61

Symposium : Newer Diagnostic Tests

HPLC Studies in Hemoglobinopathies

R.B. Colah, R. Surve, P. Sawant, E. D’Souza, K. Italia, S. Phanasgaonkar, A.H. Nadkarni and
A.C. Gorakshakar

Institute of Immunohaematology (ICMR), 13th Floor, New Multistoreyed Building, KEM Hospital Campus, Parel,
Mumbai, India

ABSTRACT
An accurate diagnosis of β-thalassemia carriers, homozygous patients and identification of different structural hemoglobin
variants is important for epidemiological studies as well as for management and prevention of the major hemoglobin disorders.
There are many electrophoretic and chromatographic approaches for estimation of HbA2 and Hb F but cation exchange HPLC
(CE-HPLC)using automated dedicated machines like the Variant Hb testing system have become the method of choice for these
investigations. CE-HPLC also helps in the presumptive identification of many abnormal hemoglobin variants and has been useful
for both neonatal screening of sickle cell disease as well as second trimester prenatal diagnosis of thalassemia by fetal blood
analysis. Other applications of HPLC in hemoglobinopathies include separation of globin chains, measuring the ratio of gamma
globin chains (Gγ/Aγ) and the recently described denaturing HPLC for detecting mutations in both α and β globin genes. [Indian
J Pediatr 2007; 74 (7) : 657-662] E-mail: colahrb@gmail.com

Key words : CE-HPLC; Hemoglobinopathies; Diagnosis; Neonatal screening; Prenatal diagnosis

The thalassemias are an autosomal recessively inherited
group of disorders of hemoglobin synthesis characterized
by the absence or reduction in output of one or more of
the globin chains of hemoglobin. The structural variants
result from substitution of one or more amino acids in the
globin chains of the hemoglobin molecule.1
The β thalassemias and their interaction with
structural hemoglobin (Hb) variants like Hb S and Hb E
are a major public health problem in India. Children
inheriting these β-thalassemia major syndromes most
often have a severe disease and a transfusion dependent
survival from early childhood. It is therefore important to
accurately identify carriers of these disorders and offer
the option of preventive measures by prenatal diagnosis
to couples at risk of having a child with severe disease. 2
The severe and fatal form of α-thalassemia, Hb Bart’s
hydrops fetalis is not seen in India. The commonest α
gene defect in our population is α single a gene deletion.
The carriers of this form of α-thalassemia usually get
presumptively identified from the MCH, MCV and RBC
count done on electronic counters, after exclusion of β-
Correspondence and Reprint requests : Dr. R.B. Colah, Institute of
Immunohaematology (ICMR), 13 th Floor, New Multistoreyed
Building, KEM Hospital Campus, Parel, Mumbai – 400 012. Tel :
(022) 24138518/19; Fax (022) 24138521
[Received August 3, 2006; Accepted August 7, 2006]
thalassemia carriers and cases of iron deficiency anemia.
However, few cases of Hb H disease have been reported
where there is only one functional α gene.3
Approaches for carrier screening for thalassemias and
identification of hemglobin variants
Way back in 1975, the expert group on “Abnormal
hemoglobins and thalassemias” of the International
Committee for Standardization in Hematology
recommended 2 sets of investigations for diagnosis of
these disorders. The initial investigations included a
complete blood count, electrophoresis of the hemoglobin
at alkaline pH, tests for sickling and solubility and
quantitation of HbA 2 and Hb F. If an abnormal
hemoglobin was detected on electrophoresis or
suspected, further testing included electrophoresis of Hb
at acid pH, electrophoresis of globin chains at acid and
alkaline pH, isoelectric focusing (IEF), heat and
isopropanol stability tests for unstable hemoglobins and
tests for identifying hemoglobins with altered oxygen
affinity.4
Accurate diagnosis of β-thalassemia carriers
Carriers of β-thalassemia have a near normal Hb level
although they may be slightly anemic with hypochromic
and microcytic red cells. In most population screening
programmes an MCV of < 80fl and MCH of < 27 pg are
generally used as cut off points for further screening. The
hallmark of diagnosis for classical β-thalassemia carriers
is a raised HbA 2 varying between 3.5% and 4%

