Analyzing cell mechanics in hematologic diseases with microfluidic biophysical flow cytometry

Abstract

Pathological processes in hematologic diseases originate at the single-cell level, often making measurements on individual cells more clinically relevant than population averages from bulk analysis. For this reason, flow cytometry has been an effective tool for single-cell analysis of properties using light scattering and fluorescence labeling. However, conventional flow cytometry cannot measure cell mechanical properties, alterations of which contribute to the pathophysiology of hematologic diseases such as sepsis, diabetic retinopathy, and sickle cell anemia. Here we present a high-throughput microfluidics-based 'biophysical' flow cytometry technique that measures single-cell transit times of blood cell populations passing through in vitro capillary networks. To demonstrate clinical relevance, we use this technique to characterize biophysical changes in two model disease states in which mechanical properties of cells are thought to lead to microvascular obstruction: (i) sepsis, a process in which inflammatory mediators in the bloodstream activate neutrophils and (ii) leukostasis, an often fatal and poorly understood complication of acute leukemia. Using patient samples, we show that cell transit time through and occlusion of microfluidic channels is increased for both disease states compared to control samples, and we find that mechanical heterogeneity of blood cell populations is a better predictor of microvascular obstruction than average properties. Inflammatory mediators involved in sepsis were observed to significantly affect the shape and magnitude of the neutrophil transit time population distribution. Altered properties of leukemia cell subpopulations, rather than of the population as a whole, were found to correlate with symptoms of leukostasis in patients-a new result that may be useful for guiding leukemia therapy. By treating cells with drugs that affect the cytoskeleton, we also demonstrate that their transit times could be significantly reduced. Biophysical flow cytometry offers a low-cost and high-throughput diagnostic and drug discovery platform for hematologic diseases that affect microcirculatory flow.

A membrane-based microfluidic device for mechano-chemical cell manipulation

“…Rosenbluth et al, proposed a microfluidic-based flow cytometer to measure the transit time of HL60 cells stimulated with Cytochalasin D and Pentoxifylline. They observed that median transit time was reduced from 0.63 s to 0.23 s for 1 h exposure of 2 μM of CytoD solution and to 0.37 s for 3 h exposure of 1 mM PTX solution (Rosenbluth et al 2008).…”

“…Drugs used for vascular complications can change biophysical properties of blood cells, due to their effect on cell membrane, cell cytoskeleton and cell fate, such as apoptosis (Lam et al 2007;Ruef et al 2010;Rosenbluth et al 2008;Wuang et al 2011;Dai et al 2010). Therefore, it is of significant importance to study mechano-biological changes of diseased circulating cells in the development of a new treatment.…”

Recent advances in microfluidic techniques for single-cell biophysical characterization

“…With the use of high-speed imaging or electrical impedance measurements, constriction-channel devices are capable of achieving a higher throughput than most other deformability measurement approaches. Due to these merits, constriction channels have been used to measure the deformability of RBCs, 22,23,[39][40][41][42][43][44] leukocytes 45 and cancer cells. 46,47 Due to the human capillary-like environment and the physiological relevance of RBC deformability, RBCs are mostly studied in the majority of existing constriction channel-based devices.…”

“…Rosenbluth et al demonstrated the clinical relevance of their microfluidic constriction channel device in sepsis and leukostasis. 45 The reported device consists of a network of 64 constriction channels. High-speed imaging was used for measuring cell transit time.…”

High‐throughput linear optical stretcher for mechanical characterization of blood cells

“…One approach is the microfluidic cell deformability cytometer which are inexpensive to prototype, use small sample volumes (nanoliters), and employ laminar flow characteristics (24) allowing for predictable and controllable flow. For example, physical constrictions (25)(26)(27)(28) or inertial focusing flow (29)(30)(31) have been used to create contact or shear forces (> 1 nN (30)) capable of significantly deforming flowing cells. Large strains (>10% deformation) however, can damage cells and should be avoided when cell isolation and viability post-analysis are of interest.…”