Abnormal haemoglobins

Dr. Arvind Kumar

Introduction

  • Abnormal haemoglobins are variations in the haemoglobin molecule that result from genetic mutations affecting the globin chains.
  • These abnormalities can lead to various haematological disorders, including sickle cell disease, thalassemia, and other hemoglobinopathies.
  • Identifying and estimating abnormal haemoglobins involves specific diagnostic techniques.
  • Here’s a detailed look at these abnormal haemoglobins and how they are identified and estimated:

Abnormal haemoglobins

Hemoglobin S (HbS)

1. Definition

  • HbS is an abnormal hemoglobin variant caused by a structural defect in the β-globin chain, responsible for Sickle Cell Disease (SCD).

2. Genetic Defect

  • Point mutation in β-globin gene (codon 6).

  • Glutamic acid → Valine (GAG → GTG).

  • Leads to abnormal hydrophobic interaction among Hb molecules.

3. Biochemical Effect

  • HbS is normal when oxygenated.

  • On deoxygenation, HbS polymerizes, forming long rigid fibers → sickling of RBCs.

4. Pathophysiology

  • Sickled RBCs become rigid and fragile.

  • Hemolysis → chronic anemia.

  • Vaso-occlusion → ischemia, pain crises, organ damage.

  • Reduced RBC lifespan (10–20 days).

5. Clinical Features

  • Hemolytic anemia, jaundice

  • Painful crises

  • Splenic dysfunction → infections

  • Acute chest syndrome, stroke, avascular necrosis

6. Laboratory Diagnosis

  • Screening: Sickling test, solubility test.

  • Confirmatory:

    • Hemoglobin electrophoresis (HbS band)

    • HPLC quantification

    • Molecular testing (β-globin mutation)

  • Blood smear: sickle cells, target cells.

7. Inheritance

  • Autosomal recessive.

  • HbAS (trait): asymptomatic.

  • HbSS (disease): full clinical expression.

8. Management (Basic)

  • Hydration, oxygen

  • Analgesics for crises

  • Hydroxyurea (↑HbF)

  • Vaccination, antibiotics

  • Blood transfusions

  • Bone marrow transplant (selected cases)

Hemoglobin C (HbC)

1. Definition

  • Hemoglobin C (HbC) is an abnormal β-globin variant caused by a point mutation that leads to mild chronic hemolytic anemia.

  • It is less severe than Hemoglobin S.

2. Genetic Defect

  • Point mutation in β-globin gene at codon 6.

  • Glutamic acid → Lysine substitution (GAG → AAG).

  • Alters surface charge but does not cause polymerization like HbS.

3. Biochemical Effects

  • HbC is less soluble than HbA → forms intracellular crystals.

  • RBCs become rigid and dehydrated, resulting in mild hemolysis.

4. Pathophysiology

  • HbC disease (HbCC):

    • Mild hemolytic anemia

    • Splenomegaly

    • Target cells and HbC crystals

  • HbSC disease:

    • Combination of HbS + HbC

    • More severe than HbC disease but milder than sickle cell disease (HbSS).

5. Clinical Features

  • Mild anemia

  • Jaundice (sometimes)

  • Splenomegaly

  • Gallstones (pigment stones)

  • Usually no painful crises unless combined with HbS (HbSC disease)

6. Laboratory Diagnosis

  • Peripheral smear:

    • Target cells, folded cells

    • HbC crystals (dense, rectangular “bar-of-gold” crystals)

  • Hemoglobin electrophoresis:

    • HbC migrates slower than HbA; characteristic band pattern

  • HPLC: Quantifies HbC percentage.

  • Solubility test: Usually negative (unlike HbS).

7. Inheritance

  • Autosomal recessive

  • HbAC: Trait, asymptomatic

  • HbCC: Mild disease

  • HbSC: Moderate sickling disorder

8. Management (Basic)

  • Usually no specific treatment needed for HbCC.

  • Folic acid supplementation

  • Treat complications (e.g., gallstones)

  • For HbSC: management similar to mild sickle cell disease.