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R.B. Colah et al

depending on the method of estimation used.5
It has been shown in a recent study that while the
MCH and HbA2 levels in β-thalassemia carriers are quite
stable in samples stored at 4 oC for upto 3 wk, the MCV
levels showed a progressive rise from 62.6 fl on day 1 to
84.0 fl on day 21, suggesting that MCV is not a good
parameter if samples cannot be processed within a day 6.
HbA 2 estimation using electrophoretic or
chromatographic techniques
Hemoglobin electrophoresis
Quantitation of HbA 2 after cellulose acetate
electrophoresis at alkaline pH traditionally relies on
elution and spectrophotometry or densitometric
scanning. Accuracy largely depends on the training and
experience of the laboratory technician and the levels of
HbA2 are not always precise and reproducible.5
Microcolumn chromatography
This technique for HbA2 estimation is also used in many
laboratories, but could give problems of co-elution of
other Hb variants like Hb S, Hb E and Hb O Arab if
present.
Hb F estimation
Hb F estimation is particularly important in diagnosis of
carriers of δβ thalassemia and hereditary persistence of
fetal hemoglobin (HPFH) as well as the major thalassemia
syndromes. Conventionally Hb F is quantitated by the
alkali denaturation methods of Singer or Betke.
However, erroneously low levels may be obtained when
Hb F levels are very high.5
Cation exchange high performance liquid
chromatography (CE –HPLC)
Today CE – HPLC has become the method of choice for
quantitation of Hb A2, Hb F and other Hb subtypes. A
few automated machines have been developed by
different ionic strengths to elute the different
hemoglobins. A dual wavelength filter photometer
monitors the eluent from the cartridge as it passes
through the photometer cell. Changes in optical density
at 415 nm are measured. A secondary filter at 690 nm
corrects the effects caused by mixing buffers of different
ionic strengths. The detector signal data are reduced by
the integrator. The data is processed and the report giving
the chromatogram of time vs absorbance where the
different peaks are identified in defined windows and
their retention time, relative percentage and area are
printed out. This automated HPLC provides a more
efficient and accurate assessment of HbA 2 and Hb F
compared to manual methods.8
Cut off values of HbA2 for diagnosis of β-thalassemia
heretrozygotes by automated HPLC
Although a cut off of >4.0% of HbA2 is recommended for
identifying β-thalassemia carriers, each laboratory needs
to establish their own normal ranges. In our laboratory,
we compared the HbA2 levels on the Variant analyzer in
500 normal individuals with normal hemoglobin levels,
normal red cell indices and absence of iron deficiency
with 500 obligate β-thalassemia heterozygotes who were
parents of affected β-thalassemia homozygous children.
(Fig 1). Majority of normal individuals had HbA2 levels
between 2.0 and 3.4% while only 18 individuals had
HbA 2 levels between 3.5 and 3.9%. Among the β-
thalassemia heterozygotes, majority had HbA2 levels of
>4.0% while in 7 cases the HbA 2 levels were between
Cases (%)

different firms for quantitation of Hb A 2, Hb F and

presumptive identification of Hb variants. The most
versatile and widely used among these is the Variant
Hemoglobin Testing System (BioRad Laboratories,
Hercules, CA).4,7
The Variant operates on the principle of HPLC and
the column comprises of a small 3.0 x 0.46 cm cation
exchange cartridge. The β-thalassemia short programme
is used for screening for β-thalassemia. Only 5 µl of an
EDTA blood sample taken in 1.0 ml of the lysis buffer is
required. Upto 100 samples can be simultaneously
loaded in the autosampling chamber and each sample
takes 6 ½ minutes for analysis.
The samples are injected into the analysis stream and
separated by the cation exchange cartridge using a Fig 1. Distribution of Hb A 2 levels on the Variant hemoglobin
testing system in normal individuals and obligate β -
phosphate ion gradient generated by mixing 2 buffers of
thalassemia heterozygotes

658 Indian Journal of Pediatrics, Volume 74—July, 2007

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HPLC Studies in Hemoglobinopathies

