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International Scholarly Research Notices

Volume 2015, Article ID 498230, 1 page
http://dx.doi.org/10.1155/2015/498230

Retraction
Retracted: Detection of Abnormal Hemoglobin Variants by
HPLC Method: Common Problems with Suggested Solutions

International Scholarly Research Notices

Received 14 April 2015; Accepted 14 April 2015

Copyright © 2015 International Scholarly Research Notices. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

The paper titled “Detection of Abnormal Hemoglobin Vari-

ants by HPLC Method: Common Problems with Suggested
Solutions,” [1] published in International Scholarly Research
Notices, has been retracted as it was submitted for publication
without the knowledge and approval of one of the coauthors,
Dr. Suman Lata Mendiratta. The submitting author Dr. Dipti
Kalita plagiarized data from the Thalassemia Control Project
headed by Dr. Suman Lata Mendiratta.

References
[1] L. Pant, D. Kalita, S. Singh et al., “Detection of abnormal hemo-
globin variants by HPLC method: common problems with sug-
gested solutions,” International Scholarly Research Notices, vol.
2014, Article ID 257805, 10 pages, 2014.
Hindawi Publishing Corporation
International Scholarly Research Notices
Volume 2014, Article ID 257805, 10 pages
http://dx.doi.org/10.1155/2014/257805

Research Article
Detection of Abnormal Hemoglobin Variants by HPLC Method:
Common Problems with Suggested Solutions

Leela Pant,1 Dipti Kalita,1 Sompal Singh,1 Madhur Kudesia,1 Sumanlata Mendiratta,2
Meenakshi Mittal,2 and Alka Mathur3
1
Department of Pathology, Hindu Rao Hospital, Delhi 110007, India
2
Thalassemia Control cell, Hindu Rao Hospital, Delhi, India
3
Department of Pediatrics, Hindu Rao Hospital, Delhi, India

Correspondence should be addressed to Dipti Kalita; [email protected]

Received 13 April 2014; Accepted 16 July 2014; Published 12 October 2014

Academic Editor: Josep Esteve-Romero

Copyright © 2014 Leela Pant et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Thalassemia and thalassemic hemoglobinopathies pose serious health problem leading to severe morbidity and mortality in Indian
population. Plethora of hemoglobin variants is prevalent in multiethnic Indian population. The aim of the present study was to
analyze laboratory aspects, namely, hematological profile and HPLC findings of the hemoglobin variants detected, and to discuss
problems that we faced in diagnosis in a routine clinical laboratory. We screened a total of 4800 cases in a hospital based population
of North India in a 2-years period of by automated HPLC method using the Variant Hemoglobin Testing System (Variant II Beta
Thalassemia Short Program, Bio-Rad Laboratories) under the experimental conditions specified by the manufacturer. Whole blood
in EDTA was used and red cell indices were determined using automated hematology analyzer. We detected 290 cases with abnormal
variants in which beta thalassemia was the most common followed by hemoglobin E. Here, we discuss the laboratory aspects of
various hemoglobin disorders and diagnostic difficulties in cases like borderline HbA2 values, presence of silent mutation, alpha
thalassemia gene, and few rare variants which at times require correlation with genetic study. Special attention was given to HbA2
level even in presence of a structural variant to rule out coinheritance of beta thalassemia gene.

1. Introduction clinically significant variants. The simplicity of the automated

system with internal sample preparation, superior resolution,
Thalassemia is an autosomal recessive inherited group of rapid assay time, and accurate quantification of hemoglobin
disorders of hemoglobin synthesis characterized by the fractions makes this an ideal methodology for the routine
absence or reduction of one or more of the globin chains of clinical laboratory [2, 3].
hemoglobin. The structural variants result from substitution With increasing global awareness and mass screening
of one or more amino acids in the globin chains of the programs undertaken at various levels by health care sys-
hemoglobin molecule [1]. Plethora of hemoglobin variants is tem, the responsibility for laboratory personnel has greatly
prevalent in India owing to ethnic diversity of its population enhanced in detection and prevention of this problem.
with minimal to major clinical significance. Being recessively Awareness about the diagnostic problems as well as their
inherited from the parents, the thalassemia and thalassemic solutions is very important so that one does not miss a single
hemoglobinopathies pose serious health problem leading to case.
severe morbidity and mortality in Indian population. Many studies have been published from India on tha-
Detection of asymptomatic carriers by reliable laboratory lassemia and thalassemic hemoglobinopathies mostly putting
methods is the cornerstone of prevention of this serious emphasis on epidemiology and screening [4–6].
health problem. Cation exchange high performance liquid Very few studies are available on the approach of diagno-
chromatography (CE-HPLC) has become the preferred tech- sis and problems in routine diagnosis, especially in laborato-
nique suitable in Indian scenario, as it can detect most of the ries with limited resources.
2 International Scholarly Research Notices

