**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 [80–82].

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 [82–84]. 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 Moreover, the classical measurements of RBC osmotic fragility and criohaemolysis have been replaced by two new tests based on the measure of RBC deformability by ektacytometry and on the measure of the fluorescence intensity in RBCs after incubation with the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.

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**).

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 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.*

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

**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

**Figure 16.** *Classical stomatocytes observed in MGG stained blood smear from a patient with hereditary stomatocytosis (HSt).*

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].

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].

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].

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

#### **Figure 17.**

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

#### **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 lifelong 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.

ATP is involved in many RBC functions requiring energy, and therefore, it is essential for RBC deformability regulation [94–96]. 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.

#### **Figure 18.**

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

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

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



#### **Table 1.**

*Red blood cell enzymopathies with clinical manifestations.*

#### **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).*

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

deficiency exhibit an increased Ohyper that is significantly higher in homozygous or double-heterozygous patients than in heterozygous carriers.

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.

### **Acknowledgements**

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.

*The Erythrocyte - A Unique Cell*
