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Investigation of Variant Haemoglobins and Thalassaemias


The haemoglobin molecule

Human haemoglobin is formed from two pairs of globin chains each with a haem group attached. Seven different globin chains are synthesised in normal subjects; two, ε and ζ, are characteristic of the embryo and contribute to four transient embryonic haemoglobins referred to as haemoglobins Gower 1, Gower 2, Portland 1 and Portland 2. Haemoglobin F is the predominant haemoglobin of fetal life and comprises the major proportion of haemoglobin found at birth. Haemoglobin A is the major haemoglobin found in adults and children. Haemoglobins A 2 and F are found in small quantities in adult life (approximately 2–3.3% and 0.2–1.0%, respectively). The adult proportions of haemoglobins A, A 2 and F are usually attained by 6–12 months of age.

The individual chains synthesised in postnatal life are designated α, β, γ and δ. Haemoglobin A has two α chains and two β chains (α 2 β 2 ); haemoglobin F has two α chains and two γ chains (α 2 γ 2 ) and haemoglobin A 2 has two α chains and two δ chains (α 2 δ 2 ). The α chain is thus common to all three types of haemoglobin molecule.

α chain synthesis is directed by two α genes, α2 and α1, on chromosome 16 and β and δ chain synthesis by single β and δ genes on chromosome 11; γ chain synthesis is directed by two genes, G γ and A γ, also on chromosome 11. The globin genes are shown diagrammatically in Figure 14-1 .

Figure 14-1, The α-globin gene cluster on chromosome 16 and that of the β-globin gene cluster on chromosome 11.

The four chains are associated in the form of a tetramer: the α 1 β 1 (and equivalent α 2 β 2 ) contact is the strongest and involves many amino acids with many interlocking side chains; the α 1 β 2 (and equivalent α 2 β 1 ) contact is less extensive, and the contacts between similar chains are relatively weak. The binding of a haem group within the haem pocket in each chain is vital for the oxygen-carrying capacity of the molecule and stabilises the whole molecule. If the haem attachment is weakened, the globin chains dissociate into dimers and monomers.

There are many naturally occurring, genetically determined variants of human haemoglobin (> 1000) and although many are harmless, some have serious clinical effects. Collectively, the clinical syndromes resulting from disorders of haemoglobin synthesis are referred to as ‘haemoglobinopathies’. They can be grouped into three main categories:

  • 1.

    Those resulting from a genetically determined structural variant of haemoglobin, such as haemoglobin S.

  • 2.

    Those owing to failure to synthesise one or more of the globin chains of haemoglobin at a normal rate, as in the thalassaemias.

  • 3.

    Those owing to failure to complete the normal neonatal switch from fetal haemoglobin (haemoglobin F) to adult haemoglobin (haemoglobin A). This category comprises a group of disorders referred to as hereditary persistence of fetal haemoglobin (HPFH).

An individual can also have a combination of two or more of these abnormalities (e.g. a variant haemoglobin can be synthesised at a reduced rate).

Structural variants of haemoglobin

Alterations in the structure of haemoglobin are usually brought about by point mutations affecting one or, in some cases, two codons encoding amino acids of the globin chains. An example of such a point mutation is haemoglobin S caused by the substitution of valine for glutamic acid in position 6 of the β-globin chain (β6 Glu → Val ). Less commonly, structural change is caused by shortening or lengthening of the globin chain. For example, five amino acids are deleted in the β chain of haemoglobin Gun Hill, whereas in haemoglobin Constant Spring, 31 amino acids are added to the α chain. Mutations associated with a frame shift can also lead to synthesis of a structurally abnormal haemoglobin, which may be either shorter or longer than normal. There may also be combinations of segments of β and either γ or δ chains resulting in hybrid haemoglobins; the β and δ combinations are known as the Lepore (5′δ3′β) and anti-Lepore (5′β3′δ) haemoglobins.

Many variant haemoglobins are haematologically and clinically silent because the underlying mutation causes no alteration in the function, solubility or stability of the molecule. Many of these variants are separated from their normal counterpart using electrophoresis or chromatography, but some are not and remain undetected. Some structural variants are associated with severe clinical phenotypes in the homozygous or even heterozygous state; these mutations affect the physical or chemical properties of the haemoglobin molecule, resulting in changes in haemoglobin solubility, stability or oxygen-binding properties. Some of these variants separate on electrophoresis or chromatography, whereas others do not. It is fortunate that the common haemoglobin variants that have clinical or genetic significance (e.g. haemoglobins S, C, D Punjab , E and O Arab ) are readily detectable by electrophoretic and chromatographic techniques.

Haemoglobins with reduced solubility

Haemoglobin S

By far the most common variant haemoglobin is sickle haemoglobin or haemoglobin S. Haemoglobin S has poor solubility in the deoxygenated state and can polymerise. The red cell shows a characteristic shape change because of polymer formation and becomes distorted and rigid, the so-called sickle cell (see p. 76, Fig. 5-53 ). In addition, intracellular polymers lead to red cell membrane changes, generation of oxidant substances and abnormal adherence of red cells to vascular endothelium and neutrophils.

Clinical syndromes associated with common structural variants and those owing to their interaction with β thalassaemia are shown in Table 14-1 .