Haemoglobin D (Hb D)

1. Definition

  • Hemoglobin D (HbD) is an abnormal β-globin variant caused by a structural mutation in the β-globin gene.

  • Most common form: HbD Punjab / HbD Los Angeles.

  • Generally causes mild hemolytic features or remains asymptomatic.

2. Genetic Defect

  • Point mutation in β-globin gene at codon 121.

  • Glutamic acid → Glutamine substitution.

  • Mutation alters the hemoglobin molecule’s charge but does not cause polymerization (unlike HbS).

3. Biochemical Effect

  • HbD is relatively stable.

  • Does not produce sickling or significant crystal formation.

  • RBC survival is mostly normal; slight hemolysis may occur.

4. Pathophysiology

  • HbD trait (HbAD): asymptomatic.

  • HbD disease (HbDD): mild hemolytic anemia, if any.

  • HbSD disease (HbS + HbD):

    • Clinically similar to sickle cell disease due to interaction with HbS.

    • Causes sickling complications but usually milder than HbSS.

5. Clinical Features

  • Most individuals are asymptomatic.

  • In HbDD:

    • Mild anemia

    • Occasional splenomegaly

    • Rare hemolysis

  • In HbSD:

    • Pain crises, mild sickling complications

    • Hemolytic anemia

6. Laboratory Diagnosis

Peripheral smear:

    • May show target cells; hemolysis is usually minimal.

Hemoglobin Electrophoresis:

    • HbD migrates with HbS in alkaline electrophoresis, making differentiation essential.

Acid Electrophoresis:

    • Helps separate HbD from HbS (HbD moves with HbA in acid medium).

HPLC:

    • Accurate quantification and identification.

Genetic testing:

    • Detects the β121 Glu→Gln mutation.

7. Inheritance

  • Autosomal recessive.

  • HbAD: Carrier, asymptomatic

  • HbDD: Mild disease

  • HbSD: Clinically significant sickling disorder

8. Management

  • HbDD usually requires no treatment.

  • Folic acid may be given if mild hemolysis occurs.

  • HbSD managed similar to mild sickle cell disease:

    • Hydration

    • Pain control

    • Infection prevention

    • Monitor for complications

Hemoglobin E (HbE)

1. Definition

  • Hemoglobin E (HbE) is a structural variant of β-globin and one of the most common abnormal hemoglobins worldwide.

  • Highly prevalent in Southeast Asia and parts of India.

2. Genetic Defect

  • Point mutation in the β-globin gene at codon 26.

  • Glutamic acid → Lysine substitution (GAG → AAG).

  • This defect also causes reduced β-globin synthesis, giving HbE a mild β-thalassemia–like effect.

3. Biochemical and Cellular Effects

  • Decreases hemoglobin stability.

  • Leads to microcytosis and slight RBC membrane abnormalities.

  • Does not cause sickling.

  • Often results in mild hemolysis.

4. Pathophysiology

  • HbE trait (HbAE):

    • Asymptomatic

    • Mild microcytosis

  • HbE disease (HbEE):

    • Mild hemolytic anemia

    • Microcytosis + hypochromia

    • Usually no major symptoms

  • HbE/β-thalassemia:

    • Clinically significant

    • Moderate to severe anemia

    • Resembles thalassemia intermedia/major

    • Requires medical management

5. Clinical Features

  • Most cases are asymptomatic (HbAE, HbEE).

  • In symptomatic individuals:

    • Mild anemia

    • Jaundice

    • Splenomegaly (occasionally)

    • Fatigue

  • HbE/β-thalassemia: growth failure, hepatosplenomegaly, bone deformities.

6. Laboratory Diagnosis

Peripheral Blood Smear:

    • Microcytosis, hypochromia

    • Target cells

    • Mild anisopoikilocytosis

Hemoglobin Electrophoresis/HPLC:

    • HbE shows a characteristic peak/band.

    • Quantitative detection of HbE and HbA2 elevation.

Genetic Testing:

    • Identifies β26 Glu→Lys mutation.

7. Inheritance

  • Autosomal recessive.

