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PurposeUnraveling the pathophysiology of COVID-19 disease is of crucial importance for designing treatment. The purpose of this study is to investigate the effects of the disease on erythrocytes (RBCs) and to correlate the findings with disease severity. Materials and MethodsHospitalized patients (n = 36) with COVID-19 and control group of healthy volunteers (n = 18) were included in the study. Demographic data, clinical, laboratory and chest Computed Tomography (CT) findings at time of admission were recorded. Laboratory measurements included: Hemoglobin (H b), indirect billirubin, LDH, D-Dimers, and plasma free hemoglobin (plasma free-Hb). On RBCs were performed: osmotic fragility (MCF), Free-Hb after mechanical stress (Free-Hb-MECH), intracellular RBC concentration of calcium ions (iCa(2+)), intracellular ROS (iROS), G6PD, intracellular active caspase-3 (RBC-caspase-3), IgG immunoglobulins (RBC-IgGs), which are bound on RBCs’ senescent neo-antigen proteins and RBC surface phosphatidylserine (RBC-PS). ResultsThe percentage of males was 50 and 66% and the mean age was 65.16 +/- 14.24 and 66.33 +/- 13.48 years among patients and controls respectively (mean +/- SD, p = 0.78). Upon admission patients’ PO2/FiO(2) ratio was 305.92 +/- 76.75 and distribution of infiltration extend on chest CT was: 0-25% (N = 19), 25-50%: (N = 7), and 50-75% (N = 9). Elevated hemolysis markers (LDH and plasma free-Hb) were observed in patients compared to the control group. Patients’ RBCs were more sensitive to mechanical stress, and exhibited significantly elevated apoptotic markers (iCa(2+), RBC-PS). Plasma free Hb levels correlated with the extend of pulmonary infiltrates on chest CT in COVID-19 patients. Surprisingly, patients’ RBC-iROS were decreased, a finding possibly related with the increased G6PDH levels in this group, suggesting a possible compensatory mechanism against the virus. This compensatory mechanism seemed to be attenuated as pulmonary infiltrates on chest CT deteriorated. Furthermore, RBC-IgGs correlated with the severity of pulmonary CT imaging features as well as the abnormality of lung function, which are both associated with increased disease severity. Lastly, patients’ D-Dimers correlated with RBC surface phosphatidylserine, implying a possible contribution of the red blood cells in the thrombotic diathesis associated with the SARS-CoV-2 disease. ConclusionThis pilot study suggests that SARS-CoV-2 infection has an effect on red blood cells and there seems to be an association between RBC markers and disease severity in these patients.

BACKGROUND: Several factors contribute to the manifestation of red blood cell (RBC) storage lesions, with one of the most interesting being the “donor variation effect”. Since many haematological characteristics of blood donors are sex-dependent, sex hormones and their age-dependent variation may affect the storage profile of RBCs. MATERIALS AND METHODS: Fresh blood from 200 healthy male and female donors underwent haematological, biochemical and physiological analysis. Three selected groups of donors (men, n=8; pre-menopausal women, n=8; and post-menopausal women, n=4) exhibiting as similar as possible baseline values were recruited for blood donation in leukoreduced CPD/SAGM units. RBC indices, haemolysis and propensity for haemolysis, reactive oxygen species (ROS) and plasma antioxidant capacity were measured bi-weekly. RESULTS: Female blood was characterised by lower plasma antioxidant capacity and free haemoglobin (Hb) levels in vivo, in spite of the higher RBC osmotic fragility, compared to male blood. Comparatively low Hb concentration was also measured in stored RBCs from female donors, as in vivo. Mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), and plasma antioxidant capacity were also lower in female donors throughout storage, even though baseline levels were equal to those of the male group. There was no difference in propensity of stored RBCs for haemolysis between male and female units but intracellular ROS levels were significantly lower in female RBCs. Increased end-of-storage extracellular potassium and recruitment of protein stress markers (clusterin, Hb) to the RBC membrane were observed in the units of post- vs pre-menopausal female donors at mid-storage onwards. DISCUSSION: Donor’s sex has an impact on Hb concentration and redox parameters of stored RBCs. In addition, menopause seems to promote RBC membrane remodelling, at least during prolonged storage. Our pilot study provides new insights on the different effects on RBC storage lesion according to sex.

Prestorage filtration of blood to remove contaminating donor leukocytes and platelets has substantially increased the safety level of transfusion therapy. We have previously shown that leukoreduction has a mitigating effect on the storage lesion profile by lowering the extent of hemolysis and of RBC aging and removal phenotypes, including surface signaling and microvesiculation. Even though protein composition may determine the fate of EVs in the recipient, the probable effect of leukoreduction on the EV proteome has been scarcely investigated. In the present paired study, we characterized the proteome of EVs released in prestorage leukoreduced (L) and nonleukoreduced (N) RBC units prepared from the same donors, by immunoblotting and qualitative proteomics analyses at two storage intervals. Apart from common proteofrms typically associated with the established EV biogenesis mechanisms, the comparative proteomics analyses revealed that both leukoreduction and storage duration affect the complexity of the EV proteome. Membrane and cytoskeleton-related proteins and regulators, metabolic enzymes and plasma proteins exhibited storage duration dependent variation in L- and N-EVs. Specific proteoforms prevailed in each EV group, such as transferrin in L-units or platelet glycoproteins, leukocyte surface molecules, MHC HLA, histones and tetraspanin CD9 in N-units. Of note, several unique proteins have been associated with immunomodulatory, vasoregulatory, coagulatory and anti-bacterial activities or cell adhesion events. The substantial differences between EV composition under the two RBC preparation methods shed light in the underlying EV biogenesis mechanisms and stimuli and may lead to different EV interactions and effects to target cells post transfusion.

Extracellular vesicles or microparticles exhibiting procoagulant and thrombogenic activity may contribute to the haemostatic potential of fresh frozen plasma.Fresh frozen plasma was prepared from platelet-rich plasma at 20 °C (Group-1 donors) or directly from whole blood at 4 °C (Group-2 donors). Each unit was aseptically divided into three parts, stored frozen for specific periods of time, and analysed by flow cytometry for procoagulant activity immediately after thaw or following post-thaw storage for 24 h at 4 °C. Donors' haematologic, biochemical and life-style profiles as well as circulating microparticles were analysed in parallel.Circulating microparticles exhibited a considerable interdonor but not intergroup variation. Fresh frozen plasma units were enriched in microparticles compared to plasma in vivo. Duration of storage significantly affected platelet- and red cell-derived microparticles. Fresh frozen plasma prepared directly from whole blood contained more residual platelets and more platelet-derived microparticles compared to fresh frozen plasma prepared from platelet-rich plasma. Consequently, there was a statistically significant difference in total, platelet- and red cell-derived microparticles between the two preparation protocols over storage time in the freezer. Preservation of the thawed units for 24 h at 4 °C did not significantly alter microparticle accumulation. Microparticle accumulation and anti-oxidant capacity of fresh frozen plasma was positively or negatively correlated, respectively, with the level of circulating microparticles in individual donors.The preparation protocol and the duration of storage in the freezer, independently and in combination, influenced the accumulation of microparticles in fresh frozen plasma units. In contrast, storage of thawed units for 24 h at 4 °C had no significant effect on the concentration of microparticles.