Effect of neonatal reticulocytosis on glucose 6-phosphate dehydrogenase (G6PD) activity and G6PD deficiency detection: a cross-sectional study

Abstract

Background Screening for G6PD deficiency in newborns can help prevent severe hemolysis, hyperbilirubinemia, and bilirubin encephalopathy, as recommended by the World Health Organization (WHO). It has been speculated that the presence of a high number of reticulocytes in newborns interferes with the diagnosis of G6PD deficiency since reticulocytes contain higher amounts of G6PD enzyme than mature erythrocytes. Therefore, the purposes of this study were to assess the effect of reticulocytosis in the determination of blood G6PD activity in Thai newborns by using a novel automated UV-based enzymatic assay and to validate the performance of this assay for the detection of G6PD deficiency in newborn samples. Methods The levels of reticulocytes and G6PD activity were measured in blood samples collected from 1,015 newborns. G6PD mutations were identified using TaqMan® SNP genotyping assay, PCR–restriction fragment length polymorphism (PCR–RFLP), and direct sequencing. The correlation between the levels of reticulocytes and G6PD activity was examined. The performance of the automated method was compared with that of the fluorescent spot test (FST) and the standard quantitative assay. Results The automated assay detected G6PD deficiency in 6.5% of the total newborn subjects compared to 5.3% and 6.1% by the FST and the standard method, respectively. The minor allele frequencies (MAFs) of G6PD ViangchanG871A, G6PD MahidolG487A, and G6PD UnionC1360T were 0.066, 0.005, and 0.005, respectively. The reticulocyte counts in newborns with G6PD deficiency were significantly higher than those in normal male newborns (p < 0.001). Compared with normal newborns after controlling for thalassemias and hemoglobinopathies, G6PD-deficient patients with the G6PD ViangchanG871A mutation exhibited elevated reticulocyte counts (5.82 ± 1.73%, p < 0.001). In a group of G6PD normal newborns, the percentage of reticulocytes was positively correlated with G6PD activity (r = 0.327, p < 0.001). However, there was no correlation between G6PD activity and the levels of reticulocytes in subjects with G6PD deficiency (r = -0.019, p = 0.881). The level of agreement in the detection of G6PD deficiency was 0.999, while the area under the receiver operating characteristic (AUC) curve demonstrated that the automated method had 98.4% sensitivity, 99.5% specificity, 92.4% positive predictive value (PPV), 99.9% negative predictive value (NPV), and 99.4% accuracy. Conclusions We report that reticulocytosis does not have a statistically significant effect on the detection of G6PD deficiency in newborns by both qualitative and quantitative methods.

Utilization of Glucose-6-Phosphate Dehydrogenase Test and the Prevalence of Enzyme Deficiency in Korea

“…Furthermore, G6PD deficiency was distributed throughout all age groups. This finding was also comparable to previous studies showing that most subjects are asymptomatic, which highlights the importance of clinicians' awareness of G6PD deficiency for the diagnosis and management of patients [1,[18][19][20]. It is important to note that G6PD deficiency is relatively rare in Korea compared with some other countries in Asia and the Middle East [1,6,7].…”

“…Various molecular variants of the G6PD gene can result in diverse biochemical phenotypes, and the identification of G6PD enzyme deficiency remains the most reliable method to determine enzyme-deficient variants that may potentially lead to clinical manifestations [1,4,5]. The information regarding the presence of conditions such as thalassemia (which is another rare disease in Korea), anemia, and a high reticulocyte count, which may have affected the G6PD test results, as well as whether the parents of pediatric patients underwent screening, was also limited [19]. The study period included the COVID-19 pandemic, during which the use of G6PD activity tests may have been different from non-pandemic situations [21][22][23].…”

Single-Drop Blood Detection of Common G6PD Mutations in Thailand Based on Allele-Specific Recombinase Polymerase Amplification with CRISPR-Cas12a

“…Briefly, 20 μL of the hemolysate from packed red blood cells was added to 1 mL of distilled water and vortexed for 5 min. The G6PD activity was then measured at 37 °C according to the previously described protocol using a clinical chemistry analyzer (BS-360E, Mindray Medical International) . The rate of absorbance change (Δ A /min) was calculated by [Δ A /min – sample] – [(Δ A /min blank) and normalized with Hb (g/dL), according to the manufacturer’s instructions.…”

“…The G6PD activity was then measured at 37 °C according to the previously described protocol using a clinical chemistry analyzer (BS-360E, Mindray Medical International). 42 The rate of absorbance change (Δ A /min) was calculated by [Δ A /min – sample] – [(Δ A /min blank) and normalized with Hb (g/dL), according to the manufacturer’s instructions. A sample of G6PD activity was considered valid only if the control G6PD activity fell within the reference range.…”

Genetic variants causing G6PD deficiency: Clinical and biochemical data support new WHO classification

“…The following were excluded: (a) data on newborns, because G6PD activity may be higher in the neonatal period [21][22][23] ; (b) data on subjects with known malaria, because their G6PD activity may be higher than in non-infected subjects. 24 In order to harmonize G6PD activity values across studies, when necessary G6PD activity was converted into % of the normal value measured in the laboratory that performed the study.…”