Alpha Chain Disorders

Answer 1

==> Beta thal intermedia
Hb 60 - 100g/L, MCV ~60
HbH disease has a higher retic count and lack of nRBCs compared to beta thal intermedia.

Question 2

List possible causes of HbH disease

Answer 2

–/-α
α cs / α cs
–/α cs
–/α nd
- nd / - nd (homozygous for non-deletional alpha thal)
Non-deletional alpha thalassaemias (point mutations in a2) can give rise to a more severe reduction in alpha chain due to the lack of compensatory increase from the a1 gene.

Question 3

Most common mutations for a+ thalassaemia

Answer 3

The most common mutations are α3.7 and α4.2

Question 4

Most common mutations for a0 thalassaemia

Answer 4

–SEA, –FIL, –THAI, –MED

Question 5

Diagnosis of HbH disease (indices, morphology, stain, Hb electrophoresis/HPLC)

Answer 5

1. FBC with indices: Hb 60-100 g/L, MCV 60, raised RBC count, low MCHC
2. Film - marked anisopoik, targets, micro hypo BUT lack of nRBCs and minimal basophilic stippling
3. HbH inclusion bodies on supravital stain
4. Hb electrophoresis and HPLC shows HbH (~10-40%), Hb Barts (1-5%) and reduced or normal HbA2.

Question 6

Gene commonly mutated in acquired HbH disease

Answer 6

ATRX

Question 7

What is Hb Constant Spring?

Answer 7

Hemoglobin Constant Spring (Hb CS) is an abnormal Hb caused by a mutation at the termination codon of an α2-globin gene, TAA—CAA. This mutation leads to the synthesis of unstable and elongated α-globin chains with 172 instead of 141 amino acid residues.
Hb CS is a variant alpha globin (non-deletional alpha thal) with reduced chain synthesis (thalassaemic haemoglobinopathy). Results in anaemia with MCV in high 70s.
Prominent basophilic stippling.

Question 8

Why do non-deletional alpha thalassaemias cause a more severe anaemia?

Answer 8

The non-deletional alpha thalassaemias are usually found on α2 gene. They give rise to a more severe reduction in alpha chain production due to the lack of compensatory increase from the α1 gene.

Question 9

Pathophysiology of HbH disease and HbH inclusions

Answer 9

• Greatly reduced rate of synthesis of alpha chain leading to beta tetramers (HbH) which have very high oxygen affinity.
• These tetramers are soluble in the bone marrow and therefore erythropoiesis is more effective than in beta thalassaemia.
• HbH is unstable and precipitates in red cells as they age (HbH inclusions seen on a supravital stain such as Brilliant Cresyl Blue)
• The inclusion bodies/precipitates cause red cell membrane damage and obstruction in the spleen leading to shortened RBC survival and haemolysis.

Question 10

Causes of worsening of anaemia in HbH disease (5)

Answer 10

1. Infection
2. Oxidative drugs
3. Pregnancy
4. B12 or folate deficiency
5. Aplastic crisis from Parvovirus B19

Question 11

Red cell indices and morphology in HbE disease

Answer 11

Hb 80 up to normal
MCV 60s
Target cells, irregularly contracted cells
**ddx is beta thal trait, beta thal/HbE and iron deficiency

Question 12

What is the definition of thalassaemia?

Answer 12

Reduced rate of globin chain synthesis resulting in an imbalance of alpha and non-alpha chains

Question 13

What is a haemoglobinopathy?

Answer 13

Broad term encompassing both thalassaemia and haemoglobinopathies. Refers to a structurally abnormal globin chain which may or may not be clinically significant.

Question 14

How do you perform an HbH inclusion body stain?

Answer 14

Two drops whole blood + 2 drops of brilliant cresyl blue
Incubate 2 hours
Make 2 slides and examine for 20 mins (10mins each)
N.B. the vital stain causes oxidative denaturation and precipitation of the unstable beta tetramers

Question 15

What conditions should be detected as part of antenatal screening?

Answer 15

1. Significant maternal haemoglobinopathies (important for maternal antenatal care)
   - Sickle cell disease: SS, SC, HbS/B
   - B thal major and intermedia
   - HbH disease
2. Maternal conditions requiring partner screening that will result in a significant disorder in the fetus:
   ==> S, C, D-Punjab, E, O-Arab, A-Lepore, Bthal trait, dB trait, a0 trait (–/aa)
   - SS
   - SC
   - S/Bthal
   - SE
   - SD-Punjab
   - SO-Arab
   - S/Lepore
   - S/dBthal
   - Hb bart’s hydrops fetalis
   - B thal major
   - E/B thal

Question 16

What is the difference between a0 and a+?

Answer 16

Thalassaemias can cause a complete absence of globin chain synthesis (α0 and β0) or globin chain synthesis at a reduced rate (α+ and β+)

Question 17

Inheritance and roles of a1 and a2 genes

Answer 17

• α1 and α2 are inherited as linked pairs. If one of the genes is deleted, there is slight upregulation of the other gene.
• α2 is responsible for about 70% of α chain production

Question 18

What is a0?

Answer 18

αo = –/aa (both alpha genes (α1 and α2) are deleted on the same chromosome so no alpha chains are produced from the linked pair)

Question 19

What is a+?

Answer 19

Usually results from deletion of all or part of the α2 globin gene
α+ = -a/aa (one of the two genes is deleted on a single chromosome)
Homozygosity for α+ = -a/-a

Question 20

How is alpha thalassaemia trait diagnosed?

Answer 20

• a/aa = silent, may have mild hypochromia, requires molecular diagnosis
• a/-a = microcytic hypochromic anaemia, Hb electrophoresis and HPLC are normal, requires molecular diagnosis

–/aa = microcytic hypochromic anaemia, Hb electrophoresis and HPLC are normal, Hb Barts can be detected on alpha thal IC strip test, very occasional HbH inclusions may be seen, requires molecular diagnosis

Question 21

How is HbH diagnosed?

Answer 21

Diagnosis of HbH Disease:

1. Blood count and blood film:

Hb 60 - 100 g/L but can be as low as 30 g/L at times of stress

MCV ~60 fL

Blood film shows striking anisopoikilocytosis including targets, fragments and tear drops. Basophilic stippling and nRBCs are not prominent compared to beta thal.

Ddx = beta thal intermedia (distinguish by high retics and lack of nRBCs in HbH)

2. HbH Inclusions
   - Numerous inclusions are seen on incubation with brilliant cresyl blue.
3. Alkaline Electrophoresis and HPLC:
   - HbH usually ~10% but can be up to 40% if there is a non-deletional alpha gene defect
   - HbBarts is present in some patients (1 - 5%)
   - Normal or reduced HbA2.

Question 22

Diagnosis of Hb Constant Spring

Answer 22

More marked anaemia than a+ thal, MCV not proportionately reduced

Basophilic stippling prominent

Diagnosis of Hb CS:

• Alkaline electrophoresis = band between carbonic anhydrase and HbA2
• HPLC = band in the C window

Question 23

Diagnosis of alpha thalassaemia (from trait to HbH disease)

1. Indices
2. Film
3. Alpha thal ICT
4. Suprvital stain for HbH cells
5. HPLC