Clinical Implications of Single-Cell Microfluidic Devices for

Sep 23, 2017 - Wilbur A. Lam is an Associate Professor of Biomedical Engineering and Pediatrics at the Georgia Institute of Technology and Emory Unive...
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Clinical Implications of Single-Cell Microfluidic Devices for Hematological Disorders Caroline E. Hansen, and Wilbur A. Lam Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01013 • Publication Date (Web): 23 Sep 2017 Downloaded from http://pubs.acs.org on September 24, 2017

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Clinical Implications of Single-Cell Microfluidic Devices for Hematological Disorders Caroline E. Hansen and Wilbur A. Lam* Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Children’s Healthcare of Atlanta / Emory University School of Medicine, Atlanta, GA 30322 Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332 Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332 School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 Keywords: Single-cell, microfluidics, diagnostics, hematology, blood ABSTRACT: Single-cell microfluidic devices are poised to substantially impact the hematology field by providing a highthroughput and rapid device to analyze disease-mediated biophysical cellular changes in the clinical setting in order to monitor disease prognosis or the development of complications. In this feature article, we cover recent advances of single-cell microfluidic devices for studying and diagnosing hematological dysfunctions and the clinical impact made possible by these advances.

Microfluidic devices have proven to be valuable platforms with which to synthesize micro- and nanomaterials,1-3 develop immunological and environmental assays,4-7 fabricate in vitro models to study biological systems,8-11 as well as develop clinical diagnostics.12-14 The plethora of microfluidic fabrication techniques provides significant customization in the design of the device specific to the application.15-17 The ability to easily fabricate complex devices coupled with the ability to impart micron-sized features in the device has enabled development of single-cell microfluidics. Moreover, multiple functionalities can be imparted onto a single chip, allowing for sample purification, analysis, and readout to be performed with minimal sample processing or human intervention. Such a setup diminishes unintentional alterations of cell properties during sample processing steps as well as the introduction of human error, both of which increase fidelity of the assay or diagnostic. In particular, single-cell microfluidics have enhanced the analytical capability in regards to blood cells and, consequently, hematological disorders. Blood is a complex environment, which consists of ~60% plasma (proteins, sugars, and fats), 4-6 million/mL (~40%) red blood cells (erythrocytes), white blood cells (leukocytes), and platelets.18,19 Erythrocytes are anucleate cells and primarily consist of hemoglobin, which is responsible for oxygen transport throughout the body via coordination with the ironcontaining heme groups.18 Leukocytes are heavily involved in the body’s immune response and are categorized into monocytes, granulocytes (neutrophils, eosinophils, and basophils), and lymphocytes (T cells, B cells, and natural killer cells).19 Monocytes mediate inflammation and can leave the vascular space to phagocytose foreign bodies in tissues.19 Granulocytes attack foreign bodies through a variety of

mechanisms including phagocytosis, neutrophil extracellular traps, and release of cytotoxic or pro-inflammatory compounds.19 Lymphocytes mediate adaptive as well as innate immunity whereby T cells recruit additional leukocytes through cell secretions and kill cells presenting antigens associated with major histocompatibility complex molecules, B cells produce antibodies that bind to foreign bodies, and natural killer cells release cytotoxic granules in response to tumor or virally infected cells.19 Finally, platelets are anucleate cell fragments that function as the “first responders” to vascular injury to initiate blood clot formation and have significant roles in innate immunity and cancer metastasis as well.19,20 Numerous hematological disorders can result in biological or biophysical changes in small cellular subpopulations within the larger population of the specific blood cell type, and these minority cellular subpopulations have been shown to mediate disease pathophysiology, even before the patient demonstrates clinical symptoms or diagnosable signs of morbidity.21-23 While flow cytometry has been proven to be an extremely useful analytical tool now widely used in the hospital setting to quantitatively characterize and identify disease-mediated biological changes in cell subpopulations, both large and small, it can be cost prohibitive in low resource settings as both the instrument and antibodies are expensive.23-25 Furthermore, flow cytometry cannot evaluate biophysical cellular changes, which we are learning are a significant contributing factor in disease pathophysiology in various hematologic disorders.26-28 Several single-cell methods exist that can determine changes in the mechanical properties of patient samples (i.e. atomic force microscopy or micropipette aspiration)29,30 but suffer from limited sample sizes due to slow operation or generation of small cell deformations (i.e.

