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Assessment of erythrocyte osmotic fragility in male and female COVID-19 patients
*Corresponding author: Hind Ahmed Kenoosh, Ministry of Education, Directorate of Education in AlAnbar, Baghdad, Iraq. Hind.a.knoosh97@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Kenoosh HA, Awad MM. Assessment of erythrocyte osmotic fragility in male and female COVID-19 patients. RMC Glob J. 2026;2:74-9. doi: 10.25259/RMCGJ_58_2025
Abstract
Objectives:
At the end of December 2019, coronavirus disease (COVID-19) emerged as a global health concern, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to evaluate the osmotic fragility of red blood cells in male and female COVID-19 patients.
Material and Methods:
A total of 65 individuals with pneumonia confirmed by computed tomography and SARS-CoV-2 infection (positive reverse transcription polymerase chain reaction [RT-PCR]) were included, along with 23 healthy controls. Participants were divided into two groups: 30 females and 35 males.
Results:
The results showed a significantly higher percentage of red blood cell hemolysis in patients compared to controls. Increased hemolysis may contribute to elevated hemolysis and ferritin levels and other physiological changes, potentially influencing disease pathogenesis and prognosis.
Conclusion:
These findings suggest that red blood cell abnormalities may play a critical role in the pathophysiology of COVID-19.
Keywords
COVID-19
Hemolysis
Osmotic fragility
SARS-CoV-2
Sodium chloride
INTRODUCTION
At the end of December 2019, coronavirus disease (COVID-19) emerged as a global health concern. This disease is caused by severe acute respiratory syndrome coronavirus 2 (SARSCoV-2). Owing to its rapid worldwide transmission, the World Health Organization (WHO) declared COVID-19 a pandemic in March 2020, with the first reported cases identified in China.1 SARS-CoV-2 enters human cells through the interaction of its surface spike protein (SP) with the angiotensin-converting enzyme 2 (ACE-2) receptor. The trimeric spike protein consists of two functional subunits, S1 and S2, which play essential roles in receptor binding and membrane fusion, respectively. Similar to the two highly pathogenic coronaviruses that caused outbreaks in the past two decades, SARS-CoV and Middle East respiratory syndrome coronavirus (MERSCoV), SARS-CoV-2 belongs to the beta coronavirus genus.2 Whole-genome sequencing has demonstrated that this enveloped, single-stranded, positive-sense ribonucleic acid (RNA) virus containing a nucleocapsid is genetically related to SARS-CoV, sharing approximately 79% sequence homology across its genome.3 Molecular modeling analyses indicate that the receptor-binding domains of SARS-CoV and SARS-CoV-2, commonly referred to as spike proteins and recognized as the most immunogenic regions of the virus, exhibit considerable structural similarity. Both viruses are capable of utilizing the same cellular receptor, ACE2, to facilitate viral entry into host cells.4 This observation suggests that comparable pathogenic mechanisms are involved in the infections caused by these two viruses. Notably, ACE2 expression is not confined to type II alveolar epithelial cells5, which account for approximately 83% of ACE2-expressing cells, but is also present in multiple other tissues, including the heart, vascular endothelium, kidneys, and gastrointestinal tract.6 Although SARS-CoV-2 is associated with lower mortality compared to MERS-CoV, the majority of infected individuals remain asymptomatic or develop only mild clinical manifestations. Nevertheless, approximately 10–20% of patients—particularly older adults and individuals with underlying comorbidities—may develop severe disease characterized by disseminated intravascular coagulation, hyperserotonemia, hepatic dysfunction, and a rapid progression to acute respiratory distress syndrome (ARDS) or septic shock, accompanied by elevated levels of acute-phase reactants, as well as interstitial pneumonia.7 Oxygen delivery to peripheral tissues throughout the human body is mediated by red blood cells (RBCs). Studies have suggested that COVID-19 infection is associated with increased bone marrow activity, leading to enhanced production and release of white blood cells and platelets. Bone marrow stimulation may also influence erythrocyte motility.8 In addition, systemic inflammatory conditions can negatively affect iron absorption and bioavailability, both of which are essential for effective hematopoiesis. These alterations are thought to result from dysregulated immune responses following SARSCoV-2 infection.9 A reduction in erythrocyte production commonly leads to anemia, triggering a compensatory mechanism that increases red cell distribution width (RDW) through the premature release of immature erythroid progenitor cells into the circulation. The term “infection course” reflects disturbances in hematopoietic regulation, increased bone marrow stress, or enhanced erythrocyte destruction secondary to elevated platelet and leukocyte production. Emerging evidence suggests that SARS-CoV-2 may directly target and infect erythrocytes, and several novel mechanisms have been proposed to explain the underlying pathophysiology of COVID-19. Notably, damage to the erythrocyte membrane has been observed following infection with SARS-CoV-2.10 This study aims to evaluate the osmotic fragility (OF) of red blood cells in patients infected with the coronavirus COVID-19, in both males and females.
