Abstract
BackgroundInvestigations of Plasmodium vivax are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in in vitro cultures. Contamination of P. vivax isolates with host leukocytes and platelets is detrimental to a range of ex vivo and molecular investigations. Easy-to-produce CF11 cellulose filters have recently provided us with an inexpensive method for the removal of leukocytes and platelets. This contrasted with previous reports of unacceptably high levels of infected red blood cell (IRBC) retention by CF11. The aims of this study were to compare the ability of CF11 cellulose filters and the commercial filter Plasmodipur at removing leukocyte and platelet, and to investigate the retention of P. vivax IRBCs by CF11 cellulose filtration.Methods and ResultsSide-by-side comparison of six leukocyte removal methods using blood samples from five healthy donor showed that CF11 filtration reduced the mean initial leukocyte counts from 9.4 × 103 per μl [95%CI 5.2–13.5] to 0.01 × 103 [95%CI 0.01–0.03]. The CF11 was particularly effective at removing neutrophils. CF11 treatment also reduced initial platelet counts from 211.6 × 103 per μl [95%CI 107.5–315.7] to 0.8 × 103 per μl [95%CI -0.7–2.2]. Analysis of 30 P. vivax blood samples before and after CF11 filtration showed only a minor loss in parasitaemia (≤ 7.1% of initial counts). Stage specific retention of P. vivax IRBCs was not observed.ConclusionCF11 filtration is the most cost and time efficient method for the production of leukocyte- and platelet-free P. vivax-infected erythrocytes from field isolates.
Highlights
Investigations of Plasmodium vivax are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in in vitro cultures
The two aims of this study were to compare the efficacy of leukocyte and platelet removal efficacy of CF11-based filters and of Plasmodipur, and to investigate whether stage specific retention of P. vivax infected red blood cell (IRBC) occurs through CF11 cellulose filtration
By pre-processing the blood sample with Lymphoprep prior to Plasmodipur filtration was it possible to reduce the mean leukocyte (0.3 × 103 per μl [95%CI -1.1–1.8]) and platelet (0.0 × 103 per μl) contamination to the same levels as that obtained by CF11 filtration
Summary
Investigations of Plasmodium vivax are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in in vitro cultures. Easy-to-produce CF11 cellulose filters have recently provided us with an inexpensive method for the removal of leukocytes and platelets. This contrasted with previous reports of unacceptably high levels of infected red blood cell (IRBC) retention by CF11. Exploiting the recently published genome[3] and transcriptome[4] of Plasmodium vivax still relies on the use of infected blood samples collected from patients or experimentally infected simians, because it is not yet possible to continuously culture this parasite. Platelets often bind to and degranulate on contact with infected red blood cells (IRBCs) adversely affecting the parasite's ex vivo development[7]
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