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

Dai Trang,Tohru Kariu,Nguyen Huy,Kunihiko Tajima,Kaeko Kamei

doi:10.1186/1475-2875-3-7

Dai Trang, Tohru Kariu + Show 3 more

Open Access

https://doi.org/10.1186/1475-2875-3-7

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Journal: Malaria journal	Publication Date: Jan 1, 2004
Citations: 129	License type: cc-by

Affiliation: Kyoto Institute of Technology

Abstract

BackgroundIsolation 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.MethodsThe 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.ResultsThe 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.ConclusionThe 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.

Highlights

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 study further indicates that a single step using only a commercial magnetic LD column (MACS) can concentrate Plasmodium berghei PRBCs and remove almost all of the contaminating WBCs
This study compared the efficiency of three methods for the concentration and purification of PRBCs: density gradient centrifugation methods using either Percoll or Nycodenz and a magnetic method using an LD column

Summary

Introduction

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. The combined use of the two steps produces sufficient PRBCs, but the method is laborious

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