"The history of science knows scores of instances where an investigator was in the possession of all the important facts for a new theory but simply failed to ask the right questions. "

Ernst Mayr

One of the main goals of this NIH-funded research program, Inflammation and the Host Response to Injury, is to make our results available to the general public and investigators who are not direct participants. Information accessible via this web site includes descriptions of standardized laboratory and clinical procedures. Analytical protocols describe experimental procedures such as isolating RNA and profiling gene expression in clinical samples via microarray technology. Clinical protocols, describing standardized procedures for collecting blood, patient data, and managing the care of injured patients, are also freely available. Protocols are posted on this web site shortly after being validated and certified by the participating investigators. A brief description of each numbered protocol is listed below.

Protocols on this page beginning with G are Genomics protocols; those beginning with MVC are Model Validation Core protocols; and those beginning with P are Protein Analyses and Cell Biology (Proteomics) protocols.

You must be a Consortium member to view the full text of the protocols. Consortium members may click here to go to the login page. Once you are logged in you may view the protocols available for your group.

G1.03 - Buffy Coat Isolation from Human Whole Blood for Total RNA

This protocol describes the preparation of total RNA from a buffy coat (white blood cell) preparation obtained from whole blood. Blood volumes are drawn from an existing arterial or venous line and collected in Vacutainer™ blood collection tubes containing sodium heparin as an anticoagulant. This procedure utilizes the RNeasy® Kit. After lysis of the buffy coat preparation with guanidine isothiocyanate to inactivate RNases, nucleic acids are removed with ethanol. Application of the sample to an RNeasy column retains RNA, which is eluted with distilled water.

[revised June 2002]

G2.04 - Buffy Coat Isolation from Human Whole Blood for Plasma and Total RNA

This protocol describes the preparation of plasma and total RNA from a buffy coat (white blood cell) preparation obtained from whole blood, for subsequent analysis of plasma protein and mRNA. Blood volumes are drawn from an existing arterial or venous line (or venipuncture, if skilled personnel are available) and collected in Vacutainer™ blood collection tubes containing EDTA as an anticoagulant. After plasma has been removed, aliquotted, bar-coded, and stored at -80°C, red blood cells (erythrocytes) are lysed and remaining leukocytes are recovered by centrifugation. After washing, the leukocyte pellet is processed for subsequent isolation and purification of total cellular RNA, using the RNeasy® Kit. Tubes should be bar-coded and stored at -80°C until ready to ship to the Sample Collection and Coordination Site.

[revised February 2003]

G2.05 - Buffy Coat Isolation from Human Whole Blood for Plasma and Total RNA (with Platelet Depletion)

This protocol describes the preparation of plasma and total RNA from a buffy coat (white blood cell) preparation obtained from whole blood, for subsequent analysis of plasma protein and mRNA. Blood volumes are drawn from an existing arterial or venous line and collected in Vacutainer™ blood collection tubes containing EDTA as an anticoagulant. Plasma should be removed, aliquotted, bar-coded, and stored at -80°C, and platelets should be centrifuged out. Red blood cells (erythrocytes) are lysed, and remaining leukocytes are recovered by centrifugation. After washing, the leukocyte pellet is processed for subsequent isolation and purification of total cellular RNA, using the RNeasy® Kit.

[revised February 2003]

G3.03 - RNA Isolation from Human Whole Blood: PAXgene™

This protocol describes the isolation of total RNA from human whole blood. Blood volumes are drawn from an existing arterial or venous line ( and collected in a room temperature (18-25°C) PAXgene™ Blood RNA Tube. Subsequent analyses of mRNA samples will be performed by polymerase chain reaction (after reverse transcription) or hybridization (microarray studies). In this procedure, leukocytes are not separated initially. Rather, proteins in whole blood are denatured after which nucleic acids are precipitated directly. After lysis, total cellular RNA is separated from DNA and proteins by precipitation and chromatographic methods, according to the PAXgene™ Blood RNA Kit. Samples should be labeled and stored at -80°C until further analysis.

