Haemoglobinopathy Diagnosis E-Book

Barbara J. Bain

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 971

Veröffentlichungsjahr: 2020

Beschreibung

An updated, essential guide for the laboratory diagnosis of haemoglobin disorders This revised and updated third edition of Haemoglobinopathy Diagnosis offers a comprehensive review of the practical information needed for an understanding of the laboratory diagnosis of haemoglobin disorders. Written in a concise and approachable format, the book includes an overview of clinical and laboratory features of these disorders. The author focuses on the selection, performance, and interpretation of the tests that are offered by the majority of diagnostic laboratories. The book also explains when more specialist tests are required and explores what specialist referral centres will accomplish. The information on diagnosis is set in a clinical context. The third edition is written by a leading haematologist with a reputation for educational excellence. Designed as a practical resource, the book is filled with illustrative examples and helpful questions that can aide in the retention of the material presented. Additionally, the author includes information on the most recent advances in the field. This important text: * Contains a practical, highly illustrated, approach to the laboratory diagnosis of haemoglobin disorders * Includes "test-yourself" questions and provides an indispensable tool for learning and teaching * Presents new material on antenatal screening/prenatal diagnostic services * Offers myriad self-assessment case studies that are ideal for the trainee Written for trainees and residents in haematology, practicing haematologists, and laboratory scientists, Haemoglobinopathy Diagnosis is an essential reference and learning tool that provides a clear basis for understanding the diagnosis of haemoglobin disorders.

Leseprobe

Table of Contents

Cover

Preface

Abbreviations and glossary

1 Haemoglobin and the genetics of haemoglobin synthesis

Haemoglobins and their structure and function

Genetics of haemoglobin synthesis

Normal haemoglobins

Variant haemoglobins and abnormalities of globin chain synthesis

Thalassaemias and variant haemoglobins

Check your knowledge

Further reading

References

Answers to questions

2 Laboratory techniques for the identification of abnormalities of globin chain synthesis

Sample collection

The blood count, film and reticulocyte count

Preparing a red cell lysate

Haemoglobin electrophoresis

Capillary electrophoresis

High performance liquid chromatography

Isoelectric focusing

Sickle solubility test and other methods for the detection of haemoglobin S

Quantification of haemoglobin A

2

Quantification and determination of distribution of haemoglobin F

Immunoassay for variant haemoglobins

Detection of haemoglobin H inclusions

Detection of an unstable haemoglobin

Detection of Heinz bodies

Osmotic fragility as a screening test for thalassaemia

Dichlorophenolindophenol test for haemoglobin E screening

Quantification of haemoglobin A

1c

Incidental detection of a variant haemoglobin

Other more specialised tests

Quality assurance

Check your knowledge

Further reading

References

Answers to questions

Appendix

3 α, β, δ and γ thalassaemias and related conditions

α thalassaemias

β thalassaemias

Haemoglobin Lepore trait

δβ, γδβ and εγδβ thalassaemias

δ thalassaemia

γ thalassaemia

Hereditary persistence of fetal haemoglobin (HPFH) and other inherited causes of an increased proportion of haemoglobin F

Check your knowledge

Further reading

References

Answers to questions

4 Sickle cell haemoglobin and its interactions with other variant haemoglobins and with thalassaemias

Sickle cell trait

Sickle cell anaemia

Sickle cell/haemoglobin C disease

Sickle cell/β thalassaemia

Other causes of sickle cell disease

Check your knowledge

References

Answers to questions

5 Other significant haemoglobinopathies

Haemoglobin C

Haemoglobin E

Haemoglobin D‐Punjab/D‐Los Angeles

Haemoglobin G‐Philadelphia

Haemoglobin O‐Arab

Unstable haemoglobins

Haemoglobin M

High affinity haemoglobins

Low affinity haemoglobins

Haemoglobin F variants

Haemoglobin A

2

variants

Check your knowledge

Further reading

References

Answers to questions

6 Acquired abnormalities of globin chain synthesis or haemoglobin structure

Acquired thalassaemia

Increased or decreased haemoglobin F

Increased or decreased haemoglobin A

2

Increased or decreased glycosylated haemoglobin

Carboxyhaemoglobinaemia

Methaemoglobinaemia

Sulphaemoglobinaemia

Check your knowledge

References

Answers to questions

7 Organisation of a haemoglobinopathy diagnostic service

General principles

Antenatal and preconceptual haemoglobinopathy/thalassaemia screening and fetal diagnosis

Neonatal screening

Pre‐anaesthetic testing

Other haemoglobinopathy investigations

Haemoglobinopathy investigations in a resource‐poor setting

Check your knowledge

Internet resources

References

Answers to questions

8 Self‐assessment: test cases

Answers

Appendix: electronic resources

Sickle cell disease (specific)

Thalassaemia (specific)

General (both sickle cell and thalassaemia, or other haemoglobinopathy)

Genes

Index

End User License Agreement

List of Tables

Chapter 1

Table 1.1 Haemoglobins normally present during adult, fetal and embryonic per...

Table 1.2 The sequences showing CACCC, CCAAT and TATA homology in the promote...

Table 1.3 Types of mutation that can occur in globin genes and adjoining sequ...

Table 1.4 Mutations and polymorphisms occurring outside the globin gene clust...

Table 1.5 Consequences of mutation of globin genes.

Chapter 2

Table 2.1 Characteristics of haemoglobin S and some variant haemoglobins with...

Table 2.2 Characteristics of haemoglobin C and some variant haemoglobins with...

Table 2.3 Retention times of common normal and variant haemoglobins on the Bi...

Table 2.4 α:β globin chain synthesis ratio in normal subjects and in various ...

Table 2.5 Techniques for diagnosis of thalassaemias and haemoglobinopathies b...

Chapter 3

Table 3.1 Classification of the thalassaemias.

Table 3.2 Classification of deletional α thalassaemia.

Table 3.3 Classification of non‐deletional α thalassaemia.

Table 3.4 Types of mutation most often responsible for α thalassaemia in diff...

Table 3.5 The prevalence of α

0

, α

+

and β thalassaemia heterozygosity in diffe...

Table 3.6 Examples of genetic abnormalities leading to haemoglobin H disease ...

Table 3.7 Types of mutation that can result in the phenotype of β thalassaemi...

Table 3.8 Some inherited causes of an increased percentage of haemoglobin A

2

...

Table 3.9 Haemoglobin A

2

percentage in normal babies and babies with β thalas...

Table 3.10 Causes of normal haemoglobin A

2

β thalassaemia.

Table 3.11 Genotypes that can produce the phenotype of β thalassaemia interme...

Table 3.12 Inherited causes of a low haemoglobin A

2

.

Table 3.13 Inherited conditions associated with high haemoglobin F percentage...

Table 3.14 Distribution of haemoglobin F in various conditions associated wit...

Table 3.15 Haematological features of heterozygosity for deletional hereditar...

Table 3.16 Haematological features of non‐deletional hereditary persistence o...

Chapter 4

Table 4.1 Prevalence (%) of haemoglobins S and C in different populations. (F...

Table 4.2 Variant haemoglobins in which the mutation of haemoglobin S is one ...

Table 4.3 Causes of sickle cell disease [41, 43, 51–54].

Table 4.4 The red cell indices and percentage of haemoglobin S reported in si...

