CADAE1405C

YBLRE-00309; No of Pages 12
Blood Reviews xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Blood Reviews
journal homepage: www.elsevier.com/locate/blre
Review
Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders☆

Lydie Da Costa a, b, c, d,⁎, Julie Galimand a, 1, Odile Fenneteau a, Narla Mohandas e, 2
a
AP-HP, Service d'Hématologie Biologique, Hôpital R. Debré, Paris, F-75019, France
b
Université Paris Diderot, Sorbonne Paris Cité, Paris, F-75010, France
c
Unité INSERM U773, Faculté de Médecine Bichat-Claude Bernard, Paris, F-75018, France
d
Laboratoire d'Excellence GR-Ex, France
e
New York Blood Center, New York, USA
a r t i c l e i n f o a b s t r a c t
Available online xxxx Hereditary spherocytosis and elliptocytosis are the two most common inherited red cell membrane disorders
resulting from mutations in genes encoding various red cell membrane and skeletal proteins. Red cell membrane,
Keywords: a composite structure composed of lipid bilayer linked to spectrin-based membrane skeleton is responsible for
Hereditary spherocytosis the unique features of flexibility and mechanical stability of the cell. Defects in various proteins involved in
Elliptocytosis
linking the lipid bilayer to membrane skeleton result in loss in membrane cohesion leading to surface area loss
Pyropoikilocytosis
and hereditary spherocytosis while defects in proteins involved in lateral interactions of the spectrin-based skel-
Stomatocytosis
Red cell membrane
eton lead to decreased mechanical stability, membrane fragmentation and hereditary elliptocytosis. The disease
Hemolysis severity is primarily dependent on the extent of membrane surface area loss. Both these diseases can be readily
diagnosed by various laboratory approaches that include red blood cell cytology, flow cytometry, ektacytometry,
electrophoresis of the red cell membrane proteins, and mutational analysis of gene encoding red cell membrane
proteins.
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction the splenic reticulo-endothelial system 7,8 resulting in regenerative

hemolytic anemia, splenomegaly, and ictera with increased free biliru-
A number of inherited red cell disorders due to altered membrane bin level. The severity of the disease depends on the extent of surface
function have been identified. These include hereditary spherocytosis area loss and ranges from asymptomatic forms to severe neonatal or
(HS), hereditary elliptocytosis (HE), hereditary ovalocytosis (SAO), prenatal forms responsible for rare hydrops fetalis cases requiring
and hereditary stomatocytosis (HSt). transfusion in utero. In this review, we will summarize the substantial
HS and HE1–6 are the most common red cell membrane disorders in progress that has been made in our understanding of i) structural orga-
the world with a prevalence of 1 out of 2000 affected cases in North nization of the red cell membrane including comprehensive character-
America and Northern European countries and are likely to be even ization of a large number of membrane proteins, ii) structural basis for
higher due to underdiagnosis of asymptomatic forms. Both diseases the interactions between various membrane and skeletal proteins and
result from defects in genes encoding various membrane and skeletal how defects in these interactions due to mutations in genes encoding
proteins that play a role in regulating membrane cohesion and mem- the various proteins lead to defective membrane function, and iii) ap-
brane mechanical stability. 1–6 In both HS and HE, red cell life span is propriate methodologies including genetic analysis that enable the
shortened as result of splenic sequestration of red cells. The abnormal diagnosis of various red cell membrane disorders.
red cells with decreased membrane surface area and increased spheric-
ity are trapped in the billroth canals in the spleen and phagocytozed by 2. Red cell membrane structure
☆ Author contributions: L. Da Costa wrote the article. J. Galimand acquired the The red cell membrane is a composite structure consisting of a
ektacytometer data. O. Fenneteau performed microscopic analysis of the red cells. N. lipid bilayer anchored to the spectrin-based membrane skeleton
Mohandas worked on the article with L. Da Costa.
through linking proteins interacting with cytoplasmic domains of
⁎ Corresponding author at: Service d'Hématologie Biologique, Hôpital R. Debré, 48 bou-
levard Sérurier, Paris, F-75019, France. Tel.: +33 1 40 03 41 66; fax: +33 1 40 03 47 95. membrane proteins embedded in the lipid bilayer. In addition, an-
E-mail addresses: [email protected] (L. Da Costa), ionic phospholipids in the inner lipid monolayer interact directly
[email protected] (J. Galimand), [email protected] (O. Fenneteau), with skeletal proteins spectrin and protein 4.1 and these interactions
[email protected] (N. Mohandas). modulate membrane function. 9–13 In addition to phospholipids, the
1
Service d'Hématologie Biologique, Hôpital R. Debré, 48 boulevard Sérurier, Paris,
France, F-75019. Tel.: +33 1 40 03 41 94; fax: +33 1 40 03 47 95.
red cell membrane contains a large panel of proteins (more than 50
2
Red Cell Physiology Laboratory, New York Blood Center, 310 East 67th street, New trans-membrane proteins and 10 skeletal proteins) that interact
York, 10021, NY, USA. Tel.: +1 212 570 3056; fax: +1 212 570 3264. with each other, and which are responsible for the antigenic
0268-960X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.blre.2013.04.003
Please cite this article as: Da Costa L, et al, Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders, Blood Rev (2013),
2 L. Da Costa et al. / Blood Reviews xxx (2013) xxx–xxx
proprieties, the transport function and the mechanical proprieties interactions results in elliptocytosis and decreased membrane mechani-
of the red cell membrane 5,14–16 (Fig. 1). The red cell membrane is cal stability leading to membrane fragmentation.