[revised May 2002]

G4 Experimental Protocol: Sample Validation v2.0

Experiment G4

[February 2002]

G5 - Staphylococcus Enterotoxin B Stimulation of Whole Blood

Gram-positive sepsis is becoming an increasingly prevalent threat in hospitalized patients. Bacterially produced substances, such as lipoteichoic acid, peptidoglycans, superantigen toxins, and enterotoxins, are strong inducers of inflammation in response to a Gram positive bacterial infection. This protocol is designed to vigorously stimulate whole blood with a purified Gram positive bacterial product, in order to evaluate leukocyte response to this challenge. Blood volumes are drawn from an existing arterial or venous line (or venipuncture, if skilled personnel are available). Whole blood is treated with a 2.0 mg/ml solution of Staphylococcal enterotoxin B, mixed gently, and incubated for two hours in a water-jacketed, 5% carbon dioxide, 95% room air incubator at 37°C. Further processing of blood should be performed as described in other SOPs defined by the program. It is expected that stimulation of whole blood with Staphylococcus enterotoxin B should activate both T cell and monocyte populations, in contrast to lipopolysaccharide treatment, which primarily activates monocytes.

[revised February 2002]

G6.01 Experimental Protocol: Murine Sample Validation, v1, revised

[revised May 2002]

G7.09 - Affymetrix GeneChip™ Hybridization of Isolated Total Human Blood RNA: 2 µg Starting Material

The purpose of this protocol is to prepare biotinylated cRNA from human whole blood total RNA, for the subsequent analysis of mRNA by hybridization (microarray studies). This protocol recommends starting with 2 µg total cellular RNA. Polyadenylated RNA is reverse transcribed to single-, then double-stranded cDNA. An in vitro transcription reaction with labeled ribonucleotides produces a biotinylated cRNA strand that can be fragmented and hybridized to an Affymetrix GeneChip™ for gene expression analyses.

[revised May 2004]

G8 Double-Stranded cDNA Sythesis, in vitro Transcription, and Affy GeneChip Hybridization of Murine Tissue RNA

[in preparation]

G9.01 Reconstitution Experiment for Determination of Whole Blood Platform, modified to include an RBC-Depleted Arm

[revised May 2002]

G11 Isolation of Murine Total RNA from Murine Tissues

[in preparation]

G12.01 Evaluation of Varying Quantities of Starting Total RNA for Microarray Analysis

[revised May 2002]

G13.02 Microarray Analysis of Isolated Whole Blood Leukocyte Populations

[revised August 2002]

G14.01 Evaluation of Leukocyte Stabilization Protocols

[revised June 2002]

G15.01 - Blood Collection Protocol from Human Volunteers for RNA Isolation of Leukocyte Subpopulations

This procedure describes the process of collecting whole blood from a human volunteer, for the purposes of isolating RNA from leukocyte subpopulations. Blood volumes should be obtained by venipuncture by personnel experienced in the technique and familiar with infectious (universal) precautions, using a Vacutainer™ blood collection system. Venous whole blood is collected at room temperature (18°-25°C) into sixteen 7-ml lavender-top Vacutainer™ tubes. Following gentle mixing of blood with anticoagulant, twelve tubes will be used for monocyte isolation and T cells, and four tubes will be used for isolation of the buffy coat fraction. Both processes should begin simultaneously. Leukocyte subpopulations are obtained through standardized centrifugation and erythrocyte lysis steps, and samples are subsequently stimulated (two hours, in a humidified incubator) with either lipopolysaccharide (LPS) or Staphylococcus enterotoxin B (SEB). For each cell preparation and each treatment, RNA is isolated using the RNeasy® Kit. RNA should be labeled and stored at -80°C until further analysis.

[revised September 2002]

G16 Determination of Effects of Cell Separation and Plating on Microarray Expression

[revised April 2003]

G17.04 - Evaluation of RNA Yields from Injured Patients: Comparison Between PAXGene™ and Buffy Coat/Lysis Protocols

To accomplish the goals of the Inflammation and the Host Response to Injury research program, the collection of blood and tissue samples must be standardized to the extent possible, but also be realistic for a real world trauma center setting. Sample collection techniques must be cost-effective and able to be performed reproducibly by adequately trained research nurses or technicians. In addition, the quality and quantity of RNA obtained from blood samples must be sufficient to conduct reproducible gene expression studies. This procedure will determine the relative feasibility of the three sample preparation protocols when tested on blood samples obtained from injured patients. The Sample Collection and Coordination Site (University of Florida) will be responsible for providing the analytical sites performing the comparative studies with the required proprietary reagents and the unique identifiers and instructions for processing and shipping of samples.

