Poorly deformable RBC flow-through microbeads: setup and results. Metal beads from a single batch were purchased as calibrated mixtures of 5- to 15-μm and 15- to 25-μm-diameter (IPS Industrie des Poudres Sphériques). Optimal results were obtained with a 50/50 mixture of 5- to 15-μm and 15- to 25-μm-diameter beads deposited onto the antiaerosol filter of a commercial 1000-μL tip. (A) Decantation of microbeads after their introduction into an inverted 1000-μL tip, leading to the formation of a 5-mm-thick bead layer (black arrow) above the antiaerosol filter (Ai1). RBCs from a volunteer or a patient blood or from a P falciparum culture were resuspended at 2% hematocrit in medium (PBS/1% albumin) and then introduced in the tubing upstream from the microbead layer (①). An electric pump containing medium was then immediately switched on (②), gently flowing RBCs through the microbead layer (③). On rinsing of the microbead layer with 7 mL of medium, the downstream sample was retrieved (④). The RBC subset retained in the microbead layer at the end of the rinsing procedure was separated from the microbeads by a 3-step decantation procedure. The proportion of rigid RBCs in “upstream,” “downstream,” and “retained” RBC samples was determined by PKH or Giemsa staining. (B) Retention rate of heated, HS, and Pf-RBCs: Heated normal RBCs (50°C for 20 minutes) or HS-RBCs were PKH-labeled and retention rate expressed as the Δ = {[(% of PKH-positive RBC in downstream sample) − (% of PKH-positive RBC in upstream sample)]/(% of PKH-positive in the upstream sample)} ×100. The same procedure was applied to determine parasitized RBC retention (parasitemia determined on Giemsa-stained blood films). Retention in the microbeads was partial at the ring stage (R), subtotal at the trophozoite stage (T), and complete at the schizont stage (S). When ring-RBCs from patients' blood were immediately processed (ex vivo ring), retention rate was low; but on culture to the next generation of ring-RBCs (cultured ring), the mean retention rate was similar to that observed with reference laboratory isolates maintained in long-term culture. (C-G) Typical upstream to downstream decrease of heated (C1-C2), or parasitized (D-E) RBC proportion as observed by PKH fluorescence or on Giemsa-stained smears. (F) Correlation rate between elongation index (LORCA) and retention rate for HS-RBCs.