CHAPTER 29
The haemoglobinopathies
David C. Rees  •  Roopen Arya
CHAPTER OUTLINE
INTRODUCTION  550
The structure and function of
haemoglobin  550
The genetic control of haemoglobin
synthesis  551
THE THALASSAEMIAS  552
α Thalassaemia  552
β Thalassaemia  553
INTRODUCTION
Since the discovery of its central role in oxygen trans-
port in the 19th century, haemoglobin has proved to
be a source of fascination to scientists and physicians
alike, and is arguably the most widely studied protein
in man. Its principal disorders, the thalassaemias and
sickle cell disease, quantitative and qualitative defects
of synthesis, respectively, are among the most common
serious inherited diseases in man and have been pivotal
in efforts to understand the molecular basis of human
diseases.
It is estimated that about 7% of the world’s popula-
tion carry the mutation for at least one type of hae-
moglobinopathy, and that 400 000 babies are born
each year with a severe form. Approximately 230 000
babies are born each year with sickle cell disease in
sub-Saharan Africa, and 120 000 are born with severe
thalassaemia in southern and South-East Asia. There is
good epidemiological and experimental evidence that
the extraordinarily high frequency of haemoglobin-
opathies and their geographical distribution are due
to the relative protection that they provide against
­malaria and its serious complications. This principally
involves improved survival and reproductive success for
heterozygotes compared with the normal population,
which outweighs the reduced reproductive success of
homozygotes. Thalassaemia reaches polymorphic fre-
quencies (>1%) in nearly all populations, except those
of northern Europe, northern Asia and the indigenous
peoples of Australia, the Americas and the Arctic. The
common abnormal haemoglobins have more limited
distributions (Fig. 29.1).
Since there is considerable overlap in the world distri-
bution of thalassaemias as well as the common structural
haemoglobin variants, co-inheritance of more than one
haemoglobin abnormality is common. This generates an
extremely diverse range of clinical phenotypes.
550
STRUCTURAL HAEMOGLOBIN VARIANTS  554
Sickle cell anaemia  555
Other structural haemoglobin
variants  556
LABORATORY DIAGNOSIS OF
HAEMOGLOBINOPATHIES  557
CONCLUSION  559
The structure and function of haemoglobin
Haemoglobin is a tetramer comprising two α and two β
globin chains (α2β2). One molecule of haem, which con-
tains iron and can bind to oxygen, is attached to each
globin chain, lying in a hydrophobic cleft. The amino
acid sequence of this haem pocket shows marked h ­ omology
between different animal species, suggesting a specific
and important function. Through its iron atom, each
haem group is capable of binding one molecule of oxy-
gen. The reversible binding of oxygen to haemoglobin
is allosteric, giving rise to the characteristic shape of the
oxygen dissociation curve (Fig. 29.2). On binding
­oxygen, conformational changes occur in both individual
globin chains and the tetrameric structure of haemoglo-
bin. The predominant change is closure of the gap be-
tween the two β-chains. The site at which this occurs is of
critical functional importance. 2,3-Diphosphoglycerate
(2,­3-DPG), the principal regulator of oxygen affinity in
red cells, binds to this part of the haemoglobin molecule.
This serves to separate the two β-chains, favouring the de-
oxygenated conformation and thereby reducing the oxygen
affinity of haemoglobin. The observation that oxygen affin-
ity is often reduced in anaemic patients is explained by the
adaptive increase in intracellular 2,3-DPG concentration.
This enhances oxygen delivery to the tissues and is one
of the important reasons why patients frequently tolerate
chronic anaemia with very few symptoms. Conversely,
the higher oxygen affinity of fetal blood necessary to
maintain adequate maternal–fetal oxygen transport is, in
large measure, due to the lower affinity of 2,3-DPG for
fetal haemoglobin (HbF).
The other main physiological influence on haemoglo-
bin oxygen affinity is the Bohr effect. First recognized
at the turn of the century through the effect of carbon
dioxide on lowering the oxygen affinity of whole blood, it
is now known to reflect the sensitivity of oxygen binding
by haemoglobin to changes in blood pH. An increase in
 29 
The haemoglobinopathies 551
HbC
HbS
FIGURE 29.1  ■  Primary geographic dis-
tribution of haemoglobins (Hb) S, C
and E.
FIGURE 29.2  ■ The oxygen dissociation curves of normal (AA)
and sickle (SS) haemoglobin (Hb).
hydrogen ion concentration stabilizes the ­deoxygenated
conformation of the haemoglobin molecule, and so physio-
logical acidosis leads to a reduction in the oxygen affinity.
If acidosis is sustained, this effect may be counterbalanced
by the reduction in intracellular 2,3-DPG concentration
due to modulation of red cell glycolysis. Several inherited
variants of haemoglobin have been described that stabi-
lize the molecule in either the oxy or deoxy conforma-
tion. In some cases, the interaction with 2,3-DPG or the
Bohr effect is modified.
A further factor affecting haemoglobin function is
temperature. An increase in temperature reduces oxy-
gen affinity, whereas a fall increases it. As the human is
normally isothermic, this is usually of little consequence
in terms of physiological adaptation, though during ex-
ercise, when oxygen consumption is increased, a raised
temperature in the muscles together with an increase in
hydrogen ion concentration favours release of oxygen by
HbE
haemoglobin. Extreme hypothermia has many adverse
consequences, including increased haemoglobin oxygen
affinity causing reduced oxygen delivery to tissues.
The genetic control of haemoglobin synthesis
Human globins are encoded by the α and β gene clus-
ters located on chromosomes 16 and 11, respectively (see
Fig. 29.3). The α-like genes include an embryonic gene
(ζ) and two adult genes (α1 and α2). The β-like genes com-
prise an embryonic gene (ε), duplicated fetal (γ) and adult
(β and δ) genes. Expression of these genes is developmen-
tally regulated during fetal life. The array of α- and β-like
genes on their respective chromosomes reflects the order
in which they are expressed. The control of the switch
from embryonic, to fetal and then to adult globin syn-
thesis, seems to be controlled by the interaction between
several transcription factors, the locus control regions
upstream of the gene cluster and the promoter regions of
the individual genes.
Normally, the synthesis of α- and β-like chains is care-
fully balanced to avoid the accumulation of free globin
chains, although it is not clear how this balance is main-
tained. In the thalassaemias, where this balance is severely
disturbed, the excess of one type of globin chain is central
to the pathophysiology.
The switch in globin synthesis during development
has important implications for clinical expression of the
haemoglobinopathies. Complete failure of α-chain syn-
thesis, which, since the α genes are duplicated on each
chromosome, occurs only when there is loss of function
involving all four α genes, becomes evident early in fe-
tal life. By contrast, even if there is little or no normal
β-chain synthesis, the effects will not be manifest until
the switch from fetal γ to adult β gene expression is com-
pleted after birth. This switch is gradual, and abnormali-
ties of β globin synthesis rarely result in clinical problems
552 Clinical biochemistry

