Isolation of late-stage Plasmodium falciparum infected red blood cells using a new cost- and time-saving magnetic cell separation kit
Abstract number: O414
Bhakdi S.C., Hartmann A., Sratogno P., Chuncharunee A., Neumann H.P., Malasit P., Pattanapanyasat K.
Objectives: In malaria research, a large variety of assays require highly purified infected red blood cells (iRBCs). Magnetic separation relies on intrinsic magnetic properties of trophozoite and schizont (late-stage) iRBCs and offers an alternative to conventional separation by Percoll® gradients. Concentration of late-stage iRBCs by high gradient magnetic separation (HGMS) was first reported in 1981. In the nineties, commercial HGMS columns with a polymer coated matrix became available for high purity separation of malaria-iRBCs. Unfortunately, high costs render their use unattractive for many laboratories. We adapted a new low-cost HGMS kit for isolation of late-stage iRBCs from Plasmodium falciparum cultures and compared costs to polymer-coated HGMS columns and separation with Percoll® gradients.
Methods: Isolation of late-stage iRBCs from P. falciparum TM 267 cultures was performed in a Skypure HGMS system. 70500 ml of packed RBCs with late-stage parasitaemias of 1.717.2% were used (total parasitaemias 8.529.9%). This corresponded to app. 750×108 total RBCs containing 2.649.4×107 late-stage iRBCs. Purification results were analysed by flow cytometer and microscopy. Viability was evaluated by calculation of infection rate after re-culture of isolates.
Results: Purity of iRBC isolates consistently ranged from 93.2% to 99.0% (mean 95.4%). Under optimised conditions, over 90% of isolated iRBCs contained segmented schizonts (Figure 1). Maximum column capacity was found to be 1 to 1.2×107 iRBCs. Processing time was less than 45 min. Infection rate ranged from 21.0% to 56.4%. Cost comparison showed 1.53 Euros for Percoll® gradient separation and 1820 Euros for HGMS systems employing polymer-coated columns, compared to 56 Euros for the HGMS system tested in this study (cost of consumables per separation of 200 ml packed cells from malaria cultures).
Conclusion: Compared to separation by Percoll® gradient, the new kit is about 30% more time efficient and offers highly consistent results, even for isolation of segmented schizont iRBCs. When compared to polymer coated columns, it offers app. 70% cost savings for consumables. Therefore, the HGMS kit examined is considered highly suitable for purification of late-stage iRBCs.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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