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Red Blood Cells

Human Blood Smear *Magnification: 100x Stain: Leishman Stain *Contains: Erythrocytes, Platelets [1]

Introduction

Red blood cells (RBCs), also known as erythrocytes, are the most abundant blood cell type found in the body, with each cubic millimetre of blood containing 4-6 million cells [2]. RBCs are derived from haematopoietic stem cells in the bone marrow and then released into the blood circulation where they perform their primary task of transporting dissolved oxygen from the lungs to the periphery, and metabolically produced carbon dioxide from the periphery back to the lungs [3]. All mammalian RBCs are enucleated at maturity, and instead have multi-subunit proteins called haemoglobin (Hb) which allow the cells to carry oxygen and also gives them their characteristic red colour due to the iron prosthetic groups within the protein [3]. With a diameter of only 6 microns RBCs are able to travel through the smallest capillaries, and their biconcave shape provides the greatest surface-area to volume ratio for maximum diffusion efficiency [2]. After approximately 120 days of circulation, RBCs begin to lose their flexibility and are then removed from the circulation and processed by specialised macrophages in both the spleen and liver [2].

Because of their vital role, and ubiquitous distribution, any red blood cell disorders will have far-reaching effects within the body. There are many abnormalities affecting RBCs, the most common of which are anaemias involving a decrease in either the number of circulating RBCs or levels of haemoglobin, both resulting in a reduced capacity for gaseous exchange and transport to cells [3]. Conversely an overproduction of RBCs, known as polycythaemia, can also have detrimental effects including hypertension, thromboembolism, and in rare cases advance to leukaemia [3].

This page also discusses some current research being performed on red blood cells, such as using eryptosis of RBCs to detect Parkinson's Disease, and new gene sequencing technologies to detect RBC mutations in patients.

History

Year Finding
ca. 1590 Hans and Zacharias Jannsen (Holland) invent the compound microscope, allowing the content of blood to be observed. Prior to this, only naked-eye inspection of the blood was possible (as reviewed by Haidju (2003) [4])
1668 Jan Swammerdam is the first person to observe and describe red blood cells under the microscope (as reviewed by Mohandas & Gallagher (2008) [5])
1675 Antoni van Leeuwenhoek described the size and illustrated the shape of what he called 'red corpuscles' (as reviewed by Mohandas & Gallagher (2008) [5])
1840 Friedrich Ludwig Hünefeld discovers the haemoglobin molecule in red blood cells [6]
1862 George Gulliver published the primary features of red blood cell membranes (as reviewed by Mohandas & Gallagher (2008) [5])
ca. 1870 Claude Bernard postulates haemoglobins role as an oxygen-carrying protein [6]
1879 Paul Ehrlich publishes a technique for staining blood smears and a method for differential blood cell counting, breaking a 20 year halt in the advancement of haematological knowledge (as reviewed by Haidju (2003) [4])
ca. 1900 Karl Landsteiner (Austria) observed patterns of blood agglutination and developed the first ABO blood group system (as reviewed by Dean (2005) [2])
1902 Alfred von Decastello and Adriano Sturli discovered the fourth blood type AB [7]
1906 Paul Carnot and Deflandre postulate the hormonal regulation of erythropoiesis (as reviewed by Jelkmann (2007) [8])
1910 James Herrick provides the first clinical description of sickle cell anaemia (as reviewed by Thein (2011) [9])
1925 Gorter and Grendel provided the first insights into the structure of the lipid bilayer membrane of red blood cells (as reviewed by Mohandas & Gallagher (2008) [5])
1925 Thomas Cooley and Pearl Lee first describe the blood disorder Thalassaemia (as reviewed by Thein (2011) [9])
1937 Karl Landsteiner and Alexander Wiener discover the Rhesus blood group [10]
1946 The Kell blood group system was discovered as the third most potent group of antigens (After the ABO and Rh groups) (as reviewed by Dean (2005) [2])
1950 The Duffy blood group is discovered, and later shown to be the receptor antigen for Plasmodium vivax (as reviewed by Dean (2005) [2])
1952 Allan Erslev provides definitive evidence for the existence of Erythropoietin as the hormone stimulant of erythropoiesis (as reviewed by Jelkmann (2007) [8])
1956 The specific biomolecular structure of sickle haemoglobin is determined [11]
1957 The specific gene mutation responsible for sickle haemoglobin is discovered [12]
1959 Max Perutz illustrates the three-dimensional structure of the haemoglobin protein using X-ray crystallography (as reviewed by Thein (2011) [9])
1968 The membrane protein Spectrin is discovered and isolated from red blood cells [13]
1971 The topology of red blood cell membrane proteins is defined [14]
2014 Red blood cells are discovered to release Sphingosine-1-Phosphate (S1P), a lipid critical for angiogenesis during embryonic development [15]
2016 Red blood cells are discovered to be an intravascular source of ATP and reactive oxygen species [16]

Structure

Scanning electron micrograph of an RBC from a healthy individual showing the typical morphology. Scale = 1 μm [17]

Erythrocytes are typically biconcave in shape and contain endovesicles which are intracellular membrane vesicles [18]. These types of cells lack cell nucleus and some organelles which accounts for its thin biconcave shape. The mean cell diameter of an adult erythrocyte is 7.5µm, however, this differs to that of foetal erythrocytes which has a mean cell diameter of 8µm [19]. A suggested reason for this difference in cell diameter is due to the lack of nucleus. This allows the adult erythrocyte to efficiently transport gases through very narrow capillaries.