not a static structure but is highly dynamic enabling it to undergo
extensive deformations necessary for traversing the vascular bed 3. Hereditary spherocytosis
performing its function of oxygen delivery. The ordered and specific
structural organization of various membrane components is responsi- Hereditary spherocytosis (HS) (known as well as the Minkowski
ble for the unique features of extensive deformability and mechanical Chauffard disease) is the most common inherited red cell membrane
stability of the membrane that are needed for the red cell to perform disorder with one case out of 2000–3000 individuals, and probably
its physiologic function during its long life span of 120 days. Altered even higher prevalence due to underdiagnosis of minor or moderate
structural membrane organization due to various protein defects is forms of HS (Table 1). Although more often diagnosed in Europe
responsible for a large panel of human disorders either constitutional and North America, HS is reported in other continents and countries,
or acquired.17–25 In addition to their function as structural proteins, without a founder effect. The inheritance is dominant in 75% of cases.
various membrane proteins play other important functional roles. For
example, i) transport function (band 3: anion transporter, aquaporin 3.1. Clinical and biological feature
1: water transporter, GLUT1: glucose and L-dehydroascorbic acid trans-
porter,26 Kidd: urea transporter, RhAG: gas transporter in particular HS may be revealed early in infancy, even in the neonatal period
CO2, various cation pumps and transporters including, Na +–K +-ATPase, or during pregnancy in the severe forms. HS is responsible for hemo-
Ca++-ATPase, Na+–K +–2Cl− and Na+–Cl−, Na +–K +, K+–Cl− lytic anemia. The clinical features are pallor due to anemia, jaundice
cotransporters and Gardos channel), ii) adhesion molecules (ICAM-4, due to the hyperbilirubinemia and splenomegaly, the spleen being
Lu/BCAM, CD36, α4 and β1 integrins) and iii) the antigenic functions the site of sequestration and phagocytosis of the undeformable HS red
(Blood group antigens) and cell signaling such as β2 adrenergic blood cells. The jaundice may be the most important sign in the neo-
receptor. 16,27–29 nates (splenomegaly is often absent) and may require an exsanguino-
Two protein complexes serve to anchor the spectrin/actin mem- transfusion in order to avoid the nuclear ictera, but most often photo-
brane skeleton to the phospholipid bilayer: the ankyrin complex therapy is sufficient to eliminate excess bilirubin. Hydrops fetalis of HS
and the 4.1R complex 5,30 (Fig. 1). The ankyrin complex is composed is rare and is likely due defects in either band 335 or spectrin.36,37 Late
of the anion exchanger band 3 tetramers, 31 blood group antigens in infancy and in adult, the classical triad (pallor/regenerative anemia,
(Rh, RhAG), 16,27,32 CD47 (thrombospondin receptor), glycophorin A jaundice and splenomegaly) of the hemolytic anemia, in association
(GPA), and Lansteiner Wiener (LW) antigen.33 RhAG and band 3 link with gallstones is noted. Careful examination of the blood smear,
the phospholipid bilayer to red cell membrane skeleton by interacting with the presence of spherocytic red cells usually confirms the diag-
with peripheral protein 4.2 and ankyrin. The 4.1R complex 34 is com- nosis. A family history of HS, splenectomy and/or cholecystectomy
posed of band 3 dimers, blood group antigens (Rh, Duffy, Kell, XK),27 can also be very helpful for the diagnosis since spherocytic red cells
and glycophorin C (GPC).14 The GPC, Rh, Duffy, and XK interact directly are also a feature of certain form of auto-immune hemolytic anemias
with 4.1R while band 3 binds to adducin.30 The loss of linkages between (AIHA). The clinical manifestations of HS are highly variable from very
the spectrin-based skeleton and the lipid bilayer leads to loss of mem- mild to severe (Table 1). As a consequence, the anemia may vary from
brane cohesion resulting in lipid vesicle formation responsible for the absent to mild or severe, with a reticulocyte count >150 × 109/l. Of
loss of surface area, a mechanism leading to the spherocyte generation. note, only 35% of the HS neonates exhibit an increased reticulocyte
Spectrin-based membrane skeleton is composed of spectrin tetramers count of more than 10%.38 Spherocytic red cells on the blood smear re-
formed by head to head association of αβ spectrin heterodimers and the sult from a reduced surface to volume ratio primarily related to loss of
junctional complex composed of αβ spectrin tails interacting with actin, membrane surface area,39 the main characteristic of the HS. The loss
protein 4.1R, adducin, dematin, tropomyosin and tropomodulin.18 The of surface area in HS is due to the disruption of the vertical linkages be-
spectrin dimer–dimer interaction and the spectrin–actin–protein 4.1R tween the phospholipid bilayer and the membrane skeleton. The de-
junctional complex play a key role in regulating membrane deformability creased surface area is a feature of both the reticulocytes and mature
and membrane mechanical stability. Weakening of either of these lateral red cells in HS.39 Mutations in genes encoding for various red cell
Fig. 1. Red cell membrane network with the two complexes, ankyrin and 4.1R embedded in the phospholipid bilayer.
L. Da Costa et al. / Blood Reviews xxx (2013) xxx–xxx 3
Table 1
HS classification.
Minor HS Moderate HS Moderate to severe HS Severe HS
Hb (g/l) Normal >80 60–80 b60

Reticulocytes (%) b6% 6–10% >10% >10%
Bilirubin (μmol/l) 17.1–34.2 >34.2 >34.2–51.3 >51.3
Red blood smear Few spherocytes Spherocytes Spherocytes Microspherocytes
and poikilocytosis
Osmotic fragility Normal or slight increased Increased Increased Increased
(fresh blood)
Osmotic fragility Increased Increased Increased Increased
(incubation at 37 °C)
Splenectomy Rarely If the capacity level is decreased and Necessary >5 y-old Necessary >2–3 y-old
depending on certain cases
Transfusions 0–1 0–2 >2 Regularly
SDS-PAGE (protein defect) Normal Sp, Ank + Sp, band 3, protein 4.2 Sp, Ank + Sp, band 3 Sp, Ank + Sp, band 3
Inheritance AD AD, de novo, AD, de novo AR
membrane protein including ankyrin, band 3, protein 4.2, α or CBC needs a thorough understanding of the technologies used to derive
β-spectrin and RhAg40–48 (Table 2) result in the assembly of an intrinsi- the various cellular parameters.
