Indian J Hematol Blood Transfus (July-Sept 2020) 36(3):535–541
https://doi.org/10.1007/s12288-019-01235-1
ORIGINAL ARTICLE
Efficacy of Dichlorophenolindophenol (DCIP) as Screening Test
for Hb E: Revisited
Prakas Kumar Mandal1,2 • K. S. Nataraj3 • Shuvra Neel Baul4
Tuphan Kanti Dolai4
Received: 20 August 2019 / Accepted: 21 November 2019 / Published online: 4 December 2019
Ó Indian Society of Hematology and Blood Transfusion 2019
Abstract Hb E-beta thalassemia is a major public health
problem in West Bengal, India and is the predominant
symptom producing thalassemia in this part of the country.
To search for an easy, reliable and cost effective screening
method for HbE that can be used at the community level
where more sophisticated methods are not readily avail-
able. And the DCIP test was performed for the purpose.
Blood samples of 425 asymptomatic family members from
80 diagnosed cases of HbE beta Thalassemia patients were
tested for Hb, RBC indices, DCIP test, HPLC, and in dis-
cordant cases confirmed by DNA mutation analysis. The
present study shows DCIP screening test to have a sensi-
tivity, specificity, positive predictive value and negative
predictive value of 96.39%, 97.43%, 96.39% and 97.43%
respectively. It also shows a false positive rate and false
negative rate in 2.56% and 4.6% cases respectively. The
advantage with DCIP over HPLC is that it can be easily
performed at the community level by a person with mini-
mum technical skill, few samples (even a single sample)
can be tested at time, at a low cost.
& Shuvra Neel Baul
shuvraneelb@gmail.com
Prakas Kumar Mandal
prakas70@gmail.com
1
Department of Hematology, CSTM and NRS Medical
College, Kolkata 700014, India
2
8C/1/N, Roy Para Road, P.S.:- Sinthee, Kolkata 700050,
India
3
Mazumdar Shaw Cancer Center, Narayana Health,
Bengaluru, India
4
Department of Hematology, NRS Medical College,
Kolkata 700014, India
• Malay Kumar Ghosh4 •
Keywords Hemoglobin E  Screening test  HPLC  DCIP
Introduction
Haemoglobin E (HbE) is a common variant hemoglobin
caused by point mutation in 26th position of b-chain that
activates cryptic splice site at the 25th codon resulting in
qualitative and quantitative defect of beta-globin chain [1].
HbE is the most frequent b-thalassemic hemoglobinopathy
in Southeast Asia and parts of the Indian sub-continent [2].
It has a significant prevalence in North East India, Ban-
gladesh, Indonesia and Sri Lanka. In India, in some foci in
the North East the prevalence reaches 80%. The prevalence
rate of HbE in Kolkata is as high as 22% [1].
Hemoglobin E in homozygous and heterozygous state is
asymptomatic. But, if they get married with beta-tha-
lassemia carriers, there is a chance of having Hb E-beta
thalassemia offsprings, which is a form of symptomatic
thalassemia. To prevent this, carrier detection by a cost-
effective and robust method is important.
HPLC is used as gold standard to diagnose thalassemia
and other hemoglobinopathies. But, it is costlier, large
number of samples are needed at a time to make it cheaper,
needs expertise skilled personnel and a well equipped
laboratory set up. For the reasons mentioned above, it is not
readily available at the community level.
Dichlorophenolindolphenol (DCIP) test is used to detect
the presence of Hb E which is easily and quickly oxidized
by DCIP reagent [3, 4]. In search of a screening test which
is much cheaper, easy to perform, requires less expertise
and can be done at the community level; we performed
DCIP test.
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Materials and Methods
The work conducted over a period of 5 years (January,
2012 to December, 2016) in asymptomatic family members
of E-beta thalassemia patients attending thalassemia OPD
and Thalassemia day care unit in the Department of
Hematology, NRS Medical College, Kolkata. A total of
425 asymptomatic family members of 80 (eighty) E-beta
thalassemia patients attending the thalassemia OPD and
Day care center were studied. The parameters studied
include detailed history and clinical examination, personal
history of blood (RBC) transfusion and history of transfu-
sion dependant anemia in the family; history of consan-
guineous marriage was noted carefully. The study excluded
