The permanent availability of RBC for transfusion depends on refrigerated storage, during which morphologically-altered RBC accumulate. Among those, a subpopulation of small RBC, comprising echinocytes III, sphero-echinocytes, and spherocytes, and defined as "storage-induced micro-erythrocytes" (SME) could be rapidly cleared from the circulation after transfusion. We quantified the proportion of SME in RBC concentrates from healthy human volunteers and assessed their correlation with transfusion recovery. We then investigated the fate of SME upon perfusion through human spleens ex vivo. Finally, we explored where and how SME are cleared in a mouse model of blood storage and transfusion. In healthy human volunteers, high proportions of SME in long-stored RBC concentrates correlated with poor transfusion recoveries. When perfused through human spleens, 15% and 61% of long-stored RBC and SME were cleared in 70 minutes, respectively. High initial proportions of SME also correlated with high retention of RBC by perfused human spleens. In the mouse model, SME accumulated during storage. Transfusion of long-stored RBC displayed reduced post-transfusion recovery, which was mostly due to clearance of SME. Following transfusions of mice, long-stored RBC accumulated predominantly in the spleen, and were ingested mainly by splenic and hepatic macrophages. In macrophage-depleted mice, splenic accumulation and the clearance of SME were delayed and transfusion recovery was improved. In healthy hosts, SME are cleared predominantly by macrophages in the spleen and liver. When this well-demarcated subpopulation of altered RBC is abundant in RBC concentrates, transfusion recovery is diminished. Quantifying SME has the potential to improve blood product quality assessment.

In acute malaria, the bulk of erythrocyte loss occurs after therapy, with a nadir of hemoglobin generally observed 3–7 days after treatment. The fine mechanisms leading to this early post-treatment anemia are still elusive. We explored pathological changes in RBC subpopulations by quantifying biochemical and mechanical alterations during severe malaria treated with artemisinin derivatives, a drug family that induce “pitting” in the spleen. In this study, the hemoglobin concentration dropped by 1.93 G/dl during therapy. During the same period, iRBC accounting for 6.12% of all RBC before therapy (BT) were replaced by pitted-RBC, accounting for 5.33% of RBC after therapy (AT). RBC loss was thus of 15.9%, of which only a minor part was due to the loss of iRBC or pitted-RBC. When comparing RBC BT and AT to normal controls, lipidomics revealed an increase in the cholesterol/phosphatidylethanolamine ratio (0.17 versus 0.24, p < 0.001) and cholesterol/phosphatidylinositol ratio (0.36 versus 0.67, p = 0.001). Using ektacytometry, we observed a reduced deformability of circulating RBC, similar BT and AT, compared to health control donors. The mean Elongation Index at 1.69Pa was 0.24 BT and 0.23 AT vs. 0.28 in controls (p < 0.0001). At 30Pa EI was 0.56 BT and 0.56 AT vs. 0.60 in controls (p < 0.001). The retention rate (rr) of RBC subpopulations in spleen-mimetic microsphere layers was higher for iRBC (rr = 20% p = 0.0033) and pitted-RBC (rr = 19%, p = 0.0031) than for healthy RBC (0.12%). Somewhat surprisingly, the post-treatment anemia in malaria results from the elimination of RBC that were never infected.

Background Intravenous artesunate is the WHO-recommended first-line treatment for severe malaria worldwide, but it is still not fully licensed in Europe. Observational studies documenting its safety and efficacy in imported malaria are thus essential. Methods We prospectively collected clinical and epidemiological features of 1391 artesunate-treated patients among 110 participant centers during the first seven years (2011-2017) of a national program implemented by the French Drug Agency. Results Artesunate became the most frequent treatment for severe malaria in France rising from 9.9% in 2011 to 71.4% in 2017. Mortality was estimated at 4.1%. Treatment failure was recorded in 27 patients but mutations in the Kelch-13 gene were not observed. Main reported adverse events (AE) were anemia (136 cases), cardiac events (24, including 20 episodes of conduction disorders and/or arrhythmia) and liver enzyme elevation (23). Mortality and AE were similar in the general population and in HIV-infected, overweight or pregnant patients, but the only pregnant woman treated in the first trimester experimented a hemorrhagic miscarriage. The incidence of post-artesunate delayed hemolysis (PADH) was 42.8% when specifically assessed in a 98-patient subgroup but was not associated with fatal outcomes or sequelae. PADH was twice as frequent in patients of European compared to African origin Conclusion Artesunate was rapidly deployed and displayed a robust clinical benefit in patients with severe imported malaria, despite a high frequency of mild to moderate PADH. Further explorations in the context of importation should assess outcomes during the first-trimester of pregnancy and collect rare but potentially severe cardiac AE.

