Red Blood Cell Abnormalities as the Mirror of SARS-CoV-2 Disease Severity: A Pilot Study

Purpose Unraveling 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 Methods Hospitalized 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 (iCa2+), 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). Results The 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/FiO2 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 (iCa2+, 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. Conclusion This 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. Graphical Abstract SARS-CoV-2 infection has significant effects on red blood cells that seems to be associated with disease severity in these patients. (A) Non-Intensive care unit hospitalized patients due to severe COVID-19 disease were included in this study (n = 36). (B) The control group included healthy subjects without renal or hepatic impairment, no diagnosis of anemia or myeloid malignancy, and no blood transfusion over the past 3 months (n = 18). (C) Demographic data, clinical, laboratory and chest Computed Tomography (CT) findings at time of admission were recorded. Hematological tests, standard biochemical analysis of serum components were performed both in the patient and in the control group and coagulation analysis were evaluated in the patient group. A peripheral blood smear examination was also performed in all patients and control samples. (D) Plasma free hemoglobin and red blood cell (RBC) osmotic fragility and free hemoglobin (Free-Hb) after mechanical stress (MECH) by mechanical hemolysis were assesed in both patients and controls. Intracellular (i) calcium (iCa2 +) levels, RBCs phosphatidylserine exposure (PS), RBC membrane-bound immunoglobulins G (IgGs), and activate RBC caspase-3 levels as cell clearance and apoptotic markers were measured by flow cytometry. A spectrophotometric assay was used to quantify the activity of G6PD enzyme. Intracellular RBC reactive oxidative species (iROS) were assesed in both patients and controls. (E) Elevated hemolysis markers (Free-Hb) and Lactate Dehydrogenase (LDH) were observed in patients compared to the control group. Patients’ RBCs were more vulnerable to mechanical stress, and exhibited significantly elevated apoptotic markers (iCa2 + and RBC-PS+). Erythrocyte iROS levels were significantly lower in the patients’ group compared to healthy controls. This finding possibly correlates with the increased G6PDH activity levels in this group, suggesting a compensatory mechanism against the virus. (F) Patients’ D-Dimers correlated with RBC-PS +, implying a possible contribution of the erythrocytes in the thrombotic diathesis associated with the SARS-CoV-2 disease. (G) Free-Hb levels correlated with the extend of pulmonary infiltrates on chest CT in COVID-19 patients. RBC-IgGs+ correlated with the severity of pulmonary CT imaging features as well as the abnormality of lung function, expressed by the PO2/FiO 2 ratio. This finding suggests that hypoxia may lead to RBC senescence.