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 Original Article

To Determine the Prevalence of Glucose‑6‑Phosphate

Dehydrogenase Deficiency Using a Novel Water‑Soluble
Tetrazolium‑8 Formazan Method’ for Neonatal Screening in
Region of Himachal Pradesh, India
Seema Sharma, Milap Sharma
Department of Pediatrics, Dr. Rajendra Prasad Government Medical College, Kangra, Himachal Pradesh, India

Abstract
Background: Glucose‑6‑phosphate dehydrogenase (G6PD) deficiency is the most significant enzyme defect in India with an incidence ranging
from 2% to 27.9% in different communities. Prolonged neonatal jaundice and haemolytic crisis are known to occur in children with G6PD
deficiency. Hence, screening of a population for G6PD deficiency is paramount. A Novel water soluble tetrazolium‑8 (WST‑8) Formazan Method
has been used in this study for in‑field mass‑screening of G6PD in the region of Himachal Pradesh, India. Materials and Methods: In this
prospective study, 5652 neonates were screened to assay G6PD activity using WST8/1‑methoxy phenazine methosulphate method within the
first 48 h of life. Orange colour at the end of the procedure indicated normal G6PD activity while pink or colourless appearance indicated
G6PD deficiency. Results: After the screening of 5652 neonates, the prevalence of G6PD deficiency was 12.4%. 45 newborns (6%) had
a severe G6PD deficiency. Males were more affected than females (70:30). Furthermore, males had higher prevalence of deficiency than
females (64% [n = 29] and 16% [n = 16]). Conclusions: G6PD deficiency assessment by the method used for population screening in the study
was easy to do and quite simple. Following this, the high prevalence of this deficiency was noted in Himachal Pradesh. This study highlights
the need to do neonatal screening of G6PD deficiency in population so that untowards complications like haemolytic crisis, complications
due to neonatal jaundice can be avoided.

Keywords: Glucose‑6‑phosphate dehydrogenase deficiency, neonate, qualitative, screening

Introduction is undergoing an epidemiological transition as congenital

Glucose‑6‑phosphate dehydrogenase  (G6PD) deficiency is
an X‑linked disorder (q28 locus) having a high prevalence
worldwide, males more commonly affected than females.[1‑4]
G6PD is an important enzyme in the cellular metabolism of the
pentose phosphate pathway, which protects erythrocytes from
oxidative stress by maintaining levels of reduced glutathione
in cells.
On the basis of percentage of G6PD enzyme activity, the
deficiency is categorised as moderate  (<30% activity) or
severe  (<10% activity).[5,6] Certain triggers in the form of
foods (Fava beans), pollen inhalation, drugs (primaquine,
chloramphenicol and aspirin) or chemicals (Henna, Naphthalene)
or infections may lead to severe haemolysis in such a situation
and urgent blood transfusion may be required.[7‑14] India
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malformations and genetic disorders are contributing
increasingly to neo and perinatal mortality than before. G6PD
deficiency is the most significant enzyme defect in India with a
varying incidence across various populations (2%–27.9%).[15‑17]
Worldwide 400 different variants and 90 different mutations of
this disease have been noted.[18] Some of the common mutations
prevalent in India are G6PD Kerala‑Kalyan mutation (949
G‑>A) reported from Punjab, Maharashtra, Andhra Pradesh,
Address for correspondence: Dr. Seema Sharma,
House No. 23, Block‑B, Type‑V, Dr. Rajendra Prasad Government Medical
College, Tanda, Kangra, Himachal Pradesh, India.
E‑mail: seema406@rediffmail.com
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How to cite this article: Sharma S, Sharma M. To determine the

DOI: prevalence of glucose‑6‑phosphate dehydrogenase deficiency using a novel
10.4103/AIHB.AIHB_11_18 water‑soluble tetrazolium‑8 formazan method’ for neonatal screening in the
region of Himachal Pradesh, India. Adv Hum Biol 2019;9:37-41.