The aim of the present study was to analyze laboratory Table 1: Distribution of hemoglobin variants.
aspects, namely, hematological profile and HPLC findings of
the hemoglobin variants detected, and to discuss problems Hemoglobin variant Number (%)
that we faced in diagnosis in a routine clinical laboratory. Beta thalassemia trait (BTT) 216 (74.48)
Beta thalassemia intermedia/major (BTI/M) 9 (3.10)
Hb E trait (HbAE) 28 (9.65)
2. Material and Methods
Hb E disease (HbEE) 2 (0.69)
This was a prospective study carried out in the Department Hb E beta thalassemia (HbE-BT) 2 (0.69)
of Pathology and Thalassemia Control Cell, Hindu Rao Hb D trait (HbAD) 15 (5.17)
Hospital, Delhi, for 2 years’ period. A total of 4800 cases HbD-beta thalassemia (HbD-BT) 4 (1.38)
were screened for presence of thalassemia or any structural HbS trait (HbAS) 9 (3.10)
variant. These included all cases of microcytic hypochromic Delta beta thalassemia (deltaBTT) 3 (1.03)
anaemia (MCV < 80 fl, MCH < 27 pg, and RBC count
Hb J Meerut 1 (0.34)
> 5 million/𝜇l) not responding to conventional treatment,
Hb Hope 1 (0.34)
clinically suspected cases of hemoglobinopathy, antenatal,
and other cases coming to the department for thalassemia Total 290
screening. A 5 mL intravenous blood sample was collected in
EDTA anticoagulant. Red cell indices were measured on an
automated haematology analyzer (Sysmex KX 21). (4) Wash/diluent solution: deionized water.
HbA2, HbF, and other haemoglobin variants were studied (5) Control: normal (HbF 1-2%, HbA2 1.8–3.2%) and
by HPLC method used for chromatographic separation of abnormal (HbF 5–10%, HbA2 4–6%) controls.
human hemoglobin [7–9].
We used the Variant Hemoglobin Testing System (Variant 2.3. Sample Collection and Preparation. Five milliliters
II Beta Thalassemia Short Program, Bio-Rad Laboratories (5 mL) of whole blood was collected in a vacuum collection
Inc., Hercules, CA, USA) under the experimental conditions tube containing EDTA which can be stored at 2–8 degrees C
specified by the manufacturer [10]. for maximum 7 days if processing is delayed. No preparation
was required unless the sample was in a tube other than the
2.1. Principle. The Variant II Beta Thalassemia Short Pro- recommended tube or there was less than 500 𝜇L of sample
gram utilizes principles of ion-exchange high-performance in the tube. In such case, sample was manually prediluted.
liquid chromatography (HPLC). The samples are automati- Predilution was carried out by mixing 1.0 mL wash/diluents
cally mixed and diluted on the Variant II Sampling Station with 5 𝜇L of whole blood sample.
(VSS) and injected to the analytical cartridge. The Variant HbA2/F calibrators and normal and abnormal controls
II Chromatographic Station (VCS) dual pumps deliver a were analyzed at the beginning of each run.
programmed buffer gradient of increasing ionic strength to
the cartridge, where HbA2/F are separated based on their
2.4. Interpretation of Reports. Reports and chromatograms
ionic interaction with the cartridge material. The separated
generated were studied and interpreted by observing HbA2
HbA2/F then pass through the flow cell of the filter photome-
and F concentration for beta thalassemia and retention
ter where the changes in absorbance at 415 nm are measured.
time and area percentage of other peaks and windows for
An additional filter at 690 nm corrects the background
structural variants. Each chromatogram shows peaks of Hb
absorbance. The Variant II CDM (CDM) Software performs
A0, A2, and Hb F along with C window, D window, S window,
reduction of raw data collected from each analysis. To aid in
and two minor peaks, P2 and P3. Several hemoglobin variants
the interpretation of results, windows have been established
elute same window; they were provisionally diagnosed by
for the most frequently occurring hemoglobins based on the
retention time and area percentage keeping in mind the
characteristic retention time. For each sample a sample report
ethnicity of the patients.
and a chromatogram are generated by CDM showing all
Other relevant tests were done, for example, sickling test
hemoglobin fractions eluted, their retention times, the area
as supporting evidence of Hb S. Family study was carried
of the peaks, and values of the fractions.
out whenever possible and correlation with findings of Hb
electrophoresis result was done in few cases.
2.2. Reagents
(1) Elution buffers (1,2): sodium phosphate buffer. 3. Results and Discussion
(2) Whole blood primer: lyophilized human red blood 3.1. Results. Among 4800 cases screened, 290 (6.04%) cases
cell hemolysate with gentamicin, tobramycin, and were detected with abnormal hemoglobin in this study.
EDTA as preservative. Presumptive identification of hemoglobin variants was made
(3) HbA2/F calibrator/diluent set: lyophilized human red primarily by retention time (RT) windows and area percent;
blood cell hemolysate with gentamicin, tobramycin, however geographical factor, ethnicity, and clinical presen-
and EDTA as preservative analytical cartridge. Dilu- tation were also taken into consideration. Distribution of
ent contains deionized water. hemoglobin variants identified is shown in Table 1. Their
International Scholarly Research Notices 3

