5. Rare Anaemias due to red blood cell defects

RBC defects can be classified into two main groups: (a) Hereditary or intrinsic defects due to structural or functional abnormalities of RBC components haemoglobin (haemoglobinopathies), membrane (membranopathies) and enzymes of metabolism (enzymopathies), and (b) Acquired or extrinsic defects due to blood plasma or vascular abnormalities. In both cases the consequence is a haemolytic syndrome characterized by anaemia of variable severity associated with a compensatory increase of bone marrow erythropoiesis and of circulating reticulocytes. In addition to anaemia, the haemolytic syndrome is characterized by three main clinical manifestations 1. reticulocytosis, 2. splenomegaly and 3. jaundice [18].

## 5.1 Haemoglobinopathies

Haemoglobinopathies are the most frequent RBC defects in comparison with membranopathies and erythroenzymopathies that in many cases can be considered ultra-rare anaemias (prevalence <1 case per 10<sup>6</sup> inhabitants). All these diseases are more frequent in Southern Europe than in Central or Northern Europe, and their clinical expression is always an hereditary haemolytic anaemia [19, 20]. Haemoglobinopathies are the consequence of globin gene mutations that can alter the synthesis (thalassaemias) or the structure of haemoglobin (structural haemoglobinopathies). Its worldwide prevalence is around 269 million carriers, and in Europe there are risk populations, especially for thalassemia, which are located in the geographical regions surrounding the Mediterranean basin (Mediterranean Anaemia).

The most frequent haemoglobinopathy is HbS (OMIM 603903), prevalent in African populations due to the protection that it has offered against malaria. HbS is the result of substitution of valine for glutamic acid in the sixth position of the globin beta chain and in its homozygous form or combined with other haemoglobinopathies, is responsible for sickle cell disease (SCD) that consists in haemolytic anaemia associated with severe vaso-occlusive crises and pain due to multiple micro-infarcts [21]. These crises are triggered by hypoxia that decreases HbS

Figure 3. Sickle-cells in a SCD patient with vaso-occlusive crisis.

solubility leading to a characteristic RBC shape distortion (sickle cell) and to a drastic decrease of deformability (Figure 3) Over the last 30 years, it has been observed a significant increase due to the immigration impact of populations from other geographical areas, mainly from Sub-Saharan African regions but also from Asia and Central America, where this disease is very prevalent. Currently, the neonatal screening programs allow an early diagnosis of the disease and its preventive treatment from the first years of life and, as a consequence, the frequency of complications have significantly reduced, and mortality during early childhood dramatically decreased [22]. There is no specific treatment for SCD, although in severe cases, the administration of hydroxyurea (HU) is recommended since, as the concentration of HbF increases, the frequency of vaso-occlusive crises, the need for transfusions, and especially the onset of the acute thoracic syndrome decrease. In addition to HbS, there are other structural haemoglobinopathies of clinical interest such as HbS, HbJ HbD and unstable haemoglobins that precipitate, and inclusion bodies or Heinz bodies are formed (Figure 4). Clinically, they present with a chronic haemolytic syndrome of variable severity and unlike the SCD, patients presents an autosomal dominant pattern of inheritance.

Figure 4. Howell Jolly bodies in a patient with unstable haemoglobin.

#### 5.2 Thalassaemias

Thalasaemias are due to the decrease in the synthesis of a globin chain (alpha or beta), due to absence, diminution or defective translation of specific messenger RNA (mRNA) caused by 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 intensity of the synthesis decrease, the severity of the clinical picture can be more or less intense [23]. In beta thalassemia the milder forms consist of a slight or moderate hypochromic and microcytic anaemia (thalassemia trait) whereas the more severe clinical forms can be classified as "thalassemia major" or "thalassemia intermedia" depending on the severity of the anaemia and the periodic transfusion requirements, respectively. In alpha thalassemia, as the genetic cluster has two genes, the mutation of a single allele, relatively common in Southern Europe in characterized by a moderate microcytosis (MCV around 80 fl) without anaemia alpha thalassemia trait, whereas if more than one allele is affected more severe forms of alpha-thalassemia occur The mutation of three alleles gives rise to the so-called haemoglobinopathy H, a clinical form very similar to intermediate beta thalassemia but with the presence of HbH or beta-globin tetramers, a result of the excess or imbalance of beta chains due to the decrease of alpha chains. The complete loss of the four alleles (homozygous alpha-thalassemia) is incompatible with life (hydrops faetalis).

