Hemoglobin variants and highperformance liquid

International Journal of Laboratory Hematology
The Official journal of the International Society for Laboratory Hematology
LETTER TO THE EDITOR
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Letter to the Editor
Hemoglobin variants and high-performance liquid
chromatography
Sir,
We read with great interest the recently published article
by Suberski et al. [1] on ‘A comprehensive analysis of
hemoglobin variants by HPLC’. In this article, the authors
have mentioned that high-performance liquid chromatography (HPLC), though a very powerful tool in the
evaluation of hemoglobin variants, cannot be used as a
sole method for identification of hemoglobin variants
and that it has to be combined with other techniques like
acid or alkaline electrophoresis or isoelectric focusing
(IEF). We completely agree with the authors that HPLC
along with cellulose electrophoresis (pH 8.9) or IEF is
very important in identification of different hemoglobin
variants and HPLC when used as a stand-alone method
may lead to missing out of the detection of some variants. Besides these valid separation methods, the ideal
combination for the modern laboratory today would be
HPLC/capillary electrophoresis (CE). This combination
allows an alternative to automated HPLC with an automated separation superior to alkaline electrophoresis.
Moreover, CE offers an alternative estimation of the fractions allowing the detection of artifacts like overestimation or underestimations of HbF, HbA2, HbH, and Hb
Bart’s and make possible the detection of large or small
abnormal fractions that may remain undetected on HPLC
or vice versa [2].
Our institute has been involved in the diagnosis of
hemoglobinopathies and we are most often referred
cases presenting with varying degrees of microcytosis
and high red cell counts for diagnosis. During our
screening, using HPLC along with cellulose acetate
electrophoresis at alkaline pH, we could identify many
hemoglobin variants like Hb S, Hb D Punjab, Hb D
Iran, Hb E, Hb Lepore, Hb Jackson, Hb J Meerut, Hb J
Paris I, Hb Koya Dora, Hb O Indonesia, Hb Fontainebleu, and Hb Koln in our population [3–6]. Two new
hemoglobin variants in the b chain (Hb Vellore and Hb
Haaglanden) have been recently reported mimicking
© 2013 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
Hb S (b6 glutamine → valine), which is commonly
present in many populations. Both the variants elute
in the Hb S window on HPLC and would have been
misdiagnosed if no other tests (sickling test, DNA
sequencing) were used to confirm the variants [7, 8].
Although the authors have excluded cases of Hb H
from the study, we feel from our experience that HPLC
along with cellulose acetate electrophoresis will help in
diagnosis of Hb H disease due to the nondeletional
hemoglobin variant Hb Sallanches when present in the
homozygous state [9]. Some variants like Hb Sallanches
show no abnormalities in the heterozygous condition
but are clinically significant when present in the
homozygous condition or when another alpha thalassemia determinant is present and results in HbH disease.
Co-inheritance of b thalassemia trait and Hb H disease
shows low levels of Hb A2 on HPLC and can be misdiagnosed if only HPLC is used [10]. Moreover, in case
of using HPLC alone, Hb E-Lepore may be wrongly
diagnosed as Hb E-b thalassemia based on the chromatogram as both Hb E and Hb Lepore will elute in
the same window [11]. However, if hemoglobin electrophoresis (pH 8.9) is also performed, Hb E and Hb Lepore
will have different mobilities. Family studies are also
useful in such situations. The authors have also said
that the relative position of the hemoglobin variants
should be analogous regardless of the system and the
data should be similar on the Bio-Rad Variant IIe
system also. We agree with the authors because during
our study we have some of the analyzed the variants
we identified on both the Bio-Rad Classic and Bio-Rad
Variant IIe systems and we did not find any difference
in the position of the variants. It is very important that
the system is accurately calibrated before each run and
the same calibrated pipette be used for loading the
hemoglobin samples (in case of Bio-Rad Variant Classic); otherwise, the discrepancy in the retention time as
mentioned by the authors will arise which will create
problems in identifying the variants. As suggested by
the authors, the hemoglobin variant peak shape,
position, and their retention time indeed act as a finger
1
2
LETTER TO THE EDITOR
print and are very helpful in detecting different variants. However, ultimately DNA sequencing has to be
carried out in all the cases to make a final and confirmed diagnosis. Thus, a combination of chromatographic and electrophoretic techniques along with
hemoglobin instability tests is needed in identifying the
variants [12].