Like genes

Like genes
Kilobases

Haemoglobins

Portland

Developmental Embryonic Fetal Adult

period

FIGURE 29.3  ■  Organization of the human globin gene cluster and developmental changes in haemoglobin.

before three months of age. Reversing this switch, and
reactivating fetal haemoglobin is also potentially curative
for diseases caused by mutations of the β globin genes.
THE THALASSAEMIAS
The thalassaemias are one of the commonest human au-
tosomal recessive disorders, with approximately 120 000
severely affected individuals born annually worldwide.
The most common and clinically significant forms are α
and β thalassaemia and the β thalassaemia-like structural
variant, haemoglobin E. These may be further subdivided
according to whether there is no output of globin (α0 or
β0 thalassaemia) or some preservation of globin chain
synthesis (α+ and β+ thalassaemia). In the case of α thal-
assaemia, this is complicated further by the duplication of
expressed α genes. α Thalassaemia is clinically s­ ignificant
only when three or four α globin genes are lost. The
clinical consequences of thalassaemia in the homozygous
(or compound heterozygous) state may be understood
in terms of the imbalance in globin chain synthesis that
results from absent or reduced synthesis of either α or
β globin. Unpaired α or β globin chains are toxic, and
damage the developing red cell, causing it to die whilst
still in the marrow, which is characteristic of thalassaemia
and called ineffective erythropoiesis. Heterozygotes are
unaffected clinically, and manifest the condition as slight
anaemia with reduced red cell size and haemoglobin con-
tent. Increasing globin chain imbalance, as occurs in ho-
mozygous and compound heterozygous states, results in
more marked anaemia and bone marrow expansion, with
corresponding development of symptoms.
A characteristic of thalassaemia syndromes is their
marked phenotypic heterogeneity. For over 20 years now,
the molecular basis of thalassaemia has been studied
and understood in great detail. The number of mutations
identified as causing thalassaemia is large and continues
to grow. However, in most populations, a small range of
5–10 different thalassaemia mutations accounts for about
90% of the mutant alleles found in that particular area.
α Thalassaemia
Though particularly common in South-East Asia, where
carrier rates may reach 50%, α thalassaemia is widely
distributed in all major populations apart from northern
Europeans (see Fig. 29.1). The pathophysiological effects
of α thalassaemia reflect the degree of impairment in α
globin production (see Table 29.1). The majority of cases
are due to deletions involving one or both α globin genes.
Haemoglobin Bart’s hydrops fetalis is the most severe
form of α thalassaemia, where all four α genes are af-
fected, abolishing or severely diminished α chain synthesis.

TABLE 29.1  The α thalassaemia syndromes

Number of %Hb Bart’s
Phenotype functional α genes Genotypea at birth HbH inclusions
Normal 4 αα/αα 0 None
α Thalassaemia trait (minor) 3 −α/αα 0–2 None
2 −α/−α or −−/αα 2–8 Occasional
HbH disease 1 −−/−α 10–40 Numerous
Hb Bart’s hydrops fetalis 0 −−/−− 80 Present in some cases

−α, denotes loss of function of one α gene on a chromosome, i.e. α+ thalassaemia.

a

−−, denotes loss of function of both α genes on a chromosome, i.e. α0 thalassaemia.