Pitting, the removal of dead parasites from their host erythrocyte, has been studied in patients with severe malaria treated parenterally with quinine or artesunate, and was recently shown to contribute to delayed hemolysis, a frequent adverse event of artesunate. We quantified pitting in 81 travelers treated with oral antimalarial therapy. Pitting rate was high (55.8%) with artemisinin-based combinations, but <10% with the nonartemisinin drugs quinine, mefloquine, and atovaquone-proguanil. This may, in part, explain the slower parasite clearance in patients treated with antimalarial drugs lacking an artemisinin component, as well as the absence of posttreatment hemolysis with these drugs.

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Background Hypothermic storage of red blood cell (RBC) concentrates for up to 42 days is associated with biochemical, molecular, morphological, and mechanical modifications. This "storage lesion" increases with storage duration and is associated with increased clearance of transfused storage-damaged RBCs from the recipient's circulation in the first few hours post-transfusion. This rapid clearance reduces transfusion efficacy, but how it occurs is not fully elucidated. RBCs with reduced surface area called "storage-induced micro-erythrocytes" (SMEs) were recently described. Their proportion increases from 2% to 23% during storage. Their reduced surface-to-volume ratio is expected to induce rapid mechanical clearance by the spleen. We aimed to evaluate whether SMEs can be used as a marker of transfusion efficacy, if this subpopulation of RBCs is preferentially cleared by the spleen after transfusion, and if so, by which mechanisms. Methods We evaluated the proportion of SMEs in stored RBC concentrates in vitro using ImageStream and correlated it to the 51Chromium-labeled 24h post-transfusion recovery (24hPTR) in vivo in 31 healthy human volunteers. We then investigated the fate of SMEs during 8 ex vivo perfusions of human spleens (16 RBC concentrates stored for 35-42 days). Finally, we developed a mouse transfusion model to assess the fate of SMEs in vivo and determine their main mechanisms of clearance. Results The proportion of SMEs in RBC concentrates at day 42 of storage correlated negatively with 24hPTR in healthy volunteers (r=-0.42, P&lt;0.01). When perfused ex vivo into human spleens, 15% of stored RBCs (35-42 days of storage) were cleared during the first 40 min of perfusion in a 2-step process: 7% of circulating RBCs disappeared in the first 2 min (1-2 passages through the spleen) while 8% were cleared between 10 and 40 min after initiating perfusion (&gt;5 passages through the spleen). The percentage of SMEs correlated with splenic retention rate ex vivo (r=0.46, p&lt;0.05). Morphological analysis of 6 stored RBC concentrates showed a mean decrease in the proportion of SMEs from 20.2% to 7.8% between the beginning and end of splenic perfusions. In our mouse transfusion model, SMEs accumulated during RBC storage. The 24hPTR also decreased with storage duration (64% on Day 14 vs. 95% on Day 1). The decrease in 24hPTR of long-stored RBCs was mostly due to clearance of the SME subpopulation. SME and morphologically normal long-stored RBC subpopulations displayed clearances of 83% and 13%, respectively. Stored RBCs accumulated predominantly in the spleen post-transfusion, and were mainly ingested by macrophages. In macrophage-depleted mice, 24hPTR improved (from 64% to 79%), splenic accumulation and clearance of SMEs were delayed, and the proportion of inflammatory monocytes increased and mediated clearance. In splenectomized mice, clearance of SMEs was not delayed, but increased accumulation was observed in the liver and bone marrow, and increased erythrophagocytosis by inflammatory monocytes was also observed. Conclusions We show that the proportion of SMEs correlates with 24hPTR in healthy human volunteers and with retention in human spleens perfused ex vivo. In vivo mouse data confirms these findings, showing that SMEs are cleared from the recipient circulation during the 24h following transfusion. Clearance of SMEs is delayed in macrophage-depleted mice, suggesting a central role of macrophages in this process. The human spleen is also likely to clear SMEs from the recipient's circulation, as suggested by experiments with human spleens perfused ex vivo. However, the spleen is not required, because SME clearance is not affected in splenectomized mice. This suggests that other organs may compensate to remove SMEs and highlights the importance of eliminating these morphologically-altered RBCs. Finally, quantification of SMEs is an operator-independent, reproducible marker of transfusion efficacy. It can be used to assess the potential of new processes to prepare and store RBC concentrates. Pre-transfusion quantification of SMEs could benefit chronically transfused patients, for whom improved transfusion efficacy is expected to reduce transfusion-induced iron overload. Disclosures Roussel: Zimmer Biomet: Research Funding. MARIN:Zimmer Biomet: Research Funding. Spitalnik:Hemanext: Membership on an entity's Board of Directors or advisory committees; Tioma, Inc.: Consultancy. Hermine:AB science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. Buffet:Zimmer Biomet: Research Funding. Amireault:Zimmer Biomet: Research Funding.

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