Single-cell measurement of red blood cell oxygen affinity Giuseppe Di Caprioa,1, Chris Stokesa, John M. Higginsb,c, and Ethan Schonbruna aThe Rowland Institute, Harvard University, Cambridge, MA 02142; bCenter for Systems Biology, Massachusetts General Hospital, Boston, MA 02115; and cDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115 Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved July 1, 2015 (received for review May 11, 2015) Oxygen is transported throughout the body by hemoglobin (Hb) in oxygen binding (21). This method allows us to quantify RBC red blood cells (RBCs). Although the oxygen affinity of blood is volume, Hb concentration (HbC), and oxygen affinity for cells well-understood and routinely assessed in patients by pulse while they are in a fluidic environment similar to the circulatory oximetry, variability at the single-cell level has not been previously system. A few previous studies have explored the measurement measured. In contrast, single-cell measurements of RBC volume and of single-cell saturation (22–24), but to our knowledge, saturation Hb concentration are taken millions of times per day by clinical measurements have never been performed on a large cell pop- hematology analyzers, and they are important factors in determining ulation or under accurate control of oxygen partial pressure. the health of the hematologic system. To better understand the In this paper, we first describe the physical properties of the variability and determinants of oxygen affinity on a cellular level, we microfluidic chip followed by the optical measurement system for have developed a system that quantifies the oxygen saturation, cell obtaining cell volume and Hb mass. We then discuss the spectro- volume, and Hb concentration for individual RBCs in high throughput. scopic measurement system and the quantification of single-cell We find that the variability in single-cell saturation peaks at an saturation from the measured multispectral absorption. The tem- oxygen partial pressure of 2.9%, which corresponds to the maximum poral dynamics of the system are then characterized to better un- – slope of the oxygen Hb dissociation curve. In addition, single-cell derstand the time and length scales required for equilibrium oxygen affinity is positively correlated with Hb concentration but deoxygenation. We use our system to capture an Hb–oxygen dis- independent of osmolarity, which suggests variation in the Hb to – sociationcurveandanSDcurveasafunctionofoxygenpartial 2,3-diphosphoglycerate (2 3 DPG) ratio on a cellular level. By quanti- pressure. At each oxygen partial pressure, we can retrieve a full fying the functional behavior of a cellular population, our system distribution of single-cell oxygen saturation and observe its corre- adds a dimension to blood cell analysis and other measurements of lation to total HbC. In addition, using the measured saturation single-cell variability. values, we can retrieve the oxygen partial pressure where the sat- uration is 50% (P ) value for each cell in the population using the hematology | medical sciences | flow cytometry | organ on chip | 50 Hill equation as a model. single-cell variability Functional RBC Analyzer ed blood cells (RBCs) are the most common type of blood cell The microfluidic device to control the oxygen partial pressure ’ Rand constitute approximately one-half of the human body s and deliver cells to the measurement region is shown in Fig. 1 A total cell count (1). They take up oxygen in the lungs and deliver it and B. The device is composed of three layers: one layer in which throughout the body, taking, on average, 20 s to complete one the RBCs flow, a thin gas-permeable membrane in poly(dimethylsi- circuit through the circulation (2). Each cell is densely packaged loxane) (PDMS; thickness = 90 μm), and the gas layer (section with hemoglobin (Hb) that binds and releases oxygen based on the showninFig.1B). Cells are driven through the microfluidic device local oxygen partial pressure. The fraction of occupied binding sites using a syringe pump and flow through a gas exchange region that relative to the total number of binding sites is called the oxygen is 1-cm long, 1-mm wide, and 6-μm thick. As they flow, cells saturation and can be described by the Hb–oxygen dissociation curve. Although it is known that several factors affect the oxygen Significance affinity of Hb and consequently, shift the dissociation curve, such as pH, temperature, and 2,3-diphosphoglycerate (2–3DPG)(3),itis not known how much variation these factors cause on a cellular Oxygen transport is the most important function of red blood level within individuals. cells (RBCs). We describe a microfluidic single-cell assay that Recently, there has been significant progress in developing quantifies the oxygen saturation of individual RBCs in high biomimetic environments