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

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 (1–3Pa), 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**<sup>0</sup> **-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

**Figure 3.** *Classical sickle-cell observed in MGG stained blood smear of a patient with sickle-cell disease (SCD).*

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].