 29 
The haemoglobinopathies 553
The disease becomes manifest in fetal life. With the fail-
ure of normal production of fetal haemoglobin (α2 γ2),
surplus fetal γ-chains combine to form tetramers (γ4) rec-
ognized electrophoretically as haemoglobin Bart’s. The
fetus is only able to survive at all in utero because of the
presence of increased amounts of the embryonic haemo-
globin Hb Portland (ζ2γ2). Haemoglobin Bart’s possesses
markedly increased oxygen affinity and is ineffective as
an oxygen transporter. In an attempt to compensate for
impaired tissue oxygen delivery, there is erythroblastosis
with extramedullary haemopoiesis, leading to hepatic and
splenic enlargement. Severe functional anaemia leads to
tissue hypoxia, increased capillary permeability, and car-
diac failure, which lead ultimately to fetal hydrops and
death in utero or stillbirth in late pregnancy.
Haemoglobin H disease occurs when there is loss of
function of three out of four α genes. There is sufficient
residual α-chain synthesis to allow production of some
normal fetal and adult haemoglobin, and fetal develop-
ment is generally normal. A variable amount (10–40%)
of Hb Bart’s is detectable at birth. After birth, the excess
β-chains form tetramers detectable electrophoretically
as the fast variant haemoglobin H (β4). Haemoglobin H
(HbH) precipitates within red cells with the formation
of inclusion bodies leading to shortened red cell survival.
Staining of these inclusions with the redox dye brilliant
cresyl blue serves as a useful diagnostic test. The clinical
effects of HbH disease vary considerably. Most patients
have a mild to moderate haemolytic anaemia, which may
be exacerbated during pregnancy or parvovirus B19 in-
fection, accompanied by splenomegaly. Growth and
development are usually normal. As in other congenital
haemolytic anaemias, there is an increased tendency to
form pigment gallstones.
It follows that Hb Bart’s hydrops fetalis and HbH dis-
ease occur only when at least one parent carries the α0
genotype (–/αα). This provides an explanation for the ob-
servation that these disorders occur primarily in southern
China, South-East Asia and the eastern Mediterranean
where α0 thalassaemia is prevalent, and are not seen in
other parts of the world, for example Africa and India,
where α0 thalassaemia is very rare.
Individuals in whom one or two α genes are dysfunc-
tional are clinically unaffected, although they show thal-
assaemic red cell indices and traces of Hb Bart’s at birth
(see Table 29.1). This form of α thalassaemia trait is very
common in most populations, with a 3.7 kb deletion ac-
counting for most cases. Non-deletional forms of α thal-
assaemia are increasingly recognized as DNA analysis
becomes more widespread and sophisticated.
β Thalassaemia
β Thalassaemia is prevalent throughout tropical and
North Africa, the Mediterranean, Middle East and large
parts of south and South-East Asia, including India
(see Fig. 29.1). In some parts of the world, for example
Cyprus, the heterozygote (carrier) frequency may reach
15%. Over 300 different mutations of the β globin gene
or its promoter are known to cause β thalassaemia, the
majority being single nucleotide substitutions (point
mutations) or small deletions. Geographically, these
segregate non-randomly, so that four or five specific mu-
tations account for the majority of β thalassaemia genes
in individual ethnic or geographic groups. Each mutation
typically occurs on a single β globin gene haplotype, sug-
gesting that in most instances individual β thalassaemia
mutations have arisen historically on a single occasion.
Though both α and β thalassaemia share reduced glo-
bin chain synthesis, the pathogenesis of anaemia in se-
vere forms of these conditions is distinct. In severe forms
of β thalassaemia (β thalassaemia major), there is severe
impairment or absence of β-chain production. As a con-
sequence, excess α-chains accumulate and precipitate in
red cell precursors leading to their destruction within
the bone marrow. There is also a degree of shortened
red cell survival, although ineffective erythropoiesis pre-
dominates. Red cells containing fetal haemoglobin (HbF)
survive preferentially since there is less globin chain im-
balance. The anaemia produces tissue hypoxia, which
stimulates erythropoietin production and massive expan-
sion of erythropoiesis in the bone marrow and extramed-
ullary sites.
If untreated, β thalassaemia major leads to severe
anaemia, failure to thrive, impaired growth and bony
distortion, with death in early childhood. As a result of
the expansion in erythropoiesis, there are bone deformi-
ties and enlargement of the liver and spleen. Medullary
expansion of the facial bones and skull gives rise to a
characteristic ‘thalassaemic’ facies, splaying of the teeth
and frontal bossing. With the advent of effective therapy,
this picture is now seldom seen in the developed world.
The treatment of β thalassaemia major comprises regu-
lar blood transfusion to maintain the haemoglobin above
95 g/L. At this level of haemoglobin, bony deformity and
hypersplenism due to anaemia are minimized. Leukocyte-
depleted blood should be used to prevent alloimmuniza-
tion against white cell antigens. The blood should be
matched for an extended range of blood groups, includ-
ing ABO, Rh and Kell, to reduce the frequency of alloan-
tibody formation.
An inevitable complication of multiple red cell trans-
fusions is iron accumulation. If untreated, this leads to
iron deposition throughout the body, which may cause
endocrine dysfunction with diabetes, hypogonadotrophic
hypogonadism and hypoparathyroidism, and, ultimately,
death due to cardiac failure and liver damage. This may be
prevented by the use of iron chelators. Desferrioxamine,
which increases both urinary and faecal iron excretion,
is administered parenterally, usually by continuous over-
night subcutaneous infusion, since it is ineffective orally.
Ascorbic acid may enhance urinary iron excretion when
given with desferrioxamine. The clinical efficacy of des-
ferrioxamine is limited by its subcutaneous route of ad-
ministration, and two oral iron chelators are currently
licensed for use. Deferiprone is probably less effective
than desferrioxamine at removing hepatic iron, but may
