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

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.

Haematological profile of malaria patients with G6PD and PKLR variants (erythrocytic enzymopathies): a cross-sectional study in Thailand.

BACKGROUND: Glucose 6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PKLR) deficiencies are common causes of erythrocyte haemolysis in the presence of antimalarial drugs such as primaquine and tafenoquine. The present study aimed to elucidate such an association by thoroughly investigating the haematological indices in malaria patients with G6PD and PKLRR41Q variants. METHODS: Blood samples from 255 malaria patients from Thailand, Myanmar, Laos, and Cambodia were collected to determine haematological profile, G6PD enzyme activity and G6PD deficiency variants. The multivariate analysis was performed to investigate the association between anaemia and G6PD MahidolG487A, the most common mutation in this study. RESULTS: The prevalence of G6PD deficiency was 11.1% (27/244) in males and 9.1% (1/11) in female. The MAFs of the G6PD MahidolG487A and PKLRR41Q variants were 7.1% and 2.6%, respectively. Compared with patients with wildtype G6PD after controlling for haemoglobinopathies, G6PD-deficient patients with hemizygous and homozygous G6PD MahidolG487A exhibited anaemia with low levels of haemoglobin (11.16 ± 2.65 g/dl, p = 0.041). These patients also exhibited high levels of reticulocytes (3.60%). The median value of G6PD activity before treatment (Day 0) was significantly lower than that of after treatment (Day 28) (5.51 ± 2.54 U/g Hb vs. 6.68 ± 2.45 U/g Hb; p < 0.001). Reticulocyte levels on Day 28 were significantly increased compared to that of on Day 0 (2.14 ± 0.92% vs 1.57 ± 1.06%; p < 0.001). PKLRR41Q had no correlation with anaemia in malaria patients. The risk of anaemia inpatients with G6PD MahidolG487A was higher than wildtype patients (OR = 3.48, CI% 1.24-9.75, p = 0.018). Univariate and multivariate analyses confirmed that G6PD MahidolG487A independently associated with anaemia (< 11 g/dl) after adjusted by age, gender, Plasmodium species, parasite density, PKLRR41Q, and haemoglobinopathies (p < 0.001). CONCLUSIONS: This study revealed that malaria patients with G6PD MahidolG487A, but not with PKLRR41Q, had anaemia during infection. As a compensatory response to haemolytic anaemia after malaria infection, these patients generated more reticulocytes. The findings emphasize the effect of host genetic background on haemolytic anaemia and the importance of screening patients for erythrocyte enzymopathies and related mutations prior to anti-malarial therapy.

Evaluating the performance of automated UV enzymatic assay for screening of glucose 6-phosphate dehydrogenase deficiency.

INTRODUCTION: A precise and reliable screening assay for glucose 6-phosphate dehydrogenase (G6PD) deficiency would greatly help avoiding unwanted outcomes due to bilirubin neurotoxicity in neonatal jaundice and antimalarial-induced haemolytic anaemia in malaria patients. Currently, available assays are laborious and require sophisticated laboratory expertise. This study aimed to evaluate the performance of a recently introduced automated screening assay for G6PD deficiency by comparing with a routine spectrophotometric assay. METHODS: An automated UV-based enzymatic (Mindray, PRC) and spectrophotometric assays were performed simultaneously in parallel to determine G6PD activity in 251 blood samples from the subjects. RESULTS: The median G6PD activity value from spectrophotometric assay was significantly lower than that of from the automated assay. The mean difference was -2.0 U/g haemoglobin (-7.3 to 3.2; P < 0.0001). The mean activity values of both assays were strongly correlated with Pearson's correlation coefficient of r = 0.8. Cohen's kappa statistics between assays was 0.77 (0.70-0.83). The sensitivity, specificity, positive and negative predictive values of the automated assay were 85.7%, 99.2%, 85.7%, 99.2%, respectively. The sensitivity and positive predictive values of the automated assay for identifying intermediate G6PD activity levels were 40.0% and 25.0%, respectively. Genotyping was performed to confirm G6PD deficient and intermediate samples. The turnaround time for 40 samples was 60 minutes for the automated assay and 300 minutes for spectrophotometric assay. CONCLUSION: The automated assay for the detection of G6PD deficiency is comparable to a routine spectrophotometric assay and help reducing sample handling time. However, the assay shows limitation in identifying individuals with G6PD intermediate.

Retinol-binding protein 4 and its potential roles in hypercholesterolemia revealed by proteomics.

Effects of hypercholesterolemia on alterations of serum proteins have not been fully elucidated. Herein, using two-dimensional gel electrophoresis (2-DE) in conjunction with LC-MS searching has successfully been carried out to investigate the change of protein expression profiles as consequences of raised blood cholesterol at different levels (normal group: total cholesterol 200 mg/dL; borderline high group: total cholesterol 200-239 mg/dL; and high group: total cholesterol ≥ 240 mg/dL) (n = 45). Results revealed that down-regulation of retinol-binding protein 4 (RBP4) (-2.26 fold), transthyretin (-1.25 fold) and gelsolin (-1.47 fold) was observed in the high group. Meanwhile, the other proteins such as haptoglobin, complement factor B and CD5 antigen-like protein were up-regulated upto +3.24, +1.96 and +2.04 fold, respectively. Confirmation by Western blotting revealed a significant reduction of RBP4 (approximately 50 %) in individual samples derived from the high group. Presumptive conclusion can be drawn that down-regulation of RBP4 might be attributable to the inflammation of adipocytes caused by the release of proinflammatory cytokines (e.g. tumor necrosis factor α and interleukin-1ß) from adipose tissues. Moreover, the decrease of transthyretin might also be taken into accounts since it is known that the transthyretin usually forms complex with RBP4 to prevent glomerular filtration and excretion through the kidney. The suppressing effect on RBP4 should be potentiated by the increase of complement factor B and CD5 antigen-like protein, which rendered the adipose tissues to overwhelm the liberation of RBP4 to blood circulation by metabolic and inflammatory processes. Such inflammation could further modulate the induction of cytokine release (e.g. IL-6 and IL-1ß), resulting in the synthesis of acute phase protein, in particular, haptoglobin and C-reactive proteins from hepatocytes. However, the mechanism of gelsolin reduction remains unclear. Among these differentially expressed proteins, the RBP4 has been proposed as a major linkage between hypercholesterolemia, adipose tissues, liver and kidney, which is believed to be a potential biomarker for metabolic and cardiovascular disorders associated with dyslipidemia in the future.