Molecular characteristics of hereditary red blood cell membrane disorders in Thailand: a multi-center registry.

Red blood cell (RBC) membrane disorders represent a significant category of hereditary hemolytic anemia; however, information from Southeast Asia is limited. We established a national registry aiming to characterize RBC membrane disorders and their molecular features in Thailand. A total of 100 patients (99 kindreds) diagnosed with RBC membrane disorders between 2011 and 2020 from seven university hospitals were enrolled. The most prevalent disorders observed were hereditary elliptocytosis (HE; n=33), hereditary pyropoikilocytosis (HPP; n=28), hereditary spherocytosis (HS; n=19), Southeast Asian ovalocytosis (SAO; n=10 of 9 kindreds), and two cases of homozygous SAO. The remaining cases were grouped as unclassified membrane disorder. Seventy-six patients (76%) were molecularly confirmed by PCR, direct DNA sequencing, or hi-throughput sequencing. The primary causative gene for HE and HPP was SPTB, accounting for 28 out of 29 studied alleles for HE and 56 of 56 studied alleles for HPP. In the case of HS, dominant sporadic mutations in the ANK1 gene (n=4) and SPTB gene (n=3) were identified as the underlying cause. Notably, the four most common variants causing HE and HPP were SPTB Providence (c.6055 T>C), SPTB Buffalo (c.6074 T>G), SPTB Chiang Mai (c.6224 A>G), and SPTB c.6171__82delins TGCCCAGCT. These recurrent SPTB mutations accounted for 79 out of 84 mutated SPTB alleles (94%). In summary, HE and hereditary HPP associated with recurrent SPTB mutations are the predominant types of RBC membrane disorders observed in Thailand. These findings have significant implications for the clinical management and future research of RBC membrane disorders in the region.

Molecular and haematological characterisation of haemolytic anaemia associated with biallelic KLF1 mutations: a case series.

AIMS: Krüppel-like factor 1 (KLF1) is an erythroid-specific transcription factor playing an important role in erythropoiesis and haemoglobin (Hb) switching. Biallelic KLF1 mutations can cause haemolytic anaemia with thalassaemia-like syndromes but are rarely reported. We explore the KLF1 mutations in Thai subjects with unexplainable haemolytic anaemia. METHODS: The study was done on 57 subjects presented with haemolytic anaemia and elevated Hb F without ß-thalassaemia diseases. Hb analysis was performed using capillary electrophoresis. Analyses of α-thalassaemia, ß-thalassaemia and KLF1 genes were performed using PCR-based methods and DNA sequencing. RESULTS: Thirteen subjects with compound heterozygous for a known and five new genetic KLF1 interactions were identified, including KLF1:c.519_525dupCGGCGCC/c.892G>C with class 3/2 (n=8), and each subject with new genetic interaction, including KLF1:c.-154C>T;643C>T/c.983G>A with class 3/2, KLF1:c.-154C>T;643C>T/c.809C>G with class 3/2, KLF1:c892G>C/c.983G>A with class 2/2, KLF1:c.892G>C/c.1001C>G with class 2/2 and KLF1:c.1001C>G/c.1003G>A with class 2/2. Most of them had anaemia with Hb levels ranging from 45 to 110 g/L, hypochromic microcytosis, aniso-poikilocytosis, increased Hb F levels (17.9%-47.4%), small amounts of Hb Bart's, regular blood transfusion, hyperbilirubinaemia, increased serum ferritin and nucleated red blood cell. CONCLUSIONS: Biallelic KLF1 mutations associated with anaemia may not be uncommon in Thailand. Characteristics of haemolytic anaemia, abnormal red cell morphology with nucleated red blood cells and elevated Hb F, and presenting small amounts of Hb Bart's without thalassaemia diseases are useful markers to further investigation of the KLF1 gene.

Thalassemia-related complications in pediatric, adolescent, and young adult patients with transfusion-dependent thalassemia: A multicenter study in Thailand.

