Haemoglobinopathies

Question 1

Thal/haemoglobinopathy pathophysiology symptoms

Answer 1

Decreased red blood cell survival   + Hb production  

 Excess of the unaffected globin chain forms unstable homotetramers that precipitate  

Result in earlier precipitation (in the RBC life span) –> marker RBC damage and severe haemolysis associated with ineffective erythropoiesis (IE) and extramedullary haemolysis  

1. Ineffective erythropoiesis:   with extramedullary haematopoeosis:  
   Distortion of the cranium, facial and long bones  
   LAD  
   Hepatosplenomegaly 
   Extramedullary tumors (some cases) 

2.Chronic hypoxia and anemia  
- Resultant increased iron absorption  (from the GI tract)  

3. Iron overload  
   - Ineffective erythropoiesis 
   - Chronic transfusions  

  

Question 2

HbH disease

Answer 2

Alpha thal
3 gene deletion

Formation of B tetramers (HbH)

HbH - high oxygen affinity and ineffective at O2 delivery and reduced red cell survival

Blood film:
- Marked hypochromic, microcytic anaemia
Anisopoikilocytosis with target cells, teardrop cells and fragments
Basophilic stippling
Polychromasia

HPLC
- Low A2
- variant Hb with very short retention time

Question 3

Gel electrophoresis

Answer 3

Utilises the charged properties of haemoglobin (zwitter ion) and its variants to replicate characteristic mobility patterns on both alkaline and acidic gels.

Cellulose acetate is then stained and comparing the distance each Hb has migrated with known Hb motilities, the bands can be identified.

Question 4

Alkaline gel electrophoresis

Answer 4

pH 8.2-8.6

Hb is negatively charged and migrates towards anode.

C S F A

C: C E O-Arab, A2

S: S, G, D (iran and punjab), Lepore, Q India

Slow band : constant Spring

Fast bands - H, Barts

Question 5

Acid gel electrophoresis

Answer 5

pH 6.2

Hb behaves as positively charged

Able to separate out Hb C and S

F A S C

Most things now run with A!
A: A, A2, E, D, G , Lepore, H, J

Question 6

Haemoglobins during life

Answer 6

HbF - 2alpha 2gamma
> embryo., fetus and neonate

HbA - 2alpha2beta
> minor component in fetus, major hb as adult

HbA2 - 2alpha2delta
> low levels in infancy and as adult

By 6 months of age = majority of Hb is HbA

Question 7

Causes of increased A2

Answer 7

Inherited
- beta thal traits
- HbS and HbE
- hereditary high HbA 2
- unstable hb
- CDA

Acquired
- thyrotoxicosis
- megaloblastic anaemia (b12/folate def)
- HIV infection

Question 8

Causes of reduced A2

Answer 8

Inherited
- HbH disease
- delta thal
- a+ thal homo and a0 hetero

Acquired
- iron def
-AoCD
- sideroblastic anaemia
- AML/aplastic anaemia
- hypothyroidism

Question 9

Major Haeomoglobinopathies

Answer 9

Question 10

Alpha thalassemia

Answer 10

4 alpha genes
2 a1 and 2 a2

Will have microcytic indices

Mutations in a2 gene have more severe phenotypes than mutations in a1 genes

When alpha thal trait is coinherited with a beta abnormalities of globin chain synthesis –> preferentially bind to normal beta chains –> reducing proportion of betal thal variants

Question 11

Beta thalassemia

Answer 11

B0 and B+

Beta thal trait
- B thal heterozygosity

Beta thal major - transfusion dependent
- Homozygosity B0/B0
- Compound heterozygote

Beta intermedia - clinical features between trait and major
- Heterozygoust B0 with coexisting alpha thal trait
- Homozygosity or compound heterozygosity of mild B+

Question 12

Beta thal trait

Answer 12

Mildly reduced Hb, increased RBC, reduced MCH and MCV. MCHC normal

Increased A2
Can have increased HbF

Can also have silent beta thal (normal indices and normal hbA2)

Question 13

Beta thalassemia major

Answer 13

Homozygous beta thal or compound heterozygous beta/variants (B0/Bvariant)

No normal beta globin chain synthesis –> free alpha chains accumulate in erythrocytes which are unstable and cytotoxic causing ineffective erythropoiesis and premature haemolysis.

