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


CONCLUSIONS
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.
Bhakdi
Sebastian C
SC
Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand. sbhakdi@gmail.com
Ottinger
Annette
A
Somsri
Sangdao
S
Sratongno
Panudda
P
Pannadaporn
Peeranad
P
Chimma
Pattamawan
P
Malasit
Prida
P
Pattanapanyasat
Kovit
K
Neumann
Hartmut P H
HP
D000818:Animals; D002448:Cell Adhesion; D002469:Cell Separation / Q000379:methods*; D004903:Erythrocyte Aggregation; D004912:Erythrocytes / Q000502:physiology*; D005434:Flow Cytometry; D008280:Magnetics / Q000295:instrumentation*; D008288:Malaria / Q000097:blood* / Q000469:parasitology; D009996:Osmotic Fragility; D010316:Particle Size; D010961:Plasmodium / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification*; D010963:Plasmodium falciparum / Q000254:growth & development / Q000302:isolation & purification*; D052939:Schizonts / Q000502:physiology