INTRODUCTION: Management of transfusion-dependent thalassemia (TDT) can be challenging due to numerous potential disease-related complications and comorbidities in particular age groups. The objective of this study was to report thalassemia-related complications and risk factors in pediatric, adolescent, and young adult patients with TDT. METHODS: A multicenter web-based registry was conducted in patients with TDT aged 25 years and younger from eight university hospitals covering all parts of Thailand. Factors significantly associated with each complication were analyzed by logistic regression methods. RESULTS: Of 605 patients, 267 thalassemia-related complications were reported from 231 pediatric, adolescent, and young adult patients with TDT patients (38.2%). The most common complications were infections, followed by cholelithiasis and growth failure. Splenectomy and elevated pre-transfusion hemoglobin were statistically significant risk factors for infections (adjusted odds ratio [AOR] = 2.3, 95% confidence interval [CI]: 1.2-4.5, p-value = .01 and AOR = 1.5, 95% CI: 1.2-1.7, p-value < .005, respectively). There were two statistically significant risk factors conferred endocrinopathies, including older age (AOR = 1.06, 95% CI: 1.01-1.1, p-value = .01) and being male (AOR = 2.4, 95% CI: 1.4-4.0, p-value = .002). CONCLUSION: Nearly 40% of the patients in this cohort had thalassemia-related complications. Periodic surveillance and optimal care for respective complications may minimize comorbidities in pediatric, adolescent, and young adult patients with TDT.

The association of growth differentiation factor-15 levels and osteoporosis in patients with thalassemia.

BACKGROUND: Ineffective erythropoiesis (IE) is a significant risk factor for osteoporosis in individuals with thalassemia. Growth differentiation factor-15 (GDF15), a biomarker of IE, was found to be elevated in thalassemia patients. This study aimed to examine the association between GDF15 levels and osteoporosis in patients with thalassemia. METHODS: A cross-sectional study was conducted in 130 adult patients with thalassemia in Thailand. Bone mineral density (BMD) at the lumbar spine was evaluated by dual-energy X-ray absorptiometry (DXA), and with a Z-score of less than -2.0 SD was defined as osteoporosis. GDF-15 was measured using the enzyme-linked immunosorbent assay (ELISA). Logistic regression analysis was used to examine the associated factors with the development of osteoporosis. Receiver operator characteristic (ROC) curve analysis was used to estimate the threshold of GDF15 in predicting osteoporosis. RESULTS: Osteoporosis was detected in 55.4% (72/130) of the patients. Advanced age and high GDF15 levels were positively associated with osteoporosis, while an increased hemoglobin level was negatively associated with osteoporosis in patients with thalassemia. In this study, the GDF15 level's ROC demonstrated a good performance in predicting osteoporosis (AUC=0.77). CONCLUSIONS: The prevalence of osteoporosis is high among adult thalassemia patients. Age and high GDF15 levels were significantly associated with osteoporosis in this study. A higher hemoglobin level is associated with a lower risk of osteoporosis. This study suggest that GDF15 could be used as a predictive biomarker for osteoporosis in patients with thalassemia. Adequate red blood cell transfusions and suppression of GDF15 function may be beneficial in preventing osteoporosis.

Clinical features and risk factors of renal dysfunctions in thalassemic patients.