family members having symptomatic anemia, jaundice
and/or hepato-splenomegaly. Samples of EDTA anticoag-
ulated whole blood tested for complete hemogram includ-
ing RBC indices, DCIP (dichloro-phenolindophenol) test
and HPLC (high performance liquid chromatography). The
results obtained from DCIP (dichloro-phenolindophenol)
test were evaluated and compared with HPLC data. The
cases showing discordance by the above mentioned meth-
ods were finally diagnosed and confirmed by DNA muta-
tion analysis. ARMS-PCR (amplification refractory
mutation system—polymerase chain reaction) technique
was used for mutation analysis. Thus, effectiveness of
DCIP (a screening test) e.g. sensitivity, specificity, positive
predictive value and negative predictive value were com-
pared with that of other costlier test e.g. HPLC.
Complete Hemogram
Done by automated cell counters using Sysmex KX-21
automated hematology analyzer. Cell counter parameters
studied included hemoglobin (Hb%), hematocrit (Hct), Red
blood cell (RBC) count and RBC indices e.g. MCV (mean
corpuscular volume), MCH (mean corpuscular hemoglo-
bin), MCHC (mean corpuscular hemoglobin concentration)
and RDW-CV (red cell distribution width- coefficient of
variation). Peripheral blood smears were examined to look
for RBC morphology and also for presence of nucleated
red blood cells (NRBCs), abnormal cells, if any.
DCIP (Dichloro-Phenolindophenol) Test (Also Known
as DCPIP Test) [5]
2,6-Dichlorophenol-indophenol (DCIP) is a blue chemical
compound used as a redox dye. HbE and other unsta-
ble haemoglobin molecules (such as HbH) are precipitated
when exposed to this dye at 37 °C. Precipitated hae-
moglobin (if any) visualized by the naked eye at the bottom
of the tube. In homozygous HbE, heavy sediment is
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Indian J Hematol Blood Transfus (July-Sept 2020) 36(3):535–541
formed; in HbE trait and Hb E/b thalassemia, the precipi-
tation of Hb E produces a cloudy or an evenly distributed
particulate appearance.
HPLC (high performance liquid chromatography)
Done by BIO-RAD ‘beta thalassemia short Programme
VARIANT’ instrument. Typical ‘retention times’ observed
were noted carefully and the hemoglobins are assigned [2]
to retention time ‘windows’ which are designated as F, P2,
P3, A, A2, S, C and D.
ARMS-PCR (Amplification Refractory Mutation System–
Polymerase Chain Reaction)
DNA was extracted from peripheral blood leucocytes by
Phenol–chloroform method [6]. ARMS-PCR technique [7]
was applied to confirm the presence of HbE mutation.
Primers used to detect the presence of Hb E and beta
thalassemia mutations (commonly found in this part of the
country) [8] are shown in Table 1.
Results
A total of 425 asymptomatic family members from 80
(eighty) diagnosed cases of HbE-beta thalassemia patients
included in the study. A total of 15 (fifteen) members from
those families were never available for study because 9
(nine) members were elderly (unable to attend) and others
(6 members) didn’t turn up in spite of every efforts. Thus,
including all, average asymptomatic member per family
was calculated as: (425 ? 15)/80 = 440/80 = 5.5 asymp-
tomatic members per family (range 2–16). Out of 425
cases, there were 198 (46.59%) male and 227 (53.41%)
females. Median age was 23.95 years (range 1–75).
Highest number of screened subjects (59.53%) belonged to
the age group of 10–40 years.
In hemogram, MCH value \ 27 pg was found in 256
(60.23%) cases and MCV \ 80 fl seen in 241 (56.7%)
cases. Smears examined for RBC morphology and com-
pared with the RBC indices and finally with HPLC reports.
Results of mean values of hemogram in normal, HbE trait
and beta thalassemia trait shown in Fig. 1. Mean hemo-
globin level, MCV, MCH, MCHC and RDW values of
HbE/HbEE (heterozygous and homozygous states of
Hemoglobin E) cases were in between that of normal
subjects and beta thalassemia traits. The mean RBC count
in Hb E/EE cases was much higher in comparison to nor-
mal and beta thalassemia trait cases. Peripheral blood
smear examined carefully for RBC morphology showed
microcytic, hypochromic RBC’s in beta thalassemia trait
and HbE/HbEE cases. Out of 425 samples tested for DCIP
Indian J Hematol Blood Transfus (July-Sept 2020) 36(3):535–541 537