Diagnosis of both forms of thalassemia is based on the data provided by the CBC and the study of haemoglobins by electrophoresis or high-performance liquid chromatography (HPLC). In the case of beta thalassemia trait an increase in the HbA2 fraction is always observed, whereas in alpha thalassaemia trait the haemoglobin pattern is normal and a molecular study is mandatory. An accurate family study is also very important to prevent diagnostic errors and the application of unnecessary treatments. In addition, an appropriate identification of the carrier condition allows the identification of couples at risk.

The treatment of severe clinical forms of β-thalassemias has been historically based on blood transfusions and iron chelation therapy. The only curative therapy currently available is allogeneic haematopoietic stem cell transplant (HSCT) from suitable donors. However, with the limited pool of suitable donors, HSCT remains unavailable for many thalassemic patients. They may instead benefit from globin gene therapy and other modalities, which exploit recent advances in understanding of globin gene regulation [24].

#### 5.3 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. Hereditary spherocytosis (HS; OMIM 182870, 182,900, 270,970, 612,653, 612,690) is the most frequent cause of HHA in Caucasians due to a defect of membrane skeletal proteins that cause vesiculation and partial loss of the same with the decrease in surface/volume ratio and formation of spherocytes (Figure 5). Proteins affected in HS are beta-Spectrin (SPTB-1) Ankyrin and Band 3, and haemolysis occurs almost exclusively in the spleen, leading to frequent severe splenomegaly. Along with splenomegaly, several complications of HHA can be frequently observed in HS such as, intermittent jaundice and increased bilirubin pigments leading to premature gallstone formation, transient erythroblastopenia crisis due to parvovirus B19 infection, folic acid deficiency and torpid malleolar ulcers [25]. In general, physicians become more familiar with diagnosing HS in the newborn period, fewer neonates with HS will develop

Figure 5. Circulating peripheral blood spherocytes in a patient with hereditary spherocytosis.

hazardous hyperbilirubinemia or present to emergency departments with unanticipated symptomatic anaemia. The early suspicion, prompt diagnosis and treatment, using anticipatory guidance, will prevent adverse outcomes in neonates with HS [26].

The diagnosis of HS is based on the triad: (1) anaemia with jaundice, (2) severe splenomegaly and (3) spherocytosis, easily demonstrated by the peripheral blood morphological examination (Figure 5). Currently the diagnosis of HS can be easily performed by measuring RBC deformability and osmotic gradient ektacytometry (OGE) using the new generation LoRRca Osmoscan from Mechatronics [27]. Curves obtained with this device allow a clear distinction between hereditary spherocytosis and the other RBC hereditary membranopathies, elliptocytosis and xerocytosis (Figure 6). The implementation of the automated haematological analysers that determine the CCMH by means of a direct system, allows us to use this magnitude as a criterion of HS when it is increased in the presence of an elevated reticulocyte count. Finally, the use of the EMA-binding test technique is currently being implemented as a reference technique, especially in the diagnosis of HS, together with ektacytometry. This test is based on the measurement of the fluorescence intensity in RBCs after incubation with a fluorochrome, eosin-5 maleimide (EMA) that binds specifically to the anion transporter (Band 3) and decreases when Band 3 decreases.

Another hereditary membranopathy is hereditary elliptocytosis (HE, OMIM 109270, 130,600, 179,650, 225,450, 611,804), with milder clinical expression when compared to HS, but with the presence of more than 30% of circulating elliptocytes in peripheral blood (Figure 7). Like HS, 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 [28]. There is no loss of membrane and, therefore, CCMH is normal. OGE profile (Osmoscan) shows here a characteristic curve that is different from HS (Figure 6). In severe clinical forms of HE known as hereditary pyropoikilocytosis (HPP) the SPTA-1 gene mutation in heterozygous state is associated "in trans" with a SPTA-1 "Lely" mutation leading to severe HHA with markedly abnormal RBC morphology and decreased heat stability (Figure 8).

