Capsule Summary

The overall goal of the Cell Separation and Sample Preparation (CSSP) Core is to develop technologies for the collection and isolation of enriched blood leukocyte cell populations that are applicable to critically ill patient populations for subsequent high throughput proteomic and genomic analyses, as well as for functional proteomics. The Core also assists with the collection, processing and archival of solid tissue samples obtained at surgical interventions in the trauma and burn patient populations.

The Core has several responsibilities within the Program:

1. the development of new technologies for the isolation of leukocyte subpopulations from critically-ill trauma and burn patients for subsequent genomic and proteomic analyses, initially employing macroscale techniques, but rapidly moving to a microfluidics approach
2. the implementation and support of macroscale and microfluidics protocols at the clinical sites, including quality control and quality assurance
3. the long-term storage and archival of plasma, leukocyte and solid tissue samples (RNA, protein) for future analyses.

Because of these distinct functions, the Core has both development and service components similar to the Genomics Core.

The Challenge

The challenge to the CSSP Core is to successfully balance the development of protocols that yield both a high quality, enriched leukocyte product and at the same time, are feasible in a critically ill patient population under adverse conditions. The Program investigators realize that technologies exist that can generate essentially pure cell populations for proteomic and genomic analyses, but because of blood volume, time, cost, training, equipment and other feasibility considerations, could not be applied to a multi-center clinical study. Therefore, protocols must be developed that are sufficiently robust and automated to yield highly enriched cell populations from small blood collection volumes that can be performed by trained nursing or research staff, within the time and effort constraints associated with a critically ill patient population.

Our Approach

The keys to success during the first five years of the Program were:

1. to develop protocols whose successful application was dependent on performing standard laboratory procedures (universal precautions, pipetting, centrifugation, labeling)
2. provide centrally prepared reagents in the form of Sample Collection Kits that standardized all reagents, collection tubes and procedures
3. require centralized and/or on-site training with real samples, followed by close monitoring of the product for quality assurance and quality control

During this time, macroscale approaches for the collection and isolation of leukocytes and leukocyte subpopulations from whole blood for genomic analyses have been developed and implemented within the Program. As the Program matured, it became apparent that to obtain meaningful biological information from the genomics and proteomics data, ever increasingly enriched cell populations would be required. It is our belief that microfluidics represents the future direction for the isolation of these enriched populations.

Simultaneous with the implementation of these macroscale approaches to the collection and isolation of leukocytes and leukocyte subpopulations, the Program has developed a highly productive microfluidics approach to cell separation and nucleic acid recovery, whose primary goal over the next funding period is their implementation at the clinical sites.

The program has three microfluidics modules in varying stages of development or implementation:

1. an erythrocyte lysis module
2. a leukocyte subpopulation enrichment module
3. two sample preparation modules for on-chip recovery of nucleic acids or peptide fragments.

Major Functions of this Core

As the Program moves forward in Years six to ten, the primary goals of the CSSP Core are:

1. to implement at the clinical sites macroscale protocols for the collection of plasma and enriched T-cell, monocyte, and neutrophil populations in blood samples collected from severely injured patients for subsequent genomic and proteomic determinations
2. to develop and implement at the clinical sites microfluidics-based modules for the depletion of erythrocytes and enrichment of specific leukocyte subpopulations (initially, T-cells, monocytes and neutrophils, and subsequently, more enriched regulatory cell populations [to be determined]) from whole blood and where required for genomic analyses, the extraction of nucleic acids from the samples

These enriched cell populations will then be used for both proteomic (high throughput proteomic determination of cellular proteins, and functional phenotypic characterization of the cell populations) and genomic analyses.

Participants

This Core consists of two centers of excellence at the University of Florida College of Medicine and Massachusetts General Hospital/Harvard Medical School. Dr. Lyle Moldawer of UFL is the Director of the CSSP Core.

Laboratory of Inflammation Biology and Surgical Science at the Department of Surgery, University of Florida School of Medicine under the leadership of Lyle Moldawer, PhD (administrative oversight for sample collection, archival and distribution to the analytical sites, and quality assurance/control for sample collection and processing)

Center for Engineering in Medicine at the Department of Surgery, Massachusetts General Hospital and Harvard Medical School under the leadership of Mehmet Toner, PhD (development and implementation of the microfluidics approaches for cell separation and nucleic acid recovery).