HAEMATOLOGY
Thalassaemias: detection,
characterisation and
laboratory interpretation
The detection and characterisation of thalassaemia is one of the more
complex roles undertaken by the haematology department.
Here, Everdina Youssef provides an overview of current practice.
Laboratory tests for the thalassaemias range
from inexpensive, rapid screening tests to
innovative research and the application of
proteomics. The thalassaemias refer to a
diverse group of haemoglobin disorders
characterised by reduced synthesis of one or
more of the globin chains (α, β, γ, δβ, γδβ, δ
and εγδβ‚). Screening may be carried out as
part of a well-defined screening programme
or be an ad hoc or opportunistic test.
Automated high-performance liquid
chromatography (HPLC) is increasingly used
and haemoglobin electrophoresis less with
isoelectric focusing (IEF) being largely
confined to screening programmes and
referral centres, particularly in newborns.
Capillary electrophoresis (CE) is being
increasingly used.
These laboratory tests, associated with
additional data, permit only a presumptive
diagnosis with definitive diagnosis requiring
either DNA or protein analysis (eg tandem
mass spectrometry). Analysis may identify
deletional or non-deletional globin chain
synthesis defects and/or α-globin gene
triplications. Additional family studies may be
indicated for clarification of the genetics and
characterisation of the thalassaemias.1,2
SCREENING TESTS
The single-tube osmotic fragility test is
potentially useful in under-resourced
laboratories although it cannot replace
automated red cell indices using electronic
counters.3 The microcytic red cells in
thalassaemia have a low surface area to
JUNE 2012
volume ratio and therefore resist lysis when
placed in a hypotonic saline solution.
Erythrocytosis is a feature of thalassaemia,
and red cells tend to be more microcytic than
in iron deficiency anaemia (IDA), while the
level of anisocytosis is higher in IDA patients.
Electronic blood counter indices incorporate
mean cell volume (MCV), mean cell
haemoglobin (MCH), red blood cell count
(RBC), haemoglobin (Hb) or red cell
Hb E trait
β-thal trait
Hb AE Barts/CS
Hb H/CS
Hb H
Hb E/ββ-thal
Fig 1. Starch gel electrophoresis at pH 8.6
showing HbH as a fast-moving band.
ARTICLE
distribution width (RDW) in various
combinations to amplify the differences of the
single parameters in both anaemias. These
indices can be effective for use as a
preliminary screening tool4 when an
appropriate cut-off threshold is chosen.
Screening by automatic cell counter-based
indices and formula is rapid and allows
selection of samples for further investigation.
The effectiveness of the URIT-2900
haematology analyser for screening
haemoglobinopathies commonly found in
South East Asian populations was examined.
Appropriate cut-off values of MCV and MCH
for screening α0- and β-thalassaemias were
derived from the receiver operator
characteristic (ROC) curve conducted
initially on 279 subjects with various
thalassaemia genotypes. Validation was
performed additionally in a cohort of another
unrelated 313 subjects. The best cut-off
values of MCV and MCH were found to be
78 fL and 27 pg, respectively.5
In countries with inadequate diagnostic
resources, babies with β-thalassaemia major
may be transfused repeatedly without a
diagnosis having been made. When optimal
diagnostic pathways have not been followed, it
may be a bone marrow aspirate6 that first
suggests the diagnosis of β-thalassaemia
major, the marrow aspirate showing erythroid
hyperplasia, dyserythropoiesis, basophilic
stippling and the presence of α-chain
inclusions in erythroblasts.
PRIMARY DIAGNOSTIC
LABORATORY TESTS
Firstline tests include HPLC, cellulose acetate
membrane electrophoresis at alkaline pH
(CAM) (Fig 1) or capillary electrophoresis
(CE), with quantification of haemoglobins F
and A2;2 δ chains are specific for HbA2, and
differ from the similar β chains by only 10
residues between positions 22 and 115,
THE BIOMEDICAL SCIENTIST 363
 ARTICLE
HbS carriers
δ-thalassaemia and δ-variants
δ/ββ-thalassaemia
α-thalassaemia combinations
β/α
‘Classic’ high HbA2 β-thalassaemia carriers
Normal HbA2 β-thalassaemia carriers
α-/--) α-thalassaemia
(α
αα/--) α-thalassaemia
(α
α-/-α
(α α) α-thalassaemia
αα/-α
(α α) α-thalassaemia
Iron depletion
Reference range
0 1
Fig 2. Specificity and overlapping of HbA2 values measured in different cohorts of patients.9
It should be noted that the normal range depends on the method used.
resulting in a higher isoelectric point with
respect to Hb A.7
Several Hb variants display electrophoretic
and cation exchange properties similar to
those of HbS. A solubility test specific for
HbS is therefore recommended. CAM at pH
