Improved methods for magnetic purification of malaria parasites and haemozoin
© Kim et al; licensee BioMed Central Ltd. 2010
Received: 14 September 2009
Accepted: 14 January 2010
Published: 14 January 2010
Malaria parasites generate free haem upon catabolism of host haemoglobin during their intraerythrocytic growth cycle. In order to minimize oxidative toxicity of the ferric iron, the free haem molecules are polymerized into the biomineral beta-haematin (commonly referred to as haemozoin). Haemozoin crystals are paramagnetic, and this property can be exploited for the purification of late stage parasites as they contain larger haemozoin crystals than early stage parasites and uninfected cells. Commercially available magnets that were originally developed for the purpose of antibody-mediated cell purification are widely used for this purpose. As these methods are not necessarily optimized for parasite purification, the relationship between magnetic field strength and the quantity and quality of yield during parasite purification was explored.
Inexpensive rare-earth neodymium magnets with commercially available disposable columns were employed to explore the relationship between magnetic field strength and recovery of free haemozoin and infected erythrocytes (iRBCs).
Yields of free haemozoin increased nearly linearly with increasing magnetic field strength to the strongest fields tested (8,500 Gauss). Stronger magnetic fields also improved the recovery of iRBCs with no detrimental effects on parasite viability. An in-house constructed magnetic stand, built for $75 in materials, produced superior results when compared with much more expensive commercial products.
Existing protocols for the magnetic purification of free haemozoin and iRBCs result in sub-optimal yields. Inexpensive high-strength neodymium magnets offer a better option, resulting in higher yields with no detrimental effects on parasite viability.
Malaria parasites catabolize the proteinaceous component of haemoglobin as a nutrient source during their intracellular growth cycle in erythrocytes . The haem moiety is not metabolized and accumulates in the parasite digestive vacuole. In order to reduce oxidative toxicity of the ferric iron, the excess haem is polymerized into the crystalline biomineral beta-haematin, more commonly referred to as haemozoin . The haemozoin crystal becomes visible by microscopic examination during the trophozoite and schizont stages roughly halfway through the intraerythrocytic developmental cycle and continues to grow in size until the parasites are mature enough to egress from the erythrocyte.
The progressive accumulation of haemozoin throughout the life cycle has previously been exploited to preferentially isolate the late trophozoite and schizont stages. Due to the paramagnetic nature of haemozoin, passage of parasites over another paramagnetic material subjected to a strong magnetic field results in specific retention of late-stage parasites while allowing erythrocytes with no parasites or early-stage parasites to flow through. This approach was first reported over half a century ago using electromagnets to purify Plasmodium vivax parasites for use in vaccination efforts . The problems of slow processing time and requirement for specialized equipment were subsequently circumvented through the use of 7,000 Gauss (G) permanent magnets , which were also employed for the direct purification of haemozoin . More recently, commercial systems that are used for purification of immuno-labelled cells have been adapted for purification of parasite-infected red blood cells (iRBCs) [5, 6] and haemozoin . These systems have been demonstrated to work with many Plasmodium species and have been readily employed in field studies .
Despite the fact that magnetic purification of iRBCs has been in use for decades, a systematic study of the effects of varying the magnetic field strength has not been reported. This report demonstrates that magnetic purification of iRBCs and haemozoin can be accomplished using inexpensive and widely available rare-earth magnets with purity and yields that are superior to widely employed commercially available systems.
Plasmodium falciparum W2 (MRA-157) was cultured using previously described methods . Briefly, parasites were grown in human erythrocytes in RPMI 1640 media supplemented with 0.25 % Albumax II (GIBCO, Life Technologies, San Diego, California, United States), 2 g/L sodium bicarbonate, 0.1 mM hypoxanthine, 25 mM HEPES (pH 7.4), and 50 μg/L gentamycin, at 37°C, 5% O2, and 6% CO2. Parasites were loosely synchronized with a single round of sorbitol treatment prior to magnetic purification.
The following neodymium rare-earth magnets were purchased from Applied Magnets (Plano, TX): paired 1.5 in OD × 1.25 in ID × 0.75 in long × 90 degree grade N42 arcs (NA005), a diametrically magnetized grade N42 1 in diameter cylinder with a 0.25 in center hole (ND062-D), diametrically magnetized grade N42 1 in diameter cylinders (ND062-TD), and grade N52 1 in cubes (NB041). Magnetic fields were measured using a DC magnetometer (AlphaLab Inc., Salt Lake City, UT).