Calibrated Retention Peak Peak name Calibrated Area (%) Retention Peak
Peak name area (%) Area (%) time (min) area area (%) time (min) area
Unknown — 0.0 0.96 1170 P1 — 0.1 0.74 1290
F 0.4 — 1.08 13120 F 29.1∗ — 1.13 326472
Unknown — 1.0 1.20 32945 P2 — 4.2 1.32 48837
P2 — 3.7 1.32 116596 P3 — 2.5 1.67 28868
Unknown — 0.6 1.48 17382 Unknown — 0.1 2.06 753
P3 — 4.1 1.72 129129 A0 — 63.0 2.54 738847
2.46 2647621 ∗
A0 —
— 83.8 A2 2.2 — 3.58 28242
A2 5.6∗ — 3.68 201552 Total area: 1,173,30
F concentration = 0.4% Total area: 3,159,5 F concentration = 29.1∗ %
A2 concentration = 5.6∗ % A2 concentration = 2.2∗ %
∗ ∗
Values outside of expected ranges Values outside of expected ranges
Analysis comments:
Analysis comments:
45.0 45.0

37.5 37.5

30.0 30.0
(%)

(%)
22.5 22.5
3.68

15.0

3.58
15.0

1.32
1.37

1.67
0.96
1.08

2.06
0.74
1.20

1.72

A2

7.5
1.48

7.5

2.54
2.46

A2
1.13
F

0.0 0.0

F
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Time (min) Time (min)
(a) (b)

Figure 1: (a) Chromatogram of beta thalassemia trait showing elevated HbA2 5.6% (RT 3.68 min) and HbF 0.4%. (b) Chromatogram showing
elevated Hb F (29.1%) suggestive of HPFH.

Table 2: Mean values (mean ± SD) of red cell parameters and hemoglobin fractions in variants detected by HPLC.

Hb (g/dL) RBC ×106 cumm MCV (fL) MCH (pg) MCHC (g/dL) HbA (%) HbA2 (%) HbF (%) Other (%) Hb variant
10.0 ± 2.1 4.8 ± 1.02 70.7 ± 9.7 21.05 ± 3.2 29.7 ± 2.7 89.0 ± 2.5 5.2 ± 0.75 1.08 ± 0.7 BTT
4.78 ± 3.4 2.2 ± 1.3 73.2 ± 5.1 20.9 ± 2.1 28.5 ± 2.8 23.5 ± 22.6 4.06 ± 1.2 64 ± 28.6 BTM/I
10.7 ± 2.4 4.2 ± 1.05 81.8 ± 1.2 25.3 ± 3.4 31.5 ± 2.2 60.9 ± 6.6 26.6 ± 4.5 1.08 ± 1.2 HbAE
11.6 ± 1.0 5.9 ± 0.7 60.4 ± 0.7 19.8 ± 1.5 32.3 ± 2.1 3.8 ± 0.86 79.9 ± 2.5 3.92 ± 0.1 HbEE
8.5 ± 1.9 5.16 ± 1.5 63.4 ± 2.3 20.8 ± 1.3 30.3 ± 2.3 17.5 ± 9.65 61.6 ± 8.1 9.75 ± 4.7 HbE-BT
11.6 ± 2.0 4.3 ± 0.41 81.4 ± 7.3 26.2 ± 3.5 32.2 ± 2.8 52.4 ± 2.6 2.14 ± 0.5 1.02 ± 1.6 HbD36.7 ± 2 HbAD
9.65 ± 1.6 4.4 ± 0.97 67.9 ± 4.3 21.8 ± 1.8 32.1 ± 0.7 3.95 ± 2.3 4.67 ± 0.87 3.95 ± 2.3 HbD 79.7 ± 1.6 HbD-BT
9.6 ± 3.4 3.8 ± 1.4 79.6 ± 13.3 23.7 ± 5.16 30.4 ± 2.03 30.4 ± 2.03 3.25 ± 0.4 2.4 ± 2.9 HbS 29.7 ± 10.4 HbAS
11.7 ± 0.46 4.9 ± 0.49 76.3 ± 3 23.3 ± 1.5 31.4 ± 0.17 75.3 ± 0.19 2.43 ± 0.17 15.7 ± 2.6 deltaBTT
5.9 4.23 66.9 13.9 20.8 75.2 2 1.1 P3 20.0 Hb J Meerut
16.7 5.31 94.5 31.4 33.3 44.8 2.0 <1 P2 48.8 Hb Hope

relevant RBC parameters and HPLC findings are given in Majority were asymptomatic and detected during carrier
Table 2. Chromatograms of some important variants are screening and family studies.
shown in figures (Figures 1, 2, 3, 4, 5, and 6). Nine cases of beta thalassemia were detected which were
Genotypes of some structural variants are shown in classified as either major or intermedia depending upon
Table 3. clinical severity. HbF was raised (20.7–97.1%), with variable
As expected, beta thalassemia trait (BTT) was the most HbA2 (2.4–5.7%). Cases of thalassemia major presented
common hemoglobin variant (74.48%) detected in our study within 1st year of life. Parental study was done to find out
with elevated HbA2 level (>3.5%) and RT 3.63–3.69 min. carrier status and to confirm the diagnosis.
4 International Scholarly Research Notices