8.4–8.6 is a simple, reliable and rapid test.
Differentiation between haemoglobins
migrating to a similar position can be
obtained using electrophoresis on acid
(agarose) gels, HPLC or IEF.1
Immunoelectric focusing has better
resolution and the advantage that it separates
more variants than CAM. It can be semi-
automated and is thus suitable for screening
large numbers of samples. It also has the
disadvantage that it separates haemoglobin
into its post-translational derivatives; for
example, HbF separates into F1 (acetylated)
and F11; HbA can separate into A0, A1,
A(αmet), A(βmet) and A(αβmet), and
similarly for other haemoglobins. This makes
interpretation more difficult. Identification
of variants is still only provisional, and
second-line methods should be used for
further analysis.1
Considerable advantages in terms of
precise quantification, savings in time and full
automation are provided by HPLC methods
over the labour-intensive conventional
techniques such as elution of Hb bands from
cellulose acetate electrophoresis or anion
exchange microcolumn chromatography.1,2
The increase in HbA2 level is the most
significant parameter in the identification of
β-thalassaemia (thal) carriers. However, in
some cases the level of HbA2 is not typically
elevated and some difficulties may arise in
making the diagnosis. For these reasons the
quantification of HbA2 has to be performed
364 THE BIOMEDICAL SCIENTIST
2 3 4 5
HbA2 (%)
with great accuracy and the results must be
interpreted with other haematological and
biochemical evidence.8
Despite the vast heterogeneity of
mutations, the increased levels of HbA2
observed in heterozygotes for the different
β-thalassaemia alleles in different ethnic
groups are remarkably uniform, usually
3.5–5.5% and rarely exceeding 6%. Unusually
high levels of HbA2 over 6.5% characterise a
subgroup of β-thalassaemia caused by
deletions that remove the regulatory elements
in the β promoter. The unusually high HbA2,
often accompanied by modest increases in
HbF, may be related to the removal of
competition for the upstream LCR, allowing
an increased interaction with the cis δ and
γ genes (Fig 2).7
The relative percentage (%) of total
haemoglobin, although not in line with the
international SI unit system, is well
established and uniformly used worldwide.
The presence of Hb variants may interfere
with the quantification of HbA2 on dedicated
HPLC systems; the presence of 25–30%
‘HbA2’ indicates HbE and about 15%
indicates Hb Lepore, a δ/β hybrid chain with
a β-thalassaemia minor phenotype.8,9 To date,
the only routine method able to separate
HbA2 from HbE and Hb Lepore is probably
capillary electrophoresis.8 Combining two
systems improves sensitivity.9
δ-chain variants were first described more
than 40 years ago with the recognition of
HbA’2 (HbB2), the result of a glycine>arginine
substitution at position 16, a silent mutation
with a high frequency in Africans. It is usually
identified using HPLC as a reduction in HbA’2
and the presence of a later running peak of
approximately equal concentration (HbA2).
6 7 8 9
The clinical importance of HbA’2 lies in the
possibility that HbA2 may be underestimated
and a diagnosis of β-thalassaemia trait
missed. It is essential to obtain a total HbA2 by
addition of HbA’2 and HbA2.10 HbA2 is barely
detectable at birth, while the β-gene is already
active at the eighth week of gestation;8 normal
newborns present with 20–30% HbA, which
gradually increases while γ-gene expression
is reduced.
The most probable diagnostic conclusions
are based on the HbA2 determination and
the observation of the basic haematological
and iron parameters; however, HbA2
concentration values may differ depending
on the method used (Fig 3).
α-thalassaemia is associated with a
variable degree of α-globin chain deficit that
reflects the number of the affected α-globin
genes. Normal individuals have two α-globin
genes (αα) linked on each chromosome 16
with the annotation that the more telomeric
copy is designated as the α2 gene, and
adjacent centromeric copy as the α1 gene.
The majority of mutations that result
in complete loss of α-globin production
(α0-thalassaemia) occur from deletions of
the duplicated α-globin genes (--) on one
chromosome.12
Hb Constant Spring (HbCS; α142,
Term→Gln [TAA>CAA in α2]) is a non-
deletional form of α-thalassaemia that is most
prevalent in southern Chinese and South
East Asian populations. The α-globin gene of
this variant, located on the α2-globin allele,
contains a point mutation at the termination
codon (TAA>CAA), permitting glutamine to
be incorporated and the α-globin chain
extended by a further 31 amino acid residues.
This mutation is associated with reduced
JUNE 2012

