Pitting of malaria parasites and spherocyte formation

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/mu L) that of RESA+, PI+ RBCs ( 28,365/mu L) ( 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.