**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].


*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.*

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**).

*Congenital Defects with Impaired Red Blood Cell Deformability – The Role of Next-Generation… DOI: http://dx.doi.org/10.5772/intechopen.109637*


#### **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.


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 [47–49]. 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.

c. **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

#### **Figure 7.**

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

*Congenital Defects with Impaired Red Blood Cell Deformability – The Role of Next-Generation… DOI: http://dx.doi.org/10.5772/intechopen.109637*

#### **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.*

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 laserdiffraction 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].

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**).


#### **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.*

*Congenital Defects with Impaired Red Blood Cell Deformability – The Role of Next-Generation… DOI: http://dx.doi.org/10.5772/intechopen.109637*

## **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 [53–57], (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 [58–60].

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 [62–64].

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

*Congenital Defects with Impaired Red Blood Cell Deformability – The Role of Next-Generation… DOI: http://dx.doi.org/10.5772/intechopen.109637*

#### **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.*

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].

#### **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 (**β**4) 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 alphathalassaemia) 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].

*Congenital Defects with Impaired Red Blood Cell Deformability – The Role of Next-Generation… DOI: http://dx.doi.org/10.5772/intechopen.109637*
