Congenital Defects with Impaired Red Blood Cell Deformability – The Role of Next-Generation Ektacytometry

Abstract

The red blood cells (RBCs) carry oxygen from the lungs to the tissues, and for this, they must be able to deform. Accordingly, an impairment of RBC deformability is the cause of RBCs trapping and removal by the spleen and haemolysis. The most common causes for the decline in red cell deformability are the RBC membrane defects (abnormal shape or ionic transport imbalance), haemoglobinopathies (increased rigidity), or enzyme deficiencies (decreased anti-oxidant defences or ATP content). The most common cause of hereditary anaemia in childhood is hereditary spherocytosis (HS), characterised by a marked RBC deformabiity. A decreased RBC deformability has been found in hereditary haemolytic anaemias (HHAs) using the new-generation osmotic gradient ektacytometry (OGE), probably due to a combination of membrane protein defects and ionic imbalance. Therefore, OGE is currently considered the gold standard for the measurement of RBC deformability and the most useful complementary tool for the differential diagnosis of HHAs. Moreover, since several new forms of treatment are currently developed for hereditary RBC defects, the clinical interest of OGE is increasing. The aim of this chapter is to provide further information about the use of RBC deformability in clinical diagnosis and the OGE as a new challenge to decrease the frequency of undiagnosed rare anaemias.

1. Introduction

The red blood cell (RBC) lives in the bloodstream about 120 days (4 months) providing oxygen and nutrients to all the tissue cells. A large part of their passage is through capillaries with a diameter ranging from 3 to 8 μm which is less than its mean cell diameter, of about 7.5 μm. Accordingly to reach all the cells, the RBCs have to adapt their shape to the dynamically changing flow conditions especially in microcirculation. This is due to its ability to deform and pass through small capillaries and recover their initial shape [1]. The basis of this extreme deformability is its characteristic discoid shape with a biconcave profile (Figure 1a, b). RBC deformability significantly affects blood viscosity as its decrease elevates the blood viscosity, the flow resistance and, in turn, the blood pressure [3].

Figure 1.

Red blood cell (RBC) shape and sizes (a and b). Structure on RBC membrane skeleton (c). Source: [2].

RBC deformability is the result of three cell properties: (a) shape (bi-concave discocyte) depending on the surface/volume (S/V) ratio, (b) viscosity depending on intracellular haemoglobin concentration and its physicochemical state and (c) viscoelasticity largely determined by the cytoskeleton, an actin-spectrin network that underlies the lipid leafet of the membrane [4]. This structure consists of long twisted strands of alpha and beta spectrin and actin filaments that form the inner shell of the RBC and provides the basis of cell deformability (Figure 1c). Spectrin is bound to the lipid bilayer of the membrane at sites containing the anion exchanger, band 3 via cytoskeletal proteins, ankyrin and adducin. Some of the transmembrane proteins (such as glycophorin A) are RBC antigens and contribute to the blood group system [5]. Due to their discoid form, the normal RBCs have an S/V ratio of about 1.56, indicating an excess of the surface (membrane) with regard to the volume. This allows RBCs to change their shape without increasing the surface and, subsequently, to be highly deformable in the bloodstream and in the spleen. The spleen is a highly vascularised organ, with a blood flow that represents around 6% of the cardiac flow. Its vasculature is formed by a complex network of capillaries and sinuses with endothelial cells that are anastomosed together without junction. Under the changes of the capillary diameter, the inter-endothelial slits (IESs) of about 0.2–0.4 μm of diameter are created, and the RBCs are obliged to pass through these IESs suffering an extreme, but reversible, deformation (Figure 2). Only the cells with normal deformability can overcome the IES, and due to this, this system is considered a particular splenic barrier for RBC defects [6]. When the S/V ratio for healthy RBCs is beyond the “physical fitness test” to pass through the IES, the cells are unable to adapt and are easily trapped and destroyed by the spleen [7].

Figure 2.

Spleen red pulp observed with scanning electron microscope. RBCs trespassing the inter-endothelial slits.

2. General factors influencing RBC deformability

RBC deformability is the key physical property to ensure suitable tissue oxygenation, and thereby, it has been recognised as a sensitive indicator of RBC functionality. As shown in Figure 1, it depends on the structural properties of the ‘horizontal’ cytoskeletal components (spectrin-action-band 4.1) and the ‘vertical’ cytoskeletal proteins (spectrin-ankyrin-band 4.2) and their interaction with the cytosolic domain of band 3 protein (anion exchanger AE 1) or glycophorin C/D, respectively [8]. This essential deformability mechanism can, in turn, be affected by various physiological and pathological factors mainly due to intrinsic defects of cell membrane skeletal architecture [9, 10, 11], haemoglobin defects [12], mechanical damage [13] or normal RBC ageing [14, 15, 16]. There are many factors that can influence RBC deformability, but in practice, they can be summarised as follows: (a) RBC shape, (b) intracellular haemoglobin concentration (MCHC), (c) temperature, (d) osmotic pressure, (e) ATP depletion, (f) nitric oxide concentration and (g) membrane lipids and/or proteins abnormalities.

2.1 RBC shape

Due to its deformability, by shearing flow, the biconcave disk form of the RBC changes to an ellipsoid form, facilitating large reversible elastic transformation into arbitrary shapes. This enables RBCs’ large deformations in the blood stream exhibiting diverse morphological features depending on physiological and physiopathological conditions [8, 14]. As mentioned before, the principal factor that makes possible the large deformations of normal RBCs is the high surface/volume (S/V) ratio, and when it changes, the abnormal deformability correlates with the pathogenesis of several RBC morphological disorders, including spherocytosis, acanthocytosis, stomatocytosis, schizocytosis and tear drop cells [15]. Since an abnormal RBC shape is the most important cause of the decreased deformability and haemolysis [16], RBC morphology examination is very helpful for the differential diagnosis of HHAs. In normal circumstances, RBC ageing is also an important cause of morphological alterations and decreased S/V ratio, as a consequence of the decrease of cellular ATP content [17].