INTRODUCTION: Chronic anemia, iron overload, and iron chelation therapy are the main contributing factors for renal complications in thalassemia, e.g., nephrolithiasis, glomerular disease, and renal tubular dysfunction. The prevalence and associated factors for developing renal dysfunctions in Thai patients with thalassemia, however, remained limited. This study aimed to determine the prevalence and risk factors of renal dysfunctions in patients with thalassemia. METHODS: A cross-sectional study was conducted on adult patients with thalassemia disease at Srinagarind Hospital, Khon Kaen University, Thailand. All patients were evaluated for complete blood count, blood chemistry, urinalysis, and urine biochemistry. Renal tubular dysfunction was defined as existing in at least one of the following parameters including; proteinuria, hypercalciuria, hypouricemia with uricosuria, or hypophosphatemia with phosphaturia. Logistic regression analysis was used to identify associated factors for renal dysfunctions. RESULTS: Of 105 patients, renal tubular dysfunction was found in 60 patients (57.1%). In multivariate analysis of the clinical risk factors for renal tubular dysfunction in thalassemia patients, age per 10 year increase (adjusted odds ratio [AOR] = 1.4, 95% CI: 1.0-2.0, p value 0.01) and Hb E/beta-thalassemia (AOR = 3.6, 95% CI: 1.3-10.3, p value 0.01) were statistically proven to be associated with renal tubular dysfunction. Hyperuricosuria was a significantly associated factor for microhematuria. (AOR = 2.9, 95% CI: 1.1-8.0, p value 0.03). CONCLUSIONS: Renal dysfunctions are prevalent in thalassemia patients, with older age and Hb E/beta-thalassemia genotype as significant risk factors for renal tubular dysfunction. Hyperuricosuria is a risk factor for microhematuria. Renal dysfunctions should be recognized and monitored in aging patients with Hb E/beta-thalassemia.

FLT3 Gene Mutations in Acute Myeloid Leukemia Patients in Northeast Thailand.

BACKGROUND Mutations in the FLT3 gene are associated with acute myeloid leukemia (AML). FLT3 mutations have been identified in approximately 30% of de novo AML patients, particularly those with typical karyotype and inferior prognosis. Therefore, we examined the frequencies of an internal tandem duplication (ITD) and missense mutations of the FLT3 gene and their associated clinical characteristics in patients with AML in northeast Thailand. MATERIAL AND METHODS The leftover bone marrow and/or peripheral blood specimens of 65 newly diagnosed AML patients recruited from Srinagarind Hospital, Khon Kaen University, northeast Thailand, between January 2020 and May 2021 were included in this study. FLT3-ITD and FLT3- tyrosine kinase domain (TKD) were amplified using PCR-related techniques. RESULTS The frequencies of FLT3-ITD and TKD were found to be 16.9% (11/65) and 10.8% (7/65), respectively. One patient had ITD and TKD mutations. The white blood cell count and peripheral blast percentage of FLT3-ITD-positive patients were statistically significantly higher than those of the FLT3-wild type patients, while the peripheral blast percentage of FLT3-TKD-positive patients was significantly lower. No other clinical characteristics among FLT3-positive and FLT3-wild-type patients were observed. DNA sequencing identified 4 FLT3-TKD mutations. The c.2504A>T; Asp835Val and c.2503G>C; Asp835His mutations were predicted as pathogenic mutations while the 2 novel mutations, c.2508C>A; Ile836= and c.2508C>G; Ile836Met were predicted as neutral mutations. CONCLUSIONS This study showed for the first time that FLT3-TKD mutation is common among northeast Thai AML patients. The data should prove useful for selecting efficacious targeted treatment plans for the patients.

Phosphatidylserine-exposed red blood cells and ineffective erythropoiesis biomarkers in patients with thalassemia.

