Efficacy and Safety of a Dispersible Tablet of GPO-Deferasirox Monotherapy among Children with Transfusion-Dependent Thalassemia and Iron Overload.

The study aimed to determine efficacy and safety of generic deferasirox monotherapy. Deferasirox was administered in transfusion-induced iron overloaded thalassemia. Efficacy was defined as responders and nonresponders by ≤ 15 reduced serum ferritin from baseline. Adverse events were also monitored. Fifty-two patients with mainly Hb E/ß-thalassemia at the mean (SD) age of 8.7 (4.1) years, were enrolled. The mean (SD) daily transfusion iron load was 0.47 (0.1) mg/kg and maximum daily deferasirox was 35.0 (6.2) mg/kg. Altogether, 52, 40 and 18 patients completed the first, second and third years of study, respectively. The median baseline serum ferritin 2,383 ng/mL decreased to 1,478, 1,038 and 1,268 ng/mL at the end of first, second and third years, respectively, with overall response rate at 73.1% (38/52). Patients with baseline serum ferritin >2,500 ng/mL showed a change in serum ferritin higher than those ≤2,500 ng/mL starting from the 9th month of chelation. Adverse events were found in 5 of 52 patients (9.6%) including transaminitis (n = 2), one each of proteinuria, rash and proximal tubular dysfunction which resolved after transient stopping or decreasing the chelation dose. Generic deferasirox was effective and safe among pediatric patients with transfusion-induced iron overloaded thalassemia.

Whole-exome sequencing uncovered genetic diagnosis of severe inherited haemolytic anaemia: Correlation with clinical phenotypes.

Next-generation sequencing has shed light on the diagnosis of previously unsolved cases of inherited haemolytic anaemia (IHA). We employed whole-exome sequencing to explore the molecular diagnostic spectrum of 21 unrelated Thai paediatric patients with non-thalassemic IHA, presenting hydrops fetalis and/or becoming transfusion-dependent for 1 year or more or throughout their lifespan. Anaemia was detected prenatally, within the first month and the fifth year of life in three, 12 and six patients respectively. Molecular diagnosis obtained from all patients revealed SPTB as the most frequently mutated gene (four reported, three novel), found in 31 of 42 studied alleles. The other two mutated genes identified were ANK1 (three novel) and KLF1 (two reported). Four recurring mutations within exon 29/30 (NM_001024858.2) accounted for the vast majority (90%) of mutated SPTB alleles, biallelic inheritance of which resulted in the most severe phenotypes: hydrops fetalis and life-long transfusion dependency. Dominant ANK1 (n = 3) and SPTB (n = 2) mutations and biallelic class 2 KLF1 mutations (n = 1) led to a shorter period of transfusion dependency. Our study demonstrated that mutated SPTB causing red-cell membranopathy is likely the most common cause of severe non-thalassemic IHA among Thai patients. This urges carrier screening in the population to prevent subsequent, severely affected births.

No differences in hemostatic and endothelial activations between haploidentical and matched-donor hematopoietic stem cell transplantation in thalassemia disease.

Hemostatic changes and endothelial activations have been recognized in ß-thalassemic patients after matched-donor hematopoietic stem cell transplantation (HSCT) but there are limited studies for haploidentical HSCT. This report demonstrates that the levels of hemostatic and endothelial markers, including thrombin antithrombin complex, prothrombin fragment, D-dimer, von Willebrand factor antigen and thrombomodulin levels, were not significantly different between haploidentical and matched-donor HSCT patients.

Increased endothelial activation in α-thalassemia disease.

