key: cord-0924368-6mdzjb1e
authors: Grau, Marijke; Ibershoff, Lars; Zacher, Jonas; Bros, Janina; Tomschi, Fabian; Diebold, Katharina Felicitas; Predel, Hans‐Georg; Bloch, Wilhelm
title: Even patients with mild COVID‐19 symptoms after SARS‐CoV‐2 infection show prolonged altered red blood cell morphology and rheological parameters
date: 2022-04-13
journal: J Cell Mol Med
DOI: 10.1111/jcmm.17320
sha: 242bc2019c4a4da0f095a20a5a9d3f495366b9a8
doc_id: 924368
cord_uid: 6mdzjb1e

Infection with the novel severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and the associated coronavirus disease‐19 (COVID‐19) might affect red blood cells (RBC); possibly altering oxygen supply. However, investigations of cell morphology and RBC rheological parameters during a mild disease course are lacking and thus, the aim of the study. Fifty individuals with mild COVID‐19 disease process were tested after the acute phase of SARS‐CoV‐2 infection (37males/13 females), and the data were compared to n = 42 healthy controls (30 males/12 females). Analysis of venous blood samples, taken at rest, revealed a higher percentage of permanently elongated RBC and membrane extensions in COVID‐19 patients. Haematological parameters and haemoglobin concentration, MCH and MCV in particular, were highly altered in COVID‐19. RBC deformability and deformability under an osmotic gradient were significantly reduced in COVID‐19 patients. Higher RBC‐NOS activation was not capable to at least in part counteract these reductions. Impaired RBC deformability might also be related to morphological changes and/or increased oxidative state. RBC aggregation index remained unaffected. However, higher shear rates were necessary to balance the aggregation‐disaggregation in COVID‐19 patients which might be, among others, related to morphological changes. The data suggest prolonged modifications of the RBC system even during a mild COVID‐19 disease course.

the infection were described to include breathlessness, cough, fever, ageusia or loss of smell, but infections might also be asymptomatic.

Infections with SARS-CoV-2 affect all age groups with men and women being equally affected. 5 Breathlessness and a reduced fitness were reported by most COVID-19 patients, and it is suggested that this might be related to altered oxygen uptake into the red blood cells (RBC) and/or oxygen binding and/or oxygen release in this disease. These phenomena might be associated to damages of the beta-chain of the haemoglobin or an increased formation of methaemoglobin, which increases the oxygen affinity of the undamaged haemoglobin. 6, 7 An altered haematological profile including reduced RBC count or shifted RBC distribution width, 8 but also changes in the RBC morphology, structure and function might occur during the acute phase of the infection and might provide a further explanation for the described symptoms. 9,10 COVID-19 is also described to augment RBC rheology. 11 In particular, RBC deformability was reduced and RBC aggregation parameters were increased, indicating that key determinants of the blood flow in the microcirculation are limited by COVID-19 following SARS-CoV-2 infection. 12 This might be associated to structural protein damages and membrane lipid remodelling, which might also affect the cytoskeleton that is of major importance for proper RBC deformability. 13 RBC deformability is crucial for the oxygen supply within the microcirculation and is determined by the surface-to-volume ratio, intracellular viscosity, membrane elasticity 14, 15 and nitric oxide (NO) availability. 16 RBC NO synthase (RBC-NOS) activity is one source of NO generation within RBC and RBC NO has been linked to S-nitrosylation of the cytoskeletal spectrins and RBC deformability changes. 17 Whether the RBC-NOS signalling pathway is affected by SARS-CoV-2 remains unknown. Moreover, whether RBC rheology is altered in a mild course of this disease has yet not been described. Thus, the aim of the study was to investigate morphological changes, RBC rheology, RBC-NOS activation and marker for oxidative stress in men and women after recovery from COVID-19 with mild symptomatic in order to further understand the deleterious impact of SARS-CoV-2 on the blood system.

A total of n = 50 participants (n = 37 male; n = 13 female) were tested after an average of 60.7 days after positive PCR result on SARS-CoV-2 infection. We thus aimed to avoid acute effects of the infection but also post-COVID-19 effects which include symptoms that persist longer than or occur after 12 weeks. An additional n = 42 healthy controls (n = 30 male; n = 12 female) were investigated.

