2.2. Hematological and Oxygenscan Parameters

Hb concentration, mean cell volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and percentage of reticulocytes (Retic) were determined with a hematology analyzer (Advia, Siemens, Rungis, France). Lactate dehydrogenase levels (LDH) were determined by standard biochemical method. Blood viscosity was measured at native hematocrit (Hct), 25 °C, and at a shear rate of 90 s⁻¹ using a cone/plate viscometer (Brookfield DVII+ with CPE40 spindle, Brookfield Engineering Labs, Natick, MA, USA). Ektacytometry was carried out with the laser-assisted optical rotational red cell analyzer (Lorrca, RR Mechatronics, The Netherlands) with the oxygenscan module to measure RBC deformability over an oxygen gradient. A volume of 50 µL of blood, standardized to a fixed RBC count of 200 × 10⁶, was mixed with 5 mL of high viscous (30 cP) Oxy-Iso polyvinylpyrrolidone (PVP) suspension [20]. The suspension was sheared at 30 Pa and 37 °C into the Couette system made of glass of the ektacytometer. The oxygen partial pressure (pO₂) was gradually decreased from 160 mmHg to 20 mmHg (deoxygenation) and then re-increased to normoxic values [16,20,21]. The diffraction pattern was analyzed by the computer, and an elongation index (EI) that reflects RBC deformability was calculated. Several parameters can be derived: (1) EImax, the RBC deformability at normoxia; (2) EImin, the lowest RBC deformability reached upon deoxygenation; (3) point of sickling (PoS), the pO₂ at which RBC deformability decreases below 5% of EImax during deoxygenation; and (4) delta EI, the difference between EImin and EImax. All measurements were standardized as recommended [20]. RBC aggregation was determined at 37 °C by the laser backscatter method, using the Lorrca MaxSis (RR Mechatronics, Hoorn, The Netherlands) after adjustment of the Hct to 40% with autologous plasma [22]. RBC aggregate strength was determined using a re-iteration procedure [22]: 7 separate pre-defined shear rates between 7.5 s⁻¹ and 800 s⁻¹ were applied on the RBC suspension, with or without alternating disaggregation shear rate, to locate the minimal shear rate needed to prevent RBC aggregation.