Chapter 16

Automated Capillary Electrophoresis in the Screening

for Hemoglobinopathies
Frédéric Cotton, Fleur Wolff, and Béatrice Gulbis

Abstract
Hemoglobinopathies are genetic disorders of globin chains characterized by the decreased expression of
α- or β-globin chains (thalassemias) or by the synthesis of an abnormal protein (hemoglobin variants in,
e.g., sickle cell disease). The screening of most hemoglobinopathies relies, together with hematological
results and clinical elements, on the separation and quantification of normal and abnormal hemoglobin
fractions. Gel electrophoresis, isoelectric focusing, and HPLC have been the methods of choice for many
years. For about 20 years, capillary electrophoresis has appeared as a strong alternative method. Since the
early 2000s, automated instruments are commercially available for the analysis of Hb fractions in adult
patients but also for neonatal screening.

Key words: Hemoglobin, Variant, Hemoglobinopathy, Thalassemia, Sickle cell disease, Capillary
electrophoresis, Automation, Neonatal screening

1. Introduction

Hemoglobin (Hb) is a tetramer composed of 2 α-type and 2 β-type

globin chains. During the fetal life, red blood cells mainly contain
Hb F (α2γ2). At birth, it still represents about 80% of total Hb, the
rest being Hb A (α2β2). After 6 months, Hb A reaches 95% and Hb
F is less than 1.5%. At that time, a third fraction, Hb A2 (α2δ2), is
seen at a level of around 2.0–3.5%. Hemoglobinopathies are
genetic disorders due to mutations of α- or β-globin genes and
leading to a decreased expression of these genes (quantitative
defects) or to the synthesis of an abnormal protein called Hb vari-
ant (qualitative defects).
Quantitative defects are mainly represented by α- and
β-thalassemias, where the synthesis of normal α- or β-globin chain
is decreased (1). Patients with β-thalassemia major (two mutated
β-globin genes) have very severe anemia with many complications

Nicola Volpi and Francesca Maccari (eds.), Capillary Electrophoresis of Biomolecules: Methods and Protocols,
Methods in Molecular Biology, vol. 984, DOI 10.1007/978-1-62703-296-4_16, © Springer Science+Business Media, LLC 2013

227
228 F. Cotton et al.

including growth retardation, bone marrow expansion, and iron

overload. β-Thalassemia minor (one mutated β-globin gene) is a
mild condition with microcytosis and hypochromia only. It is
important to make the differential diagnosis between this defect
and other causes of microcytic anemia (i.e., iron deficiency, α-thal-
assemia …), especially for genetic counselling. An elevated level of
Hb A2, and sometimes of Hb F, is the best diagnostic test for
β-thalassemia minor. Different forms of α-thalassemia exist,
depending on the number of α-globin genes defect. Clinical
expression is only seen with the suppression of three genes, reflected
by the presence of Hb H, a tetramer of β-globin chains (β4).
Hb Bart’s (γ4) is seen in newborns with one to three α-globin
genes defects but also in healthy newborns, at low levels (<0.2%).
Until now, 1,200 Hb variants have been described. About 10%
of them display an abnormal function (polymerization, instability,
oxidation, increased affinity…) but only a few dozens are clinically
significant. Hb S is the most frequent variant, causing sickle cell
disease at the homozygous state (2). Due to the replacement of the
glutamic acid in position 6 by a valine, Hb S tends to polymerize
when deoxygenated and red blood cells then take an abnormal
sickle shape. The lack of deformability of sickle cells is the cause of
severe hemolytic anemia and life-threatening vaso-occlusive crises.
Other Hb variants such as Hb C, Hb D-Punjab, or Hb O-Arab may
copolymerize with Hb S. Therefore, compound heterozygotes, i.e.,
Hb SC, Hb SD, and Hb SO, suffer from sickle cell syndromes.
Hydroxycarbamide induces the expression of Hb F which limits Hb
S polymerization and clinical events. The Hb F level is thus moni-
tored in sickle cell patients treated with this drug.
Hb analysis relies on the separation of different Hb fractions
based on their charge difference. Cation-exchange chromatogra-
phy has long been the method of choice for Hb A2 and Hb F
quantification, but since the late 1990s, capillary zone electropho-
resis (CZE) is an alternative method. For Hb variants, gel electro-
phoresis, isoelectric focusing (IEF), and HPLC have been used.
More recently, CZE has appeared as a powerful tool (3). The pre-
sumptive identification and accurate quantification of Hb variants
are mandatory to make the difference between traits and clinically
significant forms. However, secondary and sometimes tertiary
methods, including genetic analysis, are needed to confirm the
variant identity (4–6).
Newborn screening for hemoglobinopathies is recommended
in order to start preventive treatments. In sickle cell syndromes, it
was proved that early antipneumococcal vaccination and prophy-
lactic penicillin therapy dramatically decreases child mortality.
Another advantage of newborn screening is the detection of healthy
carriers for genetic counselling in the family. Hb analysis can be
done on liquid cord blood or heel blood collected and dried on
paper filter (Guthrie card) (7).
 16 Automated Capillary Electrophoresis of Haemoglobin 229