messenger RNA (mRNA) stability and
impaired binding of the αCS-globin with
α-haemoglobin-stabilising protein (AHSP)
such that the resulting Hb variant constitutes
only a minor proportion (1–2%) of the total
Hb in heterozygotes.
The interaction of the HbCS gene with
deletional α-thalassaemia is the major cause
of non-deletional HbH (β4) (--/αCS α) disease
in southern China and is usually more severe
than deletional HbH disease (--/-α).
Haemoglobin H (HbH) should be suspected
when there is a microcytic anaemia with
splenomegaly and an increased reticulocyte
count. Some patients present with symptoms
of anaemia or with splenic discomfort,
including splenic infarction, while in others
the diagnosis is incidental. Inspection of the
chromatogram is needed for the recognition
of HbH by HPLC, there being a characteristic
early double peak.2 If a diagnosis of HbH
disease is made in a person in the
reproductive age range, DNA analysis may be
indicated to aid in reproductive counselling.
Most patients with HbH disease have one
chromosome with no α genes and thus there
is a risk of a fetus with haemoglobin Barts
hydrops fetalis if a partner is heterozygous
for α0-thalassaemia.2
The problem becomes more complex
when δ-thalassaemia mutations, undetectable
on HPLC, coexist with β-thalassaemia trait.
In these cases, one will need to sequence the
U ltra L ow R etention
Pipette Tips and Filter Tips
The new pipette tips from BRAND
with patented surface treatment
– reduce costly sample loss!
 Ultra-hydrophobic
Ideal for biological samples that
contain detergents e. g.
Triton® X-100, SDS, Tween etc.
 Special patented process
No coatings that might lead to
sample contamination.
 Highly homogeneous surface
No surface defects, thus no non-
specific binding.
 High accuracy
Achema: hall 4.1, booth G35
BRAND GMBH + CO KG
97877 Wertheim (Germany)
Tel.: +49 9342 808-0
www.brand.de · info@brand.de
BRAND_BiomedScient_ULR_PipTips_engl_June12_exhib.indd 1
JUNE 2012
‘In countries with inadequate diagnostic resources, babies
with β-thalassaemia major may be transfused repeatedly
without a diagnosis having been made’
β- and δ-genes10 to prove or exclude the
presence of β-thalassaemia. The A2 level is
lowered to a normal range by a δ-thalassaemia
mutation that may occur in any ethnic
group.13
THALASSAEMIA DELETIONAL
DETERMINANTS
Since publication of the entire sequence of
the human α-globin gene cluster, multiplex
polymerase chain reaction (PCR) methods
have been developed to diagnose different
subsets of α-thalassaemia deletional
determinants. A single-tube multiplex PCR
assay14 can detect heterozygosity,
homozygosity and compound heterozygosity of
seven α-globin gene deletions which account
for the vast majority of β-thalassaemia alleles.
The most widely occurring of these are the
-α3.7 and -α4.2 single α-globin gene deletions,
while double α-globin gene deletions in cis,
such as the --SEA, --FIL and --THAI alleles are very
common in South East Asia, and the --MED
and -(α)20.5 double gene deletions occur more
frequently in the Mediterranean area.
A deletion of about 19.3 kb of the DNA
More liquid repellent than PTFE!
ARTICLE
removes both linked α-globin genes but leaves
the ζ gene, an embryonic α-like globin gene
intact (--SEA). The THAI deletion (--THAI)
removes a larger DNA segment (33.5 kb)
including the embryonic ζ-globin genes.14
Early in gestation, embryonic
haemoglobins (Gower1, Gower2 and
Portland), which do not contain α-globin
chains, are the predominant haemoglobins.
They are rapidly replaced by fetal and then
adult haemoglobin, which contain α-globin
chains. Therefore, α-thalassaemia mutations
become phenotypically evident by 12 weeks
of gestation.15
The modified multiplex PCR assay can
identify, in a single reaction, the seven most
prevalent α-globin gene deletions as well as
the most common α-globin gene triplication.16
The α- and β-thalassaemias are genetic blood
diseases that are the result of reduced or
deficient synthesis of either the α- or β-globin
chains of haemoglobin. The surplus of
unaffected complementary globin chains
precipitates. This results in ineffective
