Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers Ashok A. Kumar, 1 Caeul Lim, 2 Yovany Moreno, 2 Charles R. Mace, 3 Abeer Syed, 3 Daria Van Tyne, 2 Dyann F. Wirth, 2 Manoj T. Duraisingh, 2 and George M. Whitesides 3,4 * This paper demonstrates the enrichment of reticulocytes by centrifuging whole blood through aqueous multiphase systems (AMPSs)—immiscible phases of solutions of polymers that form step-gradients in density. The interfaces of an AMPS concentrate cells; this concentration facilitates the extraction of blood enriched for reticulocytes. AMPS enrich reticulocytes from blood from both healthy and hemochromatosis donors. Varying the osmolality and density of the phases of AMPS provides different levels of enrichment and yield of reticulocytes. A maximum enrichment of reticulocytemia of 64 6 3% was obtained from donors with hemochromatosis. When used on peripheral blood from normal donors, AMPS can provide a higher yield of enriched reticulocytes and a higher proportion of reticulocytes expressing CD71 than differential centrifugation followed by centrifugation over Percoll. Blood enriched for reticulocytes by AMPS could be useful for research on malaria. Several species of malaria parasites show a preference to invade young erythrocytes and reticulocytes; this preference complicates in vitro cultivation of these species in human blood. Plasmodium knowlesi malaria parasites invade normal human blood enriched for reticulocytes by AMPSs at a rate 2.2 times greater (P < 0.01) than they invade unenriched blood. Parasite invasion in normal blood enriched by AMPS was 1.8 times greater (P < 0.05) than in blood enriched to a similar reticulocytemia by differential centrifugation followed by centrifugation over Percoll. The enrichment of reticulocytes that are invaded by malaria parasites demonstrates that AMPSs can provide a label-free method to enrich cells for biological research. Am. J. Hematol. 90:31–36, 2015. V C 2014 Wiley Periodicals, Inc.  Introduction The characterization of different cellular components of human whole blood plays an important role in medical diagnoses and research [1]. This task is often accomplished by isolating or enriching a specific cellular sub-population from the complex mixture of cells found in whole blood. Whole blood is composed of cells—leukocytes, platelets, and erythrocytes—suspended in protein-rich plasma; each type of cell is useful in the evaluation of the health of a patient. These cells can be further subdivided into important components. For example, the isolation of reticulo- cytes—immature erythrocytes—is important for research in malaria. Plasmodium (P.) vivax, P. ovale, and P. knowlesi—three causative agents of malaria in humans—preferentially invade human reticulocytes [2–4]. The study of these species of malaria suffers from the practical difficulty of in vitro cultures in whole human blood. Their cultivation in blood enriched for reticulocytes would provide concentrated host cells in which the parasites proliferate; continuous cultures of these parasites would make their study much more practical [4–7]. Enrichment is difficult because reticulocytes exist at a low concentration (0.5–2.5% of erythro- cytes) in whole blood [8] and have a short half-life in culture (30 hr) [9]. Cryopreservation provides a method to store samples that have been enriched with reticulocytes [10], but enriched samples must be obtained prior to freezing. A method to routinely enrich reticulocytes from normal, whole blood with high yields and reproducibility would enable more scientists to perform research with reticulocytes. This paper describes a new method to obtain samples of cells that are enriched for reticulocytes using centrifugation through aqueous multi- phase systems (AMPSs). AMPSs are systems of polymers in aqueous solutions that generate immiscible phases when mixed. These phases provide self-assembling step-gradients in density [11]. We demonstrate that centrifugation through AMPSs separates cells based on their density, and con- centrates them at interfaces. AMPS enrich reticulocytes when used on blood from donors with hemochromatosis as well as when used on blood Additional Supporting Information may be found in the online version of this article. 1 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts; 2 Harvard School of Public Health, Harvard University, Boston, Massachu- setts; 3 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts; 4 Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts. Conflict of interest statement is mandatory. *Correspondence to: G.M. Whitesides; Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, MA 02138. E-mail: gwhitesides@gmwgroup.harvard.edu Contract grant sponsor: Bill and Melinda Gates Foundation; Contract grant number: OPP1016360 and OPP1023594. Contract grant sponsors: Office of Naval Research [NDSEG Fellowship to A.A.K.]; the Louis-Berlinguiet Program Postdoctoral Research Scholarship in Genomics—Fonds the Recherche du Quebec, Nature et Technologies to Y.M.; the Royal Society of Edinburgh’s J.M. Lessell’s Scholarship to A.S and the Mac Roberston Postgraduate Travel Scholarship to A.S.. Received for publication: 16 September 2014; Accepted: 24 September 2014 Am. J. Hematol. 90:31–36, 2015. Published online: 27 September 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ajh.23860 V C 2014 Wiley Periodicals, Inc. doi:10.1002/ajh.23860 American Journal of Hematology, Vol. 90, No. 1, January 2015 31 RESEARCH ARTICLE A JH A JH