Age (range) of the study groups were as follows: COVID-19 male:

24.0 ± 4.4 years (14-30); female: 24.1 ± 5.5 years (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) ; control male: 24.1 ± 5.6 years (17-37); female: 23.8 ± 6.4 years (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) .

the exception of four asymptomatic participants, all reported a mild course of disease with typical SARS-CoV-2 associated symptoms.

None were hospitalized or reported other illnesses. None of the participants was vaccinated against COVID-19 at the time of blood sampling. Fitness level was above average for all participants. The described protocols align with the Declaration of Helsinki, and all participants gave written informed consent to participate in this study. The project was approved by the local ethics committee of the German Sport University (087/2020).

Venous blood samples were collected at rest from the vena mediana cubiti into EDTA vacutainer (Becton Dickinson GmbH) and further processed immediately. Described parameters were analysed of all tested participants unless otherwise stated.

Basal RBC parameters including RBC count, haemoglobin concentration (hb), haematocrit (hct), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and RBC distribution width (RDW) were directly determined in whole blood using the hematology analyzer Sysmex Digitana KX-21N (Sysmex).

Red blood cells were separated by centrifugation (3600 g, 5 min, room temperature), dispersed on a glass slide, heat fixed and Pappenheim staining was conducted to better illustrate and analyse morphological changes of the RBC. Images of the stained slides were taken using a Zeiss microscope coupled to a CCD-camera (DXC-1850P, Sony). Magnification of the images was 200-fold. A total of five images were taken of each slide, all RBC (average 500 cells per image) were counted and morphological abnormalities were put in proportion as a percentage.

Red blood cells deformability was measured by ektacytometry using the LORRCA MaxSis (RR Mechatronics) 18 after dilution of 100*10 6 /µl RBC in polyvinylpyrrolidone (PVP) solution (1:250; 29cP at 37°C, RR Mechatronics). The samples were sheared in a Couette system and exposed to nine consecutive shear stresses between 0.3 and 30 Pa, and the diffraction pattern of a laser beam directed through the samples was analysed by the LORRCA software of each applied shear stress resulting in a respective elongation index (EI).

The LORRCA software then calculated the maximum elongation index (EImax), representing the maximum deformability at infinite shear stress and SS1/2 which represents the shear stress at one-half EImax. Finally, the SS1/2:EImax ratio was calculated and higher values display reduced RBC deformability.

The osmotic gradient ektacytometry (osmoscan) was performed using the LORRCA MaxSis and measured deformability under various osmotic conditions. Measured RBC number was standardized for each sample using the following equation: 1000/RBC count = x µl sample mixed with 5 ml PVP. Thus, the deformation of RBC shape at a defined shear stress and constant temperature (37°C) is measured during an osmotic gradient and the following parameters were provided by the LORRCA software: Omin which corresponds to the osmolality at which RBC deformability reaches its minimum in the hypotonic environment and which is influenced by the mean cellular surface-to-volume ratio. Below this point, most RBC rupture if the osmolality is further decreased. EImax corresponds to the maximum deformability near the isotonic osmolality and relates to the RBC membrane surface. Ohyper, the osmolality in the hypertonic region, corresponds to 50% of EImax and reflects the hydration status of the RBC. Ohyper is affected by the cytoplasmic viscosity and the cell volume. [19] [20] [21] Red blood cells aggregation was measured at 37°C by syllectometry using the LORRCA MaxSis after the hct of the samples was adjusted to 40% using autologous plasma. All samples were fully ox- prior to the measurement. 22 Oxygenated samples were transferred to the Couette system and changes of backscattered light were recorded over 120 s using two photodiodes and presented as a graph (syllectogram) to calculate an Aggregation-Index (AI%). The threshold shear rate balancing RBC aggregation and disaggregation was obtained after an iteration procedure was performed to primarily calculate dIsc min. This parameter defines the minimum change in backscatter intensity during the iteration procedure, representing the minimum shear rate where RBC aggregates start to disaggregate (y at dIsc min (s −1 )).

