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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 9  |  Issue : 1  |  Page : 37-41

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


Department of Pediatrics, Dr. Rajendra Prasad Government Medical College, Kangra, Himachal Pradesh, India

Date of Web Publication4-Jan-2019

Correspondence Address:
Seema Sharma
House No. 23, Block-B, Type-V, Dr. Rajendra Prasad Government Medical College, Tanda, Kangra, Himachal Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AIHB.AIHB_11_18

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  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


How to cite this article:
Sharma S, Sharma M. 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. Adv Hum Biol 2019;9:37-41

How to cite this URL:
Sharma S, Sharma M. 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. Adv Hum Biol [serial online] 2019 [cited 2020 Jul 2];9:37-41. Available from: http://www.aihbonline.com/text.asp?2019/9/1/37/249514




  Introduction Top


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],[2],[3],[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],[8],[9],[10],[11],[12],[13],[14] India is undergoing an epidemiological transition as congenital 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],[16],[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, Tamil Nadu, Kerala; G6PD Mediterranean (563 C->T) in the Vatalia Prajapatis of North India and the Parsis and the G6PD Orissa (131 C->G) found in the tribals of southern India. Prolonged neonatal jaundice and haemolytic crisis are known to occur in children with G6PD deficiency. Early screening of neonates for this enzymatic deficiency and safeguarding neonates with this deficiency from triggers is the only way to avoid adverse outcomes. Hence, a neonatal screening programme for G6PD deficiency is warranted.

Tantular and Kawamoto used a modified method using a tetrazolium salt water-soluble tetrazolium (WST8), and 1-methoxy phenazine methosulphate (PMS) (less 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 Top


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.
Figure 1: Study design.

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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 which serial colour photographs were taken, and these values were compared with G-6-P controls.
Figure 2: Principle of the water soluble tetrazolium-8 formazan method.

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Table 1: Components of the reaction mixtures

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Chemicals

WST-8 and 1-methoxyphenazine methosulphate (1-methoxy PMS) were available in a CCK-8 kit from Sigma-Aldrich Co. (Tokyo, Japan) at concentrations of 5-mm WST-8 and 0.2 mm 1-methoxy PMS in 0.15 m NaCl. Glucose-6-phosphate (G6P) and NADP were procured from Sisco Research Laboratories (Mumbai, India). The controls of known normal G6PD activity and known G6PD deficient were obtained from Trinity Biotech USA (NY, USA) which were having lyophylised G6PD in human red cell haemolysate base with stabilisers and preservatives. All G6PD deficient patients need a definitive, quantitative test for which an enzyme level <100 U/trillion red blood cells has been defined as the cut off for classifying the neonate as G6PD deficient.

Blood spots were collected with the heel-prick method onto 3 MM filter paper (Whatman) and were dried at room temperature. The dried blood spots were stored in zip-lock bags. At the time of processing, a set of controls (normal, deficient) were taken in microtubes and also on 3 MM filter paper. From each dried blood spot, a single 1/16 inch diameter disc was punched out and placed in a single well of a 96-well flat bottom microplate and in the microtubes. For no-substrate control, control spots and extra blood spots were also punched and placed in allocated wells and microtubes. One empty well served as a 'no sample' negative control. A volume of 200 μL of working reaction mixture was mixed into each well/microtube, except the substrate negative control well/microtube, into which 200 μL of no-substrate reaction mixture was added. Serial colour changes were observed. Normal G6PD activity gave a dark yellow to orange colouration, whereas severe and moderate deficiency appeared as almost colourless, and faintly pink colouration, respectively [Figure 3]. It has been observed that it was possible to distinguish intermediate from normal activity by their intermediate yellow colour and absorbance. Those neonates who were G6PD deficient on the screening test observed up to 1 week of age for the appearance of hyperbilirubinaemia and discharged after explaining the underlying disease and precautions to be taken to prevent haemolysis. Statistical analysis was performed using percentages.
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 blood; 7-normal blood©

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  Results Top


Results from 5652 newborns were analysed [Table 2], comprising 3000 males and 2652 females. Overall 703 newborns (12.4%) had G6PD deficiency, of these 45 newborns (6%) had a severe deficiency (<10% of normal enzyme activity). Males had higher prevalence of deficiency than females (males 70% [n = 491], females 30% [n = 212]). Severe deficiency occur in 64% (n = 29) males and 16% (n = 16) females. This analysis identified that severe G6PD deficiency do occur in females. Out of 703 G6PD deficient neonates 45% (n = 345) had hyperbilirubinaemia requiring phototherapy in 75% (n = 260) newborns and 13% (n = 45) newborns required exchange transfusion. Post-exchange mortality rate in G6pd deficient group is 1.55%.
Table 2: Morbidity profile of glucose-6-phosphate dehydrogenase deficient newborns

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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.
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©.

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Figure 5: Qualitative assay in well plates. 1-Normal©; 2-Moderately deficient; 3-Severely deficient; 4-Severely deficient; 5-Severely deficient©.

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  Discussion Top


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 [Figure 2] and [Figure 3]. To make test suitable for mass screening, blood spots dried on filter paper on a 96-well 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].

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],[22],[23],[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 Top


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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