be particularly effective at removing cardiac iron, and can
be used in combination with desferrioxamine. Side-effects
include arthropathy and unpredictable neutropenia, and
weekly full blood count monitoring is recommended
for those taking it. Deferasirox is a newer oral chelator
with similar efficacy to desferrioxamine and a good safety
profile. The degree of iron overload and the efficacy of
554 Clinical biochemistry
chelation can be monitored by measuring serum ferritin
concentration, liver biopsy to quantitate iron and non-­
invasive measurements using magnetic resonance ­imaging
(MRI) and the superconducting quantum interference
device (SQUID). Cardiac and hepatic MRI using T2*
and R2 protocols are increasingly used. Accurate quanti-
tation of the volume of transfused blood is also valuable
in assessing the degree of iron overload.
With adequate transfusion and chelation therapy, most
children with β thalassaemia major attain normal growth
and sexual development and survive well into adult life.
Allogeneic bone marrow transplantation from human
leukocyte antigen (HLA)-matched siblings has been used
with considerable success for treatment of β thalassaemia
major. The best results, with disease-free survival rates
of up to 95%, have been obtained in younger children
with little or no evidence of end-organ damage due to
iron overload. Trials of gene therapy are taking place in
France and the USA, with one patient to date success-
fully rendered transfusion-independent, although with
one cell clone predominating, owing to the inadvertent
activation of a potential oncogene.
As in other thalassaemia syndromes, there is consider-
able heterogeneity in the clinical expression of β globin
gene mutations. Much of this heterogeneity is unex-
plained, but two factors have been identified: first, the
extent to which β globin production is impaired, which
reflects the nature of the underlying gene mutation, and
second, co-inheritance of other interacting genetic de-
terminants that modify clinical severity. Mutations that
severely disrupt or abolish β globin synthesis (β0 thalassae-
mia) include those that prevent normal splicing of mRNA
(splice junction or cryptic splice site mutations) or gen-
erate a non-functional mRNA by premature translation
termination (nonsense mutations), and small nucleotide
deletions or insertions that result in the normal triplet
­genetic code being read out of frame (frameshift mutations).
Other mutations, for example those of the β globin chain
promoter, which alter the level of gene expression (tran-
scriptional mutations), usually result in reduced rather
than absent β globin synthesis (β+ or β++ thalassaemia).
Among the independent genetic factors that ameliorate
the clinical effects of β thalassaemia are co-inheritance of
α thalassaemia, which partially redresses the imbalance in
globin chain synthesis and therefore results in less inef-
fective erythropoiesis, and hereditary persistence of fe-
tal haemoglobin (HPFH). HPFH can be directly linked
to the β globin cluster, and its causes include large de-
letions and point mutations in the promoter regions of
the γ globin genes. Three other major loci which control
HbF concentrations have now been identified: the Xmn1
TABLE 29.2  The β thalassaemia syndromes
Genotype Heterozygote
MCH and MCV a
% HbA2 % HbF
β0 Reduced Raised <4
β+ Reduced Raised <4
β++ Slightly reduced Raised or <2
borderline
a
MCH, mean cell haemoglobin; MCV, mean cell volume.
BOX 29.1 Causes of β thalassaemia intermedia
Homozygous β thalassaemia
• Homozygous β++ thalassaemia
• Compound heterozygote β+/β++ thalassaemia
• Co-inheritance of α thalassaemia
• Co-inheritance of factors promoting increased HbF
synthesis
Heterozygous β thalassaemia
• Co-inheritance of triplicated α globin genes (ααα/αα
or ααα/ααα)
• Dominant β thalassaemia
δβ Thalassaemia
• Homozygous δβ thalassaemia
• Heterozygous δβ thalassaemia/β thalassaemia
polymorphism in promoter region of the Gγ globin genes,
the HMIP locus on chromosome 6q23.3 and BCL11A on
chromosome 2. These factors account for some cases of
the clinical syndrome thalassemia intermedia, in which
there is a variable degree of anaemia, without dependence
on regular blood transfusions (Box 29.1).
The heterozygous (carrier) state for β thalassaemia
(β thalassaemia trait) is usually harmless. Some of the
phenotype characteristics of β thalassaemia heterozygotes
are shown in Table 29.2. Most individuals have slightly
reduced haemoglobin concentrations and an elevated red
cell count. A raised HbA2 (α2δ2) concentration is an impor-
tant diagnostic marker and distinguishes β thalassaemia
trait from α thalassaemia, in which the HbA2 is normal or
low. This presumably reflects a higher output from the δ
globin gene, which normally plays a minor role in adult
haemoglobin synthesis (normal HbA2 <3.2%) in the face
of reduced β globin production. Similarly, HbF concen-
trations are frequently slightly raised in β thalassaemia
heterozygotes. Rarely, dominant β thalassaemia occurs.
STRUCTURAL HAEMOGLOBIN VARIANTS
Over 700 qualitative variants have been identified, in which
the haemoglobin molecule is structurally, and in some cases
functionally, altered. These arise from diverse molecular
defects, most commonly single amino acid substitutions,
but also insertion or deletion of amino acids and polypep-
tide fusion as a result of recombination between globin
genes, for example haemoglobin Lepore. The most com-
mon structural variants are haemoglobins S, C, DPunjab and
E, which each affect many millions of people worldwide.
Homozygote
% HbA % HbF Clinical status
0 >94 Thalassaemia major
5–50 50–90 Thalassaemia major
50–90 10–50 Thalassaemia
intermedia
 29 
The haemoglobinopathies 555
Sickle cell anaemia
Sickle cell anaemia (HbSS), caused by homozygous in-
heritance of structurally abnormal haemoglobin, is one
of the commonest genetic disorders with an estimated 60
million carriers worldwide and 230 000 affected infants
born annually in Africa alone. The prototypic molecu-
lar disease, it was the first human disorder to be under-
stood at a molecular level. The landmark discovery of
sickle haemoglobin (HbS) more than half a century ago
by Pauling and Itano, was succeeded by demonstration