A combination of electron and light microscopy in the stages of erythropoiesis has shown the difference between a foetal erythrocyte and an adult erythrocyte in that:

  • Cell size and volume decreases due to fragmentation and further disintegration of nucleolar fragments into the surrounding karyoplasm
  • Ribosome population decreased to compensate for haemoglobin synthesis

Throughout its lifecycle, erythrocytes undergo vesiculation which may be a protective mechanisms to prevent premature erythrocyte removal. This is apparent when a person suffers from inflammation-associated diseases [18].

Membrane Composition

Topography of the RBC plasma membrane proteins [20]

The erythrocyte membrane is comprised of a mixture of lipids and spectrin cytoskeletal proteins which provide it the structural ability to deform cell shape whilst maintaining its functions. [21] As cited by Zhu et al. (2007), myriads of experiments have been performed by Byers and Branton (1985), Shen et al. (1986) and Liu et al. (1987) in which major proteins were identified, such as α- and β-spectrins, ankyrin, band 3, protein 4.1, protein 4.2, and actin [22].

Membrane and Cytoskeletal Proteins

Table mostly based on review by Daniels (2007) [23]

Protein name Protein function
Urea transporter (UT)-B Urea transport
Aquaporin 1 Water channel
Aquaporin 3 Water and glycerol channel
Band 3 (CD233) HCO3-/Cl- exchanger. Links membrane to membrane skeleton
Protein 4.1 Laterally interacts with spectrin and actin. Vertically interacts with glycophorin C and band 3. Maintains membrane deformability and stability [24]
Protein 4.2 Interacts with band 3 and ankyrin. Maintains association between cytoskeleton and cell membrane [25]
Spectrin Interacts with actin, ankyrin and adducin. Maintains the stability, structure and shape of the cell membrane. Contributes to cell adhesion, cell spreading and cell cycle [26]
Adducin Binds calmodulin. Substrate for protein kinase C and Rho-associated kinase [27]
Rh-associated glycoprotein (CD241) Unknown. Possibly involved in NH4+/NH3 or CO2/O2 transport
RhD (CD240D) Possibly involved in NH4+/NH3 or CO2/O2 transport
RhCcEe (CD240CE) Possibly involved in NH4+/NH3 or CO2/O2 transport
Xk protein Unknown. Homology with neurotransmitter transporters
Duffy antigen receptor for chemokines (DARC) (CD234) Binds chemokines, possibly for removal from peripheral blood
Lutheran glycoprotein (CD329) Unknown. Probably adhesion/receptor. Binds laminin 10/11
Intercellular adhesion molecule-4 (ICAM4) (CD242) Unknown. Probably adhesion/receptor involved in stability of erythroblastic islands. Binds integrins
Erythroblast membrane-associated protein (ERMAP) Unknown. Probably adhesion/receptor
Basigin (CD147) Unknown. Probably adhesion/receptor
CD47 Unknown. Probably adhesion/receptor. May be marker of self
Decay accelerating factor for complement (CD55) Inhibits activity of C3 convertases. Protects cell from lysis by autologous complement
CD59 Inhibits assembly of membrane attack complex. Protects cell from lysis by autologous complement
Complement component (3b/4b) receptor 1 (CD35) Binds and processes immune complexes
CD44 Unknown. Probably adhesion/receptor. Binds hyaluronan
Xg glycoprotein Unknown. Possibly adhesion/receptor
CD99 Unknown. Possibly adhesion/receptor
Semaphorin 7A (CD108) Unknown. Possibly adhesion/receptor
CD151 Unknown. Might associate with integrins to generate laminin-binding complexes
Acetylcholinesterase Unknown. Enzymatic breakdown of acetylcholine
Kell glycoprotein (CD238) Unknown. Endopeptidase that cleaves big endothelin-3
ADP-ribosyltransferase 4 (CD297) Unknown. Possibly transfer of ADP-ribose, if active
Glycophorins C (CD236C) & D (CD236D) Links membrane to membrane skeleton. Could contribute to glycocalyx
Glycophorin A (CD235A) Could contribute to glycocalyx. Interacts with band 3 to enhance anion transport and band 3 trafficking
Glycophorin B (CD235B) Could contribute to glycocalyx

Deformability/ Fluidity

This deformation ability is what allows different substances such as antibiotics to allow diffusion of several ions that are essential to treatment of diseases.[28] An example of this is the use of the antifungal ionophore nystatin which alters the Sodium- Potassium pump in such as way that activates eryptosis (apoptosis-like erythrocyte death) [29]. Deformability will significantly decrease upon transfusion of erythrocytes to patients post-operation subsequently after 3 days. This property is not restored over time despite the thought associated biochemical changes such as the restoration of nitric oxide and ATP levels [30].

Structure

Changes to the erythrocyte shape also affects the surface area to volume ratio. The biconcave shape of RBCs are crucial to traversing throughout the spleen as they are sequestered and removed from circulation [5].

