Red Blood Cells in Health and Disease

Abstract

Red blood cells (RBCs) play a crucial role in the normal functioning of the human body, primarily through their ability to transport oxygen and carbon dioxide. Various diseases, including anemia and other hemolytic disorders, can arise when there is an abnormality in RBC structure or function. The pathophysiology of other conditions, such as cancer and cardiovascular disease can also involve changes in RBCs. Advances in RBC research have led to a better understanding of their structure, function, and pathophysiology. The COVID-19 pandemic has also highlighted the critical role of RBCs in disease pathology, with research suggesting that RBCs may be directly affected by the virus. This review provides a comprehensive overview of the current state of RBCs in health and disease, including recent advances in diagnosis, treatment, and the role of RBCs in disease pathology.

INTRODUCTION

Blood is a complex and dynamic fluid that connects all the systems in the human body. It is essential for life and plays a critical role in maintaining overall health. The analysis of blood provides important insights into the physiological state of the body and can help in the diagnosis and treatment of various diseases.

Red blood cells (RBCs), or erythrocytes, are the most abundant cells in the blood and are responsible for oxygen transport throughout the body. Their unique structure and function are essential for maintaining tissue oxygenation and ensuring the proper functioning of every cell, tissue, and organ.

This paper aims to review the current research on erythrocytes, including their histology, function, biochemistry, scanning electron microscope (SEM) features, histological stains, diseases, immunologic function, and recent advances.

A BRIEF HISTORY OF RED BLOOD CELLS

In 1678, Dutch scientist Jan Swammerdam was the first to observe and describe RBCs under a microscope. However, their first complete account was made by Antonie van Leeuwenhoek in 1674 using a simple microscope.[1] It was not until 1842 that the Scottish pathologist, John Hughes Bennett, coined the term “blood corpuscle” to describe RBCs.[2] Moreover, later in the 19^th century, it was discovered that RBCs contain the iron-rich protein hemoglobin (Hb), which is responsible for their oxygen-carrying capacity. The discovery of the ABO blood group system in 1901 paved the way for blood transfusions, and the use of RBCs in clinical medicine.[2] Later, the development of blood banking and the identification of Rh factors allowed for safe and effective transfusions.[3]

Red blood cells in health

Histology

RBCs have a unique biconcave discoid structure that allows for a faster exchange of oxygen and CO₂. They are small, measuring about 7–8 µm in diameter,[4] and are composed primarily of Hb (a protein that binds to oxygen and carbon dioxide), which makes up about one-third of their volume. In addition to Hb, RBCs also contain a number of enzymes involved in metabolic pathways, including those involved in glycolysis and the pentose phosphate pathway.[5] They lack a nucleus, which means they have no DNA or RNA, and rely on their cytoplasmic enzymes for metabolism.[6] The cytoskeleton of RBCs comprises spectrin, actin, and other proteins, which maintain cell shape and stability.[7] Their shape also plays a crucial role in their function, as it allows them to pass through narrow capillaries and distribute oxygen efficiently.[8]

RBCs contain various enzymes, including carbonic anhydrase, which helps regulate pH by catalyzing the reversible reaction between CO₂ and water to form bicarbonate and H+. The regulation of pH is critical for maintaining the proper functioning of enzymes and metabolic pathways. Abnormalities in RBC shape or cytoskeletal proteins can lead to various diseases such as hereditary spherocytosis, elliptocytosis, and other hemolytic anemias.[9]

Function

In addition to their role in oxygen transport, RBCs also have several other important functions [Figure 1 and Video 1];[9]

Figure 1.

Three-dimensional image of red blood cells

Oxygen transport

The primary function of RBCs is to transport oxygen from the lungs to the tissues and remove carbon dioxide from the tissues to the lungs.[10] This is made possible by the presence of Hb in RBCs which binds to oxygen and enables its transport through the body.[11]

Blood viscosity regulation

RBCs also play a critical role in regulating blood viscosity and blood flow in the microcirculation. They can change their shape and deform to fit through narrow capillaries and maintain blood flow.[12]

pH regulation

RBCs play an important role in maintaining systemic acid-base equilibria by regulating pH levels in the blood. They contain carbonic anhydrase, an enzyme that catalyzes the conversion of carbon dioxide and water to bicarbonate ions and protons, which helps maintain the pH of the blood.[12]