  • HbAE: Carrier

  • HbEE: Mild disease

  • HbE/β-thalassemia: Severe or moderate disease based on thalassemia mutation.

8. Management

  • HbAE and HbEE: No specific treatment needed.

  • Folic acid supplementation if hemolysis present.

  • HbE/β-thalassemia:

    • Regular transfusions (if moderate/severe)

    • Iron chelation

    • Splenectomy (selected cases)

    • Genetic counseling

Hemoglobin F (HbF)

1. Definition

  • Hemoglobin F (HbF) is the fetal form of hemoglobin, predominant during intrauterine life.

  • It gradually declines after birth and is replaced by adult hemoglobin (HbA).

2. Structure

  • HbF consists of 2 α-chains + 2 γ-chains (α₂γ₂).

  • The γ-chains differ from β-chains by multiple amino acid substitutions.

3. Physiological Role

  • HbF has a higher affinity for oxygen than HbA.

  • This allows efficient transfer of oxygen from mother to fetus across the placenta.

4. Normal Levels

  • Fetus/newborn: 70–90% of total Hb.

  • 6 months of age: <2%.

  • Adults: <1% (usually restricted to specific bone marrow cells).

5. Biochemical Characteristics

  • HbF binds 2,3-BPG (DPG) poorly, increasing oxygen affinity.

  • Enhances oxygen loading in low-oxygen fetal environment.

6. Conditions with Increased HbF

Physiological:

    • Newborns

    • Pregnancy (slight increase)

Pathological:

    • β-thalassemia major

    • Hereditary persistence of fetal hemoglobin (HPFH)

    • Sickle cell disease (especially during hydroxyurea therapy)

    • Leukemias

    • Aplastic anemia

7. Clinical Significance

  • Elevated HbF reduces sickling in sickle cell disease, since HbF inhibits polymerization of HbS.

  • High HbF improves anemia in thalassemias, but does not fully correct the disorder.

8. Laboratory Detection

  • HPLC: Quantifies HbF percentage.

  • Hemoglobin electrophoresis: Shows distinct migration of HbF.

  • Flow cytometry: Detects “F-cells” (RBCs containing HbF).

  • Kleihauer–Betke test: Detects fetal RBCs in maternal blood.

9. Inheritance

  • HbF synthesis is genetically regulated by γ-globin genes (Gγ and Aγ) on chromosome 11.

  • HPFH is inherited in an autosomal dominant pattern.

10. Clinical Relevance in Therapy

  • Hydroxyurea, decitabine, and L-glutamine increase HbF levels in sickle cell disease → reduce crises and hemolysis.

  • Gene therapy targets γ-globin reactivation to treat β-hemoglobinopathies.

Hemoglobin M (HbM)

1. Definition

  • Hemoglobin M (HbM) is an abnormal hemoglobin variant in which iron of the heme group is oxidized to the ferric (Fe³⁺) state, leading to methemoglobinemia.

  • HbM is unable to bind oxygen normally → causes impaired oxygen delivery to tissues.

2. Molecular Defect

  • Caused by point mutations in globin genes (α or β).

  • These mutations stabilize iron in the Fe³⁺ (methemoglobin) state.

  • Common variants: HbM Boston (α-chain mutation), HbM Iwate, HbM Hyde Park (β-chain mutation).

3. Biochemical Characteristics

  • Fe³⁺-containing hemoglobin cannot bind O₂.

  • Remaining normal Hb has increased oxygen affinity, shifting the oxygen dissociation curve to the left → tissues receive less oxygen.

  • Blood appears chocolate-brown or slate-blue.

4. Pathophysiology

  • Leads to congenital methemoglobinemia.

  • Persistent cyanosis from birth.

  • Usually mild symptoms, because total methemoglobin levels remain stable.

  • Oxygen therapy does not improve cyanosis.