cally unstable membrane leading to vesiculation of the lipid bilayer
resulting in increased cell sphericity and reduced cellular deformability. 3.2. Confirmation of HS diagnosis
The sequestration of the non-deformable spherocytes in the spleen and
their subsequent phagocytosis by the splenic macrophages is responsi- Patients with a family history of HS and typical HS biological mani-
ble for the anemia and splenomegaly. The number of spherocytes is festations (hemolytic anemia with high CHCM >36 g/dl, high percent-
highly variable from patient to patient: very few in 25 to 35% of mild age of hyperdense cells >4%, spherocytic cells on the blood smears) do
cases of HS and in 33% of HS neonates to very large numbers in the not require any additional tests (grade 1 recommendation, grade A
more severe forms of HS. The severity of the HS is directly related to evidence on the latest 2011 guidelines for the diagnosis and manage-
extent of membrane surface area loss and consequently the severity of ment of HS53).
spherocytosis.38 Spherocytic red cells are associated with polychro- More specific biological tests may be required in cases where the
matophilia and various red cell shape abnormalities depending on the HS diagnosis is not readily evident (lack of HS family history, lack of
associated membrane defect (Table 2)49 (Fig. 2A, B,C). For example, expression of typical biological manifestation including normal osmotic
“mushroom” shaped red cells are generally a feature of band 3 defects fragility when the test is performed and iron deficiency, which may
(Fig. 2A, green arrow). The Mean Cell Volume (MCV) is decreased mask the regeneration and the increased reticulocyte count).
variably in HS with largest decreases noted in severe forms of HS due to
significant decreases in the spectrin content of the red cell membrane.38 3.2.1. EMA-binding and other tests
Importantly, the reticulocyte MCV (MCVr) is also decreased to variable The osmotic fragility, 54 glycerol lysis 55 or Pink tests 56 may be
extent depending on the severity of anemia. This feature is useful used as first line of clinical laboratory tests. However, the sensitivity
for distinguishing HS from autoimmune hemolytic anemia (AIHA) or of these tests for diagnosis is low (68% for osmotic fragility test
ABO incompatibility, which is also associated with spherocytic red performed on fresh blood, 61% for the glycerol lysis test and 91% for
cells38,39,50 but does not exhibit decreases in MCVr. Another important the Pink test). 38,57,58 As a result, flow cytometry measurement of
feature of HS red cells is cell dehydration as revealed by an increased the mean red cell fluorescence, associated red cells following labeling
percentage of hyperdense cells (cells with a hemoglobin concentration with the dye eosin-5′ maleimide (EMA) to document surface area loss
of more than 41 g/dl)38,39,51,52 in association with increased Mean is being used an alternate test for diagnosis of HS. 53,58–71 The EMA
Corpuscular Hemoglobin Concentration (CHCM) values of >36 g/dl of binds covalently to the Lys430 on the first extracellular loop of the
mature red cells as well as of reticulocytes (CHCMr). In contrast, CHCMr band 3 protein predominantly but it also interacts with sulfhydryl
values are normal in AIHA. It is important to note that the red cell and groups expressed by Rh, RhAg and CD47. This test is able to detect
reticulocyte indices derived depend on the technologies used by various HS with a sensitivity of 92.7% and a specificity of 99.1%, with a positive
hematological analyzers and as such appropriate interpretation of predictive value of 97.8% (meaning that if the test is positive, the
Table 2
Molecular defects in HS.
Molecular Prevalence in HS population Inheritance Prevalent mutation Protein low expressed Disease severity Cytologic feature
defect (MGG coloration of the blood smears)
Ankyrin-1 40–65% in Europe and USA AD, AR, AD or de novo: null Spectrin and ankyrin: Minor to Spherocytes
(ANK1) de novo mutation 11% à 50% moderate form
AR: missense or
promoter mutation
Band 3 20–35% AD Functionally null Band 3: 31%–35% Minor to Spherocytes, rare mushroom red cells
(SLC4A1) mutation moderate form
α spectrin b5% AR α-LEPRA and null α spectrin: 50–75% Severe Spherocytes, poikilocytosis, contracted
(SPTA1) mutation red cells
β spectrin 15–30% AD, de Null mutation β spectrin: 15–40% Minor to Spherocytes, 5–10% acanthocytes
(SPTB) novo moderate form
4.2 Protein b5% in Europe and USA AR Missense mutation 4.2 Protein: 95–100% Minor to Spherocytes, ovalostomatocytes
(EPB42) (45–50% in Japan) moderate form
AD: autosomal dominant, AR: autosomal recessive, LEPRA: Low expression allele Prague.
Fig. 2. Red cells under microscopic examination in HS. A) Band 3 defect with the classical mushroom feature (green arrow) in association with typical spherocytic red cells, which
appear dense and hyperchromic (black arrows), few acanthocytes (red arrows) and basophilic red cell due to the hematopoietic stress (blue arrow). B) and C) β spectrin (SPTB)
defect with a lot of acanthocytes (red arrows) in association with spherocytic red cells (black arrows). D) Ektacytometry curve of two different HS patients, dark line the typical
feature with a decreased in the DImax as a sign of a decreased red cell deformability due to the surface area loss, a shift to the right of the Omin, which corresponds to the reduced
surface to volume ratio and a shift to the left of the O'/Hyper point, which corresponds to the dehydration of the red cells.
