Hereditary spherocytosis is caused by inherited defects in the red blood cell cytoskeleton that weaken its interaction with the cell membrane. This leads to a spherical shape as the cell loses membrane area to maintain its volume. The most common defects involve ankyrin, band 3, and spectrin proteins. Patients present with signs of hemolytic anemia ranging from mild to severe. Diagnosis is based on spherocytes seen on blood smear, elevated osmotic fragility, and defects found in membrane skeletal proteins. Splenectomy improves anemia but does not cure the underlying defect.

1. Hemolytic disorders due to Inherited
abnormalities in Red cell
cytoskeleton
Guvera Vasireddy
Department of Pathology
OMC

2. RED CELL CYTOSKELETON
 The remarkable elasticity and durability of the red cell are
due to the properties of its specialized membrane
skeleton
 Lies closely apposed to the internal surface of the plasma
membrane.
 Its chief protein component, spectrin, consists of two
polypeptide chains, α and β, which form intertwined
(helical) flexible heterodimers.
 The “head” regions of spectrin dimers self-associate to
form tetramers, while the “tails” associate with actin
oligomers

3. INTERACTIONS BETWEEN VARIOUS
CYTOSKELETAL PROTEINS
 Each actin oligomer binds multiple spectrin tetramers
creating a two-dimensional spectrin-actin skeleton that is
connected to the cell membrane by two distinct
interactions.
 The first, involving the proteins ankyrin and band 4.2,
binds spectrin to the transmembrane ion transporter, band
3.
 The second, involving protein 4.1, binds the “tail” of
spectrin to another transmembrane protein, glycophorin A.

4. RED CELL CYTOSKELETON

5. Protein
composition of the
red blood cell
membrane
skeleton.
The major
components of the
erythrocyte
membrane as
separated by sodium
dodecyl sulfate–
polyacrylamide gel
electrophoresis and
revealed by
Coomassie blue
staining.
G3PD, glucose 3-
phosphate
dehydrogenase.

6. HEREDITARY INTRINSIC MEMBRANE DEFECTS
 Hereditary
spherocytosis
 Spherocytic
elliptocytosis
 Hereditary
elliptocytosis
 Southeast Asian
ovalocytosis
 Hereditary
pyropoikilocytosis
 Hereditary
stomatocytosis
 Hereditary xerocytosis
 Rh antigen deficiency
 Hereditary
acanthocytosis
 Abetalipoproteinemia
 McLeod Syndrome
(Ke11 antigen
deficiency)
 Chorea-acanthocytosis
syndrome
 In(Lu)

7. ACQUIRED MEMBRANE DEFECTS
Acquired spherocytosis
 Clostridia septicemia
 Thermal burn
 Hypophosphatemia
 Zieve’s syndrome
 Snake, spider, and
insect bites
Acquired acanthocytosis
 Spur cell anemia
 Vitamin E deficiency
 Infantile pyknocytosis
 Paroxysmal nocturnal
hemoglobinuria

8. ERYTHROCYTE MEMBRANE PROTEIN DEFECTS IN INHERITED
DISORDERS OF RED CELL SHAPE
Protein Disorder Comment
Ankyrin HS Most common cause of typical dominant HS
Band 3 HS, SAO,
NIHF, HAc
"Pincered" HS spherocytes seen on blood film
presplenectomy; SAO results from 9 amino acid
deletion
β-Spectrin HS, HE,
HPP, NIHF
"Acanthocytic" spherocytes seen on blood film
presplenectomy; location of mutation in β-spectrin
determines clinical phenotype
α-Spectrin HS, HE,
HPP, NIHF
Location of mutation in α-spectrin determines clinical
phenotype; α-spectrin mutations most common
cause of typical HE
Protein 4.2 HS Primarily found in Japanese patients
Protein 4.1 HE Found in certain European and Arab populations
GPC HE Concomitant protein 4.1 deficiency is basis of HE in
GPC defects

10. HEREDITARY SPHEROCYTOSIS (HS)
 The pathogenic mutations most commonly affect ankyrin, band 3,
spectrin, or band 4.2, the proteins involved in the first of the two
tethering interactions.
 Most mutations cause shifts in reading frame or introduce premature
stop codons, such that the mutated allele fails to produce any protein.
 The prevalence of HS is highest in northern Europe, where rates of 1
in 5000 are reported.
 An autosomal dominant inheritance pattern is seen in about 75% of
cases.
 The remaining patients have a more severe form of the disease that is
usually caused by the inheritance of two different defects (a state
known as compound heterozygosity).