References
1. Szuberski J, Oliveira JL, Hoyer JD. A comprehensive analysis of hemoglobin variants by
high-performance liquid chromatography
(HPLC). Int Jnl Lab Hem 2012;20.
2. Van Delft P, Lenters E, Bakker-Verweij M,
de Korte M, Baylan U, Harteveld CL,
Giordano PC. Evaluating five dedicated
automatic devices of hemoglobinopathy
diagnostics in multi ethnic populations. Int
Jnl Lab Hem 2009;31:484–95.
3. Colah RB, Surve R, Sawant P, D’Souza E,
Italia K, Phanasgaonkar S, Nadkarni AH,
Gorakshakar AC. HPLC studies in Hemoglobinopathies. Indian J Pediatr 2007;74:657–62.
4. Nair S, Nadkarni AH, Warang P, Bhave A,
Ghosh K, Colah RB. Five a globin chain
variants identified during screening for
hemoglobinopathies. Eur J Clin Invest
2010;40:226–32.
S. Nair, A. H. Nadkarni, K. Ghosh, R. Colah
National Institute of Immunohematology, Mumbai, India
E-mail: [email protected]
doi: 10.1111/ijlh.12052
5. Upadhye DS, Jain D, Nair SB, Nadkarni
AH, Ghosh K, Colah RB. First case of Hb
Fontainebleau with sickle hemoglobin and
other non-deletional a gene variants identified in neonates during newborn screening
for sickle cell disorders. J Clin Pathol
2012;65:654–9.
6. Warang P, Kedar P, Nair S, Nadkarni A,
Ghosh K, Bhave A, Colah R. Hb Koln [b98
(FG5) [GTGATG, Val Meth]: the first report
from India. Indian J Hematol Blood Transfus 2012 (in press).
7. Edison ES, Sathya M, Rajkumar SV, Nair
SC, Srivastava A, Shaji RV. A novel b
globin gene mutation HBB.c.22G>C produces a hemoglobin variant (Hb Vellore)
mimicking Hb S in HPLC. Int Jnl Lab
Hem 2012;34:556–8.
8. Harteveld CL, Ponjee G, Bakker-Verweij M,
Arkesteijn SGJ, Phylipsen M. Hb Haaglanden: a new non sickling b7Glu>Val variant.
Consequences for basic diagnostics, screen-
9.
10.
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12.
ing and risk assessment when dealing with
Hb S like variants. Int Jnl Lab Hem
2012;34:551–5.
Warang P, Nair S, Nadkarni A, Ghosh K,
Colah RB. HbH disease due to homozygosity for a rare a2 globin variant, Hb Sallanches. Hemoglobin 2010;34:45–8.
Yin XL, Wu ZK, Zhou XY, Zhou TH, Zhou
YL, Wang L, Huan J, Zhang XH. Co-inherited b-thalassemia trait and Hb H disease:
clinical characteristic and interference in
diagnosis of thalassaemia by high performance liquid chromatography. Int Jnl Lab
Hem 2012;34:427–31.
Italia K, Sheth J, Sawant P, Nadkarni A,
Ghosh K, Colah R. Prenatal diagnosis of Hb
E-Lepore and Hb Lepore-b-thalassemia: the
importance of accurate genotyping of the
couple at risk. Prenat Diagn 2012;32:703–7.
Giordano PC. Editorial: measurement of
HbA2. Int Jnl Lab Hem 2012;34:335.
© 2013 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.

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