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.

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.

Three-Decade Successive Establishment of Care for Women/Girls from Families with Haemophilia.

Objective: The study aimed to report a 3-decade successive establishment of care for women/girls from families with haemophilia. Methods: A retrospective analysis was conducted on 462 women/girls from 243 families from 1987 to 2021. Results: Combining phenotypic analysis of coagulation factor and genotypic analysis of either linkage analysis or mutation detection confirmed the status of all obligate haemophilia carriers (A118, B19). For potential carrier, 159 proven carriers (A130, B29) and 146 noncarrier status (A126, B20) were diagnosed except 20 potential carriers (A16, B4). Only 54 prenatal diagnoses were requested resulting in normal males (n = 21), males with haemophilia A (n = 12) and females with either normal or carrier status (n = 21). Additionally, 40 women/girls with haemophilia carrier received a diagnosis of severe haemophilia A with Turner's syndrome (n = 2) and mild haemophilia (A31, B7). The skewed X-chromosome inactivation of the nonmutant factor VIII/IX carrying X-chromosome of 8% (2/25) was found in mild haemophilia. Factor concentrate and desmopressin are prescribed for these affected women/girls. The response of women/girls with either haemophilia carrier or haemophilia was amazement with their religious beliefs and cultural acceptance. Conclusion: Appropriate care for women/girls from families with haemophilia concerning diagnosis and management of haemophilia and carrier has been successively established.

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.

Prominent Mutation of Intron 22 Inversion in Sporadic Hemophilia: Is It Worth the Antenatal Screening?

Background: Adequate replacement for patients with hemophilia is costly, especially in countries with limited resources. Objective: Factor VIII gene mutations among Thai patients with hemophilia A were analyzed for the most common mutation. The cost-effectiveness of finding one female without family history of hemophilia possessing the most common factor VIII mutation was compared with the cost of treating one patient with hemophilia. Methods: In all, 109 unrelated patients with hemophilia A, defined as sporadic cases (n=58) and hereditary cases (n=51), were enrolled for genotypic analysis. Results: Intron 22 inversion was prominently found in 34 sporadic (58.6%) and 27 hereditary (51.9%) cases. The screening for intron 22 inversion among females without family history of hemophilia at antenatal care has been optionally suggested. A female with a positive result will undergo further prenatal diagnosis of hemophilia in her male offspring. On the contrary, a female with a negative test result remains at risk to have a hemophiliac son caused by other factor VIII gene mutations not included in the screening but the risk is not as high as intron 22 inversion. Although the screening of factor VIII mutation among females without family history of hemophilia is against the current practice, it has been initiated due to the inadequate treatment provided to patients with hemophilia in countries with limited resources. The study calculated approximately one female with intron 22 inversion would exist among 17,064 females without family history of hemophilia. The cost of screening (194,870 USD) was much less than that of treating one patient with hemophilia from birth to 40 years of age by the current regimen (378,000 USD). Conclusion: Implementing antenatal screening of intron 22 inversion among females without family history of hemophilia is optionally suggested, especially in economically less-developed countries with inadequate treatment service for patients with hemophilia.

A Simple and Applicable Method for Human Platelet Lysate Preparation Using Citrate Blood.

OBJECTIVES: To determine and compare the platelet growth factors in human platelet lysate (HPL) prepared from citrated whole blood, with final centrifugations at 4oC and 25oC. METHODS: We collected specimens of citrated whole blood from 27 healthy volunteers. The platelet-rich plasma (PRP) was separated to prepare the HPL, which was further divided into 2 portions for the final centrifugation, at 4oC and 25oC, respectively. Platelet growth factors were measured and compared between the 2 groups. RESULTS: All platelet growth factors were higher than those in PRP prepared from citrated whole blood. Moreover, the final centrifugation at 25oC resulted in noninferiority of platelet-growth-factor level. CONCLUSION: This study provided a simple method for small-volume of HPL preparation using only 10-15 mL of citrated whole blood. Further, the entire process of centrifugation can be performed at room temperature of 25oC, which is more applicable than lower temperatures for other laboratories.

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.

The use of immature platelet fraction to predict time to platelet recovery in patients with dengue infection.