5. Clinical Features

  • Slate-blue or chocolate-colored blood

  • Cyanosis that does not resolve with oxygen

  • Mild anemia (sometimes)

  • Headache, dizziness if methemoglobin levels are high

  • Generally no severe symptoms in heterozygous individuals

6. Laboratory Diagnosis

1. Methemoglobin levels:

    • Elevated (normally <1%)

    • HbM causes chronic elevation

2. Pulse oximetry:

    • Low oxygen saturation (~85%)

    • ABG shows normal PaO₂ → “saturation gap”

3. Hemoglobin Electrophoresis:

    • Characteristic migration patterns depending on variant

4. Spectrophotometry:

    • Absorption peak at 630 nm confirms methemoglobin

5. Genetic Testing:

    • Identifies specific α- or β-chain mutation

7. Inheritance

  • Autosomal dominant.

  • Homozygous state is rare and more severe.

8. Management

  • Most HbM variants require no specific treatment.

  • Avoid oxidant drugs (e.g., sulfonamides, dapsone, nitrates).

  • Methylene blue is ineffective in HbM (unlike acquired methemoglobinemia), because Fe³⁺ is stabilized by the structural mutation.

  • Ascorbic acid may reduce symptoms in some cases.

9. Clinical Significance

  • Important in the differential diagnosis of cyanosis with normal PaO₂.

  • Distinguished from acquired methemoglobinemia by lifelong cyanosis + family history + resistance to methylene blue.

 

Identification and Estimation Techniques

Hemoglobin Electrophoresis

  • Technique:
    • Preparation: Blood is mixed with a buffer and applied to an electrophoresis medium (e.g., agarose or cellulose acetate).
    • Separation: An electric field is applied, causing haemoglobins to migrate based on their charge and size.
    • Visualization: Separated haemoglobin fractions are stained or visualized to identify different types.
    • Quantification: Band intensity is compared to known standards or reference curves to estimate the proportion of each haemoglobin type.
  • Advantages: Reliable for identifying and quantifying abnormal haemoglobins; used for screening and diagnostic purposes.
  • Disadvantages: Requires expertise and can be affected by multiple haemoglobin variants.

High-Performance Liquid Chromatography (HPLC)

  • Technique:
    • Preparation: Blood is processed to separate haemoglobin from other blood components.
    • Separation: Hemoglobin is separated as it passes through a chromatographic column under high pressure.
    • Detection: Detected using UV or fluorescence spectroscopy as they exit the column.
    • Quantification: Peak areas or heights are used to estimate the concentration of each haemoglobin type.
  • Advantages: Highly sensitive and specific; capable of distinguishing between various haemoglobin variants.
  • Disadvantages: Requires specialized equipment and trained personnel.

Capillary Electrophoresis

  • Technique:
    • Preparation: Blood is processed and loaded into a capillary tube.
    • Separation: An electric field is applied, causing haemoglobins to migrate through the capillary based on size and charge.
    • Detection: Hemoglobins are detected as they exit the capillary tube.
    • Quantification: Peak areas or heights are analyzed to estimate the amount of each haemoglobin type.
  • Advantages: Provides high-resolution separation and rapid results.
  • Disadvantages: Requires specialized equipment and interpretation.

DNA Analysis

  • Technique:
    • Extraction: DNA is extracted from blood or tissue samples.
    • Amplification: Specific regions of the β-globin gene are amplified using PCR.
    • Sequencing or Mutation Detection: PCR products are sequenced or analyzed for known mutations using restriction fragment length polymorphism (RFLP) or allele-specific PCR techniques.
  • Advantages: Provides a definitive diagnosis by identifying genetic mutations; useful for carrier screening and prenatal diagnosis.
  • Disadvantages: Requires advanced laboratory facilities and technical expertise.

Solubility Test for Hemoglobin S

  • Technique:
    • Preparation: Blood is mixed with a reagent that causes HbS to precipitate while HbA remains soluble.
    • Detection: The appearance of a turbid solution indicates the presence of HbS.
  • Advantages: Simple and quick screening test for sickle cell disease.
  • Disadvantages: Less specific; positive results should be confirmed with more precise methods like electrophoresis or HPLC.