erythropoiesis and haemolysis typical of
thalassaemia.
Product Video
W !
NE
09.05.2012 14:44:22
THE BIOMEDICAL SCIENTIST 365
 ARTICLE
The vast majority of α-thalassaemias are
attributable to deletions that eliminate entire
α-globin genes. The most common of these is
the -α3.7 deletion. The cause of this deletion is
an unequal crossover event during meiosis that
results in chromosomes with either a single
active hybrid α-globin gene (-α3.7) or an
αααanti 3.7 α-globin gene triplication. An α-globin
gene triplication can also originate
complementary to an -α4.2 deletion but is much
less common. Triplication of the α-globin
genes leads to increased production of α-globin
chains. This is not a problem in otherwise
healthy individuals, but it may exacerbate the
α- and β- chain imbalance in β-thalassaemia.
As an α-globin gene triplication can compensate
and thus mask α-globin gene deletions in
α-thalassaemia carriers, it can interfere with
identification of couples at risk of conceiving
a fetus with Hb Barts (γ4) hydrops fetalis.
The detection of an α-globin gene
triplication during antenatal screening is also
important for carriers of mutant β-globin genes
as it can aggravate the clinical manifestations
of β-thalassaemia in their offspring. This PCR
assay cannot distinguish how many αααanti 3.7
α-globin gene triplications are present, but it
can detect both α-globin gene deletions and the
αααanti 3.7 α–globin gene triplication in a single
tube reaction, making it a less-laborious and
less-expensive technique than other assays.16
Multiplex ligation-dependent probe
amplification (MPLA) is based on the ligation
and PCR amplification of two adjacently
hybridising oligonucleotides. Each
oligonucleotide pair is designed to give a
product of a unique length, and, using
common ends, all probes can be amplified
with one primer pair. Using a fluorescent
label allows probe separation on a capillary
sequencing system. Discrimination of
probes based on chemically synthesised
oligonucleotides (~40–60 nt) has been
doubled using two universal primer sets, each
labelled with a different fluorophore, allowing
up to 40 probes to be used in a single reaction.
Multiplex ligation-dependent probe
amplification uses standard technology (ie
hybridisation, ligation, PCR and CE) and is a
rapid and sensitive method for high-resolution
analysis of the globin gene clusters.17
DNA ANALYSIS
Confirmation at the DNA level is imperative
in case of risk assessment and genotype/
phenotype correlation but is also needed to
identify fractions with normal isoelectric points
remaining under the HbA fractions or
hyperunstable Hbs that may remain invisible or
disappear quickly. In these cases, the complete
haematological and biochemical picture will
provide an indication of whether or not to
proceed with molecular analyses.9
Pyrosequencing is a real-time sequencing
technique, based on the detection of light;
however, the α-globin genes can be challenging
to study as they are highly homologous and
reside in a GC-rich cluster with other highly
homologous genes, making specific gene
366 THE BIOMEDICAL SCIENTIST
‘Mass spectrometry is a
powerful complementary
technique that can detect
and identify the majority
of phenotypically silent
mutations’
amplification problematic. An added difficulty
is the extremely high population frequency of
the deletional forms of α-thalassaemia. This
means that it is not unusual for individuals to
have co-existing deletional and non-deletional
α-thalassaemia mutations which can make
data interpretation complex.
Against these technical difficulties,
pyrosequencing has been investigated to see
whether or not it can be used as a rapid
screening method to distinguish the 10 most
common clinically significant mutations in
the α1- and α2-globin genes.18 A total of 105
patients with non-deletional α-thalassaemia
showed 100% concordance with known
genotype as identified by Sanger sequencing.
Pyrosequencing prove to be simpler, more
robust, quicker and cheaper than
conventional sequencing, making it a good
choice for rapid and cost-effective diagnosis of