Film:
marked microcytic hypochromic anaemia with increased poly, nRBC and basophilic stippling

HbA absent, HbA2 usually normal or can be reduced

Question 14

Alpha thalassemia vs Beta thalassemia

Answer 14

Question 15

Hereditary persistence of fetal haemoglobin (HPFH)

Answer 15

Increased HbF, persisting after 2 years
> failure to silence y-globin gene expression

Results from deletions within beta globin cluster or from promoter mutatioons.

Little or no imbalance of globin chain synthesis

NORMAL red cell indicies

Heterozygous HPFH shows 15-35% of HbF

Has therapeutic relevance because HbF can reduce disease severity in conditions like sickle cell anemia.

Question 16

HbS and its interaction with other variant haemoglobins and with thalassemia

Answer 16

HbS - on deoxygenation –> solubility is reduced –> polymerisation –> sickle shape.
Sickle cells show reduced deformability and increased adhesion to endothelial cells –> vascular occlusion

Partial protection from falciparum malaria infection in heterozygote.

Sickle cell trait: HbS (BA and BS)

Sickle cell anaemia: HbS (BSBS); sickle cell disease

Compound heterozygotes: HBSC or HbS/B

Question 17

Sickle cell trait

Answer 17

BA/BS

FBE film normal
(unless with concurrent alpha thal)

Diagnosis:
> HbA and HbS on HPLC

Interactions:
Thalassemias -
- alpha thal: % of HbS is lower
- beta thal: % of HbS is more than 50%

Hb Variants:
- alpha variant: Hybrid Hb forms
- beta variant; compound heterozygote with no HbA

Question 18

HPLC (High Performance Liquid Chromatography) MoA

Answer 18

EDTA sample, lyse red cells

All Hb released from red cells into reagent

haemolysate injected into HPLC column. Column contains a stationary phase (weakly cationic) which interacts with the haemoglobins as Hb weakly negative.

Separation by charge: A buffer solution (mobile phase) flows through column, as buffer composition changes (pH or salt conc) different Hbs detach from column at different retention times.

Time at which elution occurs is recorded - and graphed on chromatogram.

Peaks are identified by retention time and AUC - correlated with a specific Hb type

Question 18

HPLC vs CE

Answer 18

Question 18

Capillary electrophoresis

Answer 18

Separate haemoglobins based on their charge-to-size ratio in an electric field, using a thin capillary tube filled with buffer.

Whole EDTA sample is lysed to release Hb.
Lysate diluted with buffer and loaded into capillary tube with electrodes places at both ends of capillary.

Hemoglobins which have different charges and sizes, migrate through the buffer at different speeds toward the detector

As hemoglobins reach the detector window, they are recorded via UV absorbance,

Results are displayed as peaks (electropherogram)

Question 19

Triple alpha

Answer 19

ie aaa/aa

Usually of no consequence unless co-inherited with a beta thal trait - can increase severity of beta thal

Question 19

Alpha thalassaemia

Answer 19

Reduced synthesis of alpha chains

Deletional alpha chains: can be a0 or a+, depending on the length and nature of the deletion

Non-deletional alpha thal: most mutations in a2 genes.

Non-deletional alpha thals show MORE severe phenotypes than deletional alpha thalassaemias as NO upregulation of the alpha 1gene.

Mutations in a2 gene have more severe phenotypes than mutations in the a1 gene.

Question 20

Alpha thal table summary

Answer 20

Question 21

Alpha thal trait

Answer 21

a+ heterozygosity, homozygosity and a0 heterozygosity.

Thalassaemic red cell indices, normal HbA2 and F, MCH<25

In neonatal period - elevated HbBarts (gamma tetramer) suggestive of a0 trait or a+ homozygosity. Hb Barts will then disappear by 3-6 months of age when HbF reaches adult levels.