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Table 1. Summary of single-cell microfluidic devices, the blood cells investigated, and their clinical implications.

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optical stretchers).31 Bulk analysis methods (i.e. rheology) can be conducted in shorted time frames but can mask the small cell populations in which the disease-mediated changes have occurred. These single-cell and bulk analysis methods require expensive and specialized instrumentation setups, which can limit experimental design for expanded applications. As such, single-cell microfluidic devices are uniquely poised to function as an analytical device to determine diseased-mediate biochemical or biophysical changes of cells for fundamental analysis, disease diagnosis, or design of patient-specific treatment (Table 1). The ability to easily tailor the device and system setup for a specific experimental design makes them apt platforms for a vast array of single-cell investigations. These devices can elucidate sample heterogeneity due to diseased states in a high-throughput manner for both biochemical or biophysical cell properties. Furthermore, these microfluidic devices can be made portable,

expanding their utility to point-of-care (PoC) in low resource settings for global health applications. This feature article discusses recent advances in single-cell microfluidic devices to study or diagnose hematologic disorders.

LEUKOCYTES Leukocytes marginate to the periphery of vascular spaces during circulation, in part, due to increased cell stiffness compared to erythrocytes (~400 Pa compared to ~150 Pa).32 Margination aids in their functional behavior of regulating inflammation by enabling interactions with endothelial cells. These interactions first begin with leukocyte rolling along and adhering to the endothelium, mediated by endothelial cell adhesive proteins P- and E-selectins and leukocyte L-selectin, P-selectin glycoprotein ligand-1 (PSGL-1), CD44, CD43, and E-selectin ligand-1.33 Leukocyte arrest then occurs in the presence of chemoattractants/chemokines by activating α4β1, lymphocyte function-associated antigen 1 (LFA-1), and

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Analytical Chemistry opportunity for early management, as well as the need for therapeutic intervention. Development of sepsis in patients who sustain severe burns is another pathology that is difficult to identify early-on due to inflammation, specifically, the development of systemic inflammatory response syndrome (SIRS), which is a common development in burn patients but, problematically, is also used as an indication of sepsis. Hence, for this patient population, SIRS can “mask” clinically used signs of sepsis development. Once sepsis is suspected, a 12-24 hour blood culture is required before sample analysis and initiation of aggressive antibiotic treatment, which significantly prolongs treatment and decreases probability of survival.45,46 Furthermore, the majority of patient deaths due to sepsis are caused by antibiotic resistant strains, making treatment once diagnosed difficult and contributing to its status as the leading cause of death for burn victims.47 Like CVD for diabetes mellitus patients, a clinical tool to monitor patients at risk for developing sepsis (i.e. burn patients with SIRS) for early detection would be extremely useful in preventing patient mortality. Indeed, the current method for diagnosing sepsis relies solely on subjective clinical signs and symptoms, and a significant need exists for more objective diagnostic markers. Upon tissue injury or infection, neutrophils undergo chemotaxis to locate the site, whereupon phagocytosis, release of cytokines or lytic enzymes occurs.48-50 As chemotaxis is the first neutrophil behavior in response to injury or infection, analyzing changes in this behavior may be a powerful biomarker for identifying sepsis in burn patients. Jones et al. met this need by developing a microfluidic device that monitors neutrophil chemotaxis through straight channels with and without biophysical challenges (i.e. posts or bifurcations).51 A total of 74 patient samples were studied, which corresponded to burn patients in various conditions during their hospital stay, to investigate 18 parameters of neutrophil chemotaxis. The investigators were able to identify a spontaneous neutrophil migration phenotype in the absence of a chemoattractant that is specific to development of sepsis. Impressively, this phenotypic behavior could be identified in patients two days before the onset and traditional diagnosis of sepsis and disappeared concurrent with successful treatment. The findings made possible by this microfluidic device motivate the need for further research into the early role of neutrophils in sepsis development, which can have implications for discovering better treatment modalities – earlier detection and alternative therapeutics to overcome antibiotic resistant strains. Pathologic changes to neutrophil chemotaxis was also utilized to develop a single-cell microfluidic device to diagnose asthma. Traditional diagnostic approaches rely on patients presenting signs of asthma during the diagnostic test and require strict patient compliance, which can lead to substantial misdiagnosis in certain patient populations.52,53 Instead of relying on the symptoms of asthma, Sackmann et al. developed a microfluidic device that analyzes one of the key effector cells in asthma pathology, specifically neutrophil chemotaxis.54,55 A patient sample of whole blood, collected via a finger prick, is loaded in a microfluidic device (Figure 1) without the need of a syringe pump. Neutrophils are captured by the Pselectin-coated surface of the device. After washing the device, a gel with embedded chemoattractant, is adhered to the