The levels of Adenosine Triphosphate (ATP) within Red Blood Cells is integral in the maintenance and integrity of their membranes. The depletion of ATP within Red Blood Cells results in a deformity in the cell membrane from the Biconcave Disc Shape to a Disc-Sphere Shape. [31] This is due to the fact that membrane deformability is determined and reliant upon the interface between the cell membrane and cell interior where there are ATP-Calcium-Dependent Sol-Gel changes. The balance between Sol-Gel in this region determines the stability or deformability of the cell membrane. [32]

Cross-section through an RBC showing the cortical actin-spectrin network.[33]

Cytoskeleton

Erythrocytes have a cytoskeleton which is composed of a spectrin-actin lattice which has the ability to contract in response to electrolytes and changes in pH [34]. This elasticity and mechanical strength is crucial for the red blood cell to be able to traverse the microvasculature of the body as it constantly needs to deform and reshape. The cytoskeleton of Red Blood Cells are able to exert various forces resulting in the softening of the Red Blood Cell membrane, assisting the ability to deform and traverse through the microvasculature. This structure is crucial also to maintain a constant internal volume as well as surface area. [35] [36]

Nans et al. (2011) used cryo-electron tomography to characterize and evaluate the cytoskeletons of mouse erythrocytes. Their resulting models and images depicted a network of spectrin filaments which converged at actin nodes which the average length between junctional complexes being 31-61nm. It also seemed that this lattice structure was the most dense and thick at the centre of their cytoskeletons and tapered off in both density and thickness towards the periphery. [36]

Left: Quaternary structure of haemoglobin. Right: Haem, in its oxy state [37]

Haemoglobin

Haemoglobin (Hb) is a metalloprotein (protein which contains metal cofactors such as haem) that is attached to vertebral erythrocytes [38]. Haemoglobin has a quaternary form that is comprised of two αβ dimers[39][40] . It has a high tendency to bind with oxygen due to the iron (Fe2+) groups in a haemoglobin. It was confirmed by Gibson and Harrison (1946), that each gram of iron is capable of carrying 393mL of O2 [41].

ABO Blood Groups and Rhesus Factor

Blood types are distinguished through blood group antigens located on the surface of red blood cells. There are approximately 700 erythrocyte antigens that have been discovered, some of which have been further categorised in blood group systems [42]. In particular, ABO and Rhesus (Rh) factor groups systems are quintessential to blood type classification. Through recent research, it has been discovered that certain blood antigens predisposes individuals to an increased risk to certain diseases [43]. For example, non-type O blood type individuals have an increased risk for osteopenia (decrease in protein and mineral content of bone tissue) [44].

ABO Genotypes and Phenotypes

Table based on review by Hosoi E (2008) [45]

*Note the frequencies of ABO blood groups in other countries differ to those of Japan

Phenotype Genotype Frequency (%)
A A/A 8
A A/O 31
O O/O 29
B B/B 3
B B/O 19
AB AB 10

Rhesus Factor

Individuals with the Rhesus Factor or Rh-positive produce anti-Rh antibodies, leading to the agglutination of red blood cells. On the other hand, individuals that are Rh-negative did not have agglutinated blood [46].

The Rh chromosome contains three genes that are expressed in two different allelomorphs: C/c, D/d and E/e. A triplet of these allelomorphs such as cDE correlate to a subgroup such as Rh2. Furthermore, a pair of these genes will determine the phenotype of the Rhesus factor. i.e. if the individual's blood is Rh-positive or Rh-negative.

Gradual change in the phenotype of an individual from Rh-positive to Rh-negative occurs through:[47]

  • Blood transfusions
  • Blood group chimerism
  • Leukemia
  • Dispermy
  • Somatic mutation due to defects in the chromosome containing the Rh gene

Function

Gaseous Exchange

Erythrocyte Gaseous Exchange [48]

The characteristic function of RBCs is their ability to transport oxygen from the capillaries within the pulmonary system, to the capillaries supplying the tissues of the body. During nutrient exchange between capillaries and tissues, oxygen is transferred and exchanged for carbon dioxide. This function is able to occur because of the iron rich molecule haemoglobin which resides within the cytoplasm of the red blood cells. For haemoglobin to perform gaseous exchange it must have the ability to bind O2 to such a degree that it is able to draw it out of the pulmonary capillaries and release the O2 molecules when it reaches the site of delivery at the tissues. RBCs bind O2 in the pulmonary capillaries (an area of high O2 tension) and release O2 in the periphery where there is low O2 tension. [49] [50] [51]

Each haemoglobin molecule is made up of 4 subunits which contain an iron and haem complex at it's core. Each of these subunits can bind to one oxygen molecule, so a haemoglobin molecule can bind to 4 oxygen molecules at once. The initial binding of O2 enhances the binding for the other O2 molecules by causing conformational changes to the haemoglobin molecule, allowing this disposition towards binding [52] [53] [54]. Iron is crucial for O2 transport as helps make up the structure of haemoglobin as a metal cofactor. Transferrins are plasma proteins that transport iron into immature RBCs. Transferrin receptors one the surface of erythrocytes do not interact with iron, but regulate and control its uptake by most cells. [55]

