This study describes a rapid, sensitive, reproducible and accurate assay to quantify phagocytosis of p- and np-RBCs in the same sample using CF-SE- and EB-labelled target cells and the human phagocytic monocyte cell line THP-1. Phagocytes were previously activated with TNF and IFNγ to upregulate Fc- and complement receptors and enhance phagocytic efficiency without loss of specificity [10, 11]. Target RBCs were double-labelled with CF-SE and EB. CF-SE is the fluorescent derivative of CFDA-SE, a non-fluorescent lipophilic molecule that passively diffuses into the cell where it is activated by esterase cleavage of its acetyl groups to the brightly fluorescent derivative CF-SE. The latter, a non-toxic molecule, is stably retained in the cell-forming covalent conjugates to free amino groups, emits stable and homogeneous fluorescence and does not interfere with RBC functionality [7, 15]. CF-SE does not localize in cell membranes and does not elicit membrane alterations that may induce phagocytic recognition of the labelled cells. EB, a widely used DNA fluorescent label, allows to discriminate p-RBCs that contain parasitic DNA from the np-RBCs totally devoid of DNA.
The phagocytic efficiency of the THP-1 cells was checked with np-RBCs modified by two treatments known to maximally enhance phagocytosis: opsonization with anti-D IgG to stimulate IgG-mediated phagocytosis, and RBCs treatment with zinc-BS3, that generates stable band-3 clusters and induce a predominantly IgG/complement-mediated phagocytosis . Phagocytosis by activated THP-1 cells was lower compared to phagocytosis by adherent human monocytes [9, 16]. However, THP-1 cells displayed the same specificity and relative phagocytic activity with respect to p-RBCs as human monocytes, while presenting a number of advantages, such as independence from availability of human buffy-coats, constant performance, low cost and easy maintenance.
Removal of large numbers of np-RBCs accompanies the rupture of p-RBCs at schizogony, as shown by the simultaneous presence of p-and np-RBCs in the same phagocytic cell in peripheral blood and organ phagocytes [17–19] and by studies on the clearance kinetics of p- and np-RBCs [20, 21]. It is generally maintained that np-RBCs are cleared in approximately 10-fold excess compared to elimination of p-RBC at schizogony. However, this estimate is based on a single study , performed by modelling 12 neurosyphilitic patients who underwent malaria therapy. Those authors observed a substantial destruction of np-RBCs occurring during two cycles of parasitaemia with peak values at 20,000-40,000 parasites per μl and estimated that an average of 8.5 np-RBCs were destroyed per rupturing schizont. An analogous study  has described an even larger excess removal of np-RBCs in vivax malaria anaemia, a frequent and severe complication of vivax infection occurring by largely undisclosed mechanism [23, 24].
Apart from RBC lysis at schizogony, it is assumed that phagocytosis is the predominant mechanism of removal of np- and p-RBC. This assertion is based on a balance study in acute SMA where a quantitative comparison was performed between the loss of blood haemoglobin (Hb) and increase of Hb in plasma and urine. Increase in plasma Hb was less than 1% and excretion of Hb in urine was less than 0.5% of total Hb loss . This data tends to exclude complement lysis  and is in line with the common paradigm of phagocytic removal of senescent, variously damaged RBCs and falciparum p-RBCs as the consequence of membrane damage and opsonization through enhanced binding of complement factor C3b and (anti-band 3)-IgG [9, 27, 28].
Most available cytofluorimetric methods (for example: [14, 29]) do not allow to discriminate the relative contribution of np- vs p-RBC removal. One exception, however, is the method by Tippett et al designed to assess phagocytosis by patient monocytes or by THP-1 cells of p- and np-RBCs simultaneously labelled by EB and fluorescein isothiocyanate (FITC). Comparison of our results with Tippett et al’ is difficult because they used unstimulated THP-1 cells with low phagocytic activity and FITC, a membrane-standing molecule known to interfere with RBC membrane transport systems and surface carbohydrates [31, 32]. In few cases though, a comparison was possible. For example, the percentage of phagocytically active cells was between nil and 10% in unstimulated , and 45% in pre-stimulated [this study] THP-1 cells when both TPH-1 cells were challenged with serum-opsonized p-RBCs. Higher phagocytic activity was also noted when RBC suspensions with 3-8% p-RBCs were challenged by unstimulated  or pre-stimulated [this study] THP-1 cells. Phagocytically active cells were approx. 2% in unstimulated and approx. 24% pre-stimulated THP-1 cells.
In conclusion, due to the lack of reliable quantitative data on the relative role of phagocytosis of p- vs np-RBCs, the method presented here may help to fill gaps particularly in the pathogenesis of SMA. For example, it will be useful to analyse the factors or conditions that modulate the share of np-RBC removal in vitro and in vivo. It appears that np-RBC removal may vary within broad limits as documented in human SMA and in humanized murine models and thus be an important determinant of SMA [33, 34]. The causes for this variability are unknown. Possible factors are bystander modifications that induce RBC membrane modifications, transfer of toxic parasite-produced molecules in rosettes , malaria- or age-related variations in phagocytosis-enhancing complement factors [26, 36] and surface IgG , and upregulation of phagocytic activity of host phagocytes . Phagocytosis studies ex vivo may also help to clarify the mechanism of malaria protection against severe anaemia observed in correlative studies in alpha-thalassaemia patients characterized by minimal alterations of RBC morphology, functionality and RBC lifespan in the np-status.