Pitting of malaria parasites and spherocyte formation

Abstract

Background: A high prevalence of spherocytes was detected in blood smears of children enrolled in a case control study conducted in the malaria holoendemic Lake Victoria basin. It was speculated that the spherocytes reflect intraerythrocytic removal of malarial parasites with a concurrent removal of RBC membrane through a process analogous to pitting of intraerythrocytic inclusion bodies. Pitting and re-circulation of RBCs devoid of malaria parasites could be a host mechanism for parasite clearance while minimizing the anaemia that would occur were the entire parasitized RBC removed. The prior demonstration of RBCs containing ring-infected erythrocyte surface antigen (pf 155 or RESA) but no intracellular parasites, support the idea of pitting.

Methods: An in vitro model was developed to examine the phenomenon of pitting and spherocyte formation in Plasmodium falciparum infected RBCs (iRBC) co-incubated with human macrophages. In vivo application of this model was evaluated using blood specimens from patients attending Kisumu Ditrict Hospital. RBCs were probed with anti-RESA monoclonal antibody and a DNA stain (propidium iodide). Flow cytometry and fluorescent microscopy was used to compare RBCs containing both the antigen and the parasites to those that were only RESA positive.

Results: Co-incubation of iRBC and tumor necrosis factor-alpha activated macrophages led to pitting (14% +/- 1.31% macrophages with engulfed trophozoites) as opposed to erythrophagocytosis (5.33% +/- 0.95%) (P < 0.01). Following the interaction, 26.9% +/- 8.1% of the RBCs were spherocytes as determined by flow cytometric reduction in eosin-5-maleimide binding which detects RBC membrane band 3. The median of patient RBCs with pitted parasites (RESA+, PI-) was more than 3 times (95,275/muL) that of RESA+, PI+ RBCs (28,365/muL) (P < 0.01). RBCs with pitted parasites showed other morphological abnormalities, including spherocyte formation.

Conclusion: It is proposed that in malaria holoendemic areas where prevalence of asexual stage parasites approaches 100% in children, RBCs with pitted parasites are re-circulated and pitting may produce spherocytes.

Figure 1

Extraction of P. falciparum and pitting of infected red cells by THP-1 monocytes. Trophozoite infected human group O⁺ red cells (1 × 10⁶) in RPMI 1640 medium were co-incubated with TNF-α activated THP-1 monocytes (3 × 10⁶) for 1 hour at 37°C. Giemsa stained thin smears of the cultures were prepared and examined under oil immersion using ×100/1.3 objective of an Olympus microscope (Olympus America, NY). Pitting was preferred to erythrophagocytosis (panel A). The photomicrographs show monocytes with six pitted malaria parasites (B), phagocytosed infected RBCs (C) and spherocytic RBCs (D, arrow). Photomicrographs were captured using an Olympus MagnaFire camera (Olympus America, NY) using MagnaFire acquisition software.

Figure 2

Flow cytometric analysis of eosin-5-maleimide (EMA)-labelled P. falciparum infected (iRBC) and unifected RBC (nRBC) before and after interactions with THP-1 monocytes. Trophozoite infected human group O⁺ red cells (1 × 10⁶) in RPMI 1640 medium were co-incubated with TNF-α activated THP-1 monocytes (3 × 10⁶) for 1 hour at 37°C. Following the interaction, the cell mixture was stained with EMA by incubation in the dark for one hour at room temperature. Panel A is an overlay histogram of fluorescent profile of EMA labeled iRBC before (dashed lines) and after interaction (continuous lines) for the best of the 6 experiments. Panel B is a bar graph of median channel fluorescence (MCF, ± SEM) for 6 experiments of iRBC and nRBC following the indicated treatments.

Figure 3

Flow cytometric analysis of patient RBCs labeled with anti-RESA Mab and propidium iodide. Patient RBCs were fixed with 0.05% glutaldehyde for 30 min prior to permeabilization with 1% Saponin for 5 minutes at room temperature. After two washes, the cells were stained with either RESA Mab, propidium iodide or both. The cells were then washed twice with PBS and a FITC conjugated goat anti-mouse IgG-Fab fragment added and cells incubated for 30 minutes at room temperature. (A) Flow cytometry dot plots of RBC labeled with FITC-anti-RESA (FL1-positive) and propidium iodide (FL-3 positive) showing proportion of RBC with pitted cells (RESA+, PI-) and those with malaria parasites (RESA+, PI+). (B) Box plotting demonstrate higher proportions of the once infected RBCs (RESA+) compared to those with malaria parasites (PI+).

Figure 4

Fluorescence microscopy images illustrating RESA and propidium iodide staining of patient RBC. Thin blood smears were fixed with methanol and stained with either RESA Mab, propidium iodide or both. Images of the same field are shown in sets of three: a) green, b) red, and c) red + green composite. The number of RBCs showing pitted parasites (RESA+, panel A) were more than those with patent parasitaemia (PI+, panel B) and panel C. RBCs with pitted parasites showed other morphological abnormalities, including spherocyte formation (D).