The current paradigm in Plasmodium falciparum malaria pathogenesis states that young, ring-infected erythrocytes (rings) circulate in peripheral blood and that mature stages are sequestered in the vasculature, avoiding clearance by the spleen. Through ex vivo perfusion of human spleens, we examined the interaction of this unique blood-filtering organ with P falciparum-infected erythrocytes. As predicted, mature stages were retained. However, more than 50% of rings were also retained and accumulated upstream from endothelial sinus wall slits of the open, slow red pulp microcirculation. Ten percent of rings were retained at each spleen passage, a rate matching the proportion of blood flowing through the slow circulatory compartment established in parallel using spleen contrast-enhanced ultrasonography in healthy volunteers. Rings displayed a mildly but significantly reduced elongation index, consistent with a retention process, due to their altered mechanical properties. This raises the new paradigm of a heterogeneous ring population, the less deformable subset being retained in the spleen, thereby reducing the parasite biomass that will sequester in vital organs, influencing the risk of severe complications, such as cerebral malaria or severe anemia. Cryptic ring retention uncovers a new role for the spleen in the control of parasite density, opening novel intervention opportunities. (Blood. 2008;112:2520-2528) IntroductionThe pathogenesis of malaria involves multiple parasite and host factors. 1 Spleen filtering and immune functions have a major impact on the course of plasmodial infection in experimental models. 2,3 In malaria-endemic countries, splenectomy predisposes to fever, to more frequent and higher parasitemia (including circulating mature forms), and may reactivate latent plasmodial infections. 4,5 Despite relatively few published data (reviewed by Bach et al 5 ), clinicians include malaria in the list of infectious diseases justifying increased awareness in splenectomized nonimmune patients. 6 Because key features may differ between animal and human plasmodial infection and because detailed exploration of the human spleen is limited by ethical and technical constraints, 7 the fine interactions between Plasmodium falciparum-infected red blood cells (iRBCs) and the human spleen microcirculatory structures have been explored only indirectly 8,9 or postmortem. 10 Therefore, the mechanisms underlying the putative spleen protective or pathogenic effects during human malaria remain essentially speculative.The architecture of the spleen red pulp (RP) permits intimate scrutiny of red blood cells (RBCs), leading to selective retention of abnormal or senescent RBCs within the RP. 11 To reenter the venous system, RBCs leaving the reticular meshwork of the RP must cross the narrow interendothelial slits in walls of the venous sinuses. This process requires RBCs to undergo considerable deformation: if cells are not sufficiently deformable, they are retained upstream from the venous sinus wall. 11 Such RBC-processin...

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The spleen plays a central role in the pathophysiology of several potentially severe diseases such as inherited red cell membrane disorders, hemolytic anemias, and malaria. Research on these diseases is hampered by ethical constraints that limit human spleen tissue explorations. We identified a surgical situation-left splenopancreatectomy for benign pancreas tumors-allowing spleen retrieval at no risk for patients. Ex vivo perfusion of retrieved intact spleens for 4 to 6 hours maintained a preserved parenchymal structure, vascular flow, and metabolic activity. Function preservation was assessed by testing the ability of isolatedperfused spleens to retain Plasmodium falciparum-infected erythrocytes preexposed to the antimalarial drug artesunate (Art-iRBCs). More than 95% of Art-iRBCs were cleared from the perfusate in 2 hours. At each transit through isolated-perfused spleens, parasite remnants were removed from 0.2% to 0.23% of Art-iRBCs, a proportion consistent with the 0.02% to 1% pitting rate previously established in artesunatetreated patients. Histologic analysis showed that more than 90% of Art-iRBCs were retained and processed in the red pulp, providing the first direct evidence of a zonedependent parasite clearance by the human spleen. Human-specific physiologic or pathophysiologic mechanisms involving clearing or processing functions of the spleen can now be experimentally explored in a human tissue context. IntroductionThe spleen plays a key role in innate and adaptative immune responses, in selective clearance of aged or abnormal red blood cells (RBCs), and in removal of pathogens present in the blood. 1 It is also central to the pathophysiology of several frequent and potentially severe diseases such as inherited red cell membrane disorders, hemolytic anemias, and malaria. [2][3][4][5] Detailed studies on the mechanisms involved are hampered by 2 linked factors: the human spleen displays anatomic and physiologic features not observed in rodent models, and the human spleen is difficult to explore. 1,6 The risk of life-threatening intraperitoneal bleeding makes spleen biopsy or needle-aspirate an unethical method for human research purposes. [7][8] Histology studies from postmortem or splenectomy samples provide crucial information, but their scope is limited to late or complicated disease stages. 9-10 So far, explorations of the human spleen have been restricted to "black box" approaches, in which intrasplenic tissue processing was inferred from the kinetics of elements circulating in the peripheral blood. [11][12][13][14][15][16][17] Humanized mouse models provide an interesting research tool, but are limited by the rapid clearance of normal human RBCs and by the different spleen anatomy, vascularization/circulatory bed, and cellular subsets compared with humans. 1,6,[18][19][20][21] The spleen is one of the few organs that may be removed while healthy. Left pancreas resection for tumor removal usually includes a splenectomy for vascular-related constraints. 22 Retrieval of intact spleen is then pos...