One complication of thalassemia is thromboembolism (TE), which is caused by an abnormal red blood cell surface, as well as endothelial and platelet activation. These findings are commonly observed in severe ß-thalassemia. However, limited information on α-thalassemia exists. This study enrolled subjects with deletional and non-deletional α-thalassemia and normal controls (NC). Plasma and serum of subjects were tested for endothelial activation markers including thrombomodulin (TM), vascular cell adhesion molecule-1 (VCAM-1), and von Willebrand factor antigen as well as platelet activation markers including thromboxane B2 and platelet factor 4. A total of 179 subjects were enrolled: 29 in the deletional group (mean age 13.3 ± 4.4 years), 31 in the non-deletional group (mean age 12.9 ± 4.8 years), and 119 in the NC group (mean age 13.6 ± 3.0 years). Twenty nine percent of subjects in the non-deletional group received regular red blood cell transfusion and iron chelator administration. Serum ferritin level was higher in the non-deletional group than that in the deletional group. Multivariate analysis demonstrated that VCAM-1 and TM levels were increased significantly in α-thalassemia compared with NC group (816.8 ± 131.0 vs 593.9 ± 49.0 ng/ml, and 4.9 ± 0.7 vs 4.0 ± 0.4 ng/ml, P < 0.001 respectively). VCAM-1 and TM levels in the non-deletional group were significantly higher than that in the deletional group. The present study demonstrated endothelial activation in children with α-thalassemia disease, especially those in the non-deletional group, which might be one risk factor for TE in α-thalassemia disease.

Reticulocyte hemoglobin equivalent in a thalassemia-prevalent area.

BACKGROUND: Reticulocyte hemoglobin equivalent (Ret-He), a direct measure of the hemoglobin (Hb) in the young red blood cells, has been reported to be useful in the diagnosis of iron deficiency anemia (IDA) but may have some limitations in thalassemia trait. This study evaluated the differences in Ret-He in school-aged children, and assessed the diagnostic value of Ret-He in identifying IDA in a thalassemia-prevalent area. METHODS: Blood samples underwent complete blood count analysis, including Ret-He, ferritin, serum iron and total iron binding capacity. Blood samples also underwent Hb typing and a molecular study for α-thalassemia. Receiver operating characteristic analysis was performed to determine the predictive capacity of Ret-He in the diagnosis of IDA. ID was defined as serum ferritin <30 ng/mL and/or transferrin saturation (TSAT) <16%; IDA was defined as serum ferritin <12 ng/mL and/or TSAT <16% with low Hb for age. Normal healthy children (normal controls: NC) had normal iron study, without the thalassemia trait. RESULTS: Ninety-eight children with a mean age of 12.9 ± 0.6 years were included. Ret-He in the thalassemia trait group (26.7 ± 2.4 pg), ID group (29.0 ± 2.9 pg), IDA group (25.4 ± 2.7 pg), ID + thalassemia trait group (26.6 ± 2.8 pg), and the IDA + thalassemia trait group (24.6 ± 2.3 pg) was significantly lower than in the NC group (30.8 ± 1.7 pg; P < 0.001, 0.01, 0.006, 0.002 and <0.001, respectively). Ret-He had an area under the curve of 0.904 in diagnostic ability for IDA, while a cut-off ≤27 pg had a sensitivity of 91.7% and a specificity of 81%. CONCLUSION: Ret-He was lowest in subjects with IDA + thalassemia trait. A Ret-He cut-off ≤27 pg was suggestive of IDA in the present study.

The Effect of Blood Transfusion on Growth of Patients with Hb E/ß-Thalassemia.

A retrospective evaluation of growth in 112 patients (68 males, 44 females) with Hb E (HBB: c.79G>A)/ß-thalassemia (ß-thal), classified as 88 transfusion-dependent thalassemia (TDT) and 24 non transfusion-dependent thalassemia (NTDT), is reported. Patients with TDT have received regular transfusions of red blood cells (RBCs) 15 mL/kg every 4 weeks to maintain pre transfusion hemoglobin (Hb) levels of at least 9.0 g/dL and were categorized according to age at initiation of regular RBC transfusion as subgroup 1, <4 years; subgroup 2, 4-10 years, and subgroup 3, >10 years. Iron chelation was initiated at the mean age of 7 years. The results revealed that patients in subgroups 1 and 2, receiving RBC transfusions at a young age (2.9 and 6.9 years, respectively), had normal prepubertal growth at enrollment and last follow-up. Patients in subgroup 3, with the lowest initial height Z-score of -2.10, were able to achieve comparable final adult height as those in subgroups 1 and 2. The mean final height of 21 males and 13 females with TDT at the ages of 18.9 and 18.7 years was 168.1 and 157.7 cm, respectively, which did not significantly differ from their midparental height and those with NTDT. Early initiation of optimal transfusion and iron chelation promoted normal prepubertal growth. However, delayed initiation of transfusion at age 12 years impaired prepubertal growth but they could achieve normal final adult height.