Table 1 Following primers were used to detect the presence of Hb E and beta thalassemia mutations
Mutation Primer sequence 50 ? 30 Second primer Fragment size (bp)

Codon26 (G ? A) HbE N- TAA CCT TGA TAC CA ACCT GCC CAG GGC GTC C1 236
M- TAA CCT TGA TAC CA ACCT GCC CAG GGC GTT C1 236
IVS1-5 (G ? C) N- CTC CTT AAA CCT GTC TTG TAA CCT TGT TAC C1 285
M- CTC CTT AAA CCT GTC TTG TAA CCT TGT TAG C1 285
IVS1-1 (G ? T) N- GAT GAA GTT GGT GGT GAG GCC CTG GGT AGG C2 454
M- TTA AAC CTG TCT TGT AAC CTT GAT ACG AAA C1 281
Codon41/42 (-CTTT) N- GAG TGG ACA GAT CCC CAA AGG ACT CAA AGA C1 443
M- GAG TGG ACA GAT CCC CAA AGG ACT CAA CCT C1 439
Codon15 (G ? A) N- TGA GGA GAA GTC TGC CGT TAC TGC CCA GTG C2 500
M- TGA GGA GAA GTC TGC CGT TAC TGC CCA GTA C2 500
Codon30 (G ? C) N- TAA ACC TGT CTT GTA ACC TTG ATA CCT ACC C1 280
M- TAA ACC TGT CTT GTA ACC TTG ATA CCT ACG C1 280
Codon8/9 (? G) N- CCT TGC CCC ACA GGG CAG TAA CGG CAC ACT C1 214
M- CCT TGC CCC ACA GGG CAG TAA CGG CAC ACC C1 215
C1 and C2 are common primes used in PCR reaction to check for amplification. All the primers used for ARMS are HPLC purified. Common
primer 1 (C1):- 50 ACC TCA CCC TGT GGA GCC AC30 . Common primer 2 (C2):- 50 CCC CTT CCT ATG ACA TGA ACT TAA 30

Fig. 1 comparison of mean values of hemogram in normal, HbE trait and Hb beta trait

(Fig. 2), 152 (32.76%) samples were positive for the test appearance. In 4 cases, heavy sediment was formed at the
and 273 (67.24%) samples showed negative results. In bottom of the test tube. The results of HPLC reports are
majority of the positive cases there was a cloudy particulate summarized in Fig. 3 showing comparison of mean values

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Fig. 2 tube at the center is blank, other two tubes showing precipitate
at the bottom indicating positive result with DCIP
of HPLC in normal, HbE trait, beta thalassemia trait and
HbEE cases. In 137 (32.23%) cases HPLC studies showed
normal chromatographic pattern; 154 (36.24%) and 134
(31.53%) cases showed chromatographic pattern sugges-
tive of HbE/HbEE and beta thalassemia traits respectively.
Thus, HPLC study of family members showed a ratio of
normal: HbE: beta thalassemia trait = 137: 154:
134 = 1.02: 1.15: 1.
Fig. 3 Comparison of mean values of HPLC in normal, Hb beta trait, HbE trait and HbEE
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Indian J Hematol Blood Transfus (July-Sept 2020) 36(3):535–541
The test results of DCIP screening test and HPLC were
now compared and summarized in Table 2. Out of 425
blood samples tested with both the procedures, in 14
(3.29%) cases there were discordance/discrepancy in
results. As compared to HPLC, DCIP showed false positive
and false negative results in 7 (1.65%) cases and 7 cases
(1.65%) respectively.
Blood samples of all those 14 cases showing discor-
dance/discrepancy in results between DCIP screening test
and HPLC test, processed for DNA analysis by ARMS-
PCR. All the samples were first tested for HbE mutations;
the mutation was detected in 7 cases (heterozygous-6 cases
and homozygous-1 case) which gave false negative results
in DCIP test as compared to HPLC. DNA samples showing
no HbE mutation (7 cases) were now subjected to studies
for the common beta thalassemia mutations; in 3 cases
there was beta thalassemia mutation and in rest 4 cases no
mutation was detected (hence, reported as normal). Sum-
mary of tested results of DCIP, HPLC and ARMS-PCR
result are shown in Table 2.
Validation of DCIP Screening Test Results
Among 425 samples tested, there was discordance in 14
(3.29%) cases (false positive-7 cases and false negative-7
Indian J Hematol Blood Transfus (July-Sept 2020) 36(3):535–541 539