Nitrotyrosine and phosphorylation of the RBC-NOS serine 1177 residue were measured by immunohistochemistry. 17 Thus, nitrotyrosine represents a marker for the generation of free radicals, NO in particular, and a marker for oxidative stress. Phosphorylation of the RBC-NOS serine 1177 residue has been described as activation site, thus representing increased enzyme activity and hence NO production. 16, 17 Briefly, separated RBC were immediately fixed in Statistical analyses of the data were performed using GraphPadPrism software 8.0. Data were analysed for normal distribution of the data, and one-way ANOVA was performed to detect effects between the tested groups. Differences were considered as significant with * p < 0.05, ** p < 0.01 and *** p < 0.001. Presented data are mean ± standard deviation (SD). TA B L E 1 Red blood cell parameters of male and female COVID-19 participants compared with respective female and male healthy control group to an apex which can be observed at the RBC ( Figure 1A ). These changes were significantly higher after SARS-CoV-2 infection in both, males and females compared with healthy controls ( Figure 1C ).

Permanent elongated RBC ( Figure 1B) were also significantly higher in both COVID-19 groups compared to the respective control groups ( Figure 1D ).

Red blood cell deformability was represented as SS1/2 to EImax ratio. SS1/2:EImax values were significantly higher and thus, RBC deformability was significantly lower in both COVID-19 groups.

Comparison of male and female controls revealed higher RBC deformability in females compared to males (Figure 2A ). ( Figure 2B ). The threshold shear rate balancing RBC aggregation and disaggregation was significantly higher in male COVID-19 compared with the respective control group. In the female test group, values tend to be higher compared with the control cohort (effect size: 0.41; Figure 2C ).

Omin was reduced after SARS-CoV-2 infection in both, female and male COVID-19 cohorts ( Figure 2D ). EImax (osmoscan) was significantly reduced in males but not in females with COVID-19 ( Figure 2E) and Ohyper was also significantly reduced in both COVID-19 groups compared with their respective control groups. Comparison of male and female controls showed higher Ohyper values in females than in males ( Figure 2F ).

Higher RBC-NOS serine 1177 phosphorylation is related to an increase in enzyme activation. 17 

Severe SARS-CoV-2 dependent COVID-19 disease was shown to affect the RBC system 24, 25 and RBC rheological parameters in particular. 12 This might be related to morphological changes 9 and might impact oxygen transport and supply. The recent study aimed to address the question whether a mild COVID-19 disease progress is related to prolonged alterations in RBC morphology and rheological parameters. patients showed membrane defects, which was a higher percentage than observed in females with about 1% of RBC defects. This means that 200-500 billion circulating cells show membrane defects given the fact that about 20 trillion RBC circulate through the human body.

Because blood sampling described herein was scheduled approximately 60 days after infection to exclude the acute infection phase, it seems plausible that the amount of RBC defects was most likely higher during acute infection state. It seem reasonable that these structural changes limit RBC deformation which is one reason for erythrophagocytosis by macrophages 37 *** * * * COVID-19 infection. Indeed, RBC deformability was significantly lower in both COVID-19 groups underlining previous findings. 11, 12 Moreover, deformability under various osmotic conditions showed significant differences between COVID-19 and healthy participants.

EImax (osmoscan), Omin and Ohyper were significantly lower in male and female COVID-19 patients respectively. Thus, COVID-19 dependent changes of the RBC shifted the osmsocan curve to the left and downwards as described for stiffened cells 19 concluding that the described haematological and morphological changes impairs RBC deformability even after recovery from mild COVID-19 disease.

Since RBC deformability is crucial for the passage of the small capillaries and oxygen supply within the microcirculation, it is suspected that stiffening of RBC, next to haemoglobin alterations, might also be associated to the described hypoxemia 38 during COVID-19 infection. RBC aggregation represents another crucial rheological parameter which was described to be affected by acute severe COVID-19 disease. 12, 39 The present data could not support these findings, possibly because of the mild nature of the disease course reported herein or because the enrolled subjects did not show additional diseases which were described for the COVID-19 patients by Renoux and colleagues. 12 Though, shear rate balancing aggregation and disaggregation was remarkably higher in the COVID-19 groups and it seems plausible that the membrane evaginations and elongation of the cells described earlier increase the contact area between adjacent RBC thus increasing forces connecting these cells.