2.2 Haemoglobin concentration

Mean cell haemoglobin concentration (MCHC) determines cytoplasmic viscosity, and it is another crucial factor determining RBC deformability. An increase of MCHC is always associated with a decrease of RBC deformability, and the two classical examples of this situation are hereditary spherocytosis (HS) and hereditary xerocytosis (HX) [1, 4, 18]. Additionally, reduced deformability in aged cells is also correlated with an increase of MCHC and RBC cytoplasmic viscosity due to cell dehydration [8].

2.3 Temperature

Waugh and Evans [19] demonstrated that the temperature plays an important role in RBC deformability. Below 25°C, deformability decreases as temperature decreases, whereas no apparent change in RBC deformability is observed between 25 and 37°C [20]. Moreover, body or febril temperature may be particularly important since the increase of body temperature decreases RBCs’ elasticity and filterability. However, this has no clinical effect on the pathophysiology of haemolytic anaemia [21].

2.4 Osmotic pressure

Different osmolalities of extracellular medium can bring significant changes on RBC shape and, in turn, on its deformability. At normal physiological osmotic pressure (295 mOsm/kg H₂O), the RBCs maintain their biconcave shape and deformability, but in a hypotonic medium (< 295 mOsm/kg H₂O), they are swollen due to water intake and lyse (haemolysis). On the contrary, in a hypertonic medium (>295 mOsm/kg H₂O), the RBCs suffer a cell shrinkage and become less deformable. Although the total number of Hb molecules in RBCs, or the MCHC, does not significantly change with osmolality, the value of Hb concentration can considerably change due to water influx (Hb dilution) or efflux (Hb concentration). RBCs exhibit their maximum deformability at physiological osmotic pressure; but under either hypertonic or hypotonic conditions, their deformability decreases [22]. Interestingly, this has demonstrated that at low shear stress (1–3 Pa), the RBC deformability was maximal in hypotonic conditions (225–250 mOsm/kg H₂O), which is lower than the normal plasma osmolality (290–310 mOsm/kg H₂O). This may play an important role in microcirculation processes [22].

2.5 Adenosine 5′-triphosphate (ATP) depletion

The metabolic dependence of RBC deformability has been described many years ago by Weed et al. [23], and it has been also demonstrated by techniques measuring the mechanical properties of ATP-depleted RBCs [24]. Accordingly, the ATP concentration seems to be crucial for maintaining the biconcave shape of normal RBCs, and its decrease affects the RBC shape, inducing the change from its classical biconcave shape to a flattened echinocytic shape with decreased deformability [25]. When the ATP content of RBC decreases, three factors become altered: (a) ion handling by pumps and passive transport pathways [26], (b) proteolytic activity of Ca++dependent protease calpain [27] and (c) structural integrity of the membrane architecture [28].

2.6 Nitric oxide

Nitric oxide (NO) is an important cardiovascular regulator that has an action on the vascular smooth muscle, but also as a regulatory factor in RBC deformability and aggregation [29, 30]. It has been demonstrated that the decrease of NO concentration due to the effect of NO synthase inhibitors is accompanied by a decrease of RBC deformability [31].

2.7 Disturbances of membrane lipids and/or proteins

The membrane lipids that form the double-layered surface of RBCs (the lipid bilayer) are classified as phospholipids, glycolipids and cholesterol. An increase in the cholesterol-to-phospholipid ratio (C/PL) from 1.28 to 2.0 results in a decrease in RBC filterability due to an increase of membrane rigidity [32, 33]. RBC deformability may be also affected by abnormalities of the membrane skeletal proteins such as Band 3 and glycophorin, but the most important cause of decreased RBC deformability is the existence of abnormal RBC membrane cytoskeletal proteins due to genetic defects. The best example is hereditary spherocytosis (HS), due to the decreased S/V ratio, but in homozygous hereditary elliptocytosis (HE) and pyropoikilocytosis (HPP), the deformability changes are closely related to the reduced levels of band 4.1 protein. This protein participates in the maintenance of normal membrane skeletal equilibrium and shape [34].

Haemoglobinopathies such as sickle cell disease (SCD) and thalassaemia can also alter the RBC deformability and decrease the RBC life span. SCD is an autosomal recessive inherited blood disorder due to a point mutation in β-globin gene that results in the production of HbS that under deoxygenated conditions becomes self-assembled and grows to fibres inside RBCs up to a few micrometre lengths. Due to these highly stiff HbS fibres, RBCs become elongated and sickled (sickle cells) with a significantly increase of rigidity and a decrease of deformability (Figure 3). After repeated sicklings, a fraction of RBCs become irreversible sickle cells (ISC), with a static rigidity that is strongly affected by the haemoglobin concentration [35]. ISCs exhibit the highest loss in deformability, and they are trapped by the spleen and retained in the microcirculation leading to severe painful vaso-occlusive crises (VOCs). Thalassaemia is characterised by a partial or total absence of one of the globin chains (α, β, γ or δ), and when this is associated with an excess of uncoupled free globin chains, these precipitate leading to RBCs’ inclusions composed of denatured Hb called Heinz bodies (Figure 4, arrow). The limited synthesis of the globin chain, and Heinz bodies formation, may result into a local rigidification of RBC membrane [12].

Figure 3.

Classical sickle-cell observed in MGG stained blood smear of a patient with sickle-cell disease (SCD).

Figure 4.

Intracellular RBC hemoglobin precipitates (Heinz bodies) in a patient with HbH disease.