Table 14-1
Clinical syndromes encountered with β S and β C
Haemoglobin Genotype Name Clinical Problems
S β A /β S Sickle cell trait Usually none
β S /β S Sickle cell anaemia Moderate anaemia, significant haemolysis, vaso-occlusive episodes and their consequences
C β A /β C C trait None
β C /β C C disease Mild haemolytic anaemia, increased incidence of gallstones
D Punjab β A /β D Punjab D trait None
β D /β D Punjab D disease Occasional mild anaemia
O Arab β A /β O Arab O trait None
β O Arab /β O Arab O disease Haemolytic anaemia
Interactions β S /β C Sickle cell/haemoglobin C disease Mild anaemia, haemolysis, vaso-occlusive episodes
β S /β D Punjab Sickle cell/haemoglobin D disease As for sickle cell anaemia
β S /β O Arab Sickle cell/haemoglobin O disease As for sickle cell anaemia
β S /β 0 thalassaemia Sickle/β 0 thalassaemia As for sickle cell anaemia
β S /β + thalassaemia Sickle/β + thalassaemia Milder than sickle cell anaemia, severity being determined by the percentage of haemoglobins A and F
β C /β thalassaemia C/β thalassaemia Mild haemolytic anaemia
β D Punjab /β thalassaemia D/β thalassaemia Mild haemolytic anaemia
β O Arab /β thalassaemia O/β thalassaemia Thalassaemia intermedia

Sickle cell disease

Sickle cell disease is a collective name for a group of conditions causing clinical symptoms that result from the formation of sickled red cells. It is common in people originating from Africa, but it is also found in considerable numbers of people of Indian, Arabic and Greek descent.

The homozygous state or sickle cell anaemia (β genotype SS) causes moderate anaemia resulting largely from the reduced oxygen affinity of haemoglobin S, although there is also significant haemolysis. The main clinical disability arises from repeated episodes of vascular occlusion by sickled red cells resulting in acute crises and eventually in end organ damage. The clinical severity of sickle cell anaemia is extremely variable. This is partly due to the effects of inherited modifying factors, such as interaction with α thalassaemia or increased synthesis of haemoglobin F, and partly to socioeconomic conditions and other factors that influence general health.

Sickle cell trait (β genotype AS), the heterozygous state, is very common, affecting millions of people worldwide. There are no associated haematological abnormalities other than occasional target cells. In vivo sickling occurs only at very high altitudes and when oxygen pressure is low for other reasons. Spontaneous haematuria, owing to sickling in the renal papillae, is found in about 1% of people with sickle cell trait.

Sickle cell/haemoglobin C disease is a compound heterozygous state for haemoglobins S and C. The abbreviation ‘SC disease’ is ambiguous and should be avoided; however, the term haemoglobin SC disease is acceptable. This compound heterozygous state usually results in a milder form of sickle cell disease.

Sickle/β thalassaemia arises as a result of inheritance of one β S and one β thalassaemia gene. Africans and Afro-Caribbeans with this condition are often heterozygous for a mild β + thalassaemia allele resulting in the production of about 20% of haemoglobin A. This gives rise to a mild sickling disorder. The less common coinheritance of β S and β 0 thalassaemia is associated with severe sickle cell disease. Interaction of haemoglobin S with haemoglobin D Punjab (haemoglobin D Los Angeles ) or with Hb O Arab gives rise to severe sickle cell disease.

Haemoglobin C

Haemoglobin C is the second most common structural haemoglobin variant in people of African descent. The replacement of glutamic acid in position 6 of the β chain by lysine results in a haemoglobin molecule with a highly positive charge, decreased solubility and a tendency to crystallise. Haemoglobin C does not give a positive sickle solubility test. Heterozygotes are asymptomatic, but target cells and irregularly contracted cells may be present in blood films. Homozygotes may have mild haemolytic anaemia with numerous target cells and irregularly contracted cells (see Chapter 5, Fig. 5-49 ). Coinheritance of β C and β 0 or β + thalassaemia results in mild or moderate haemolytic anaemia.

Other sickling haemoglobins

In addition to haemoglobin S, there are at least thirteen haemoglobins (haemoglobins C Ziquinchor , C Harlem , C Ndjamena , S Antilles , S Cameroon , S Clichy , S Jamaica Plain , S Oman , S Providence , S San Martin , S South End , S Travis , S Wake ) that have both the β6 glutamic acid to valine substitution and an additional substitution, resulting from a second point mutation, in the same β globin chain. These haemoglobins also have a positive solubility test since they are prone to polymerise but generally exhibit different electrophoretic and chromatographic properties from haemoglobin S. They have clinical significance similar – but not necessarily identical – to that of haemoglobin S (e.g. haemoglobin S Antilles and haemoglobin S Oman are associated with an even greater propensity to sickling than haemoglobin S).

Unstable haemoglobins

Amino acid substitutions close to the haem group, or at the points of contact between globin chains, can affect protein stability and result in intracellular precipitation of globin chains. The precipitated globin chains attach to the red cell membrane giving rise to Heinz bodies, and the associated clinical syndromes were originally called the congenital Heinz body haemolytic anaemias. Changes in membrane properties may lead to haemolysis, often aggravated by oxidant drugs. There is considerable heterogeneity in the haematological and clinical effects of unstable haemoglobins. Many are almost silent and are detected only by specific tests, whereas others are more unstable, causing haemolytic anaemia in the heterozygous state. Haemoglobin Köln is the most common variant in this rare group of disorders. ,

Haemoglobins with altered oxygen affinity

Haemoglobin variants with altered oxygen affinity are a rare group of variants that result in increased or reduced oxygen affinity. Mutations that increase oxygen affinity are generally associated with benign lifelong erythrocytosis. This may be confused with polycythaemia vera and in the past has been inappropriately treated with cytotoxic drugs and 32 P.

Haemoglobin variants with decreased oxygen affinity are, with the exception of haemoglobin S, even less common and are usually associated with mild anaemia and cyanosis. However, owing to the reduced oxygen affinity, these patients are not functionally anaemic despite the reduced haemoglobin concentration (Hb). The low steady-state Hb in haemoglobin S homozygotes is, to a considerable extent, a result of its reduced oxygen affinity.

Measurement of oxygen dissociation is described on p. 251.

Haemoglobin M

The haemoglobin M group is another rare group of variants. Such haemoglobins have a propensity to form methaemoglobin, generated by the oxidation of ferrous iron (Fe ++ ) in haem to ferric (Fe +++ ) iron, which is incapable of binding oxygen. Despite marked cyanosis, there are few clinical problems. Most are associated with substitutions that disrupt the normal six-ligand state of haem iron.