Table 4.5 Features of sickle cell anaemia linked to either haemolysis or hype...

Table 4.6 Causes of anaemia in sickle cell anaemia.

Table 4.7 Factors ameliorating or aggravating features of sickle cell anaemia...

Table 4.8 Haematological characteristics and percentages of various haemoglob...

Table 4.9 The blood film features of sickle cell anaemia, sickle cell/haemogl...

Table 4.10 Percentage of haemoglobin A in compound heterozygosity for S and β

Table 4.11 Making a distinction between haemoglobin O‐Arab and haemoglobin C‐...

Chapter 5

Table 5.1 The types of functional abnormality that can occur as a result of m...

Table 5.2 Prevalence of haemoglobin E carriers in various countries. (From re...

Table 5.3 M haemoglobins [2, 136–141].

Chapter 6

Table 6.1 Acquired β, δβ and γδβ thalassaemia. (From references 3–6, 27, 28.)

Table 6.2 Some acquired causes of an increased percentage of haemoglobin F. (...

Table 6.3 Some acquired causes of an increased or decreased percentage of hae...

Table 6.4 Causes of real and apparent increased and decreased percentages of ...

Table 6.5 Some causes of an increased concentration of methaemoglobin. (Deriv...

Table 6.6 Causes of an increased concentration of sulphaemoglobin [42, 119–12...

Chapter 7

Table 7.1 Haemoglobinopathies of such severity that the likelihood of their o...

Table 7.2 Disorders of globin chain synthesis that should be detected in pros...

Table 7.3 Less severe haemoglobinopathies for which the prediction of the con...

Table 7.4 Methods applicable to neonatal haemoglobinopathy diagnosis.

List of Illustrations

Chapter 1

Fig. 1.1 Diagrammatic representation of the quaternary structure of haemoglo...

Fig. 1.2 Diagrammatic representation of the sites and rates of synthesis of ...

Fig. 1.3 Diagrammatic representation of the average percentages of various h...

Fig. 1.4 Diagrammatic representation of a haemoglobin molecule with a haem g...

Fig. 1.5 Oxygen dissociation curve: (a) normal oxygen dissociation curve ind...

Fig. 1.6 Diagrammatic representation of the effect of oxygenation and deoxyg...

Fig. 1.7 Diagrammatic representation of haem synthesis. Tf, transferrin; TfR...

Fig. 1.8 Diagram of chromosomes 11 and 16 showing the positions of the β and...

Fig. 1.9 Diagrammatic representation of the α and β globin gene clusters on ...

Fig. 1.10 Diagrammatic representation of ribonucleic acid (RNA) synthesis an...

Fig. 1.11 Diagram showing the rate of rise of haemoglobin A

2

in haematologic...

Fig. 1.12 High performance liquid chromatography (HPLC) chromatogram (Bio‐Ra...

Fig. 1.13 The rate of fall of haemoglobin F percentage post‐natally in norma...

Fig. 1.14 Some examples of fusion genes produced by non‐homologous crossover...

Chapter 2

Fig. 2.1 Reticulocyte preparation showing an increased reticulocyte count in...

Fig. 2.2 Diagram of apparatus for performing haemoglobin electrophoresis.

Fig. 2.3 Haemoglobin electrophoresis on cellulose acetate at pH 8.3 showing:...

Fig. 2.4 Haemoglobin electrophoresis on cellulose acetate at pH 8.3 showing ...

Fig. 2.5 Scanning densitometry of a cellulose acetate electrophoretic strip ...

Fig. 2.6 Diagram showing the mobility of normal and variant haemoglobins on ...

Fig. 2.7 Haemoglobin electrophoresis on agarose gel at alkaline pH (pH 8.6):...

Fig. 2.8 Haemoglobin electrophoresis on citrate agar at pH 6.0–6.2 showing f...

Fig. 2.9 Haemoglobin electrophoresis on agarose gel at pH 6.0–6.2 (with vari...

Fig. 2.10 Diagram showing the mobility of normal and variant haemoglobins on...

Fig. 2.11 Capillary electrophoresis electropherogram (Sebia Capillarys 3) sh...

Fig. 2.12 Capillary electrophoresis electropherogram (Sebia Capillarys 3) of...

Fig. 2.13 Annotated screen shots showing electropherograms (Sebia Capillarys...

Fig. 2.14 Capillary electrophoresis electropherogram (Sebia Capillarys 3) in...

Fig. 2.15 HPLC chromatogram (Bio‐Rad Variant II) showing increased haemoglob...

Fig. 2.16 HPLC chromatogram (Bio‐Rad Variant II) in a patient with sickle ce...

Fig. 2.17 HPLC chromatogram (Bio‐Rad Variant II) in a patient with a bilirub...

Fig. 2.18 Artificial mixture showing the retention times of normal and impor...

Fig. 2.19 Typical elution patterns on HPLC with the Bio‐Rad Variant II syste...

Fig. 2.20 Typical elution patterns for normal and variant haemoglobins with ...

Fig. 2.21 Typical elution patterns for normal and variant haemoglobins with ...

Fig. 2.22 Photograph of isoelectric focusing (IEF) plate showing, from left ...

Fig. 2.23 Densitometric scanning of one lane of an IEF plate from a sample s...

Fig. 2.24 Diagram showing the mobilities of various haemoglobins on an IEF p...

Fig. 2.25 Sickle solubility test showing a positive control, a negative cont...

Fig. 2.26 False positive sickle solubility test caused by increased plasma p...

Fig. 2.27 Capillary electrophoresis, electropherogram of a normal sample, Se...

Fig. 2.28 HPLC chromatogram (Bio‐Rad Variant II) in a carrier of haemoglobin...

Fig. 2.29 Diagrammatic representation of the principle of microcolumn chroma...

Fig. 2.30 Kleihauer test showing: (a) heterogeneous distribution of haemoglo...

Fig. 2.31 A haemoglobin H preparation from a patient with haemoglobin H dise...

Fig. 2.32 A haemoglobin H preparation in a patient with haemoglobin H diseas...

Fig. 2.33 An isopropanol test for an unstable haemoglobin.

Fig. 2.34 A Heinz body preparation (incubation with methyl violet) in a pati...

Fig. 2.35 A 2,6‐dichlorophenolindophenol (DCIP) test being read on a light b...

Fig. 2.36 HPLC chromatogram (Bio‐Rad Variant II) in a patient with haemoglob...

Fig. 2.37 An oxygen dissociation curve showing haemoglobin A (broken line) a...

Fig. 2.38 Results of globin chain synthesis analysis showing: (1) α and β ch...

Fig. 2.39 Globin chain synthesis studies in a patient with haemoglobin C/β

+

...

Fig. 2.40 Southern blot analysis following hybridisation of a ζ globin gene ...

Fig. 2.41 Polymerase chain reaction (PCR) in the diagnosis of α thalassaemia...

Fig. 2.42 Reverse dot blot analysis for the detection of four non‐deletional...

Chapter 3

Fig. 3.1 Diagrammatic representation of the likely mechanism for the occurre...

Fig. 3.2 Diagrammatic representation of some common deletions that can lead ...

Fig. 3.3 Blood film of an adult male α

+

thalassaemia homozygote (genotype −α

Fig. 3.4 Red cell cytograms and histograms on a Technicon H2 instrument of (...