MCQs

1. Hemoglobinopathies are disorders affecting:

A. Structure only
B. Function only
C. Production only
D. Structure, function, or production

2. The most common adult hemoglobin is:

A. HbF
B. HbA
C. HbA2
D. HbS

3. Normal adult hemoglobin HbA has the globin composition:

A. α₂β₂
B. α₂γ₂
C. α₂δ₂
D. β₂γ₂

4. Fetal hemoglobin (HbF) is composed of:

A. α₂β₂
B. α₂δ₂
C. α₂γ₂
D. γ₂β₂

5. Abnormal hemoglobin variant HbS causes:

A. Thalassemia
B. Sickle cell anemia
C. Iron deficiency anemia
D. Hemolytic anemia unrelated to Hb

6. The amino acid substitution in HbS is:

A. Glu → Lys
B. Val → Glu
C. Glu → Val
D. Lys → Glu

7. HbC is caused by substitution of glutamic acid by:

A. Lysine
B. Valine
C. Histidine
D. Arginine

8. Hemoglobin E (HbE) results from a β chain mutation causing:

A. Glu → Lys substitution
B. Val → Glu substitution
C. Lys → Glu substitution
D. No chain change

9. Hb Barts consists of:

A. α₄
B. β₄
C. γ₄
D. δ₄

10. Hb Barts has very high affinity for oxygen, making it:

A. Efficient at releasing O₂
B. Inefficient at releasing O₂
C. Normal in function
D. Only present in adults

11. Thalassemias arise from:

A. Structural change in Hb
B. Defective globin synthesis
C. Iron overload only
D. Viral infection

12. α-Thalassemia is caused by:

A. Mutations in β-globin only
B. Deletions of α-globin genes
C. Iron deficiency
D. Mutations in δ-globin gene

13. β-Thalassemia major results when:

A. One β-globin gene is deleted
B. Both β-globin genes are severely mutated
C. Only α-globin genes are deleted
D. Only HbF is increased