Immune function

RBCs have been shown to interact with immune cells and modulate immune responses, and RBC-derived extracellular vesicles have been implicated in intercellular communication and immune regulation.[13,14] Furthermore, RBCs may play a role in the clearance of pathogens and immune complexes and in the regulation of inflammation.[15] Understanding the role of RBCs in immune function may have important implications for the diagnosis and treatment of various immune-related disorders.[16]

Metabolism

RBCs have a unique metabolism that involves glycolysis, the pentose phosphate pathway, and the methemoglobin reductase pathway. These pathways allow for energy production and maintenance of cellular integrity.[17]

Biochemistry

RBCs are metabolically active cells that produce adenosine triphosphate (ATP) through the glycolytic pathway. RBC metabolism is regulated by various enzymes and cofactors, such as glucose-6-phosphate dehydrogenase, pyruvate kinase, and NADPH.[18]

They lack nuclei and organelles, such as mitochondria, but contain a high concentration of Hb that binds oxygen and carbon dioxide.[19,20] Hb is a tetrameric protein composed of four subunits, each containing a heme group that can bind one molecule of oxygen.[21] RBCs also play a role in the immune system, with some evidence suggesting that they may contribute to antigen presentation and T-cell activation.[22,23] The cytoplasm also contains other enzymes involved in the glycolytic pathway, which generates ATP for energy production.[24] The structure and mechanical properties of RBCs are crucial for their function and involve a complex interplay of membrane proteins, cytoskeletal components, and lipid bilayers.[25] Their membrane is composed of several proteins, such as spectrin, actin, ankyrin, and band 3, which regulate cell deformability, stability, and adhesion.[26] The lipid composition of RBCs also plays a critical role in their function, particularly in maintaining membrane fluidity and flexibility.[27,28] RBCs are constantly exposed to oxidative stress, and their antioxidant systems are essential for protecting the cells from damage.[29] The main antioxidant enzymes in RBCs are superoxide dismutase, catalase, and glutathione peroxidase.[30] Abnormalities in RBC biochemistry or contents can lead to various diseases, such as anemia, hemolytic disorders, and metabolic disorders.[31]

Scanning electron microscope features

Under a SEM, a normal RBC appears as a biconcave disc with a diameter of approximately 7.5 µm and a thickness of 2–3 µm.[32] The surface of the RBC is covered by a lipid bilayer membrane, which appears as a smooth, continuous layer under SEM. The membrane is supported by a network of cytoskeletal proteins, including spectrin and actin, which form a mesh-like structure just beneath the membrane.[33] The cytoplasm of the RBC appears as a central area devoid of organelles, containing mostly Hb molecules and enzymes involved in glycolysis. The RBC’s biconcave shape and deformability are critical for its function, allowing it to pass through narrow capillaries and maximize its surface area for oxygen diffusion.[34]

SEM was used to visualize the surface topography of sickle RBCs, revealing the presence of long, needle-like protrusions that are absent in healthy RBCs. SEM analysis also showed that RBCs from malaria-infected patients have altered surface features, such as knobs and ridges, that facilitate parasite adhesion and invasion.[35] In another study, SEM was used to examine the mechanical properties of RBCs in diabetes mellitus, a disease known to affect RBC deformability and oxygen delivery. They found that RBCs from diabetic patients had reduced surface area and increased stiffness, which could contribute to impaired microvascular circulation and tissue hypoxia.[36,37]

Histological stains

In the context of RBCs, several stains are commonly used to diagnose diseases and identify abnormal RBC morphology.[8]

1. Wright’s stain, also known as Wright-Giemsa stain, helps identify abnormal RBC morphology, such as sickle cells (SCs), target cells, and schistocytes, which are indicative of various diseases[38]

2. Giemsa stain is particularly for diagnosing malaria. This stain binds to the DNA of malaria parasites, allowing them to be identified under a microscope[39]

3. Prussian blue stain binds to excess iron in tissues, allowing it to be visualized under a microscope. It is particularly useful for detecting iron deposits in the liver, which is a hallmark of hemochromatosis

4. Sudan black B stain is used to identify lipid-rich structures in RBCs, such as Heinz bodies, which are indicative of various diseases