[revised November 2002]

G18.01A - DNA Isolation and Storage: FlexiGene™ DNA Kit

This protocol details one of two procedures for isolating and storing DNA from human whole blood. This method utilizes the FlexiGene™ DNA Kit. To avoid potential data interpretation problems associated with microchimerism, it is essential that blood be drawn from trauma patients before allogeneic blood transfusion. The protocol suggests that discarded, EDTA-anticoagulated blood should be used for DNA analyses. This blood may be obtained directly in the emergency room, or alternatively, from a hospital clinical (hematology) laboratory that stores collected blood at 4°C for 24-48 hours. DNA must be obtained from the fresh (never frozen) buffy coat layer of whole blood that has been refrigerated up to seven days.

[revised December 2002]

G18.01B - DNA Isolation and Storage: QIAamp® DNA Blood Midi Kit

This protocol details one of two procedures for isolating and storing DNA from human whole blood. This method utilizes the QIAamp® DNA Blood Midi Kit. To avoid potential data interpretation problems associated with microchimerism, it is essential that blood be drawn from trauma patients before allogeneic blood transfusion. The protocol suggests that discarded, EDTA-anticoagulated blood should be used for DNA analyses. This blood may be obtained directly in the emergency room, or alternatively, from a hospital clinical (hematology) laboratory that stores collected blood at 4°C for 24-48 hours. DNA must be obtained from the fresh (never frozen) buffy coat layer of whole blood that has been refrigerated up to seven days.

[revised January 2003]

G19.02 - Gene Expression Profiling from Whole Blood Leukocytes and Enriched T-Cell and Monocytes

A major goal of theInflammation and the Host Response to Injuryresearch program is to ascertain the effects of traumatic and burn injury on gene expression and proteomic profiles in leukocytes. To accomplish this goal, a simple, reproducible protocol for isolating and enriching leukocyte populations is necessary, and this procedure must be adaptable to a multicenter study setting. Since previous analyses suggest distinct and significant variation in whole blood leukocyte expression patterns depending on the method used for RNA isolation, there is a critical need to identify the contribution of individual leukocyte populations toward observed gene expression patterns from buffy coat or whole blood leukocyte samples. This protocol compares gene expression profiles from T-cell and monocyte enriched populations (obtained using protocol P003) with gene expression profiles from essentially pure (>95%) monocyte and T-cell populations. If the expression profiles obtained via the two methods prove to be equivalent, then leukocyte enrichment achieved with the P003 protocol will be sufficient to reflect the behavior of each individual cell population.

[revised April 2003]

G20 - Synthesis of Alpha RNA: Affymetrix® Clean-Up

Trauma and sepsis alter gene expression profiles in leukocyte populations from whole blood. This protocol describes the preparation of RNA from human whole blood for subsequent microarray analyses. Recent improvements in molecular biological methods permit the use of increasingly smaller quantities of starting total RNA to yield sufficient amounts of cRNA for microarray studies. This procedure involves selecting mRNA from total RNA (200 ng) through a double amplification step. The mRNA is reverse transcribed into single-, then double-stranded cDNA, followed by a clean-up step using the Affymetrix® GeneChip Sample Cleanup Module. After clean-up, the cDNA is in vitro transcribed to yield non-biotin-labeled cRNA (alpha RNA). This alpha RNA is then reverse transcribed to yield cDNA, which is then in vitro transcribed using biotinylated ribonucleotides to yield labeled cRNA for use in hybridization studies. The yield of alpha RNA produced will be quantitated, and sample quality will be assessed by agarose gel electrophoresis.

[revised April 2003]

G21 - Synthesis of Alpha RNA: MicroSpin® Clean-Up

Trauma and sepsis alter gene expression profiles in leukocyte populations from whole blood. This protocol describes the preparation of RNA from human whole blood for subsequent microarray analyses. Recent improvements in molecular biological methods permit the use of increasingly smaller quantities of starting total RNA to yield sufficient amounts of cRNA for microarray studies. This procedure involves selecting mRNA from total RNA (200 ng) through a double amplification step. The mRNA is reverse transcribed into single-, then double-stranded cDNA, followed by a clean-up step using a BioGel P-6 MicroSpin® Column. After clean-up, the cDNA is in vitro transcribed to yield non-biotin-labeled cRNA (alpha RNA). This alpha RNA is then reverse transcribed to yield cDNA, which is then in vitro transcribed using biotinylated ribonucleotides to yield labeled cRNA for use in hybridization studies. The yield of alpha RNA produced will be quantitated, and sample quality will be assessed by agarose gel electrophoresis.