© 2019 Advances in Human Biology | Published by Wolters Kluwer - Medknow 37

 Sharma and Sharma: Neonatal Screening for G6PD Deficiency

Tamil Nadu, Kerala; G6PD Mediterranean (563 C‑>T) in the which serial colour photographs were taken, and these values
Vatalia Prajapatis of North India and the Parsis and the G6PD were compared with G‑6‑P controls.
Orissa  (131 C‑>G) found in the tribals of southern India.
Prolonged neonatal jaundice and haemolytic crisis are known Chemicals
to occur in children with G6PD deficiency. Early screening WST‑8 and 1‑methoxyphenazine methosulphate (1‑methoxy
of neonates for this enzymatic deficiency and safeguarding PMS) were available in a CCK‑8 kit from Sigma‑Aldrich Co.
neonates with this deficiency from triggers is the only way (Tokyo, Japan) at concentrations of 5‑mm WST‑8 and 0.2 mm
to avoid adverse outcomes. Hence, a neonatal screening 1‑methoxy PMS in 0.15 m NaCl. Glucose‑6‑phosphate (G6P)
programme for G6PD deficiency is warranted. and NADP were procured from Sisco Research
Laboratories (Mumbai, India). The controls of known normal
Tantular and Kawamoto used a modified method using G6PD activity and known G6PD deficient were obtained
a tetrazolium salt water‑soluble tetrazolium (WST8), from Trinity Biotech USA (NY, USA) which were having
and 1‑methoxy phenazine methosulphate (PMS) (less
lyophylised G6PD in human red cell haemolysate base with
light‑sensitive PMS hydrogen carrier) for this test.[19] The
test was colourimetric in nature and could be done on dry
blood spot. In the present study, neonatal screening for
G6PD deficiency is done by WST8 method with the help of
microtubes as well as modified form of the WST8 method
by using dried blood spots in a 96‑well plate. In the present
study, we have evaluated the modified method for screening of
neonates for G6PD deficiency and ascertaining its prevalence
in population from Himachal Pradesh.

Materials and Methods

This was a prospective descriptive study in which a total of
5652 newborns were screened within 48 h after birth for G6PD
over a period of 1 year [Figure 1]. Due ethical approval from
the Institutional Review Board and consent of parents was
taken before conducting the study.
Principle of the water‑soluble tetrazolium‑8 formazan
method and preparation of reaction mixtures
For the detection of G6PD activity, the hydrogen of NADPH
produced by G6PD converts WST‑8 to WST‑8 formazan in the
presence of a hydrogen carrier, 1‑methoxy PMS [Figure 2]. The
reaction mixtures required are 0.05 m Tris‑HCl buffer, adjusted
pH to 7.2–7.5, which contained 5 mm MgCl2 and 0.1% saponin,
the substrate mixtures of 2.5 mm G6P, 0.2 mm nicotinamide
adenine dinucleotide phosphate (NADP) in H2O and the
WST‑8/1‑methoxy PMS mixture. The WST‑8/1‑methoxy PMS
mixture can be stored in for 12 months at −20°C, and other
reagents for 6–12 months at 4°C in the dark [Table 1]. G6PD
activity was observed by carrying out a standard method in a
microtube as well as economical method in well plate along
with controls, at room temperature. Five microlitres of blood
was taken and mixed with the reaction mixtures, followed by Figure 1: Study design.

Table 1: Components of the reaction mixtures

Reaction mixtures Qualitative assay for field surveys Quantitative assay for mass screening using an ELISA reader (µl)
Standard (µl) Economic (µl)
Tris‑HCl buffer with 0.1% saponin 400 200 100
Substrate mixtures of G6P and NADP 400 200 100
WST‑8/1‑methoxy PMS mixture 30 15 10
Whole blood 5 2 or 3 1 or 2
G6P: Glucose‑6‑phosphate, ELISA: Enzyme‑linked immunosorbent assay, NADP: Nicotinamide adenine dinucleotide phosphate, PMS: Phenazine
methosulphate, WST: Water soluble tetrazolium salt