Fig. 3.5 Blood film of a haemoglobin Constant Spring heterozygote showing ba...

Fig. 3.6 Blood film of a female α

0

thalassaemia heterozygote (genotype −−

SEA

Fig. 3.7 (a) Haemoglobin electrophoresis on cellulose acetate at alkaline pH...

Fig. 3.8 Haemoglobin H preparation in a patient with α thalassaemia trait sh...

Fig. 3.9 Red cell cytograms and histograms on a Technicon H2 instrument in t...

Fig. 3.10 Blood films of four patients with haemoglobin H disease showing th...

Fig. 3.11 Bone marrow aspirate in haemoglobin H disease; there is erythroid ...

Fig. 3.12 Ultrastructural examination of bone marrow erythroblast in a patie...

Fig. 3.13 Haemoglobin electrophoresis on cellulose acetate at alkaline pH in...

Fig. 3.14 HPLC chromatogram (Bio‐Rad Variant) in two patients with haemoglob...

Fig. 3.15 Haemoglobin H preparation in a patient with haemoglobin H disease ...

Fig. 3.16 Ultrastructural examination of erythrocytes in a patient with haem...

Fig. 3.17 Blood film of a patient with haemoglobin H disease who has had a s...

Fig. 3.18 Haemoglobin H preparation in a patient with haemoglobin H disease ...

Fig. 3.19 Fetus with haemoglobin Bart’s hydrops fetalis.

Fig. 3.20 Blood films in two cases of haemoglobin Bart’s hydrops fetalis sho...

Fig. 3.21 Haemoglobin electrophoresis in haemoglobin Bart’s hydrops fetalis,...

Fig. 3.22 HPLC chromatogram (Bio‐Rad Variant) in (a) a normal neonate showin...

Fig. 3.23 Sites of some mutations giving rise to β

0

thalassaemia, including ...

Fig. 3.24 Sites of some mutations giving rise to β

+

thalassaemia. Introns ar...

Fig. 3.25 Blood films of three patients with β thalassaemia trait showing th...

Fig. 3.26 Blood film of a patient with β thalassaemia trait who had been spl...

Fig. 3.27 Bone marrow aspirate in β thalassaemia trait showing mild dyseryth...

Fig. 3.28 Ultrastructural examination of the bone marrow in β thalassaemia t...

Fig. 3.29 Haemoglobin electrophoresis on cellulose acetate at alkaline pH sh...

Fig. 3.30 Capillary electrophoresis, electropherogram (Sebia Capillarys 3) s...

Fig. 3.31 HPLC chromatogram (Bio‐Rad Variant II) in a patient with homozygos...

Fig. 3.32 Densitometric scan of an electrophoretic strip in β thalassaemia t...

Fig. 3.33 Blood film in dominant β thalassaemia showing anisocytosis, poikil...

Fig. 3.34 Bone marrow aspirate in dominant β thalassaemia showing erythroid ...

Fig. 3.35 Ultrastructural examination in dominant β thalassaemia.

Fig. 3.36 Blood film in haemoglobin Lepore trait showing hypochromia, microc...

Fig. 3.37 Haemoglobin electrophoresis on cellulose acetate at alkaline pH in...

Fig. 3.38 HPLC chromatogram (Bio‐Rad Variant II) in haemoglobin Lepore trait...

Fig. 3.39 Radiograph of the hands of a patient with β thalassaemia intermedi...

Fig. 3.40 Computed tomography (CT) scan of abdomen showing a tumour‐like mas...

Fig. 3.41 Blood films of four patients with thalassaemia intermedia: (a) adu...

Fig. 3.42 Bone marrow film of a patient with thalassaemia intermedia (same p...

Fig. 3.43 The face of a child with β thalassaemia major showing frontal boss...

Fig. 3.44 Skull radiograph of a child with β thalassaemia major showing a ‘h...

Fig. 3.45 An undertransfused child with β thalassaemia major showing abdomin...

Fig. 3.46 Blood films of four patients with β thalassaemia major; patients (...

Fig. 3.47 Bone marrow aspirates of two patients with β thalassaemia major: (...

Fig. 3.48 HPLC chromatogram (Bio‐Rad Variant II) in a baby at 4 months of ag...

Fig. 3.49 Blood film of an adult male with δβ thalassaemia trait; the red ce...

Fig. 3.50 Haemoglobin electrophoresis on cellulose acetate at alkaline pH (l...

Fig. 3.51 HPLC (Bio‐Rad Variant II) of a 10‐year‐old Iraqi girl with heteroz...

Fig. 3.52 Blood film of an 11‐year‐old Iraqi girl with homozygosity for δβ t...

Fig. 3.53 HPLC (Bio‐Rad Variant II) of an 11‐year‐old Iraqi girl with homozy...

Fig. 3.54 Blood film in a neonate with γδβ thalassaemia trait showing microc...

Fig. 3.55 HPLC chromatogram (Bio‐Rad Variant II) in δ

0

thalassaemia homozygo...

Fig. 3.56 Deletions resulting in hereditary persistence of fetal haemoglobin...

Fig. 3.57 Blood film of an adult African woman with hereditary persistence o...

Chapter 4

Fig. 4.1 Transmission electron micrography showing polymerisation of haemogl...

Fig. 4.2 The multifocal origin and spread of the β

S

gene.

Fig. 4.3 Blood films of three patients with sickle cell trait showing the ra...

Fig. 4.4 Haemoglobin electrophoresis on cellulose acetate at alkaline pH sho...

Fig. 4.5 High performance liquid chromatography (HPLC) chromatogram (Bio‐Rad...

Fig. 4.6 Capillary electrophoresis electropherogram (Sebia Capillarys 3) fro...

Fig. 4.7 Blood film of a patient with sickle cell trait and the genotype of ...

Fig. 4.8 HPLC chromatogram (Bio‐Rad Variant II) in sickle cell trait plus ha...

Fig. 4.9 Haemoglobin electrophoresis on agarose gel at pH 8.6 showing three ...

Fig. 4.10 HPLC chromatogram in a patient with heterozygosity for both haemog...

Fig. 4.11 Long term result of ‘dactylitis’ in sickle cell anaemia: (a) the h...

Fig. 4.12 Radiography in sickle cell anaemia: (a) radiograph of the head of ...

Fig. 4.13 Face of a child with sickle cell anaemia showing pallor and jaundi...

Fig. 4.14 Cholecystogram showing gallstones (negative images), caused by inc...

Fig. 4.15 Skull radiograph in sickle cell anaemia showing expansion of the b...

Fig. 4.16 Red cell cytogram and histograms from a Technicon H2 instrument sh...

Fig. 4.17 Blood film of a neonate with sickle cell anaemia showing: (a) one ...

Fig. 4.18 Blood film of a child with sickle cell anaemia: (a) at the age of ...

Fig. 4.19 Blood films of four patients with sickle cell anaemia showing the ...

Fig. 4.20 Blood film showing dyserythropoiesis as a feature of ‘stress eryth...

Fig. 4.21 Blood film of an Arab patient with a high haemoglobin F percentage...

Fig. 4.22 Haemoglobin electrophoresis on cellulose acetate at alkaline pH sh...

Fig. 4.23 Capillary electrophoresis in sickle cell anaemia (Sebia Capillarys...