Methaemoglobinaemia is also found in congenital nicotinamide adenine dinucleotide (NAD) H methaemoglobin reductase deficiency, as well as after exposure to oxidant drugs and chemicals (nitrates, nitrites, quinones, chlorates, phenacetin, dapsone and many others).

Thalassaemia syndromes

The thalassaemia syndromes are a heterogeneous group of inherited conditions characterised by defects in the synthesis of one or more of the globin chains that form the haemoglobin tetramer. The clinical syndromes associated with thalassaemia arise from the combined consequences of inadequate haemoglobin production and of unbalanced accumulation of one type of globin chain. The former causes anaemia with hypochromia and microcytosis; the latter leads to ineffective erythropoiesis and haemolysis. Clinicopathological manifestations range from completely asymptomatic microcytosis to profound anaemia that is incompatible with life and can cause death in utero ( Table 14-2 ). This clinical heterogeneity arises as a result of the variable severity of the primary genetic defect in haemoglobin synthesis and the coinheritance of moderating factors, such as the capacity to synthesise increased amounts of haemoglobin F.

Table 14-2
Clinical syndromes of thalassaemia
Clinically asymptomatic
Silent carriers
α + thalassaemia heterozygotes (some cases)
Rare forms of β thalassaemia heterozygosity
Thalassaemia minor (low MCH and MCV, with or without mild anaemia)
α + thalassaemia heterozygotes (some cases)
α 0 thalassaemia heterozygotes
α + thalassaemia homozygotes
β 0 thalassaemia trait
β + thalassaemia trait
Some cases of haemoglobin E/β thalassaemia
Thalassaemia intermedia (transfusion independent) *
Some β + /β + thalassaemia homozygotes and compound heterozygotes
Interaction of β 0 /β 0 , β 0 /β + or β + /β + with α thalassaemia
Interaction of β 0 /β or β + /β with triple α
Haemoglobin H disease
α 0 /haemoglobin Constant Spring thalassaemia
β 0 /δβ or β + /δβ thalassaemia compound heterozygotes
δβ/δβ thalassaemia
Some cases of haemoglobin E/β thalassaemia and haemoglobin Lepore/β thalassaemia
Rare cases of heterozygotes for β thalassaemia mutation, particularly involving exon 3 (‘dominant β thalassaemia’)
Thalassaemia major (transfusion dependent)
β 0 /β 0 thalassaemia
β + /β + thalassaemia
β 0 /β + thalassaemia
Some cases of β 0 /haemoglobin Lepore and β + /haemoglobin Lepore thalassaemia
Some cases of β 0 /haemoglobin E and β + /haemoglobin E thalassaemia
MCH, mean cell haemoglobin; MCV, mean cell volume.

* β thalassaemia intermedia is defined as a symptomatic condition in which regular transfusion is not essential to maintain life; however some patients have a poor quality of life and may benefit from transfusion as their condition progresses (e.g. when there is increasing splenomegaly).

Thalassaemias are generally inherited as alleles of one or more of the globin genes located on either chromosome 11 (for β, γ and δ chains) or on chromosome 16 (for α chains). They are encountered in every population in the world but are most common in the Mediterranean littoral and near equatorial regions of Africa and Asia. Gene frequencies for the α and β thalassaemias on a global basis range from 1% to more than 80% in areas where malaria is endemic.

β thalassaemia syndromes

Many different mutations cause β thalassaemia and related disorders. These mutations can affect every step in the pathway of globin gene expression: transcription, processing of the messenger ribonucleic acid (mRNA) precursor, translation of mature mRNA and preservation of post-translational integrity of the β chain. More than 400 mutations have been described. Most types of β thalassaemia are the result of point mutations affecting the globin gene, but some large deletions are also known. Certain mutations are particularly common in some communities. This helps to simplify prenatal diagnosis, which is carried out by detection or exclusion of a particular mutation in fetal deoxyribonucleic acid (DNA).

The effect of different mutations varies greatly. At one end of the spectrum are a group of rare mutations, mainly involving exon 3 of the β globin gene, which are so severe that they can produce the clinical syndrome of thalassaemia intermedia in the heterozygous state. At the other end are mild alleles that produce thalassaemia intermedia in the homozygous or compound heterozygous state and some that are so mild that they are haematologically completely silent, with normal mean cell volume (MCV), mean cell haemoglobin (MCH) and haemoglobin A 2 in the heterozygous state. In between are the great majority of β + and β 0 alleles, which cause β thalassaemia major in the homozygous or compound heterozygous state and in the heterozygous state give rise to a mild anaemia or Hb at the low end of the normal range, with microcytosis and a raised haemoglobin A 2 .

β thalassaemia major is a severe, transfusion-dependent, inherited anaemia. There is a profound defect of β chain production. Excess α chains accumulate and precipitate in the red cell precursors in the bone marrow resulting in ineffective erythropoiesis. The few cells that leave the marrow are laden with precipitated α chains and are rapidly removed by the reticuloendothelial system. The constant erythropoietic drive causes massive expansion of bone marrow and extramedullary erythropoiesis. If untreated, 80% of children with β thalassaemia major die within the first 5 years of life.

Heterozygotes for β thalassaemia alleles usually have either a normal Hb with microcytosis or a mild hypochromic microcytic anaemia; haemoglobin A 2 is elevated and haemoglobin F is sometimes also elevated. Laboratory features of various β thalassaemia syndromes are shown in Table 14-3 .