Preventable Severe Thalassemia among Children.

This retrospective study analyzed 27 children with preventable severe thalassemia born to 24 at-risk couples between 1997 and 2017. The couples were categorized into two groups: the prenatal diagnosis (PND) group (n = 8) and the non PND group (n = 16). In the PND group, following comprehensive counseling on having a fetus with thalassemia, six couples decided to continue the pregnancy (n = 6). Termination of the two remaining fetuses was excluded as the thalassemia status was reported at a gestational age of 24 weeks. In the non PND group, medical errors were found in the misdiagnosis of couples as non thalassemia carriers (n = 4) and not offering PND to couples with known thalassemia carrier status when attending the antenatal clinic (ANC) (n = 2). Additionally, parental ignorance was found in parents experiencing their own thalassemia, or that of their spouse or child (n = 6). The remaining couples (n = 4) with known carrier status either directly refused PND or were ineligible for it. A total of five divorces (5/24 = 20.8%) occurred in the PND (n = 2) and the non PND (n = 3) groups. Knowledge, beliefs, religion, experience of thalassemia, as well as the sex of the at-risk fetus all influenced parental decisions. Therefore, both medical personnel and parents are key in preventing new cases of thalassemia. Parents should be aware of the consequences of having children with severe thalassemia, while medical personnel should provide accurate carrier detection and PND.

A molecular study on the role of alpha-hemoglobin-stabilizing protein in hemoglobin H disease.

The clinical course of hemoglobin H (HbH) disease is remarkably variable. It is not completely clear how genetic and environmental factors interplay to modify clinical severity in affected individuals. Previous studies suggested that altered structure or function of alpha-hemoglobin-stabilizing protein (AHSP) could modify the clinical phenotypes of thalassemias. The present study attempted to explore the potential role of AHSP in the pathophysiology of HbH disease in 95 Chinese and Thai/Sino-Thai patients with deletional and non-deletional form of this disease. We identified six polymorphic sites in AHSP which were subgrouped into major haplotype clades. No association between AHSP genotypes or haplotypes and clinical phenotypes was observed. Instead, multiple linear regression analysis indicated that expression of AHSP correlated negatively with age (P < 0.001) and hemoglobin (P = 0.007), but positively with reticulocyte count (P = 0.003) and severity score (P = 0.003). Subgroup analysis showed that AHSP expression was higher in the non-deletional form than in the deletional form (P < 0.001). Moreover, specific types of non-deletional HbH disease with production of mutant alpha-globin chains that do not bind to AHSP (Hb Constant Spring and Hb Pakse) showed the highest AHSP expression. The present findings demonstrate that AHSP expression is a biomarker of HbH disease severity and infer an important role of AHSP in modulating the pathophysiology of this disease. Pharmacological or genetic means to alter AHSP expression may be a novel approach for amelioration of disease severity in HbH disease.

Effect of Iron Chelation Therapy on Glucose Metabolism in Non-Transfusion-Dependent Thalassaemia.

AIMS: To compare insulin sensitivity, ß-cell function and iron status biomarkers in non-transfusion-dependent thalassaemia (NTDT) with iron excess during pre- and post-iron chelation. METHODS: Subjects with NTDT, aged older than 10 years, with serum ferritin >300 ng/ml, were included. Iron chelation with deferasirox (10 mg/kg/day) was prescribed daily for 6 months. RESULTS: Ten patients with a median age of 17.4 years were enrolled. The comparison between pre- and post-chelation demonstrated significantly lower iron load: median serum ferritin (551.4 vs. 486.2 ng/ml, p = 0.047), median TIBC (211.5 vs. 233.5 µg/dl, p = 0.009) and median non-transferrin binding iron (5.5 vs. 1.4 µM, p = 0.005). All patients had a normal oral glucose tolerance test (OGTT) both pre- and post-chelation. However, fasting plasma glucose was significantly reduced after iron chelation (85.0 vs.79.5 mg/dl, p = 0.047). MRI revealed no significant changes of iron accumulation in the heart and liver after chelation, but there was a significantly lower iron load in the pancreas, assessed by higher T2* at post-chelation compared with pre-chelation (41.9 vs. 36.7 ms, p = 0.047). No adverse events were detected. CONCLUSIONS: A trend towards improving insulin sensitivity and ß-cell function as well as a reduced pancreatic iron load was observed following 6 months of iron chelation (TCTR20160523003).