Calibrated Area (%) Retention Peak

Peak name area (%) time (min) area
Unknown — 0.0 0.98 1013 Peak name Calibrated Area (%) Retention Peak
F 0.2 — 1.07 6584 area (%) time (min) area
Unknown — 0.7 1.25 19627 P1 — 0.1 0.73 866
∗
P2 — 2.7 1.35 76646 F 30.1 — 1.12 470514
18463 P3 — 2.7 1.83 42498
Unknown — 0.7 1.52
2.0 55348 Unknown — 0.8 2.03 12667
Unknown — 1.74 Unknown — 4.0 2.25 62942
P3 — 2.2 1.83 62326
2.42 A0 — 7.0 2.48 109207
A0 — 63.3 1778805 A2 51.4∗ — 3.75 868101
A2 24.8∗ — 3.72 791409
Total area: 2,810,221 Total area: 1,566,79
F concentration = 0.2% F concentration = 30.1∗ %
A2 concentration = 24.8∗ % A2 concentration = 51.4∗ %
∗ ∗
Values outside of expected ranges Values outside of expected ranges
Analysis comments: Analysis comments:
45.0 45.0

37.5
3.72
37.5

30.0 30.0
A2

(%)
22.5 22.5
(%)

2.48
1.35

15.0 15.0
1.74
1.63

2.25
1.52
1.25
1.07

1.83
2.03
0.98

0.73
1.12

3.75
2.43

7.5 7.5
F

0.0 0.0

A2
F
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Time (min) Time (min)
(a) (b)

Figure 2: (a) Chromatogram of HbE trait showing HbA2 24.8% (RT 3.72 min). (b) Chromatogram of E beta thalassemia showing elevated
HbA2 51.4%, Hb F 30%.

Table 3: Genotypes of some common structural variants. with raised RBC counts and reduced MCV and MCH values.
Parental study was carried out for confirmation.
Abnormal variants Genotype Two cases of double heterozygous of HbE and beta
Hb E beta26(B8)Glu → Lys, GAG → AAG thalassemia trait were found showing peak in HbA2 window
Hb D Punjab beta121(GH4)Glu → Gln, GAA → CAA (53.5–69.7%) and raised HbF level (5–14.5%). Again, parental
Hb S beta6(A3)Glu → Val, GAG → GTG and family study was carried out for confirmation.
Hb D Iran beta22Glu → Gln, GAA → CAA Nineteen cases showed peak in the D window (RT
Hb Hope beta136(H14)Gly → Asp (GGT → GAT) 4.13–4.15 min), indicating presence of structural variant
Hb OArab beta121(GH4)Glu → Lys, (GAA → AAA) hemoglobin D (Hb D) Punjab. Among these, 15 were HbD
Hb C beta6(A3)Glu → Lys, GAG → AAG Punjab heterozygote showing Hb D level 31–40% and near
Hb J Meerut alpha120(H3)Ala → Glu, GCG → GAG
normal RBC parameters. Four cases had HbD level 77–
81.3%, with raised HbA2 levels (3.8–6%). These cases showed
Hb Q India alpha64(E13)Asp → His, GAC → CAC
reduced hemoglobin, MCV, and MCH levels. They were
provisionally diagnosed as double heterozygous of Hb D and
beta thalassemia trait. Parental study and genetic study were
done for confirmation.
Three cases showed raised HbF (13.7–19.7%), normal Nine cases had peak in S window (RT 4.27-4.28 min),
HbA2 values (2.2-2.3%), and normal Hb level, raised RBC indicating presence of hemoglobin S (HbS). Eight of them
count, and reduced MCV and MCH levels. Parental study was had HbS level 25–40%, HbF-0.2–7.4%. Mildly raised HbA2
done with a provisional diagnosis of delta beta thalassemia (3.3–3.7%) level was seen in 5 of them. There was one case
trait and DNA study was advised. with marked reduction in Hb, MCV, and MCH levels and
Hemoglobin E (HbE) was found to be the most common HbA2 10% and HbS 65.4%. Parental study was advised to
structural variant with raised peak in A2 window (RT 3.76– confirm the double heterozygous status. Sickling test was
3.78 min). A total of 28 cases of Hb E trait were detected with found to be positive in all these cases.
peak area ranging from 18.5 to 39% and mild elevation of HbF One case showed elevated P3 peak 20%, with RT 1.81 min,
level. Two cases of homozygous Hb E were detected showing suggesting Hb J Meerut with reduced MCV and MCH values.
77–83% HbE and 2–5% HbF. Hb level was mildly reduced Iron studies and family study were correlated.
International Scholarly Research Notices 5