diagnosis of HS is confirmatory in 97.8% of the cases) and a negative and additional studies are needed to confirm or rule out the diagnosis of
predictive value of 96.9% (meaning that if the test is negative, HS can HS.72 The cut-off point in the extent of decreased fluorescence to validate
be ruled out in 96.9% of the cases). This test is able to detect HS due to diagnosis of HS is still a matter of debate. For Crisp et al.,71 it is at 17% of
defects in band 3, spectrin and 4.2 but less effective for HS due to mean channel fluorescence decrease and recently, Bianchi et al.,58 de-
ankyrin defects.70 The test can also be positive in pyropoikilocytosis scribed a lower cut-off at 11% to separate 150 HS patients from normal
(HPP) due to significant extent of cell fragmentation leading to genera- controls, which may lead to over-diagnose of HS if the laboratories per-
tion of red cells with decreased surface area. However, if fragmented red form only flow cytometry. Additional large-scale studies are needed in
cells are gated out by their distinct forward scatter signal, the non- order to define the best cut-off and the best strategy to adopt for the diag-
fragmented red cells do not express decreased membrane associated nosis of HS. However, this test should replace the much lower sensitive
fluorescence, which is in contrast to HS where the entire population and specific tests since: 1) it is easy to perform particularly in neonates,
of red cells exhibit decreased membrane associated fluorescence. since only 5 μl of peripheral blood is needed and since a flow cytometer
The EMA test may be problematic in the case of South East Asian is available in most hematology laboratories; 2) the test results are avail-
ovalocytosis (SAO) due to a nine amino acid deletion in the band 3 pro- able in 2 to 3 h; 3) the samples may be analyzed up to 7 days after the
tein, with mutant protein failing to bind EMA. King et al. described de- blood sampling72; and 4) the gating on the abnormal red cells allows
creased membrane associated fluorescence in patients with congenital the avoidance of the bias due to presence of transfused red cell enabling
dyserythropoietic anemia type II (CDAII) with abnormal glycosylation the diagnosis of HS in patients with a recent transfusion history.67
of band 3 and in 2 patients with cryohydrocytosis (positive test). How-
ever, it is to be noted that these two latter patients carried the band 3 3.2.2. Ektacytometry
memphis polymorphism.70 The EMA-binding is not dependent on the Bianchi et al. 58 point out the fact that the combination of different
phenotype and is positive also in compensated HS. However, the sensitiv- diagnosis tests can increase test sensitivity for diagnosis of HS when
ity is increased in splenectomized HS compared to non-splenectomized the EMA-binding test is combined with the acidified glycerol lysis
HS.58 It is also independent of the molecular defect in the red cell mem- test (AGLT) 73 (100% sensitivity) or with the Pink test (99% sensitivi-
brane protein but it may be less sensitive for diagnosis if HS is with ty). In our experience and since we have access in our laboratory to
undefined molecular defects and with ankyrin defects.58,70 The cut-off an ektacytometer, 23,74,75 we prefer to combine the EMA-binding test
above which the test is considered positive is much debated. Classically, to the ektacytometry, generally acknowledged to be the reference
the mean fluorescence of patient red cells is compared to the mean fluo- technique for diagnosis of red cell membrane disorders. 21,38,39,57 In
rescence of red cells obtained from 6 age-matched controls on the same the 2011 guidelines, the recommended screening tests if the HS diag-
day. Ratio of mean fluorescence of the patient red cells to mean fluores- nosis is equivocal, are the cryohemolysis test 71 and EMA-binding test
cence derived for the 6 controls is derived and when the ratio is decreased (grade 1 recommendation, grade A evidence) 53 and the osmotic fra-
by >21%, the test is considered positive while a value of b 16% is consid- gility test is not recommended for routine use. However, the tech-
ered negative. Values between 16 and 21% are considered indeterminate nique of osmotic gradient ektacytometry has not been evaluated in
formulating the guidelines because of lack of its general availability, mutations, 10 nonsense or in non-coding sequence, 5 missense) are
in spite of its recognition as the reference technique for diagnosis of reported53 amongst them the spectrin Kissimmee, which alters the
red cell membrane disorders. The ektacytometer is a viscometer, in β-spectrin binding to 4.1 (p.Trp202Arg) or the spectrin Primassao,
which the deformation of red cells suspended in a viscous PVP solution which modifies the translational initiation start site.
at defined values of applied shear stress is monitored as a continuous – Mutations in SLC4A1 gene, encoding for the anion exchanger protein
function of suspending medium osmolality. Three distinct features band 3, are identified in ~15 to 20% of the HS cases that include mis-
of the osmotic gradient ektacytometry profiles are: the Omin point, sense (23), nonsense (18), and larger mutant proteins (3).53 Band 3
which corresponds to the osmolarity at which 50% of the red cells are and protein 4.2 are decreased on the SDS-PAGE gels to different ex-
lysed in the classical osmotic fragility test and represents the surface tents depending on the mutation. A case of homozygous null muta-
area to volume ratio, the maximal deformability index (DImax) value, tion (Coimbra mutation)35 results in complete deficiency of band 3
which represents the maximal cellular deformability of the red cell pop- and very severe anemia. Inheritance of two heterozygous SLC4A1 mu-
ulation, and the O' or hyper point, which corresponds to the osmolarity tations with different levels of expression has been shown to worsen
at DImax/2, which reflects the hydration status of the red cells. In the the clinical phenotype in one affected HS patient.80,81 Transmission is
case of HS,38,39 the characteristic features are a decrease in the DImax, dominant and no sporadic mutations have been reported.
in conjunction with a shift of the Omin point to the right (reduced – Mutations in EPB42 gene, encoding for protein 4.2 are rare and the
surface to volume ratio) and a shift of the O' or hyper point to left SDS-PAGE exhibits the absence of protein 4.2 as a result of homozy-
(increased dehydration of the red cells) (Fig. 2D). However, either gous or compound heterozygous mutations. Only 10 mutations
Omin or O' may be in the normal range without ruling out the HS have been described, which include missense (4), nonsense or dele-
diagnosis (Fig. 2D, dashed curve). The amount of blood required to tion (3) and splicing (2) mutations. 53,82 All the known mutations are
perform the test is small (100 μl) and as with EMA dye test ektacytometry reported in the NH2-terminal region of protein 4.2, which binds to
can be performed on blood samples from neonates. The limitations of band 3. Mutations in EPB42 gene are often described in the Japanese
ektacytometry are the limited availability of the instrumentation population.