3. Measurement of RBC deformability

The diagnostic procedures for the etiological investigation of HHAs have experienced a great progress in recent years [2]. The easy accessibility of RBCs from a single blood sample and the improvement of the methods used for their study have contributed to bring together the most recent knowledge in disciplines as diverse as optical and ultrastructural morphology, enzymology, metabolomics, proteomics, membrane ionic transport and, more recently, rheology or the behaviour of RBCs in suspension [36, 37]. However, the morphological examination of MGG stained blood smear still remains the simplest procedure and, in many cases, the most effective procedure for the diagnosis of HHA [1]. In the general laboratory, the classical RBC morphology examination is complemented by other simple laboratory tests such as the RBC osmotic fragility test (OFT) or the RBCs vital staining procedures with new methylene blue (NMB) for the reticulocyte count and the brilliant cresyl blue (ACB) for Heinz-bodies examination. Moreover, the incubation of a blood sample with oxidising agents, such as acetylphenylhydrazine (APH), is a very simple procedure to assess the RBC oxidant-reducing capacity. In this context, the implementation of the measure of RBC deformability may become another relatively simple diagnostic haemolysis tests. Unfortunately, the use of RBCs’ deformability has not yet been implemented in the diagnostic laboratories due to the several reasons that we describe here. One of these reasons is the different procedures that can be used currently to measure the deformability in both, individual or multiple RBCs.

3.1 Techniques for the measurement of individual cells

The most used procedures for measuring individual RBC deformability are based on direct measurements of single cells. This includes micropipette aspiration technique (MAT), atomic force microscopy (AFM), optical tweezers (OT) and quantitative phase imaging (QPI). A further very simple method to measure individual RBC deformability is the readout of RBC filterability after the passage of RBCs through cellulose columns [38].

1. The micropipette aspiration technique (MAT) was developed in the early 1980 s, and since then it has been extensively used to measure the mechanical properties of RBC membranes. The measurement system consists of a micropipette, a manometer system that controls aspiration pressure and a chamber on a microscope stage from which erythrocytes are aspirated into the micropipette (Figure 5). By applying negative pressure, the RBC is aspirated into the micropipette, and the corresponding convexity (L) is visible in the capillary allowing measurement of the membrane elasticity [39].

2. The atomic force microscopy (AFM) allows viewing high-resolution topographies of materials at the atomic or molecular scale when a sharp-probe mounted at the end of a flexible cantilever deflects when interacting with the surface of a sample [40, 41]. This is detected by photodetectors, which are associated with the position of a laser beam reflected from the tip and can provide three-dimensional topographical images and their local mechanical properties that can be quantitatively determined from force vs. distance curves (Figure 6).

3. Optical tweezers (originally called single-beam gradient force trap) use a highly focused laser beam to hold and move the microscopic and sub-microscopic objects (nanoparticles and droplets), in a manner similar to tweezers. Optical tweezers are capable of manipulating nanometre and micron-sized dielectric particles by exerting extremely small forces via a highly focused laser beam [42]. RBC deformability can be measured by sending this highly focused laser bean through a microscope objective, and the light refraction induces trapping forces that comprise light scattering and gradient forces caused by the interaction of the light and the RBCs. The trapping forces can be determined by measuring the refractive indexes of the trapped particles and the ambient medium, laser power and particle size. When trapped particles are much smaller than the laser wavelength, the optical force can be determined by Rayleigh scattering theory; and for larger particles, such as RBCs, the Mie scattering theory is applied [43].

4. Quantitative phase imaging (QPI) is an optical microscopy technique in which the optical field, consisting of amplitude and phase information, is measured [44]. Since optical phase information is quantitatively related to the physical and chemical properties of a sample, QPI enables to directly analyse RBCs and quantify its size, morphology, cellular behaviour and viscoelasticity. One QPI technique used to investigate RBC deformability is the diffraction phase microscopy (DPM), and more recently, the combination of DPM analysis and mathematical modelling has been used to determine the mechanical properties of individual RBCs, such as shear modulus, bending modulus, area expansion modulus and cytoplasmic viscosity [45].

Figure 5.

The micropipette aspiration technique to measure individual RBC deformability. Source: [2].

Figure 6.

Using a laser beam and a photodiode, the reflected light generated by scanning a small cantilever over the surface of the sample is collected and the images processed. Source: S Faith Mokobi. Atomic Force Microscope (AFM)- Definition, Principle, Parts, Uses. Sagar Aryal and Wikipedia. Created with biorender.com.

3.2 Techniques for the measurement of multiple cells

In addition to the old microfluidic approaches, some techniques, based on the measurement of RBC deformability as a function of shear stress, have become increased popularity to investigate RBC deformability and are potential tools for the routine RBC deformability measurements in clinical practice. These include the filtration method, the microfluidic filtration and the laser diffractometry.

1. The filtration method examines the ability of RBCs to pass through membrane filters, and it has been the first method used to measure the RBC deformability. For this, whole blood is passed through holes in a membrane filter by using the force of gravity or by applying positive or negative pressure [4, 8]. Considering the dimensions of RBCs, the pore diameters in membrane filters are 3–5 μm (e.g. Nucleopore; Corning, Acton, MA, USA). Quantification of the process is achieved either by measuring the time required to pass a certain volume of RBCs through the filter or by the pressure-flow relationship. Due to the simplicity of its components and operating principle, the filtration method has been widely used for the measurement of RBC deformability in clinical practice [46]. Unfortunately, its reproductibility is limited due to the blockage of the pores by the more rigid leukocytes or by platelet microaggregates and due to the inability to standardise the size of the pores.