Calibrated Area (%) Retention Peak

Peak name area (%) time (min) area
F 2.0 ∗ — 1.10 25943 Peak name
Calibrated
area (%) Area (%) Retention
time (min) Peak
area
Unknown — 0.8 1.74 11676 F 10.8∗ — 1.12 99893
P3 — 3.4 1.88 48007 P2 — 3.2 1.35 29854
Unknown — 0.6 2.09 7847 P3 — 2.7 1.71 25154
A0 — 3.4 2.32 46975 A0 — 54.5 2.54 513793
A2 77.5∗ — 3.73 1255408 A2 3.0 — 3.63 30726
Total area: 1,395,856 S window — 25.9 4.42 244100
F concentration = 2.0∗ % Total area: 943,519
F concentration = 10.8 ∗ %
A2 concentration = 77.5∗ %
A2 concentration = 3.0%
∗
Values outside of expected ranges ∗
Values outside of expected ranges
Analysis comments: Analysis comments:
45.0 45.0

4.42
37.5 37.5

30.0 30.0

1.12
(%)

(%)
22.5 22.5

F
15.0 15.0

3.63
1.86

1.35
1.71
1.10

1.74

2.32

3.73
2.09

7.5 7.5

A2
2.54
F

0.0 0.0
A2

0 1 2 3 4 5 6 0 1 2 3 4 5 6
Time (min) Time (min)
(a) (b)

Figure 3: (a) Chromatogram of HbE homozygous showing HbA2 77.5% (RT 3.73 min). (b) Chromatogram of Hb S trait showing Hb S 25.9%
(RT 4.42 min).

One case showed elevated peak (RT 1.37 min), 48.8% with None of the above-mentioned methods can detect multiple
normal hematological parameters. It was provisionally diag- hemoglobin fractions in a single step procedure. HPLC has
nosed as Hb Hope after further confirmation by hemoglobin many advantages over these methods and over the past
electrophoresis. Family study and DNA analysis were advised. decades it has evolved as an excellent and powerful diagnostic
tool for identification of most of the clinically significant
3.2. Discussion. India is an ethnically diverse country with Hb variants especially to beta thalassemia trait owing to
marked regional variation. This diversity is reflected in the its quantitative power and automation. HPLC is sensitive,
presence of different hemoglobin variants in different ethnic specific, reproducible, and less time consuming and requires
groups. Due to migration, there is constant mixing of peoples less man power. Hence, it is ideal for a routine clinical
from different regions. Many of these abnormal variants are laboratory with high work load.
of little clinical significance in heterozygous state, but when Our study was carried out in a tertiary care hospital in
combined with other variants they may give rise to severe Delhi, which represented not only north Indian population
disease. Therefore there is always a need for a screening but a large migrant population of eastern and north east-
method which can detect maximum variants. HPLC has ern parts of the country also. Apart from 𝛽-thalassaemia,
the advantage of quantifying Hb F and Hb A2 along with common variants were encountered with various incidences:
detecting other variants in a single screening test. HbE, HbD Punjab, and HbS—being the most common along
Besides HPLC, there are other analytical procedures used with a rare variant Hb Hope.
for detection of thalassemia and hemoglobinopathies such Thalassemia being the major concern in this study,
as alkaline and acid electrophoresis, Hb A2 quantification quantitation of HbA2 and Hb F levels by HPLC was of prime
by ion-exchange column chromatography, and Hb F quan- importance in our laboratory where facilities for genetic
tification by alkali denaturation and radial immunodiffusion studies are not available. However, parental study is also
(9). Electrophoretic method does not separate all variants of great help in arriving at a conclusive diagnosis before
from each other, and it is recommended by screening pro- referring patients for costly genetic studies.
grams such that further tests should always be carried out HbA2 is constantly elevated in heterozygous beta tha-
to confirm the presumed identity of an abnormal variant. lassemia carriers with values ranging from 3.5 to 7% with a
6 International Scholarly Research Notices