2. Hemo-
globinopathies
and Capillary
Electrophoresis CZE at alkaline pH is an efficient method for the separation of Hb
fractions. In 1991, Chen developed a CZE method using fused
silica capillaries filled with a pH 8.6 proprietary buffer that could
separate Hb A, Hb S, and Hb C within a few minutes (8). In 1997,
Jenkins published a similar method using a pH 9.98 borate buffer
that was able to separate and quantify common variants (Hb S, Hb
D, Hb E, and Hb A but not Hb F) and Hb A2 with a precision of
8.4% (9). Later, Shihabi described a method using a pH 8.5 Tris
buffer showing a precision of 5.7% for Hb A2 quantification (10).
At the same time, a seducing approach was developed by the Analis
company for two commercial kits. Their patented dynamic coating
of the capillary wall with charged polymers in alkaline or acidic
conditions allowed to control and increase the electroosmotic flow
in the capillary and also prevented protein adhesion to the capillary
wall. The advantages were short run times, high reproducibility,
and long capillary lifetime. We could show that these methods
allowed the separation of most common Hb variants and the accu-
rate and precise quantification of Hb A2, Hb F, and other Hb frac-
tions (11–13). Capillary IEF and micellar electrokinetic
chromatography can also be used for Hb analysis but are limited to
specialized investigations (14, 15).

3. Automated CZE

3.1. Instrument In the early 2000s, automated instruments such as the Capillarys 2
system (Sebia Benelux, Vilvoorde, Belgium) appeared on the mar-
ket. This instrument is a fully automated CZE instrument using
eight parallel fused silica capillaries (100 μm ID × 17 cm length)
and handling eight-tube racks with barcode reading. The Capillarys
hemoglobin assay is dedicated to adult blood samples. Hemolysates
are automatically prepared from red cells pellets. After hydrody-
namic injection, Hb fractions are separated under a voltage of
7.8 kV in a pH 9.4 buffer and detected at 415 nm. Migration times
are given in arbitrary units, the retention time of Hb A being fixed
at 150. The throughput is 34 samples per hour. Liquid cord blood
can be analyzed in “cord blood mode” where the retention time of
Hb F is fixed at 180. Typical results are shown on Figs. 1 and 2.
The Capillary 2 Neonat Fast instrument is similar to the
Capillarys 2 but it includes an automated puncher for dried blood
on filter paper with barcode reading of the cards. With the Neonat
Fast Hb assay, punched spots are automatically eluted and Hb frac-
tions are then separated in the same manner as for the Capillarys
Hemoglobin assay, with an adjustment of the Hb F peak to 180.
The throughput is 48 samples per hour.
230 F. Cotton et al.

Fig. 1. Separation profiles obtained on blood samples analyzed with the Capillarys Hemoglobin assay. (a) Normal profile,
(b) Hb S heterozygote, and (c) Hb G-Philadelphia heterozygote. The mutated α-chain of Hb G-Philadelphia is also included
in Hb A2, which appears as a double peak (Hb A2 + Hb X2)

Fig. 2. Separation profiles obtained on liquid cord blood samples analyzed with the Capillarys Hemoglobin assay in “cord
blood mode.” (a) Normal profile, (b) Hb C heterozygote, and (c) Hb S heterozygote.

3.2. Methods
3.2.1. Diagnosis
of Hemoglobinopathies
in Adult Patients
Many studies evaluated the ability of the Capillarys Hemoglobin
assay to detect Hb variants and to quantify Hb fractions.
Louahabi et al. showed that the analytical performances of the
method were good with imprecision lower than 5 and 10% for Hb
A2 and Hb F quantification, respectively, and lower than 2.5% for
migration times (16). In other studies, better results with lower
imprecision were given (17–19). Several studies showed that Hb
A2 and Hb F results obtained with CZE were slightly lower from
those obtained with other methods such as HPLC. Therefore,
reference ranges had to be adapted to 2.1–3.2% for Hb A2 and
<0.8% for Hb F (16). However, CZE was superior to HPLC for
quantifying Hb A2 in the presence of various Hb variants except
Hb C (17, 20, 21).
Several papers showed that the Capillarys Hemoglobin method
was suitable for the presumptive identification and quantification
of common Hb variants, including Hb H, although quantitative
results were slightly different from those obtained with HPLC in
some cases (16, 22, 23). Several researchers showed that this
method was better than HPLC for the detection of Hb Constant
Spring, an important unstable α-globin variant (18, 24). Fucharoen
et al. studied the identification of 14 α- and β-globin chain variants
 16 Automated Capillary Electrophoresis of Haemoglobin 231

frequent in Southeast Asia, such as Hb Q-Thailand, Hb Siam, Hb

Paksé, Hb Tak, and Hb Pyrgos (24). We previously published the
migration times of 18 variants identified by genetic analysis (17).
An updated list is now presented on Table 1. Some variants (Hb
Anderlecht, Hb Köln, Hb Loire, Hb Marseille, Hb Raleigh, and
Hb Saclay) were not separated from Hb A. Hb Köln is unstable
and could be detected thanks to an abnormally broad peak shape.
The other variants not separated from Hb A have no clinical con-
sequence. Some variants such as Hb S and Hb Stanleyville II had
close migration times, but α-globin chain mutants could be
detected as Hb A2 was split into two peaks (Fig. 1c).