Table 2 Summary of results of DCIP, HPLC and ARMS-PCR result

Case no. DCIP HPLC Inference (DCIP ARMS-PCR result Final diagnosis
(sample no.) test report compared to HPLC)

19 (-) ve E trait False - ve Codon26 (G ? A) HbE Heterozygous for HbE mutation

31 (-) ve E trait False - ve Codon26 (G ? A) HbE Heterozygous for HbE mutation
36 (?) ve Beta trait False ? ve Codon8/9 (? G) Heterozygous for beta mutation
84 (-) ve E trait False - ve Codon26 (G ? A) HbE Heterozygous for HbE mutation
86 (?) ve Beta trait False ? ve IVS1-5 (G ? C) Heterozygous for beta mutation
131 (-) ve E trait False - ve Codon26 (G ? A) HbE Homozygous EE
206 (?) ve Normal False ? ve No mutation detected Normal
269 (-) ve E trait False - ve Codon26 (G ? A) HbE heterozygous for HbE mutation
277 (?) ve Normal False ? ve No mutation detected Normal
298 (?) ve Beta trait False ? ve Codon8/9 (?G) heterozygous for beta mutation
307 (?) ve Normal False ? ve No mutation detected Normal
356 (-) ve E trait False - ve Codon26 (G ? A) HbE Heterozygous for HbE mutation
395 (?) ve Normal False ? ve No mutation detected Normal
401 (-) ve E trait False - ve Codon26 (G ? A) HbE Heterozygous for HbE mutation