11. PATHOGENESIS
 Young HS red cells are normal in shape.
 Deficiency of membrane skeleton reduces the stability of
the lipid bilayer, leading to the loss of membrane
fragments as red cells age in the circulation.
 The loss of membrane relative to cytoplasm “forces” the
cells to assume the smallest possible diameter for a given
volume, namely, a sphere.
 Life span of the affected red cells is decreased on
average to 10 to 20 days from the normal 120 days

12. The left panel shows the
normal organization of the
major red cell membrane
skeletal proteins. Various
mutations involving α-
spectrin, β-spectrin,
ankyrin, band 4.2, or band
3 that weaken the
interactions between these
proteins cause red cells to
lose membrane fragments.
To accommodate the
resultant change in the
ratio of surface area to
volume these cells adopt a
spherical shape.
Spherocytic cells are less
deformable than normal
ones and therefore
become trapped in the
splenic cords, where they
are phagocytosed by
macrophages. GP,
glycophorin.
Role of the red cell membrane skeleton in
hereditary spherocytosis.

13. PATHOPHYSIOLOGYOFHEREDITARYSPHEROCYTOSIS.

14. MEMBRANE DEFECTS THAT LEAD TO HS

15. CLINICAL PRESENTATION
 Presents at any age.
 Highly variable from asymptomatic to severely anaemic,
but usually there are few symptoms.
 Well-compensated haemolysis;
 Features of haemolytic anaemia: splenomegaly,
gallstones, mild jaundice may be present.
 Occasional aplastic crises occur, e.g. with parvovirus B19
infection.

16. LABORATORY DIAGNOSIS

17. BLOOD FILM
 Erythrocyte morphology in HS is variable.
 Typical HS patients have blood films with easily identifiable
spherocytes lacking central pallor
 May present with only a few spherocytes on the film or with
numerous small, dense spherocytes and bizarre erythrocyte
morphology with anisocytosis and poikilocytosis.
 Rarely, spherostomatocytes are seen.
 Specific morphologic findings have been identified in patients
with certain membrane protein defects, such as pincered
erythrocytes (band 3) or spherocytic acanthocytes (-spectrin).

18. Peripheral blood film
of spherocytic
hemolysis.
Spherocytes are
round, are slightly
smaller than normal
red blood cells, and
lack central pallor.
Note the nucleated
red blood cells and
polychromatophilic
cells.

19. Note the
anisocytosis
and several
dark-appearing
spherocytes
with no central
pallor. Howell-
Jolly bodies
(small dark
nuclear
remnants) are
also present in
red cells of this
asplenic patient.
PERIPHERAL SMEAR IN HS

20. Marrow smear
from a patient with
hemolytic anemia.
The marrow
reveals greatly
increased
numbers of
maturing erythroid
progenitors
(normoblasts).

21. OSMOTIC FRAGILITY
 Osmotic fragility is tested by adding increasingly hypotonic
concentrations of saline solution to red cells.
 Spherocytes, which already are at maximum volume for surface area,
burst at higher than normal saline concentrations.
 Some HS individuals have a normal osmotic fragility on freshly drawn
red blood cells, with the osmotic fragility curve approximating the
number of spherocytes.
 After incubation at 37°C for 24 hours, HS red cells lose membrane
surface area more readily than normal because their membranes are
leaky and unstable.
 Incubation accentuates the defect in HS erythrocytes and brings out
the defect in osmotic fragility, making incubated osmotic fragility the
standard test in diagnosing HS.

22. A. Histograms of the
distribution of (top)
MCV and (bottom)
MCHC in red cells of a
patient with HS before
splenectomy. Vertical
lines mark the normal
limits of the
distributions.
B. Osmotic fragility
testing. The shaded
area is the normal
range. Results
representative of both
typical and severe
spherocytosis are
shown.
A "tail," representing
very fragile
erythrocytes that have
been conditioned by
the spleen, is common
in many HS patients
prior to splenectomy.