38 Advances in Human Biology  ¦  Volume 9  ¦  Issue 1  ¦  January‑April 2019

 Sharma and Sharma: Neonatal Screening for G6PD Deficiency

stabilisers and preservatives. All G6PD deficient patients females. This analysis identified that severe G6PD deficiency
need a definitive, quantitative test for which an enzyme do occur in females. Out of 703 G6PD deficient neonates
level <100 U/trillion red blood cells has been defined as the 45% (n = 345) had hyperbilirubinaemia requiring phototherapy
cut off for classifying the neonate as G6PD deficient. in 75%  (n  =  260) newborns and 13%  (n  = 45) newborns
Blood spots were collected with the heel‑prick method
onto 3 MM filter paper  (Whatman) and were dried at room Table 2: Morbidity profile of glucose‑6‑phosphate
temperature. The dried blood spots were stored in zip‑lock bags. dehydrogenase deficient newborns
At the time of processing, a set of controls (normal, deficient)
Profile Status Percentage
were taken in microtubes and also on 3 MM filter paper. From
Total (n=5652) Sex
each dried blood spot, a single 1/16 inch diameter disc was
Male 3000 (53.1)
punched out and placed in a single well of a 96‑well flat bottom
Female 2652 (46.9)
microplate and in the microtubes. For no‑substrate control, Screening for G6PD G6PD deficient (n=703) 12.4%
control spots and extra blood spots were also punched and placed Male 491 (70)
in allocated wells and microtubes. One empty well served as a Female 212 (30)
‘no sample’ negative control. A volume of 200 μL of working Normal (n=4949) 87.6%
reaction mixture was mixed into each well/microtube, except the Male 2602 (53)
substrate negative control well/microtube, into which 200 μL of Female 2347 (47)
no‑substrate reaction mixture was added. Serial colour changes Hyperbilirubinaemia G6PD deficient (n=345) 49%
were observed. Normal G6PD activity gave a dark yellow to Male 244 (71)
orange colouration, whereas severe and moderate deficiency Female 101 (29)
appeared as almost colourless, and faintly pink colouration, Normal (n=138) 2.8%
respectively [Figure 3]. It has been observed that it was possible Male 64 (46)
to distinguish intermediate from normal activity by their Female 72 (54)
intermediate yellow colour and absorbance. Those neonates who Phototherapy G6PD deficient (n=260) 75%
were G6PD deficient on the screening test observed up to 1 week Male 186 (72)
of age for the appearance of hyperbilirubinaemia and discharged Female 74 (28)
after explaining the underlying disease and precautions to be Normal (n=128) 93%
taken to prevent haemolysis. Statistical analysis was performed Male 62 (48)
using percentages. Female 64 (52)
Exchange G6PD deficient (n=45) 13%
transfusion Male 29 (64)
Results Female 16 (36)
Results from 5652 newborns were analysed [Table 2], Normal (n=18) 13%
comprising 3000  males and 2652  females. Overall Male 8 (44)
703 newborns  (12.4%) had G6PD deficiency, of these Female 10 (56)
45 newborns (6%) had a severe deficiency (<10% of normal Mortality after G6PD deficient (n=5) 1.5%
exchange transfusion Male 4 (80)
enzyme activity). Males had higher prevalence of deficiency
than females (males 70% [n = 491], females 30% [n = 212]). Female 1 (20)
Severe deficiency occur in 64% (n = 29) males and 16% (n = 16) Normal (n=2) 1.5%
Male 1 (50)
Female 1 (50)
G6PD: Glucose‑6‑phosphate dehydrogenase

Figure 3: Qualitative assay in microtubes at 15 min. 1-Reaction mixtures

without blood©; 2-glucose-6-phosphate dehydrogenase-deficient
blood©; 3-glucose-6-phosphate dehydrogenase-severe deficient blood;
4-5-glucose-6-phosphate dehydrogenase-deficient blood; 6-normal
Figure 2: Principle of the water soluble tetrazolium-8 formazan method. blood; 7-normal blood©