of the underlying single amino acid substitution and the
gene mutation responsible. Sickle haemoglobin polym-
erizes on deoxygenation to produce long molecules that
damage and distort the red cell, producing the classic
sickle-shaped erythrocytes that give the disease its name.
The resulting microvascular occlusion and shortened red
cell survival combine to produce a clinical syndrome that
comprises anaemia, vaso-occlusion, vasculopathy, hae-
molysis, inflammation, hypercoagulability, organ damage
and susceptibility to infection due to hyposplenism.
A single base change (CAG→CTG) in the sixth codon
of the β globin gene determines the substitution of valine
for glutamic acid, which results in the structural variant
sickle haemoglobin S. While the sickle mutation occurs
predominantly in those of African origin, with smaller
numbers affected in the countries of the Mediterranean
littoral, Saudi Arabia and India, population migration has
resulted in its spread globally. The allele frequency of βs
is thought to be high because of protection against the
complications of severe malaria in the heterozygous state,
as described earlier for thalassaemia mutations.
Although HbSS is the commonest cause of sickle cell
disease, this condition occurs with other genotypes, in
which there is co-inheritance of a different β globin mu-
tation. The most important of these compound hetero-
zygous states are: HbSC disease, HbSβ+ thalassaemia and
HbSβ0thalassaemia, although about 15 different geno-
types have been identified as causing sickle cell disease.
Among sickling disorders, HbSS and Sβ0 thalassaemia
are the more severe clinically, with a greater degree of
anaemia (haemoglobin 60–80 g/L) and more severe organ
damage. While HbSC and Sβ+ thalassaemia are perceived
as milder genotypes than HbSS, there is considerable
overlap and some types of end-organ damage, particu-
larly proliferative retinopathy, occur more frequently,
particularly in HbSC disease. Other genetic factors that
may influence disease expression include co-inheritance
of α+ thalassaemia and high fetal haemoglobin (HbF)
­levels, both of which may ameliorate the severity of sickle
cell disease.
The primary pathophysiological event in sickling is
intracellular polymerization of deoxy HbS. The β6 valine
substitution alters the surface charge of haemoglobin,
resulting in interaction between haemoglobin tetramers
and the formation of 14-stranded polymers. These are
then ordered into parallel arrays of fibre bundles, which
can be visualized by electron microscopy. The formation
of polymer fibres (or gelation) is affected by four main
variables: oxygen tension, HbS concentration, tempera-
ture and the presence of non-sickling haemoglobins. The
mean corpuscular haemoglobin concentration (MCHC)
has a profound effect on the kinetics of sickling through
its effect on the delay period between deoxygenation and
polymerization. Small increases in haemoglobin concen-
tration, as occur with cellular dehydration, may therefore
act as a trigger for the sickling process.
Polymer formation is accompanied by several changes
in the membrane of sickle erythrocytes, making them
less deformable, increasingly fragile and leaky to cations.
These changes are thought to result not only from inter-
actions between sickle haemoglobin and the membrane
but also from oxidative damage caused by free radicals,
concentrations of which have been shown to be increased
in these cells. These membrane changes are initially re-
versible but become increasingly pronounced as the cell
undergoes repeated cycles of sickling and unsickling, cul-
minating in an irreversibly sickled cell (ISC). The per-
centage of ISCs has been used as a marker for the sickling
process but correlates poorly with the clinical condition
of the patient. Red cell membrane phospholipids are ab-
normally distributed in these cells, with the normal in-
ner leaflet amino-phospholipids being rearranged on the
outer leaflet of the lipid bilayer. Cation homoeostasis is
also disordered, with potassium loss exceeding sodium
gain, resulting in cellular dehydration and increased
concentration of intracellular haemoglobin. This is ac-
companied by an up to four-fold increase in intracellu-
lar calcium. There is increased intravascular haemolysis,
leading to a high plasma concentration of free haemo-
globin, which binds nitric oxide avidly and results in a
functional nitric oxide deficiency; this is implicated in the
development of vasculopathy, which contributes to the
complications of pulmonary hypertension, priapism and
cerebrovascular disease. Other pathological processes in-
clude hypercoagulability, inflammation, oxidative stress
and reperfusion injury, all of which contribute to the mul-
tisystem nature of the disease.
The polymerization of HbS and associated membrane
changes cause a marked decrease in the ability of these
cells to flow through the microvascular circulation, re-
sulting in compromised oxygen delivery and setting up a
vicious circle of worsening vaso-occlusion. There is also
evidence of increased adherence of these red cells to vas-
cular endothelium, which would increase deoxygenation
and obstruct the flow of other red and white cells through
the microvasculature.
The clinical manifestations of sickle cell disease are
protean and include both acute and chronic complica-
tions. The majority of symptoms are attributable to the
effects of vascular occlusion, since the anaemia is usually
well tolerated. Sickle cell disease is a multisystem disease.
Acute episodes or ‘crises’ can take several forms, includ-
ing pain or severe anaemia; acute worsening of anaemia
can be due to splenic or hepatic sequestration, or tran-
sient erythroid hypoplasia secondary to parvovirus B19
infection. The most common symptom is acute, severe
pain, which may be preceded by infection, dehydration
and cold exposure, though often no particular trigger is
identified. Vaso-occlusion, which commonly involves the
bones, results in avascular necrosis of the bone marrow
with associated inflammation and increased intramedul-
lary pressure. This results in painful swelling of the hands
and feet (dactylitis) in early infancy. In older children and
556 Clinical biochemistry