5. Acid-fast stains are also used to identify Mycobacterium tuberculosis in RBCs, which can be helpful in the diagnosis of tuberculosis.[40]

Immunohistochemistry

Immunohistochemistry (IHC) is a staining technique used to visualize specific proteins or antigens in tissues. In the context of RBCs, IHC can be used to identify various cell surface markers and cytoskeletal proteins. For example, CD34 is a cell surface marker expressed on hematopoietic stem cells and progenitor cells but not on mature RBCs. Using IHC, it is possible to differentiate between immature and mature RBCs based on their CD34 expression levels.[41]

IHC can also be used to detect cytoskeletal proteins, such as spectrin and actin, which are important for maintaining RBC shape and function. In diseases such as hereditary spherocytosis and elliptocytosis, mutations in spectrin or other cytoskeletal proteins can lead to abnormal RBC morphology and increased fragility. IHC can be used to identify these mutations and characterize their effects on RBC structure and function.[42]

Erythropoiesis

RBCs have an average life span of 120 days and are formed by a process called erythropoiesis, which occurs primarily in the bone marrow. In humans, bone marrow consists of two types: Red bone marrow (RBM) and yellow bone marrow (YBM). RBM is responsible for hematopoiesis, including erythropoiesis, while YBM mainly stores adipose tissue.[43] The erythroid progenitor cells in the bone marrow differentiate and mature through several stages to become RBCs. This process is regulated by erythropoietin (EPO), a hormone produced mainly by the kidneys. EPO stimulates erythroid progenitor cells to divide and differentiate, leading to an increase in RBC production.[44]

In healthy individuals, the majority of erythropoiesis occurs in RBM. The process of erythropoiesis is regulated by various factors, including cytokines, growth factors, and transcription factors. The regulation of erythropoiesis is a complex process, involving the interaction of various cell types and signaling pathways.[45]

Red blood cells in disease

RBCs are crucial for human health, and their shape and biomechanical properties are essential for their functions. In health, RBCs have a characteristic biconcave disc shape, which enables them to deform and pass through narrow capillaries to deliver oxygen to tissues. However, various diseases can alter RBC shape, biochemistry, and other properties, leading to impaired oxygen delivery and other complications.[4]

Anemias

Anemia is a condition characterized by a reduced number of RBCs or Hb levels, which can be caused by various factors such as nutritional deficiencies, genetic disorders, or chronic diseases.[46] Some commonly found anemic conditions include:

Iron-deficiency anemia

Iron-deficiency anemia is the most common type of anemia worldwide.

Cause: Lack of iron in the diet or poor absorption of iron by the body.[47]

Systemic features: Fatigue, weakness, pale skin, and shortness of breath.

Oral manifestations: May include smooth, sore, or red tongue, and brittle or spoon-shaped nails.

Diagnostic investigations: Complete blood count (CBC), serum iron studies, ferritin levels, and stool tests to rule out gastrointestinal bleeding.[9]

Vitamin-deficiency anemia

Cause: Lack of certain vitamins in the diet, such as Vitamin B12 and folate.

Systemic features: Fatigue, weakness, shortness of breath, and neurological symptoms.

Oral manifestations: Sore or red tongue, and oral ulcerations.

Diagnostic investigations: CBC, serum Vitamin B12 and folate levels, and tests for intrinsic factor antibodies.[48]

Hemolytic anaemia

Hemolytic anemia occurs when RBCs are destroyed faster than they are produced.

Cause: Autoimmune disorders, infections, medications, and inherited conditions such as hereditary spherocytosis.

Systemic features: Jaundice, dark urine, fatigue, and weakness. Oral manifestations may include palatal petechiae, gingival bleeding, and ulcerations.

Diagnostic investigations: CBC, reticulocyte count, Coombs test, and Hb levels.[49]

Sickle cell anemia

Cause: Genetic disorder that affects the production of Hb. This causes RBCs to become rigid, sticky, and crescent-shaped, leading to a decrease in oxygen supply to tissues and organs.

Systemic features: Pain crises, fatigue, shortness of breath, and increased susceptibility to infections.

Oral manifestations: Delayed tooth eruption, delayed wound healing, and periodontal disease.