patients with suspected non-deletional α-
thalassaemia.
The amplification primers designed to
detect the Hb Adana mutation amplify the
α1 and α2 genes, which means that, unlike
other pyrosequencing reactions which only
amplify the α2 gene, the expected allelic ratio
between the mutant and wild-type allele is
1:3 for heterozygotes, 1:1 for homozygotes,
and 1:1 or 1:2 for hemizygotes (depending
on whether it is a one-gene or two-gene
α-globin gene deletion on the other allele).
Four of the samples tested for Hb Adana
were amniotic fluid samples that had been
referred for prenatal diagnosis from two
separate couples at risk of a pregnancy
affected by HbH hydrops fetalis. Both mothers
had α0-thalassaemia trait (-FIL/αα), while
both fathers were carriers of Hb Adana.
Pyrosequencing confirmed that all fetuses
were compound heterozygous for both
mutations and thus were affected by HbH
hydrops fetalis.18
There are 10 non-deletional α-
thalassaemia mutations that are clinically
significant and relatively common in the UK
population. These mutations consist of the α2
termination codon mutations (Hb Constant
Spring, Hb Paksé, Hb Icaria and Hb Seal
Rock), the α2 polyadenylation signal
mutations (Saudi, Turkish and Indian
mutations), and the mutations that give rise
to the hyperunstable α-globin chain variants
Sun Prairie, Quong Sze and Adana.
Unlike the common deletional
α-thalassaemia mutations which can be
identified by simple and inexpensive
techniques (eg Gap-PCR), non-deletional
mutations usually require identification by
DNA sequencing which is more expensive
and labour-intensive. Thus, there is a demand
for technologies which can quickly and
accurately screen for these mutations.
DETECTION AT THE DNA
OR RNA LEVEL
Dominantly inherited β-thalassaemia
(inclusion body β-thalassaemia) is
heterogeneous at the molecular level.
Many cases involve mutations of exon 3 of
the β-globin gene, and include frame shifts,
premature chain termination (non-sense)
mutations, and complex rearrangements that
lead to the synthesis of truncated or elongated
and highly unstable β-globin gene products.
The resulting β-chain variants are very
unstable, and in many cases the products of
the dominantly inherited β-thalassaemia are
not detectable. Non-sense or frame shift
mutations that would produce truncated‚
chains up to 72 residues in length are usually
associated with a mild phenotype in
heterozygotes. It is believed that the mRNAs
associated with these mutations are not
transported to the cytoplasm, and hence no
gene product is made. On the other hand,
mRNAs with mutations in exon 3 are
transported and translated normally. They
produce long and highly unstable globin gene
products that are capable of binding haem,
but not of combining with α chains to
produce any stable Hb tetramer. Hence, these
large truncated products tend to precipitate
in the red cell precursors, together with
excess α chains, to produce large inclusion
bodies. This is the basis for the dominant
forms of β-thalassaemia.
In transfusion-dependent haemolytic
anaemia, haemoglobin analysis reveals no
abnormalities, except the presence of
inclusion (Heinz) bodies. The HbA2 and
HbF levels are normal. No abnormal Hb is
detected by starch gel electrophoresis. An Hb
variant that is highly unstable is likely to be
degraded soon after translation. Therefore,
the molecular characterisation is carried out
at the DNA level, sequencing the β-globin
gene. For example, in a patient who had a
normal complement of four α-globin genes
the sequence of exon 3 was compared with
the sequence of a normal β-globin gene and
a deletion of 6 bp was found. This mutation
created an HphI site that was used to identify
Hb Stara Zagora.19
QUANTIFICATION BY TANDEM
MASS SPECTROMETRY
Peptide-based analysis of whole blood using
electrospray tandem mass spectrometry
(MS/MS) in multiple reaction monitoring
(MRM) mode enables the rapid detection and
sequence confirmation of clinically significant
Hb variants.20 A quantitative approach to the
measurement of δ:β-globin peptide ratios as
potential surrogate markers of HbA2, a
JUNE 2012
 ARTICLE