Finally, there is a very rare form of membrane disease called hereditary stomatocytosis (OMIM 194380, 185,000) which most relevant characteristic is the presence of RBC with an elongated central pallor instead of a round one (Figure 9). Its genetic and molecular mechanism is poorly understood, although it is known that in all forms there is a disorder of the permeability to sodium or potassium ions by which the RBC can be hydrated or dehydrated. These disorders of erythrocyte

#### Figure 6.

Osmotic gradient ektacytometry (OGE) profiles from Osmoscan (LoRRca) in different hereditary membranopathies. EI: Elongation Index.

hydration are classified as primary, due to inherent disorders of erythrocyte volume regulation, and secondary, due to other disorders affecting the erythrocyte that secondarily influence cell hydration. In general, the degree of perturbation of water and ion content parallels the degree of haemolytic anaemia [29].

Overhydrated stomatocytosis (OHSt) or hereditary hydrocytosis refers to a rare, heterogeneous group of disorders with haemolytic anaemia and large numbers of stomatocytes on peripheral blood smear (Figure 9). OHSt is associated with markedly increased sodium permeability of the membrane from 10 to 40 times normal. This leads to a significant increase in intracellular sodium with a lesser decrease in intracellular potassium, resulting in increased total mono-valent cation content and increased intracellular water. Erythrocyte membranes from many OHSt patients

Figure 7. Ellyptocytes in a patient with hereditary elliptocytosis.

Figure 8. Peripheral blood MGG stained examination of a newborn with hereditary pyropoikylocytosis (HPP).

lack stomatin, but mutations have not been found in the gene of affected patients and erythrocytes from stomatin knockout mice are normal. There is a variant of OHSt variant known as Cryohydrocytosis in which patient erythrocytes exhibit minimal to mild changes in cation leak at physiologic temperatures, but a marked increase in monovalent cation permeability at low temperature, typically 5°C. Erythrocytes demonstrate a sphero-stomatocytic morphology on peripheral smear. Heterozygous missense mutations in band 3, the anion exchanger (SLC4A1), have been identified in some cryohydrocytosis patients [29]. Dehydrated stomatocytosis or hereditary xerocytosis (HX) syndromes, are the most common primary disorders of erythrocyte hydration and are the most clinically heterogeneous. HX erythrocytes are dehydrated due to a cation leak, primarily of potassium, that leads to decreased potassium concentration. Because the cation leak is not accompanied by a proportional net gain of sodium and water, cellular dehydration ensues. Peripheral blood smear reveals few target cells, and occasional dessicytes (erythrocytes with haemoglobin puddled to one side), and rare stomatocytes. MCHC is increased (34–38 g/dL), and RBC osmotic fragility is decreased, both reflecting cellular dehydration [30]. Osmotic gradient ektacytometry (LoRRca Osmoscan) reflects a characteristic pattern of mixed reduced deformability index and dehydration given by a leftward shift of the minimal osmolality point (Figure 6).

When studied at the genetic level, in most cases of HX, mutations in PIEZO1 have been identified. PIEZO proteins are ion channels mediating mechanic sensory transduction in mammalian cells, and PIEZO1 is a large integral membrane protein

Figure 9. Typical RBC morphology in a patient with hereditary stomatocytosis.

with numerous transmembrane domains that assemble into a homo-multimeric complex fully active as a mechanic sensitive cation channel [29]. A few HX patients do not have mutations in PIEZO1, but instead have mutations in the Gardos channel, encoded by KCNN4. The role of Gardos channel in normal erythrocytes has not yet been defined, butin HX-associated mutant KCNN4 channels demonstrate alterations in channel kinetics and trafficking. Clinically, HX patients with KCNN4 mutations experience variable degree of anaemia and RBC dehydration with typically more severe forms than in PIEZO1-mutant HX patients [29].

Treatment of HHA due to RBC membrane defects are always palliative, depending on the severity of anaemia. In HS, splenectomy allows a complete recover of haemoglobin concentration, whereas in the HE this recovery is only partial. In HS, splenectomy is not recommended because it facilitates thrombotic events due to an increased thrombophilia.