Table 14-3
Laboratory findings in thalassaemia
Phenotype Genotype Usual MCV Usual MCH Haemoglobin A 2 Haemoglobin H Inclusions
α thalassaemia
α + thalassaemia heterozygosity − α/αα N N N or ↓ −
α + thalassaemia homozygosity − α/− α N or ↓ N or ↓ N or ↓ ±
α 0 thalassaemia heterozygosity − −/αα ↓ ↓ N or ↓ +
Haemoglobin H disease
Mild or moderate − −/− α ↓ ↓ N or ↓ +++
Severe − −/α T α ↓ ↓ N or ↓ +++
Haemoglobin Bart’s hydrops fetalis (α thalassaemia major) α 0 homozygosity − −/− − ↓ ↓ − −
β thalassaemia
β thalassaemia trait β 0 /β or β + /β ↓ ↓ ↑ −
β thalassaemia trait with normal Hb A 2 β + /β ↓ ↓ N −
δβ thalassaemia trait δβ 0 /β ↓ ↓ N or ↓ −
Hb Lepore trait δβ Lepore /β ↓ ↓ N or ↓ −
β thalassaemia intermedia Heterogeneous ↓ ↓ ↑ or N −
β thalassaemia major β 0 /β 0 , β 0 /β + , β + /β + ↓ ↓ ↑ or N
MCH, mean cell haemoglobin; MCV, mean cell volume; N, normal.

α thalassaemia syndromes

There are four α thalassaemia syndromes: α + thalassaemia trait, where one of the two globin genes on a single chromosome is absent or fails to function; α 0 thalassaemia trait, where a chromosome has no functional α genes; haemoglobin H disease, usually with three genes absent or defective; and haemoglobin Bart’s hydrops fetalis, where all four genes are absent or severely defective. These syndromes are usually a result of deletions of one or more genes, although approximately 20% of the mutations described are nondeletional; nondeletional α thalassaemias include some where a variant haemoglobin is synthesised at a greatly reduced rate (e.g. haemoglobin Constant Spring). α + thalassaemia is particularly common in Africa and Southeast Asia, whereas α 0 thalassaemia is common in Southeast Asia and also occurs in certain Mediterranean countries. The laboratory features are shown in Table 14-3 .

Haemoglobin Bart’s hydrops fetalis occurs mainly in people from Southeast Asia but is also occasionally observed in people from Greece, Turkey and Cyprus. An affected fetus will be stillborn or will die shortly after birth. Severe anaemia and oedema are the hallmarks of this condition. Women carrying a hydropic fetus have a high incidence of complications of pregnancy. Prenatal diagnosis should be offered for women at risk of having a fetus with haemoglobin Bart’s hydrops fetalis.

Haemoglobin H disease gives rise to haemolytic anaemia; rarely patients require transfusion or splenectomy.

α 0 thalassaemia trait is characterised by microcytosis. The Hb may be normal or slightly reduced. α + thalassaemia trait can be completely silent, or there may be borderline microcytosis with a slightly reduced or normal MCH. Haematologically, homozygosity for α + thalassaemia trait resembles heterozygosity for α 0 thalassaemia trait, but the genetic implications are very different. Both α + thalassaemia trait and α 0 thalassaemia trait are more difficult to diagnose than β thalassaemia trait because there is no characteristic elevation in haemoglobin A 2 , and haemoglobin H inclusions may not be demonstrated. Definitive diagnosis of α thalassaemia trait is more reliably made with the use of DNA techniques or globin chain biosynthesis studies.

Thalassaemic structural variants

These are abnormal haemoglobins, such as the Lepore haemoglobins, characterised by both a biosynthetic defect and an abnormal structure ( Table 14-4 ).

Table 14-4
Thalassaemic structural variants *
Haemoglobin Structure When Heterozygous When Homozygous In Combination with Other Haemoglobinopathies ,
Lepore Hybrid of part of δ and part of β chain owing to unequal crossover Microcytosis, sometimes mild anaemia Thalassaemia major or intermedia With β thalassaemia gives thalassaemia major or intermedia
E † β 26Glu → Lys resulting in a structural variant and a false splice site Microcytosis, sometimes mild anaemia Microcytosis, mild anaemia With β thalassaemia gives thalassaemia major or intermedia
Constant Spring or Paksé Elongated α chain owing to incorporation of 31 extra amino acids Microcytosis, mild anaemia Microcytosis, mild anaemia or haemoglobin H disease With α 0 thalassaemia gives haemoglobin H disease

* Many other thalassaemic structural variants have been described but are much rarer than the three shown in this table.

† 13–30% frequency in Cambodia, Thailand, Vietnam and some parts of China.

Increased haemoglobin F in adult life

Haemoglobin production in man is characterised by two major switches in the haemoglobin composition of the red cells. During the first 3 months of gestation, human red cells contain embryonic haemoglobins (see p. 283), whereas during the last 6 months of gestation, red cells contain predominantly fetal haemoglobin. The major transition from fetal to adult haemoglobin synthesis occurs in the perinatal period, and by the end of the first year of life, red cells have a haemoglobin composition that usually remains constant throughout adult life. The major haemoglobin is then haemoglobin A, but there are small amounts of haemoglobins A 2 and F. Only 0.2–1.0% of total haemoglobin in adult human red cells is haemoglobin F and it is restricted to a few cells called ‘F cells’. Both the number of F cells and the amount of haemoglobin F per cell can be increased in various conditions, particularly if there is rapid bone marrow regeneration.

The general organisation of human globin gene clusters is shown in Figure 14-1 . The products of two γ genes differ in only one amino acid: G γ has glycine in position 136, whereas A γ has alanine. In fetal red cells, the ratio of G γ to A γ is approximately 3:1; in adult red cells, it is approximately 2:3.

In recent years there has been much interest in the attempts to manipulate the fetal switch pharmacologically. If it were possible to reactivate haemoglobin F synthesis reliably beyond the perinatal period, both β thalassaemia major and sickle cell disease would be ameliorated.