2. The microfluidic filtration can resolve the issue of non-uniform pore size in membrane filters using a micromachining technique that produces an array of parallel microchannels.With this technique, the deformation of whole cells can be observed and measured with a microscope while they pass through the microchannels (Figure 7). Therefore, microfluidics filtration represents a promising, cost-effective and high-throughput method for measuring RBC deformability, with a minimum amount of blood required for the test [47, 48, 49]. The microfluidic device mimics the in vivo capillary blood flow system (with internal diameters measuring only a few micrometres), and RBC deformability can be measured by passing a blood sample through a funnel-shaped microconstriction. It is worth mentioning that microfluidic measurements can provide both individual RBC and population assessments of cellular deformability.

3. Laser diffractometry is a technique that uses light diffraction patterns produced by a laser beam traversing a sheared low haematocrit RBC suspension. When a laser beam is incident on diluted RBC suspensions, the light is scattered by the RBCs population and creates a single image or diffraction pattern. The shape of the diffraction pattern reflects the average shape of hundreds or thousands of cells analysed. Due to the shape analysis of the laser diffraction pattern, laser diffractometry is also known as ektacytometry. Currently, laser diffractometry has become the primary method for testing RBC deformability, and three commercially available ektacytometers exist, using the same laser-diffraction principle but different shearing geometries [2, 50] (Figure 8). With these instruments, a whole blood RBC suspension in a high viscous medium is subjected to varying shear stresses that deform RBCs and different diffraction parameters are obtained. The most important diffraction parameter is the elongation index (EI) that measures the RBC deformability [50].

Figure 7.

Inverted microscopiy with an array of parallel microchanes to measure the RBC deformability by microfluidic filtration technique. Source: [47].

Figure 8.

Ektacytometers are using the same laser-diffraction principle but different shearing geometries: (a) concentric cylinders, (b) cone and plate, (c) parallel disks, and (d) Poiseullie slit flow.

Due to its precision, sensitivity and convenience, laser diffractometry has become the primary method for testing RBC deformability in clinical practice and currently is represented by a new generation ektacytometer called ‘Laser-assisted Optical Rotational Cell Analyser (LoRRca) MaxSis (RR Mechatronics)’ (Figure 9). Through its Osmoscan module, which measures the RBC deformability under an osmotic gradient (OGE), the LoRRca allows to obtain a well-standardised measure of RBC deformability depending on both the shape and the position along the osmolality axis [51]. For this test, 200 μl of whole blood is needed, and four RBC parameters are defined: 1. Deformability (EImax), 2. Osmotic fragility (Omin), 3. Cellular hydration (Ohyper) and 4. Area under the curve (AUC) (Figure 10).

1. EImax is the maximum elongation index and represents the maximal RBC deformability at a physiological value of osmolality (Omax). EImax value is an expression of RBC membrane shape and rigidity.

2. Omin is the value of osmolality at which the EI is minimal and corresponds to the 50% lysis point as determined by the classical osmotical fragility test (OFT). Omin reflects cellular surface-to-volume ratio (S/V).

3. Ohyper is the value of osmolality in the hypertonic region that corresponds to the 50% of the EImax and reflects the cellular hydration status or intracellular viscosity. Ohyper correlates with the reciprocal function of the MCHC, and during normal RBC ageing, increased MCHC correlates with decreased RBC deformability.

4. AUC is the area under the curve (AUC) with a starting point in the hypo-osmolar region (Omin) and an ending point in the hyper-osmolar region (500 mOsm/kg).

Figure 9.

Laser-assisted Optical Rotation Cell Analyzer (LoRRca MaxSis, Mechatronics, Hoorn, The Netherlands) to measure the osmotic gradient ektacytometry (OGE) parameters in hereditary hemolytic anemias.

Figure 10.

Osmotic Gradient Ektacytometry (OGE) curve provides information on RBCs deformability (EI), osmotic fragility (Omin) and cell hydration (Ohyper). EI values and also the membrane rigidity are depending on both the RBCs shape and their position along the osmolality axis.

4. Congenital defects and decreased RBC deformability

The congenital defects associated with a decreased RBC deformability are an important group of rare diseases (RDs) with anaemia as their most relevant clinical manifestation. For this reason, this group of RDs are also called rare anaemias (RAs) and have been largely studied in the context of the European Network for Rare and Congenital Anemias (ENERCA). ENERCA was launched in 2002 by the European Commission (EC) to create a multidisciplinary approach for the diagnosis and clinical follow-up of patients with RAs [52]. After 2017, ENERCA has become a member of the Independent Advisory Board (IAB) of the European Reference Network (ERN) for rare haematological diseases or EuroBloodNet. Five years later, in 2022, the Thalassemia International Federation (TIF) has launched the Rare Anemias International Network (RAIN), a global community-based organisation of patient advocacy groups and industry partners which aims to advocate for the rights of people living with rare and ultra-rare anaemias worldwide. RAIN will work to raise RAs awareness through education and collaboration and to enable timely diagnosis, access to basic treatment and advanced therapies, development of specific healthcare policies and exchange best practices through more targeted and personalised services for patients with RAs (https://thalassaemia.org.cy/projects/rain/).

The most important causes of HHAs are the defects of RBC structural components: haemoglobin (haemoglobinopathies), membrane (membranopathies) and enzymes (enzymopathies). Haemoglobinopathies and enzymopathies are, in general, easily diagnosed by conventional laboratory tests such as electrophoresis, high-performance liquid chromatography (HPLC) and RBC enzyme activity measurements, respectively. On the contrary, membranopathies, despite the morphological examination of stained blood smear, allow the diagnosis in a relatively important number of cases; it is frequently hampered by several interferences. Examples of these interferences are the following: (a) the coinheritance of more than one RBC defect [18], (b) the existence of de novo mutations [53, 54, 55, 56, 57], (c) the overlapping of clinical variability and (d), the degree of reticulocytosis and/or to the frequent blood transfusion requirements especially in newborns and children [58, 59, 60].

