One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells

BACKGROUND: Isolation of a concentrated, living preparation of malarial parasite-infected red blood cells (PRBCs) that have low contamination of white blood cells (WBCs) facilitates research on the molecular, biochemical and immunological aspects of malarial parasites. This is currently carried out by a two-step method, including the concentration of PRBCs using density gradient centrifugation through Percoll or Nycodenz, followed by the removal of host WBCs using a cellulose powder column or a commercially available filtration unit. These two-step methods can help isolate sufficient PRBCs, but they are laborious. In this study, a simplified one-step procedure that takes advantage of the difference between diamagnetic low-spin oxyhaemoglobin and paramagnetic haemozoin (haem polymer) was described. The paramagnetic polymer is deposited in the food vacuoles of the parasite, allowing the use of magnetic separation to efficiently and rapidly concentrate PRBCs while removing contaminating host WBCs. METHODS: The magnetic removal of WBCs using a commercial LD column (MACS) was evaluated as a new method for concentrating and purifying PRBCs. To compare this method with the two density gradient centrifugation methods using Percoll or Nycodenz, we analysed the quantities of enriched PRBCs and contaminating host WBCs as well as the viability of malarial parasites in the final preparations. RESULTS: The quantity of PRBCs and the viability of malarial parasites in the isolated PRBCs were similar between magnetic and centrifugation methods. However, 90–99% of the contaminating WBCs were removed from the starting material using a magnetic column, whereas WBC content did not change using the Percoll or Nycodenz methods. CONCLUSION: The use of a commercially available magnetic LD column is effective, safe and easy for the one-step purification of PRBCs. This simple method does not affect the viability of malarial parasites.