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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

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:

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

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

Indian Journal of Pediatrics, Volume 74—July, 2007 657


R.B. Colah et al

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

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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

In this technique the hemoglobins migrate in a pH P2

gradient prepared by using a combination of different Hb F
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 Hb H Faster than Hb A
Hb Bart's
sharp bands which makes quantitation also more
accurate. Fig 2. Retention times of hemoglobin variants on CE – HPLC
(Biorad Variant Hb Testing System) and their electrophoretic
Capillary IEF (cIEF) mobilities
Here, the sensitivity of detection of capillary Problems in interpretation
electrophoresis is combined with the automated sampling
and data acquisition of HPLC. Quantiation of HbA2, Hb Automated HPLC using the Variant machine has some
F and other variants compare well with HPLC and some intrinsic problems. Some hemoglobins co-elute in the Hb
variants like Hb D Punjab and Hb C Harlem give a better A 2 window like Hb E, Hb D Iran, Hb G Copenhagen,
resolution.4,11 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) HbD Iran -Thal Hb D Punjab trait HbS Homozygous

Hb Q India Thal Hb S Thal β Thal trait Normal HbE trait (7 mths child)
Fig. 3. CE-HPLC Chromatograms of some hemoglobin variants seen in Indians

impossible to quantitate HbA2 in the presence of these sickling and solubility test.15 Thus, definitive diagnosis
hemoglobins.4 does require alternative methods.
In the presence of HbS, CE – HPLC gives falsely Variants eluting in the HbF window
increased HbA 2 levels due to the presence of Hb S
Some α and β chain variants may elute in the Hb F
adducts and in the presence of Hb D, HbA 2 is falsely
window and whenever there is an unexpected increase in
reduced due to an integration error.12, 13
Hb F levels usually >10%, further studies should be
Hb H and Hb Bart’s can only be suspected by visual undertaken.14
analysis as they elute as a sharp peak at the start of the
Variants eluting in the P2 window
chromatogram. The software does not integrate elution
peaks that have a retention time of <0.63 min and hence Hb A1c which is increased in diabetes elutes in the P 2
they are not identified in the chromatogram.4, 10 window. Only one variant, Hb Hope with a percentage
of >40% elutes in this window.14
A large prospective study was undertaken in the
United States of America on over 60,000 samples Variants eluting in the P3 window
analysed by CE-HPLC on the Variant II machine over a
Most of the α and β chain Hb J variants elute in the P3
32 months period in a multiethnic population. They
window, besides some other abnormal hemoglobins like
encountered 34 unique Hb variants and 2 tetramers. Of
Hb Fannin –Lubbock and Hb N Baltimore. Hb
these, 18 variants and 2 tetramers were identified by
electrophoresis is helpful for identification of some of
retention time alone while a few variants also required
these variants.14
electrophoretic mobility analysis. In this study one
variant, Hb New York was missed on HPLC.14 Variants eluting in the Ao window
We recently reported a new abnormal hemoglobin Hb One variant Hb Twin Peaks shows a characteristic hump
D Agri with 2 amino acid substitutions in the same β in the A0 peak. Hb New York has an identical retention
globin chain [β9(A6)Ser → Tyr, β121 (GH-4) Glu →Gln]. time as HbAo.14
This variant would have been mistaken for Hb S based on
HPLC alone. Further analysis was undertaken as this Variants eluting in the A2 window
hemoglobin eluting in the Hb S window had a negative As mentioned earlier, Hb E, Hb D Iran and Hb Lepore all