Inherited abnormalities that increase haemoglobin F concentration

More than 50 mutations that increase haemoglobin F synthesis have been described. , They result in one of two phenotypes, hereditary persistence of HPFH or δβ thalassaemia; differentiation between these two phenotypes is not always simple but has clinical relevance. In general, HPFH has a higher percentage of haemoglobin F and much more balanced chain synthesis. The most common, the African type of HPFH, is associated with a high concentration of haemoglobin F (15–45%), pancellular distribution on Kleihauer staining and normal red cell indices. Mutations causing increased synthesis of haemoglobin F are mostly deletions, but some nondeletional mutations have also been described. In contrast, subjects with δβ thalassaemia have lower levels of haemoglobin F accompanied by microcytosis. The major clinical significance of these abnormalities is their interaction with β thalassaemia and haemoglobin S. Compound heterozygotes for either of these conditions and HPFH have a much milder clinical syndrome than homozygotes for haemoglobin S or β thalassaemia. Compound heterozygotes for either of these conditions and δβ thalassaemia have a condition much closer in severity to the homozygous states for HPFH or δβ thalassaemia.

Increased haemoglobin F is also found in many other haematological conditions, including inherited red cell aplasia and inherited aplastic anaemia (Blackfan–Diamond syndrome and Fanconi anaemia, respectively), juvenile myelomonocytic leukaemia and some myelodysplastic syndromes. A small but significant increase in haemoglobin F may occur in the presence of erythropoietic stress (haemolysis, bleeding, recovery from acute bone marrow failure) and in pregnancy.

Investigation of patients with a suspected haemoglobinopathy

Investigation of a person at risk of a haemoglobinopathy encompasses the confirmation or exclusion of the presence of a structural variant, thalassaemia or both. If a structural haemoglobin variant is present, it is necessary to ascertain the clinical significance of the particular variant so that the patient is appropriately managed. If it is confirmed that thalassaemia trait is present, it is not usually necessary to determine the precise mutation because the clinical significance is usually negligible. The exception to this is an antenatal patient whose partner has confirmed or suspected thalassaemia trait. If prenatal diagnosis is being considered, mutation analysis is necessary to predict fetal risk accurately and to facilitate prenatal diagnosis (see p. 134).

Because the inheritance of a haemoglobinopathy per se has genetic implications, it is important that genetic counselling is available for these patients.

In the majority of patients, the presence of a haemoglobinopathy can be diagnosed with sufficient accuracy for clinical purposes from knowledge of the patient’s ethnic origin and clinical history (including family history) and the results of physical examination combined with relatively simple haematological tests. Initial investigations should include determination of Hb and red cell indices. A detailed examination of a well-stained blood film should be carried out. In some instances, a reticulocyte count and a search for red cell inclusions gives valuable information. Assessment of iron status by estimation of serum ferritin is sometimes necessary to exclude iron deficiency. Other important basic tests are high performance liquid chromatography (HPLC), capillary electrophoresis (CE) or cellulose acetate electrophoresis (CAE), a sickle solubility test and measurement of haemoglobin A 2 and F percentages. In the case of common haemoglobin variants and classic β thalassaemia trait, accurate data from these tests will permit a reliable diagnosis without the need for more sophisticated investigations. However, definitive diagnosis of some thalassaemia syndromes can only be obtained using DNA technology (see p. 134 and p. 135). Similarly, in particular situations, haemoglobin variants will require unequivocal identification by the use of DNA technology or protein analysis by mass spectrometry. Individuals or families who require such investigation must be carefully selected on the basis of family history and on the results of the basic investigations described later in this chapter. Large-scale screening programmes are increasingly being undertaken in some countries where individual case histories and the results of other laboratory tests are not usually available. The problems of such programmes are discussed on p. 299.

The majority of errors occurring in the detection and identification of a haemoglobinopathy are the result of either failure to obtain correct laboratory data or failure to interpret data correctly. In this chapter, a sequence of investigations is proposed based on procedures that should be available in the laboratory of any major hospital. Automated HPLC has largely replaced haemoglobin electrophoresis as the initial investigative procedure in laboratories analysing large numbers of samples. Capillary electrophoresis is currently less used than HPLC. Isoelectric focusing (IEF) is, in general, used only to a limited extent, mainly for neonatal screening or in specialist laboratories, and it is only briefly described here.

Laboratory investigation of a suspected haemoglobinopathy should follow a defined protocol, which should be devised to suit individual local requirements. The data obtained from the clinical findings, blood picture and electrophoresis or HPLC will usually indicate in which direction to proceed. The investigation for a structural variant is described in the first section and that for a suspected thalassaemia syndrome in the second section of this chapter. Screening tests for thalassemia trait and haemoglobin E trait that may be especially applicable in under-resourced areas are described in Chapter 26 .

Laboratory detection of haemoglobin variants

A proposed scheme of investigation is shown in Figure 14-2 and a list of procedures follows: ,

  • 1.

    Blood count and film examination (see below)

  • 2.

    Collection of blood and preparation of haemolysates (see p. 291)

  • 3.

    Cellulose acetate electrophoresis, Tris buffer, pH 8.5 (see p. 292)

  • 4.

    Acid agarose gel electrophoresis, pH 6.0 (see p. 294)

  • 5.

    Automated high performance liquid chromatography (see p. 294)

  • 6.

    Capillary electrophoresis (see p. 296)

  • 7.

    Isoelectric focusing (see p. 297)

  • 8.

    Tests for haemoglobin S (see p. 297)

  • 9.

    Detection of unstable haemoglobins (see p. 299)

  • 10.

    Detection of haemoglobin Ms (see p. 300)

  • 11.

    Detection of altered affinity haemoglobins (see p. 301)

  • 12.

    Differentiation of common structural variants (see p. 301)

  • 13.

    Neonatal screening (see p. 299)

  • 14.

    Tests, such as zinc protoporphyrin estimation, to exclude iron deficiency as a cause of microcytosis (see Chapter 9 )

  • 15.

    Molecular techniques (see Chapter 8 )

  • 16.

    Procedures for use in under-resourced laboratories (see Chapter 26 ).