macrophage-1 antigen (Mac-1), which bind to endothelial cell ligands vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1).33 Leukocytes can then transmigrate through the paracellular space, which is heavily mediated by platelet endotheleial cell adhesion molecule (PECAM), enabling leukocytes to reach the interstitial space to mediate inflammation or infection.34 Patients with diabetes mellitus have been at a historically high risk for developing cardiovascular disease (CVD), a condition we now understand to be heavily mediated by inflammatory pathways and functions as the leading cause of death for these patients.35-38 Screenings for traditional risk factors (i.e. hypertension and dyslipidemia) are not always predictive for this patient population because diabetes functions as an independent risk factor for CVD.39 Significant work has been conducted towards understanding the etiology of CVD in diabetes patients with the hope of identifying effective predictive factors and developing robust screening methodologies, enabling early management and risk mitigation.40 Due to cross-talk between insulin-signaling and inflammatory pathways, patients with diabetes are in a state of chronic, low grade inflammation.41-43 Due to the relationship between inflammation and CVD, investigations into inflammatory markers represents a promising avenue towards uncovering a clinically relevant early detection marker of CVD in diabetes patients. Recently, a microfluidic device has been reported that can be used to screen for the risk of CVD in type 2 diabetes mellitus (T2DM) patients based upon neutrophil biomechanics caused by chronic, low-grade inflammation. Impressively, it only requires a finger prick of patient blood, minimal sample processing, and can be conducted in 20 minutes. Here, Hou et al. used dean flow fractionation to rapidly separate neutrophils from RBC-lysed whole blood samples with 90% yield.44 Isolated neutrophils were then perfused into a straight microchannel coated with E-selectin to examine rolling of single neutrophils in order to determine functional phenotype and activation. This device setup thereby mimics neutrophil rolling along an endothelium activated by inflammation. As neutrophils exhibit substantial heterogeneity across cell type, single-cell microfluidic devices offer the opportunity for greater sensitivity in determining low-grade activation. Brightfield microscopy was used to monitor rolling speed and trajectory. The investigators found that rolling speed was significantly higher for T2DM patients compared to healthy patients. The trajectory of rolling was also more discontinuous in T2DM patients than that of healthy patients. Interestingly, when comparing rolling speed within T2DM patients, the investigators found correlations in speed with presence of inflammatory indicators (reactive oxygen species and Pselectin glycoprotein ligand 1) as well as traditional indicators of CVD risk (cholesterol, high-sensitive C-reactive protein, and HbA1c), thereby suggesting a correlation. The authors suggested this device could thus be used to prescreen T2DM patients for risk of cardiovascular events by analyzing rolling speed and trajectory of neutrophils. While further, large-scale analysis across a diverse T2DM patient population is required to correlate adverse cardiovascular events to neutrophil rolling speed and trajectory, this device presents as an exciting new platform with which to monitor disease risk and provides the