HbA2 Reduced Normal Borderline Increased

(<2.3%) (2.3–3.5%) (3.6–4.0%) (4.1–8.0%)

MCV, MCH Reduced Normal or Normal Reduced Normal or Reduced Reduced

reduced reduced

RBC Normal or Normal or Normal or Normal Normal or Normal or Normal Normal, Normal or
reduced reduced reduced reduced increased increased increased
or reduced

HbF <1% <1% <1% <1% 3–16% <1% <1% 3–16% <1% or <1% or increased <1–8%
increased

Iron Altered Normal Normal Normal Normal Altered Normal Normal Normal Normal Normal
markers

Possible Iron α-thal δ or α Normal HPFH Iron α-thal carrier δβ-thal Normal HbA2 β-thal carriers High
δ+ββ-thal
diagnosis deficiency traits gene deficiency large β-thal γδβ-thal α gene triplications HbA2
defects deletions Hb variants with β or β-thal
α-thalassaemia phenotype carrier
some unstable Hb variants

Molecular α Gap-PCR β Gap-PCR Gap-PCR β Gap-PCR β or α sequence β sequence

confirmation α or δ sequence γ sequence α or δ sequence γ sequence α Gap-PCR
α MLPA β MLPA α and β MLPA β MLPA α MLPA

Fig 3. Diagnostic flowchart for the interpretation of HbA2.

biomarker used in population screening for β-
thalassaemia trait has been described. δ:β
peptide ratios were calculated using the
World Health Organization (WHO) 1st
International Reference Reagent HbA2, with
a nominal value of 5.3% (by weight) of total
Hb, as a calibrator.21
In contrast to existing techniques of Hb
analysis measuring intact tetramers, MS/MS
measures individual denatured globin
proteins or, after tryptic digestion, specific
peptides. HbA2 is a tetramer of equal α-
and δ-globins, suggesting the possibility that
measuring specific δ protein peptides by
MS/MS might provide a useful surrogate
biomarker. The results for each peptide are
numerically different, primarily because of
differences in ionisation relative to control
peptides.
After calibration against the WHO HbA2
reference reagent, the MS/MS peptide results
are not identical with HPLC, confirming that
the two methods measure fundamentally
different molecular targets.21
It is possible for a person to inherit two
different δ-chain mutations. This is rare
but indicates the need for multiple peptide
analysis in quantitative proteomics. It might
be argued that because the most common
δ-chain mutation is clinically silent and
occurs in the T2 peptide then it should not
be included; however, it is essential for the
detection of all three reported Hb Lepore
variants, which are the result of δ-β fusion
processes (Hb Lepore Hollandia: δ 1–22;
Hb Lepore Baltimore: δ 1–50; Hb Lepore
Boston-Washington: δ 1–87).
It is predicted that an increased proportion
of δ-chain peptides should be found before
the fusion point. The T2 peptide (9–17) is the
only peptide with a difference from the
δ sequence before amino acid 22. The effect
on the respective δ-chain ratios is impressive,
with significant increases, to 12–17%, in the
T2 and T3 peptides and essentially normal
results for T13. The pattern is diagnostic of
an Hb Lepore but does not distinguish
between the three variants. Unequivocal
characterisation may be obtained using
whole-molecule MS/MS.22
Antenatal screening for
haemoglobinopathies is usually a local service,
owing to the requirement for fresh blood
samples for HbA2 measurement and
interpretation together with erythrocyte
indices. In contrast, newborn screening is
centralised, using blood spot cards that are
easily transported. Haemoglobin spotted
onto filter paper is oxidised rapidly to form
methaemoglobin, which has a greater
positive charge than that of the original
oxyhaemoglobin and is a major problem
for the quantification of HbA2 by HPLC.
The methaemoglobin appears as a secondary
peak, similar to that of an δ-chain variant,
and thus renders the chromatogram difficult
to interpret. In contrast, using MS/MS
measurement of δ-chain ratios, it is possible
to quantify an equivalent HbA2 in blood spots
up to a month old. It is essential, however,
that results be interpreted in conjunction with
erythrocyte indices and ethnic origin. In the
current health economic environment, a
method offering centralisation of antenatal
screening is of considerable interest.21
ELECTROSPRAY IONISATION
MASS SPECTROMETRY
Born after an uneventful pregnancy at 39
weeks, a one-year-old male child of Pakistani
parents underwent routine screening and
full blood count, which indicated fairly severe
anaemia (MCV 46.0 fL; MCH 12.0 pg).23
High-performance liquid chromatography
of the proband showed a major component
eluting at the same time as HbA0 and
an elevated HbA2 (5.8%), suggesting
β-thalassaemia trait. There was also a minor
component (Y) that eluted just prior to the
major component and was not present in the
proband’s mother with β-thalassaemia trait.
This minor component was also present at
a lower level (3.3%) in the trace from the
proband’s father, who subsequently was
found to carry the same variant as his son.
Immunoelectric focusing presented a similar
picture with a minor band that was slightly
faster (more anodal) than the major band at
the position of HbA0.
The red cell parameters were inconsistent
with those expected for a simple
β-thalassaemia trait and a blood sample was
submitted for electrospray ionisation mass
spectrometry (ESI-MS).23 This revealed a
variant β-chain that was heavier than normal
by 46 Da with no normal β chain. This mass
increase strongly suggested the mutation
Gly→Cys, as it is the only mutation that can
give a 46 Da mass increase over normal by a
single base change in the nucleotide codon.
Furthermore, the glutathionylated variant
β-chain was significantly more intense
than the glutathionylated normal β-chain.
This supports the creation of a new cysteine
residue in the β-chain that is accessible to
reaction with glutathione to form an adduct
via a disulphide bond.
Also present was a minor component
(~3% of non-α-chains) at mass 31824.6 Da,
which corresponds to a disulphide-linked

