Does G6PD deficiency cause further damage to red blood cells of patients with sickle cell anaemia?

Glucose6dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in the world,1 which leads to a lower level of reduced glutathione, an antioxidant, in red blood cells (RBCs). Most of the time, those who are affected have no symptoms. However, they should avoid specific triggers that may promote oxidative stress such as fava beans, that may fragilize RBCs and cause haemolysis. G6PD deficiency has been proposed to be a modulator of certain complications in sickle cell anaemia (SCA), such as stroke2 and vasoocclusive crises,3 but others found no major role of G6PD deficiency in the clinical manifestations and haematological phenotype of SCA.4– 6 The present study investigated the effects of G6PD deficiency on RBC deformability, RBC senescence markers, haematological parameters and clinical severity in SCA. We hypothesized that RBC oxidative stress would be greater in SCA patients with G6PD deficiency than those without, and this would be accompanied by greater alterations in RBC rheology. Seventyone patients with homozygous sickle cell disease (SCA, HbSS), no recent blood transfusion or acute complication participated in the present study after giving written informed consent. Medical records were reviewed by two physicians to identify patients who had acute chest syndrome (ACS) or vasoocclusive crises (VOC) in the four years preceding the study, as well as a diagnosis of osteonecrosis, glomerulopathy, retinopathy and leg ulcers. A patient was considered as having frequent VOC when the number of VOC per year was greater or equal to two. This study was performed in accordance with the Declaration of Helsinki and approved by the HCL Ethics committee (L14127). Screening for G6PD deficiency in SCA patients was first done at the enzyme level in all patients. Both G6PD and pyruvate kinase (PK) activities were measured and the ratio G6PD/PK calculated. A ratio comprised between 0.5 and 1.5 indicates no deficiency. For hemizygotes or homozygotes, the ratio is very low (<0.10). When the ratio was lower than 0.5, high resolution melting (HRM) analysis was performed focusing on the Med and A(−) variants7 to detect a possible heterozygous carrier state. Haematological parameters were determined with a haematology analyser (Advia, Siemens, Rungis, France). Ektacytometry was carried out with the Laserassisted optical rotational red cell analyser (Lorrca, RRMechatronics) with the oxygenscan module to measure RBC deformability (Elongation Index; EI) over an oxygen gradient and at 30 Pa, as previously described.8 Several parameters were derived: (1) EImax (normoxic RBC deformability), (2) EImin (the lowest RBC deformability reached during deoxygenation) and (3) PoS (the pO2 at which RBC deformability decreases below 95% of EImax during deoxygenation), which reflects the pO2 at which RBCs start to sickle during deoxygenation. Phosphatidylserine (PS) exposure at the outer membrane leaflet of RBCs and CD47 were assessed using Annexin VPE (Miltenyi, 130118363) and antiCD47PE antibody (Miltenyi 130101348), respectively.9 Intracellular RBC reactive oxygen species (ROS) were determined using 2′,7′– dichlorofluorescin diacetate (DCFDA, SigmaAldrich, SaintQuentinFallavier, France).9 DCFDA is a nonspecific probe that allows the determination of the degree of overall oxidative stress in the cells by reacting with various reactive oxygen species. Despite the fact that nitric oxide may oxidize DCFDA, this probe seems sensitive enough to detect intracellular oxidative stress levels in RBCs in a pathophysiological context and for in vitro pharmacological modulation assessments.9 Ten patients had G6PD deficiency (six homozygotes, four hemizygotes), all A(−) variants. Age was not different between the two groups (23.7 ± 16.8 vs 24.9 ± 13.8 years in patients with and without G6PD deficiency, respectively). Biological results (Table 1) showed greater intracellular ROS content and higher percentage of positive RBCs for ROS in SCA patients with G6PD deficiency. RBC ROS level was also analysed in a group of healthy subjects (36.9 ± 7.8 years): positive RBCs = 55.0% ± 24.3%; median fluorescence intensity (MFI) = 0.57 ± 0.14. Both parameters were below the values found in the two SCA groups. The other markers were not different between the two groups. Table 2 shows no difference in the clinical parameters between the two groups. Bouguerra et al.10 previously showed higher numbers of PSexposing RBCs and higher ROS level in the RBCs from nonSCA G6PDdeficient patients than from healthy volunteers. Indeed, it is not surprising that greater intracellular ROS levels were observed in SCA patients with G6PD deficiency than in those without in the present study. However, no difference was observed between the two groups for RBC senescence markers (percentage of RBCs exposing PS and CD47 expression), RBC deformability in both normoxia and hypoxia, the propensity of sickling (i.e., PoS), the level Received: 10 June 2022 | Accepted: 7 August 2022