Calibrated Retention Peak Calibrated Area (%) Retention

Peak name area Peak
area (%) Area (%) time (min) Peak name area (%) time (min) area
Unknown — 0.0 0.96 720 F 0.1∗ — 1.06 2002
F 0.1∗ — 1.06 3545 Unknown — 0.5 1.21 6574
Unknown — 0.9 1.21 20718 P2 — 0.1 1.40 1014
P2 — 1.9 1.34 46113 P3 — 0.3 1.79 3598
Unknown — 0.4 1.52 9265 A0 — 4.2 2.13 60542
P3 — 2.1 1.72 51629 Unknown — 0.6 2.36 9102
Unknown — 0.5 1.98 12855 Unknown — 1.1 2.52 15738
Unknown — 2.0 2.13 47777 Unknown — 2.6 2.88 36721
A0 — 49.1 2.46 1194355 A2 2.4 — 3.67 38898
A2 2.3 — 3.69 60767 D window — 87.9 4.20 1263052
D window — 40.5 4.15 985084
Total area: 1,437,242
∗
F concentration = 0.1 % Total area: 2,432,82 F concentration = 0.1∗ %

A2 concentration = 2.3% A2 concentration = 2.4%

∗ ∗
Values outside of expected ranges Values outside of expected ranges
Analysis comments: Analysis comments:
45.0 45.0
37.5 37.5
30.0 30.0
(%)

(%)
22.5 22.5

3.67
3.69

15.0 15.0

2.23
2.13
F 1.06
1.34
1.73
1.21

1.79
1.92
1.98

2.36
2.52
F 1.06
1.21
1.40

2.88
0.96

2.46

4.20
3.15

7.5 7.5

A2
A2

0.0 0.0

0 1 2 3 4 5 6 0 1 2 3 4 5 6
Time (min) Time (min)
(a) (b)

Figure 4: (a) Chromatogram of HbD Punjab trait showing HbD 40.5% (RT 4.15 min). (b) Chromatogram of Hb D Punjab homozygous
showing Hb D 87.9%.

mean of 5% [1]. During routine reporting, we faced certain was 2.0–3.3%. Few cases with macrocytic RBC indices had
problems, regarding the cut off value of HbA2 for beta values as high as 3.4%.
thalassemia trait. Different authors have established different The variation of cut off value as well as borderline
cut off values for HbA2 for diagnosis of beta thalassemia value by the same method leads to confusion resulting in
trait, which ranges from 3.5 to 4%, although it has been underdiagnosis or overdiagnosis. In such situations, genetic
recommended that each laboratory needs to establish their counseling with DNA analysis should be recommended. But
own normal ranges [4, 11]. Rangan et al. used the term in a resource-limited country where population is huge but
borderline to HbA2 levels 3.0-4.0% and found mutations in facilities of genetic or molecular tests are available only in a
32% people with HbA2 3.4–3.9% [12]. Similar findings are few research laboratories, we may refer only antenatal cases
described by Colah et al. [11]. In regions with high prevalence for genetic studies if one partner is a trait. For other cases, we
of beta thalassemia researchers have taken variable values as can opt for parental study if possible or iron studies before
referring them for costly genetic test.
borderline, for example, 3.3–3.7% [13] and 3.1–3.9% [14].
On the other hand, there were few cases with symp-
Iron deficiency anaemia is a very common occurrence in tomatic refractory microcytic hypochromic anaemia with
most of the screening populations, namely, school children borderline, normal, or reduced HbA2 levels. These cases
and pregnant women in India. There are several studies should be investigated for presence of alpha thalassemia or its
regarding the impact of iron deficiency on HbA2 level and coinheritance with beta thalassemia gene. Alpha thalassemia
controversy over its significance in screening of beta tha- is by far the commonest hemoglobinopathy in India, with
lassemia trait [4, 15, 16]. Like Denic et al., we also suggest that highest prevalence in Punjabi population in the northern
concurrent iron deficiency anaemia should be considered region [17]. Molecular genotyping of 𝛼-thalassemia helps
in cases of borderline HbA2 with microcytic hypochromic to diagnose unexplained microcytosis and thus prevents
anemia. On the other hand, we came across few cases with unnecessary iron supplementation [18].
3.4–3.6% HbA2 with normal MCV and MCH values. It is Hb E, the most common structural variant in our study, is
difficult to determine whether they are carriers of silent one of the world’s important mutations. Hb E trait, homozy-
mutations or high normal HbA2 without genetic test. In our gous Hb E disease (Hb EE), and Hb E beta thalassemia are
laboratory, range of HbA2 value with normal RBC parameter common in north eastern part of the country [19]. Hb E trait
International Scholarly Research Notices 7

Peak name Calibrated Area (%) Retention Peak

area (%) time (min) area
Unknown — 0.2 0.91 6267
F 0.2 — 1.04 5136
Peak name Calibrated Area (%) Retention Peak Unknown — 0.6 1.21 17393
area (%) time (min) area P2 — 2.3 1.32 69227
F 5.8∗ — 1.08 87528 P3 — 5.0 1.71 150318
P2 — 6.4 1.32 99803 Unknown — 0.9 2.14 28115
P3 — 4.3 1.70 67364 A0 — 46.4 2.44 1397825
72.9 2.44 1145749 ∗ 3.60 1337204
A0 —
∗
A2 41.5 —
A2 10.1 — 3.67 170271
Total area: 3,011,48
F concentration = 5.8∗ % Total area: 1,570,71
F concentration = 0.2%
∗
A2 concentration = 10.1 %
∗
A2 concentration = 41.5∗ %
Values outside of expected ranges ∗
Values outside of expected ranges
Analysis comments:
Analysis comments:

45.0 45.0

37.5 37.5

30.0 30.0
(%)

22.5 22.5
(%)
3.67
F 1.06

15.0 15.0

1.71
1.32

1.211.32
A2

2.14
1.70

1.04

3.60
7.5
0.91
7.5
2.44

2.44
F
0.0

A2
0.0

0 1 2 3 4 5 6 0 1 2 3 4 5 6
Time (min) Time (min)
(a) (b)

Figure 5: (a) Chromatogram of showing HbA2 10.1% presumptive diagnosis of Hb Lepore. (b) Chromatogram of Hb D Iran showing HbA2
41%.

and Hb EE are mild disorders. Detection of this variant is very It has been reported that coinheritance of beta tha-
important because when combined with thalassemia or HbS, lassemia and HbD Punjab may result in symptomatic disease
it gives rise to moderate to severe disease. and its detection is important in prenatal diagnosis. In cases,
We observed that distinction between homozygous HbE where HPLC or Hb electrophoresis shows very high HbD
disease and HbE beta thalassemia sometimes become diffi- level and negligible HbA, one should look for raised HbA2
cult as both of these conditions show variably elevated HbE level and do parental study or DNA analysis for confirmation
and Hb F with marked microcytic hypochromic picture. Hb of coinheritance.
D Iran also comes as a differential diagnosis. Compared to Hb A total of nine cases of Hb S were detected of which 5 cases
E trait, Hb D Iran tends to have more area percentage at the showed elevated Hb A2 level (3.3−3.7%) with 25–40% HbS
window of Hb A2 (usually more than 40%). Parental study, if and moderate to severe anaemia. The mean values of HbA2
possible, may easily resolve the problem. Otherwise, genetic have been reported to be elevated in sickle cell syndrome in
study should be done. previous reports [23, 24]. The reason may be HbS adducts
Hb D Punjab was the second most common structural (carbamylated and glycated) coeluting with HbA2. Parental
variant in our study, mostly presented as asymptomatic het- study should be done in suspicious cases before reporting
erozygous condition with normal hematological parameters. such case as sickle-beta thalassemia.
Hb D Punjab occurs with greatest prevalence, that is, 2% We reported 3 cases of delta-beta thalassemia trait.
among Sikhs in Punjab, and in Gujarat, reported prevalence Delta-beta thalassemia and hereditary persistence of fetal
is 1% [20]. Hb D Punjab in the form of heterozygote hemoglobin (HPFH) constitute a heterogeneous group of
Hb D trait, Hb S-D disease, and Hb D-thalassemia are disorders characterized by absent or reduced synthesis of
commoner forms; however, homozygous form is very rare adult hemoglobin (Hb A) and increased synthesis of fetal
[20, 21]. Few detailed studies are available on clinical, hema- hemoglobin (Hb F). The distinction between HPFH and
tological, and molecular analyses of Hb D Punjab in India delta-beta thalassemia is subtle and should be confirmed by
[21, 22]. alpha-beta-globin chain synthesis ratio and DNA analysis
8 International Scholarly Research Notices

Calibrated Retention Peak

Peak name area (%) Area (%) time (min) area
Calibrated Retention Peak P1 — 0.4 0.71 4107
Peak name area (%) Area (%) time (min) area Unknown — 0.1 0.97 810
P1 — 0.9 0.74 26261 F 0.8 — 1.08 8958
F 0.2 — 1.10 6469 Unknown — 0.9 1.23 9703
P2 — 48.4 1.37 1387977 P2 — 2.1 1.34 23468
P3 — 2.0 1.73 58281 P3 — 25.5 1.69 290435
A0 — 45.0 2.50 1290273 A0 — 68.7 2.37 783063
A2 3.0 — 3.69 96326 A2 1.5∗ — 3.61 19024
Total area: 2,865,588 Total area: 1,139,569
F concentration = 0.2%
A0 concentration = 3.0% F concentration = 0.8%
∗
Values outside of expected ranges A0 concentration = 1.5∗ %
∗
Values outside of expected ranges
Analysis comments: Analysis comments:
45.0 45.0

37.5 37.5
2.50

30.0 30.0
(%)

22.5 22.5

15.0 (%) 15.0

3.61
3.69

1.34
1.09
0.71
0.07
1.23
F 1.10
0.74

1.73

7.5 7.5

2.37
1.69
A2
1.37

A2
F
0.0 0.0

0 1 2 3 4 5 6 0 1 2 3 4 5 6
Time (min) Time (min)
(a) (b)

Figure 6: (a) Chromatogram of Hb Hope showing elevated P2 peak (48.4%). (b) Chromatogram of Hb J showing elevated P3 peak.