adults, the juxta-articular areas of long bones, flat bones
like the ribs and pelvis and the vertebral column are most
commonly affected. The management of acute pain is
supportive, ensuring adequate analgesia, hydration and
oxygenation. Other acute complications include pria-
pism, retinal artery occlusion and sudden death.
Sickle cell patients are at increased risk of bacterial in-
fection due largely to loss of splenic function and, histori-
cally, overwhelming pneumococcal sepsis has been the
major cause of early mortality. In developed countries,
this risk has been significantly reduced by the introduc-
tion of pneumococcal prophylaxis in the form of pneu-
mococcal vaccines and daily oral penicillin. The major
cause of mortality after early childhood is the acute chest
syndrome, which results from a combination of infection,
infarction and fat embolism in the lungs, and is character-
ized by fever, severe chest pain, dyspnoea and pulmonary
infiltrates. This complication needs prompt and vigorous
management including oxygenation, hydration, intrave-
nous antibiotics and blood transfusion. Increasing num-
bers of patients in developed countries are surviving to
late middle and old age, and progressive multi-organ fail-
ure, particularly involving the kidneys, is an increasingly
common form of death. From a very early age, most chil-
dren with SCD display a number of renal abnormalities,
including glomerular hyperfiltration and nephrogenic
diabetes insipidus. Many go on to develop significant
­albuminuria in later childhood, with progressive renal
failure contributing to death in about 30% of adults.
Stroke due to occlusion of the large cerebral vessels
affects about 11% of patients by the age of 20, with the
highest incidence between the ages of two and five years.
Without treatment, there is a high rate of recurrence.
Long-term transfusion significantly reduces the risk
of a further stroke but carries with it the necessity for
iron chelation to prevent siderosis. Transcranial Doppler
scanning identifies children with early vasculopathy who
are at high risk of stroke. Regular blood transfusion of
children with such cerebral vasculopathy has been shown
to be an effective form of primary stroke prevention, both
in clinical trials and practice.
Pulmonary hypertension affects up to 5% of adults
with sickle cell disease, and is thought to be associated
with an increased risk of premature death. Increased rates
of haemolysis have been linked to the pathology, and it
may be part of a more general vasculopathy.
Chronic organ damage due to sickling may take several
forms. Ischaemic damage to the bones and joints results
in progressive destruction which, in the case of avascular
necrosis of the hip, may lead to severe disability. Chronic
restrictive lung disease may follow recurrent episodes of
infection and infarction. As in other chronic haemolytic
states, gallstones are common and found in nearly one-
third of young adults with sickle cell disease. Proliferative
retinopathy can result in bleeding, retinal detachment
and blindness. Stasis and occlusion of the small vessels in
the lower limbs may cause leg ulceration.
The prognosis for patients with sickle cell disease in
developed countries has been transformed by early diag-
nosis, improved supportive care and, most importantly,
prophylaxis against pneumococcal infection. At least
85% of HbSS patients and 95% of HbSC patients in the
USA now survive to 20 years and 50% of patients survive
beyond the fifth decade. Relatively little is known about
the natural history of the condition in African countries,
but the majority of children with sickle cell disease are
thought to die before the age of five years. This mark-
edly higher mortality in Africa reflects the importance of
environmental factors, and is probably mainly related to
malaria, pneumococcal and other infections. Sickle cell
disease remains a disease without a cure. Haematopoietic
stem cell transplantation has been successfully performed
in a few patients but carries a procedure-related risk
with 5% mortality. In the absence of reliable predictors
of clinical severity, which varies widely among affected
patients, this is difficult to justify in most cases. The role
of transplantation is likely to increase as the procedure
becomes safer and able to draw from a wider range of do-
nors. Human trials of gene therapy are currently planned,
but as yet, no patients have been successfully treated.
The search for effective anti-sickling agents has tar-
geted the essential steps in the pathophysiology of sickle
cell disease: polymer formation, membrane changes and
interactions with the microvasculature. The first ap-
proach, aimed at combating polymer formation, either by
altering oxygen affinity or increasing HbF concentrations,
seems most likely to be successful. Hydroxycarbamide
(hydroxyurea), which increases HbF concentrations, has
been shown to reduce the frequency of acute pain and
acute chest syndrome in randomized controlled trials,
and is now an important therapeutic option in adults and
children suffering frequent episodes of pain or severe
chest problems.
Other structural haemoglobin variants
There are several hundred less common structural hae-
moglobin variants, most of which are very rare and have
no clinical or functional consequences. An exception
is the β globin variant haemoglobin E (β26 Glu→Lys),
which is probably the most prevalent haemoglobin vari-
ant, being carried by an estimated 84 million individuals
worldwide, mainly in South-East Asia. Haemoglobin E
has an electrophoretic mobility similar to that of HbC
and HbA2 at pH 8.9 but can be differentiated by citrate
agar electrophoresis at acid pH. Unlike most other struc-
tural variants, inheritance of HbE is associated with a β
thalassaemia phenotype with microcytosis and hypochro-
mia. This is because the βE mutation activates a cryptic
splice site that inhibits the normal splicing mechanism.
Haemoglobin E is also unstable, which may contribute
to the unexpectedly severe phenotype that occurs when
HbE is co-inherited with β thalassaemia mutations. This
compound heterozygous state results in thalassaemia
­major in about half of cases.
The other common abnormal haemoglobins are C