3.0% and 3.9%. We then looked at the β-thalassemia Automated HPLC for detection of Hb variants
mutation in these 7 cases. (Table 1). Five cases were
silent carriers having the Capsite +1 (A→C) mutation Specific elution windows are defined for abnormal
which can cause normal HbA2 β-thalassemia while two hemoglobins like Hb S, Hb D, Hb E and Hb C using the β
individuals with an HbA 2 of 4.0 % had the common IVS thalassemia short programme. Nevertheless, it is
1 – 5 (G→C) mutation and the CD 16 (-C) mutation advisable to use another method also for a definitive
repectively. diagnosis of an abnormal variant to avoid missing some
rare variants which may have similar elution profiles.4,10
Identification of abnormal hemoglobin variants Fig. 2 shows the retention times of different Hb variants
seen in India along with their electrophoretic mobilities at
Hb Variants are usually identified from their
alkaline pH. The chromatograms of some of these
electrophoretic mobility, the quantity of the abnormal
abnormal variants are shown in Fig 3.
hemoglobin and the ethnic background of the individual
which can provide a useful clue.9 CE - HPLC
Hemoglobin Retention Hemoglobin Electrophoretic
The most common Hb variants encountered in any Windows Time (min) Variants Mobility at
laboratory undertaking investigations for Hb C
Alkaline pH
hemoglobinopathies are Hb S, Hb E, Hb D Punjab and Hb Q India Hb S retion
Hb C. Hb S Hb D Agri Hb S region
Hb D
Electrophoretic methods
Most of the common variants can be identified using a
combination of alkali and acid Hb electrophoresis on Hb E Hb A2 retion
Hb A 2 Hb Lepore Hb S region
cellulose acetate. At alkali pH, some hemoglobin variants
Hb D Iran Hb S region
co-migrate with Hb A2 (e.g Hb C, Hb E, Hb O Arab) while
some co-migrate in the Hb S region (e.g Hb D Punjab, Hb
D Iran, Hb G). At acid pH, it is possible to separate Hb C
from Hb E, Hb O Arab and Hb S from Hb D and Hb G. Hb A
Hb E cannot be separated from Hb O Arab and Hb D
from Hb G.4,10
Hb J
Isoelectric focusing (IEF) Variants Faster than Hb A
P3

In this technique the hemoglobins migrate in a pH P2

gradient prepared by using a combination of different
ampholines and focus at their iso-electric point, i.e at the
position of zero net charge. Very small differences in
electrophoretic mobility can be picked up which help to
resolve Hb C from Hb E and Hb S from Hb D as very
sharp bands which makes quantitation also more
accurate.
Capillary IEF (cIEF)
Here, the sensitivity of detection of capillary
electrophoresis is combined with the automated sampling
and data acquisition of HPLC. Quantiation of HbA2, Hb
F and other variants compare well with HPLC and some
variants like Hb D Punjab and Hb C Harlem give a better
resolution.4,11
Hb F
Hb H Faster than Hb A
Hb Bart's
Fig 2. Retention times of hemoglobin variants on CE – HPLC
(Biorad Variant Hb Testing System) and their electrophoretic
mobilities
Problems in interpretation
Automated HPLC using the Variant machine has some
intrinsic problems. Some hemoglobins co-elute in the Hb
A 2 window like Hb E, Hb D Iran, Hb G Copenhagen,
Hb Lepore and Hb Osu Christianbourg making it

TABLE 1. β thalassemia Heterozygotes with Normal or Borderline Hb A2 Levels on CE-HPLC

S.No Hb (g/dl) RBC (x 1012/L) MCV (fl) MCH(pg) HbA2 (%) Hb F (%) β thalassemia mutations

1. 14.2 5.11 79.1 27.8 4.0 0.0 CD 16 (- C)

2. 9.9 4.39 71.8 22.6 3.2 0.0 Cap site + 1 (A→C)
3. 12.5 4.49 78.0 27.8 3.6 0.0 Cap site + 1 (A→C)
4. 6.7 4.55 51.6 14.7 4.0 0.0 IVS 1–5 (G→C) hetero
5. 15.2 5.34 82.2 28.7 4.0 0.0 Cap site + 1 (A→C)
6. 14.0 5.66 77.4 24.7 3.6 0.0 Cap site + 1 (A→C)
7. 12.4 4.36 78.9 28.4 3.6 0.0 Cap site + 1 (A→C)

