Stanford School of Medicine
Institute for Immunity Transplantation and Infection–

The Human Immune Monitoring Center - Protocols

CCSR
The Center for Clinical Sciences Research

Operational Plan Phase 1

The HIMC is pleased to offer an integrated package of complementary immune assays.  This consists of: 
1) Cytokine analysis for 28 different biomarkers  in serum or plasma using a Luminex system, 2) analysis of leukocyte subsets by florescence activated cell sorting  using a customized Becton Dickenson LSRII analyzer  and 3) Comprehensive gene expression analysis of peripheral blood mononuclear cells (PBMC) with the Agilent microarray platform.

Samples can be collected as whole blood or processed according to the protocols shown here and transfered to the HIMC on dry ice.  Additional material can be cataloged and archived at -80C or in liquid nitrogen if requested.

One of the objectives of the HIMC is to create a HIPPA compliant database of immune system information across a wide spectrum of clinical indications. One year after the results of these assays will be released to the investigator (or earlier if requested), the data will be released for all registered users of the facility to access.  We also plan to analyze large numbers of healthy individuals using these assays and to make this data available to HIMC users.

Part of Phase II will feature fully integrated data analysis that will allow integrated analysis across all  platforms as well as offering additional analysis options.

1.  Multiplex cytokine assay (Luminex 200 IS platform)

Secreted cytokines are characteristic of many types of immune response and inflammatory conditions. To measure large numbers of cytokines simultaneously in clinical isolates, we are using the Luminex 200 IS System, which is a very flexible analysis platform based on flow cytometry. The system enables you to multiplex (simultaneously measure) up to 100 cytokines in a single microplate well, using very small (~40 microliter) sample volumes. The system can deliver fast and cost-effective bioassay results in many assay formats including nucleic acid assays, receptor-ligand assays, immunoassays and enzymatic assays.

Current Status: The platform is installed and we currently have a 28-plex human panel that is running in triplicate.  We can measure: IL-1β, IL-2,, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17, Eotaxin, IFN-γ, GM-CSF, MIP-1β, MCP-1, G-CSF, FGF basic, VEGF, TNF-a, RANTES, Leptin, TNFβ, TGFβ, and NGF in the pg/ml range from human serum, plasma or from cellular supernatants. We are currently testing panels that measure five additional cytokines (for 30 total) and expect to have that validated shortly. We can also add cytokine or other soluble factor assays to this system on a custom basis, i.e. CRP.

2. Characterization of major white blood cell populations by flow cytometry (BD LSR II )

Human blood contains a variety of different types of white blood cells that mediate different immunological and inflammatory responses. Flow cytometry using labeled antibodies to different cell surface markers is the standard way to analyse  the frequency of different cell types in a blood sample and the LSR II is an excellent instrument with which to do this. Ours has been configured with four fixed alignment lasers and can detect up to 18 colors. In this set of assays, we will determine the relative frequency of the different types of white blood cells in a given individual’s blood sample.  As some of these cell types can vary widely (>10X) between different individuals this analysis can be used to both flag unusual cellular anomalies and to normalize the gene expression and cytokine data.  The following table lists the cells we will analyze routinely.  Other markers can be added on a custom basis, as can intracellular cytokine stains as well as specific phosphoproteins.   

Cell population

Marker

Pan hematopoietic cell marker

CD45

T cells (all types)

CD3 CD4 CD8

T cell naïve vs memory

CD45RA CCR7

B cells

CD19 or CD20

NK cells / NK T cells

CD16 CD56

Monocytes

CD14

DC’s

Class II MHC, CD11c, CD123

T cell activation

CD38 intensity

DC activation

CD83 CD86 intensity

Gamma Delta T cells

Pan Gamma Delta

Neutrophils

Coulter counter assay

 

3.   Gene expression microarray assay (Agilent Platform). It has become routine to conduct gene expression studies on a genome-wide scale to broadly survey gene activity. The HIMC is offering gene expression profiling as part of the standard package of analyses done on blood samples.  The Agilent 44K probe human array covers all expressed genes across the human genome.   The probes are synthesized using piezo printers as 60 mers with one probe for each potential RNA transcript.    The Agilent DNA microarray scanner is a 48-slide scanning system that can read any mix of 1 x 3” glass slide microarrays (both Agilent and non-Agilent) .  RNA samples are first  checked by bioanalyzer and nanodrop to determine their quality prior to microarray analysis. 

  These analysis platforms are robust and will allow us to add more capabilities as we move forward to accommodate user specific needs.  Ideally we would like to be involved in the planning stages of a study so that we can contribute to the experimental design.  Alternatively, potential users may have samples from on going studies and we would be happy to analyze these. 

Sample collection
It is critical that samples and accompanying information be collected and handled properly.  The HIMC would be happy to assist in training personal in collecting and handling samples so as to minimize the change of introducing variability that is not biologically relevant.  We are also building a web based interface that will allow data input for the samples that will be critical for tracking and analysis.  All blood samples collected for the HIMC will be processed according to the following SOPs below. 

Existing protocols in place are for blood plasma and serum, nucleic acid purification and whole cells (PBMC’s):

Sample handling
Clinical personnel will be instructed on how to handle sample tubes after collection.  To bring in sample, contact our staff 2 days in advance so we can prepare to receive and process the samples within 1-3 hours of collection. 

Data entry
Data entry begins with the assigning of a barcode at blood draw.  The barcode will be the only way that the sample is linked to assay results and additional information.  The data will be entered to the database using a web based interface which is designed to collect de-identified patient data that is relevant to the data set.  The barcode is scanned into the system when the blood arrives and each tube or 96 well plate location is assigned that barcode as the samples are process.  Authorized personal will be able to log into the system and track sample progress and also view assay results in a simplified html format.  This software will also be able to show statistics of the number of samples collected and processed as well as the amounts and location in the archives.  

Sample Results
More detailed assay results will be stored in the databases that are connected to the various data analysis programs.  All these programs are able to output a report in several common formats (excel, pict, word etc) and the HIMC will set up a data base that will be able to access and compare data.  We anticipate that we will have a completely integrated system up and running soon. 

Dyes that can be used with the LSRII current filter set and lasers

FITC/GFP/A488

FITC/GFP/A488

PE-TEXAS RED

PE

PE-CY5

PERCP- CY5.5

PE CY7

PACIFIC BLUE

AMCYAN

QDOT 545

QDOT 565

QDOT 585

QDOT 605

QDOT 655

QDOT 705

APC

Alexa 700

APC-CY7

 

For more information contact:
David L Hirschberg, PhD
Director, Center for Immune Monitoring
email:
phone: +1.650.723.1671
Stanford School of Medicine
CCSR Building 0125A
269 Campus Drive
Stanford, CA 94305-5166
fax: +1.650.498.6345

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