Advances in Human Biology  ¦  Volume 9  ¦  Issue 1  ¦  January‑April 2019 39

 Sharma and Sharma: Neonatal Screening for G6PD Deficiency
required exchange transfusion. Post‑exchange mortality rate
in G6pd deficient group is 1.55%.
Qualitative assay using the standard method [Figure 3]
showed different activities of G6PD in the form of orange
colour development. Orange colour in normal blood samples
could be observed at 15 min after incubation at room
temperature [Figure  3; Tube 7]. This orange colour was
distinct from the pinkish colour in the two negative controls,
i.e., normal blood without the substrate mixtures [Tube 2],
or G6PD deficient blood [Tube 3]. In normal blood sample,
colour reaction was maximum at 1.5 h, whereas in two
negative controls, colour development was not noticed even
at 2 h incubation. The negative controls showed no colour
development after incubation for 24 h. Blood samples with
25%–50% residual activities [Figure 4; Tubes 4 and 5] showed
a slow colour development visible by naked eye. Increase
concentration of the WST‑8/1‑methoxy PMS mixture to
40 µl can be used for rapid screening. On the other hand, use
of plate wells for testing of G6PD activity from dried blood
spots showed distinct yellow colour (1st well) indicates a
sample with normal G6PD activity, the well with pale yellow
colour (2nd well) represents a sample with moderate deficiency
and the 3rd and 4th almost colourless wells are indicative of
severe deficiency [Figure 5]. Assay validation was done by
quantitative biochemical testing in 100 newborns which
showed a high level of agreement.
Discussion
Until date, various tests have been reported for detection of
G6PD‑deficiency status. In our assay, WST‑8 did not react with
haemoglobin, and its formazan was water soluble, which made it
possible for the whole reaction to being carried out in an aqueous
reaction mixture in a tube or a well. Apart from measuring G6PD
activity qualitatively by change in colour, we can measure it
quantitatively by reading absorbance. Development of strong
orange colour in a few minutes (10–15 min) indicates normal
G6PD activity whereas pink or colourless solution indicates
G6PD deficient activity [Figures 2 and 3]. To make test suitable
for mass screening, blood spots dried on filter paper on a 96‑well
Figure 4: Qualitative assay in microtubes at 60 min. 1-Reaction mixtures
without blood©; 2-glucose-6-phosphate dehydrogenase-deficient
blood©; 3-glucose-6-phosphate dehydrogenase-severe deficient blood;
4-5-glucose-6-phosphate dehydrogenase-deficient blood; 6-normal
blood; 7-normal blood©.
40
microtitre plate have been used. This enables assessment of
G6PD deficiency in large number of neonates even in places
with little hospital facilities. Subjects with a milder deficiency
could also be identified, this could further be confirmed with a
cytochemical assay.[20]
Blood sample obtained from heel prick method is convenient
and can be stored in bulk using filter paper. This study
has indicated the prevalence of G6PD deficiency  (12.4%)
in Himachal Pradesh with males having a higher
prevalence  (70%) than females  (30%). This could be
because of the presence of a predominating G6PD variant
that phenotypically expresses a severe form of deficiency
resulting in a large number of heterozygous females. These
heterozygous females have extensively different enzyme
levels because of the variant and level of mosaicism which
results in difficulty in their identification using enzymatic
assays. It suggests that there has been a different genetic
variation causing distinct patterns of G6PD deficiency
prevalence in Himachal Pradesh, which has to be established.
Further, the effect of levels of haematocrit on this assay may
be taken into consideration in future studies.
All the samples (n = 5652) were collected and processed by
only 1 operator in a short time. No specific storage conditions
and the temperature was required. All test reagents and
mixtures were stored at room temperature. Even prepared
mixtures could be used up to 2 weeks when kept at room
temperature in the dark, making it extremely useful especially
in field settings without any refrigeration facilities. Blood spots
on filter paper could be stored up to 5 days at room temperature
and up to 10 days at 4°C. Therefore, it would be possible to
undertake multiple assay at a single time with the help of a
suitable number of operators. There was no difference in the
Qualitative assay by both modified method in 96 well plate
and standard method in microtubes. This enzyme method
can be adopted for the screening of G6PD deficiency in India
as it can be easily incorporated along with routine newborn
screening for inborn errors of metabolism. The test is easy to
perform, and the results are available within 24 h. It should
be easily acceptable to the population for mass screening.
The higher cost of WST‑8/1‑methoxy PMS mixture than that
of the MTT/PMS mixture pose a disadvantage, but this can
be overcome by using an economical method by carrying the
assay in a 0.5 ml tube [Table 1].
Figure 5: Qualitative assay in well plates. 1-Normal©; 2-Moderately
deficient; 3-Severely deficient; 4-Severely deficient; 5-Severely deficient©.
Advances in Human Biology  ¦  Volume 9  ¦  Issue 1  ¦  January‑April 2019
 Sharma and Sharma: Neonatal Screening for G6PD Deficiency
With the help of screening programmes we can identify
the neonates at risk. In India, 55% of G6PD deficient
neonates were apparently normal but had a higher risk of
haemolytic anaemia and hyperbilirubinaemia if exposed to
triggers; whereas 32% of the G6PD deficient neonates had
hyperbilirubinaemia.
Due to these reasons, neonatal screening programmes for
the detection of G6PD deficiency are running all over the
globe such as the United States, Europe and the Middle East.
Countries such as Singapore and Greece have shown great
promise with these screening programmes as significant
declines in kernicterus and haemolytic crisis have been noted.
Hence, implementation of such screening programmes goes a
long way in less hospital admissions for the haemolytic crisis,
less requirement of blood transfusions, dialysis. Savings on
medical care and patient morbidity make these programmes
cost‑effective.
Till date, many screening tests fluorescent ones and formazan
methods have been mentioned in literature for detecting G6PD
deficiency.[21‑24] Here, we have come up with an improved,
single‑step formazan method using WST‑8. This method is
qualitative as well as quantitative. The quantitative assay is done
by measurement of absorbance (to detect NADPH production).
This test is easy to administer and promising for mass screenings.
Conclusions
The study highlighted that G6PD deficiency was prevalent in
Himachal Pradesh. This factor needed to be considered while
planning future strategies for neonatal screening. Rapid mass
screening for G6PD deficiency can be easily implemented
with single step formazan method using WST‑8. After initial
trials focusing on institutional deliveries, the programme can
involve home deliveries also.
Acknowledgement
The authors acknowledge with thanks financial support from
ICMR, New Delhi for conducting this research.
Financial support and sponsorship
ICMR, New Delhi has funded this project under extra mural
ad hoc projects.
Conflicts of interest
There are no conflicts of interest.
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