Fig. 4.24 Capillary electrophoresis in sickle cell anaemia (Sebia Capillarys...

Fig. 4.25 HPLC chromatogram (Bio‐Rad Variant II) in a neonate with sickle ce...

Fig. 4.26 Bone marrow aspirate in sickle cell anaemia showing erythroid hype...

Fig. 4.27 Bone marrow aspirate in sickle cell anaemia showing a sea‐blue his...

Fig. 4.28 Trephine biopsy section in sickle cell anaemia showing numerous ps...

Fig. 4.29 Trephine biopsy section in sickle cell anaemia showing erythroid h...

Fig. 4.30 Radiograph of hips and pelvis in a patient with sickle cell/haemog...

Fig. 4.31 Blood films of four patients with sickle cell/haemoglobin C compou...

Fig. 4.32 Haemoglobin electrophoresis on agarose gel at pH 6.2 showing a pat...

Fig. 4.33 HPLC chromatogram (Bio‐Rad Variant II) in sickle cell/haemoglobin ...

Fig. 4.34 Capillary electrophoresis in sickle cell/haemoglobin C disease sho...

Fig. 4.35 Blood films of two patients with sickle cell/β

0

thalassaemia showi...

Fig. 4.36 Blood films of two patients with sickle cell/β

+

thalassaemia showi...

Fig. 4.37 Haemoglobin electrophoresis on cellulose acetate at alkaline pH in...

Fig. 4.38 HPLC chromatogram (Bio‐Rad Variant II) of haemoglobin S/β

+

thalass...

Fig. 4.39 Capillary electrophoresis (Sebia Capillarys 3) in sickle cell/β

+

t...

Fig. 4.40 Bone marrow aspirate in sickle cell/β

0

thalassaemia compound heter...

Fig. 4.41 Blood film in sickle cell/haemoglobin D‐Punjab compound heterozygo...

Fig. 4.42 Bone marrow aspirate in sickle cell/haemoglobin D‐Punjab compound ...

Fig. 4.43 HPLC chromatogram (Bio‐Rad Variant II) in sickle cell/haemoglobin ...

Fig. 4.44 Blood film in sickle cell/haemoglobin O‐Arab compound heterozygosi...

Fig. 4.45 Haemoglobin electrophoresis on cellulose acetate at alkaline pH in...

Fig. 4.46 Haemoglobin electrophoresis on agarose gel at acid pH in sickle ce...

Fig. 4.47 HPLC chromatogram (Bio‐Rad Variant II) in a patient with haemoglob...

Fig. 4.48 HPLC chromatogram (Bio‐Rad Variant II) in a patient with compound ...

Fig. 4.49 Blood film in sickle cell/hereditary persistence of fetal haemoglo...

Fig. 4.50 Blood film in sickle cell/haemoglobin E compound heterozygosity sh...

Fig. 4.51 Haemoglobin electrophoresis at acid and alkaline pH in sickle cell...

Fig. 4.52 HPLC chromatogram (Bio‐Rad Variant II) in sickle cell/haemoglobin ...

Fig. 4.53 Capillary electrophoresis in sickle cell/haemoglobin E compound he...

Fig. 4.54 Blood film of a 1‐year‐old child with haemoglobin S/haemoglobin Si...

Fig. 4.55 HPLC chromatogram (Bio‐Rad Variant II) in a patient with compound ...

Fig. 4.56 Blood film of a patient with compound heterozygosity for haemoglob...

Chapter 5

Fig. 5.1 Distribution of haemoglobin C in north‐west Africa.

Fig. 5.2 Blood films of four patients with haemoglobin C trait showing the r...

Fig. 5.3 Haemoglobin electrophoresis on cellulose acetate at alkaline pH sho...

Fig. 5.4 High performance liquid chromatography (HPLC) chromatogram (Bio‐Rad...

Fig. 5.5 Capillary electrophoresis (Sebia Capillarys 3) in haemoglobin C tra...

Fig. 5.6 Blood films of four patients with haemoglobin C disease showing the...

Fig. 5.7 Ultrastructural examination of an erythrocyte containing a haemoglo...

Fig. 5.8 Haemoglobin electrophoresis on cellulose acetate at alkaline pH in ...

Fig. 5.9 HPLC chromatogram (Bio‐Rad Variant II) in haemoglobin C homozygosit...

Fig. 5.10 Bone marrow aspirate of a patient with haemoglobin C disease showi...

Fig. 5.11 Ultrastructural examination showing the characteristic abnormality...

Fig. 5.12 Blood films of two patients with haemoglobin C/β thalassaemia comp...

Fig. 5.13 HPLC chromatogram (Bio‐Rad Variant II) in a patient with haemoglob...

Fig. 5.14 Capillary electrophoresis (Sebia Capillarys 2) in a patient with h...

Fig. 5.15 An 8‐month‐old baby with compound heterozygosity for haemoglobins ...

Fig. 5.16 Haemoglobin electrophoresis on cellulose acetate at alkaline pH in...

Fig. 5.17 Haemoglobin electrophoresis on agarose gel at acid pH in a patient...

Fig. 5.18 Blood films of two patients with haemoglobin E trait showing: (a) ...

Fig. 5.19 Haemoglobin electrophoresis on cellulose acetate at alkaline pH in...

Fig. 5.20 HPLC chromatogram (Bio‐Rad Variant II) in a patient with haemoglob...

Fig. 5.21 Capillary electrophoresis (Sebia Capillarys 2) in a patient with h...

Fig. 5.22 HPLC chromatogram (Bio‐Rad Variant II) in a patient with haemoglob...

Fig. 5.23 HPLC chromatogram (Bio‐Rad Variant II) in a patient with haemoglob...

Fig. 5.24 Blood film of a patient with haemoglobin E homozygosity showing mi...

Fig. 5.25 Haemoglobin electrophoresis on cellulose acetate at alkaline pH in...

Fig. 5.26 HPLC chromatogram (Bio‐Rad Variant II) in a patient with haemoglob...

Fig. 5.27 Capillary electrophoresis (Sebia Capillarys 3) in haemoglobin E ho...

Fig. 5.28 Blood film of a 6‐year‐old boy with haemoglobin E/β

+

thalassaemia ...

Fig. 5.29 Blood film of a patient with haemoglobin E/β thalassaemia compound...

Fig. 5.30 Blood film of a Thai patient with haemoglobin E heterozygosity and...

Fig. 5.31 HPLC (Bio‐Rad Variant II) in haemoglobin D‐Punjab heterozygosity. ...

Fig. 5.32 Capillary electrophoresis (Sebia Capillarys 3) in haemoglobin D‐Pu...

Fig. 5.33 HPLC (Bio‐Rad Variant II) in haemoglobin D‐Punjab homozygosity. Th...

Fig. 5.34 Capillary electrophoresis (Sebia Capillarys 3) in haemoglobin D‐Pu...

Fig. 5.35 Blood film of a patient with haemoglobin D‐Punjab/β

0

thalassaemia ...

Fig. 5.36 HPLC chromatogram (Bio‐Rad Variant II) in a patient with haemoglob...

Fig. 5.37 Capillary electrophoresis (Sebia Capillarys 3) in haemoglobin G‐Ph...

Fig. 5.38 Cellulose acetate electrophoresis at alkaline pH in a patient with...