and the need to perform the analysis within 48 h of blood sampling. Im- – Mutations in SPTA1 gene, encoding for the α-spectrin chain are ex-
portantly, the ektacytometer generates distinct osmotic deformability tremely rarely associated with HS. When present, only homozygous
profiles enabling diagnosis of not only HS but also the other red cell mutation or compound heterozygous mutations are responsible for
membrane disorders such as elliptocytosis, HPP, stomatocytosis and HS. Indeed, due to synthesis of a large excess of α-spectrin in ery-
SAO. throid cells, the defect of α-spectrin needs to be severe to lead to
a HS phenotype. Of particular interest, is the spectrin Prague muta-
3.2.3. Identification of the molecular defects (Tables 1 and 2) tion leading to the exon 37 skipping and frameshift.83,84 This muta-
The molecular defects responsible for HS are in large part detected tion is in trans of allele αLEPRA (LEPRA: Low Expression PRAgue), a
by SDS-PAGE electrophoresis performed on red cell ghosts prepared low expression recurrent allele of the SPTA1 gene (3.3%). This allele
from the fresh blood using a 4% to 12% gradient acrylamide gels accor- is silent if carried by a normal individual. Allele αLEPRA carries the
ding to Fairbanks76 and/or discontinuous buffer system of Laemmli C>T transition at position −99 of intron 30. It activates an alterna-
with linear gradient of acrylamide from 6% to 14%.77 The use of SDS- tive acceptor site at position −70 of intron 30 causing mis-splicing
PAGE is recommended 53 1) when the clinical phenotype is more severe and frameshift in about 80% of the pre-mRNA. Allele α LEPRA is asso-
than predicted from the red cell morphology; 2) when the red cell mor- ciated in cis with a functionally neutral polymorphism, Bug Hill
phology is more severe than predicted from parental blood films where (GCT>GAT codon 970; Ala>Asp). SpαLEPRA allele is prevalent in
one parent is known to have HS; 3) if the diagnosis is not clear; and 4) in non-dominant HS83,85 and can result in a marked deficiency of both
any case prior to splenectomy, in order to avoid misdiagnosis in partic- α and β spectrin bands in the affected propositus but spectrin content
ular with stomatocytosis. In this latter case, the splenectomy will lead is normal in both parents.
to lethal thrombosis, and is strictly contraindicated.78 The molecular
defect may be difficult to detect in case of ankyrin defects due to in At present, routine screening for mutations in various genes
part to large numbers of reticulocytes, which contain excess amounts accounting for analysis of the affected genes in HS is not well
of ankyrin and mask the reduction in ankyrin content of mature red established although progress is made in this avenue. Furthermore,
cells, and in 20% of cases the molecular defect cannot be detected knowledge of the molecular defect doesn't influence the clinical
especially in asymptomatic carriers or individuals with very mild management of HS.
HS. 41
3.2.4. Other biological indicators
– Mutations in ANK1 gene are most frequently associated with HS and It is important to emphasize the fact that cytology with a meticulous
account for 50% of the cases. Sporadic or de novo mutations are often microscopic examination of the blood smear examination be the first
described in recessive HS associated with ANK1 gene mutation while important step for the diagnosis of HS and all the red cell disorders. Oth-
transmission is dominant in the familial cases. No homozygous erwise it is likely that misdiagnosis or over-diagnosis of these red cell
mutation has been identified to date. All forms of mutations have disorders will occur.
been reported: frameshift (17), nonsense (8), abnormal splicing (4), Other biological tests that are important in the diagnosis to con-
missense (4), and even in the promoter region (mutations in trans firm the hemolytic anemia include: increased indirect bilirubine-
to mutations in the coding sequence) (2).53 Ankyrin Florianopolis, mia >17 μmol/l, increased LDH >300 IU/l, a decrease in haptoglobin
which results in one nucleotide insertion in exon 14 is a recurrent b10 mg/dl (signature of the hemolytic anemia) and a DAT test to rule
mutation, which has been found in three unrelated severe HS pa- out an immune hemolytic anemia (AIHA or ABO incompatibility).
tients.79 On SDS-PAGE electrophoresis, ankyrin defect when present We propose the flow chart for laboratory diagnosis of HS in Fig. 3A
is sometimes associated with reduced spectrin content and consis- and B.
tently with decreased levels of band 4.2.
– Mutation in SPTB gene, encoding for the β-spectrin protein is mutated 3.3. Clinical management of affected HS individuals
in 20% of the HS cases. As with ANK1 gene, transmission is dominant
but some sporadic mutations have also been identified. No homozy- In the familial HS, with the exception of some severe cases of HS,
gous mutation has been identified to date. On SDS-PAGE gels, only in which there is a need for in utero transfusion program during the
the spectrin bands are reduced. More than 20 mutations (10 null pregnancy, 86 the management of the mild to moderate HS begins at
birth, the hemoglobin being often normal at birth but decreasing includes growth measurement and medical exam is sufficient. In the ab-
rapidly after birth. The educational program of both parents is mandato- sence of symptoms, the most recent recommendations do not suggest
ry. It must emphasize the signs of anemia at this age of life (pallor, an annual blood test.53 The parents should be warned about the risk
difficulties to thrive, dyspnea). The French orphanet website (http:// of parvovirus B19 infection (enhancement of the pallor, asthenia, dys-
www.orpha.net/) has posted very helpful information for the fami- pnea, fever and the classical skin eruption) and the need for their infants
lies.57 The weekly follow-up on hemoglobin and the other red cell and children to seek immediate medical attention. Confirmation of
parameters including the reticulocyte count and blood smear exam is diagnosis leads to transfusion of red cells to correct the sudden
necessary until the hemoglobin levels stabilize with a reticulocyte aregenerative anemia, due to the erythroid tropism of the parvovirus
count corresponding to the degree of anemia and hemoglobin levels. leading to the erythroblastopenia. The HS children with severe anemia
Care of the infant by a hematologist in concert with the pediatrician need to be carefully monitored during breakout of any viral infections.
