SBIR-STTR Award

Microfluidic Processing of Leukocytes for Diagnostics Testing Summary/Abstract: New tests to quantify the numbers and functional states of key leukocyte types from blood samples offer enhanced determination and personalization of clinical diagnosis, prognosis, and treatment response. For example, labeling of >30 cell surface and intracellular target molecules can assess signaling pathway status of multiple types of normal leukocytes vs leukemia cells simultaneously, by multi- parameter flow cytometry or in the future, atomic mass spectrometry. However, current procedures to harvest blood leukocytes are expensive, time-consuming, have low cell yields, introduce cytotoxic reagents, selectively lose leukocyte subsets, and require considerable human expertise. Furthermore, multiple sequential steps are required including erythrocyte lysis, surface labeling with monoclonal antibodies (Mabs), fixation/permeablization, and intracellular labeling. Each step requires 1 or more centrifugal wash steps, resulting in cell loss and reduced research productivity. Development of an automated, reagent-free process that ensures high yield of target cells and greatly streamlines the pre-analytical workflow would be a significant benefit to research and clinical labs. Commercial goal: Market a system that combines the harvest, wash and concentration of leukocytes from blood, outperforming standard centrifugal methods. We will apply microfluidic deterministic lateral displacement (DLD), in which the paths cells take through the microfluidic system is based on size and is deterministic, i.e. absolutely determined, not subject to random processes. The key innovation of this STTR project is the use of DLD to remove leukocytes/leukemia cells from erythrocytes and also from labeling or other reagents in a continuous flow process, finally concentrating the target leukocytes/leukemia cells. This is a new use, making this application commercially novel as well as clinically beneficial. The Research Strategy leverages a collaboration among micromechanical systems experts at GPB Scientific LLC; hematopoietic cell biology and cell processing experts at University of Maryland (Civin lab); and microfluidics experts at Princeton University (Sturm-Austin lab). In Specific Aim 1, the team will create microfluidic devices to wash and concentrate leukocytes from a stream containing labeling Mabs in an automated process in a few minutes. In Specific Aim 2, the system will be applied to harvest and concentrate leukocytes labeled in whole blood, removing erythrocytes and unbound Mabs. This project focuses on leukocyte processing for flow cytometry, but the same technology could be used for many existing and new cellular and other (e.g. DNA, RNA) tests for cancer and other diseases. The value proposition is clear: by replacing erythrocyte lysis and centrifugal washing, GPB technology should increase research and clinical productivity through automated, reagent-free workflows that provide high quality cell products, enabling high value cell analyses with reproducible results.

Public Health Relevance:
Microfluidic Processing of Leukocytes for Molecular Diagnostic Testing This project is focused on achieving a breakthrough in the sample preparation of leukocytes prior to multi- parameter analysis via flow cytometry. Current methods involving centrifugation are tedious, manual processes that require expert operators and result in lost and damaged cells. Our microfluidic approach will greatly improve cell quality; streamline workflows, and lower costs. Applications include research and clinical diagnostics in cancer, infectious disease, and inflammatory disease, among other disease areas.

Public Health Relevance Statement:
Microfluidic Processing of Leukocytes for Molecular Diagnostic Testing This project is focused on achieving a breakthrough in the sample preparation of leukocytes prior to multi- parameter analysis via flow cytometry. Current methods involving centrifugation are tedious, manual processes that require expert operators and result in lost and damaged cells. Our microfluidic approach will greatly improve cell quality; streamline workflows, and lower costs. Applications include research and clinical diagnostics in cancer, infectious disease, and inflammatory disease, among other disease areas.

NIH Spending Category:
Bioengineering; Biotechnology; Cancer; Hematology

Project Terms:
abstracting; Am 80; Area; austin; base; Binding (Molecular Function); Blood; Blood Flow Cytometry; Blood specimen; cell injury; Cell physiology; Cell surface; Cells; Cellular biology; Centrifugation; Clinical; clinical Diagnosis; Collaborations; Communicable Diseases; cost; Cytolysis; cytotoxic; design; Development; Diagnostic; Diagnostic tests; Disease; DNA; Dyes; Ensure; Erythrocytes; Excision; Expert Systems; Flow Cytometry; Future; Goals; Harvest; Hematopoietic; Human; improved; Incubated; Inflammatory; innovation; Intracellular Membranes; Label; Lateral; leukemia; Leukocytes; Malignant Neoplasms; Manuals; Marketing; Maryland; Mass Spectrum Analysis; Methods; microchip; Microfluidic Microchips; Microfluidics; Molecular Diagnostic Testing; Monoclonal Antibodies; novel; Nuclear; Nucleic Acids; One-Step dentin bonding system; outcome forecast; Output; Performance; Phase; Population; Preparation; Procedures; Process; Productivity; Reagent; Recovery; Reproducibility; Research; RNA; sample fixation; Sampling; Signal Pathway; Small Business Technology Transfer Research; Solutions; Staging; Stem cells; Stream; Surface; System; Technology; Testing; Time; treatment response; Universities; Whole Blood