Figure 14-2, Suggested scheme of investigation for structural variants.

Globin chain electrophoresis at pH 8.0 and 6.3 and citrate agar electrophoresis at acid pH are now rarely performed. Methods can be found in previous editions of this book.

Blood count and film

The blood count, including Hb and red cell indices, provides valuable information useful in the diagnosis of both α and β thalassaemia interactions with structural variants (see Chapter 3 ). A film examination may reveal characteristic red cell changes such as target cells in haemoglobin C trait, sickle cells in sickle cell disease and irregularly contracted cells in the presence of haemoglobin C or an unstable haemoglobin (see Chapter 5 ).

Discriminant functions using various formulae have been proposed as a basis for further testing for thalassaemia, , but we do not advise their use. Although such functions and formulae do indicate whether thalassaemia or iron deficiency is more likely, they may lead to individuals who have both iron deficiency and thalassaemia trait not being tested promptly. Generally this is not a problem and indeed it may be preferable to keep the patient under observation until iron deficiency has been treated and then to reassess the likelihood of thalassaemia trait. However, many of the patients who require testing for thalassaemia are women who are already pregnant. In such patients the likely delay in testing is unacceptable. Moreover, these formulae have generally not been validated for use during pregnancy. For these reasons, we advise that whenever genetic counselling might be required, testing for β thalassaemia trait should be carried out in all individuals with an MCH < 27 pg and screening for α 0 thalassaemia trait should be carried out in those individuals with an MCH < 25 pg who belong to an ethnic group in which α 0 thalassaemia is prevalent.

Collection of blood and preparation of haemolysates

Ethylenediaminetetra-acetic acid (EDTA) is the most convenient anticoagulant because it is used for the initial full blood count and film (see Chapter 1 ), although samples taken into any anticoagulant are satisfactory. Cells freed from clotted blood can also be used.

Preparation of haemolysate for qualitative haemoglobin electrophoresis

See individual methods.

Preparation of haemolysate for the quantification of haemoglobins and stability tests

Preparation of haemolysate is necessary for quantification of haemoglobin A 2 or a variant haemoglobin by elution and for the quantification of haemoglobin F by alkali denaturation. It is also essential for reliable heat stability tests.

Lyse 2 volumes of washed packed cells in 1 volume of distilled water. Shake the tubes vigorously for approximately 1 min, then centrifuge at 1200 g (3000 rev/min on a standard bench-top centrifuge) for 30 min at 4 °C. Transfer the supernatant to a clean sample container and adjust the Hb to 100 ± 10 g/l with water. The quality of the haemolysate is much improved by the addition of carbon tetrachloride (1 volume) prior to shaking the haemolysate mixture. However, the use of organic solvents is not recommended in many countries and therefore should be omitted. Be aware that if an unstable haemoglobin is suspected, organic solvents should be avoided in haemolysate preparation.

Note : Whole blood samples are best stored as washed, packed cells frozen as droplets in liquid nitrogen and subsequently stored at − 20 °C, − 70 °C or over liquid nitrogen. Alternatively, haemolysates may also be frozen at − 20 °C, − 70 °C or over liquid nitrogen.

Control samples

Interpretation of migration patterns of test samples is undertaken by comparison with the migration and separation of known variant haemoglobins used as control materials. Ideally, a mixture of haemoglobins A, F, S and C should be included on each electrophoretic separation. This material can be prepared as follows:

  • 1.

    The control can be made from either the combination of a sickle cell trait sample (A + S) combined with a haemoglobin C trait sample (A + C) and normal cord blood (F + A) or the combination of normal cord blood with a sample from a person with sickle cell/haemoglobin C compound heterozygosity (S + C).

  • 2.

    Prepare lysates by the method given for a purified haemolysate.

  • 3.

    Mix equal volumes of the lysates together and add a few drops of 0.3 mol/l KCN (20 g/l).

  • 4.

    Analyse samples by electrophoresis to assess quality.

  • 5.

    Aliquot and store frozen.

Note : Repeated freezing and thawing should be avoided.

Lyophilised controls are stable for considerably longer than liquid and can be purchased from commercial sources.

Quality assurance

Because the haemoglobinopathies are inherited conditions, some of which carry considerable clinical and genetic implications, precise documentation and record-keeping are of paramount importance. The use of cumulative records when reviewing a patient’s data is very useful, because it of itself constitutes an aspect of quality assurance. In some situations, repeat sampling, family studies or both may be required to elucidate the nature of the abnormality in an individual.

In-house standard operating procedures should be followed carefully, particularly in this field of haematology, where a small difference in technique can make a significant difference in the results obtained and can lead to misdiagnosis. Many of the techniques described have attention drawn to specific technical details that are important for ensuring valid results.

It is necessary to use reference standards and control materials in each of the analyses undertaken and in some cases to use duplicate analysis to demonstrate precision. There are international standards for haemoglobins F and A 2 (see p. 536), whereas in some countries national reference preparations are also available from national standards institutions. These are extremely valuable because the target values have been established by collaborative studies. Control materials can be prepared in-house or obtained commercially. Samples stored as whole blood at 4 °C can be used reliably for several weeks. All laboratories should confirm the normal range for their particular methods and the normal range obtained should not differ significantly from published data.

All laboratories undertaking haemoglobin analysis should participate in an appropriate proficiency testing programme (see p. 539). In the UK, the National External Quality Assessment Service (NEQAS) provides samples for sickle solubility tests, for detection and quantification of variant haemoglobins and for quantification of haemoglobins A 2 , F and S.

National and international guidelines have been published for all aspects of the investigations given here. ,

Cellulose acetate electrophoresis at alkaline pH

Haemoglobin electrophoresis at pH 8.4–8.6 using cellulose acetate membrane is simple, reliable and rapid. It is satisfactory for the detection of most common, clinically important haemoglobin variants.