Further, nitrotyrosine levels were measured to investigate a possible role of oxidative stress in the observed functional changes.

Nitrotyrosine originates from the reaction of NO and superoxide resulting in peroxynitrite which in turn nitrates tyrosine residues of proteins. Thus, nitrotyrosine represents a marker for cellular damage and oxidative stress. 40 Elevated levels of RBC free radical content have been previously described to induce a reorganization of the membrane structure as well as water and ion imbalance 41 which was also linked to reduced RBC deformability. [42] [43] [44] Damage of RBC proteins and membrane lipid remodelling in COVID-19 patients was reported by Thomas et al. 13 The observed changes include fragmentation of crucial RBC cytoskeleton proteins such as spectrin which is essential for RBC deformability, 17 but also of the anion exchanger 1/band 3 essential for membrane stability. 45 The authors concluded that modifications of the RBC proteins by COVID-19 might explain the lack of the cells to respond to environmental oxygen saturation and/or oxidative stress. 13 In the present study, nitrotyrosine levels were increased in female COVID-19 patients confirming, at least in part, higher free radical content in RBC of COVID-19 patients which promotes the decline in RBC deformability. Such a relation was not observed in male COVID-19 patients. However, since a large number of different free radical species were described to be present in RBC, further studies including the investigation of other free radical species need to be carried out. High free radical content was also described to reduce activation of the NO producing enzyme RBC-NOS. 42 RBC-NOS produced NO has been linked to S-nitrosylation of RBC cytoskeletal proteins including spectrin, thus positively affecting RBC deformability 17 and possibly preserving deformability in conditions of oxidative stress. 46 Surprisingly, RBC-NOS activation, represented by the phosphorylation state of the serine 1177 residue, 16 was significantly higher in both COVID-19 groups. Thus, also higher NO levels might be hypothesized in RBC of COVID-19 patients because higher RBC-NOS activation was associated to increased levels of RBC NO. 16, 17, 47 In addition, the generation of nitrotyrosine requires the presence of NO as described above. A study by Mortaz and colleagues report increased NO levels in RBC of severe COVID-19 patients 48 which might apply to the finding of the present study. Given the reduced RBC deformability in COVID-19 patients despite higher RBC-NOS activation, it is assumed that this observation might represent a compensatory mechanism, which, however, cannot maintain RBC deformability. 49, 50 It seems plausible that the observed morphological changes were caused by damages of the cytoskeleton, the membrane or both and that increased RBC-NOS produced NO is not capable to affect deformability because of the structural damages.

In conclusion, to the best of our knowledge, these are the first data revealing prolonged prominent RBC structural and rheological changes in patients after a mild COVID-19 disease. These changes were observed in both, young female and young male COVID-19 patients although certain haematological parameters and morphological changes seemed to be more pronounced in male patients. Impairment of RBC deformability and aggregate strength, morphological changes and oxidative stress seem to be highly interrelated.

Impaired rheological parameters were shown to affect blood flow dynamics and, together with the reported left-shift of the oxygen dissociation curve, 6 possibly oxygen supply in the microcirculation.

Investigations of the RBC system post-COVID-19 (>12 weeks after infection) but also investigations on the exact underlying mechanisms of altered RBC structure and rheological parameters are needed to develop specific therapies and/ or therapeutic agents.

The authors thank Ms Bianca Collins, Ms Emily Zollmann, Ms Anke Schmitz and Mr Alexander Floß for technical and organizational support. Open Access funding enabled and organized by Projekt DEAL.

The authors declare no conflict of interest. 

All patients gave written informed consent to participate in this study.

