Plasmion

The parasite culture was centrifuged at 500g for 5 min and the pellet resuspended in a ratio of 2 volumes pellet to 3 volumes RPMI-based growth media without human serum (incomplete medium) and 5 volumes Plasmion (Fresenius Kabi), and allowed to settle for 20–30 min at 37 °C. Trophozoite stage pRBC in the top layer were then washed three times in incomplete medium and the parasitaemia assessed by Giemsa-stained smear.

Asexual blood-stage 3D7 P. falciparum parasites were cultured as previously described in Lopez-Rubio et al (link)and W.H.O. (2021) . Parasites were cultured in human RBCs (obtained from the Etablissement Français du Sang with approval number HS 2016-24803) in RPMI-1640 culture medium (11875; Thermo Fisher Scientific) supplemented with 10% vol/vol Albumax I (11020; Thermo Fisher Scientific), hypoxanthine (0.1 mM final concentration, C.C.Pro Z-41-M), and 10 mg gentamicin (G1397; Sigma-Aldrich) at 4% hematocrit and under 5% O₂ and 3% CO₂ at 37°C. Parasite development was monitored by Giemsa staining. Parasites were synchronized by sorbitol (5%, S6021; Sigma-Aldrich) lysis at the ring stage, plasmagel (Plasmion; Fresenius Kabi) enrichment of late stages 24 h later, and an additional sorbitol lysis 6 h after plasmagel enrichment. Parasites were cultured under static conditions with the exception of shaking during the late schizont until an early ring stage. Parasites were harvested at 1–5% parasitemia.

Trained operators (competence in advanced critical care echocardiography) performed transthoracic echocardiography in the supine position at baseline, and during iNO and almitrine administration. They focused on global function (velocity–time integral of left ventricular outflow tract, cardiac index), and the right ventricle function as previously proposed [14 (link)]. Because of severe hypoxia, all patients had a detection of potential shunting across patent foramen ovale in four-chamber view after injection of sterile-modified fluid gelatine solution (Plasmion, Fresenius-Kabi, Sevres, France) aerated with room air to generate microbubbles as previously proposed [15 (link)].

50 µl Protein A Dynabeads (Invitrogen) were washed three times with 500 µl 1% BSA/PBS, using a magnet to retain the beads each time. The beads were resuspended in 1% BSA/PBS and 2.5 µg ICAM-1^D1D5 (an Fc fusion protein of ICAM-1 domains 1–5^{25 (link)}) added to a final volume of 400 µl and incubated at room temperature with 15 rpm rotation for 1 hour. The beads were then washed three times with 500 µl 1% BSA/PBS using a magnet to retain the beads and resuspended in 200 µl 1% BSA/PBS. Parasite cultures were enriched for trophozoite stage using Plasmion (Fresenius Kabi France) by standard techniques⁵⁶ . Enriched parasites were resuspended in 200 µl 1% BSA/PBS and added to the ICAM-1^D1D5 labelled Dynabead suspension. The mixture was rotated at 15 rpm for 45 min at room temperature. Two washes were carried out with 500 µl 1% BSA/PBS to remove unbound parasites. The beads were then transferred to a new flask and cultured as described.

This prospective two-center study received the regional ethics committee of Nord-Pas-De-Calais, France approval (No. 2011-A00990-41). All subjects received oral and written information and provided written consent prior to study enrollment. All examinations were performed in semirecumbent position with the trunk elevated 30°–45° from the horizontal lower limbs. Ultrasonographic and clinical data were recorded immediately before and after VE, performed as a 30-min infusion of 500 mL 4% gelatin (Gelofusine^® 4%, B. Braun, Melsungen, Germany or Plasmion^®, Fresenius-Kabi, Louviers, France). Relative changes in velocity time integral of aortic blood flow (VTIao) induced by VE were calculated using the formula: VE-induced change in VTIao (%) = 100 × (post-VE value − baseline value)/baseline value. Patients were classified as responders if VTIao increased by ≥ 10%, and nonresponders if VTIao increased by < 10% after VE. This threshold value seemed clinically relevant (i.e., in terms of VE-induced changes in systolic arterial pressure and pulse pressure) and was at least twice the intra-observer variability of the VTIao measured in our previous study [15 (link)].

Blood stage 3D7 P. falciparum parasites were cultured as previously described in Lopez‐Rubio et al (2009 (link)). Briefly, parasites were cultured in human RBCs supplemented with 10% v/v Albumax I (Thermo Fisher 11020), hypoxanthine (0.1 mM final concentration, C.C.Pro Z‐41‐M) and 10 mg gentamicin (Sigma G1397) at 4% hematocrit and under 5% O2, 5% CO₂ at 37°C. Parasites were synchronized by sorbitol (5%, Sigma S6021) lysis during ring stage followed by a plasmagel (Plasmion, Fresenius Kabi) enrichment for late blood stages 24 h later. Another sorbitol treatment 6 h afterward places the 0 h time point 3 h after the plasmagel enrichment. Thus, the window of synchronicity for cultures is ± 3 h. Parasite development was monitored by Giemsa staining. Parasites were harvested at 1–5% parasitemia.