cases) as compared to HPLC and ARMS-PCR results.
Thus, the present study with DCIP test shows sensitivity of
96.39% and specificity of 97.43%. In assessing the diag-
nostic power of the (DCIP screening) test, it showed pos-
itive predictive value, negative predictive value, percentage
of false positives and percentage of false negatives of
96.39%, 97.43%, 2.56% and 4.6% respectively.
Discussion
Haemoglobin E, a hereditary abnormality of human
hemoglobin was first described by Chernoff et al. [8].
Independently in the same year it was also described by
Itano et al. [9] as fourth abnormal hemoglobin. Since its
classic description by Chernoff et al. [8], it has been found
to be an important public health problem in the Indian
subcontinent and Southeast Asia.
The first case of Hb E/b-thalassemia in India was
reported by Chatterjea et al. [10] from Calcutta. Tha-
lassemia and other hemoglobinopathies is a major public
health problem in West Bengal, India; Hb E-beta tha-
lassemia is the predominant symptom producing tha-
lassemia in this part of the country. The prevalence rate of
HbE in this state [11] is 4.1% with reported very high
prevalence of 22% in Kolkata [1], the capital city of West
Bengal. These established facts are the major driving force
in searching for a suitable and ideal screening test to be
performed at the community level.
Hemoglobin E in homozygous and heterozygous state is
asymptomatic. But, if they get married with beta tha-
lassemia carriers, there is a chance of having Hb E-beta
thalassemia offsprings, which may give rise to a form of
symptomatic thalassemia. To prevent this, carrier detection
by a cost-effective and robust method is necessary.
At present in India and also in West Bengal, HPLC is
used as a screening test as well as confirmatory test for
diagnosis of HbE and also other hemoglobinopathies and
thalassemias. Though it gives reliable and reproducible
results, but it is costlier, needs expertise personnel and well
equipped laboratory set up and thus not available for
population screening at the community level. The present
study was a blinded study as the blood samples were first
tested by automated cell counter, DCIP test; then subjected
to HPLC test. The results coming out of automated cell
counter and DCIP test were then corroborated with HPLC
test results. In the present study, we evaluated the effec-
tiveness of DCIP screening test for HbE in corroboration
with the HPLC reports and any discordance thus coming
out between the two methods were finally confirmed by
DNA mutation analysis by ARMS-PCR method. In the
present study, total of 425 asymptomatic members from 80
diagnosed cases of E-beta thalassemia patients were
included in the study. Fifteen members from those families
were never available for study because 9 (nine) members
were elderly (unable to attend) and others (6 members)
didn’t turn up in spite of every efforts.
Mean Hb A2 ? E level in Hb E and HbEE were 28.3%
and 90% respectively which was at par (29.3%) with
studies by Sanchaisuriya et al. [12] done in Thai popula-
tion. Mean Hb A2 ? E level in heterozygous state was
28.3% which is slightly less in comparison to other reports
from south India (30.1%) and western countries (30%)
[13, 14]. This low level of Hb A2 ? E in eastern India and
Southeast Asia are explained by possible concurrent pres-
ence of alpha-thalassemia mutations in this part of the

