**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 [9–11], haemoglobin defects [12], mechanical damage [13] or normal RBC ageing [14–16]. 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].