Diagnostic investigations: CBC, SC test, and Hb electrophoresis.[12]

Hemoglobinopathies

Hemoglobinopathies are a group of genetic disorders that affect the production or structure of Hb, the protein responsible for carrying oxygen in RBCs.[50]

Sickle cell disease

SC disease is a common hemoglobinopathy that causes RBCs to become rigid, sticky, and crescent-shaped, leading to a decrease in oxygen supply to tissues and organs.[51]

Cause: A genetic mutation in the HBB gene that codes for the beta-globin subunit of Hb.

Systemic features: Pain crises, fatigue, shortness of breath, and increased susceptibility to infections.

Oral manifestations: Delayed tooth eruption, delayed wound healing, and periodontal disease.

Diagnostic investigations: CBC, SC test, and Hb electrophoresis.

Thalassemia

Thalassemia is another hemoglobinopathy caused by genetic mutations affecting the production of Hb.

Cause: Genetic mutations in the HBB gene or other genes involved in Hb production.

Systemic features: Fatigue, weakness, shortness of breath, and growth retardation in severe cases.

Oral manifestations: Malocclusion, maxillary protrusion, and delayed eruption of permanent teeth.

Diagnostic investigations: CBC, Hb electrophoresis, and genetic testing.[27]

Haemoglobin C disease

Cause: Hb C disease is caused by a genetic mutation affecting the production of beta-globin. In this disorder, there is a substitution of the amino acid lysine for glutamic acid at position 6 of the beta-globin chain.

Systemic features: Hb C disease is generally a mild disorder and may not cause any symptoms in some cases. However, individuals with Hb C disease may experience mild anemia, fatigue, and weakness.

Oral manifestations: There are no specific oral manifestations associated with Hb C disease.

Diagnostic investigations: CBC, Hb electrophoresis, and peripheral blood smear.[19,20]

Hemoglobin sickle cell disease

Cause: Hb SC disease is caused by a genetic mutation affecting the production of beta-globin. In this disorder, there is a substitution of the amino acid valine for glutamic acid at position 6 of the beta-globin chain.

Systemic features: Hb SC disease is usually a milder form of SC disease, but individuals with this disorder may still experience pain crises, fatigue, shortness of breath, and increased susceptibility to infections.

Oral manifestations: Similar to SC disease, delayed tooth eruption, delayed wound healing, and periodontal disease can occur in individuals with Hb SC disease.

Diagnostic investigations: CBC, SC test, and Hb electrophoresis.[51]

Cytoskeletal abnormalities

Cytoskeletal abnormalities are a group of genetic disorders that affect RBC shape and stability.[52] Some of the common cytoskeletal abnormalities of RBCs are:

Hereditary spherocytosis

Cause: Genetic mutation affecting the RBC membrane cytoskeleton, leading to loss of RBC membrane surface area, decreased deformability, and premature destruction of RBCs by the spleen.

Systemic features: Anemia, jaundice, and splenomegaly.

Oral manifestations: Gingival bleeding, periodontal disease, delayed tooth eruption, and increased incidence of caries.

Diagnostic investigations: Blood smear, osmotic fragility test, and genetic testing.[50]

Hereditary elliptocytosis

Cause: Genetic mutation affecting the RBC membrane cytoskeleton, leading to abnormal RBC shape, decreased deformability, and premature destruction of RBCs by the spleen.

Systemic features: Anemia, jaundice, and splenomegaly.

Oral manifestations: None reported.

Diagnostic investigations: Blood smear, osmotic fragility test, and genetic testing.

Other cytoskeletal abnormalities include; pyropoikilocytosis, southeast Asian ovalocytosis, hereditary stomatocytosis, etc.[49]

Segregation, storage, transport and transfusion of red blood cells

Segregation

The segregation of RBCs from blood is an essential aspect of blood banking and transfusion medicine. The process involves the separation of whole blood into its components, including RBCs, plasma, and platelets, using a coagulant. The segregation of RBCs is typically performed using centrifugation-based techniques, such as differential density centrifugation, which separates RBCs from other blood components based on their density.[1] The segregation process must be performed carefully to prevent RBC damage or contamination, which can affect their quality and safety during storage and transfusion.[53]

Storage

The storage of RBCs is necessary for future use in transfusions, but it can also lead to a decline in RBC function and viability over time due to various factors. RBCs stored in citrate-phosphate-dextrose (CPD) solution are typically transfused within 4 h of collection.[1] During storage, RBCs undergo changes such as the loss of deformability, the release of Hb, and alterations in cell membrane properties. These changes can affect the viability, functionality, and safety of the transfused RBCs. Therefore, the quality of stored RBCs must be carefully monitored to ensure their safety and efficacy during transfusion.[54]