Blood Coagulation and Asthma Exacerbation in Children.

BACKGROUND: Recent studies have demonstrated the activation of coagulation pathways in asthmatic airways. This study aimed to determine systemic blood coagulation during asthma exacerbation compared with the stable state in children. METHODS: Pediatric patients (aged between 5 and 15 years) suffering from asthma exacerbation were enrolled. von Willebrand factor (vWF), plasminogen activator inhibitor type-1 (PAI-1), protein C, D-dimer, prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin complex (TAT), and C-reactive protein (CRP) levels were measured during asthma exacerbation and stable state. RESULTS: A total of 22 patients were enrolled. The median vWF, PAI-1, and CRP during asthma exacerbation were significantly higher than those of the stable state: 147.5% (interquartile range, IQR: 111.05-196.57) versus 94% (IQR: 69.72-109.62, p < 0.001), 41.9 ng/ml (IQR: 21.91-48.61) versus 26.17 ng/ml (IQR: 15.89-34.44, p < 0.03), and 4.46 mg/l (IQR: 2.15-16.23) versus 0.87 mg/l (IQR: 0.20-3.89, p < 0.015), respectively. However, the median protein C during asthma exacerbation was significantly lower than that of the stable state: 99.5% (IQR: 86.75-117) versus 113% (IQR: 94-115.25), p = 0.01. No significant difference was found between the levels of D-dimer, F1 + 2, and TAT during asthma exacerbation and stable state. Ultimately, D-dimer was positively correlated with asthma exacerbation score (R = 0.466, p = 0.027). A significant correlation was observed between vWF and CRP (R = 0.527, p = 0.012). CONCLUSION: Evidence was found of increased endothelial activation and increased PAI-1 during asthma exacerbation. This may emphasize the potential role of blood coagulation in asthma exacerbation.

Response to propranolol in infantile hemangioma.

Propranolol, 2 mg/kg/day, is effective in the treatment of infantile hemangioma. We report the response to propranolol in infants with hemangioma at a dose of 1 mg/kg/day. Sixteen infants with newly diagnosed infantile hemangioma were given propranolol at a dose titrated from 0.5 mg/kg/day then increased to 1 or 2 mg/kg/day based on response to treatment until the lesions showed clinical stability for 3 consecutive months. Five out of 16 patients (31.2%) responded to propranolol at 1 mg/kg/day, while the remainder required 2 mg/kg/day for response. Vascular endothelial growth factor significantly decreased after treatment (median, 117.8 pg/mL; range, 35.3-468.7 pg/mL vs 59.2 pg/mL; range, 26.3-133.0 pg/mL; P = 0.016). Therefore, we recommend initiating treatment at 0.5 mg/kg/day for 2 days, then 1 mg/kg/day for 1 month. If the hemangioma has not decreased in size by 1 month follow up, the dose is subsequently increased to 2 mg/kg/day.

Combined chelation therapy with daily oral deferiprone and twice-weekly subcutaneous infusion of desferrioxamine in children with ß-thalassemia: 3-year experience.