23. OSMOTIC FRAGILITY
HS Trait
or Carrier
Mild
Spherocytosis
Moderate
Spherocyto
sis
Moderately
Severe
Spherocyto
sis*
Severe
Spherocyto
sis
Fresh
blood
Normal Normal or
slightly
increased
Distinctly
increased
Distinctly
increased
Distinctly
increased
Incubated
blood
Slightly
increased
Distinctly
increased
Distinctly
increased
Distinctly
increased
Markedly
increased

24. Laboratory
Findings
HS Trait
or
Carrier
Mild
Spherocytosis
Moderate
Spherocytos
is
Moderately
Severe
Spherocytosi
s*
Severe
Spherocyto
sis
Hemoglobin
(g/dL)
Normal 11–15 8–12 6–8 <6
Reticulocytes
(%)
1–2 3–8 ± 8 ≥10 ≥10
Bilirubin
(mg/dL)
0–1 1–2 ± 2 2–3 ≥3
Spectrin
content (% of
normal)‡
100 80–100 50–80 40–80§ 20–50
Blood film Normal Mild
spherocytosis
Spherocytosi
s
Spherocytosis Spherocytosi
s and
poikilocytosis

25. DIFFERENTIAL DIAGNOSIS OF HS
 HS must be differentiated from acquired hemolytic anemias that
produce circulating spherocytes and abnormal osmotic fragility.
 Autoimmune hemolytic anemia (AHA) usually produces spherocytes.
The direct antiglobulin test (direct Coombs test), readily distinguishes
most AHA from HS.
 On microscopic examination, HS usually produces more uniform
spherocytosis than does AHA.
 An elevated MCHC may also help differentiate HS from AHA.
 Other causes of acquired spherocytosis such as transfusion
reactions, AB0 incompatibility, oxidant erythrocyte damage, thermal
bums, snake venom, and Clostridia sepsis are distinguished from HS
by the clinical setting and lack of chronicity.
 Unusual inherited conditions occasionally confused with HS include
Rh antigen deficiency, hereditary stomatocytosis, unstable
hemoglobins, and the oxidant hemolysis of Wilson’s disease.

26. Peripheral blood film of
microspherocytes seen
in Clostridium
perfringens sepsis.
Although regular
spherocytes are
usually smaller than
normocytic red blood
cells, microspherocytes
are even smaller than
that. This finding is
usually seen in critically
ill, septic patients with
severe C. perfringens
infection.

27. MANAGEMENT
 Patients with HS may require red cell transfusions for a plastic crisis,
chronic folate administration to ward off megaloblastic crisis, or
cholecystectomy for biliary lithiasis.
 The most significant therapeutic decision, however, centers on the
issue of splenectomy.
 Splenectomy does not eliminate the spherocytic defect but
dramatically improves the rate of hemolysis in HS patients.
 In very mild cases (older age, normal hemoglobin, minimal hemolysis,
and no complications), there is no need for splenectomy.
 In more severely affected individuals (young age, moderate anemia,
active hemolysis, and complications), splenectomy is clearly indicated
and beneficial.
 Newer surgical techniques such as laparoscopic splenectomy and
partial splenectomy are beginning to impact on the treatment of HS,
but there are insufficient data to ascertain whether the indication for
splenectomy has changed.
 Vaccination against , pneumoniae prior to splenectomy is strongly
recommended.

28. COMPLICATIONS

29. NEONATAL PERIOD AND IMMUNO
COMPROMISED PATIENTS
 Parvovirus B19 selectively infects erythropoietic
progenitor cells and inhibits their growth.
 Parvovirus infections frequently are associated with
mild neutropenia, thrombocytopenia, or
pancytopenia.
 Parvovirus infection presents with fever, chills,
lethargy, vomiting, diarrhea, myalgia, and a
maculopapular rash on the face (slapped cheek
syndrome), trunk, and extremities.

30. APLASTIC CRISIS
 During the aplastic phase, hematocrit level and reticulocyte
count fall, marrow erythroblasts disappear, and, as the plasma
iron turnover decreases, plasma iron level increases.
 Giant pronormoblasts, a hallmark of the cytopathic effects of
parvovirus B19, often appear in the marrow. As production of
new red cells declines, the remaining cells age, and
microspherocytosis and osmotic fragility increase.
 Return of marrow function is heralded by a fall in serum iron
concentration and emergence of granulocytes, platelets, and,
finally, reticulocytes.
 Aplastic crises usually last 10 to 14 days (about half the life
span of typical HS red cells), the hemoglobin value usually falls
to about half its usual level before recovery occurs.

31. HEMOLYTIC CRISIS
 Hemolytic crises usually are associated with viral
illnesses and typically occur in childhood.
 They generally are mild but during severe hemolytic
crises, marked jaundice, anemia, lethargy, abdominal
pain, and tender splenomegaly occur.
 Hospitalization and erythrocyte transfusion may be
required.
 The most common etiologic agent in these cases is
parvovirus B19, which causes erythema infectiosum.