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R.B. Colah et al
Hb Lepore trait 19 wks fetus(BTT)
Hb Q India Thal Hb S Thal
Fig. 3. CE-HPLC Chromatograms of some hemoglobin variants seen in Indians
impossible to quantitate HbA2 in the presence of these
hemoglobins.4
In the presence of HbS, CE – HPLC gives falsely
increased HbA 2 levels due to the presence of Hb S
adducts and in the presence of Hb D, HbA 2 is falsely
reduced due to an integration error.12, 13
Hb H and Hb Bart’s can only be suspected by visual
analysis as they elute as a sharp peak at the start of the
chromatogram. The software does not integrate elution
peaks that have a retention time of <0.63 min and hence
they are not identified in the chromatogram.4, 10
A large prospective study was undertaken in the
United States of America on over 60,000 samples
analysed by CE-HPLC on the Variant II machine over a
32 months period in a multiethnic population. They
encountered 34 unique Hb variants and 2 tetramers. Of
these, 18 variants and 2 tetramers were identified by
retention time alone while a few variants also required
electrophoretic mobility analysis. In this study one
variant, Hb New York was missed on HPLC.14
We recently reported a new abnormal hemoglobin Hb
D Agri with 2 amino acid substitutions in the same β
globin chain [β9(A6)Ser → Tyr, β121 (GH-4) Glu →Gln].
This variant would have been mistaken for Hb S based on
HPLC alone. Further analysis was undertaken as this
hemoglobin eluting in the Hb S window had a negative
660
64
HbD Iran -Thal Hb D Punjab trait HbS Homozygous
β Thal trait Normal HbE trait (7 mths child)
sickling and solubility test.15 Thus, definitive diagnosis
does require alternative methods.
Variants eluting in the HbF window
Some α and β chain variants may elute in the Hb F
window and whenever there is an unexpected increase in
Hb F levels usually >10%, further studies should be
undertaken.14
Variants eluting in the P2 window
Hb A1c which is increased in diabetes elutes in the P 2
window. Only one variant, Hb Hope with a percentage
of >40% elutes in this window.14
Variants eluting in the P3 window
Most of the α and β chain Hb J variants elute in the P3
window, besides some other abnormal hemoglobins like
Hb Fannin –Lubbock and Hb N Baltimore. Hb
electrophoresis is helpful for identification of some of
these variants.14
Variants eluting in the Ao window
One variant Hb Twin Peaks shows a characteristic hump
in the A0 peak. Hb New York has an identical retention
time as HbAo.14
Variants eluting in the A2 window
As mentioned earlier, Hb E, Hb D Iran and Hb Lepore all
Indian Journal of Pediatrics, Volume 74—July, 2007
65
HPLC Studies in Hemoglobinopathies
elute in the HbA2 window. Hb Lepore has a characteristic
hump in the peak.14
Variants eluting in the Hb C window
Few variants like Hb O Arab and Hb Constant Spring
elute in the Hb C window.10 HbQ India elutes as a sharp
peak just before the Hb C window.
The library of hemoglobin variants with their
characteristic retention times is helpful for presumptive
identification of some of the rare variants which are
occasionally encountered.
Reverse phase HPLC of globin chains
This is an additional tool often helpful for identifying a
few Hb variants although its major application is in
measuring the γ chain ratios (Gγ/Aγ) in different
hemoglobin disorders. The polypeptide chains are
separated according to their hydrophobicity. The
separation and quantitation of the normal and variant
globin chains often helps in the presumptive diagnosis of
some uncommon variants.16
CE-HPLC for newborn screening of hemoglo-
binopathies
Newborn screening is primarily useful to identify infants
with sickle cell disease so that early intervention with
prophylactic penicillin can ameliorate complications and
comprehensive care can reduce morbidity and
mortality.17
Hb electrophoresis has fallen out as a laboratory
approach for neonatal screening although it was used in
early studies. IEF and CE – HPLC are now the methods
of choice. 18 The sickle cell short programme on the
Variant machine provides a 3 minutes analysis for
detection and quantitation of Hb S. The β-thal short
programme can also be used for neonatal screening for
both sickle cell disease and β-thalassemia major.
A liquid blood sample collected in EDTA from the
umbilical cord or by heel prick can be used. Alternately
a dried blood spot (DBS) taken by heel prick on a Guthrie
card can be used. The latter has the advantage of ease of
