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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

Simultaneous determination of phagocytosis of Plasmodium falciparum-parasitized and non-parasitized red blood cells by flow cytometry

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Valentina Gallo, Oleksii A Skorokhod, Evelin Schwarzer, Paolo Arese
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-428) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

VG, PA, OAS and ES designed experiments; VG and OA performed experiments; VG and PA drafted the manuscript; all authors discussed experiments and results and commented on the manuscript. All authors read and approved the final manuscript.

Abstract

Background

Severe falciparum malaria anaemia (SMA) is a frequent cause of mortality in children and pregnant women. The most important determinant of SMA appears to be the loss of non-parasitized red blood cells (np-RBCs) in excess of loss of parasitized (p-) RBCs at schizogony. Based on data from acute SMA where excretion of haemoglobin in urine and increased plasma haemoglobin represented respectively less than 1% and 0.5% of total Hb loss, phagocytosis appears to be the predominant mechanism of removal of np- and p-RBC.
Estimates indicate that np-RBCs are cleared in approximately 10-fold excess compared to p-RBCs. An even larger removal of np-RBCs has been described in vivax malaria anaemia. Estimates were based on two single studies both performed on neurosyphilitic patients who underwent malaria therapy. As the share of np-RBC removal is likely to vary between wide limits, it is important to assess the contribution of both np- and p-RBC populations to overall RBC loss, and disclose the mechanism of such variability. As available methods do not discriminate between the removal of np- vs p-RBCs, the purpose of this study was to set up a system allowing the simultaneous determination of phagocytosis of p- and np-RBC in the same sample.

Methods and Results

Phagocytosis of p- and np-RBCs was quantified in the same sample using double-labelled target cells and the human phagocytic cell-line THP-1, pre-activated by TNF and IFNγ to enhance their phagocytic activity. Target RBCs were double-labelled with fluorescent carboxyfluorescein-succinimidyl ester (CF-SE) and the DNA label ethidium bromide (EB). EB, a DNA label, allowed to discriminate p-RBCs that contain parasitic DNA from the np-RBCs devoid of DNA. FACS analysis of THP-1 cells fed with double-labelled RBCs showed that p- and np-RBCs were phagocytosed in different proportions in relation to parasitaemia.

Conclusions

The assay allowed the analysis of phagocytosis rapidly and with low subjective error, and the differentiation between phagocytosed p- and np-RBCs in the same sample. The presented method may help to analyse the factors or conditions that modulate the share of np-RBC removal in vitro and in vivo and lead to a better understanding of the pathogenesis of SMA.
Zusatzmaterial
Additional file 1: Optimization of phagocytosis assay. (A) Dependence of phagocytosis on phagocytosis time. Cell ratio: 50 np-RBCs per THP-1 cell. (B) Dependence of phagocytosis on np-RBCs/THP-1 cell ratio. Phagocytosis time: 150 min. CF-SE-labelled, IgG anti-D-opsonized np-RBCs were exposed at indicated cell ratios to pre-activated THP-1 cells for various phagocytosis periods. Phagocytosis is expressed as percentage of phagocytically-active THP-1 (phTHP-1). Mean values ± SD (N = 4). For details, see Methods. (JPEG 53 KB)
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