JUNE 2012 THE BIOMEDICAL SCIENTIST 367

 ARTICLE
dimer of the variant β-chain. These data,
together with the raised HbA2 on HPLC,
are consistent with the proband being a
double heterozygote for the variant and
β0-thalassaemia. A 30-minute tryptic digest
was analysed by ESI-MS, which showed that
the mutation occurred in the βT-5 peptide,
which contains two glycine residues.
However, the mutation at β46 (CD5)
Gly→Cys was unlikely because the codon for
glycine at this position (GGG) requires two
base changes to produce cysteine (TGT or
TGC). The codon for 56Gly (GGC) requires
only one base change. Hence, the mutation
was probably β56 (D7) Gly→Cys. This was
confirmed by undertaking MS/MS on the
normal and variant βT-52+ ions. The 46 Da
mass increase between normal and variant at
y”4, and all subsequent y” ions, confirmed the
mutation β56 (D7) Gly→Cys: Hb Leeds.23
Mass spectrometry is a powerful
complementary technique that can detect
and identify the majority of phenotypically
silent mutations, as well as those that are
easily detected by phenotypic means. Peptide
mutations, except Leu→Ile, are detectable
in heterozygotes by ESI-MS provided their
abundance is >10% of total haemoglobin.
The Leu→Ile mutation cannot be detected by
ESI-MS because it produces no mass change.
CONCLUDING COMBINATIONS
No screening programme is 100% specific
and sensitive.24 The diagnostic procedure
implies full haematological, biochemical and
molecular analysis, including Hb separation
on HPLC and CE, globin chain separation
on reverse-phase chromatography, globin
chain synthesis, sequencing of all globin
genes, including the δ-globin genes, and
gap-PCR for the common α globin gene
deletions. Multiplex ligation-dependent
probe amplification is performed for the
analysis of (large) deletions that may involve
the α- or the γ-, δ- and β-genes, resulting
in reduced HbA2 expression;13 however,
technical and interpretive problems can lead
to misdiagnosis.2
Combined HPLC and CE analysis gives
an advantage in recognising artefacts such
as over-estimation of HbA2 and HbF and
fractions not separated on either one or
the other system.9 The identification of
haemoglobins is often presumptive,
based on electrophoretic mobility or other
characteristics in an individual of appropriate
family origin and should be based on a
minimum of two techniques based on
different principles. Definitive identification
usually requires DNA analysis, mass
spectrometry or protein sequencing, but
family studies are also of considerable
importance in elucidating the nature of
disorders.1,23
Finally, organisational pitfalls can lead
to erroneous interpretation of results, such
as when the diagnostic laboratory is not
informed that a patient has been transfused
or is taking hydroxycarbamide (which raises
368 THE BIOMEDICAL SCIENTIST
haemoglobin F percentage), or when there is
failure to provide the laboratory with accurate
information on ethnic origin.2 r 14 Tan AS, Quah TC, Low PS, Chong SS.
REFERENCES
1 Ryan K, Bain BJ, Worthington D et al.;
British Committee for Standards in
Haematology. Significant
haemoglobinopathies: guidelines for
screening and diagnosis. Br J Haematol
2010; 149 (1): 35–49.
2 Bain BJ. Haemoglobinopathy diagnosis:
algorithms, lessons and pitfalls. Blood Rev
2011; 25 (5): 205–13.
3 Chow J, Phelan L, Bain BJ. Evaluation of
single-tube osmotic fragility as a screening
test for thalassemia. Am J Hematol 2005;
79 (3): 198–201.
4 Urrechaga E, Borque L, Escanero JF. The
role of automated measurement of red cell
subpopulations on the Sysmex XE 5000
analyzer in the differential diagnosis of
microcytic anemia. Int J Lab Hematol
2011; 33 (1): 30–6.
5 Karnpean R, Pansuwan A, Fucharoen G,
Fucharoen S. Evaluation of the URIT-