transportation and stability of proteins and DNA if
required for analysis upto 1 year.19
CE-HPLC in prenatal diagnosis of hemoglobinopathies
Population screening, genetic counseling and prenatal
diagnosis programmes by analysis of fetal blood in
second trimester fetuses started in Mediterranean
countries in the 1970s resulting in considerable reduction
in birth rates of affected children.20
In Iran, premarital screening was initiated in 1996.
Within 5 years, 10,000 couples were screened for β-
thalassemia and there was a 70% reduction in the
expected annual births of affected infants with β-
thalassemia major.21
Indian Journal of Pediatrics, Volume 74—July, 2007
Today first trimester fetal diagnosis by CVS and DNA
analysis is the method of choice for prenatal diagnosis of
hemoglobinopathies. However, in India and some other
countries couples at risk get identified late and second
trimester diagnosis becomes necessary.22
Experience in Thailand, where Hb E thalassemia is
common, showed that while cordocentesis and HPLC
analysis of fetal blood using the β thal short programme
was useful for prenatal diagnosis of β o thalassemia
homozygotes and β o / Hb E thalassemia double
heterozygotes, β + thalassemia homozygotes may be
misdiagnosed as thalassemia carriers. Further, HPLC
may not differentiate βo thalassemia/ Hb E from Hb E
homozygotes as both would show the absence of Hb A
and the amount of Hb E may overlap. However, they
found that the pattern obtained for Hb Bart’s hydrops
fetalis, which is also seen in Thailand was very specific
making this approach simple, reliable and inexpensive.23
Our own experience with fetal blood analysis by
automated HPLC for second trimester prenatal diagnosis
has shown that it is possible to diagnose β o and β +
thalassemia homozygotes as well as fetuses with sickle
cell disease using the β-thalassemia short programme.
The Hb Ao levels ranged from O to 0.6% in the βo and β+
thalassemia homozygotes while heterozygotes had levels
of A o greater than 1.7%. These findings could be
correlated with the molecular data. However, there was
an overlap between the HbA o levels in β-thalassemia
heterozygous and normal fetuses.22,24 Although CE-HPLC
analysis of fetal blood obtained by cordocentesis between
18 and 20 week gestation is technically simple, some
precautions need to be followed. The fetal blood sample
should be free of any maternal contamination. Controls
must always be run and the baseline and retention times
of different hemoglobin types checked. A distilled water
sample must be run before the fetal sample to ensure that
there is no carry over of Ao and other hemoglobins from
a previous sample. The fetal sample must always be run
in duplicate and in case of borderline values of HbA o (0.7
to 1.7%), a second method must be used for confirmation
of diagnosis.
Thus CE-HPLC has proved to be an accurate and
simple technique for quantitation of HbA2 and Hb F and
other hemoglobin subtypes. It helps in the presumptive
identification of many abnormal hemoglobin variants and
can also be applied for newborn screening and prenatal
diagnosis of hemoglobinopathies.
Denaturing HPLC (DHPLC)
A recently described powerful technique for detection of
mutations in DNA is denaturing HPLC (DHPLC). This
has been used for detection of single nucleotide
substitutions or small insertions and deletions in both α
and β globin genes. Small regions of the genes are
amplified by PCR and analyzed using either partially or
661

R.B. Colah et al
fully denaturing HPLC. The method is based on the
differential retention time of homoduplexes and
heteroduplexes in a mixture of denatured and re-
annealed PCR products. The eluted DNA is monitored
using UV or fluorescent detectors. DHPLC has been
shown to be a quick, sensitive and specific means of
detecting mutations and polymorphisms, however, DNA
sequencing may be required sometimes for a definitive
characterization of the molecular defect 25-27.
Defining the mutations by molecular analysis
DNA analysis is sometimes required for identifying
carriers of silent β-thalassemia or normal HbA 2 β-
thalassemia. Molecular methods are also required for
identification of some of the novel and rare hemoglobin
variants. Different methods like covalent reverse dot blot
hybridization, amplification refractory mutation system
(ARMS) or DNA sequencing can be used for direct
detection of mutations. 28
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Indian Journal of Pediatrics, Volume 74—July, 2007