Optimized high gradient magnetic separation for isolation of Plasmodium-infected red blood cells

Abstract

Background: Highly purified infected red blood cells (irbc), or highly synchronized parasite cultures, are regularly required in malaria research. Conventional isolation and synchronization rely on density and osmotic fragility of irbc, respectively. High gradient magnetic separation (HGMS) offers an alternative based on intrinsic magnetic properties of irbc, avoiding exposure to chemicals and osmotic stress. Successful HGMS concentration in malaria research was previously reported using polymer coated columns, while HGMS depletion has not been described yet. This study presents a new approach to both HGMS concentration and depletion in malaria research, rendering polymer coating unnecessary.

Methods: A dipole magnet generating a strong homogenous field was custom assembled. Polypropylene syringes were fitted with one-way stopcocks and filled with stainless steel wool. Rbc from Plasmodium falciparum cultures were resuspended in density and viscosity optimized HGMS buffers and HGMS processed. Purification and depletion results were analysed by flow cytometer and light microscopy. Viability was evaluated by calculating the infection rate after re-culturing of isolates.

Results: In HGMS concentration, purity of irbc isolates from asynchronous cultures consistently ranged from 94.8% to 98.4% (mean 95.7%). With further optimization, over 90% of isolated irbc contained segmented schizonts. Processing time was less than 45 min. Reinfection rates ranged from 21.0% to 56.4%. In HGMS depletion, results were comparable to treatment with sorbitol, as demonstrated by essentially identical development of cultures.

Conclusion: The novel HGMS concentration procedure achieves high purities of segmented stage irbc from standard asynchronous cultures, and is the first HGMS depletion alternative to sorbitol lysis. It represents a simple and highly efficient alternative to conventional irbc concentration and synchronization methods.

Figure 1

Morphology and corresponding flow cytometric analysis of P. falciparum irbc before and after buffer optimized HGMS. 5 × 10⁸ rbc from culture were applied to a HGMS column as described above. Parasitemia was measured by flow-cytometer after staining with Acridine Orange (100,000 events counted). Histograms (A1 and B1) and corresponding blood smears (A2 and B2) of one representative experiment are shown. M1 non infected rbc, M2 infected rbc. A1 and A2: Culture with a total parasitemia of 10.9%. B1 and B2: Result of buffer optimized HGMS of irbc from the culture: irbc containing late trophzoites and schizonts are enriched to a concentration of 98.4%.

Figure 2

Schizont-irbc fraction of isolates in eleven independent experiments. Total numbers of 2.5 × 10⁸ to 3 × 10⁹ rbc from cultures with 5-15% total parasitemia were applied to the HGMS column. Increasing the total number or schizont-irbc applied to the column resulted in higher fractions of schizont-irbc in total irbc. Total irbc purities ranged from 93.20 to 98.40% (Mean 95.69%, S.D. 1.52).

Figure 3

Isolation of segmented stage irbc by increased irbc loading of buffer optimized HGMS columns. In this representative experiment, 3 × 10⁹ rbc from an asynchronized culture containing app. 3% schizont irbc were applied to a HGMS column. Over 90% of segmented stage irbc are obtained, total irbc purity is >98%. Differential parasitemias were counted microscopically (1000 cells per sample).

Figure 4

Infection rate of re-cultured HGMS purified parasites. Initial parasitemias of re-cultures were adjusted between 0.12% and 2.58%. Infection rate was calculated assuming 10 merozoites per schizont. The apparent influence of initial parasitemia on re-infection rate was not significant.

Figure 5

Comparison of culture development after HGMS-depletion and sorbitol lysis. Bars represent the fraction of ring-stage irbc out of total irbc. Percentage of ring-stage irbc decrease over time from 97.7% for HGMS depletion and 98.9% for sorbitol lysis on day 0 (day of experiment and re-culture) to 79.3% for HGMS depletion and 84.8% for sorbitol lysis on day 6. No significant difference was observed between both methods. Data are from 5 independent experiments for HGMS-depletion and from 3 independent experiments for sorbitol lysis.