Fig. 5.39 Agarose gel electrophoresis at acid pH in a patient with haemoglob...

Fig. 5.40 HPLC chromatogram (Bio‐Rad Variant II) in a patient with haemoglob...

Fig. 5.41 Blood film of a patient with compound heterozygosity for haemoglob...

Fig. 5.42 Blood films of three patients with unstable haemoglobins showing: ...

Fig. 5.43 Haemoglobin electrophoresis on cellulose acetate at alkaline pH in...

Fig. 5.44 HPLC chromatogram (Bio‐Rad Variant II) in a patient with haemoglob...

Fig. 5.45 Bone marrow aspirate in haemoglobin St Mary’s: (a) MGG ×100 showin...

Fig. 5.46 Clinical photograph of a patient with haemoglobin M‐Saskatoon show...

Fig. 5.47 Diagram showing the relationship of haem to the proximal and dista...

Fig. 5.48 Absorption spectra of haemoglobins M‐Iwate, M‐Boston, M‐Hyde Park ...

Fig. 5.49 Oxygen dissociation curves showing two high affinity haemoglobins ...

Fig. 5.50 HPLC chromatogram (Bio‐Rad Variant II) showing a double peak resul...

Fig. 5.51 HPLC chromatogram (Bio‐Rad Variant II) in a patient with both haem...

Fig. 5.52 Capillary electrophoresis (Sebia Capillarys 3) in a patient with b...

Chapter 6

Fig. 6.1 Blood film of a 60‐year‐old man with acquired haemoglobin H disease...

Fig. 6.2 Acquired haemoglobin H disease in a patient with erythroleukaemia: ...

Fig. 6.3 Juvenile myelomonocytic leukaemia showing: (a) blood film MGG ×100;...

Fig. 6.4 High performance liquid chromatography (HPLC) chromatogram (Bio‐Rad...

Fig. 6.5 HPLC chromatogram showing: (a) increased haemoglobin A

1c

in a diabe...

Fig. 6.6 Spectroscopy showing carboxyhaemoglobin (COHb), oxyhaemoglobin (O

2

H...

Fig. 6.7 Hands of a patient with anaemia and mild methaemoglobinaemia caused...

Fig. 6.8 The tongue of a patient with methaemoglobinaemia.

Fig. 6.9 A bag of blood removed during exchange transfusion (bottom) from a ...

Fig. 6.10 Blood film of a patient with dapsone‐induced methaemoglobinaemia s...

Fig. 6.11 Spectroscopy showing methaemoglobin (MetHb), oxyhaemoglobin (O

2

Hb)...

Fig. 6.12 Spectroscopy showing sulphaemoglobin (sulfHb), oxyhaemoglobin (O

2

H...

Chapter 7

Fig. 7.1 Flow chart for universal antenatal screening for variant haemoglobi...

Fig. 7.2 Flow chart for universal antenatal screening for variant haemoglobi...

Fig. 7.3 Flow chart for selective antenatal testing in a low prevalence area...

Fig. 7.4 A protocol for neonatal screening. IEF, isoelectric focusing; MS, m...

Fig. 7.5 A protocol for pre‐anaesthetic testing to detect patients with sick...

Guide

Cover

Table of Contents

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Haemoglobinopathy Diagnosis

Barbara J. Bain MBBS FRACP FRCPath

Professor of Diagnostic HaematologySt Mary’s Hospital Campus of Imperial College London, UK

Honorary Consultant HaematologistSt Mary’s Hospital, London, UK

Third Edition

This edition first published 2020© 2020 John Wiley & Sons Ltd

Edition History© Barbara J. Bain (2e, 2006) Published by Blackwell Publishing Ltd.

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Library of Congress Cataloging‐in‐Publication Data

Names: Bain, Barbara J., author.Title: Haemoglobinopathy diagnosis / Barbara J. Bain, MBBS, FRACP, FRCPath, Professor of Diagnostic Haematology, St. Mary’s Hospital Campus of Imperial College, London, UK, Honorary Consultant Haematologist, St. Mary’s Hospital, London, UK.Description: Third edition. | Hoboken : Wiley, 2020. | Includes bibliographical references and index.Identifiers: LCCN 2020000661 (print) | LCCN 2020000662 (ebook) | ISBN 9781119579953 (hardback) | ISBN 9781119579984 (adobe pdf) | ISBN 9781119579991 (epub)Subjects: LCSH: Hemoglobinopathy–Diagnosis.Classification: LCC RC641.7.H35 B73 2020 (print) | LCC RC641.7.H35 (ebook) | DDC 616.1/51075–dc23LC record available at https://lccn.loc.gov/2020000661LC ebook record available at https://lccn.loc.gov/2020000662

Cover images: blue purple abstract © Pobytov/Getty Images, graph on purple background courtesy of Barbara BainCover design by Wiley

Preface

This book is dedicated to the past and present scientific staff of the haematology departments of Princess Alexandra Hospital, Brisbane, Australia and St Mary’s Hospital, Paddington, London. It was the former group who first awakened my interest in this field. They also suggested that there was a need for a practical book on the laboratory diagnosis of haemoglobinopathies and that I might be the person to write it. The second group, and in particular Lorry Phelan, for over four decades shared my pleasure in solving diagnostic problems and, at the same time, providing an accurate, clinically relevant diagnostic service. It is with much pleasure that I dedicate this new edition to these colleagues and friends.

I should also like to acknowledge many other colleagues throughout the world who have helped in diverse ways, including those who have contributed images. They are individually acknowledged in the figure legends. Dr Barbara Wild kindly reviewed Chapter 2.

Barbara J. BainLondon, 2019

Abbreviations and glossary

α

the Greek letter, alpha

α chain

the α globin chain, which is required for synthesis of haemoglobins A, F and A

2

and also the embryonic haemoglobin, Gower 2

α gene

one of a pair of genes on chromosome 16,

HBA1

and

HBA2

, that encode α globin

α thalassaemia

a group of thalassaemias characterised by absent or reduced α globin chain synthesis, usually resulting from deletion of one or more of the α globin genes; less often it results from altered structure of an α gene or mutation of the locus control gene,

LCRA

, or genes encoding

trans

‐acting factors

α

0

thalassaemia

a thalassaemic condition in which there is no α globin chain translation from one or both copies of chromosome 16

α

+

thalassaemia

a thalassaemic condition in which there is reduced translation of α chain from one or both copies of chromosome 16

β

the Greek letter, beta

β chain

the β globin chain, which forms part of haemoglobin A and haemoglobin Portland 2 and is the only globin chain in the abnormal haemoglobin, haemoglobin H

β gene

the gene on chromosome 11,

HBB

, that encodes β globin

β thalassaemia

a thalassaemia characterised by reduced β globin synthesis, usually caused by mutation of a β globin gene; less often it results from gene deletion or from deletion or mutation of the locus control region,

LCRB

γ

the Greek letter, gamma

γ chain

the γ globin chain, which forms part of fetal haemoglobin (haemoglobin F) and the embryonic haemoglobin, haemoglobin Portland 1, and is the only globin chain in the abnormal variant haemoglobin Bart’s