According to British Committee for Standards guidelines [61], in a high percentage of cases, the consideration of patient’s family history of HHA associated with typical clinical and laboratory features allows an accurate phenotypic diagnosis of RBC membranopathies. However, the recent implementation of next-generation sequencing (NGS) has drastically changed the diagnostic workflow of HHA and significantly decreased the frequency of undiagnosed cases [62, 63, 64].

From the clinical point of view, the RAs are classified into two categories: hereditary and acquired, but according to their pathophysiology, they can be classified into five groups: 1. Bone marrow (erythropoietic) defects, 2. RBC defects, 3. Iron metabolism (sideroblastic and non-sideroblastic anaemia), 4. Blood plasma discrasias (autoimmune haemolytic anaemia and related syndromes) and 5. Microcirculation diseases (haemolytic uremic syndrome and other microangiopathic disorders).

RBC deformability is affected in the RAs dealing with hereditary abnormalities of RBC components (membrane, haemoglobin or enzymes), and the acquired abnormalities are mainly due to the presence of abnormal plasma components that act on the RBC membrane (i.e. autoantibodies, crioagglutinins, plasma complement in PNH, bacteria or parasites such as plasmodium falciparum in malaria infection), to blood vessels or cardiac abnormalities (mechanic haemolysis) or to microcirculation and capillary defects (microangiopathic haemolysis). The anaemia due to haemolysis is always accompanied by a compensatory increase of bone marrow erythropoiesis and of circulating reticulocytes. If the haemolysis is fair, the increase of bone marrow erythropoiesis can maintain the haemoglobin concentration within the normal range, and there is no anaemia (compensated haemolysis). However, when bone marrow erythropoiesis is unable to compensate the intensity of the haemolysis, a typical haemolytic syndrome appears, characterised by anaemia and reticulocytosis, associated in most cases with jaundice and splenomegaly [18].

4.1 Haemoglobinopathies (structural)

These are the most frequent RBC defects when compared with membranopathies and enzymopathies and are the consequence of globin gene mutations that can alter the synthesis (thalassaemias) or the structure of haemoglobin molecule (structural haemoglobinopathies). The most frequent worldwide haemoglobinopathy is sickle cell disease (SCD), characterised by the presence of circulating sickle cells (Figure 3). In its homozygous form (HbSS), or combined with other haemoglobinopathies (HbSC, HbSD, HbSthal, etc.), SCD is characterised by a haemolytic syndrome of variable intensity associated with severe painful vaso-occlusive crises (VOCs) as the consequence of multiple organ micro-infarcts [65]. These VOCs are triggered by hypoxia that decreases HbS solubility, disrupts the RBC shape (sickle cells) and increases their rigidity facilitating the obliteraction of small vessels (capillaries), local intravascular haemolysis and VOC.

Haemoglobinopathies have a worldwide prevalence of about 300 million carriers, and in Europe, there are populations at risk, especially for thalassaemia, which are located in the geographical regions surrounding the Mediterranean basin (Mediterranean anaemia). HbS is not present in Caucasian individuals, but its presence in Europe is the consequence of the migration impact from people coming from Asia or African Sub-Saharan geographical regions [66]. Due to this, SCD has become one of the most important health problems in Europe and has promoted the wide implementation of neonatal screening programmes for its early detection in almost all European countries. These programmes allow to start the treatment since the first years of life, decreasing the morbidity and the mortality during early childhood [67]. Earlier studies using filtration techniques and primitive ektacytometers reported decreased deformability of sickle RBCs even under oxygenated conditions, and quantitative phase microscopy measurements demonstrated decreased membrane fluctuations on sickle RBCs [68, 69]. Recently, using membrane fluctuations, measurements of four important mechanical properties of sickle RBCs have been retrieved, and interestingly, it has been observed that in individuals with sickle cell trait (with only one abnormal allele of the Hb beta gene), their RBCs also exhibit decreased deformability when compared with healthy RBCs [70, 71].

The osmoscan curves from patients with different haemoglobinopathies are shown in Figure 11. They have in common a left shift of both curve tails, suggesting the existence of a different degree of RBCs dehydration depending on the type of haemoglobinopathy [72]. The most severe decrease of EImax and left shift of the osmoscan curve is observed in patients with Hb SS and HbSC, all associated with severe vase-occlusive crises. Despite the osmoscan module not considering the oxygenation of the sample, the possible deoxygenation during the analytical process may explain a partial Hb S polymerisation and, in turn, the increase of red cells dehydration and rigidity [73]. The AUC, which is an important marker of decreased deformability in RBC membranopathies [72], is also decreased in all the haemoglobinopathies studied by us. Carriers for Hb S, Hb C and β-thal show a similar osmoscan profile with an intermediate left shift of the curve and a less decrease of EImax (deformability) at normal osmotic value, suggesting the existence of a less degree of dehydration when compared with Hb SS and HbSC. Moreover, Hb D and Hb E show an almost normal osmoscan profile with a slight decrease of EImax and Ohyper in accordance with their low or absent clinical expression. In addition to SCD, HbD, HbC, HbE and HbO-Arab, other structural haemoglobinopathies such as the unstable haemoglobins with intracellular haemoglobin precipitates or Heinz bodies exhibit a CNSHA of variable severity, but unlike SCD, the inheritance has an autosomal dominant pattern [72]. Interestingly, we have recently described one patient with the hyperunstable haemoglobin Bristol-Alesha, associated with severe haemolytic anaemia that exhibited the same OGE profile as β-thalassaemia [74].

Figure 11.