14. Hydrops fetalis with Hb Barts occurs when:

A. One α gene is deleted
B. Two α genes are deleted
C. Three α genes are deleted
D. Four α genes are deleted

15. Hemoglobin H disease occurs with deletion of:

A. One α-globin gene
B. Two α-globin genes
C. Three α-globin genes
D. Four α-globin genes

16. Patients with β-thalassemia trait typically show increased:

A. HbA only
B. HbA₂
C. HbF only
D. HbS

17. Hemoglobin electrophoresis is used to:

A. Measure iron levels
B. Identify abnormal hemoglobin variants
C. Determine blood type
D. Detect white cell counts

18. A compound heterozygote for HbS and HbC results in:

A. Normal phenotype
B. HbSC disease
C. β-Thalassemia
D. Iron deficiency anemia

19. Hereditary persistence of fetal hemoglobin (HPFH) is:

A. A benign condition
B. Severe anemia
C. Iron overload disorder
D. Acquired during adulthood

20. Increased HbF in sickle cell disease tends to:

A. Worsen symptoms
B. Improve symptoms
C. Cause iron deficiency
D. Cause thalassemia

21. Hemoglobin variants can be detected by:

A. PCR only
B. Electrophoresis or HPLC
C. CBC only
D. Iron studies

22. Hemoglobin S (HbS) polymerizes when:

A. Fully oxygenated
B. Deoxygenated
C. In the presence of iron
D. In high pH

23. Sickle cell trait (heterozygous HbAS) typically has:

A. Severe disease
B. No symptoms or mild symptoms
C. Iron deficiency
D. Thalassemia

24. HbE is most common in populations from:

A. Europe
B. Southeast Asia and South Asia
C. North America
D. Australia

25. HbC can cause mild hemolytic anemia when:

A. Homozygous
B. Heterozygous
C. Only with iron deficiency
D. Only with thalassemia

26. The term “hemoglobinopathy” refers to:

A. Any anemia
B. Any blood disorder
C. Disorders of Hb structure or synthesis
D. Infectious diseases

27. Thalassemia major is characterized by:

A. Mild anemia
B. Severe anemia requiring transfusions
C. High HbA levels
D. No symptoms

28. Target cells on peripheral smear are seen in:

A. Thalassemia
B. Iron deficiency only
C. Leukemia only
D. Aplastic anemia

29. Sickle cell crises are precipitated by:

A. Alkalosis
B. Hypoxia and acidosis
C. Low temperature only
D. High oxygen levels

30. Bone deformities in thalassemia patients are due to:

A. Iron deficiency
B. Marrow hyperplasia
C. Infection
D. Vitamin D deficiency

31. Hemoglobin D is a variant that is:

A. Always severe
B. Usually mild or asymptomatic
C. Only found in thalassemia
D. Only found with iron deficiency

32. Deletions of α-globin genes are best detected by:

A. CBC only
B. Molecular genetic tests
C. Serum iron studies
D. Bone marrow biopsy

33. HbA₂ is composed of:

A. α₂δ₂
B. α₂β₂
C. α₂γ₂
D. β₄

34. Hemoglobinopathy distributions often overlap with:

A. Malaria-endemic regions
B. Arctic regions
C. North Europe only
D. High altitude areas

35. HbF binds oxygen more strongly because:

A. It has more iron
B. Less interaction with 2,3-BPG
C. It is larger
D. It is only in adults

36. A high level of HbF in an adult may be due to:

A. Iron deficiency
B. HPFH
C. Acute infection
D. Vitamin B12 deficiency

37. Beta-thalassemia minor usually presents with:

A. Microcytic anemia
B. Macrocytic anemia
C. Normocytic anemia
D. Leukocytosis

38. In β-thalassemia major, the majority of hemoglobin is:

A. HbA
B. HbF
C. HbA₂
D. HbS

39. Hemoglobinopathies are typically inherited in:

A. Autosomal dominant
B. Autosomal recessive
C. X-linked recessive
D. Mitochondrial

40. Hemoglobin S polymer formation leads to:

A. RBC dehydration
B. Sickling and vaso-occlusion
C. Increased oxygen delivery
D. Iron overload

41. HbO is a rare hemoglobin variant characterized by:

A. Substitution of Glu by Lys in β chain
B. Excess γ chains
C. Iron deficiency
D. No functional change

42. HbH (β₄) forms in:

A. β-thalassemia
B. α-thalassemia with three gene deletions
C. Normal adults
D. Sickle cell trait

43. Hemoglobin electrophoresis separates variants based on:

A. Size only
B. Charge differences
C. Oxygen affinity
D. Iron content

44. Hb Bart’s (γ₄) appears in:

A. Normal adults
B. Severe α-thalassemia (hydrops fetalis)
C. Mild thalassemia
D. Iron deficiency anemia

45. A compound heterozygote for HbE and β-thalassemia may present as:

A. Normal
B. Moderate to severe anemia
C. Iron deficiency
D. Only in children

46. Distinct hemoglobin variants may affect:

A. HbA1c assays
B. Oxygen affinity
C. RBC lifespan
D. All of the above

47. HbSC disease is usually:

A. More severe than HbSS
B. Milder than HbSS
C. Unrelated to sickle disease
D. Only in females

48. Increased HbA₂ is a marker of:

A. β-thalassemia trait
B. α-thalassemia
C. Sickle cell trait
D. Iron deficiency only

49. Hemoglobin variants are best diagnosed by:

A. CBC only
B. Hb electrophoresis/HPLC
C. Bone marrow biopsy
D. Serum iron

50. Hemoglobinopathies can be prevented by:

A. Iron supplements
B. Genetic counseling and carrier screening
C. Vaccines
D. Blood transfusions

Answer Key

1-D
2-B
3-A
4-C
5-B
6-C
7-A
8-A
9-C
10-B

11-B
12-B
13-B
14-D
15-C
16-B
17-B
18-B
19-A
20-B

21-B
22-B
23-B
24-B
25-A
26-C
27-B
28-A
29-B
30-B

31-B
32-B
33-A
34-A
35-B
36-B
37-A
38-B
39-B
40-B

41-A
42-B
43-B
44-B
45-B
46-D
47-B
48-A
49-B
50-B