and DPunjab, which affect millions, predominantly in West
Africa and the Punjab region of India, respectively. In the
homozygous state, there is mild haemolysis, with few if
any symptoms. Co-inheritance of both of these variants
with HbS results in sickle cell disease.
The unstable haemoglobin variants usually result
from neutral substitutions affecting amino acid residues
that contact the haem group and generally present as a
 29 
The haemoglobinopathies 557
congenital Heinz body haemolytic anaemia, for example
Hb Köln and Hb Bristol. Heinz bodies are inclusion bod-
ies seen in some red cells consisting of degraded haemo-
globin. The diagnosis is made by the heat denaturation
or isopropanol precipitation tests and identification of
the globin mutation by protein or DNA analysis. Owing
partly to the instability of the variant haemoglobin, only
half of these variants are detectable by electrophoresis.
Amino acid substitutions involving either α- or β-chains
in the vicinity of the haem group can also result in altered
oxygen affinity or a propensity to methaemoglobin forma-
tion. Haemoglobin variants in which oxygen affinity is sig-
nificantly increased (e.g. Hb Chesapeake, Hb San Diego)
are associated with erythrocytosis (polycythaemia). Low-
affinity haemoglobins (e.g. Hb Kansas) and HbM variants
(e.g. HbM Boston) cause cyanosis, usually with no associated
signs or symptoms of disease. The possible consequences
of abnormal haemoglobins are summarized in Table 29.3.
LABORATORY DIAGNOSIS OF
HAEMOGLOBINOPATHIES
The primary investigation of a haemoglobinopathy should
include a full blood count, peripheral blood film and
haemoglobin electrophoresis (see Fig. 29.4). The full
­ ference from methaemoglobin present in such samples.
blood count allows assessment of haemoglobin formation,
as judged by the red cell indices, the mean cell volume
(MCV) and mean cell haemoglobin (MCH). Microcytosis
(MCV <76 fL) and hypochromia (MCH <27 pg), in the
face of a normal or raised red cell count (>5.5 × 1012/L) in
an iron-replete patient, suggest a diagnosis of thalassae-
mia. In the case of β thalassaemia major or intermedia,
this is associated with a significant degree of anaemia,
whereas in thalassaemia trait the haemoglobin is usually
normal or only marginally reduced. Examination of a
blood film stained by a Giemsa method may be helpful in
confirming the diagnosis. However, definitive diagnosis
often rests on electrophoretic or chromatographic analy-
sis of haemoglobin in red cell haemolysates. Cellulose ac-
etate electrophoresis at alkaline pH (8.9–9.1) is the most
widely used method, being simple, rapid, inexpensive and
effective in separating the common haemoglobin variants.
In homozygous sickle cell anaemia, HbS predominates. A
variable amount of HbF is present, higher proportions
TABLE 29.3  The functional and clinical consequences of abnormal haemoglobins
Clinically
Functional abnormality expresseda Clinical disorder
None No None
Polymerization with Hom/Co het Sickle cell disease
reduced solubility
Unstable Het Haemolytic anaemia
Increased O2 affinity Het Erythrocytosis
Decreased O2 affinity Het Cyanosis
Methaemoglobinaemia Het Blue discolouration
α Thalassaemia Hom/Co het Thalassaemia intermedia
β Thalassaemia Co het Thalassaemia intermedia/
major
a
The abnormality may be expressed in the homozygous (Hom), heterozygous (Het) or compound heterozygous (Co het) state.
b
This chain terminator mutation results in an α globin chain with an additional 31 amino acids.
(>10%) generally being associated with a milder clinical
course. Haemoglobin A2 concentration is usually normal.
Solubility testing based on the reduced solubility of
deoxy-HbS in the presence of reducing agents, for ex-
ample sodium dithionite, has a limited role in the diagno-
sis of sickle cell disease since it does not differentiate the
homozygous disease and carrier states. In an emergency
situation, a positive solubility test taken in conjunction
with a significantly reduced haemoglobin and typical red
cell morphology points strongly towards a diagnosis of
sickle cell disease. This should be confirmed by haemo-
globin analysis at the earliest opportunity. Several other
variants, including HbD, HbG and Hb Lepore, have an
electrophoretic mobility identical to that of HbS on cel-
lulose acetate but may be distinguished by the negative
sickle solubility test and citrate agar gel electrophoresis
at acid pH (6.0). Similarly, haemoglobins C, E and O,
which co-migrate on cellulose acetate at alkaline pH, can
be differentiated by citrate agar electrophoresis. Both
HbE and Hb Lepore are associated with thalassaemic
red cell indices, which further aids their distinction from
electrophoretically similar variants. Isoelectric focusing
improves the resolution of some structural variants and
can also be used for neonatal screening of eluates from
Guthrie (dried blood spot) cards, since it reduces inter-
High performance liquid chromatography (HPLC) is
a fast and sensitive method for separation and quantita-
tion of haemoglobins that, in some cases, allows identifi-
cation of variants not possible by other techniques. Since
it is largely automated and requires minute quantities
of sample, HPLC has become the method of choice for
large-scale population testing such as neonatal screen-
ing programmes. Universal neonatal screening for sickle
cell disease has been used in the USA and England for
some time, and similar programmes are being introduced
into other European, Middle Eastern and African coun-
tries, depending on the prevalence of the conditions and
the resources available. Such programmes have resulted
in significant benefits in terms of reduced mortality and
morbidity due to improved care, early ­implementation
of prophylaxis against pneumococcal infection and
­parental education. In β thalassaemia, the proportion of
­individual haemoglobins varies with the underlying gen-
otype. Homozygous β0 thalassaemia is associated with
Example Molecular abnormality
HbG Philadelphia α68 Asn→Lys
HbS β6 Glu→Val
Hb Köln β98 Val→Met
Hb Chesapeake α92 Arg→Leu
Hb Kansas β102 Asn→Thr
HbM Saskatoon β63 His→Tyr
Hb Constant Spring α + 31Cb (142 Stop→Gln)
Hb Lepore–Boston δ (1–87) β (116–146) fusion
558 Clinical biochemistry

