimulation.

Flow Cytometric Analysis of Cell Division by Dye Dilution

This assay uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to tag proliferating cells. As stimulated cells divide, covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The assay can resolve up to eight successive generations. CFSE is long lived, permitting several batches of assays to be run, frozen and stored for several weeks.  The assay can then be run on large batches of samples.  The HIMC includes other markers in the assay that allow the specific phenotyping of dividing cells.

Firefly System for Signaling Analysis of Tissue and Cell Samples

The Firefly 3000 Protein Biomarker Analysis System (Cell Biosciences) is an automated, rapid and quantitative protein measurement apparatus that combines nanofluidic isoelectric focusing with antibody detection to measure specific protein amounts and degrees of modification, such as phosphorylation. This method provides an ability to analyze the degree of signaling protein modification in tumor biopsies of just a few hundred cells and thus could be described as the “phosphoflow” equivalent for solid tissues. Its great sensitivity makes it an excellent alternative to Western blotting, especially when there are limited amounts of tissue available. 

Centralized Assay Results Database and Analysis Support

The HIMC has developed a repository for all patient samples that is linked to all archived samples, all data generated by the assays and all assays performed on data generated in other projects with a unique barcode identifier.  This ensures all data and samples handled by the HIMC are de-identified.   The HIMC has a database manager who curates the HIMC database and develops programs to allow users to relate different datasets to each other within a particular study and between studies. The database manager also works with individual users to help them analyze their data.

Operation

The HIMC is located in the basement of the CCSR building. The lab is ~1200 square feet including a tissue culture facility with two biological safety hoods. This lab is a fully functional laboratory with state of the art equipment to provide cutting edge at the genomic, proteomic and cellular assays that measure the health of the immune system. HIMC is at: http://himc.stanford.edu

Procedures

Requests for services can be made by directly contacting the Facility Director, Holden Maecker, PhD.

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