for studying physiological processes throughput. Although single-RBC measurements of volume and of cells in vitro. Analogs to the lung (4), heart (5), bone marrow mass are routinely performed in hospitals by hematology ana- (6), and gut (7) have all been developed in microfabricated lyzers, measurements that characterize the primary function of RBCs, the delivery of oxygen, have not been made. With this chips that enable ex vivo studies to closely replicate an in vivo system, we find measurable variation that is positively corre- environment. In addition to adherent cell cultures, chips for lated with cellular hemoglobin concentration but independent studying flowing blood have been developed that enable control of osmolarity. These results imply that the cytoplasmic envi- of oxygen partial pressure with high spatial resolution (8–12). ronment of each cell is different and that these differences We take advantage of this new technology and combine it with modulate the function that each cell performs. a recently developed microfluidic cytometry method that en- ables us to quantify cell volume and Hb mass for individual Author contributions: G.D.C., J.M.H., and E.S. designed research; G.D.C. performed re- flowing cells in high throughput (13). search; C.S. contributed new reagents/analytic tools; G.D.C. and E.S. analyzed data; and Our system for measuring RBC mass is based on the optical G.D.C., J.M.H., and E.S. wrote the paper. absorption of Hb (14, 15). Unlike other recently developed The authors declare no conflict of interest. single-cell mass measurements (16–20), however, it is straight- This article is a PNAS Direct Submission. forward to extend this method to resolving the mass of both 1To whom correspondence should be addressed. Email: [email protected]. oxygenated and deoxygenated species because of the well- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. known modification of the absorption spectra of Hb caused by 1073/pnas.1509252112/-/DCSupplemental. 9984–9989 | PNAS | August 11, 2015 | vol. 112 | no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1509252112 Downloaded by guest on September 26, 2021 Fig. 1. Gas exchange RBC cytometry. A shows a side view of the microfluidic chip. The gas and the sample channel are filled with blue and red dye, re- spectively. The sample channel is 2.2-cm long. Left Inset shows a picture of the gas exchange region, where the pillar structure is visible as well as the overlay between the gas and the sample channel. Right Inset shows the parallel channels in the measurement region. (Scale bars: Insets, 200 μm.) (B) Sketch of the cross-section of the three layers composing the chip. C shows the optical setup. Two blue (L1 and L2) and one red (R) LEDs are combined using dichroic mirrors. The light transmitted by the cells flowing in the microfluidic channel is then projected onto a color camera by a microscope objective. equilibrate with the oxygen partial pressure in the gas channel lo- that the system has reached a steady-state condition. After cells cated above a gas-permeable membrane. A hexagonal lattice of pass through the gas exchange region, they are imaged in the PDMS pillars (50-μm diameter separated by 100 μm) sustains the measurement region, which is composed of 16 parallel chan- channel (Fig. 1A, Left Inset). Traveling at a mean velocity of 2 mm/s, nels (width of 30 μm)showninFig.1A, Right Inset. Cells are cells spend ∼5 s in the gas exchange region, which is comparable measured within 100 μm of the last gas channel to minimize with the time that they spend in the microcirculation (25). The gas reoxygenation, and we have verified that oxygen saturation channel is serpentine and has dimensions of 250-μmwideand35- values in this region are similar to those measured underneath μm thick. The oxygen partial pressure of the input gas is controlled the last gas channel itself. off chip by mixing a tank of pure N2 with a tank of air (21% O2 and The optical setup is shown in Fig. 1C. Cells are suspended in a 79% N2). The partial pressure of the gas mixture is measured by refractive index-matching buffer, into which a nonmembrane-per- an oxygen sensor (GS-Yuasa Oxygen Sensors KE-Series) be- meable absorbing dye has been added (AB9; concentration of 0.8 fore and after flowing in the gas serpentine channel to verify g/dL) (13, 26). The cells are illuminated using a red light-emitting MEDICAL SCIENCES ENGINEERING Fig. 2. RBC oxygen binding kinetics. A shows a schematic of the chip geometry used to characterize cellular binding kinetics. Blood flows in the red straight channel from left to right, and the saturation is measured at different intersections with the gas channel (in blue). B shows the saturation (Sat) at different intersections for two different gas mixtures plotted vs. intersection number and time for a cell velocity of 2 mm/s.