Haemoglobinopathy Diagnosis (Thalassaemia International, GTG 2014)
N.B. Limited text functions on this site have made this page a challenge, all Greek characters have been swapped back into the Latin.
Hbpathies are the commonest recessive monogenic disorders worldwide
Thalassaemias – Reduced production of haemoglobin E.g. a/b/d/e/g-thal
Haemoglobin variant – Altered structure of haemoglobin. E.g. HbS/D/O (b), HbG (a)
Thalassaemic Hb variant – E.g. Hb Constant Spring (a-chain), Hb Lepore, Hb E (b-chain)
Chromosome 16 - a1, a2, z genes
Chromsome 11 - b, d, e, g genes
Homozygous a0 - China, Taiwan, SE Asia, Cyprus, Greece, Turkey, Sardinia
b-Thal - Anywhere other than North Europe
HbS - Africa, Greece, Southern Italy, Turkey, Middle East, India
HbE - SE Asia
HbC - West Africa
-a/aa a+ Trait
Hb normal, MCV low, MCH low. Normal electrophoresis
-a/-a Homozygous a+ Trait
Hb normal, MCV low, MCH low (<25). Normal electrophoresis
--/aa a0 Trait
Hb normal, MCV low, MCH low (<25). Normal electrophoresis
--/-a+ HbH Disease
Hb 70-110, microcytic, hypochromic
Results in production of HbH from 4 b chains. Detectable by electrophoresis, unlike the traits.
Spectrum of clinical phenotype from asymptomatic to severe anaemia and even hydrops.
--/-- Hydrops Fetalis
Incompatible with fetal life due to absence of a chain synthesis.
Results in production of Hb Barts from 4 g chains.
aTa/aTa Non-deletional a-thal
E.g. Saudi T
In ‘standard’ deletional a+ thal the remaining genes are upregulated to compensate.
In homozygous aTa/aTa this does not happen --> as a result HbH disease can occur even with two functioning a genes.
-/b b-Thal Trait
Hypochromic, microcytic anaemia with a raised HbA2
-/- (b0) or -/partial (b+) b-Thal Major
1 in 4 births where both parents are -/b
Mutations --> deletion of b gene, d&b genes or even b, d & g genes
Frequently the result of inheritance of two different mutations, i.e. compound heterozygotes.
a chains still produced in normal quantities and so become present in excess --> pathology of b-Thal
Severe anaemia at 3-6 months when the switch from HbF to HbA occurs
Hepatosplenomegaly, Bone expansion, Iron Overload, Infection, Osteoporosis
?When to start – case by case decision
Vol/Freq – aim pre-Tx Hb 90-100, often equates to 2-3 units per month in adults.
Classified as d0 and d+, similar to b-Thal.
No clinical significance other lowering the HbA2 and so compromising the diagnosis of a co-inherited b-Thal.
Classified as db0 and db+, depending on residual output of chains from the affected chromosome.
Includes Hb Lepore – a fusion mutation of the d&b genes.
Rare, results from large deletions
Severe haemolytic, hypochromic anaemia at birth. Improves after age of 3-6 months.
Only reported in heterozygotes. Homozygous state thought to be lethal at early fetal stage
Recommended to offer to at risk groups, i.e. most non-Northern European ethnicities.
If abnormality detected, partner should be tested
Purpose is to Detect:
1. Significant maternal hbpathy - SS, SC, S/b, HbH, b-thal intermedia
2. Maternal carrier states - AS, AC, AD, AE, AO, ALepore, b-trait, db-trait, HPFH,
homozygous a0 trait
High prevalence areas = ≥1.5 SCD per 10,000 pregnancies --> universal lab screening
Low prevalence areas = <1.5 SCD per 10,000 pregnancies --> FOQ to direct lab screening
Screening reports must be available within 3 working days
All newborns screened for SCD, and any <1 year old arriving in the country.
Aimed at early diagnosis to prevent long term morbidity
May or may not detect other hbpathies.