or the general practitioner is important for good clinical management. The vaccination against hepatitis B is recommended since the HS
After the stabilization of hemoglobin values, the duration of which patients may need regular transfusions, in addition to the usually
may be different from one patient to another, a once a year visit that recommended vaccinations. During the neonate period, treatment is
Fig. 3. Flow chart proposal for laboratory diagnosis of HS and other red cell membrane disorders (A) and in particular HS (B).
necessary for moderate to severe forms of HS with folate supplemen- bladder is left in situ even after gallstones extraction, a close follow-up
tation at the dose of 2.5 mg/D to manage nutritional requirements of using ultrasound is necessary. The other scenario of whether the
stress erythropoiesis. Recombinant erythropoietin (EPO) treatment at spleen should be removed if cholecystectomy is needed in case of
the dose of 1000 IU/kg/sem in 3 subcutaneous injections has been symptomatic gallstones in mild HS is still being debated. Recently,
evaluated in 16 transfusion-dependent neonates and infants and Alizai et al. 104 reported that only 3 mild HS individuals out of 16
resulted in avoidance of transfusions in 10 of 16 and the need for who underwent cholecystectomy without splenectomy have been
only one transfusion in 3 of 6 HS infants. 87 However, use recombinant splenectomized subsequently within 2 to 5 years.
EPO is still debated and large and multicenter clinical studies are
needed to validate its effectiveness. 4. Hereditary elliptocytosis (HE), pyropoikilocytosis (HPP), South
Coinheritance of a hemochromatosis gene can aggravate the iron East Asian ovalocytosis (SAO)
overload in a chronic hemolytic anemia, in which the iron absorption
is already increased. Red cells matched for antigens in the Rhesus and Hereditary elliptocytosis is another red cell membrane disorder char-
Kell systems should be used for transfusion when the hemoglobin acterized by mutations in genes encoding membrane or skeletal proteins,
falls below a non-tolerable level, but there is no set threshold of hemo- which alters membrane function and reduces red cell deformability.
globin value below, which transfusion is needed. Only the tolerance of HE is due to defects in the horizontal protein connections of the red cell
anemia should be an important determinant in the decision to transfuse membrane skeletal network including the spectrin dimer–dimer interac-
or not. tion or the spectrin–actin–protein 4.1R junctional complex. The genes
Splenectomy should not be performed simply based on HS diagnosis mutated in HE are thus the α-spectrin (SPTA1), the β-spectrin (SPTB) or
but only on the basis of severity of anemia. Thus, splenectomy is indicat- protein 4.1R genes.105–117 An acquired deficit in 4.1R is reported in
ed in severe HS, with significant anemia and gallstone complications, myelodysplastic syndromes and HE diagnosis in adults, with no history
while splenectomy should be considered in moderate HS if anemia of hemolytic anemia during infancy should consider this possibility in
has an important effect on quality of life. Indeed, splenectomy abolishes differential diagnosis.118–120
anemia and gallstone complication in such cases. But, it should not HE is a common red cell hemolytic anemia (3 to 5 affected individ-
be performed in cases of mild HS except in specific cases with large re- uals for 10,000), with a worldwide distribution but a higher preva-
ductions in exercise tolerance. The laparoscopic surgical approach is lence like for other red cell defects in malaria endemic regions. 121
recommended but is dependent on the availability of appropriately The red cell shape is classically elliptic, with different features from
trained surgeons and suitable equipment.88 In any case, splenectomy the short stick shape (4.1 deficiency) (Fig. 4A,B, red arrows) to the
should be considered only after 6 years of age53 with the usual precau- shape more oblong on the blood smears (Fig. 4A, B, blue arrows). 49
tions (vaccinations according to the national guidelines, penicillin The phenotype and genotype are heterogeneous with autosomal
antibiotics and parent education in case of fever >38 °C) due to the dominant inheritance with the exception of the pyropoikilocytosis
infectious risks with some encapsulated germs such as haemophilus B, (HPP). The vast majority of the HE affected individuals are asymp-
meningococcus, and Streptococcus pneumonia bacteria. It is important tomatic and the HE is discovered fortuitously on a blood smear,
to ascertain that the splenectomized patients adhere to the latest while some patients exhibit hemolytic anemia with anemia, jaundice
vaccination guidelines (Price et al., for US 89). The patients and par- and splenomegaly. Neonatal jaundice, hemolytic anemia and hydrops
ents should be aware of life-long risk of overwhelming sepsis follow- fetalis are also reported. 37,122 It may be difficult to distinguish HE
ing splenectomy. However, there is no consensus regarding the need with neonatal poikilocytosis from HPP (Fig. 4B, black arrows). In HE
for reimmunization and its frequency, the type and the duration of with neonatal poikilocytosis, fragmentation and hemolysis decline
the prophylactic post-splenectomy antibiotics. 53 The risk of throm- with time and the phenotype after 4 months to 2 year of age is a
bosis in HS patients following splenectomy is the same as in the gen- mild common HE. The worsening of the hemolysis in the first months
eral population and the prophylactic anticoagulation to prevent of life has been attributed to the particularities of the fetal erythropoi-
thrombosis should adhere to standard protocols. However, the HS esis and the large amount of the fetal hemoglobin in the first months
diagnosis needs to be fully validated prior to splenectomy since in cer- in the red cells, which is responsible for the increase in 2,3-DPG con-
tain red cell disorders, such as stomatocytosis splenectomy is accompa- centration in the cell, which destabilizes the spectrin–actin–protein
nied by lethal thrombosis or because splenectomy is less effective in 4.1 complex, enhancing membrane instability. 123 Splenectomy with
disorders such as CDAII. the usual precautions may be a good option in order to increase the
Partial splenectomy can be performed instead of total splenecto- red cell life-span and increase the hemoglobin levels but it should
my in order to reduce the infectious risk, while eliminating most of be considered only for severe forms of elliptocytosis and after
the red cell destruction site, reducing anemia, reticulocytosis and 5 years of age. In HPP, splenectomy reduces but does not eliminate
hyperbilirubinemia. 90–100 The largest multicenter study so far on hemolysis completely.
