Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers

Abstract

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. IntroductionThe 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 reticulocytes-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][3][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][5][6][7]. Enrichment is difficult because reticulocytes exist at a low concentration (0.5-2.5% of erythrocytes) in whole blood [8] and have a short half-life in culture ( 30 hr) [9]. Cryopreservation provides a method to store samples that hav...

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