Centrifugal Elutriation for the Removal of Leukocytes from Malaria-Infected Monkey Blood

Abstract

Centrifugal elutriation has been employed for the removal of leukocytes from the blood of owl monkeys (Aotus trivirgatus) infected with the malarial parasite, Plasmodium falciparum. The process has proved to be effective and reproducible for this purpose. Approximately 99% leukocyte removal was achieved. In addition, the separation has been carried out under aseptic conditions and the resulting preparation was shown to be infective to an owl monkey and to be free of bacterial contamination. The use of the elutriator for removal of leukocytes is discussed in relation to other techniques. Research in parasitology often presents unique problems in separating the parasitic organisms from other biological entities. In order to study the metabolism of the erythrocytes of owl monkeys (Aotus trivirgatus) infected with the malarial parasite, Plasmodium falciparum, removal of the leukocytes was desired. Conventional centrifugation does not produce an adequate separation in this instance, and has the disadvantage of subjecting the parasitized erythrocyte to potentially damaging forces. The technique of centrifugal elutriation has been used for the separation of various blood cells from normal whole blood (Lindahl and Lindahl, 1955; McEwen, Stallard, and Juhos, 1968). This report describes the use of centrifugal elutriation for the removal of leukocytes from blood samples taken from P. falciparum-infected owl monkeys. Centrifugal elutriation is a process in which particles sediment through a liquid flowing in a direction opposite to the sedimentation direction. A separation takes place when the sedimentation rate of a portion of the particles is less than the fluid flow velocity, and these slowly sedimenting particles are washed out of the rotor, while more rapidly sedimenting particles are retained in the rotor chambers. The separation process can be controlled by altering the fluid velocity and rate of rotation. Lindahl (1948) developed the first system for centrifugal elutriation and Lindahl and Lindahl (1955) described its use for a number of bioReceived for publication 9 February 1971. *Department of Preventive Medicine, Stanford University School of Medicine, Stanford, California 94305. logical ystems, including the concentration of eosinoph lic leukocytes of the horse. Recently, McEwen, Stallard, and Juhos (1968) reported on e development of a centrifugal elutriation system intended for use in a standard laboratory centrifuge. An advanced version of this instrum nt was used in this study for the fractionation of blood of malaria-infected monkeys. MATERIALS AND METHODS The CR-3 rotor used for these experiments is illustrated in Figure 1. Procedures for use of this rotor have been described by McEwen et al. (1968). Rotor temperature during the separations was 5 to 10 C. The buffer used as elutriating fluid and the overflow fractions collected in centrifuge tubes were also maintained at 5 to 10 C. A combination of rate of rotation and liquid flow rate was chosen which would retain most of the leukocytes in the rotor while the erythrocytes were carried in the overflow fraction. When the rotor speed is increased, a corresponding increase in the liquid flow rate is required to obtain the same separation. The use of higher rotor speeds and fluid flow can appreciably shorten the time required to process a sample. However, these conditions increase the chance of plugging the narrow end of the rotor chambers. Preliminary results indicated that the blood sample used should contain less than 1010 cells. Thus, the sample size was adjusted according to the cell count of the blood specimen. In practice, samples of 1 to 4 ml were convenient. After the sample was introduced into the system, overflow fractions were collected until the predetermined volume was reached. When the process was completed, heavy particles, including most of the leukocytes, were flushed from the rotor chambers at high liquid flow (approximately 140 ml/min) before the next sample was added. As many as 8 samples (22 ml of blood) have been processed in 1 day. The elutriating fluid used for these studies was the Versene buffer described by Paul (1960). It has a pH of about 7.4 and has the following compo-