OBJECTIVE: The degree of ineffective erythropoiesis is known to be associated with clinical severity among individuals with thalassemia. The association of ineffective erythropoiesis biomarker levels with different thalassemia genotypes, however, remains limited. The aim of this study was to explore the level of phosphatidylserine-exposed red blood cells (PS-exposed RBCs) and ineffective erythropoiesis biomarkers (growth-differentiation factor-15 and soluble transferrin receptors) in patients with different genotypes. METHODS: A cross-sectional study was conducted on 139 patients of age 18 years and above with different genotypes at Srinagarind Hospital, Khon Kaen University, Thailand. The levels of PS-exposed RBCs were determined using flow cytometry. Measurements of growth-differentiation factor-15 (GDF-15) and soluble transferrin receptors (sTfR) were evaluated by the ELISA method. RESULTS: The PS-exposed RBCs levels were found to be significantly higher in splenectomized beta-thalassemia patients. Patients with beta-thalassemia had the highest GDF-15 levels, followed by patients with non-deletional alpha-thalassemia. Patients with non-deletional alpha-thalassemia showed elevated hemoglobin levels and reduced GDF-15 levels after splenectomy. Patients with beta-thalassemia and non-deletional alpha-thalassemia had the highest levels of PS-exposed RBCs and ineffective erythropoiesis biomarkers, which correlated with the clinical severity of thalassemia. CONCLUSIONS: The levels of ineffective erythropoiesis biomarkers were different across thalassemia genotypes. Splenectomy may improve clinical symptoms of patients with non-deletional alpha thalassemia but not of patients with beta-thalassemia. These findings demonstrate differences in the degree of ineffective erythropoiesis in thalassemia, which emphasizes the need for different treatment approaches among patients with different thalassemia genotypes.

Red blood cell alloimmunization and other transfusion-related complications in patients with transfusion-dependent thalassemia: A multi-center study in Thailand.

BACKGROUND: Thalassemia is a common genetic disease in Southeast Asia. Red blood cell (RBC) transfusion is an essential treatment for severe forms of thalassemia. We performed a study to demonstrate RBC alloimmunization and other transfusion-related complications in patients with transfusion-dependent thalassemia (TDT). STUDY DESIGN AND METHODS: A multi-center web-based registry of TDT was conducted in eight medical centers across Thailand. Thalassemia information, transfusion therapy, and transfusion-related complications were collected. Factors associated with each complication were demonstrated using the logistic regression analysis. RESULTS: Of 1000 patients recruited for the study, 449 were males (44.9%). The mean age was 23.9 ± 15.4 years. The majority of patients, 738 (73.8%) had hemoglobin E/beta-thalassemia. In the study, 421 transfusion-related complications were reported from 357 patients (35.7%). Alloimmunization was the most common complication which was found in 156 patients (15.6%) with 284 positive antibody tests. The most frequent antibodies against RBC were anti-E (80/284, 28.2%) followed by anti-Mia (45/284, 15.8%) and anti-c (32/284, 11.3%). Age ≥3 years at initial blood transfusion, splenomegaly, higher frequencies, and volumes of transfusion were significant factors associated with alloimmunization. None of the patients had to terminate blood transfusion due to multiple alloantibodies. Other commonly seen complications were allergic reactions (130, 13.0%), autoimmune hemolytic anemia (70, 7.0%) and febrile non-hemolytic transfusion reaction (54, 5.4%). CONCLUSIONS: Transfusion-related complications, especially alloimmunization, were common among Thai patients with TDT. Extended RBC antigen-matching for the Rh system and Mia should be implemented to prevent the development of alloantibodies in multi-transfused patients.

Clinical factors predictive of recurrent febrile neutropenia in adult patients with acute leukemia.

Febrile neutropenia (FN) is considered an oncologic emergency in acute leukemia. There were 250 FN events in 124 hospitalized patients with hematologic malignancy. These data imply that two FN events may occur per patient, yet data on the prevalence, risk factors, and outcomes of recurrent FN in adult patients with leukemia are limited. A retrospective cohort study was conducted that enrolled adult patients diagnosed with acute leukemia who developed FN. The eligible patients were categorized as with or without recurrent FN. A stepwise, multivariate logistic regression analysis was performed to identify predictors of recurrent FN. A total of 203 patients met the study criteria; of these, 46 (22.66%) had recurrent FN, and this group had a median of three recurrent FN emergencies. After adjusted, three independent factors remained in the final model including ALL, FN at admission, and treatment with idarubicin (3 days) and cytarabine (7 days). The three factors were positively associated with recurrent FN with adjusted odds ratios of 6.253, 4.068, and 10.757, respectively. No significant differences were found between the two groups in terms of other sources of infection, other pathogens, ICU stay, hospital stay, and mortality. ALL and FN at admission and treatment with idarubicin (3 days) and cytarabine (7 days) were associated with recurrent FN in acute leukemia patients with FN. Clinical outcomes for patients with or without recurrent FN were mostly comparable; however, due to its small sample size, further studies are required to confirm the results of this study.