Osmoscan curve profile of different hemoglobinopathies. They have in common a left shift of both curve tails, suggesting the existence of different degree of RBCs dehydration. Patients with Hb SS and HbSC, were associated with severe anemia and vase-occlusive crises.

4.2 Thalassaemia

Thalassaemia is the consequence of a decrease in the synthesis of a globin chain (alpha or beta) with normal Hb molecule. It is caused by the absence, decrease or defective translation of specific messenger RNA (mRNA) due to deletions or point mutations of the globin genes. While point mutations predominate in beta genes, large deletions are more frequent in alpha genes. According to the type of mutation and the severity of the decrease of globin chain, the clinical phenotype can be more or less severe [75]. In beta thalassaemia, the milder forms consist of a slight or moderate hypochromic and microcytic anaemia (thalassaemia trait), whereas the more severe clinical forms can be classified as ‘thalassaemia major’ or ‘thalassaemia intermedia’, depending on the periodicity of transfusion requirement. In alpha thalassaemia, as the genetic cluster has two genes, the mutation of a single allele, relatively common in Southern Europe, is characterised by a moderate microcytosis (MCV of about 80 fl) without anaemia (alpha thalassaemia traït), whereas if more than one allele is affected, more severe forms of alpha-thalassaemia appear like the haemoglobinopathy H (HbH) due to the formation of beta globin tetràmers (β4) as result of the excess or imbalance of beta chains. HbH has a similar clinical phenotype to the intermediate beta thalassaemia, but with the presence of HbH that due to its instability is sometimes undetectable. The complete loss of the four alleles (homozygous alpha-thalassaemia) is not compatible with life, leading to hydrops faetalis, abortion and death.

The differential diagnosis of thalassaemia is based on the CBC and the study of haemoglobins by electrophoresis or high-performance liquid chromatography (HPLC). In beta thalassaemia trait, there is always a characteristic increase of HbA2 fraction except in patients with concomitant iron deficiency because this condition decreases HbA2. In alpha thalassaemia trait, the haemoglobin profile is normal, and a genetic study is required for the diagnosis [75]. Concerning treatment, for the most severe cases of β-thalassaemia, it has been historically based on blood transfusions and iron chelation therapy. The only curative therapy available is allogeneic haematopoietic stem cell transplant (HSCT) from suitable donors. However, with the limited pool of donors, HSCT remains unavailable for many thalassaemic patients who may instead benefit from globin gene therapy and other modalities, which exploit recent advances in understanding of globin gene regulation [76].

RBC deformability in thalassaemia is not well known. Recently, we have demonstrated that beta-thalassaemia (β-thal and δβ-thal) shows a characteristic left shift of osmoscan curve that is different from iron deficiency anaemia [77, 78]. Probably, the decrease of one globin chain synthesis may lead to the imbalance of the α/β chains equilibrium and to the overproduction of the normal chain that may increase RBC dehydration and rigidity [8, 78, 79].

4.3 Membranopathies

Membranopathies are due to structural or functional defects of the RBC membrane proteins. In general, they are inherited as autosomal dominant pattern but transmitted with a recessive character [80, 81, 82].

Hereditary spherocytosis (HS) is the most frequent cause of HHA in Caucasians, and the most frequent proteins affected in HS are beta-spectrin (SPTB-1) Ankyrin (ANK) and Band 3 (Anion exchanger 1, AE1). Haemolysis occurs almost exclusively in the spleen, leading to splenomegaly, intermittent jaundice and cholelithiasis [57, 83]. In some patients, several complications can occur: transient erythroblastopenia crisis due to parvovirus B19 infection, severe folic acid deficiency and torpid malleolar ulcers [7, 18]. Newborns with HS and fewer can develop hazardous hyperbilirubinaemia and jaundice (neonatal icterus) associated or not with a severe anaemia. The early suspicion is essential for a prompt diagnosis and treatment, and using anticipatory guidance, adverse outcomes can be prevented [82, 83, 84]. The diagnosis of HS is based on the triad: (1) anaemia and jaundice, (2) splenomegaly and (3) spherocytosis, easily demonstrated by the peripheral blood morphological examination (Figure 12). The implementation of the automated haematological analysers, which perform a direct measure of the MCHC, has facilitated the use of this parameter in HS when it is increased in the presence of a high reticulocyte count. Moreover, the classical measurements of RBC osmotic fragility and criohaemolysis have been replaced by two new tests based on the measure of RBC deformability by ektacytometry and on the measure of the fluorescence intensity in RBCs after incubation with the fluorochrome, eosin-5- maleimide (EMA) by flow cytometry (EMA-binding test). EMA binds specifically to the anion transporter (Band 3) and decreases when Band 3 decreases.

Figure 12.

Classical spherocytes observed in MGG stained blood smear from a patient with hereditary spherocytosis (HS).

The measurement of RBC deformability using the osmoscan module of the new generation LoRRca Osmoscan from Mechatronics (Figure 9) has become the most sensible, accurate and reproductible method for the diagnosis of hereditary membranopathies [63]. Accordingly, when used together with the EMA-binding test, the OGE has become a reference procedure for the diagnosis of HS and an extremely useful tool for other membranopathies (Figure 13).

Figure 13.

Osmoscan curve profile of different membranopathies. A clear distinction between normal control (green line), hereditary spherocytosis (HS), hereditary elliptocytosis (HE), hereditary pyropoikylocytosis (HPP) and hereditary xerocytosis (HX) can be observed.