The data that support the findings of this study are available on reasonable request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

https://orcid.org/0000-0003-1980-9195

Characteristics of SARS-CoV-2 and COVID-19

Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T-cell interactions

Treatment for COVID-19: An overview

COVID-19: molecular diagnostics overview

The oxygen dissociation curve of blood in COVID-19

COVID-19: Attacks the 1-beta chain of hemoglobin and captures the porphyrin to inhibit human heme metabolism

Dynamic changes in routine blood parameters of a severe COVID-19 case

Are mushroomshaped erythrocytes an indicator of COVID-19?

Antemortem vs postmortem histopathologic and ultrastructural findings in paired transbronchial biopsy specimens and lung autopsy samples from three patients with confirmed SARS-CoV-2

Physical phenotype of blood cells is altered in COVID-19

Impact of COVID-19 on red blood cell rheology

Evidence of structural protein damage and membrane lipid remodeling in red blood cells from COVID-19 patients

Red cell deformability and its relevance to blood flow

Red blood cell deformability, membrane material properties and shape: regulation by transmembrane, skeletal and cytosolic proteins and lipids

Red blood cells express a functional endothelial nitric oxide synthase

RBC-NOS-dependent S-nitrosylation of cytoskeletal proteins improves RBC deformability

The Laser-assisted Optical Rotational Cell Analyzer (LORCA) as red blood cell aggregometer

Interpretation of osmotic gradient ektacytometry (osmoscan) data: a comparative study for methodological standards

Diagnostic tool for red blood cell membrane disorders: assessment of a new generation ektacytometer

Use of Laser Assisted optical rotational cell analyzer (LoRRca MaxSis) in the diagnosis of RBC membrane disorders, enzyme defects, and congenital dyserythropoietic anemias: a monocentric study on 202 patients

Comparison of three instruments for measuring red blood cell aggregation

Shear stress activation of nitric oxide synthase and increased nitric oxide levels in human red blood cells

COVID-19 detection from red blood cells using highly comparative time-series analysis (HCTSA) in digital holographic microscopy

Digital holographic deep learning of red blood cells for field-portable, rapid COVID-19 screening

Comparisons of blood parameters, red blood cell deformability and circulating nitric oxide between males and females considering hormonal contraception: a longitudinal gender study

Impact of type of sport, gender and age on red blood cell deformability of elite athletes

Etiology of differences in hematocrit between males and females: sequence-based polymorphisms in erythropoietin and its receptor

Gender difference in rheologic properties of blood and risk of cardiovascular diseases

Increase in red blood cell-nitric oxide synthase dependent nitric oxide production during red blood cell aging in health and disease: a study on age dependent changes of rheologic and enzymatic properties in red blood cells

Hematologic changes predict clinical outcome in recovered patients with COVID-19

Role of hematological parameters in the stratification of COVID-19 disease severity

COVID-19) patients

COVID-19: hemoglobin, iron, and hypoxia beyond inflammation. A narrative review

Anemia and iron metabolism in COVID-19: a systematic review and meta-analysis

Changes of hematological and immunological parameters in COVID-19 patients

How do red blood cells know when to die?

The pathophysiology of 'happy' hypoxemia in COVID-19

Increased blood viscosity and red blood cell aggregation in patients with COVID-19

Red blood cells in the metabolism of nitric oxide-derived peroxynitrite

Structural and functional changes in the membrane and membrane skeleton of red blood cells induced by peroxynitrite

Shear stress ameliorates superoxide impairment to erythrocyte deformability with concurrent nitric oxide synthase activation

Assessment of oxidant susceptibility of red blood cells in various species based on cell deformability

Effect of superoxide anions on red blood cell rheologic properties

Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton

On the effects of reactive oxygen species and nitric oxide on red blood cell deformability

Erythrocytes may synthesize their own nitric oxide

Silent hypoxia: higher NO in red blood cells of COVID-19 patients

Effect of acute exercise on RBC deformability and RBC nitric oxide synthase signalling pathway in young sickle cell anaemia patients

High red blood cell nitric oxide synthase activation is not associated with improved vascular function and red blood cell deformability in sickle cell anaemia

Even patients with mild COVID-19 symptoms after SARS-CoV-2 infection show prolonged altered red blood cell morphology and rheological parameters