since the distinction between these two conditions is not start of integration [29]. We, however, did not find any such
always possible from routine hematologic analyses. It is case.
important to differentiate between these two conditions This automated HPLC system is intended for separation
especially in antenatal screening because HPFH is clinically and determination of area percentage for HbA2 and HbF and
asymptomatic, but interaction of 𝛿𝛽-thalassemia with 𝛽- for qualitative detection of abnormal hemoglobins. With the
thalassemia can result in a severe disorder. integration of proper algorithms involving retention time, %
Hb J Meerut was another rare variant detected in our Hb, and peak characteristics, a clinical laboratory is capable of
study. This clinically asymptomatic variant has been reported identifying 75% of the common variants encountered without
to interfere with HbA1c estimation [25] and to mask beta the need for confirmatory studies such as alkaline and acid
thalassemia trait [26]. electrophoresis [9]. In the light of family study, ethnicity and
Among the rarely encountered variants we detected one clinical data, we can detect most of the frequently occurring
case of Hb Hope. In cation exchange HPLC method, it clinically significant variants by HPLC method alone.
may be mistaken as high HbA1c, as it comes in the same HPLC has some limitations, including falsely decreased
position as HbA1c. Hb Hope (beta 136(H14) Gly → Asp HbA2 levels in patients with the HbD Punjab trait, falsely
(GGT → GAT)) is one of the unstable haemoglobin variants increased HbA2 levels in patients with HbS, and coelution
of the beta-globin chain, which has been demonstrated in of various Hbs, including HbE, Hb Osu Christianborg, HbG
people of various ethnic backgrounds. Few case reports of Coushatta, and Hb Lepore with HbA2 [30]. Interference
Hb hope and its association with thalassemia and Hb E have from endogenous compounds or from drugs is not frequently
been published from southeast Asia [27, 28]. This variant reported in literature. Howanitz et al. reported unknown
is clinically silent and when associated with thalassemia tall peaks with elution times and shapes of hemoglobin
may produce mild to moderate symptoms depending upon Barts on hemoglobin chromatograms that could not be
genotype. confirmed by alkaline and acid gel electrophoresis mostly
It has been reported that one can detect HbH form in patients with hemoglobin SS [31]. The authors provided
of alpha thalassemia on HPLC by visual analysis of the evidence that the peak is not hemoglobin Barts, but rather
chromatogram plot as it produces a sharp peak before the bilirubin, and recommended exclusion of bilirubin before
International Scholarly Research Notices 9

chromatographic results are interpreted as consistent with chromatography: its use in diagnosis and in the assessment of
hemoglobin Barts. Determination of HbA2 and HbF is not cellular distribution of hemoglobin variants,” American Journal
interfered by endogenous substances like triglyceride and of Hematology, vol. 17, no. 1, pp. 39–53, 1984.
bilirubin levels up to 4600 mg/dL and 20 mg/dL, respectively [9] A. Joutovsky, J. Hadzi-Nesic, and M. A. Nardi, “HPLC reten-
[10]. In diabetic patient labile HbA1c greater than 2.5% may tion time as a diagnostic tool for hemoglobin variants and
adversely affect HbF quantitation [10]. In our study we did hemoglobinopathies: a study of 60000 samples in a clinical
not experience such interference. diagnostic laboratory,” Clinical Chemistry, vol. 50, no. 10, pp.
1736–1747, 2004.
[10] VARIANT II b thalassemia short program instruction manual.
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globinopathies,” Indian Journal of Pediatrics, vol. 74, no. 7, pp.
To conclude, RBC indices, HPLC finding, and family study
657–662, 2007.
are sufficient to detect and manage most of the hemoglobin
[12] A. Rangan, P. Sharma, T. Dadu, R. Saxena, I. C. Verma, and M.
variants prevalent in this country. However, one has to be
Bhargava, “B-Thalassemia mutations in subjects with borderline
aware of the limitations and problems associated with the HbA 2 values: a pilot study in North India,” Clinical Chemistry
diagnostic methods to avoid false negative diagnosis in day and Laboratory Medicine, vol. 49, no. 12, pp. 2069–2072, 2011.
to day practice. Genetic studies are indicated to confirm bor- [13] A. Mosca, R. Paleari, R. Galanello et al., “Occurrence of
derline cases and to detect silent carriers of beta thalassemia, HbA2 borderlinephenotypes in areas with high prevalence of
alpha thalassemia, and rare and novel variants in routine thalassemia,” in Proceedings of the 16th Meeting on EARCR,
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Conflict of Interests vol. 97, no. 3, pp. 472–473, 2012.
The authors declare that there is no conflict of interests [16] S. Denic, M. M. Agarwal, B. Al Dabbagh et al., “Hemoglobin A2
regarding the publication of this paper. lowered by iron deficiency and 𝛼-thalassemia: should screening
recommendation for 𝛽-thalassemia change?” ISRN Hematol-
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