Principle

At alkaline pH, haemoglobin is a negatively charged protein, and when subjected to electrophoresis will migrate toward the anode (+). Structural variants that have a change in the charge on the surface of the molecule at alkaline pH will separate from haemoglobin A. Haemoglobin variants that have an amino acid substitution that is internally sited may not separate, and those that have an amino acid substitution that has no effect on overall charge will not separate by electrophoresis.

Equipment

  • Electrophoresis tank and power pack. Any horizontal electrophoresis tank that will allow a bridge gap of 7 cm. A direct current power supply capable of delivering 350 V at 50 mA is suitable for both cellulose acetate and acid agarose gels.

  • Wicks of filter or chromatography paper

  • Blotting paper

  • Applicators . These are available from most manufacturers of electrophoresis equipment, but fine microcapillaries are also satisfactory.

  • Cellulose acetate membranes . Plastic-backed membranes (7.6 × 6.0 cm) are recommended for ease of use and storage.

  • Staining equipment

Reagents

  • Electrophoresis buffer. Tris/EDTA/borate (TEB), pH 8.5. Tris-(hydroxymethyl)aminomethane (Tris), 10.2 g; EDTA (disodium salt), 0.6 g; boric acid, 3.2 g; water to 1 litre. The buffer should be stored at 4 °C and can be used up to 10 times without deterioration.

  • Wetting agent. For example, Zip Zone Prep solution (Helena Laboratories, www.helena.com ): 1 drop of Zip Zone Prep in 100 ml water

  • Fixative/stain solution. Ponceau S ( www.helena.com ), 5 g; trichloroacetic acid, 7.5 g; water to 1 litre

  • Destaining solution. 3% (v/v) acetic acid, 30 ml; water to 1 litre

  • Haemolysing reagent. 0.5% (v/v) Triton X-100 ( www.helena.com ) in 100 mg/l potassium cyanide.

Method

  • 1.

    Centrifuge samples at 1200 g for 5 min. Dilute 20 μl of the packed red cells with 150 μl of the haemolysing reagent. Mix gently and leave for at least 5 min. If purified haemolysates are used, dilute 40 μl of 100 g/l haemolysate with 150 μl of lysing reagent.

  • 2.

    With the power supply disconnected, prepare the electrophoresis tank by placing equal amounts of TEB buffer in each of the outer buffer compartments. Wet two chamber wicks in the buffer, and place one along each divider/bridge support ensuring that they make good contact with the buffer.

  • 3.

    Soak the cellulose acetate by lowering it slowly into a reservoir of buffer. Leave the cellulose acetate to soak for at least 5 min before use.

  • 4.

    Fill the sample well plate with 5 μl of each diluted sample or control and cover with a 50 mm coverslip or a ‘short’ glass slide to prevent evaporation. Load a second sample well plate with Zip Zone Prep solution.

  • 5.

    Clean the applicator tips immediately prior to use by loading with Zip Zone Prep solution and then applying them to a blotter.

  • 6.

    Remove the cellulose acetate strip from the buffer and blot twice between two layers of clean blotting paper. Do not allow the cellulose acetate to dry.

  • 7.

    Load the applicator by depressing the tips into the sample wells twice and apply this first loading onto some clean blotting paper. Reload the applicator and apply the samples to the cellulose acetate.

  • 8.

    Place the cellulose acetate plates across the bridges, with the plastic side uppermost. Place two glass slides across the strip to maintain good contact. Electrophorese at 350 V for 25 min.

  • 9.

    After 25 min electrophoresis, immediately transfer the cellulose acetate to Ponceau S and fix and stain for 5 min.

  • 10.

    Remove excess stain by washing for 5 min in the first acetic acid reservoir and for 10 min in each of the remaining two. Blot once, using clean blotting paper and leave to dry.

  • 11.

    Label the membranes and store in a protective plastic envelope.

Interpretation and comments

Figure 14-3 shows the relative electrophoretic mobilities of some common haemoglobin variants at pH 8.5 on cellulose acetate. Satisfactory separation of haemoglobins C, S, F, A and J is obtained ( Fig. 14-4 ). In general, haemoglobins S, D and G migrate closely together, as do haemoglobins C, E and O Arab . Differentiation between these haemoglobins can be obtained by using acid agarose gels, HPLC or IEF. There are slight differences in mobility between haemoglobins S, Lepore and D Punjab and also between haemoglobins C and E. Generally, the Lepore haemoglobins and Hb D Punjab migrate slightly anodal to haemoglobin S (i.e. they are slightly faster than S); haemoglobin C migrates slightly cathodal to haemoglobin E (i.e. it is slightly slower than E). These differences are, however, too subtle to avoid the necessity for a second confirmatory technique.

Figure 14-3, Schematic representation of relative mobilities of some abnormal haemoglobins.

Figure 14-4, Relative mobilities of some abnormal haemoglobins.

All samples showing a single band in either the S or C position should be analysed further using acid agarose gel electrophoresis, HPLC or IEF to exclude the possibility of a compound heterozygote state such as SD, SG, CE or CO Arab .

The quality of separation resulting from this procedure is affected primarily by both the amount of haemoglobin applied and the positioning of the origin. Also, delays between application of the sample and commencement of the electrophoresis, delay in staining after electrophoresis or inadequate blotting of the acetate prior to application will cause poor results. This technique is sensitive enough to separate haemoglobin F from haemoglobin A and to detect haemoglobin A 2 variants as long as the strip is inspected carefully.

If an abnormal haemoglobin is present, the detection of a haemoglobin A 2 variant band in conjunction with the abnormal fraction is evidence that the variant is an α chain variant.

When an abnormal haemoglobin is found, it may be of diagnostic importance to measure the percentage of the variant; this can be done by the electrophoresis with elution procedure for haemoglobin A 2 estimation given on p. 303. Quantification of haemoglobin S is often clinically useful, both in patients with sickle cell disease who are being treated by transfusion and for the diagnosis of conditions in which haemoglobin S is co-inherited with α and β thalassaemia, as outlined in Table 14-5 . Quantification of haemoglobin S can be done with HPLC, electrophoresis with elution or microcolumn chromatography.