Once the erythrocytes have reached the peripheral capillaries, CO2 diffuses from the surrounding tissue regions into erythrocytes. The CO2 is then converted into bicarbonate HCO3- and a proton by Carbonic Anhydrase. This free proton is then able to bind onto the globin which stabilizes the deoxyhaemoglobin conformation, promoting the release of oxygen into the surrounding tissue regions. Helping this process further occurs when the CO2 binds directly to oxyhaemoglobin, forming carbaminohaemoglobin which further stimulates O2 release. This is the process by which O2 and CO2 are exchanged, with O2 being taken from pulmonary capillaries and delivered to the peripheral capillaries and CO2 taken up by haemoglobin to be carried away by erythrocytes.[56] [57] (See Diagram : Erythrocyte Gaseous Exchange)

Regulatory Functions

RBCs are able to preserve and keep haemoglobin in the circulation. Haemoglobin has a free half life of several hours, but within erythrocytes are able to circulate for the life span of the cell. This is key for the constant function of gas exchange and conservation of energy. [58]

Erythrocytes are able to respond to changes in the metabolic activity around them and therefore are able to act as metabolic sensors. They are able to detect the changes and pressures of oxygen gradients in the areas around them and respond accordingly. For example, in hypoxia or tissue acidosis, haemoglobin unbinds oxygen and releasing it into the oxygen deprived region around it. The erythrocytes are then able to produce an intermediate glycolytic product which is able to bind to the newly formed deoxyhaemoglobin to stabilize it and support the release of oxygen to the surrounding area. Through this, RBCs are able to detect, sense and respond to varying oxygenation needs. [59]

Erythrocytes are able to also respond to changes in sheer stress around them. One way they do this, is as they are compressed and subjected to the stress of thin microcirculation or pores, they are able to synthesise Nitric Oxide which is a vasodilator. In addition to this the glycolytic ATP that is produced and formed by the erythrocytes are exported and stimulate endothelial formation of Nitric Oxide as well as other vasodilators. [60]

Buffering Ability

Erythrocytes are able to extract CO2 from the tissues and areas that it supplies O2 with because CO2 is membrane permeable. Carbonic Anhydrase II (CAII) which is within Erythrocytes are able to convert the CO2 to HCO3-. HCO3- is a buffering agent and assists to maintain the pH within the body, keeping it within homeostasis allowing for the body to properly function [56]. (See Diagram : Erythrocyte Gaseous Exchange)

Haemoglobin is a weak acid as it corresponds to a lower pH. Through Astrup, Rorth & Thorshauge's (1970) research, it was found that the lower the ratio of moles of intraerythrocytic 2,3-Diphosphoglycerate (DPG): moles of haemoglobin, the lower the pH and hence higher acidity. [61] 2,3-diphosphoglycerate (DPG)

Inflammation

Khoory et al. showed that RBCs in acute inflammatory circumstances can increase levels of Reactive Oxygen Species (ROS) and ATP. The increased level of ROS that builds up within the RBCs from their role in oxygen transport can result in shortened red blood cell life span as well as deformability in the membrane leading to an inability to traverse through microvasculature. They showed that acute and chronic inflammation encourage a significant increase in the total number of intravascular ROS, and that RBCs could potentially be the cause for the increase of intravascular reactive oxygen species.[16]
Erythrocyte Immune Complex Clearing Pathway. [62]

Immunological

Erythrocytes have an immunological function where they are able to act as opsonising agents (that is to mark a foreign particle for phagocytosis). This is because erythrocytes have a receptor on their membranes called C1R which is able to bind to a type of molecule which are called opsonins, in particular C3b. It is also able to bind to C3bi and C4b with less efficacy than C3b. These opsonin molecules are then able to bind to foreign material which have been bound by specific antigens. These conglomerates of erythrocytes, antigens, antibodies and opsonin molecules are called an immune complex which are then transported by erythrocytes to the monocyte phagocytic system where the immune complexes are engulfed by phagocytes [62]. (See Diagram : Erythrocyte Immune Complex Clearing Pathway)

Synthesis and Regulation

Illustration of the red blood cell lifecycle

Overview of RBC life cycle

YouTube Link

Life cycle

The process of erythropoiesis. Erythroid progenitors in the bone marrow that depend on Epo and EpoR for differentiation into mature red blood cells (a). The signaling pathways stimulated by EpoR upon binding to Epo (b). EpoR erythropoietin receptor, Epo erythropoietin, RBCs red blood cells [63]

Once erythrocytes have entered the bloodstream, they are able to stay there for around 120days [64]. As these cells have no cellular organelles or nucleus, they are unable to undergo cell division, and are instead manufactured within the bone marrow. As erythrocytes can die whilst in circulation, it is essential that they are replaced very frequently. It is approximated that they are produces at 2-3million per second. Erythrocytes are produced by a process known as erythropoiesis and are removed from circulation within the spleen [64].

Erythrocyte production

Erythrocytes come to full maturity in the bone marrow, and develop from hematopoietic stem cells. The maturity and development of red blood cells is based on hematopoietic growth factors, specifically involving erythropoietin[65]. This growth factor is released from kidney based on cues detected and the response of low oxygen levels, such as haemorrhage, destruction of bone marrow, diseases involving the circulation and being the high altitude regions (low atmospheric oxygen). Once erythropoietin is released from the kidneys, it travels through the blood stream to the bone marrow where it triggers differentiation of pluripotent stem cells to erythrocytes [66]. During the differentiation process, the erythrocyte goes through developmental stages or changes (known as erythropoiesis), where it loses its nucleus and cell organelles, forming a reticulocyte as the final form before a mature red blood cell. It is during this stage where ribosomes are still present within the cell. During particular events that require a very quick erythrocyte synthesis, it is known that reticulocytes enter the blood stream as well, where normally, approximately 0.5-2.5% of circulating red blood cells are reticulocytes, in events of rapid erythropoeisis, this percentage can vary.

