Malaria remains one of the world's major health burdens. With 2.5 billion people at risk it affects an estimated 500 million people annually, causing one to three million deaths, the majority of which occurs in children under five years of age. Improved methods facilitating research in the field are urgently needed [1, 2].
Isolation of infected red blood cells (irbc) is a crucial step in basic and applied malaria research. For the past three decades, isolation has been performed mostly by Percoll® density gradient separation, exploiting the fact that density of irbc decreases with parasite maturation . A further refinement of this method are hypertonic, discontinuous Percoll®-sorbitol gradients, where particular fractions of irbc can be obtained. Hypertonicity causes cell shrinkage of rbc, while irbc swell back due to an influx of sorbitol through new permeability pathways. This increases the density gaps between the different developmental stages and allows better separation than in pure Percoll® gradients . Purification results, however, depend on a variety of factors, including individual research experience. Gelatin sedimentation is used as an alternative concentration method, however, it is useful only for parasite strains exhibiting knobs .
Frequently, not only highly purified but also stage-synchronized parasite cultures and isolates are required. Synchronization of cultures is performed by isotonic sorbitol lysis of late-stage irbc, as described 30 years ago . Time-consuming synchronization cycles by repeated sorbitol lysis and/or Percoll® isolation are required to obtain synchronized and pure irbc suitable for downstream applications [6–8]. While sorbitol selectively lyses late-stage irbc, it imposes sub-lytic osmotic stress in younger stage irbc and likely enters these cells . Whether or not exposure of irbc to synthetic chemicals and osmotic stress, respectively, has unwanted consequences on parasites remains an open question.
In principle, high gradient magnetic separation (HGMS) offers a way to concentrate or deplete malaria irbc from suspensions, relying solely on their intrinsic magnetic properties. Particularly late-stage irbc are known to behave as paramagnetic particles . In a paramagnetic particle, magnetic poles are induced only when exposing the particle to a magnetic field, the removal of which leads to immediate de-magnetization. Due to the very small distance separating the particle's respective north- and south-poles, very high magnetic field gradients are required to create a net magnetic force, which is able to attract or repel the particle. Such gradients are generated by placing thin filamentous or spherous ferromagnetic material as a matrix into a strong homogenous magnetic field, which is usually provided by rare-earth dipole magnets or electromagnets. With this technology, magnetic gradients up to 100 Tesla/cm can be created at the surface of the matrix .
Paramagnetism in malaria irbc results from the hemoglobin catabolism of intra-erythrocytic malaria parasites. Free haem as a toxic by-product is de-toxified by polymerization and by oxidation of the molecule's central iron atom . Oxidized iron [Fe+3] carries five unpaired electrons in its d-orbitals, rendering the molecule and the resulting polymer paramagnetic [10, 13, 14]. Deposition and accumulation of polymerized haem (haemozoin) in the parasite's food vacuole result in a continuously increasing magnetic susceptibility of the irbc .
Successful, but not highly efficient HGMS of late-stage irbc from malaria cultures was first described in 1981 . Later, commercially available, polymer coated HGMS columns were shown to offer improved results [15–18]. Recently, successful synchronization was demonstrated with polymer coated HGMS columns. However, sorbitol-pretreatment of Plasmodium cultures was essential . Generally, the use of HGMS in malaria research is still hampered by limited column capacity, inconsistent separation purities and high costs.
Firstly, this study presents a buffer-optimized HGMS concentration system for irbc, as an alternative to Percoll® density gradient separation. Secondly, a HGMS-BSA gradient system is introduced for HGMS depletion of late-stage irbc, as an alternative to Sorbitol lysis.
Buffer-optimized HGMS was developed by identifying the three key variable parameters relevant to HGMS for cell separation. Briefly, these are derived as follows:
Particles in an HGMS device can be captured only if
where is the magnetic force, the drag force and the gravitational force acting on the particle.
The gravitational force is defined by Newton's law:
where m is the mass of the particle and the gravitational acceleration.
The drag force is defined by Stoke's law:
where η is the viscosity of the carrier fluid, r the radius and the velocity of the particle. While the radius of the rbc is constant and the viscosity of the carrier fluid depends on its defined chemical properties, the velocity of the particle is further determined by two vectors:
where is the velocity of the carrier fluid and the sedimentation velocity of the particle. Here, while the velocity of the carrier fluid can be easily adjusted, the sedimentation velocity deserves further discussion:
is the density of the particle, d
the density of the carrier fluid, the gravitational acceleration, r the radius of the particle and η the viscosity of the carrier fluid .
Analysing equation (1)-(5), it becomes obvious that apart from the constant intrinsic parameters of the particle, there are only four extrinsic parameters governing the HGMS system. These are a) the gravitational acceleration, b) the velocity of the carrier fluid, c) the viscosity of the carrier fluid, and d) the density of the carrier fluid.
It was hypothesized that in an ideal buffer-optimized HGMS system, the density of the carrier fluid can be adjusted to the density of rbc, which would render the influence of the gravitational acceleration on the particle negligible, setting the particle sedimentation velocity close to zero. In this case, particle velocity would be perfectly controllable solely by adjusting the velocity of the carrier fluid. Additionally, the density-concurrent increase in viscosity η would favourably support laminar flow conditions in the column.
To test this hypothesis, commonly available raw materials were employed to perform selective, buffer-optimized HGMS of P. falciparum late-stage irbc from asynchronous, standard routine malaria cultures, avoiding exposure to anything but incomplete malaria culture medium (RPMI 1640) or isotonic sucrose solution, substituted with either BSA or gelatine, respectively. A concentration protocol was developed as an alternative to Percoll® separation and HGMS with polymer-coated columns. We show that with further refinement, optimized HGMS results in unprecedented purities of segmented-stage irbc from standard asynchronous cultures.
The second protocol demonstrates efficient depletion of late-stage irbc from Plasmodium cultures. Here, a BSA density gradient was introduced within the HGMS separation column, which prevented uncontrolled sedimentation of rbc during incubation within the matrix of the column. It is demonstrated that HGMS depletion alone, without HGMS concentration, can be used as an alternative to Sorbitol lysis for culture synchronization, potentially further widening the application of HGMS in malaria reserach.