Table 14-5
Results of laboratory investigations in interactions of haemoglobin S and α or β thalassaemia in adults
MCV % S % A % A 2 % F
AS N 35–38 62–65 < 3.5 < 1
SS N 88–93 0 < 3.5 5–10
S/β 0 thalassaemia L 88–93 0 > 3.5 5–10
S/β + thalassaemia L 50–93 3–30 > 3.5 1–10
S/HPFH N 65–80 0 < 3.5 20–35
AS/α + thalassaemia N/L 28–35 62–70 < 3.5 < 1
AS/α 0 thalassaemia L 20–30 68–78 < 3.5 < 1
SS/α thalassaemia N/L 88–93 0 < 3.5 1–10
HPFH, hereditary persistence of fetal haemoglobin; L, low; MCV, mean cell volume; N, normal.

Agarose gel electrophoresis

Agarose gels are commercially available as for both alkaline and acid electrophoresis. They are simple to use and particularly useful in laboratories that process small numbers of samples.

Reagents and method

The manufacturer’s method should be followed.

Interpretation

With alkaline systems, in general the same separation patterns are obtained as for CAE, but where individual application notes are available these should be used for reference. Because not all kits provide these, laboratories may need to build up their own data on known variants. At acid pH, the migration pattern helps to distinguish haemoglobin S from D/G and haemoglobin C from E ( Fig. 14-5 ).

Figure 14-5, Schematic representation of relative mobilities of some abnormal haemoglobins.

Automated high performance liquid chromatography

Automated cation-exchange HPLC is being used increasingly as the initial diagnostic method in haemoglobinopathy laboratories with a high workload. Both capital and consumable costs are higher than with haemoglobin electrophoresis, but labour costs are less; overall costs may be similar. In comparison with haemoglobin electrophoresis, HPLC has four advantages:

  • 1.

    The analysers are automated and thus require less staff time and permit processing of large batches.

  • 2.

    Very small samples (5 μl) are sufficient for analysis; this is especially useful in paediatric work.

  • 3.

    Quantification of normal haemoglobins (including haemoglobin A 2 ) and variant haemoglobins is available on every sample.

  • 4.

    A provisional identification of a larger proportion of variant haemoglobins can be made.

Principle

HPLC depends on the interaction of charged groups on the ion exchange material with charged groups on the haemoglobin molecule. A typical column packing is 5 μm spherical silica gel. The surface of the support is modified by carboxyl groups to have a weakly cationic charge, which allows the separation of haemoglobin molecules with different charges by ion exchange. When a haemolysate containing a mixture of haemoglobins is adsorbed onto the resin, the rate of elution of different haemoglobins is determined by the pH and ionic strength of any buffer applied to the column. With automated systems now in use, elution of the charged molecules is achieved by a continually changing salt gradient; fractions are detected as they pass through an ultraviolet/visible light detector and are recorded on an integrating computer system. Analysis of the area under these absorption peaks gives the percentage of the fraction detected. The time of elution (retention time) of any normal or variant haemoglobin present is compared with that of known haemoglobins, providing quantification of both normal haemoglobins (A, F and A 2 ) and many variants.

Figure 14-6 shows a schematic representation of an HPLC system and Figure 14-7 shows a chromatogram of a mixture of different haemoglobins. Systems are available from various manufacturers.

Figure 14-6, Diagrammatic representation of high performance liquid chromatography (HPLC) showing the flow of sample and buffers.

Figure 14-7, A mixture of haemoglobins separated by HPLC.

Method

The manufacturer’s procedure should be followed. To prolong the life of the column it is important to follow the manufacturer’s instructions with regard to the concentration of haemoglobin in the sample to be injected.

Interpretation and comments

Results are generally accurate and reproducible, but as with every method of haemoglobin analysis, controls should be run with every batch. Controls are provided by manufacturers and should be used according to their instructions. If the system is being used for the detection of haemoglobin variants, elution times can be compared with those of known controls; actual times, however, are affected by the batch of buffer and column, the age of the column and the laboratory temperature. A better comparison may be obtained using the relative elution time, which is calculated by dividing the elution time of the variant with that of the main haemoglobin A fraction. It should be noted that haemoglobin A is separated into its component fractions of A 0 and A 1 , and the A 1 fraction frequently subdivides into several peaks. Skill is required in interpretation of the results because various normal and abnormal haemoglobins may have the same retention time, and a glycosylated variant haemoglobin will have a different retention time from the nonglycosylated form. HPLC usually separates haemoglobins A, A 2 , F, S, C, D Punjab and G Philadelphia from each other. , However, both haemoglobin E and haemoglobin Lepore co-elute with haemoglobin A 2 (as other haemoglobins co-elute with A, S and F). The retention time of glycosylated and other derivatives of haemoglobin S can be the same as those of haemoglobins A 0 and A 2 . Since derivatives of haemoglobin S co-elute with haemoglobin A 2 , percentages of A 2 by this method are inaccurate in the presence of S, and therefore do not have the same significance as percentage of haemoglobin A 2 measured by alternative methods. For these reasons, and because there are more than 1000 known variants, HPLC can never definitively identify any haemoglobin. It is important to analyse variants found using second-line techniques, such as a sickle solubility test, alkaline and acid electrophoresis, or iso-electric focusing.

HPLC is also applicable for the quantification of Hb A 1c for the monitoring of diabetes mellitus; to make optimal use of staff and equipment, this procedure is sometimes carried out in haematology laboratories. In fact, an increased glycosylated fraction is not infrequently noted when HPLC is performed for investigation of a suspected haemoglobinopathy. Unless the patient is already known to suffer from diabetes mellitus, this abnormality should be drawn to the attention of clinical staff.

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