γ gene

one of a pair of very similar genes on chromosome 11,

HBG1

and

HBG2

, encoding γ globin chain

γ thalassaemia

a thalassaemic condition resulting from reduced synthesis of γ globin chain

δ

the Greek letter, delta

δ chain

a β‐like globin chain, which forms part of haemoglobin A

2

δ gene

a gene of the β cluster on chromosome 11,

HBD

, that encodes δ globin

δ thalassaemia

reduced synthesis of δ globin and therefore of haemoglobin A

2

ε

the Greek letter, epsilon

ε chain

the epsilon globin chain, which is synthesised during early embryonic life and forms part of haemoglobins Gower 1 and Gower 2

ε gene

a gene of the α globin cluster on chromosome 16,

HBE1

, that encodes ε globin chain

ψ

the Greek letter, psi, used to indicate a pseudogene

ζ

the Greek letter, zeta

ζ chain

the ζ globin chain that is synthesized in intrauterine life and that forms part of haemoglobins Gower 1, Portland 1 and Portland 2

ζ gene

a gene of the α globin gene cluster on chromosome 16,

HBZ

, that encodes ζ globin chain

2,3

‐

DPG

2,3‐diphosphoglycerate; a small molecule that interacts with haemoglobin, decreasing its oxygen affinity

3′

the end of a gene where transcription ceases

5′

the end of a gene where transcription starts

acquired

a condition that is not present at birth or is not inherited

affinity

the avidity of haemoglobin for oxygen

AIDS

acquired immune deficiency syndrome

ala

δ‐aminolaevulinic acid, the first compound formed during the process of haem synthesis

AML

acute myeloid leukaemia

ARMS

amplification refractory mutation system, a PCR technique used, for example, for the detection of mutations causing β thalassaemia; it employs two primer sets, one amplifying normal sequences and one abnormal sequences

balanced polymorphism

the stable persistence of two or more alleles of a gene in a significant proportion of a population; a potentially deleterious allele may show balanced polymorphism if the heterozygous state conveys an advantage

base

a ring‐shaped organic molecule containing nitrogen, which is a constituent of DNA and RNA; DNA contains four bases – adenine, guanine, cytosine and thymine; RNA contains four bases – adenine, guanine, cytosine and uracil

Bohr effect

the effect of pH on oxygen affinity; the alkaline Bohr effect is the reduction of oxygen affinity of haemoglobin as pH falls from above to below the physiological pH; there is also an acid Bohr effect which is a rise of oxygen affinity as the pH falls further, at a pH level that is incompatible with life

bp

base pair, the pairing of specific bases, e.g. adenine with thymine, in the complementary strands of the DNA double helix

CAP

7‐methyl guanosine cap, added to RNA molecule during processing

carbonic anhydrase

a red cell enzyme that is the second most abundant red cell protein after haemoglobin; it may be apparent on haemoglobin electrophoretic strips if a protein rather than a haem stain is used

carboxyhaemoglobin

haemoglobin that has been chemically altered by combination with carbon monoxide

CE

capillary electrophoresis

CE‐HPLC

cation‐exchange high performance liquid chromatography

chromatography

a method of separating proteins from each other by means of physical characteristics, such as molecular weight, charge or hydrophobicity, or by means of differing affinity for lectins, antibodies or other proteins; in column chromatography the proteins move through an absorbent column and emerge after different periods of time

cis

on the same chromosome (

see also

trans

)

cis

‐

acting

a DNA sequence that affects the expression of a gene on the same chromosome but not on the homologous chromosome (

see also

trans

‐

acting

)

CO

carbon monoxide, the molecule composed of one carbon atom and one oxygen atom, formed by combustion of hydrocarbons

CO

2

carbon dioxide, the molecule composed of one atom of carbon combined with two atoms of oxygen

codon

a triplet of nucleotides that encodes a specific amino acid or serves as a termination signal; there are 61 codons encoding 20 amino acids and 3 codons that act as termination or STOP codons

congenital

present at birth, often but not necessarily inherited

cooperativity

the interaction between the four globin monomers that makes possible the Bohr effect and the sigmoid shape of the oxygen dissociation curve

CT

computed tomography

CV

coefficient of variation

DCIP test

a screening test for haemoglobin E using dichlorophenolindophenol

deletion

loss of part of a chromosome, which may include all or part of a globin gene

deoxyhaemoglobin

haemoglobin that is not combined with O

2

DGGE

denaturing gradient gel electrophoresis, a molecular genetic technique for locating a mutation prior to precise analysis

DNA

deoxyribonucleic acid, the major constituent of the nucleus of a cell; a polynucleotide strand that is able to replicate and that codes for the majority of proteins synthesised by the cell; the DNA molecule is a double helix of two complementary intertwined polynucleotides

EDTA

ethylene diamine tetra‐acetic acid

EKLF

erythroid Krüppel‐like factor

electrophoresis

separation of charged suspended particles such as proteins by application to a membrane or gel followed by exposure to a charge gradient, e.g. haemoglobin electrophoresis

ELISA

enzyme‐linked immunosorbent assay

elution

removal of an absorbed substance from a chromatography column or membrane

enhancer

a DNA sequence that influences the promoter of a nearby gene to increase transcription; an enhancer acts on a gene in

cis

and may be sited upstream, downstream or within a gene

exon

a part of a gene that is represented in mature messenger RNA; most genes are composed of exons and non‐translated introns

FAB classification

French–American–British classification

FBC

full blood count

Fe

iron

Fe

2+

ferrous or bivalent iron

Fe

3+

ferric or trivalent iron

fetal haemoglobin

see

haemoglobin F

G6PD

glucose‐6‐phosphate dehydrogenase

GAP‐PCR

a PCR technique in which there is amplification across a ‘gap’ created by a deletion

GATA1

an erythroid‐specific transcription factor

gene

the segment of DNA that is involved in producing a polypeptide chain; it includes regions preceding and following the coding region (5′ and 3′ untranslated regions) as well as intervening sequences (introns) between individual coding segments (exons); genes mediate inheritance; they are located on nuclear chromosomes or, for a minority of genes, in a mitochondrion

genetic code

the relationship between a triplet of bases, called a codon, and the amino acid that it encodes

genotype

the genetic constitution of an individual (cf. phenotype)

globin

the protein part of the haemoglobin molecule, usually composed of two pairs of non‐identical chains, e.g. two α chains and two β chains

H

+

a proton

haem

a porphyrin structure that contains iron and that forms part of the haemoglobin molecule

haemoglobin

a complex molecule composed of four globin chains, each one enclosing a haem group

haemoglobin A

the major haemoglobin component present in most adults, having two α and two β chains

haemoglobin A

1c

glycosylated haemoglobin A

haemoglobin A

2

a minor haemoglobin component present in most adults and, as an even lower proportion of total haemoglobin, in neonates and infants, having two α chains and two δ chains

haemoglobin A

2

′

a haemoglobin A

2

variant, also known as haemoglobin B

2

haemoglobin Bart’s

an abnormal haemoglobin with four γ chains and no α chains, present as the major haemoglobin component in haemoglobin Bart’s hydrops fetalis and as a minor component in neonates with haemoglobin H disease or α thalassaemia trait

haemoglobin Bart’s hydrops fetalis

a fatal condition of a fetus or neonate with no α genes and consequently no production of haemoglobins A, A