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Figure 1. The microfluidic device developed to analyze neutrophil chemotaxis for asthma characterization requires less blood, time, and human resources than traditional transwell assay used to monitor chemotaxis. Traditional transwell methods used to monitor neutrophil chemotaxis (top) requires large whole blood volumes and nontrivial neutrophil isolation to first prepare the sample. Neutrophils are then added to the well with a surround chemoattractant, and then the well is manually monitoring and neutrophils tracked, requiring 2 hours to track approximately 20 neutrophils. Conversely, the developed microfluidic device (bottom) requires a finger prick of whole blood and no intricate neutrophil isolation procedure. An automated system is required to track the neutrophils once loaded into the device, which takes approximately 3 minutes to track over 20 neutrophils. This figure was modified from the original version originally published in Blood. Sackmann, E. K.; Berthier, E.; Young, E. W. K.; Shelef, M. A.; Wernimont, S. A.; Huttenlocher, A.; Beebe, D. J. Microfluidic Kit-on-a-Lid: A Versatile Platform for Neutrophil Chemotaxis Assays. Blood. 2012; 120: e45-e53 (ref. 55). Copyright 2012 the American Society of Hematology. constricted region was monitored and used to assess if top of the device, such that the chemoattractant diffuses into the channel through one of the inlets. Neutrophil chemotaxis is cytokines from the serum of ARDS patients altered cell then monitored with a microscope and quantified with a stiffness, with longer entry times indicating stiffer cells. custom tracking software. The investigators were able to Pathologic leukocytes (leukocytes incubated with ARDS identify chemotaxis velocity as a significant differentiating serum) showed stiffness heterogeneity demonstrated by two parameter between asthmatic and non-asthmatic patients, dominating populations of microchannel entry times, both of specifically a chemotactic velocity below 1.545 µm/minute which were longer than entry times of cells incubated in was indicative of asthma. They demonstrated the sensitivity healthy patient serum. The investigators further used their and specificity of their device by accurately identifying 22 of microfluidic device to show that interleukin-8 (IL-8) and tissue 23 asthmatic patients and 8 of 11 patients as non-asthmatic. necrotic factor-α (TNF-α) increased THP-1 stiffness, Moreover, this is the first report indicating a low neutrophil identifying these cytokines from the ARDS serum as possible chemotaxis velocity corresponds to asthma, a finding triggers for cellular stiffening. Cell stiffness was mediated with warranting further investigation. the addition of blocking antibodies to mitigate the cellstiffening effects of cytokines from ARDS patient serum, Sepsis, along with other conditions like severe trauma or thereby significantly decreasing microchannel entry time pneumonia, have been known to cause acute respiratory towards the healthy patient regime. While clinical trials distress syndrome (ARDS), a condition characterized by treating sepsis patients with blocking antibodies to TNF-α widespread pulmonary inflammation with a mortality rate of have shown poor efficacy,65 the authors suggest further 45%.56-58 The complex nature of this condition makes it investigating treatment regimens employing multiple cytokine difficult to identify and treat, resulting in diffuse alveolar blockers to mediate leukocyte stiffness and possible vasodamage and pulmonary microvascular endothelial injury, occlusive crises for ARDS patients. which is primarily mediated by neutrophils.59 Pharmacological interventions have yet to realize major success in clinical Pathologic alterations in the biophysical properties of trials;60,61 therefore, current treatment options rely on early leukocytes can also occur in the setting of leukemia, or cancer identification and implementation of supportive measures (i.e. of blood cell and blood cell precursors. Acute myeloid low tidal volume ventilation or extracorporeal membrane leukemia (AML) is a cancer of precursor blood cells oxygenation), while undergoing treatment of the underlying characterized by the rapid growth of abnormal white blood 62,63 disease. cells that build up in the bone marrow and interfere with the production of normal blood cells. Similar to ARDS patients, As neutrophils heavily mediate the initial pathogenesis of for AML patients, changes in the biophysical properties of ARDS, Preira et al. utilized a microfluidic device to determine leukocytes (i.e. stiffness) are thought to contribute to acute if leukocyte stiffening contributed to the pathophysiology in complications like microvascular occlusion of vital organs. ARDS, and if treatments affecting leukocyte activation could Leukostasis is one such complication wherein leukocytes mitigate the stiffness-mediated pathophysiology of the occlude the microvasculature (primarily in the lungs and condition.64 Here, the investigators incubated neutrophils, central nervous system), leading to respiratory failure and monocytes, and THP-1 cells with the serum of ARDS patients. hemorrhagic stroke. As the cause of leukocyte occlusion Cells were perfused through two sequential microchannels of within the context of AML is poorly understood, it is a constricted geometry: the first being 6 µm wide and 12 µm complication that is extremely difficult to predict, relying on high, and second of the same width but 9 µm in height. The respiratory or neurological symptoms of occlusion to present time for full entry of individual cells into the second themselves before a diagnosis can be made.66 Development of