32. MEGALOBLASTIC CRISIS
 Megaloblastic crisis occurs in HS patients with
increased folate demands,
 Occurs in pregnant patients, growing children, or
patients recovering from an aplastic crisis.
 This complication is preventable with appropriate
folate supplementation.

33. GALLBLADDER DISEASE
 Formation of bilirubinate gallstones, the most frequently
reported complication in up to half of HS patients.
 Coinheritance of Gilbert syndrome uridine
diphosphoglucuronate glucuronosyltransferase gene
polymorphism markedly increases the risk of gallstone
formation.
 Most gallstones occur in adolescents, children, and
young adults.
 Routine management should include interval
ultrasonography to detect gallstones because many
patients with cholelithiasis and HS are asymptomatic.

34. OTHER COMPLICATIONS
 Dermatologic manifestations of HS, including skin ulceration, gouty tophi,
and chronic leg dermatitis, are uncommon.
 Findings attributable to extramedullary hematopoiesis have been
described in some HS patients.
 Thrombosis has been reported in several HS patients, usually
postsplenectomy.
 Iron overload has been described in untransfused HS patients both with
coinherited hemochromatosis and in patients without HFE
(hemochromatosis gene) mutations.
 Several HS kindred have been reported with neuromuscular
abnormalities including cardiomyopathy, slowly progressive
spinocerebellar degenerative disease, spinal cord dysfunction, and
movement disorders.
 The observation that erythrocyte ankyrin and -spectrin are also expressed
in muscle, brain, and spinal cord raises the possibility that these HS
patients suffer from defects of one of these proteins.
 Heterozygous defects of band 3 have been described in patients with
inherited distal renal tubular acidosis and normal erythrocytes.

35. THERAPY AND PROGNOSIS
 Splenectomy alleviates the anemia in the overwhelming majority of
patients.
 Postsplenectomy, spherocytosis and altered osmotic fragility persist,
but the "tail" of the osmotic fragility curve, created by conditioning of a
subpopulation of spherocytes by the spleen, disappears.
 Erythrocyte life span nearly normalizes, and reticulocyte counts fall to
normal or near-normal levels.
 Changes typical of the postsplenectomy state, including Howell-Jolly
bodies, target cells, siderocytes, and acanthocytes, become evident
on the blood film.
 Postsplenectomy, patients with the most severe forms of HS still
suffer from shortened erythrocyte survival and hemolysis, but their
clinical improvement is striking.

36. HEREDITARY ELLIPTOCYTOSIS, PYROPOIKILOCYTOSIS,
AND RELATED DISORDERS

38. HEREDITARY ELLIPTOCYTOSIS SYNDROMES
 Disorders characterized by elliptical red cells on the
peripheral blood smear
 Most are clinically silent and are discovered incidentally
when a blood smear is reviewed..
 The clinical expression of hemolytic HE ranges from a
moderate hemolytic anemia to severe, near-fatal or fatal
hemolytic anemia.
 These disorders have identified abnormalities of various
erythrocyte membrane proteins, including - and -spectrin,
protein 4.1, and GPC.
 The majority of defects occur in spectrin, the principal
structural protein of the erythrocyte membrane skeleton

39. VARIANTS OF HE
 Hereditary pyropoikilocytosis (HPP) is a severe hemolytic
anemia, with red cell fragments, poikilocytes, and
microspherocytes seen on peripheral blood smear.
 Once regarded as a separate condition, HPP is now
recognized to be a variant of the HE disorders.
 Spherocytic HE is a rare condition in which both ovalocytes
and spherocytes are present on the blood smear.
 Southeast Asian ovalocytosis (SAO), also known as
stomatocytic elliptocytosis, is an HE variant prevalent in the
malaria-infested belt of Southeast Asia and the South Pacific.
 Characterized by rigid spoon-shaped cells that have either a
longitudinal slit or a transverse ridge.

40. A: Micropoikilocytes and elliptocytes in a neonate with transient
poikilocytosis and an α-spectrin gene mutation. B: Same child at 7 months
of age, now exhibiting morphology of common hereditary elliptocytosis.

41. C: Compound heterozygous hereditary elliptocytosis due to two α-
spectrin self-association–site structural mutations. Note distorted red cell
shapes, elliptocytes, and fragments. D: Hereditary pyropoikilocytosis. Red
cell abnormalities are similar to those in (A) and (C) with prominent
budding and fragmentation.