MATERIAL AND METHODS
Study design and characteristics of patients
Samples for the research were taken from COVID-19 patients in Iraq at Fallujah Teaching Hospital. The study comprised 65 individuals with pneumonia (based on computed tomography imaging) and SARS-CoV-2 infection (confirmed by a positive RT-PCR test), as well as 23 healthy individuals serving as a control group. The study samples were divided into two groups: the first included 30 females, and the second group included 35 males. Participants aged between 20and 75 years, able to provide informed consent, and who had not received any treatment that could affect red blood cells prior to sample collection. Patients were excluded if they had chronic hematologic disorders or immune conditions affecting red blood cells, were receiving medications that could influence red blood cell fragility, were critically ill requiring intensive respiratory support, or were pregnant or lactating.
Getting samples
Strict sterilization procedures were followed during blood sample collection. Venipuncture was performed, and 4 mL of blood was drawn and transferred into ethylenediaminetetraacetic acid (EDTA) tubes for the RBC osmotic fragility test.
Determination of RBC osmotic fragility
Principle of the test
The basic principle of the osmotic fragility test depends on the degree of membrane resistance to lysis in low concentrations of a salt solution. This test is based on the intensity of light transmitted through the hemoglobin solution resulting from the suspension of red blood cells in a hypotonic medium, as hemoglobin is the only protein in red blood cells that plays a role in light absorption. The commonly used wavelength of light is 540 nm, and the results of this test are important because they provide diagnostic features for certain diseases.11
Assay procedure
A 1% sodium chloride (NaCl) solution was prepared by dissolving 10 g of NaCl in 1 liter of distilled water. Ten tubes were prepared and labeled with numbers from 1 to 10. The first tube contained distilled water only and served as a standard tube, while the last tube, with a concentration of 0.9%, represented a blank tube. The remaining tubes contained different concentrations of sodium chloride hypotonic solutions, as shown in Table 1. Then, a small volume of blood (approximately 30 µL) was added to each tube, mixed well, and the tubes were left at room temperature for ten minutes. Subsequently, the tubes were centrifuged for five minutes at a speed of 2000 rpm. After centrifugation, primary hemolysis was recorded and identified by the presence of a clear red solution in the tube, while complete hemolysis was determined by the absence of red blood cells deposited at the bottom of the tube or by the presence of a dark red color. The absorbance of each tube was then measured at a wavelength of 540 nm using a spectrophotometer. The percentage of hemolysis for each tube was calculated using the equation.12
| Tube | Tube 1 | Tube 2 | Tube 3 | Tube 4 | Tube 5 |
|---|---|---|---|---|---|
| Con. of NaCl (M) | 0 | 0.1 | 0.2 | 0.3 | 0.4 |
| Tube | Tube 6 | Tube 7 | Tube 8 | Tube 9 | Tube 10 |
| Con. of NaCl (M) | 0.5 | 0.6 | 0.7 | 0.8 | 0.9 |
NaCl: Sodium chloride.
Primary hemolysis
The early stage of RBC breakdown, where some cells rupture and release their contents, but hemolysis is not complete. It is typically indicated by a pale red color in the sodium chloride solution and the presence of a blood pellet at the bottom of the tube, or by an initial change in the optical density of the solution.
Complete hemolysis
The stage in which all RBCs are fully lysed, releasing hemoglobin completely into the sodium chloride solution. No blood pellet remains at the bottom of the tube, and the solution appears uniformly dark red, often corresponding to a maximum optical density.12
Ethical approval
According to the standards set by the Al-Anbar University Medical Research Ethics Clearance Committee, all investigations were conducted at Al-Anbar University, Iraq. The study protocol was reviewed and approved by the local Ethics Committee of Al-Anbar University. All participants provided informed consent prior to participation. Patient confidentiality was strictly maintained, and all procedures adhered to established ethical principles.