OBJECTIVE: To study the efficacy of combined treatment with oral and subcutaneous iron chelators. MATERIAL AND METHODS: 50-100 mg/kg/day of oral deferiprone (DFP) combined with 40 mg/kg/dose s.c. desferrioxamine (DFO) twice weekly were given to transfusion-dependent ß-thalassemia children. RESULTS: Enrolled patients (9 with ß-thalassemia major and 33 with ß-thalassemia hemoglobin E), ranging from 3 to 18 years in age, were divided into 3 groups; group 1 ferritin ≥1,000-2,500 ng/ml (n = 10), group 2 ferritin >2,500-4,000 ng/ml (n = 23) and group 3 ferritin >4,000 ng/ml (n = 9). Of the 42 patients, 28 reached the 36-month follow-up. Ten patients whose ferritin declined <15% while receiving 100 mg/kg/day of DFP were considered nonresponders. The median age and previous transfusion duration before enrollment were significantly higher in nonresponders than responders (p = 0.04 and 0.003, respectively). The responders exhibited a significant fall in median ferritin levels from 2,954.6 to 936.6 ng/ml (p < 0.001). Time to a significant decrease in serum ferritin among responders was 6 months. In 13 patients, 16 episodes of adverse events occurred: hemophagocytosis with cytopenia (n = 1), neutropenia (n = 2), thrombocytopenia (n = 2), elevated alanine aminotransferase (n = 5), elevated serum creatinine (n = 1), proteinuria (n = 1) and gastrointestinal discomfort (n = 4). CONCLUSION: Combination therapy with daily oral DFP and subcutaneous DFO twice weekly is a safe and effective alternative to chelation monotherapy in ß-thalassemia children.

Global fibrinolytic activity, PAI-1 level, and 4G/5G polymorphism in Thai children with arterial ischemic stroke.

BACKGROUND: Prolonged euglobulin clot lysis time (ECLT) and increased level of plasminogen activator inhibitor-1 (PAI-1) were reported to be risk factors of arterial ischemic stroke (AIS) by some studies; however, these findings were not supported by other studies. The objective of this study was to determine the association of ECLT, PAI-1 level, and polymorphisms of 4G and 5G of PAI-1 gene to the development of AIS in Thai children. METHODS: This study included patients aged 1-18 years old. Diagnosis of AIS was confirmed by imaging study. The control group was age- and sex-matched healthy subjects. Demographic data were recorded, and blood was tested for ECLT, PAI-1 level, lipid profiles, fasting blood sugar (FBS), and 4G and 5G polymorphisms of PAI-1 gene. RESULTS: There were 70 subjects participating in this study, consisting of 30 patients and 40 controls. Demographic data, lipid profiles, and FBS were similar between the 2 groups. Furthermore, ECLT and PAI-1 level did not differ between patient and control groups; however, both showed significant correlation (r = .352, P = .006). The 4G/5G polymorphism was the most common genotype in both patient and control groups (69.0% vs. 80.0%). However, 4G and 5G polymorphisms of PAI-1 gene did not correlate with PAI-1 level in this study (P = .797). CONCLUSIONS: The PAI-1 level and 4G/5G polymorphism may not be a risk factor of AIS in this population. It was also found that the 4G/5G polymorphism was the most common PAI-1 genotype in this study.

New mathematical formula for differentiating thalassemia trait and iron deficiency anemia in thalassemia prevalent area: a study in healthy school-age children.

Iron deficiency anemia (IDA) and thalassemias are common diseases especially in the Mediterranean, Middle East and Asian regions. Both conditions show the same clinical findings of hypochromic and microcytic red blood cells. Although previous studies have devised mathematical formulae to differentiate between these two conditions, the prevalence of alpha- and beta-thalassemias among the affected populations may undermine the accuracy of these formulae. This study generated a new formula that was able to differentiate IDA and thalassemia traits and to determine the incidence rates of IDA and thalassemia traits. A total of 345 healthy Thai children with a mean age (+/- SD) of 11.3 (+/- 1.7) years were enrolled. Complete blood count, iron status, hemoglobin typing and DNA for alpha-1 thalassemia identification were investigated. Discriminant analysis was used to create a new mathematical formula containing significant variables to differentiate between IDA and thalassemia traits. The new formula of (1.5 Hb-0.05 MCV >14) had a receiver operator characteristic curve of 0.92 in differentiating thalassemia traits from IDA, with sensitivity and specificity of 84.6 and 87.5%, respectively. The incidence of IDA and thalassemia traits in the study group was 12% and 32%, respectively. This formula should be useful as a screening tool to differentiate between these two conditions.

