MTT FORMAZAN REPLACED WST-8 AS A BETTER SIMPLE SCREENING METHOD FOR DETECTION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY

Indah Tantular

= http://dx.doi.org/10.20473/ijtid.v7i6.13454
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Abstract


We have previously developed the WST-8 method as a simple and rapid screening test for detection of glucose-6-phosphate dehydrogenase (G6PD) deficiency accomplished by the naked eye. However, it was little difficult to distinguish between faint orange colors developed by heterozygous females and pink colors of normal hemolyzed blood, since both have similar tones. To solve this problem, we established a new and simple screening method that utilizes another formazan substrate, MTT (3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H tetrazolium bromide) in combination with a hydrogen carrier, 1-methoxy phenazine methosulfate. MTT formazan exhibits a purple color, thus allowing for the ability to easily distinguish the pink colors of hemolyzed blood. However, MTT has been reported to react with hemoglobin non-specifically and to interfere with the interpretation of the color reaction. In our examinations by mixing MTT with hemolyzed blood, we found that the non-specific reaction was very slow, and that the addition of a small amount of blood (5~10 μl) into a reaction mixture (800 μl) did not interfere with the reaction of G6PD activity. In this new MTT method, a strong purple color was generated in normal blood samples at 20~30 min after incubation, which could be distinguished by the naked eye from G6PD-deficient blood samples with less than 50% residual activity. In addition, quantitative measurement using a spectrophotometer was also possible despite the fact that MTT formazan is water-insoluble.


Keywords


G6PD-deficiency, new screening method, formazan substrate, MTT, purple color development

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References


Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiency. Lancet.2008; 371: 64-74.

Minucci A, Moradkhani K, Hwang MJ, Zuppi C, Giardina B, Capoluongo E. Glucose-6-phosphate dehydrogenase (G6PD) mutations database: review of the"old" and update of the new mutations. Blood Cells Mol Dis. 2012; 48: 154-65.

Matsuoka H, Arai M, Yoshida S, Tantular IS, Pusarawati S, Kerong H, et al. Five different glucose-6-phophate dehydrogenase (G6PD) variantsfound among 11 G6PD-deficient persons in Flores Island, Indonesia.JHum Gen. 2003; 48:541-4.

Matsuoka H, Wang J, Hirai M, Arai M, Yoshida S, Kobayasi T. et al. Glucose-6-phosphate dehydrogenase (G6PD) mutations in Myanmar: G6PD Mahidol (487G>A) is the most common variant in the Myanmar population. J Hum Gen. 2004; 49: 544-7.

Jalloh A, van Thien H, Ferreira MU, Ohashi J, Matsuoka H, Kanbe T, et al. Rapidepidemiologic assessment of glucose-6-phosphate dehydrogenase (G6PD) deficiency in malaria-endemic areasinSoutheast Asia using a novel diagnostic kit. Trop Med Int Health. 2004; 9:615-23.

Matsuoka H, Nguon C, Kanbe T, Jalloh A, Sato H, Yoshida S, et al. Glucose-6-phosphate dehydrogenase (G6PD) mutations in Cambodia: G6PD Viangchan (871G>A) is the most commonvariant in the Cambodian population. JHumGen. 2005; 50: 468-72.

Kawamoto F, Matsuoka H, Kanbe T, Tantular IS, Pusarawati S, Kerong H, et al. Further investigations of glucose-6-phosphate dehydrogenase variants in FloresIsland, eastern Indonesia. JHum Gen. 2006; 51: 952-7.

Matsuoka H, Thuan DTV, van Thien H, Kanbe T, Jalloh A, Hirai M, et al. Seven different glucose-6-phosphate dehydrogenase variants including a new variant distributed in Lam Dong Province in southern Vietnam. Acta Med Okayama. 2007; 61: 181-5.

Tantular IS, Matsuoka H, Kasahara Y, Pusarawati S, Kanbe T, Tuda JSB, et al. Incidence and mutation analysis of glucose-6-dehydrogenase deficiency in eastern Indonesian populations. Acta Med Okayama. 2010; 64: 367-73.