3.2.2. Newborn Screening Several studies have evaluated the potential of the Capillary system
for Hemoglobinopathies for the newborn screening of sickle cell disease and other hemoglo-
binopathies. They all focused on the Neonat Fast Hb assay. Renom
et al. analyzed 6,766 newborn samples with IEF and CZE and
showed that all significant variants were detected and presumably
identified, including Hb Bart’s. The absence of Hb A was well
observed in four cases of β-thalassemia major (25). Mantikou et al.
evaluated the system on 1,600 samples analyzed with HPLC and
CZE. They also compared liquid cord blood and dried blood col-
lected on filter paper. All results were perfectly concordant although
some degradation of Hb fractions was seen when blood was col-
lected on paper (26). Murray et al. made a similar evaluation on
347 dried blood samples analyzed with HPLC and CZE. Their
conclusion was identical (27). We performed a validation of the
Capillarys Hemoglobin assay in “cord blood mode” by analyzing
962 liquid cord blood samples with IEF and CZE (Fig. 2). No
discrepancy between the results provided by both methods was
observed (28). Table 2 shows the migration times of the five most
common Hb variants and of Hb Bart’s.
The CZE methods being quantitative, some authors studied
the possibility to detect β- and α-thalassemia traits according to the
Hb Bart’s and Hb A level, respectively. Mantikou et al. showed a
relation between Hb A level and gestational age at birth and sug-
gested that below 15%, a β-thalassemia trait should be suspected
(26, 29). Srivorakun et al. correlated the Hb Bart’s level with the
genotype of 226 newborns displaying 17 different thalassemic
genotypes. Hb Bart’s was detected in all patients with two or three
α-globin genes defects. However, Hb Bart’s was detected only in a
few cases with one α-globin gene defect (30). Munkongdee et al.
made a similar study on 587 newborns including 158 cases with
one to three α-globin genes defects. Hb Bart’s was detected at
high levels in all cases of two or three α-globin genes defects but
there was an overlap between Hb Bart’s levels in normal newborns
and those carrying one α-globin gene defect. The sensitivity and
specificity for diagnosing α-thalassemia were 95 and 98%, respec-
tively, using a value of 0.2% for Hb Bart’s (31).
232 F. Cotton et al.

Table 1
Mean migration time of Hb variants with the Capillarys
Hemoglobin assay (arbitrary units)

Hb Migration time

H 29
a
J-Norfolk 75
J-Baltimore 80
J-Torontoa 91
Hofu 93
a
Grady 94
Ube IIa 103
K-Woolwich 123
Camden 130
a
Anderlecht 150
Köln 150
Loire 150
Marseille 150
Raleigh 150
Saclay 150
G-San Jose 181
G-Coushatta 197
Korle Bu 201
G-Philadelphiaa 204
D-Ouled Rabah 205
G-Philadelphia 205
D-Punjab 206
Lepore 206
Osu Christiansborg 208
S 214
Stanleyville IIa 214
O-Indonesia 215
a
Ottawa 217
Aryaa 218
(continued)
 16 Automated Capillary Electrophoresis of Haemoglobin 233

Table 1
(continued)

Hb Migration time
E 229
C-Harlem 238
O-Arab 240
C 251
a
Constant Spring 256
α-Globin chain mutant
a

Table 2
Mean migration time of frequent Hb variants with the
Capillarys Hemoglobin assay in “cord blood mode”
(arbitrary units)

Hb Migration time

Bart’s 47
D-Punjab 217
S 227
C 281
E 245
O-Arab 262

4. Discussion
and Conclusion
Capillary electrophoresis is a powerful tool for the analysis of nor-
mal and abnormal Hb fractions and thus for the screening and diag-
nosis of severe diseases like thalassemias and sickle cell disease.
The fully automated Capillarys Hemoglobin assay allows a
very good and reproducible separation of normal and abnormal
Hb fractions in short times. The quantification of Hb A2 and Hb F
is precise, even in the presence of most Hb variants. Results are
significantly lower than with HPLC and adapted reference ranges
should be used. The method detects and distinguishes most com-
mon and clinically significant Hb variants.
The Neonat Fast Hb and Capillarys Hemoglobin in “cord
blood mode” assays are fully automated and can handle dried blood
on paper filter and liquid cord blood. All clinically significant Hb
234 F. Cotton et al.
variants are easily detected and the accurate quantification of Hb A
and Hb Bart’s is useful for the detection of β- and α-thalassemia.
Thanks to all these advantages, the Capillarys instrument is
probably one of the most powerful first-line solutions for hemo-
globinopathies screening in newborn and adult patients.
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