One-step amplification refractory mutation system-PCR/high-resolution melting curve assay for carrier detection of red blood cell membranopathy caused by common SPTB mutations.

INTRODUCTION: Hereditary pyropoikilocytosis (HPP) is the most common cause of non-thalassemic severe inherited hemolytic anemia in Thai population. Up to 90% of affected patients harbor biallelic mutations of SPTB Providence (SPTB c.6055T>C), SPTB Buffalo (SPTB c.6074T>G), and SPTB Chiang Mai (SPTB c.6224A>G). This study aimed to develop a simple assay for mass screening of the three common SPTB mutations and to study their carrier frequencies in a healthy Thai population. METHODS: We combined multiplex amplification refractory mutation system-PCR (ARMS-PCR) and high-resolution melting (HRM) curve analysis to create a one-step single-tube assay. The primers were designed to generate products with different melting temperatures in the presence of 6055C, 6074G, and 6224G. Internal control primers were added for quality control. Residual samples from blood donors and healthy adolescents were collected and tested for the three common SPTB mutations using the newly developed assay. RESULTS: Optimized multiplex ARMS-PCR/HRM curve assay yielded well-separated melt curves to detect the three SPTB mutations with 4-h turnaround time. The assay was validated in screening of 2261 non-repetitive blood donors and 89 adolescents, in which 10 (0.43%), 2 (0.09%), and 3 (0.13%) individuals were identified as carriers of SPTB Providence, SPTB Buffalo, and SPTB Chiang Mai, respectively. All mutated SPTB and 20 random wild-type samples were confirmed using Sanger sequencing with 100% accuracy. CONCLUSION: The novel ARMS-PCR/HRM curve assay is simple, accurate, and time-effective for mass screening of the common SPTB mutations. This can be employed to prevent HPP birth in a Thai population.

Multicenter Study of Diagnostic Tool for Patients with Hemophilia: From Bedside to Comprehensive Investigations.

Background: Hemophilia cannot be diagnosed in most laboratories of economically less-developed countries leading to high mortality and morbidity rates. Aim: A diagnostic tool was established ranging from bleeding assessment and a simple bedside test of mixing venous clotting time (VCT) to comprehensive DNA analysis for patients with hemophilia. Methods: Patients with known (n=80) and suspected hemophilia (n=14) were included. Their bleeding symptoms were initially evaluated using verified translated-Thai ISTH bleeding assessment tool. Then, blood samples were drawn using a two-syringe technique, 2 mL each was placed in three tubes, for the mixing VCT and citrate blood was kept for coagulogram and coagulation factor assay. Finally, DNA analysis was determined. Results: A total of 94 patients with hemophilia (A68, B26) defined as severe (A 57, B 17), moderate (A 7, B 5), and mild degrees (A 4, B 4) with the mean (SD) age of 14.0 (11.7) years and 24 normal controls aged 25.5 (4.5), were enrolled in the study. The mean (SD) bleeding score of patients with hemophilia was 13.5 (5.5), which did not significantly differ between patients with hemophilia A and B. The mixing venous clotting time offered the presumptive diagnosis of hemophilia A and B, which were subsequently confirmed by the prolonged APTT, low FVIII:C and FIX:C and mutations on the factor VIII and IX genes. Conclusion: A diagnostic tool for bleeding assessment, mixing venous clotting time, coagulogram, coagulation factor assay, and DNA analysis for patients with hemophilia has been established in the existing health-care system.

Inhibin-A levels between 14 and 20 weeks of gestation in Thai women.

AIM: To construct a normal value for inhibin-A concentrations at 14-20 weeks of gestation for a Thai population. MATERIAL AND METHODS: Inhibin-A concentrations from pregnant women without Down's syndrome at 14-20(+6) weeks of gestation were measured. Maternal serum inhibin-A levels were analyzed according to the gestational age. RESULTS: Serum specimens from 727 Thai women were analyzed. Inhibin-A levels decreased from 14 to 17(+4) weeks and then gradually rose thereafter, giving a U-shape pattern. CONCLUSION: The data of inhibin-A concentration at 14-20 weeks of gestation for normal Thai pregnant women fitted well with quadratic regression.