Kawamoto F, Matsuoka H, Pham NM, Hayashi T, Kasahara Y, Dung NT, et al. Further molecular analysis of G6PD deficiency variants in southern Vietnam and a novel variant designated as G6PD Ho Chi Minh (173 A>G; 58 Asp>Gly): Frequency distributions of variants compared with those in other Southeast Asian countries. Acta Med Okayama. 2017; 71: 325-32.

Kawamoto F. Rapid diagnosis of malaria by fluorescence microscopy using light microscope and interference filter. Lancet. 1991; 337: 200-2.

Kawamoto F, Billingsley PF. Rapid diagnosis of malaria by fluorescence microscopy. Parasitol Today. 1992;8: 69-71 pmid: 15463575

Tantular IS, Kawamoto F. An improved, simple screening method for detection ofglucose-6-phosphatedehydrogenase deficiency. Trop Med Int Health. 2003; 8: 569-74.

Beutler E. A series of new screening procedures for pyruvate kinase deficiency, glucose-6-phosphate dehydrogenase deficiency, and glutathione reductase deficiency. Blood. 1966; 28: 553-62.

Beutler E, Mitchell M. Special modification of the fluorescent screening method for glucose-6-phosphate dehydrogenase deficiency. Blood. 1968; 32: 816-8.

Fujii H, Takahashi K, Miwa S. A new simple screening method for glucose-6-phosphate dehydrogenase deficiency. Acta Haematol Jap 1984:47: 185-8.

Hirono A, Fujii H, Miwa S. An improved single-step screening method for glucose-6-phosphate dehydrogenase deficiency. Jap J Trop Med Hyg. 1998; 26:1-4.

Hirono A, Ishii A, Kere N, Fujii H, Hirono K, Miwa S. Molecular analysis of glucose-6-phosphate dehydrogenase variants in the Solomon Islands. American J Hum Gen. 1995; 56:1243-5.

Tantular IS, Iwai K, Lin K, Basuki S, Horie T, Htay HH, et al. Field trials of a rapid test for G6PD deficiency in combination with a rapid diagnosis of malaria. Trop Med Int Health. 1999; 4, 245-50.

Ishii A, Nagai N, Arai M, Kawabata M, Matsuo T, Bobogare A, et al.Chemotherapeuticmalaria control as a selective primary health care activity in the Solomon Islands. Parasitologia. 1999; 41: 383-4.

Iwai K, Hirono A, Matsuoka H, Kawamoto F, Horie T, Lin K, et al. Distribution of Glucose-6-phosphate dehydrogenase mutations in Southeast Asia. Hum Gen. 2001; 108: 445-9.

Suryantoro P. Glucose-6-phosphate dehydrogenase (G6PD) deficiency in Yogyakarta and its surrounding areas. Southeast A J Trop Med Pub Health. 2003; 34 Suppl. 3: 138-9.Fairbanks VF, Beutler E. A simple method for detection of erythrocyte glucose-6-phosphate dehydrogenase deficiency (G-6-PD spot test). Blood. 1962; 20: 591-601.

Osorio L, Carter N, Arthur P, Bancone G, Gopalan S, Gupta SK, et al.Performance of BinaxNOW G6PD deficiency point-of-care diagnostic in P. vivax-infected subjects. Am J Trop Med Hyg. 2015; 92: 22-7.

Goo YK, Ji SY, Shin HI, Moon JH, Cho SH, Lee WJ, et al. First evaluation of glucose-6-phosphate dehydrogenase (G6PD) deficiency in vivax malaria endemic regions in the Republic of Korea. PLoS One. 2014; 9: e97390.

Bancone G, Gornsawun, Chu CS, Porn P, Pal S, Bansil P, et al. Validation of the quantitative point-of-care CareStart biosensor for assessment of G6PD activity in venous blood. PLoS One. 2018; 13: e0196716.


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