Atomic force microscopy (AFM) has become an important tool for quantifying mechanical properties of biological materials ranging from single molecules to cells and tissues. Current AFM techniques for measuring elastic and viscoelastic properties of whole cells are based on indentation of cells firmly adhered to a substrate, but these techniques are not appropriate for probing nonadherent cells, such as passive human leukocytes, due to a lateral instability of the cells under load. Here we present a method for characterizing nonadherent cells with AFM by mechanically immobilizing them in microfabricated wells. We apply this technique to compare the deformability of human myeloid and lymphoid leukemia cells and neutrophils at low deformation rates, and we find that the cells are well described by an elastic model based on Hertzian mechanics. Myeloid (HL60) cells were measured to be a factor of 18 times stiffer than lymphoid (Jurkat) cells and six times stiffer than human neutrophils on average (E(infinity) = 855 +/- 670 Pa for HL60 cells, E(infinity) = 48 +/- 35 Pa for Jurkat cells, E(infinity) = 156 +/- 87 for neutrophils, mean +/- SD). This work demonstrates a simple method for extending AFM mechanical property measurements to nonadherent cells and characterizes properties of human leukemia cells that may contribute to leukostasis, a complication associated with acute leukemia.

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.

Deformability of blood cells is known to influence vascular flow and contribute to vascular complications. Medications for hematologic diseases have the potential to modulate these complications if they alter blood cell deformability. Here we report the effect of chemotherapy on leukemia cell mechanical properties. Acute lymphoblastic and acute myeloid leukemia cells were incubated with standard induction chemotherapy, and individual cell stiffness was tracked with atomic force microscopy. When exposed to dexamethasone or daunorubicin, leukemia cell stiffness increased by nearly 2 orders of magnitude, which decreased their passage through microfluidic channels. This stiffness increase occurred before caspase activation and peaked after completion of cell death, and the rate of stiffness increase depended on chemotherapy type. Stiffening with cell death occurred for all cell types investigated and may be due to dynamic changes in the actin cytoskeleton. These observations suggest that chemotherapy itself may increase the risk of vascular complications in acute leukemia. IntroductionAlterations of biophysical properties of blood cells contribute to the pathophysiology of hematologic diseases. 1-3 While chemotherapy-induced cell death has been a mainstay of cancer treatment for decades and is well-studied biochemically, little is known about the mechanical effects chemotherapy may have on leukemia cells. Furthermore, since hyperleukocytosis accompanies some cases of acute leukemia, mechanical changes in leukemia cells due to chemotherapy could significantly alter the overall blood rheology.In this work, we quantified the effect of standard induction chemotherapy on the stiffness of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cells using atomic force microscopy (AFM), a tool for imaging and characterization of materials at the nanometer scale. 4 The high force sensitivity of AFM and its ability to measure properties of individual cells over long times makes the technique particularly appropriate for measuring dynamic changes in cell stiffness. We find that when exposed to chemotherapy, leukemia cell stiffness increased by nearly 2 orders of magnitude at a rate dependent on the type of chemotherapy employed. Materials and methods Leukemia cell sources and reagentsLeukemia cells were isolated via centrifugation from the blood of patients with newly diagnosed acute leukemia noted to have peripheral blast cells. Leukemic cell lines were purchased commercially (ATCC, Manassas, VA). University of California, San Francisco (UCSF) and UC Berkeley institutional review boards approved all experimental procedures. Informed consent was obtained from each human subject, in accordance with the Declaration of Helsinki, before a blood sample was obtained. Dexamethasone and daunorubicin (Sigma-Aldrich, St Louis, MO) are mainstay induction chemotherapeutic agents for ALL and AML, respectively. Accordingly, lymphoid and myeloid leukemic cells were exposed to typical treatment doses of 1 M dexamethasone and 1 M...