ความสัมพันธ์ของพารามิเตอร์เม็ดเลือดแดงกับความถูกต้องของผลการทดสอบดีซีไอพี ในผู้เป็นพาหะฮีโมโกลบินอี: แนวทางปรับปรุงวิธีการทดสอบเพื่อป้องกันการเกิดผลลบปลอม

Plubplueng  Sansai; Ekkapong  Boriboonwong

Authors

Plubplueng Sansai 1Division of Clinical Chemistry and Toxicology, Department of Medical Technology, School of Allied Health Sciences, University of Phayao, Phayao Province, Thailand
Ekkapong Boriboonwong Division of Clinical Hematology and Microscopy, Department of Medical Technology, School of Allied Health Sciences, University of Phayao, Phayao Province, Thailand

Keywords:

Hemoglobin E carrier, DCIP test, Red blood cell parameters, False-negative result, Packed red cells

Abstract

Screening for hemoglobin E (HbE) using the DCIP (Dichlorophenol Indophenol) test is a simple and effective method, however, false-negative results can still occur even under standardized testing conditions. This study aimed to investigate the relationship between red blood cell parameters, sample type, and blood volume with the accuracy of the DCIP test in HbE carriers. A total of 300 blood samples from individuals displaying EA phenotypes were analyzed. HbE mutation was confirmed using allele-specific polymerase chain reaction. The DCIP test was performed using the KKU-DCIP-Clear reagent kit. The effects of hemoglobin (Hb) concentration, sample type, and blood volume on test results were evaluated. Among the samples, 51 (17%) yielded negative results. The mean Hb concentration and HbE percentage in the false-negative group were 7.7 ± 2.2 g/dL and 19.0 ± 5.1%, respectively, compared to 10.5 ± 2.1 g/dL and 25.6 ± 4.0% in the true-positive group. Logistic regression analysis revealed a statistically significant association between Hb concentration and DCIP test results (p-value = 0.03, OR = 2.516, 95% CI = 1.073-5.897). The rate of false negatives increased when Hb levels were below 8 g/dL and reached 100% when levels dropped below 6 g/dL. Repeat testing using packed red blood cells in previously false-negative samples yielded positive results in all cases. These findings indicated that Hb concentration significantly affects the accuracy of the DCIP test. The likelihood of false-negative results increased as Hb levels decreased. Therefore, Hb concentration should be assessed prior to testing, and packed red cells should be used when Hb is below 8 g/dL to prevent false-negative results.

References

Orkin SH, Kazazian HH Jr, Antonarakis SE, Ostrer H, Goff SC, Sexton JP. Abnormal RNA processing due to the exon mutation of beta E-globin gene. Nature 1982; 300: 768-9. doi: 10.1038/300768a0.

Fucharoen S, Weatherall DJ. Progress Toward the Control and Management of the Thalassemias. Hematol Oncol Clin North Am 2016; 30: 359-71. doi: 10.1016/j.hoc.2015.12.001.

Weatherall DJ, Clegg JB. Inherited haemoglobin disorders: an increasing global health problem. Bull World Health Organ 2001; 79: 704-12.

Fucharoen S, Winichagoon P. Thalassemia in SouthEast Asia: problems and strategy for prevention and control. Southeast Asian J Trop Med Public Health 1992; 23: 647-55.

Olivieri NF, Pakbaz Z, Vichinsky E. HbE/β-thalassemia: basis of marked clinical diversity. Hematol Oncol Clin North Am 2010; 24: 1055-70. doi: 10.1016/j.hoc.2010.08.008.

Mandal PK, Nataraj KS, Baul SN, Ghosh MK, Dolai TK. Efficacy of Dichlorophenolindophenol (DCIP) as Screening Test for Hb E: Revisited. Indian J Hematol Blood Transfus 2020; 36: 535-41.

Winichagoon P, Thitivichianlert A, Lebnak T, Piankijagum A, Fucharoen S. Screening for the carriers of thalassemias and abnormal hemoglobins at the community level. Southeast Asian J Trop Med Public Health 2002; 33: 145-50.

Fucharoen S, Sanchaisuriya K, Fucharoen K, Sae-ung N. Inventer; The Thailand Research Fund, assignee. Reagent kit for simple and rapid screening of hemoglobin E. Thai patent TH 18004. 2005 Jun 01. (in Thai)

Sanchaisuriya K, Fucharoen S, Fucharoen G, et al. A reliable screening protocol for thalassemia and hemoglobinopathies in pregnancy: an alternative approach to electronic blood cell counting. Am J Clin Pathol 2005; 123: 113-8. doi: 10.1309/fuf9evgq24v1pktp.

Chapple L, Harris A, Phelan L, Bain BJ. Reassessment of a simple chemical method using DCIP for screening for haemoglobin E. J Clin Pathol 2006; 59: 74-6. doi: 10.1136/jcp.2005.027961.