2

, F or Gower 2

haemoglobin C

a variant haemoglobin with an amino acid substitution in the β chain, mainly found in those of African ancestry

haemoglobin Constant Spring

a variant haemoglobin with a structurally abnormal α chain that is synthesised at a reduced rate, leading to α thalassaemia

haemoglobin D

the designation of a group of haemoglobin variants, some α chain variants and some β chain variants, that have the same mobility as haemoglobin S on electrophoresis at alkaline pH

haemoglobin dissociation curve

a plot of percentage saturation of haemoglobin against partial pressure of oxygen

haemoglobin E

a variant haemoglobin with an amino acid substitution in the β chain, mainly found in South‐East Asia and parts of the Indian subcontinent

haemoglobin F

fetal haemoglobin, the major haemoglobin of the fetus and neonate, having two α chains and two γ chains; also present as a very minor component in most adults and as a larger proportion in a minority

haemoglobin G

the designation of a group of haemoglobin variants, some α chain variants and some β chain variants, that have the same mobility as haemoglobin S on electrophoresis at alkaline pH

haemoglobin Gower 1

an embryonic haemoglobin, having two ζ chains and two ε chains

Haemoglobin Gower 2

an embryonic haemoglobin, having two α chains and two ε chains

Haemoglobin H

a variant haemoglobin with four β chains and no α chains, present in haemoglobin H disease and, in small quantities, in α thalassaemia trait

haemoglobin H disease

a haemoglobinopathy caused by marked underproduction of α chains, often consequent on deletion of three of the four α genes

haemoglobin I

a group of variant haemoglobins that move more rapidly than haemoglobin A on electrophoresis at alkaline pH

haemoglobin J

a group of variant haemoglobins that move more rapidly than haemoglobin A but more slowly than haemoglobin I on electrophoresis at alkaline pH

haemoglobin K

a group of variant haemoglobins moving between A and J on electrophoresis at alkaline pH

haemoglobin Lepore

a number of variant haemoglobins resulting from the fusion of part of a δ globin gene with part of a β globin gene, giving a δβ fusion gene and a fusion protein that combines with α globin to form haemoglobin Lepore

haemoglobin M

a variant haemoglobin that oxidises readily to methaemoglobin

haemoglobin N

a group of variant haemoglobins moving between J and I on electrophoresis at alkaline pH

haemoglobin O‐Arab

a β chain variant haemoglobin moving near C at alkaline pH and near S at acid pH

haemoglobinopathy

an inherited disorder resulting from synthesis of a structurally abnormal haemoglobin; the term can also be used to encompass the thalassaemias in which there is a reduced rate of synthesis of one of the globin chains

haemoglobin Portland 1

an embryonic haemoglobin, having two ζ chains and two γ chains

haemoglobin Portland 2

an abnormal embryonic haemoglobin, having two ζ chains and two β chains, present in some severe α thalassaemia syndromes

haemoglobin S

sickle cell haemoglobin, a variant haemoglobin with a tendency to polymerise at low oxygen tension, causing erythrocytes to deform into the shape of a sickle

Hb

haemoglobin concentration

Hct

haematocrit

HDW

haemoglobin distribution width

heteroduplex analysis

a molecular genetic technique for locating a mutation prior to precise analysis

heterozygosity

the state of having two different alleles of a specified autosomal gene or, in a female, two different alleles of an X chromosomal gene

heterozygous

having two different alleles of a specified autosomal or X chromosome gene

HIV

human immunodeficiency virus

homologue

an equivalent or similar structure; the α1 and α2 genes are homologues as are the two copies of a chromosome

homologous

being equivalent or similar to another

homology

the presence of structural similarity, implying a common remote origin; the δ and β genes show partial homology

homozygosity

the state of having two identical alleles of a specified autosomal or X chromosome gene

homozygous

having two identical alleles of a specified autosomal gene or, in a female, two identical alleles of an X chromosome gene

HPFH

hereditary persistence of fetal haemoglobin

HPLC

high performance liquid chromatography, a method of separating proteins, such as haemoglobin variants, from each other on the basis of characteristics such as size, hydrophobicity and ionic strength; a solution of proteins is eluted from a specially designed column by exposure to various buffers, different proteins emerging after varying periods of time

HS1, HS2, HS3, HS4

hypersensitive sites 1, 2, 3, and 4, upstream of the β globin gene cluster

HS −40

an upstream enhancer of α globin gene transcription

HVR

hypervariable region

ICSH

International Council for Standardization in Haematology

IEF

isoelectric focusing, the separation of proteins in an electric field as they move through a pH gradient to their isoelectric points

inherited

a characteristic that is transmitted from a parent, by means of genes that form part of nuclear or mitochondrial DNA

initiation

(i) the process by which RNA transcription from a gene commences; (ii) the process by which protein translation from mRNA commences

initiation codon

the three nucleotide codon (ATG) at the 5′ end of a gene that is essential to permit initiation of transcription of a gene, i.e. initiation of polypeptide synthesis

insertion

the insertion of a DNA sequence, e.g. from one chromosome into another

intervening sequence (IVS)

an intron

intron

a sequence of DNA in a gene that is not represented in processed messenger RNA or in the protein product

inversion

the reversal of the normal position of a DNA sequence on a chromosome

isoelectric point

the pH at which a protein has no net charge

kb

kilobase, a unit for measuring the length of DNA; one kilobase is 1000 nucleotide base pairs

kDa

kilodalton, a unit for measuring molecular weight; one kilodalton is 1000 daltons

LCR

locus control region, a DNA sequence upstream of genes of the α or β globin cluster that enhances transcription of the genes of the cluster,

LCRA

and

LCRB

control the α and β gene clusters, respectively

LDH

lactate dehydrogenase

MCH

mean cell haemoglobin

MCHC

mean cell haemoglobin concentration

MCV

mean cell volume

MDS

myelodysplastic syndrome

methaemoglobin

oxidised haemoglobin, which does not function in oxygen transport

MGG

May–Grünwald–Giemsa (stain)

mis‐sense mutation

a mutation that leads to the encoding of a different amino acid

mRNA

messenger RNA, ribonucleic acid that is transcribed in the nucleus, on a DNA template, and moves to the cytoplasm, becoming attached to ribosomes and serving as a template for synthesis of proteins

MS

mass spectrometry, electrospray ionization mass spectrometry, a method for determining the mass and the charge of a molecule

NO

nitric oxide

nonsense mutation

a mutation that leads to no amino acid being encoded that therefore functions as a STOP or termination codon, leading to synthesis of a truncated polypeptide chain

NRBC

nucleated red blood cells

O

2

oxygen

ORF

open reading frame

oxyhaemoglobin

haemoglobin combined with O

2

P

50

P

o

2

at which haemoglobin is half saturated

P

a

O

2

partial pressure of oxygen in arterial blood

partial pressure of oxygen

that part of the total blood gas pressure exerted by oxygen

PAS stain

periodic acid–Schiff stain

PCR

polymerase chain reaction, a method of making multiple copies of a DNA sequence

PCV

packed cell volume, haematocrit

phenocopy

a condition that simulates an inherited condition; a phenocopy may be acquired or may be a genetic characteristic that simulates another

phenotype

the characteristics of an individual, which may be determined by the genotype, or may be an acquired characteristic (cf. genotype)