Background: In dengue infection, knowing time to platelet recovery is essential for optimal management.Aims: To determine a predictor for platelet recovery in patients with dengue infection.Methods: Platelet count and immature platelet fraction (IPF) from daily blood samples of patients with dengue infection during hospitalisation and 1-4 weeks after discharge were retrospectively analysed. The levels of patients' IPF were compared with normal controls recruited from healthy children with normal platelet counts.Results: A total of 244 EDTA blood samples were collected daily from 64 patients (45 males) with dengue infection (36 dengue fever, 28 dengue haemorrhagic fever) during hospitalisation and after discharge from the hospital. They did not receive any platelet concentrate transfusion. The median IPF among normal children was 3.6% with a 95 percentile of 9.9%. In dengue patients, an IPF of ≥10.0% after defervescence was associated with a subsequent platelet count of ≥60 × 109/L within 72 hours.Conclusion: In patients with dengue infection, IPF ≥10.0% after defervescence is a predictor of subsequent platelet recovery to a haemostatic level ≥60 × 109/L within 72 hours.

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.

Coagulant activity of recombinant human factor VII produced by lentiviral human F7 gene transfer in immortalized hepatocyte-like cell line.

Human mesenchymal stem cells (hMSCs) have the potential to differentiate into hepatocyte-like cells, indicating that these cells may be the new target cell of interest to produce biopharmaceuticals. Our group recently established a hMSC-derived immortalized hepatocyte-like cell line (imHC) that demonstrates several liver-specific phenotypes. However, the ability of imHC to produce coagulation factors has not been characterized. Here, we examined the potential for imHC as a source of coagulation protein production by investigating the ability of imHC to produce human factor VII (FVII) using a lentiviral transduction system. Our results showed that imHC secreted a low amount of FVII (~22 ng/mL) into culture supernatant. Moreover, FVII from the transduced imHC (0.11 ± 0.005 IU/mL) demonstrated a similar coagulant activity compared with FVII from transduced HEK293T cells (0.12 ± 0.004 IU/mL) as determined by chromogenic assay. We demonstrate for the first time, to the best of our knowledge, that imHC produced FVII, albeit at a low level, indicating the unique characteristic of hepatocytes. Our study suggests the possibility of using imHC for the production of coagulation proteins.

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.

Generation of a human induced pluripotent stem cell line (MUi010-A) from skin fibroblast of patient carrying a c.2104C>T mutation in MYH9 gene.

Mutations in MYH9 gene is one of the major causes of inherited thrombocytopenia resulted from nonfunctional myosin-9 protein. We have generated a human induced pluripotent stem cell line MUi010-A from skin fibroblasts of a patient who had a point mutation c.2104C>T (p.R702C) in the exon 16 of MYH9 gene using a non-integrative reprogramming method. The MUi010-A exhibited embryonic stem cell-like characteristics with consistent pluripotent markers expression, was capable of all three embryonic germ layers differentiation, and had a normal karyotype.

Abnormal red blood cell indices increase the risk of arterial ischemic stroke in children.

A high red cell distribution width (RDW) and low hemoglobin level increase the risk of arterial ischemic stroke (AIS), mostly in adults. The mechanisms related to AIS remain unknown. A total of 233 subjects (90 patients and 143 healthy controls [HC]) were enrolled. The mean(SD) age in patients and HC was 9.5(3.8) and 11.4(1.8) years, respectively. We found increased odds ratios (ORs) for large vessel and small vessel subtypes in patients without underlying diseases with a mean corpuscular volume (MCV) <80 fL (OR: 5.4, 95%CI 1.8-16.3 and 2.8, 95%CI 1.2-7.2), mean corpuscular hemoglobin levels <27 pg (OR: 2.9, 95%CI 1.0-6.7 and 2.6, 95%CI 1.0-6.7), and RDW >15% (OR: 5.5, 95%CI 1.3-24.5 and 2.7, 95%CI 1.0-7.3). RBC indices showed significant correlations with TM levels. Therefore, low MCV and MCH levels, and a high RDW were risk factors for AIS and associated with TM levels in this population.