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clinical tools to predict the onset of leukostasis would enable preventative treatment (i.e. leukapheresis) before occlusion can occur, thereby decreasing patient morbidity and mortality. To address this need, Rosenbluth et al. developed a singlecell microfluidic device that functions as a biophysical flow cytometer. The reported device mimics the constricted geometry of the microvascular environment with microchannels 5.89 µm wide and 13.3 µm tall.67 Leukocytes from AML patients (symptomatic and non-symptomatic of leukostasis) were perfused through the device to investigate if changes in leukocyte stiffness led to microchannel occlusion. Leukocytes from acute lymphoid leukemia (ALL) were also analyzed as a control as chemotherapy drugs have been shown to increase baseline cell stiffness. Microchannel transit times of single cells as well as microchannel occlusion events were monitored via video microscopy and determined via automated image analysis. Interestingly, the authors showed that the transit times followed a non-Gaussian distribution with a small population of cells exhibiting significantly longer transit times for both AML and ALL patients. This stiff cell population was significantly larger in AML patients symptomatic of leukostasis (29% compared to 17% in non-symptomatic AML patients and 8% and 9% in ALL patients). The investigators further showed that channel occlusion of HL60 cells (a promyelocytic leukemic cell line) treated with drugs that disrupt the actin cytoskeleton or inhibit phosphodiesterase (cytochalsin D and pentoxifylline, respectively) could return occlusion incidence to that exhibited by samples from nonsymptomatic patients, further suggesting that channel occlusion was due to leukocyte stiffening. The collection of large data sets with single-cell resolution in this investigation elucidated the heterogeneity in leukocyte stiffness seen in ALL and AML patients that were symptomatic and nonsymptomatic of leukostasis. Moreover, their findings suggest that leukostatis can be in-part attributed to substantial stiffening of a small population of leukocytes – a finding that may have been masked in low-throughput or bulk methods. While these microfluidic devices showed utility in elucidating the impacts of heterogeneity in leukocyte stiffness on disease pathophysiology, there is concern that variations in cell size and interactions between the cell and surface of the microfluidic device could influence transit times. To address this, Gossett et al. developed a high throughput microfluidic device to determine cell stiffness via hydrodynamic stretching.68 Their deformability cytometer employs inertial focusing to linearly distribute cells through a cross-junction wherein a perpendicular extensional flow deforms the individual cell, enabling deformation without confounders of cell-surface interactions (Figure 2A-C). Deformation is monitored via video microscopy and determined using an automated image analysis algorithm (Figure 2D). Cell diameter before deformation and the ratio of the long axis diameter to the short axis diameter during deformation was plotted on a graph (Figure 2E,F). Impressively, the device is capable of single-cell analysis at a rate of 2,000 cells/second, enabling rapid collection of large data sets for robust analysis.

Figure 2. The deformability cytometer determines cell deformability via hydrodynamic stretching in a highthroughput manner. (A) A macroscopic image of the hydrodynamic stretching microfluidic device with schematic of the device (B) indicating inertial focusing regions and (C) tjunction of extensional flow. Brightfield micrographs of cells flowing through the device and into the hydrodynamic stretching region (D) are shown with comparison of cell morphology and diameter (E). Initial cell diameter and the ratio of deformed diameters (deformability) are shown in a 2D scatter plot for 9,740 human embryonic stem cells. Reproduced from Proceedings of the National Academy of Sciences USA, Gossett, D. R.; Tse, H. T. K.; Lee, S. A.; Ying, Y.; Lindgren, A. G.; Yang, O. O.; Rao, J.; Clark, A. T.; Di Carlo, D. Hydrodynamic Stretching of Single Cells for Large Population Mechanical Phenotyping. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (20), 7630-7635 (ref. 68).