Clinical picture and/or blood count or film

suggestive of a haemoglobinopathy

Hb Electrophoresis

Hb variants, e.g. S,C,D,E Normal

Hb Bart’s and HbH

Estimation of HbF Estimation of HbA2

Raised N/low

Globin chain DNA

synthesis analysis

Thalassaemia Thalassaemia trait Thalassaemia Further tests,

HPFH ‘Normal A2’ e.g. unstable Hb
Thalassaemia Thalassaemia O2 affinity
Heinz bodies

FIGURE 29.4  ■  A simple algorithm for the diagnosis of haemoglobinopathies.

a predominance of HbF, absence of HbA and variable
amounts of HbA2 (range 1.0–6.0%, mean 1.7%). In in-
dividuals with homozygous β+ thalassaemia or compound
heterozygous β0/β+ thalassaemia, a variable amount of
HbA is present. Haemoglobin F is increased and distrib-
uted heterogeneously among red cells.
Accurate quantitation of HbA2 by HPLC or microcol-
umn chromatography is essential for the diagnosis of β
thalassaemia trait, in which the HbA2 is elevated, typically
>3.5%. Carriers of ‘normal A2’ or ‘silent’ β thalassaemia
due to mild β gene defects or co-inheritance of a δ gene
mutation in cis or trans cannot easily be distinguished
from α thalassaemia by conventional screening methods
and require investigation by specialized techniques, in-
cluding in vitro globin chain synthesis and DNA analysis.
Analysis of globin chain synthetic ratios by tritiated
­leucine incorporation and carboxymethylcellulose chro-
matography is the definitive way of identifying individu-
als with thalassaemia, although it is rarely used because of
its laborious nature.
The α thalassaemias are characterized electropho-
retically by the presence of the fast moving variants, Hb
Bart’s (γ4) and HbH (β4), which are most obvious in neo-
natal samples. In hydrops fetalis owing to homozygous
α0 thalassaemia, Hb Bart’s predominates and is found in
smaller amounts in other α thalassaemia syndromes in the
neonatal period. Haemoglobin H may also be detected
by staining for HbH inclusion bodies. The diagnosis of
clinically silent forms of α thalassaemia is often one of
exclusion, being made on the basis of the subject’s eth-
nic origin, microcytic hypochromic red cell indices and
a normal or low HbA2 concentration in the presence of
normal iron status. Definitive diagnosis can be made by
DNA analysis, which can also often distinguish α0 and α+
thalassaemia.
While the majority of haemoglobinopathies can be di-
agnosed by haemoglobin electrophoresis, variants caused
by amino acid substitutions that do not alter charge, such
as those found in some unstable haemoglobins or haemo-
globins with altered oxygen affinity, may escape detec-
tion. Further investigations that may be helpful in this
context include assessment of haemoglobin instability
and oxygen affinity. High throughput DNA analysis has
made this a feasible way of screening for globin mutations
in richer countries, with techniques such as multiplex
ligation-dependent probe amplification allowing identi-
fication of large gene mutations which were previously
only detectable using Southern blotting. Haemoglobin
mass spectrometry can also be used to identify abnormal
globins by measuring their mass accurately, with particu-
lar potential use in screening programmes which already
use mass spectrometry.
The identification of couples at risk for major haemo-
globinopathies by antenatal or preconceptional screening
permits informed reproductive choice with the option of
prenatal diagnosis. In most cases, this can now be accom-
plished in the first trimester by detection of mutant glo-
bin genes in chorionic villous DNA. In several countries,
 29 
The haemoglobinopathies 559
notably Cyprus where the carrier rate for β thalassaemia
reaches 12%, this has led to a marked decline in the birth
incidence of haemoglobin disorders. Preimplantation
genetic diagnosis is increasingly used to allow selection
of unaffected embryos, although it continues to be a de-
manding and expensive process that is not applicable to
most couples. Attempts continue to develop non-invasive
prenatal diagnosis using fetal cells and DNA in maternal
blood, although this is currently not technically feasible
as a routine procedure for the haemoglobinopathies.
CONCLUSION
Haemoglobin is arguably the best studied of all human
proteins. It is a tetramer of two pairs of globin chains.
Each chain binds a molecule of haem, to which a mol-
ecule of oxygen can become reversibly bound. The affin-
ity of haemoglobin for oxygen can be modified by various
physiological factors in ways that respond to the require-
ments of tissues for oxygen under different circumstances.
Inherited disorders of haemoglobin synthesis can be
qualitative or quantitative. More than 400 structural vari-
ants have been described, of which the most important
is haemoglobin S, the haemoglobin of sickle cell disease.
Quantitative abnormalities of globin chain synthesis cause
the thalassaemias. Advances in diagnosis and treatment
have dramatically altered the prognosis for many affected
patients, although there is still a limited range of treat-
ments available.
Further reading
Dacie J. The hereditary haemolytic anaemias. Part 2. 3rd ed
The haemolytic anaemias. vol. 2. Edinburgh: Churchill Livingstone;
1988.
Rees DC, Williams TN, Gladwin M. Sickle-cell disease. Lancet
2010;376:2018–31.
Serjeant GR, Serjeant BE. Sickle cell disease. 3rd ed. Oxford: Oxford
University Press; 2001.
Weatherall DJ, Clegg JB. The thalassaemia syndromes. 4th ed. Oxford:
Blackwell Science; 2001.
Each of these texts provides a comprehensive account of the pathology,
diagnosis and management of the haemoglobinopathies.