Purification of haemozoin
Synchronized cultures at 2% haematocrit were grown to a ring parasitaemia of 10% followed by two additional days of culture to allow natural egress and release of haemozoin. Each LS column (Miltenyi Bioec, Auburn, CA) was loaded with 25 ml of resuspended culture, washed three times with 5 ml water, and eluted in 5 ml water. Haemozoin was recovered by centrifugation for 15 min at 12,500 × g and resuspended in 100 ul water. The final concentration of the recovered haemozoin was determined by dissolution into monomers and spectroscopic determination at 400 nm using a molar extinction coefficient of 105, as previously described .
Purification of iRBCs
Cultures were synchronized by one round of sorbitol synchronization and purified once the parasites reached the late-trophozoite stage. Each LD column (Miltenyi Biotec) was loaded with 50 ml of parasite culture at 2% haematocrit and 5-15% parasitaemia. Columns were washed once with 5 ml complete RPMI medium (RPMIc) and eluted in 5 ml RPMIc. Recovered cells were counted on a haemocytometer and purity was assessed by Giemsa-stained blood smear.
Design and synthesis of magnetic purification stand
The purification stand was designed in SolidWorks 2009 (Dassault Systémes SolidWorks Corp., Concord, MA) as three separate components (body, lid, and legs). The body accommodates five 1 in diameter × 1 in length diametrically magnetized cylinders. Designs were exported as STL files and prototyped in acrylonitrile butadiene styrene (ABS+) plastic on a uPrint 3D printer (Dimension Inc., Eden Prairie, MN). The magnetic stand design files, appropriate for 3D printing, are available as supplementary files (Additional File 1). Updated versions are available in the 3D design repository at the DeRisi lab website .
Percentages were arcsine transformed as summarized by Zar . Means and confidence intervals were calculated on the arcsine-transformed data and converted back to linear space. All reported p-values are calculated from two-tailed t-tests assuming unequal variances on the arcsine-transformed data.
Results and Discussion
Purification of free haemozoin
Naturally generated haemozoin has traditionally been prepared through a series of extractions to remove all traces of contaminating nucleic acids, proteins, and lipids . More recent studies examining the immuno-stimulatory properties of haemozoin have turned to the use of magnetic purification in order to isolate haemozoin in a state closer to its physiological form upon release from parasites undergoing egress from erythrocytes . Although magnetic purification is increasingly employed in the field of malaria research, the relationship between magnetic field strength and haemozoin yield during magnetic purification procedures has not previously been examined.
Purification of infected erythrocytes
A previous study suggested that "better separation" of iRBCs might be achieved through the use of strong electromagnets . However, it has remained unclear how different magnetic fields might influence the purity, yield, and viability of isolated iRBCs, especially in comparison with commercially available systems that are commonly used for this purpose. A comparison of iRBC purification on slow flow-rate LD columns (Miltenyi Biotec) subjected to the magnetic fields from the MidiMACS, cylinder magnet, and double cube magnet configurations was, therefore, undertaken.
Purification of iRBCs
Parasites Recovered (% of input)
7.9 × 107 (5.0%)
1.10 × 108 (7.0%)
2 × cubes
1.13 × 108 (7.2%)
Practical application of magnetic purification
Late stage parasites from synchronous cultures of P. falciparum were isolated in parallel using both the in-house-designed purification stand and three MidiMACS separators. The recovered parasites were assessed for overall yield using a haemocytometer, purity by blood smear, and viability by culture. The cylinder magnets recovered approximately 60% more parasites than the MidiMACS separators (25.9% and 15.0% total recoveries, respectively; p = 0.0002). Purity was high and comparable for both methods (cylinders 95.3% mean with 95% CI 93.6% to 96.9%; MidiMACS 95.7% mean with 95% CI 92.5% to 98.0%; p = 0.86).
Improvements to existing protocols for magnetic purification of free haemozoin and malaria-infected erythrocytes are described. In addition to giving higher yields of haemozoin and iRBCs with no detrimental effects on viability of purified parasites, the described systems are two orders of magnitude less expensive than commercially available cell purification magnets, making the approach much more widely accessible to the malaria research community.
We thank David Hesslein and Lewis Lanier for providing us with MidiMACS separators, Wallace Marshall for providing the magnetometer, and Centdrika Dates for Plasmodium cultures. CCK is supported by an A.P. Giannini Foundation Medical Research Fellowship. This work was supported by the Howard Hughes Medical Institute.
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