Major steps for analyzing the cellular components of blood [click for a larger view]

We are building an integrated platform to automatically and systematically handle blood while avoiding artifacts, and extract scientific or clinically relevant information from target populations of cells in blood. Individual modules are being developed for depleting red blood cells, sorting leukocytes into homogenous phenotypes and interrogating cells based on phenotypic or genotypic characteristics. Integrated microsystems for rapid and comprehensive blood analysis are poised to become single use, disposable, point-of-care diagnostic tools for clinical applications or science research tools for blood analysis from small laboratory animals.

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Microfluidic lysis device: device design and construction to perform rapid lysis of whole blood to obtain pure leukocyte populations

We developed a microfluidic device for the isolation of leukocytes from whole blood by the rapid lysis of erythrocytes and can achieve complete lysis of erythrocytes within seconds, as compared with twenty-plus minutes for the regular protocols, due to significantly reduced diffusion times in microfluidic systems. After complete erythrocyte lysis, the leukocytes can be returned to physiological isotonic conditions for further separation and/or molecular analysis.

Array of single leukocytes in 15 by 15 μm microwells

Following erythrocyte lysis, leukocytes can be separated into subpopulations based on surface markers identified as relevant for clinical applications. Polyethylene glycol (PEG) microwells can be employed to organize leukocytes into high-density arrays and make these cells amenable to rapid optical characterization and subsequent sorting. Multiparametric cell analysis through direct visualization of cell morphology, cross correlation with immunostaining, and the ability for collecting dynamic information over long periods of time from the same sample are unique characteristics for cytometry in an array format. Nonetheless, individual cells with prescribed characteristics can be precisely selected and retrieved for genomic and proteomic analysis using laser capture microdissection tools.












Microfabrication Array

Schematic of prototypic microfabrication-based dynamic arrayed cytometer

Given recent advances in molecular analysis techniques, it has become very important to obtain homogeneous subpopulations of cells for basic biological research and clinical diagnosis and prognosis. A particulate handling system is proposed that has the potential to combine the powers of microscopy and flow cytometry, creating dynamic data sets of living cells and, if desired, subsequently fractionating the population into subpopulations. We have developed a sorting technology based on pneumatic actuation via heterogeneously nucleated thermal vapor microbubbles. Harnessing the power of mono-nucleated thermal vapor microbubbles, we are developing an arrayed particulate analysis device for use in particle-sensitive applications.

Scanning electron micrograph of the microfluidic device employed for single cell capture, lysis and biochemical analysis into 50pL microvials

Considering that the blood processing and cell selection and isolation steps in the microsystem are likely to result in very small samples, we are developing new techniques to handle and perform comprehensive analysis in small homogenous populations down to individual cells.

Fast and uniform lysis, simultaneous with complete inhibition of the intracellular RNA degrading enzymes, for each of the individual cells in a small sample is implemented in microfludic devices. This will allow extracting high-quality RNA with high efficiency from as little as 100 cells and, after amplification in the same device, running gene chip analysis from the selected homogenous cell population.

Surface Modification

This project examines the separation of T and B lymphocytes from mixtures using microfluidic chambers coated with antibodies, focusing on flow conditions and surface chemistry. The adhesion of both cell types decreases as shear stress increases, irrespective of the surface chemistry.

The incorporation of polyethylene glycol (PEG) chains along with the antibodies on the chamber surface is shown to improve significantly the reproducibility of cell adhesion and is thus an important part of the overall system design. Furthermore, this technique is shown to be an effective way of isolating highly pure subpopulations of lymphocytes from model mixtures, even when the target cell concentration is low.