The investigators examined cell deformability of peripheral blood mononuclear cells (PBMCs) and cells in pleural fluid from patients with chronic inflammation, acute inflammation, and malignancy. They were able to identify unique profiles in the 2D scatter plots showing increased deformability for PBMCs activated with anti-CD3 antibody or phytohaemagglutinin in vitro and samples from patients with inflammation or malignancy. Furthermore, by gating the 2D scatter plots, a sensitivity of 91% and specificity of 65% can be achieved in detecting malignancy. In a subsequent study of the deformability cytometer as a pre-screening step for cytopathologists, Tse et al. identified the likelihood of malignancy in pleural effusions from 119 patient samples using a graded scoring system from data extracted by the biophysical profile.69 The authors demonstrate that, out of 174 procedures (119 initial reads and 55 follow-ups), using the deformability cytometry to pre-screen samples could have reduced the sample load on the cytopathologist by 112 samples, thereby freeing up valuable hospital resources and advancing patient care timelines. Further decoupling confounding factors in microfluidic device-based deformability cytometry, Otto et al. developed a real-time deformability cytometry (RT-DC) wherein cells are perfused into a constricted region (20 µm x 20 µm) to measure cellular deformation without shear stresses or pressure gradients.70 Individual cells are monitored with a camera, and an algorithm executes single-cell analysis in real-time to determine contour, deformation, and size, which is then plotted

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iMCS analysis could differentiate between cells of different cancer lines or be used for hematological applications. Another single-cell microfluidic device has been reported that evaluates drug response and identifies multidrug resistance (MDR) in vitro, which utilizes same single-cell analysis (SASCA) in various cancer cells including adult AML.73-77 MDR occurs when efflux of hydrophobic drugs is enhanced by increased expression of ATP binding cassette (ABC) transporter proteins. SASCA is conducted utilizing a chamber to hold a single cancer cell in place while exposing it to a cross flow of fluorescently tagged chemotherapeutics (i.e. Danorubicin) or drug efflux transporter inhibitors (i.e. MK571, fumitremorgin C, or cyclosporine A). Fluorescence of the cell is then monitored to determine the amount of drug that is retained in the cell. The device has evolved by optimizing the assay procedure to enhance operator ease and decrease assay time (SASCA-A),74 include chambers for on-chip size-based separation of cancer cells from whole blood,76 and include a dielectrophoretic (DEP) cell trap feature77 to further enhance user ease while conducting the assay (Figure 3).

on a scatter plot. Importantly, their algorithm calculates the tangential and normal stresses on the cell surface to derive the expected deformation of an isotropic, linearly elastic sphere. The modeled shapes are consistent with the bullet-like shapes seen during experimentation and effectively decouple sizebased confounders on cell deformation calculations. The authors first demonstrated the utility of RT-DC by first investigating deformability of HL60 cells that have been treated with various concentrations of cytochalasin D, in which they were able to show a dose-dependent response with deformation plateauing at 0.1 µM cytochalasin D. Furthermore, they showed RT-DC could resolve distinct deformation populations during cell-cycle progressions corresponding to G1, S, G2, and M phases, which has yet to be demonstrated with FACS. HL60 cells were then differentiated into granulocyte, monocyte, and macrophage cell types, which RT-DC could resolve showing increased stiffness in macrophages and decreased stiffness in granulocytes and monocytes compared to undifferentiated HL60 cells. The investigators then demonstrated the ability to resolve red blood cells, PBMCs, platelets, and granulocytes from whole blood into distinct deformation populations. The high resolution and rapid real-time analysis (40,000 cells in 85 seconds) make RTDC an attractive device to further develop for diagnostic applications. A numerical model using linear elastic and neoHookean hyperelastic models was recently developed to extract mechanical data from cells analyzed with RT-DC.71 The investigators used poly(acrylamide) nanoparticles as model cells and were able to show good correlation between elastic moduli calculated from RT-DC using the developed numerical model and that determined via atomic force microscopy. This allows for further understanding into the mechanical properties (i.e. bulk and cortex elasticities) of pathologic cells in investigating etiology, pathophysiology, and in vitro screening of treatment candidates. While not expressly developed for analysis of hematological disorders, the recently reported inertial microfluidic cell stretcher (iMCS) shows great potential for further development as it does not rely on sheath flows that can clog and offers a higher cell deformation force (>1 µN) compared the RT-DC (