62 HS infants from 1990 to 2008 96 has confirmed the benefit of the The heterozygous mutated patients are classically asymptomatic
partial splenectomy with no post-splenectomy sepsis complication while the clinically evident HE patients exhibit anemia that ranges
in 18 years of follow-up and with only 4.84% of the patients undergo- from mild to severe including the severe HE variant, HPP due to a ho-
ing subsequent total splenectomy. mozygous mutation or compound heterozygous mutations. In the
The question of splenectomy and cholecystectomy has finally severe form HPP, extensive red cell fragmentation is responsible for
been resolved and cholecystectomy should not be performed if a large decrease in the MCV. Some of the fragmented red cells are counted
there is no cholelithiasis at the time of splenectomy. Splenectomy re- as platelets by the hematology analyzer and as such overestimate the
duces hyperbilirubinemia and thus no pigment stones are formed platelets counts. In these instances platelet count should be performed
following splenectomy, reducing the risk of gallstones. 101 However, manually. Another feature of red cells in HPP is their increased sensitivity
Gilbert disease in HS patients increases the risk of gallstone forma- to thermal fragmentation at a lower temperature (45° to 46 °C) rather
tion by 5 fold. 102 Ultrasound measurement is the reference proce- than normal (49 °C). The more specialized tests include ektacytometry,
dure used to detect gallstones and recommended after the age of SDS-PAGE electrophoresis and analysis of spectrin tetramer–dimer ratios
5 years. It also allows an accurate measurement of the spleen size, using non-denaturing gels. Molecular biological studies (screening of
which is important before laparoscopic splenectomy.102 Cholecystectomy the low expression polymorphism α-LELY) are not necessary for the diag-
should be performed if the gallstones are symptomatic otherwise it has nosis but may be useful in the severe and persistent forms, including
been shown that cholecystectomy alters the bile salt metabolism and in- HPP (Fig. 4B). In the typical HE forms, the ektacytometry curve exhibits
creases the risk of colon carcinoma later in life.103 However, if the gall a trapezoidal form with a decrease in the red cell deformability
Fig. 4. Red cell in hereditary elliptocytosis and other elliptic red cell membrane disorders. A) Top panel: hereditary elliptocytosis with the classical form (red arrows) and more
ovalocytic red cells (blue arrows). Bottom panel: Ektacytometry curve in HE with the classical trapezoidal feature and characterized with a decreased DImax and normal Omin
and Hyper points. B) Top panel: elliptic (red arrows) and ovalocytic red cells (red arrows) with fragmented cells (black arrows), which characterized the severe form of HE,
pyropoikilocytosis (HPP). Bottom panel: the ektacytometry curve with a large decrease in DImax and a shift to the left of both Omin and Hyper points. C) Top panel: classical feature
of ovalocytic red cells in South-east Asian ovalocytosis (SAO) (black arrows). Bottom panel: the ektacytometry curve, which exhibits the undeformable feature of the red cells with
close to null DImax.
(decreased DImax) (Fig. 4A, bottom panel). On the SDS-PAGE, in the extent of the loss of surface area. Indeed, marked red cell fragmentation
severe homozygous HE due to mutation in 4.1R gene, an absence of is a key feature of HPP. In this case, either a homozygous mutation or
4.1 and p55, and 30% of the normal content of the glycophorins C and heterozygous compound mutations in the spectrin genes are reported
D are observed. In addition the two classical forms of 4.1R defect can but also the association between the low expression V/41 polymorphism
be seen with a truncated 4.1R (4.1 65/68) or an elongated high molec- αLELY in trans and a mutation on the α-spectrin SPTA1 gene in cis.124,125
ular weight of 4.1R (4.1 95).4 In addition, HE due to 4.1R defect exhibits The hereditary ovalocytosis, also designated as South-east Asian
a large number of thin and elongated elliptocytes, such as bacillus on the ovalocytosis (SAO) has a geographical distribution mostly in the malaria
blood smear (Fig. 4A). In HE due to mutation in either α- or β-spectrin endemic regions of Indonesia, Philippines, Melanesia and Southern
there is an increase in spectrin dimer to tetramer ratio as documented Thailand as SAO phenotypes offer protection against both Plasmodium
by non-denaturing gel electrophoresis of spectrin extracted at 4 °C. In falciparum and Plasmodium vivax malaria.126,127 Deformability of SAO
HPP, additional deficiency in spectrin/band 3 ratio is observed on the red cells quantitated by either ektacytometry (Fig. 4C, bottom panel) or
SDS-PAGE electrophoresis and explains the presence of spherocytes on by the micropipette aspiration technique is dramatically decreased.20,128
the blood smears amongst with fragmented cells. Diagnosis of HPP, due Paradoxically, this extent of decreased deformability in vitro has little
to low expression LELY allele can be easily performed by the molecular effect on red cell survival in vivo and the adult affected individuals are
screening of the low expression polymorphism αLELY. SpαLELY is a very completely asymptomatic and diagnosis is usually made coincidentally
common polymorphism, with 42% heterozygosity and 9% homozygosity on examination of blood smears with the characteristic feature of oval
in European Caucasian population. Its identification in the proband and shaped red cells with 1 to 2 transverse ridges (Fig. 4C, top panel). SAO
the molecular screening analysis of both parents with a meticulous may however express as a hemolytic anemia in the neonates and requires
blood smear exam with one parent exhibiting elliptocytes, the other no phototherapy. The inheritance is autosomal dominant and to date only
red cell morphology abnormality and but carrying the α-LELY polymor- heterozygous individuals have been reported.129 SAO results a mutation
phism are sufficient to confirm the HPP diagnosis in the proband. SpαLELY in the gene encoding band 3 and 3 (SLC4A1), characterized by a deletion
is a combination of two linked mutations, one in exon 40 and one in in- of the 27 nucleotides encoding the amino acids 400 to 408 of band 3.
tron 45 on the α-spectrin gene. Exon 40 mutation, the G>C transition While various hypotheses have been proposed to explain the discrepancy
leads to the p.Leu1857Val amino acid change. The p.Leu1857Val is linked between the mild phenotype and the strong effect of the band 3 mutation
to the other allele variation C>T transition in intron 45, minus 12 nucle- on the red cell membrane rigidity, no clear explanations have emerged.
otides from the 3′ splice site of exon 46. This latter allelic variation leads to
exon 46 skipping in 50% of α spectrin mRNA, which fails to assemble into 5. Stomatocytosis
stable spectrin dimer and αLELY △exon 46 alleles which are degraded.