Statistical analysis
The data analysis was conducted using the program, Statistical Package for the Social Sciences (SPSS), based on a completely randomized design (CRD) in a one-way manner. The least significant difference (LSD) test was used to determine whether the mean differences were significant at a probability level of ≤ 0.05.13
RESULTS
The percentage of RBC osmotic fragility in COVID-19 patients, both male and female, and the control groups
The results of the current study show that there are significant differences in the arithmetic means of hemolysis percentages. It was observed that, compared to healthy individuals, COVID-19 patients exhibit a higher percentage of red blood cell hemolysis and lower resistance to hemolysis across different concentrations of sodium chloride solution. Where the hemolysis percentage was at a concentration of 0.1 in male and female COVID-19 patients (96.12 ± 0.485, and 95.59 ± 0.542% respectively), when comparable to both the male and female control groups (72.65 ± 6.251, and 77.35 ± 2.526% respectively), as shown in Figure 1. And hemolysis percentage was at a concentration of 0.2 in male and female COVID-19 patients (92.02 ± 1.086, and 93.47 ± 0.946% respectively) when comparable to both the male and female control groups (76.93 ± 2.016, and 75.84 ± 2.192% respectively) as shown in Figure 2. hemolysis percentage at a concentration of 0.3in male and female COVID-19 patients (91.33 ± 1.022, and 91.57 ± 1.322% respectively), when comparable to both the male and female control groups (74.47 ± 2.181, and 70.98 ± 1.766% respectively) as shown in Figure 3. And hemolysis percentage at a concentration of 0.4 in male and female COVID-19 patients (77.28 ± 1.621, and 74.02 ± 1.896% respectively), when comparable to both the male and female control groups (52.85 ± 2.057, and 56.32 ± 1.552% respectively), as shown in Figure 4. Hemolysis percentage at a concentration of 0.5 in male and female COVID-19 patients (9.08 ± 0.601, and 5.13 ± 0.421% respectively), when comparable to both the male and female control groups (3.41 ± 0.454, and 1.94 ± 0.281% respectively), as shown in Figure 5. And hemolysis percentage at a concentration of 0.6 in male and female COVID-19 patients (.19 ± 0.256, and 2.11 ± 0.205% respectively when comparable to both the male and female control groups (0.81 ± 0.143, and 0.95 ± 0.202% respectively), as shown in Figure 6. The hemolysis percentage of red blood cells in a 0% sodium chloride solution is 100%, serving as the standard solution, while at 0.9% it is 0%, representing the blank solution. No hemolysis was observed in saline concentrations of 0.7% and 0.8%. For COVID-19 patients, the onset of hemolysis occurred at 0.5%, with complete hemolysis at 0.4%, compared to healthy individuals, whose hemolysis started at 0.4% and was complete at 0.3%. The results also indicated no significant differences between female and male patients.
DISCUSSION
According to the findings of the current study, the rates of red blood cell breakdown in COVID-19 patients at various sodium chloride solution concentrations differ significantly from those of the control group. The most notable concentrations at which red blood cell hemolysis occurred ranged from 0.1% to 0.6%, as these are hypotonic solutions. A decrease in the resistance of red blood cells to hemolysis was observed in COVID-19 patients, resulting in a higher rate of hemolysis compared to healthy individuals. The rate of hemolysis in a 0% sodium chloride solution is 100%, serving as the standard solution, while at 0.9%, it is 0% in the blank solution, which is isotonic. No hemolysis was observed at saline concentrations of 0.7% and 0.8%, as these concentrations are close to the isosmotic solution.