[revised April 2003]

G22 - Comparison of Alpha RNA Purification Methods

Trauma and sepsis alter gene expression profiles in leukocyte populations from whole blood. Two protocols (G020 and G021) describe the preparation of RNA from human whole blood for subsequent microarray analyses. Recent improvements in molecular biological methods permit the use of increasingly smaller quantities of starting total RNA to yield sufficient amounts of cRNA for microarray studies. The two procedures involve selecting mRNA from total RNA (200 ng) through a double amplification step. The mRNA is reverse transcribed into single-, then double-stranded cDNA, followed by a clean-up step using either the Affymetrix® GeneChip Sample Cleanup Module or a BioGel P-6 MicroSpin® Column. After clean-up, the cDNA is in vitro transcribed to yield non-biotin-labeled cRNA (alpha RNA). This alpha RNA is then reverse transcribed to yield cDNA, which is then in vitro transcribed using biotinylated ribonucleotides to yield labeled cRNA for use in hybridization studies. The yield of alpha RNA produced by either method will be quantitated and compared, and sample quality will be assessed by agarose gel electrophoresis.

[revised February 2002]

G23 Murine Blood RNA

[revised February 2003]

G24 Murine Splenocyte RNA

[revised February 2003]

G25.09 - Modified Buffy Coat Isolation and Lysis

This protocol describes the isolation of plasma and total cellular RNA from leukocytes in human whole blood. Subsequent sample processing will yield plasma protein and mRNA fractions, respectively, for further analyses. To yield the buffy coat (white blood cell component), whole blood is first centrifuged to recover plasma. Bicarbonate lysis of erythrocytes (red blood cells) in the plasma-removed fraction followed by centrifugation yields the remaining leukocytes. After washing, leukocytes are stabilized through shearing in an RNA stabilization buffer solution. Resulting leukocytes are frozen (-80°C) in preparation for transport to the Genomics Core for further analyses.

[revised January 2004]

G26 - Total RNA Isolation from Human Skin, Muscle and Solid Organs

This protocol describes the isolation of total RNA from human fibrous tissue (skin, muscle, and solid organs). Subsequent analyses of mRNA samples will be performed by polymerase chain reaction (after reverse transcription) or hybridization (microarray studies). This procedure utilizes the RNeasy® Fibrous Tissue Midi Kit. After mechanical homogenization and proteinase K digestion steps, nucleic acids are removed with ethanol. Application of the sample to an RNeasy® column retains RNA, which is eluted with distilled water.

[revised March 2003]

G27 - Total RNA Isolation from Human Adipose Tissue

This protocol describes the isolation of total RNA from human adipose tissue. Subsequent analyses of mRNA samples will be performed by polymerase chain reaction (after reverse transcription) or hybridization (microarray studies). This procedure utilizes the RNeasy® Lipid Tissue Mini Kit. After mechanical homogenization and lipid extraction with chloroform, nucleic acids are removed with ethanol. Application of the sample to an RNeasy column retains RNA, which is eluted with distilled water.

[revised March 2003]

G29 Gene Expression Profiling from Whole Blood Leukocytes

Experiment G29

[revised August 2003]

G30 Comparison Study of G25 and G2

Experiment G30

[revised September 2003]

G31.2 Input RNA Comparisons

[revised August 2003]

G35 Gene Profiles from Whole Blood Leukocytes and Purified Neutrophil Populations

[revised March 2004]

G37 Tissue Collection and Preservation in RNAlater v2

[revised January 2004]

MVC0 - General Consensus on Operating Procedures

MVC1 - Murine LPS Kinetics Study

MVC2 - Blood and Spleen Tissue Harvest Procedure

MVC3 - Modified Blood and Spleen Harvest Procedure

MVC4 - Comparison of RNA Isolation Technique from Murine Whole Blood

MVC5 - Murine 2 Microgram Endotoxin Kinetic Studies

Experiment MVC5

MVC6 - Optimization of Blood Lysis Procedure

Beta testing performed at all three sites. Blood lysis procedure performed using ACK to decrease WBC loss and decrease platelet contamination in murine blood samples. Samples sent to GC for RNA analysis (q/q).