Fucharoen G, Sanchaisuriya K, Sae-ung N, et al. A simplified screening strategy for thalassemia and haemoglobin E in rural communities in south-east Asia. Bull World Health Organ 2004; 82: 364-72.

Prayongratana K, Polprasert C, Raungrongmorakot K, Tatone K, Santiwatanakul S. Low cost combination of DCIP and MCV was better than that of DCIP and OF in the screening for hemoglobin E. J Med Assoc Thai 2008; 91: 1499-504.

Jaiwang P, Meemuangthum I, Wiwanitkit V. Positive DCIP rates: A study on laboratory incidence. Chula Med J 2007; 51: 101-4. (in Thai)

Sudjaroen Y. Efficacy evaluation of new hemoglobin E screening test in community hospitals. Scientific Research and Essays 2014; 9: 528-34.

Carrell RW, Kay R. A simple method for the detection of unstable haemoglobins. Br J Haematol 1972; 23: 615-9.

Bender JW, Adachi K, Asakura T. Precipitation of oxyhemoglobins A and S by isopropanol. Hemoglobin 1981; 5: 463-74.

Khamphikham P, Sakkhachornphop S, Pongsatha S, Pornprasert S. Strong Positive Dichloropheno-lindophenol Precipitation Suggests Hb Dhonburi (or Hb Neapolis) (HBB: c.380T>G) Inheritance in a Couple at Risk for Severe β-Thalassemia. Hemoglobin 2022; 46: 184-6. doi: 10.1080/03630269.2022.2069033.

Fucharoen G, Sanchaisuriya K, Aoonjai T, et al. Screening for Hemoglobin E Using the KKU-DCIP-Clear Reagent Kit at Various Temperatures J Med Tech Assoc Thai 2004; 32: 576-84. (in Thai)

Sanchaisuriya K, Kruban P, Rusameepakai S, et al. Problem evaluation and efficiency improvement for thalassemia and hemoglobinopathies screening at community hospitals. J Med Tech Phy Ther 2007; 19: 42-54. (in Thai)

Panyasai S, Cheechang S. Efficiency of screening for carriers of severe thalassemia in a community hospital. Songkla Med J 2009; 27: 61-72. (in Thai)

Sae-ung N, Srivorakun H, Fucharoen G, Yamsri S, Sanchaisuriya K, Fucharoen S. Phenotypic expression of hemoglobins A2, E and F in various hemoglobin E related disorders. Blood Cells Mol Dis 2012; 48: 11-6. doi: 10.1016/j.bcmd.2011.09.008.

Sangkitporn S, Sangkitporn S, Sangnoi A, Supangwiput O, Tanphaichitr VS. Validation of osmotic fragility test and dichlorophenol indophenol precipitation test for screening of thalassemia and Hb E. Southeast Asian J Trop Med Public Health 2005; 36: 1538-42.

Fucharoen S, Fucharoen G, Ratanasiri T, Jetsrisuparb A, Fukumaki Y. A simple non-radioactive assay for hemoglobin E gene in prenatal diagnosis. Clin Chim Acta 1994; 229: 197-203. doi: 10.1016/0009-8981(94)90242-9.

Fucharoen G, Fucharoen S. Thalassemia screening in Thailand. J Med Tech Phy Ther 2007; 20: 165-77. (in Thai)

Fucharoen G, Canta K, Pratumchat R, et al. Thalassemia and Iron Deficiency in Populations with Positive Screening Results from the OF Test and KKU-DCIP-Clear. J Hematol Transfus Med 1999; 9: 111-8. (in Thai)

Fucharoen G, Sanchaisuriya K, Sae-ung N, Fucharoen S. Hemoglobin E and thalassemia among Khon Kaen University Population. KKU Res J 2000; 5: 61-7. (in Thai)

Wanapirak C, Sirichotiyakul S, Luewan S, Srisupundit K, Tongsong T. Comparison of the accuracy of dichlorophenolindophenol (DCIP), modified DCIP, and hemoglobin E tests to screen for the HbE trait in pregnant women. Int J Gynaecol Obstet 2009; 107: 59-60. doi: 10.1016/j.ijgo.2009.04.020.

Prayongratana K, Polprasert C, Raungrongmorakot K, Tatone K, Santiwatanakul S. Low cost combination of DCIP and MCV was better than that of DCIP and OF in the screening for hemoglobin E. J Med Assoc Thai 2008; 91: 1499-504.

Sanchaisuriya K, Fucharoen G, Sae-ung N, Jetsrisuparb A, Fucharoen S. Molecular and hematologic features of hemoglobin E heterozygotes with different forms of alpha-thalassemia in Thailand. Ann Hematol 2003; 82: 612-6. doi: 10.1007/s00277-003-0689-y.