Erythrocyte membrane alterations have been reported in several studies involving COVID-19 patients, supporting the current findings. Given the essential role of red blood cells in oxygen transport and delivery, these alterations may contribute to the severely reduced oxygen saturation observed in COVID-19 patients. This underscores the importance of determining whether SARS-CoV-2 infection affects erythrocyte metabolism directly or indirectly, thereby impacting blood circulation, structural integrity, and gas transport.14 The results of this study are also consistent with recent research indicating that elevated levels of ferritin and heme ions, both associated with poor prognosis, reflect erythrocyte lysis in COVID-19 patients. One consequence of this lysis is the release of cell-free hemoglobin (CFH), a recognized mediator of disease and adverse prognostic marker in sepsis and ARDS, which can lead to multi-organ damage.15
Data from previous studies suggest that RBC abnormalities may play a significant role in COVID-19 infection. In hospitalized patients infected with SARS-CoV-2, anemia has been associated with increased mortality.16 Moreover, blood smears from individuals with COVID-19-related anemia have revealed unusual patterns of RBC morphological abnormalities.17 Several mechanisms have been proposed to explain the increased fragility and hemolysis of RBCs in COVID-19 patients. One potential factor involves epitopes on the SARS-CoV-2 spike protein, which share significant homology with ankyrin-1, a membrane-integrating protein in red blood cells.18 Due to this structural similarity, antibodies targeting the viral spike protein may cross-react with erythrocytes, leading to their destruction and subsequent anemia.19 SARS-CoV-2 infection and the associated COVID-19 disease may also directly impact RBCs, potentially altering oxygen delivery.20 During the acute phase of infection, changes in erythrocyte morphology, structure, and function may occur.21 These alterations may be related to damage to structural proteins and membrane lipid remodeling, which can affect the cytoskeleton, an essential component for maintaining proper RBC deformability.14
Additionally, it has been demonstrated that severe COVID-19, associated with SARS-CoV-2 infection, significantly affects the erythrocyte system.22 Elevated oxidation of structural proteins and imbalances in membrane lipids have also been observed, potentially altering RBC conformation and contributing to the thromboembolic complications frequently reported in severe COVID-19 cases.23 Another mechanism involves pro-inflammatory cytokines such as IL-1, TNF-α, and IFN-γ, which may impair erythropoiesis by reducing the synthesis of renal erythropoietin (EPO), potentially leading to decreased expression of EPO receptors and programmed cell death in erythroid progenitor cells.24 Additional studies suggest that sepsis induced by COVID-19 may reduce RBC deformability, promoting systemic oxidative stress and organ damage. Systemic inflammation can further disrupt microcirculation, vascular reactivity, platelet aggregation, and leukocyte adhesion to the endothelium.25 Moreover, the inability of the bone marrow to produce healthy red blood cells in COVID-19 patients is another contributing factor. Overproduction of inflammatory cytokines in these individuals may interfere with hematopoiesis by altering erythropoietin release or responsiveness, as well as by modifying erythrocyte structure and function, thereby increasing RBC fragility and variability in cell volume.26 In the present study, we focused on evaluating RBC osmotic fragility in COVID-19 patients and observed a clear manifestation of this phenomenon. Previous research has shown that SARS-CoV-2 infection can impact RBCs and that changes in specific RBC markers are associated with disease severity. Therefore, it is reasonable to hypothesize that the degree of RBC fragility may also correlate with clinical severity in COVID-19 patients, such as mild versus severe pneumonia. Although statistical analyses to examine this potential correlation were not performed in our study, our findings are consistent with previous reports, indicating that hematologic alterations may reflect the progression and severity of COVID-19.27
CONCLUSION
In this study, we observed a higher percentage of hemolysis in red blood cells from COVID-19 patients compared to healthy individuals, suggesting a decreased resistance of red blood cell membranes to hemolysis. This elevated hemolysis may contribute to the increase in heme and ferritin levels, as well as other physiological variables, which could play a role in disease pathogenesis and poor prognosis. It has been hypothesized that SARS-CoV-2 infection could be linked to changes in red blood cell properties, which might contribute to anemia and altered oxygen transport in affected patients. These red blood cell abnormalities may play an important role in the pathophysiology of COVID-19.
Acknowledgment:
We sincerely thank the patients and healthy volunteers who participated in this study. We also extend our gratitude to all medical staff at the hospital for their invaluable support and assistance throughout the research.
Ethical approval:
The study approved by the Institutional Ethics Committee at Anbar University, number 3102, dated 6th October 2021.
Declaration of patient consent:
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given consent for clinical information to be reported in the journal. The patient understands that the patient’s names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Conflicts of interest:
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Financial support and sponsorship: Nil.
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