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


HPLC Studies in Hemoglobinopathies

elute in the HbA2 window. Hb Lepore has a characteristic Today first trimester fetal diagnosis by CVS and DNA
hump in the peak.14 analysis is the method of choice for prenatal diagnosis of
hemoglobinopathies. However, in India and some other
Variants eluting in the Hb C window
countries couples at risk get identified late and second
Few variants like Hb O Arab and Hb Constant Spring trimester diagnosis becomes necessary.22
elute in the Hb C window.10 HbQ India elutes as a sharp
Experience in Thailand, where Hb E thalassemia is
peak just before the Hb C window.
common, showed that while cordocentesis and HPLC
The library of hemoglobin variants with their analysis of fetal blood using the β thal short programme
characteristic retention times is helpful for presumptive was useful for prenatal diagnosis of β o thalassemia
identification of some of the rare variants which are homozygotes and β o / Hb E thalassemia double
occasionally encountered. heterozygotes, β + thalassemia homozygotes may be
misdiagnosed as thalassemia carriers. Further, HPLC
Reverse phase HPLC of globin chains
may not differentiate βo thalassemia/ Hb E from Hb E
This is an additional tool often helpful for identifying a homozygotes as both would show the absence of Hb A
few Hb variants although its major application is in and the amount of Hb E may overlap. However, they
measuring the γ chain ratios (Gγ/Aγ) in different found that the pattern obtained for Hb Bart’s hydrops
hemoglobin disorders. The polypeptide chains are fetalis, which is also seen in Thailand was very specific
separated according to their hydrophobicity. The making this approach simple, reliable and inexpensive.23
separation and quantitation of the normal and variant
Our own experience with fetal blood analysis by
globin chains often helps in the presumptive diagnosis of
automated HPLC for second trimester prenatal diagnosis
some uncommon variants.16
has shown that it is possible to diagnose β o and β +
CE-HPLC for newborn screening of hemoglo- thalassemia homozygotes as well as fetuses with sickle
binopathies cell disease using the β-thalassemia short programme.
The Hb Ao levels ranged from O to 0.6% in the βo and β+
Newborn screening is primarily useful to identify infants thalassemia homozygotes while heterozygotes had levels
with sickle cell disease so that early intervention with of A o greater than 1.7%. These findings could be
prophylactic penicillin can ameliorate complications and correlated with the molecular data. However, there was
comprehensive care can reduce morbidity and an overlap between the HbA o levels in β-thalassemia
mortality.17 heterozygous and normal fetuses.22,24 Although CE-HPLC
Hb electrophoresis has fallen out as a laboratory analysis of fetal blood obtained by cordocentesis between
approach for neonatal screening although it was used in 18 and 20 week gestation is technically simple, some
early studies. IEF and CE – HPLC are now the methods precautions need to be followed. The fetal blood sample
of choice. 18 The sickle cell short programme on the should be free of any maternal contamination. Controls
Variant machine provides a 3 minutes analysis for must always be run and the baseline and retention times
detection and quantitation of Hb S. The β-thal short of different hemoglobin types checked. A distilled water
programme can also be used for neonatal screening for sample must be run before the fetal sample to ensure that
both sickle cell disease and β-thalassemia major. there is no carry over of Ao and other hemoglobins from
a previous sample. The fetal sample must always be run
A liquid blood sample collected in EDTA from the in duplicate and in case of borderline values of HbA o (0.7
umbilical cord or by heel prick can be used. Alternately to 1.7%), a second method must be used for confirmation
a dried blood spot (DBS) taken by heel prick on a Guthrie of diagnosis.
card can be used. The latter has the advantage of ease of
transportation and stability of proteins and DNA if Thus CE-HPLC has proved to be an accurate and
required for analysis upto 1 year.19 simple technique for quantitation of HbA2 and Hb F and
other hemoglobin subtypes. It helps in the presumptive
CE-HPLC in prenatal diagnosis of hemoglobinopathies identification of many abnormal hemoglobin variants and
Population screening, genetic counseling and prenatal can also be applied for newborn screening and prenatal
diagnosis programmes by analysis of fetal blood in diagnosis of hemoglobinopathies.
second trimester fetuses started in Mediterranean Denaturing HPLC (DHPLC)
countries in the 1970s resulting in considerable reduction
in birth rates of affected children.20 A recently described powerful technique for detection of
mutations in DNA is denaturing HPLC (DHPLC). This
In Iran, premarital screening was initiated in 1996. has been used for detection of single nucleotide
Within 5 years, 10,000 couples were screened for β- substitutions or small insertions and deletions in both α
thalassemia and there was a 70% reduction in the and β globin genes. Small regions of the genes are
expected annual births of affected infants with β- amplified by PCR and analyzed using either partially or
thalassemia major.21
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R.B. Colah et al

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