MVC7 - RNA Yield and Quality of One versus Two Animals

Since RNA yield was not doubled between blood from 2 vs. 4 mice test blood from 1 vs. 2 mice for RNA isolation.

MVC8 - Resuscitation Fluid Comparison Following Trauma-Hemorrhage

Compare Ringer’s lactate resuscitation with blood plus Ringer’s lactate in order to mimic clinical conditions of resuscitation (being revised).

MVC9 - Resuscitation Fluid Comparison Following Trauma-Hemorrhage

Compare female vs. male responses to Ringer’s lactate vs. blood plus Ringer’s lactate resuscitation.

MVC10 - Thermal Injury Model

Remove burn wound margin, normal skin and fat and process them using RNALater, snap freeze and 70% ethanol.

MVC11 - Blood Hemavet Stability

Draw blood and perform analysis at different times to determine if delaying the measurement decreases the yield of cells.

MVC12 - Time Course Studies of Trauma-Hemorrhage and Resuscitation Studies

MVC13 - Comparison of Cytokine Release and Hematological Responses to Intravenous vs. Intraperitoneal Endotoxin Infusion

MVC14 - Comparison of the Physiological Response (Blood Pressure, Heart Rate and Body Temperature) to Intravenous vs. Intraperitoneal Endotoxin Infusion

MVC15 - Gene Expression Profile Comparison Among Different Injury Models (being revised)

MVC16 - ACK and EL Buffer Mediated Mouse RBC Lysis Procedures

The primary objective of MVC16 is to directly compare the efficiency and quality of our MVC6 ACK RBC lysis protocol with the commercially available RBC lysis reagent, EL Buffer.The scheme for MVC16 will be for the three individual MVC sites to test ACK lysis and EL lysis using the outlined protocol. We will depend on the Hemavet instrument data to provide us with an accurate measure for the initial and final blood cell differentials and yields.

Experiment MVC16

MVC17 - Comparison of ACK and EL for Lysis: Compare Blood Lysis Using ACK vs. EL at 4ºC

We will also compare the hemavet data with FACS to determine cell loss parameters

MVC18 -Optimization of Splenocyte RBC Lysis Procedure: Compare Blood Lysis Using ACK vs. EL at 4ºC.

We will also compare the hemavet data with smear to determine cell loss parameters.

MVC19 - Optimization of RBC Lysis Procedure in Different Injury Models

Study to be performed at 2 h after injury to ensure the lysis procedure also works optimally in samples following thermal injury, endotoxemia and trauma-hemorrhage.

Experiment MVC19

MVC22 -Evaluation of Change in Gene and Protein Expression in Three Injury Models at 1, 3, and 7 Days after Injury

[revised August 2004]

P1 - Patient Confidentiality and Generation of Unique Identifiers

[revised May 2002]

P2 - Sample Receipt and Acknowledgement

[revised May 2002]

P3.09/G25.09 - Flowchart

[revised April 2004]

P3.11 - Blood Collection Protocol from Human Traumatized and Burned Patients. I. 25 ml Blood Collection

This protocol outlines specific procedures for the collection of a specified blood volume (25 ml) from trauma and burn patients at fixed time points after injury: 12 hours, 1 day, and subsequent 3-day intervals as long as the patient remains hospitalized and enrolled. This "25 ml blood collection" protocol allows for sufficient amount of sample to yield the following fractions for further analyses: total RNA (gene expression), plasma (cytokine measurement and proteomic studies), lymphocyte and mononuclear pellets (phenotypic characterization), and whole blood (endotoxin stimulation). Blood volumes are drawn from an arterial or venous line and collected in blood collection tubes. Venous whole blood is collected at room temperature into two 7-ml lavender-top Vacutainer™ tubes and one 3-ml green-top Vacutainer™ tube. It is critical that two isolation protocols proceed simultaneously: the lavender-top tube protocol for isolation of leukocyte subpopulations and the green-top tube protocol for endotoxin stimulation. Investigators should alternate centrifugation steps with sample processing to permit the two protocols to take place concurrently.