The goal of this Phase II project is to optimize and validate a microfluidic chip-based process that automates and shortens the labeling, washing and collecting of human blood leukocytes (WBCs) for flow cytometric analysis of clinically important cell-surface and intracellular markers. Multi-parameter flow cytometry is an increasingly powerful and widely used technology in research and in clinical diagnostic testing for cancer and many other diseases. There is a critical need for new methods to improve the efficiency of sample preparation, which is laborious and variable and typically requires many cycles of washing collecting cells by centrifugation. GPB LLC is developing a novel microfluidic Deterministic Lateral Displacement (DLD) chip processing technology that can harvest cells from a flow of fluid on the basis of size. A mixture of fluid and particles flows through an array f microposts that are tilted at a small angle from the direction of the fluid flow. Cells within the target size range are deflected off the microposts in a process that is rapid yet gentle. The novel disposable DLD chips will allow automation and potentially combination of the processes of labeling, fixing and washing cells from very small samples of whole blood. The Phase I STTR project, a collaboration among GPB, Princeton University and the University of Maryland (UM), successfully demonstrated proof of principle by showing that prototype DLD chips can harvest monoclonal antibody (Mab)-labeled WBCs from human whole blood with high cell recovery and viability while removing the large excess of red blood cells (RBCs) and unbound Mab, and without skewing the distribution of WBC subsets. The objective of this Phase II project is to optimize a suite of DLD chips and processes as prototype commercial products. Aim 1 is to design and optimize DLD chip-based systems for washing and concentrating human blood WBCs. Aim 2 is to design and validate DLD chip-based systems for surface labeling, on-chip fixation-permeabilization, or intracellular labeling of WBCs, combined with on-chip washing. Aim 3 is to develop a DLD chip-based system to fix, permeabilize, intracellularly label, wash, and concentrate WBCs on a single chip. Aim 4 is to produce prototype application-specific chips and an automated platform product to control WBC processing via a user interface. Cell labeling will be tested with panels of Mab reagents and cocktails that are commonly used for cell surface labeling and for intracellular labeling in clinical diagnostic, prognostic and research testing. Outcomes will be measured by flow cytometry in several laboratories at UM, GBP and several collaborating large diagnostic companies. Some of these companies have expressed interest in helping to commercialize chip and processing system products.

Public Health Relevance Statement:


Public Health Relevance:
Flow cytometry for single-cell analysis is a widely used analytical method for research, clinical diagnosis and treatment monitor, but the multi-step cell staining protocols are manual, tedious and fraught with variability. The novel GPB novel microfluidic technology platform will simplify, automate, and reduce the cost of the cell processing steps and decrease sample variability. Successful completion of this Phase II project will result in a suite of products that will start with whole blood and generate purified WBC samples that are ready for flow cytometric analysis. Moreover, these products also will facilitate sample preparation for other powerful new technologies such as mass cytometry, rare cell isolation, single cell genomics, proteomics, metabolomics and a wide range of new techniques under development.

Project Terms:
Address; Am 80; analytical method; Automation; base; Binding (Molecular Function); Blood; Blood Platelets; Bone Marrow Cells; Bone Marrow Neoplasms; cancer diagnosis; Cell membrane; Cell physiology; Cell Separation; Cell surface; Cells; Centrifugation; Clinical; clinical Diagnosis; Clinical Treatment; Collaborations; Collecting Cell; cost; Cytolysis; Cytometry; design; Development; Development Plans; Devices; Diagnostic; Diagnostic tests; Disease; DNA; Drug Targeting; Dyes; Employee Strikes; Erythrocytes; Flow Cytometry; fluid flow; Genomics; Goals; Harvest; Human; improved; Incubated; interest; Killings; Label; Laboratories; Lateral; leukemia; Leukocytes; Liquid substance; Malignant Neoplasms; Manuals; Maryland; Mass Spectrum Analysis; Measures; metabolomics; Methods; Microfluidics; Molecular; Molecular Diagnostic Testing; Monitor; Monoclonal Antibodies; neoplastic cell; new technology; novel; Nuclear; Nucleic Acids; Outcome; outcome forecast; particle; Pathway interactions; Phase; Population; Preparation; Process; prognostic; Proteomics; Protocols documentation; prototype; public health relevance; Reagent; Recovery; Research; RNA; sample fixation; Sampling; Series; Signal Pathway; single cell analysis; Small Business Technology Transfer Research; Staging; Staining method; Stains; Surface; System; Techniques; Technology; Testing; Time; treatment response; Universities; Whole Blood