Filtering and destruction of erythrocytes

The degradation of haem to bilirubin [67]

This process occurs within the spleen, a lymphoid organ that stores blood cells and removes the old erythrocytes from the circulation. Some red blood cells are hemolyzed with the bloodstream (eryptosis), but majority are phagocytesed by macrophages in the spleen and also in the liver (but to a smaller extent). During the destruction of erythrocytes by macrophages, the haemoglobin is catabolised [68], where the iron is removed and the resulting heme group is converted to a yellow compound known as bilirubin. This is released into the bloodstream and can give the blood a slight yellow tinge. Bilirubin is further catabolised in the liver and the products are secreted in the bile to the small intestine and excreted in the faeces, which gives it its brown tinge. Other bilirubin metabolites that are released in the bloodstream move to the kidneys and excreted in urine and give urine the yellow colour [69]. The accumulation of bilirubin within circulation can lead to complications as it is toxic - jaundice is a common disease that can result from this [69].

Recycling of iron

The iron that was released from the haemoglobin is recycled and is able to form new haemoglobin. Iron is transported through the blood on the protein transferrin which picks up the iron from the gastrointestinal tract, or from the spleen and transports the iron to the bone marrow for synthesis of erythrocytes. Iron can also be stored within the liver bound to the ferritin protein. There are specific macrophage proteins, such as Nramp1, that can participate in the recycling of iron taken from phagocytose senescent erythrocytes [70].

Eryptosis

Eryptosis and its signalling molecules: the changes it effects on erythrocytes[71]

Erypoptosis is known as the suicidal death of red blood cells within circulation. This is another method of RBC death other than phagocytosis by macrophages within the spleen. It is commonly characterised by cell shrinkage, cell membrane blabbing and translocation of phosphatidylserine on the cell membrane surface [72].

Triggers of eryptosis include: ceramide formation, stimulation of capsizes, calpain activation (this is a growth signalling factor), energy depletion, oxidative stress and mainly calcium ion entry[72].

  • Calcium ion entrance can characterise all types of RBC death methods. As a result of this increased cytoplasmic Ca2+ due to activated Ca2+ channels, the following methods of RBC death can occur [73]:
  • Calcium ions may cause phosphatidlyserine translocation on the surface of the red blood cell membrane, this can result in cell membrane phospholipid scrambling.
  • Activation of the calcium ion sensitive channels will also result in the activation of potassium in sensitive channels, causing an influx of potassium ions. This leads to hyper polarisation of the RBC membrane. At the same time chloride ions leave the cell to create a balance in charges. This results in osmotic water flow causing cell shrinkage.
  • Entry of calcium ions, followed by the activation of calpain leads to the degradation of membrane proteins resulting in membrane blebbing [74].

Phosphatidylserine complexes are part of the phospholipid membrane. They exposure of these complexes on the surface on RBCs allow the cell to adhere to the vascular wall by binding onto endothelial receptors[72]. This binding acts as a marker - ‘scavenger receptor’ and induces a very strong chemotactic response and calcium mobilisation, thus leading to the events described above. This labelling can also result in a phagocytic response clearing these red blood cells from circulation. By this method, red blood cells are haemolysed within the bloodstream rather than within the spleen[72].

As stated in the review, [75], when eryptosis is accelerated, it can be ‘fixed’ with enhanced erythropoiesis and reticulocytosis. Specific diseases such as diabetes, sickle cell anaemia and even iron deficiency can lead to increased eryptosis [75]. It is also known however, based on the review[76] that inhibition of eryptosis mainly occurs due to nitric oxide, as this makes RBCs resistant to increased influx of cytoplasmic calcium ions; and erythropoietin, which inhibits calcium permeable cation channels.

Diseases and Abnormalities

Iron Deficiency Anaemia

Iron Deficiency Anaemia (IDA) is a type of nutritional anaemia that results when there is insufficient iron available for haemoglobin in red blood cells, myoglobin in muscle cells, and a range of other cellular processes [77]. As reviewed by Pasricha et al. (2010) [78], the WHO defines anaemia as a haemoglobin level below 130 g/L in men, 120 g/L in women, and 110 g/L in pregnant women and preschool children. IDA occurs in two main forms.

Absolute Iron Deficiency Anaemia

Absolute Iron Deficiency Anaemia (AIDA) exists when total iron body stores are low or depleted due to iron requirements or losses surpassing the rates of iron absorption and recycling [77]. Because the body's requirements for iron exceeds the maximum amount it can absorb, AIDA is actually quite common amongst young children during periods of rapid growth, adolescent girls and women during menstruation [79], pregnant women and regular blood donors [80]. In these cases it is simply treated by increasing the amount of iron-rich foods in the diet, or iron supplementation, combined with absorption enhancers such as vitamin C [77]. However AIDA can also have much more serious causes such as by blood loss through gastrointestinal bleeding, and iron malabsorption may be a sign of intestinal mucosal disorders, impaired gastric acid secretion or colonisation by Helicobacter pylori [78].