2900 automated hematology analyzer for
screening of thalassemia and
hemoglobinopathies in Southeast Asian
populations. Clin Biochem 2011;
44 (10–11): 889–93.
6 Abdulsalam AH, Sabeeh N, Bain BJ.
Diagnosis of beta thalassemia major from
bone marrow morphology. Am J Hematol
2011; 86 (2): 187.
7 Thein SL. Genetic modifiers of beta-
thalassemia. Haematologica 2005; 90 (5):
649–60.
8 Mosca A, Paleari R, G Ivaldi G, Galanello
R, Giordano PC. The role of haemoglobin
A(2) testing in the diagnosis of
thalassaemias and related
haemoglobinopathies. J Clin Pathol 2009;
62 (1): 13–7.
9 Van Delft P, Lenters E, Bakker-Verwey M
et al. Evaluating five dedicated automatic
devices for hemoglobinopathy diagnostics
in multi-ethnic populations. Int J Lab
Hematol 2009; 31 (5): 484–95.
10 Phylipsen M, Harteveld CL, de Metz M
et al. New and known β-thalassemia
determinants masked by known and new
δ gene defects [Hb A(2)-Ramallah or
δ6(A3)Glu→Gln, GAG>>CAG].
Hemoglobin 2010; 34 (5): 445–50.
11 Fucharoen S, Viprakasit V. Hb H disease:
clinical course and disease modifiers.
Hematology Am Soc Hematol Educ
Program 2009;: 26–34.
12 Liao C, Zhou JY, Xie XM, Li J, Li R, Li DZ.
Detection of Hb Constant Spring by a
capillary electrophoresis method.
Hemoglobin 2010; 34 (2):175–8.
13 Phylipsen M, Gallivan MVE, Arkesteyn SGJ,
Harteveld CL, Giordano PC. Occurrence
of common and rare δ-globin gene defects
in two multiethnic populations: thirteen
new mutations and the significance of
δ-globin gene defects in β-thalassemia
diagnostics. Int J Lab Hematol 2011;
33 (1): 85–91.
A rapid and reliable 7-deletion multiplex
polymerase chain reaction assay for
α-thalassaemia. Blood 2001; 98 (1):
250–1.
15 Vichinsky E. Complexity of alpha
thalassaemia: growing health problems
with new approaches to screening,
diagnosis, and therapy. Ann NY Acad Sci
2010; 1202: 180–7.
16 de Mare AA, Groeneger AH, Schuurman S,
van den Bergh FA, Slomp J. A rapid single-
tube multiplex polymerase chain reaction
assay for the seven most prevalent alpha-
thalassaemia deletions and
alphaalphaalpha(anti 3.7) alpha-globin
gene triplication. Hemoglobin 2010;
34 (2): 184–90.
17 Harteveld CL, Voskamp A, Phylipsen M
et al. Nine unknown rearrangements in
16p13.3 and 11p15.4 causing alpha- and
beta-thalassaemia characterised by high
resolution multiplex ligation-dependent
probe amplification. J Med Genet 2005;
42 (12): 922–31.
18 Haywood A, Dreau H, Timbs A, Schuh A,
Old J, Henderson S. Screening for
clinically significant non-deletional alpha
thalassaemia mutations by
pyrosequencing. Ann Hematol 2010;
89 (12): 1215–21.
19 Efremov GD. Dominantly inherited
β-thalassemia. Hemoglobin 2007; 31 (2):
193–207.
20 Boemer F, Ketelslegers O, Minon JM,
Bours V, Schoos R. Newborn screening for
sickle cell disease using tandem mass
spectrometry. Clin Chem 2008; 54 (12):
2036–41.
21 Daniel YA, Turner C, Haynes RM,
Hunt BJ, Dalton RN. Quantification of
hemoglobin A2 by tandem mass
spectrometry. Clin Chem 2007; 53 (8):
1448–54.
22 Rai KK, Green BN, Landin B, Alvelius G,
Griffiths WJ. Accurate mass measurement
and tandem mass spectrometry of intact
globin chains identify the low proportion
variant hemoglobin Lepore-Boston-
Washington from the blood of a
heterozygote. J Mass Spectrom 2004;
39 (3): 289–94.
23 Williams JP, Scrivens JH, Green BN,
Farrar LM, Sutcliffe M. Hb Leeds
[‚56(D7)Gly→Cys]: a new hemoglobin that
aggravates anemia in a child with beta(0)-
thalassemia trait. Hemoglobin 2007;
31 (3): 367–73.
24 NHS Sickle Cell and Thalassaemia
Screening Programme. Handbook for
Laboratories 2nd edn. London: NSC,
2009 (http://sct.screening.nhs.uk).
This article is based on an essay submitted
as part of the IBMS Structured Reading
programme.
JUNE 2012