Coinheritance with other abnormalities of globin chain synthesis:
- beta thal: lessens the laboratory and clinical abnormalities
- beta variants: proportion of variant Hb (Hb S, Hb C and Hb E trait) is reduced.
- alpha variants: proportion of variant Hb is increased

Question 22

Portion of variant haemoglobins

Answer 22

Alpha - 25% (4 genes) - >25% if from alpha 2 gene mutations - <25% if from alpha 1 gene mutations. Beta - 50% (2 genes) - <50% if variant globin chains are positively charged, and even lower if coexisiting alpha thal - >50% of variant globins are negatively as they have a higher affinity to alpha chains. Alpha2 is produced in a 3:1 ratio to alpha1 - thus alpha2 deletions (-4.2 deletions) this causes a more severe phenotype even if the same number of genes are deleted

Question 23

Conditions not detected by screening HPEPG testing

Answer 23

Alpha thal traits Silent beta thalassaemia trait Unstable or altered O2 affinity haemoglobin variants Combined alpha, beta and delta thalassaemia leading to normal MCV/MCH and normal HbA2.

Question 24

Neonatal screening

Answer 24

Main objective is for identification of thalassemia major or Sickle cell disease and improve outcome by provision of early management. Cord blood/capillary venous sample What can be detected: 1. Alpha thalassaemia (trait and major). 2. Haemoglobin variants (alpha and beta) 3. Major beta gene abnormalities What can be difficult to detect: 1. beta thal trait 2. delta thal/variants 3. HPFH 4. Compound hetero of beta gene abnormality

Question 25

Sickle solubility test

Answer 25

EDTA whole blood --> packed cells >0.5 Hct to avoid false negativity Principle: A mixture of HbS in a reducing solution (e.g. sodium dithionite) gives a turbid appearance because of precipitation of HbS vs normal Hb gives a clear solution QC: known positive and negative patient controls Interpretation: ALWAYS in conjunction with either gel electrophoresis or an alternative technique, in order to (1) distinguish sickle cell trait, anaemia and compound heterozygosity and (2) exclude false positives and negatives Does not differentiate between homozygotes, compound heterozygotes or heterozygotes False positive: leukocytosis, paraproteinaemia, unstable Hb, high lipids False negative: infants <6 months (predominance of Hb F), anaemia, Hb S<20%

Question 26

Unstable haemoglobins

Answer 26

"Unstable haemoglobin" refers to variant haemoglobins that cause clinically identifiable haemolysis with Heinz body formation Haemoglobin instability can arise from either an unstable globin chain or unstable haemoglobin molecule >100 Hb Köln is the most common Most present in the heterozygous state - except for Hb Bushwick, which presents in the homozygous state (and is clinically silent in heterozygotes) 1/3 of unstable haemoglobins occur from sporadic mutations, with no family history

Question 27

HPLC retention times

Answer 27

Question 28

Gap PCR

Answer 28

-Specific molecular testing for alpha thalassaemia - DNA extracted -Primers added for alpha globin genes -Most common alpha globin mutations are deletions (4.2 and 3.7 but there are many others such as MED, SEA etc.) -If a deletion is not present, no PCR product will be made as the resulting complementary strand is too long to create -If a deletion is present, a complementary DNA strand will be made of (likely) known length -PCR product run through gel electrophoresis to separate DNA strands by size with a control that it can be correlated to If Gap PCR is inconclusive then can proceed to MLPA or sanger sequencing

Question 29

MLPA

Answer 29

-DNA extracted from white cells from patient blood -Primers added for DNA fragments of certain lengths depending on mutation that is present -If no deletion, then primers will not be able to ligate and no PCR product made -If deletion is present, appropriate complementary strand will bind and create a complementary DNA strand that can then be measured

Question 30

Sanger sequencing

Answer 30

-Patient DNA mixed with nucleotides, some of which are fluorescently labelled that result in cessation of replication of DNA strand but emit light -Light released detected and corresponds to specific nucleotide being added at specific point of DNA replication -Can sequence entire alpha 1 and alpha 2 gene as well as flanking regions to determine mutations that may be present