Hereditary elliptocytosis (HE) has a milder clinical expression and is characterised by the presence of more than 30% of circulating elliptocytes in peripheral blood (Figure 14). HE is due to a skeletal protein defect, mainly alpha-Spectrin (SPTA-1) and Band 4.1 that alters the elasticity of the membrane preventing its recovery after elongation [8, 34]. Due to this, the resulting OGE profile is characterised by a trapezoidal curve that differs from HS (Figure 13). However, in about 20% of patients with HE, the curve falls in the area covered by HS, making not possible to differentiate HE from HS by Osmoscan only. In the most severe clinical form of HE called hereditary pyropoikilocytosis (HPP), the SPTA-1 gene mutation in heterozygous state is associated ‘in trans’ with an SPTA-1 ‘Lely’ mutation leading to severe HHA with decreased heat stability and markedly abnormal RBC morphology (Figure 15).

Figure 14.

Classical elliptocytes observed in MMG stained blood smear from a patient with hereditary elliptocytosis (HS).

Figure 15.

Marked anisopoikilocytosis in a newborn with neonatal hemolytic anemia and jaundice due to hereditary pyropoikilocytosis (HPP).

Hereditary stomatocytosis (HSt) is an ultra-rare membranopathy where RBCs show an elongated central pallor instead of a round, and due to this they are called stomatocytes (Figure 16). The genuine form of HSt is the overhydrated stomatocytosis (OHSt) or hereditary hydrocytosis with chronic haemolysis and a large number of stomatocytes on peripheral blood smear. The genetic and molecular mechanism of HSt is poorly understood, but it is known that in all forms there is a disorder of the permeability to sodium and/or potassium ions associated with a markedly increased sodium permeability of about 10–40 times of normal leading to a significant increase of total mono-valent cation and water content [85, 86]. There is a variant of HSt known as cryohydrocytosis in which patient’s RBCs exhibit minimal to mild changes in cation leak at physiologic temperatures, but a marked increase in monovalent cation permeability at low temperature. RBCs demonstrate a sphero-stomatocytic morphology and in some patients, heterozygous missense mutations in band 3, the anion exchanger (SLC4A1) [8, 87].

Figure 16.

Classical stomatocytes observed in MGG stained blood smear from a patient with hereditary stomatocytosis (HSt).

Since many years, it has been considered the existence of a second variant of HSt called dehydrated stomatocytosis (DHSt) or hereditary xerocytosis (HX). HX is the most common primary disorders of RBC ionic transport and the most clinically heterogeneous. In this disease, RBCs are dehydrated due to a cation leak, primarily of potassium, and since it is not accompanied by a proportional net gain of sodium and water, a cellular dehydration appears. Peripheral blood cell morphology is not characteristic, but few target cells and occasional erythrocytes with haemoglobin puddled to one side (eccentrocytes) can be observed [88]. When RBCs are observed in glutaraldehyde suspension, few xerocytes with the classical horse saddle shape can be seen (Figure 17). In HX, as in HS, the MCHC is almost always increased (34–38 g/dL), and RBC osmotic fragility decreased, reflecting cellular dehydration. OGE (LoRRca Osmoscan module) reflects a characteristic pattern of mixed reduced deformability index (decreased EImax) and dehydration (increased Ohyper) given by a leftward shift of the minimal osmolality point (Figure 13). The most frequent genetic mutation identified in HX affects PIEZO1 [89, 90]. but in a few HX patients, mutations in the Gardos channel, encoded by the KCNN4 gene, have been observed Clinically, HX patients with KCNN4 mutations exhibit a variable degree of anaemia associated with a higher RBC dehydration when compared with the patients with PIEZO1 gene mutations [91].

Figure 17.

RBCs in suspension with glutaraldehyde observed with optical microscopy. A xerocyte (arrow) can be observed with its classical horse ridder shape.

Treatment of HHA due to RBC membrane defects is always palliative, depending on the severity of anaemia. Whereas in HS and HE, splenectomy is followed by a full and partial recovery, respectively, in HX, but also in OHS, splenectomy is not recommended due to an unexplained association with thrombophilia [91].

4.4 Erythroenzymopathies

Hereditary red blood cell (RBC) enzyme defects (erythroenzymopathies) are, in general, enzyme deficiencies, which are associated with a metabolic defect leading to CNSHA or acute haemolytic crisis with anaemia of variable severity. Some ultra-rare erythroenzymopathies are associated with neonatal cyanosis, erythrocytosis, neurological disease and myopathy.

In the circulation, RBC lifespan depends on two main metabolic pathways: 1.The anaerobic glycolysis (Embden-Meyerhof Pathway) that uses glucose to generate ATP, necessary to meet energy requirements and 2. Hexose Monophosphate Shunt (HMS) that uses NADH and NADPH to generate reduced glutathione (GSH) necessary to detoxify hydrogen peroxide (Figure 18). The most frequent erythroenzymopathy is glucose 6 phosphate dehydrogenase (G6PD) deficiency, followed by pyruvate kinase deficiency (PKD) and glucose-6-phosphate isomerase (GPI) deficiency. The normal RBC contains about 40 different enzymes from which 14 make up the erythrocyte metabolism. Since the mature RBCs lack mitochondria, the production of energy, in form of ATP, is entirely dependent on the anaerobic glycolysis. Accordingly, the vast majority of RBC enzyme defects described so far pertain to this metabolic pathway (Table 1). G6PD deficiency is in most cases asymptomatic until the patient suffers an oxidative stress induced by the ingestion of certain drugs or fava beans (favism), by infections and by other stressing clinical situations. This generates an acute haemolytic crisis with anaemia after the oxidative stress, and only few ultra-rare cases of G6PD deficiency present a life-long CNSHA as is the case of PKD and GPI. Other ultra-rare enzymopathies such as TPI, PGK and PFK exhibit a concomitant neurological impairment or myopathy, respectively.

Figure 18.

RBC Metabolic pathways, Anaerobic glycolysis (Embden-Meyerhof Pathway) and the Hexose Monophosphate Shunt (HMS). Source: [92].