Functional Iron Deficiency Anaemia

The other form is Functional Iron Deficiency Anaemia (FIDA), a disorder which arises when total body iron stores are normal but are not being mobilised to the bone marrow for erythropoiesis [78]. This is due to the action of the hormone hepcidin, the master regulator of iron metabolism, which when upregulated, such as during inflammation, blocks the release of iron from hepatocytes and macrophages and also reduces iron absorption by enterocytes [78] [81].

3D rendered isosurfaces of RI maps of individual RBCs from (A) healthy, (B) IDA, (C) reticulocyte, and (D) HS red blood cells[82]

IDA Symptoms

Table based on review by Lopez et al. (2016) [77]

*Note many of the symptoms are common to all anaemia types

Very frequent Frequent Rare
  • Paleness
  • Fatigue
  • Dyspnoea
  • Headache
  • Diffuse/moderate alopecia
  • Atrophic glossitis
  • Restless leg syndrome
  • Dry/rough skin
  • Dry/damaged hair
  • Cardiac murmur
  • Tachycardia
  • Neurocognitive dysfunction
  • Angina pectoris
  • Vertigo
  • Haemodynamic instability
  • Syncope
  • Plummer-Vinson Syndrome

Hereditary Spherocytosis

Hereditary Spherocytosis (HS) is a group of variable inherited anaemias that results in spherical-shaped erythrocytes (spherocytes) [83]. The common cause amongst HS disorders is a loss of cohesion between the cytoskeleton and plasma membrane due to a weakening of the vertical linkages that anchor the cytoskeletal elements to the integral membrane proteins [84]. These vertical linkages include spectrin, ankyrin-1, band-3 and protein-4.2, and when compromised cause the lipid bilayer to become unstable and release "skeleton-free lipid vesicles" via one of two pathways (depending on which vertical linkages are defective), both of which result in the formation of spherocytes that have a reduced surface-area to volume ratio and capacity for deformability [83] [84].

During normal uptake by the spleen spherocytes suffer additional damage or 'splenic conditioning', such as further loss of surface area and increased cell density. This is due to the hostile environment of the spleen which consists of low pH and severely reduced concentrations of glucose and ATP, contact with phagocytic cells and the high local concentrations of oxidants [84]. Some spherocytes are able to escape the spleen and re-enter the systemic circulation, while those remain are eventually destroyed causing haemolytic anaemia [83] [84].

Defects in the vertical linkages are caused by highly variable mutations in the genes encoding those proteins which are responsible for the heterogeneous nature of HS based upon the combination of linkages that are affected. For example, Bogusławska et al. [85] reported a novel ankyrin-1 missense mutation that resulted in a single nucleotide substitution (CTG → CCG) which correlated with a HS phenotype in a Polish family. The clinical characteristics of HS are wide ranging due to the variability of the disease and thus patients can be asymptomatic, but the common haemolysis typically results in anaemia, jaundice, reticulocytosis, gallstones and splenomegaly which can be almost effectively cured with a splenectomy (as reviewed by Perrotta et al. (2005) [84]).

Sickle Cell Anaemia

Normal and Sickled Red Blood Cells[86]

Sickle cell anaemia is a genetic disorder of the haemoglobin molecule within red blood cells. The disease is homozygously inherited from both parents and is caused by a point mutation (GAT → GTT) in the ß-globin gene [87], replacing glutamic acid with valine in the ß-globin polypeptide which results in a sickle-shaped haemoglobin molecule when deoxygenated [11] [12]. Due to its shape, the sickle haemoglobin (HbS) molecule not only has a reduced capacity for oxygen uptake, but can also lead to occlusion of the micro- and macro-vasculature. Once deoxygenated, HbS molecules have a tendency to polymerise to each other resulting in distortion of the cell shape, cellular dehydration, increased rigidity and stickiness that promotes RBC adhesion to the endothelial lining of blood vessels [87], placing the patient in a thrombotic state.

This adherence is due to the α4β1 and CD36 receptors on the surface of the sickled RBCs, which bind the cells to the endothelial layer through vascular cell adhesion molecule (VCAM)-1 and thrombospondin (released from activated platelets) [88] [89]. As reviewed by Ballas (2002) [87], Matsui et al. (2001) [90] found that activated endothelial cells release a glue-like molecule called P-selectin onto their outer membrane surface where it also binds sickle RBCs, further contributing to their adherence. This vascular occlusion not only leads to ischaemia and potential infarction, but can also result in an inflammatory state within the vessels once the obstruction is cleared and reperfusion occurs due to neutrophilia and the local production of free radicals as a consequence of endothelial injury and tissue damage from ischaemia [87].

Thalassaemia

Thalassaemia is a heterogeneous group of inherited haemoglobin disorders characterised by defects in the genes encoding the α- and ß-globin polypeptides, resulting in an overall defective Hb molecule [91] but the exact phenotype involves a complex interaction between environmental and other genetic factors [92], and thus the severity of the anaemia varies between individuals . Thalassaemia is broadly categorised into two main groups based on the imbalanced rate of production of the α- and ß-globin chains: α-thalassaemia results from excess ß chains, and ß-thalassaemia from excess α chains [93].
The regulation and effects of hepcidin on iron metabolism and erythropoiesis [94]

ß-thalassaemia

Inheriting a single ß-globin mutation results in ß-thalassaemia carrier with no significant symptoms, while inheriting an allele from both parents causes clinically severe dyserythropoietic anaemia [95] as the excessive α chains damage the red blood cells and their precursors, as well as affecting bone development due to expansion of the defective marrow [93]. In addition to this, intestinal iron absorption is also increased leading to iron deposition in endocrine organs, the liver and the heart [93]. The increased iron absorption is caused by the suppression of hepcidin transcription, which is the opposite of what occurs in FIDA.