This allele variation is responsible for low allele expression. The αLELY This rare red cell disorder is divided into two different entities: the
polymorphism is completely asymptomatic at the heterozygous but xerocytosis or dehydrated hereditary stomatocytosis (DHSt) and the
also at the homozygous state, due to the large excess by 3 to 4 fold of overhydrated hereditary stomatocytosis (OHS).1,2,4,41,130 Both exhibit
the α spectrin chains, but when the αLELY is associated in trans with a mu- a cation leak to the univalent cations Na+ and K + resulting in altered in-
tation of the α spectrin gene, the mutated spectrin forms increase and tracellular cation content and cell volume alterations. The inheritance
are responsible for the HPP phenotype. The severity of HE is dependent of stomatocytosis is autosomal dominant. The phenotype can vary
on the extent of the membrane instability,112 per se dependent on the from asymptomatic to severe hemolytic form.
In the DHSt, the most frequent form, the main characteristic is red reflect the over-hydration of red cells. In this form, the number of the
cell dehydration, due to the loss of the cation content, in particular K + stomatocytes on the blood smear is usually much higher than that in
and cell water. As a consequence, the MCHC is increased (>36 g/dl) the DHSt and the diagnosis is easy to establish. Recently, mutations
and the ektacytometric osmotic deformability profile is shifted to the p.Ile61Arg and p.Phe65Ser of the RhAg (Rh-associated glycoprotein)
left. Strikingly, the red cell survival is not significantly compromised. have been identified in the OHS. RhAg is a member of the band 3 com-
The phenotype varies from mild to moderate anemia with a normal or plex and is considered as an ammonium transporter and/or gas chan-
slightly increased MCV in spite of cell dehydration, with the presence of nel.138,139 Stomatin protein has not been found mutated in OHS but
stomatocytes with the classical mouth feature on blood smears (Fig. 5A, has been found to be expressed at low levels or absent in OHS.140 As
B,C). The diagnosis may be difficult when the number of stomatocytes is no defect in gene encoding stomatin could be identified and stomatin
low. DHSt diagnosis may be difficult to diagnose when associated to protein expression appears normal it is likely that observed stomatin de-
pseudohyperkalemia or perinatal edema.131 DHSt may be responsible fect is secondary. It has been suggested that the stomatin defect increases
for hydrops fetalis.132,133 The locus of the candidate gene has been located the glucose uptake through its interacting with Glut1 transporter.
in 16q23-24134 and redefined more recently in 16q24.2-16qter from Other forms of stomatocytosis have been described: the familial
two large families from Rochester, USA and Manitoba, Canada.135 Recent- pseudohyperkalemia and the cryocytosis. Familial pseudohyperkalemia
ly, Zarychanski et al.136 by copy number analyses, linkage analysis, and is particular in that it is not associated with hemolytic anemia and
exome sequencing, have identified that mutations in PIEZO1 protein stomatocytes are uncommon but red cells exhibit a leak of K + at
encoded by FAM38A gene are associated with DHSt in all affected DHS pa- room temperature but not at physiologic temperature. The gene re-
tients from both North American families. PIEZO proteins are the pore- sponsible for this such form is yet to be defined but two different loci
forming subunits of channels that control the mechanotransduction and have been described: one involved in DHSt and the other one maps to
stretch-activated cation channel activation in mammalian cells. PIEZO chromosome 2q35-q36. 141,142 In the cryocytosis (CHC) form, affected
protein has been identified in the red cell membrane. PIEZO proteins, patients exhibit mild to moderate hemolytic anemia. Band 3 mutations
which have been only recently identified137 may play an important role (p.Ser731Pro, p.His734Gln, and others affecting the amino acids D705,
in the red cell cation and volume homeostasis. R760,143 and the p.Gly796Arg144), which transform band 3 anion ex-
In the overhydrated hereditary stomatocytosis (OHS), in contrast to changer into a cation transporter, are described. The band 3 mutation
DHSt, the red cells are hydrated due to a 20 to 40 fold increase in the p.Leu687Pro is responsible for an intermediate form between the CHC
cation leak. This form of stomatocytosis is rare (20 reported cases and the pseudohyperkalemia (Blackburn stomatocytosis).143 The com-
worldwide) but leads to the most severe phenotypes. In addition to plexity is even higher when some forms of stomatocytosis, with band 3
reticulocytosis, hemolytic anemia in OHS is characterized by a large in- mutation p.Asp705Tyr and p.Arg760Gln have been reclassified as HS with
crease in MCV (>110 fl but up to 150 fl in some cases) and decreased a low temperature leak (HS-LTL)143 or when the mutated p.Gly796Arg
MCHC (between 24 and 30 g/dl), with a shift to the right of the band 3 is associated not only with stomatocytosis but also to congenital
ektacytometric osmotic deformability profile. All these red cell features dyserythropoietic type I (CDAI) feature.144,145
Fig. 5. A, B, and C: three cytological features of dehydrated stomatocytosis in A only the mouth red cell noticed; the number of stomatocytic red cell being sometimes very low. B and
C: more stomatocytic red cells (black arrows). D) Classical ektacytometry curve of DHSt with a normal DImax and a shift to the left of Omin and Hyper point, corresponding to the
dehydrated red cells.
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YBLRE-00309; No of Pages 12

Blood Reviews xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders☆

1. Introduction the splenic reticulo-endothelial system 7,8 resulting in regenerative

Minor HS Moderate HS Moderate to severe HS Severe HS

Hb (g/l) Normal >80 60–80 b60

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