Transport

During transportation, RBCs are stored at a temperature between 1°C and 10°C to maintain their viability and functionality. It is crucial to establish and maintain the cold chain for the transport of RBCs to ensure the safety of the recipient. The cold chain includes careful monitoring of temperature and proper handling during transport to prevent damage or contamination of RBCs.[2,55]

Transfusion

During transfusion, RBCs must be transfused within 4 h after collection and stored in a CPD solution. However, transfusion of RBCs can have adverse effects such as hemolytic reactions, infections, and immunological reactions. The transfused RBCs must be carefully matched to the recipient’s blood group, and the quality of the transfused RBCs must be monitored to ensure their safety and efficacy.[3] Different techniques, such as additive solutions, modified atmosphere storage, and irradiation, have been employed to improve the quality and safety of stored RBCs. However, further research is needed to better understand the underlying mechanisms of RBC storage lesions and to develop better preservation methods to maintain the quality and safety of stored RBCs for transfusion.[56]

Red blood cell investigations

RBC investigations are crucial for diagnosing and monitoring a wide range of hematological disorders.

• CBC is the most commonly used diagnostic tool for RBCs, providing information on RBC count, Hb concentration, hematocrit, and RBC indices[1]

• Peripheral blood smear can be used to evaluate the RBC morphology and identify any structural abnormalities[2]

• Hb electrophoresis is an important diagnostic tool in patients suspected of having hemoglobinopathy[3]

• Flow cytometry and fluorescent-based assays can be used for identifying and characterizing RBCs in certain hematological disorders, such as paroxysmal nocturnal hemoglobinuria[4]

• Genotyping and next-generation sequencing are some advanced diagnostic techniques that are used for the diagnosis and characterization of certain inherited RBC disorders.[7]

Red blood cells and COVID-19

COVID-19 has been known to have various effects on the human body, including on RBCs. Several studies have investigated the impact of COVID-19 on RBCs and have come up with the following findings:

• Some studies have reported changes in the shape and size of RBCs in COVID-19 patients, which may be related to inflammation or alterations in oxygen levels, leading to anemia and other conditions

• COVID-19 patients may have an increased risk of developing blood clots, which can be related to changes in RBCs or alterations in the blood clotting system

• In some cases, COVID-19 patients may develop autoimmune hemolytic anemia, a condition where the immune system attacks and destroys RBCs

• A study also found that COVID-19 patients with severe disease had significantly lower levels of Hb and higher levels of lactate dehydrogenase, a marker of RBC damage, compared to those with mild disease.[4]

Further research is needed to fully understand the mechanisms of RBC involvement in COVID-19 and their clinical implications.

Recent advances

Recent advances in RBC research include new methods for RBC analysis, diagnosis, and treatment. A few of them are:

• Microfluidic devices used to study RBC deformability and adhesion in vitro[32]

• Novel diagnostic tools, such as Raman spectroscopy[57] and impedance flow cytometry,[58] can be used to diagnose various RBC-related diseases

• New therapeutic approaches, such as gene therapy and nanoparticle-based drug delivery, can also be used to treat RBC-related diseases[59,60]

• Development of novel methods for the isolation and analysis of specific subpopulations of RBCs, such as reticulocytes and senescent cells[61]

• Identification of new functions of RBCs, such as their role in immune regulation and inflammation[62]

• Use of RBC indices and other parameters in the diagnosis and monitoring of various diseases, including COVID-19.[63]

CONCLUSION

RBCs play a critical role in maintaining normal physiology, including oxygen transport, carbon dioxide removal, and regulation of blood viscosity and flow. Abnormalities in RBCs can lead to various diseases, such as anemia, hemoglobinopathies, and hemolytic disorders, as well as playing a critical role in the pathophysiology of other diseases, such as cancer, cardiovascular disease, and inflammation. Overall, this review highlights the crucial role of RBCs in human physiology, the potential for novel diagnostic and therapeutic approaches, and the importance of further research to improve our understanding of RBC-related diseases and their interactions with other health conditions, including COVID-19.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.