P

O

2

partial pressure of oxygen

polymorphism

the occurrence of a variant form of a gene in a significant proportion (at least 1%) of a population

promoter

a sequence of DNA at the 5′ end of a gene that is essential for initiation of transcription

pseudogene

a non‐functioning homologue of a gene

purine

one of the two types of nitrogenous base found in nucleic acids; purines have a double ring structure (

see also

pyrimidine

)

pyrimidine

one of the two types of nitrogenous base found in nucleic acids; pyrimidines have a single ring structure (

see also

purine

)

RBC

red blood cell count

RDW

red cell distribution width, a measure of anisocytosis

restriction endonuclease

an enzyme that recognises specific sequences in a DNA molecule and cleaves the molecule in or very near the recognition site

restriction fragment

a fragment of DNA produced by cleavage by a restriction endonuclease

RFLP

restriction fragment length polymorphism, variation between homologous chromosomes with regard to the length of DNA fragments produced by application of a specific restriction endonuclease; can be used for the demonstration of heterozygosity or for demonstration of a specific gene that removes or creates a specific cleavage site

ribosome

a cytoplasmic structure on which proteins are translated from messenger RNA; ribosomes may be free within the cytosol or form part of the rough endoplasmic reticulum

RNA

ribonucleic acid, a polynucleotide in which the nitrogenous bases are adenine, guanine, cytosine and uracil and the sugar is ribose; RNA is produced in the nucleus and in mitochondria from DNA templates

rRNA

ribosomal RNA, RNA that, together with protein, constitutes the ribosomes

sickle cell

an erythrocyte that becomes sickle‐ or crescent‐shaped as a result of polymerisation of haemoglobin S

sickle cell anaemia

the disease resulting from homozygosity for haemoglobin S

sickle cell disease

a group of diseases including sickle cell anaemia and various compound heterozygous states in which clinicopathological effects occur as a result of sickle cell formation (preferred definition but sometimes used as a synonym for sickle cell anaemia)

sickle cell trait

heterozygosity for the β

S

gene that encodes the β chain of haemoglobin S

SOP

standard operating procedure

splicing

the process by which RNA sequences, corresponding to introns in the gene, are removed during processing of RNA

SSP

stage selector protein

sulphaemoglobin

haemoglobin that has been irreversibly oxidised and chemically altered by drugs or chemicals with incorporation of a sulphur atom into the haemoglobin molecule

thalassaemia

a disorder, usually inherited, in which one or more of the globin chains incorporated into a haemoglobin molecule or molecules is synthesised at a reduced rate

thalassaemia intermedia

a genetically heterogeneous thalassaemic condition that is moderately severe but nevertheless does not require regular blood transfusions to sustain life

thalassaemia major

thalassaemia that is incompatible with more than a short survival in the absence of blood transfusion

thalassaemia minor

an asymptomatic thalassaemic condition, attributable to β thalassaemia heterozygosity or to deletion of one or two of the four α genes

trait

a term applied to heterozygosity for an inherited characteristic; in the case of disorders of globin genes, the term would not be used if heterozygosity were associated with a significant phenotypic abnormality; rather it is used when homozygosity or compound heterozygosity produces a clinically significant abnormality but simple heterozygosity does not

trans

having an influence on a DNA sequence on another chromosome (

see also

cis

)

trans

‐

acting

a DNA sequence that affects the expression of a gene on another chromosome (

see also

cis

‐

acting

)

transcript

an RNA molecule, corresponding to one gene, transcribed from nuclear DNA

transcription

the synthesis of RNA on a DNA template

transcription factor

a protein capable of enhancing transcription of one or more genes

translation

the synthesis of protein from a mRNA template

tRNA

transfer RNA, RNA molecules that bind to specific amino acids and transport them to ribosomes; there they bind to specific mRNA sequences, leading to incorporation of amino acids into peptide chains in the sequence specified by the mRNA

unstable

a term applied to a haemoglobin that is abnormally prone to post‐translational structural alteration, which may include loss of the normal tertiary or quaternary structure

UTR

untranslated region

variant

a term applied to any haemoglobin other than haemoglobins A, A

2

, F and the normal embryonal haemoglobins

WBC

white blood cell count

yolk sac

part of an embryo, the initial site of formation of blood cells

1Haemoglobin and the genetics of haemoglobin synthesis

Haemoglobins and their structure and function

The haemoglobin molecule contained within red blood cells is essential for human life, being the means by which oxygen is transported to the tissues. Other functions include the transport of carbon dioxide (CO2) and a buffering action (reduction of the changes in pH that would otherwise be expected when an acid or an alkali enters or is generated in a red cell). A normal haemoglobin molecule has a molecular weight of 64–64.5 kDa and is composed of two dissimilar pairs of polypeptide chains, each of which encloses an iron‐containing porphyrin designated haem (Fig. 1.1). Haem is essential for oxygen transport while globin serves to protect haem from oxidation, renders the molecule soluble and permits variation in oxygen affinity. The structure of the haemoglobin molecule produces an internal environment of hydrophobic radicals, which protects the iron of haem from water and thus from oxidation. External radicals are hydrophilic and thus render the haemoglobin molecule soluble. Both haem and globin are subject to modification. The iron of haemoglobin is normally in the ferrous form (Fe2+). Haem is able to combine reversibly with oxygen so that haemoglobin can function as an oxygen‐transporting protein. Oxidation of iron to the ferric form (Fe3+) is a less readily reversible reaction, converting haem to haematin and haemoglobin to methaemoglobin, a form of haemoglobin that cannot transport oxygen. Auto‐oxidation of haemoglobin to methaemoglobin is a normal process. About 3% of haemoglobin undergoes this process each day with about 1% (0.4–1% in one study) of haemoglobin being methaemoglobin [1, 2]. Methaemoglobin is converted back to haemoglobin mainly by the action of the enzyme NADH‐cytochrome b5‐methaemoglobin reductase.

The haemoglobin molecule can also combine with CO2 and is responsible for about 10% of CO2 transport from the tissues to the lungs; transport is by reversible carbamation of the N‐terminal groups of the α chains of haemoglobin. Because carbamated haemoglobin has a lower oxygen affinity than the non‐carbamated form, binding of the CO2 produced by the metabolic processes in tissues facilitates oxygen delivery to tissues. In addition, non‐oxygenated haemoglobin can carry more CO2 than oxygenated haemoglobin so that unloading of oxygen to the tissues facilities the uptake and transport of CO2. Because of its buffering action (mopping up of protons, H+), haemoglobin also contributes to keeping CO2 in the soluble bicarbonate form and thus transportable. The reaction CO2 + H2O → HCO3− + H+ is facilitated.

Haemoglobin also has a role in nitric oxide (NO) transport and metabolism, being both a scavenger of NO and an active transporter. NO is produced in endothelial cells and neutrophils by the action of nitric acid synthase [2–5]. It has a very high affinity for oxyhaemoglobin so that blood levels are a balance between production and removal by binding to oxyhaemoglobin. NO is a potent vasodilator, but this effect is limited by its binding to haemoglobin. The iron atom of a haem group of oxyhaemoglobin (preferentially the haem enclosed in the haem pocket of an α chain), binds NO. A haemoglobin molecule with NO bound to two haem groups strikingly favours the deoxy conformation so oxygen is readily released. NO–haemoglobin is subsequently converted to methaemoglobin with release of NO