α-thalassaemia

α-thalassaemia on the other hand is caused by gene deletions with the phenotype dependent on the number of α mutations and their interactions with each other. As there are two α genes on each chromosome 16, this results in three main phenotypes [91]:

  • An asymptomatic or slightly anaemic carrier
  • An intermediate condition causing severe haemolytic anaemia
  • A lethal condition that usually results in intrauterine or prenatal death

In general, the excessive ß chains in α-thalassaemia form β4 molecules (Haemoglobin H) which do not precipitate in the bone marrow, but do precipitate in mature RBCs [93].

Co-Inheritance and Treatment

Individuals can also co-inherit both α and ß mutations resulting in a clinically different phenotype, but the patient can be misdiagnosed as a ß-thalassaemia carrier which becomes significant when considering treatments [91]. Currently the only cure for thalassaemia is a bone marrow transplant, while ongoing treatments include regular blood transfusions and iron-chelation therapy, with a splenectomy being performed only in the rarest of cases [93].

Polycythaemia

Polycythaemia is an abnormality where the percentage volume of red blood cells within the blood, known as the Packed Cell Volume (PCV) or Haematocrit (HCT), is increased [96]. It can be broadly split into two categories.

Absolute Polycythaemia

Absolute Polycythaemia arises when there is an actual increase in the number of circulating RBCs, and can be further divided into another two categories.

  • Primary Polycythaemia: Primary polycythaemias, such as polycythaemia vera, occur as a result of myeloproliferative neoplasms within the bone marrow affecting haematopoiesis [97] and producing excessive amounts of RBCs as well as other blood cells [96]. As reviewed by Campbell et al. (2005) [98], a relationship between polycythaemia vera and a somatic V617F mutation in the Janus Kinase 2 (JAK2) gene was found to exist in most patients which up-regulated JAK2 activity, thereby allowing haematopoietic stem cells to differentiate into blood cells independently of erythropoietin (EPO) signalling. In the rarest of occasions, primary polycythaemias can progress to acute leukaemia, with potential for malignancy [96].
  • Secondary Polycythaemia: Secondary Polycythaemias are the result of an increase in EPO levels [96] which then trigger the production of RBCs through normal pathways, albeit past normal levels. While secondary polycythaemias can occur as a result of specific cancers affecting EPO producing organs (kidney and liver) to produce abnormal levels of EPO [96], they more commonly arise as a physiological compensatory mechanism for when the body is placed in a hypoxic state, such as at high altitudes and in heart and lung diseases [97]. When the body's oxygen supply is inadequate, Hypoxia-Inducible-Factors (HIFs) are activated which then upregulate EPO production [99] leading to increased RBC levels.

Relative Polycythaemia

Relative Polycythaemia is due to a decrease in the blood Plasma Volume (PV) which will elevate the PCV because the non-cellular volume of the blood has decreased [97]. It is not limited to any one cause, as a number of contributing factors can lead to a reduction in PV including obesity, hypertension, smoking and alcohol [96].

Clinical Implications

Whatever the cause, polycythaemia will result in a thickening of the blood which negatively affects blood flow [96], creates hypertension, places the patient in a hyper-coagulable state and at much greater risk of developing a thromboembolism.
Illustration of the malarial Plasmodium lifecycle [100]

Malaria

Mechanisms of resistance to malarial Plasmodium conferred by sickle cell disease [101]

Malaria is a parasitic disease caused by infection with the Plasmodium protozoan of which there are 5 different species that affect humans. They are transmitted through the female Anopheles mosquito vector during its blood feeding when malarial sporozoites are injected into the bloodstream and immediately invade the liver hepatocytes and asexually multiply through shizogony for up to 8 days to become merozoites [102]. Once they leave the hepatocytes, the merozoites then invade the erythrocytes to begin an intraerythrocytic asexual cycle where they feed on the contents of the RBC until it bursts, and then re-invade more RBCs to produce sexual gametocytes that will reproduce through meiosis if taken up by another female Anopheles[102].

The destruction of the RBC schizont, and the subsequent release of parasites and intracellular material, activates a cytokine cascade which is ultimately responsible for the clinical manifestations seen in malaria patients [103]. The Plasmodium also change the plasma membrane by inserting parasite-derived proteins and expose cryptic surface antigens which increase nutrient uptake, and discard the toxic haem waste product through "lipid-mediated crystallisation" to the biologically inactive haemozoin (a malarial pigment) (as reviewed by White et al. (2014) [102]).