Enzyme	RBC pathway	Clinical manifestations	Genetic Transmission	Reported cases (mutations)
Adenosine deaminase, hyperactivity (ADA)	Nucleotide metabolism	Chronic Haemolytic Anaemia	AD	3 families (No genetic data)
Adenylate kinase (AK)	Nucleotide metabolism	Chronic Haemolytic Anaemia	AR	12 families, (7 mutations)
Aldolase (Ald)	Glycolysis	Chronic Haemolytic Anaemia	AR	6 cases. (4 mutations)
		Neurological disease
		Myopathy
Phosphofructokinase (PFK)	Glycolysis	Chronic Haemolytic Anaemia	AR	50–100 cases (17 mutations)
		Myopathy
Phosphoglycerate kinase (PGK)	Glycolysis	Chronic Haemolytic Anaemia	X-linked	40 cases. 19 mutations
		Neurological disease
		Myopathy
Glucose phosphate isomerase (GPI)	Glycolysis	Chronic Haemolytic Anaemia	AR	>50 families (31 mutations)
		Mild neurological disease
Glucose-6-phosphate dehydrogenase (G6PD)	Hexose monophosphate shunt	Acute Haemolytic Anaemia crisis triggered by oxidant drugs,	X-linked	>400 million people (>70 mutations)
		Infection and fava beans (Favism)
		Chronic Haemolytic Anaemia (Very rare)
6-Phosphogluconatedehydrogenase (6PGD)	Hexose monophosphate shunt	Chronic Haemolytic Anaemia	AR	5 cases (No genetic data)
		Episodic Haemolytic Events
Glutathione reductase (GR)	Glutathione metabolism	Acute Haemolytic Anaemia crisis triggered by oxidant drugs, infection and fava beans (Favism)	AR	2 families, (3 mutations)
		Cataracts
Glutathione synthetase (GS)	Glutathione metabolism	Chronic Haemolytic Anaemia.	AR	>50 families (32 mutations)
		Neurological disease
Hexokinase (HK)	Glycolysis	Chronic Haemolytic Anaemia	AR	20 cases (5 mutations)
Pyrimidine-5′-nucleotidase (P5′N)	Nucleotide metabolism	Chronic Haemolytic Anaemia	AR	>60 families. (26 mutations)
Pyruvate kinase (PK)	Glycolysis	Chronic Haemolytic Anaemia	AR	>500 families (>200 mutations)
Triosephosphate Isomerase (TPI)	Glycolysis	Chronic Haemolytic Anaemia	AR	50–100 families (10 mutations)
		Severe Neurological Disease

Table 1.

Red blood cell enzymopathies with clinical manifestations.

AR: Autosomic recessive AD: Autosomic dominant. Source: [93].

ATP is involved in many RBC functions requiring energy, and therefore, it is essential for RBC deformability regulation [94, 95, 96]. Accordingly, RBC decreased viability in PKD and other enzymopathies pertaining to the glycolytic pathway have been suggested to depend on the decreased RBC deformability due to the decreased ATP content [8, 97]. However, the OGE profile has been found to be normal in erythroenzymopathies [98, 99] exception made of GPI deficiency where the osmoscan curve displays a significant shift to the right side and increased Ohyper.

The study of the OGE parameters in 14 patients with RBC enzymopathies is shown in Figure 19. Three patients had asymptomatic G6PD deficiency (Figure 19a), five patients with homozygous PKD associated with CNSHA (Figure 19b), and six patients had GPI deficiency (Figure 19c). Interestingly, all the six cases with GPI deficiency exhibit an increased Ohyper that is significantly higher in homozygous or double-heterozygous patients than in heterozygous carriers.

Figure 19.

Osmoscan curve profiles in three RBC enzymopathies: G6PD deficiency (a), PKD (b), GPI deficiency (c) and the comparison of the OGE of GPI deficiency with overhydrated HHA (membranopathies).

The finding of RBC overhydration in GPI deficiency, but not in PKD, despite both enzymes pertaining to the same glycolytic pathway is intriguing. In a previous study of a cohort of 37 patients, clinically and phenotypically diagnosed as membranopathies, we observed a particular group of six patients with a characteristic overhydrated osmoscan curve profile [100]. Three of these patients were HS type 2, according to our previous classification [63], and two patients were HHA without membranopathy phenotype but with SPTB and ANK mutations, respectively. The remaining patient was an HHA without apparent mutations and of unknown origin. In principle, our patient with GPI deficiency may be included within this group, whereas in all the patients with overhydrated HHA, the opened osmoscan profile shows an enlargement of the curve with deviation of its both sides, in GPI deficiency this enlargement affects the right side of the curve, only (Figure 19d). This means that in GPI deficiency, Omin and RBC osmotic fragility test (OFT) are normal but, as previously suggested [101], the membranopathies with overhydrated RBC profile are probably the associated with a concomitant unknown cannelopathy that may disturb the RBC ionic homeostasis.

In addition to haemolytic anaemia, GPI deficiency has been associated with neuromuscular dysfunction [18] because in human cells, the monomeric form of this enzyme is identical to neuroleukin (NLK), an important autocrine motility factor (AMF) AMF has an effect on the cell endoplasmic reticulum (ER) and intracellular Ca++ homeostasis [102], and since the mature RBCs are devoid of ER, the deficient AMF/GPI protein may activate some unknown membrane ionic channel leading to overhydration. Further studies are necessary to confirm this hypothesis.

Acknowledgments

We are indebted to the European Community’s Rare Diseases Research Programme that allowed to maintain the infrastructure necessary to perform this ektacytometry research project and book chapter. We are also grateful to the EU Equality/Equality Plus Projects for their partial financial support.

Compliance with ethical standards.

Conflict of interest

The authors declare that they have no conflict of interest.