[revised May 2005]

P6 - Blood Separation Schema for 15 ml Purple Top Collection

[revised January 2002]

P6.02 - Blood Separation Schema for 2.5 ml Pediatric GreenTop Collection

[revised March 2002]

P7 - AST Analysis (Murine)

[revised March 2002]

P9 - Murine IL-6 ELISA (U. of Florida Protocol)

[revised November 2001]

P10 - Human ELISAs (U. of Michigan Protocol)

[revised March 2002]

P11 - Small Volume Blood Collection for Proteomic Analyses

This protocol describes the collection of small blood volumes from trauma and burn patients from whom large (over 25 ml) volumes have not already been collected. Small blood volumes are drawn from an existing arterial or venous line and collected in a lavender-top EDTA Vacutainer™. Blood samples must be processed immediately. If samples cannot be centrifuged within 10 minutes, they should either be refrigerated or placed on wet ice, then processed within 4 hours. The plasma supernatant is aliquotted into bar-code-labeled microcentrifuge tubes, then stored at -80°C.

[revised March 2002]

P12 - Detection of Murine Cytokine

Changes in plasma cytokine concentration are one characteristic of the body's response to trauma and burn injury. While cytokine detection in the circulating blood of injured patients is not always possible, the presence of elevated cytokines can occasionally correlate with clinical outcome. This laboratory procedure describes an analytical procedure to measure cytokine levels in murine plasma samples. The quantitative sandwich enzyme immunoassay method employs cytokine-specific monoclonal antibody-coated 96-well microtiter plates, to which recombinant standards or plasma samples are added. The biotin/streptavidin-based ELISA assay quantitates cytokine levels spectrophotometrically.

[revised March 2002]

P13 - Processing of Paraformaldehyde-Fixed Leukocytes

This protocol describes a flow cytometric method for measuring intracellular signal transduction intermediates in relatively small volumes of paraformaldehyde-fixed leukocytes. The method utilizes a dual antibody approach to detect the presence of phosphorylated signaling proteins in a mixed population of relatively intact monocytes, lymphocytes, and neutrophils. Myeloperoxidase/lactoferrin and forward and side scatter characteristics are used to distinguish leukocyte populations.

[revised April 2002]

P14 - Leukocyte Phenotyping Human Whole Blood

This flow cytometric protocol describes the evaluation of surface markers and intracellular cytokines in previously separated and frozen peripheral blood monocytes and T cells. Cells are stimulated ex vivo, then subjected to flow cytometry using 3 or 4 distinguishing fluorophores. Due to high autofluorescence in the FITC channel of monocytes, flow cytometry in these cells will be limited to the use of 3 fluorophores. For the T cell population, surface markers have been chosen to permit discrimination between Th1 and Th2 cells, as well as to allow characterization of effector vs. memory phenotype and the possible expression of inhibitory signaling ligands. For monocytes, CD33 expression will be monitored along with alterations in the expression of TLR4 and TLR2, which are crucial elements in the body's response to infection and may be molecular markers of trauma injury.

[revised April 2004]

P15 - Freeze-Thaw and Cytokine Stability in Plasma

Cytokine levels are often increased after trauma and burn injury, and relative concentrations often reflect the severity of injury. Cytokines can be induced ex vivo by stimulation of whole blood with endotoxin and subsequently measured by collecting and analyzing plasma. Because cytokine measurements will be taken throughout the course of the five-year research program, this protocol will assess the stability of cytokine levels in plasma and tissues samples that have been subjected to repeated freezing at -80°C. This protocol will determine the number of freeze-thaw cycles that can be conducted before cytokine levels in plasma and tissue samples are adversely affected.

[revised April 2002]

P17 - Beta Testing of P3 Protocol

[revised May 2002]

P18 - Beta Testing of P3 Protocol

[revised May 2002]

P19 - Beta Testing of P3 in Critically Ill Patients

[revised November 2002]

P20 - Reevaluation of the LPS Dose Used for Leukocyte Activation

[revised February 2003]

P21 - Large Volumes of Stimulated Pooled Plasma for High-Throughput Proteomics

Recent technological advances in proteomics have raised the possibility of performing multiple assays from a small volume of plasma. To test whether such novel techniques are both durable and reproducible over long periods of time (several years), this protocol describes the generation of a large volume of blood stimulated ex vivo and aliquotted and stored in several smaller batches. Since the volume of blood required for these analyses exceeds the amount that can be obtained from volunteer donation, the protocol specifies the purchase of a single donor whole blood volume from a local commercial blood bank. Following ex vivo stimulation, investigators at the Sample Collection and Coordination Site (University of Florida) will separate plasma from cellular elements to generate the pooled standard for later high-throughput proteomic analyses.

[revised February 2003]