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globe which can only be effectively diagnosed by mutation
analysis for alpha thalassemia [15].
HbE was detected in 154 (53.1%) cases among the
family members and beta thalassemia trait was detected in
134 (46.9%); thus, the present study reports a higher
prevalence rate of HbE, possibly because of biasness in
selecting the study population including only the family
members of diagnosed HbE beta thalassemia patients. This
is in contrast to other studies [2, 11], where they performed
the study in general population. Study conducted by Balgir
[16] on ‘the genetic epidemiology of the three predominant
abnormal hemoglobins in India’ showed that HbE is mostly
restricted to the north-east part of India i.e. West Bengal,
Assam, Nagaland, Manipur, Tripura and Meghalaya with
an average allele frequency of 10.9% with occasional case
reports from other part of the country.
Mean Hb % values of 11.8 gm/dl, 10.74 gm/dl and 12.3
were found in HbE/EE, beta thalassemia trait and normal
cases respectively (Fig. 1). Mean RBC count was much
higher in both conditions (4.69 9 1012/ll and 5.29 9 1012/
ll) in comparison to normal cases (4.11 9 1012/ll) and
mean MCH of 25.35 pg and 20.7 pg was lower than the
normal cases (29.1 pg). MCH value \ 27 pg was found in
256 (60.23%) cases and MCV \ 80 fl seen in 241 (56.7%)
cases. Study by Sanchaisuriya et al. [12], considering MCH
value \ 27 pg and MCV \ 80 fl for screening, showed a
sensitivity, specificity, positive predictive value and nega-
tive predictive value of 78.9%, 79%, 72.6%, 84.15% and
72.0%, 84.3%, 76.4% and 81% respectively. Many of the
HbE heterozygotes shown normal RBC indices; therefore
gave high false negative results; and they showed that,
sensitivity of MCV and MCH screening protocol was
improved to 100% when it was combined with DCIP test.
In this study, out of 425 samples tested for DCIP, 152
(32.76%) samples were positive for the test and 273
(67.24%) samples showed negative results. The samples
then subjected to HPLC test; showed results as follows:
normal, beta thalassemia trait, HbE and HbEE in 137 cases,
134 cases, 150 cases and 4 cases respectively. Thus, DCIP
test results when corroborated with HPLC test results
Table 3 Comparison of different studies regarding effectiveness of DCIP test
Study group Sensitivity Specificity Positive predictive
(%) (%) value (%)
Present study 96.39 97.43 96.39
Prayongratana et al. [19] 97.16 98.93 99.42
Sanchaisuriya et al. [12] 100 76.2 74
Fuchareon et al. [4] 100 69.8 77.2
Chapple et al. [18] 100 92 85.7
Jaiwang et al. [20] 100 100 NA
NA not available
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Indian J Hematol Blood Transfus (July-Sept 2020) 36(3):535–541
(Table 2) gave a false positive of 7 cases and false negative
of 7 cases respectively. Tyagi et al. [17] from All India
Institute of Medical Sciences, New Delhi concluded that,
HPLC being an automated instrument is highly sensitive
and specific, has high resolution and helps in quantification
of various hemoglobins. However, in a developing country
like India where economical factors play a major role in
planning for management of patients, the role of HPLC is
limited.
The present study shows DCIP screening test to have a
sensitivity, specificity, positive predictive value and nega-
tive predictive value of 96.39%, 97.43%, 96.39% and
97.43% respectively. It also shows percentage of false
positives and percentage of false negatives in 2.56% and
4.6% cases respectively. As shown in Table 3, many other
studies also reported the effectiveness of using DCIP and
found that this test has a sensitivity of 94.4–100%, speci-
ficity of 69.8–98.2%, positive predictive value of
75.0–86.9%, and negative predictive value of 98.1–100%;
can be used as an effective screening test for detection of
Hb E [4, 12, 18–20]. The present study in comparison to
others resulted in a slightly higher value of false negative
rate which may impart a negative impact on the success of
screening programme that may possibly be overcame by
performing the study in a larger population as shown by
study [19] done with a large number of cases.
The criteria for screening are based on two considera-
tions: the disease to be screened, and the test to be applied
[21]. The disease to be screened should fulfill the following
criteria: (1) the condition sought an important public health
problem, (2) facilities available for confirmation of diag-
nosis, and (3) early detection reduces morbidity, mortality
and disease burden in the community. The test also must
satisfy the criteria of acceptability, repeatability and
validity, besides others such as simplicity, safety, rapidity
and cost [22]. Performance of a screening test is measured
by it’s predictive value which reflects the diagnostic power
of the test. The present study showed a positive predictive
value, Negative predictive value of 96.39% and 97.43%
respectively. As evident, DCIP test may be accepted an
Negative predictive False positive False negative
value (%) rates (%) rates (%)
97.43 2.56 4.6
95.19 1.07 2.66
100 NA NA
100 NA NA
100 NA NA
NA NA NA
Indian J Hematol Blood Transfus (July-Sept 2020) 36(3):535–541
effective screening test considering simplicity, rapidity,
validity and cost.
However, there were certain limitations in the study;
(a) small sample size: diagnostic power of the test e.g. pre-
dictive value can possibly be improved further by increasing
sample size working with a larger population. (b) Biasness:
the present study was conducted including a biased popula-
tion of asymptomatic family members of the HbE beta tha-
lassemia patients which can be minimized by performing the
test in general population having high prevalence of HbE,
(c) DNA mutation analysis by ARMS-PCR was performed in
those cases only showing discordance between DCIP and
HPLC for HbE and common beta thalassemia mutations
reported from this part of the country.
The advantage with DCIP over HPLC is that it can be
easily performed at the community level by a person with
minimum technical skill, few samples (even a single
sample) can be tested at time at a low cost. DCIP solution
can be stored at 4 °C for a long time in a refrigerator,
which is freely available nowadays even in the community
health centers. In contrast, HPLC is needs highly skilled
and dedicated technical personnel, a well organized labo-
ratory set up and the running cost is very high, if done in a
small number of samples.
Conclusion
DCIP test is a simple, easy to perform and cost effective
method for detecting HbE; can be used in rural areas in a
population with high prevalence with access to limited
health care facilities.
Funding None.
Compliance with Ethical Standards
Conflict of interest The authors declare no potential conflicts of
interest.
Ethical Approval The study was approved by the Institutional Eth-
ical Committee (IEC).
Patient Consent Written consent taken from each patient and/or
their legal guardians at the entry into the study.
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