The merozoites invade the RBCs through different ligand-receptor interactions depending on the particular Plasmodium species involved. In particular, P. vivax interacts with the Duffy blood group antigen Fya or Fyb, and as a result people who carry the Duffy-negative FyFy phenotype are immune to P. vivax infection [102]. Contrary to popular belief, people with sickle cell disease are not fully immune to malaria, as it is only those who are heterozygous for the sickle gene that are much more resistant to it. This is because the malarial merozoites greatly increase the rate of RBC sickling upon invasion, making them much more likely to become phagocytised by macrophages [104]. It is well known that this heterozygous mutation has created an evolutionary advantage for people living in areas prone to malaria transmission (especially parts of Africa and South Asia).

Malaria Symptoms

Based on reviews by Parmet et al. (2010) [105] and Roach (2012) [106]

Intermediate [105] Severe [106]
  • Sudden, violent chills
  • Intermittent fever
  • Sweating
  • Exhaustion
  • Headaches
  • Seizures
  • Delirium
  • Acidosis
  • Hypoglycaemia
  • Anaemia
  • Shock
  • Disseminated intravascular coagulation
  • Renal failure
  • Haemoglobinuria
  • Pulmonary oedema
  • Cerebral infarction and oedema

Current Research

Erythrocytes of Parkinson's disease patients prepared from whole blood smears[107]

Eryptosis and Parkinson's disease [107]

This study mainly involves the use of biological markers that can help in identifying at-risk people of Parkinson's disease (PD) or to track disease progression and response to specific therapies. Signalling molecules that are involved in inflammatory responses in PD pathophysiology are used to look at PD in a 'holistic' way. These signalling molecules are also important in the coagulation/haematology system. The hypothesis that is tested in this study is the possible interaction of these signalling molecules in PD and the possible effect these molecules can have on erythrocytes of PD patients. Results are observable as changes in morphology of red blood cells and of PD patients relative to healthy controls were taken. It has been shown that red blood cells of PD patients are very different in their morphology and show signs of eryptosis. This led to the possible conclusion that changes in morphological appearance of red blood cells due to eryptosis can be used as a useful diagnostic and prognostic tool.

Selected inflammatory signalling molecules associated with PD such as prostaglandins and thromboxanes are involved in the coagulation/haematology system. Erythrocytes and fibrin networks were observed in a selection of PD patients to determine if their RBCs and fibrin networks had changed. The hypothesis presented in this study was the possible interaction of these signalling molecules on PD and how these molecules may affect the coagulation/haematology system of the patients.

The question was whether signalling molecules that are up-regulated in Parkinson’s disease is implicated in eryptosis. They are closely involved in the development of the variant programmed cell death. It was originally argued that there were no reported cases of eryptosis in PD patients, however due to the changes signalling molecule profiles, it can be argued otherwise. These signalling molecules can act as triggers that lead to increased cytoplasmic Ca2+ due to the activation of Ca2+ permeable channels. This sudden influx of Ca2+ can cause cells shrinkage, membrane scrambling and membrane blebbing - all that contribute to RBC death. The main signalling molecules incites in PD that are related to eryptosis is calpain and ceramide - levels of molecules change in PD patients. These molecules are known to belong to a family of calcium-dependent cysteine proteases that play an important role in neuronal death.

Gene sequencing technique to diagnose Polycythaemia [108]

As previously discused, Polycythaemia is an abnormality where there is a higher percentage of RBC within circulation[96]. A study conducted by Tiong et al, focused on the possible use of a next generation sequencing (NGS) technique that has the ability to diagnose polycythaemia in patients that would otherwise be missed. It has been found in 95% of patients diagnosed with polycythaemia also have a somatic activating mutation in exon 14 of the JAK2 gene encoding the JAK2 non-tyrosine receptor. JAK2 gene is responsible for controlling the production of RBCs from hematopoietic stem cells, therefore a mutation in this gene can lead to the overproduction of RBCs. As described in the study, NGS of the entire coding region of JAK2 revealed mutations present in exon 14. The potential use of NGS as a diagnostic measure provides an accurate conclusion whether a patient has polycythaemia and is able to prevent unnecessary investigations, thus ruling out the possibility of the patient having erythrocytosis. Next generation sequencing of all JAK2 exons by NGS is highly sensitive and overcomes the limitations of direct sequencing, and it is likely that it will become the standard secondary assay for detection of JAK2 mutations after a specific test for the V617F mutations is negative.

Erythrocytes in nanomedicine [28]

As reviewed by Zhang (2016) [28], the issue of current drug delivery systems (DDSs) are limited in its clinical application as the use and administration of nanoparticles are recognised as foreign materials within the human body, thus leading to immunological reactions and toxic implications to the patient. Therefore in order to overcome this issue would be to find a way to administer therapeutic agents in nanoparticles and keeping them hidden from the immune system and successfully target the drug delivery regions of interest. Recent studies are focusing on the possibility of delivering nanoparticles using natural cells - in particular, erythrocytes. RBCs present a possible drug carrier due to their biocompatibility and long systemic circulation. The opened and closed property of RBC membranes also present as an advantage, as it allows the encapsulation of a wide range of biologically active substances (including nano particles).

Methods used for the administration of nano particles within the RBC include hypnotic osmotic lysis. With the use of a near-infrared laser, this would induce the formation of pores within the membrane of the RBC, allowing soluble agents (nano particles) to flow against a concentration gradient within the RBC. These pores are re-sealed under isotonic conditions to form erythrocyte based nanoparticulate DDSs for long periods of time with controlled drug release.

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