Low Prevalence Area Screening Algorithm Simplified
- Cellulose Acetate or Agarose Gel (pH 8.2-8.6)
- Hb negatively charged and will move towards the positive anode
- A split A2 band is suggestive of an a chain variant
- Citrate Agar or Agarose Gel (pH 6.0-6.2)
- Hb complexes with agaroprotein and moves towards the positive anode
- Non-complexed Hb will move toward the negative cathode.
- Used to distinguish S from D, and C from E & O-Arab
Sickle Solubility Test
Purchased as a commercial kit
Detects HbS down to a concentration of approx. 20%
False negatives – Anaemia, Infancy, Recent Transfusion
False positives – Paraprotein, Hyperlipidaemia, Heinz Bodies, Leukocytosis
HbH precipitates with brilliant cresyl blue (a ‘supravital’ stain)
Results in golf ball cells and Heinz bodies (if hyposplenic)
high performance liquid chromatography (HPLC)
1. Mixture of molecules (normal and variant Hb) with a net positive charge are adsorbed onto a negatively charged column.
2. These are then eluted off in a mobile phase – a liquid containing increasing concentrations of cations flows through the column, competing for the anionic binding sites.
3. As the positive Hb molecules are eluted off they are detected optically and the retention time is recorded. The area under the peak can quantify each Hb.
Advantages over Electrophoresis
- Less labour intensive
- Very small sample required
- Quantification possible
- Greater range of Hb’s identified
- A2 detected and quantified à easier to diagnosis b-thal
Disadvantages compared to Electrophoresis
- High capital and reagent costs
- Each haemoglobin present can undergo ‘post-translational modification’, usually glycation, which affects its charge and so appears as a separate peak to the left of the original Hb.
- Commonest example – the glycated A peak will increase in diabetes (HbA1c)
- In SCD, there is a factitious rise in A2 as the glycated S sits in the A2 window.
Low A2, 0-2%, Normal FBC
d-variant (e.g. A2 prime) will produce to 2 equal peaks, in the A2 and S windows (‘Split A2’)
a-variant (e.g. G-Philidelphia) will produce to 2 unequal peaks (‘Split A2’)
Normal A2, 2-3.5%, Normal FBC
Pitfall: Silent b-thal mutations (e.g. +1480(CàG))can have a normal HbA2
Pitfall: Delta variants (e.g. A2 prime) give a peak in the S window. Need to add the normal and variant A2 together to get total, otherwise might miss a b-thal.
Pitfall: The HPLC trace baseline can wander up, giving an underestimated A2.
Normal A2, 2-3.5%, low MCH
a-thal trait Indistinguishable from iron deficiency
a0 and a+ Indistinguishable on HPLC
egdb-Thal Once an adult, indistinguishable from a and iron def.
Therefore, if MCH <25 and a0 possible based on ethnicity à DNA Analysis
Raised A2, >3.2% (b chain disorders)
≥3.5% + MCH <27 Heterozygous b-thal
>4% + MCH normal Mild b-thal
>10% Hb Lepore heterozygous
>15% Hb E
Co-existence of a-thal will reduce A2 by 5% for each missing a gene.
E.g. HbE typically shows HbA of 30%. HbE/a+ homozygous will have 20% HbA2
Pitfall: Normal individuals with low MCH due to iron deficiency or a-thal trait, may have a HbA2 of 3.5-4% due to hyperthyroidism or drugs for HIV.
Raised HbF, >0.8%
>5% + MCH <27 Could be heterozygous db-thal
>5% + MCH normal Hereditary Persistance of Fetal Haemoglobin (HPFH)
- Deletional – results from db chain deletions
- Non-deletional – may be a beneficial modifier
Pitfall: HPFH often associated with a co-inherited a-thal and so MCH not a reliable means to differentiate between HPFH and db-thal.
40% S trait (40% not 50% as a chains prefer the normal b chains)
HbS % will rise if S trait combined with b-thal as a chains can no longer preferentially bind with the normal b chains. Recently transfused patients will have a lower HbS%
Pitfall: S, C and E can carry over from previous sample and appear in the next patient’s trace.
Extra Notes on Variants
b-variants (S, C, E, D, O) will make up roughly 50% of Hb (2 genes)
a-variants (G) will make up roughly 25% of Hb (4 genes)
If an a-variant and b-variant co-exist three peaks will form – the a, b and hybrid.