Cognitive Impairment in Thalassemia and Associated Factors.

INTRODUCTION: Patients with thalassemia increase the risk of developing cognitive impairment. Chronic anemia, oxidative stress from excess iron, and hypercoagulable state were related to this condition. The study regarding its prevalence and the associated factor in Southeast Asia is limited. Therefore, the study aimed to investigate the prevalence of cognitive impairment and associated factors. METHODS: This was a cross-sectional study of thalassemic patients aged 18 years or more at the Hematology Clinic of Srinagarind Hospital, Khon Kaen University, Thailand, from January to May 2021. The Thai version of the Mini-Cog test was used to determine the presence of cognitive impairment. The clinical and laboratory parameters indicated as potential risk factors for dementia were evaluated in all patients. A stepwise logistic regression analysis was used to determine the associated risk factors for cognitive impairment. RESULTS: Among 150 patients, cognitive impairment was found in 40 patients (26.7%). Age per 10-year increase (adjusted odds ratio [AOR] of 1.6), no iron chelation therapy (AOR of 9.8), current smoking (AOR of 5.0), hemoglobin (Hb) (AOR of 0.63), and ferritin (AOR of 1.0001) were independent factors associated with cognitive impairment. CONCLUSIONS: The prevalence of cognitive impairment was high among thalassemic patients. Increasing age, low Hb, iron overload, and current smoking were significant associated factors with cognitive impairment. Screening for dementia in these patients is recommended, particularly in patients with high-risk factors.

Prevalence of peripheral eosinophilia and clinical associations in systemic sclerosis patients.

BACKGROUND: Peripheral eosinophilia (eosinophilia) is observed among systemic sclerosis (SSc) patients. The association between eosinophilia and SSc in terms of pathogenesis remains uncertain. We aimed to determine the prevalence of the clinical, serological, and cytokine associations with eosinophilia in SSc patients. METHODS: A cross-sectional study was conducted among adult SSc patients. We excluded patients having overlap syndrome and other conditions that cause eosinophilia. Investigations into the etiology of eosinophilia were performed on the same study date, including clinical parameters, blood tests for tissue parasites, IgE, interleukin-5, and transforming growth factor-beta. Eosinophilia is defined when the total eosinophil count is > 500 cells/mm3. RESULTS: According to the sample size calculation, 185 patients were enrolled, of whom 57 (30.8%) had eosinophilia. The causes of eosinophilia were based on laboratory indicators without clinical symptoms in 21 cases (10 had a parasitic infection, 9 adrenal insufficiency, and 2 tuberculosis). After excluding suspected causes of eosinophilia, the total prevalence of eosinophilia was 21.9% (95%CI 15.9-29.1). Most of patients (164 cases; 70.6%) had diffuse cutaneous SSc. According to the logistic regression analysis, the factors associated with eosinophilia were being male (OR 3.46), duration of disease increasing every year (OR 1.16), and Raynaud's phenomenon (OR 0.27), while SSc subset, serology (i.e., anti-topoisomerase I, anti-neutrophilic cytoplasmic antibody), inflammatory markers, and cytokine levels were not. CONCLUSIONS: Eosinophilia of unknown causes was detected in 1 in 5 SSc patients, particularly in males with no vasculopathy. Eosinophilia has a nonspecific role vis-à-vis clinical relevance in SSc.

Acalypha indica -induced transient glucose-6-phosphate dehydrogenase deficiency with acute haemolysis.

Acalypha indica is a tropical herb found in Asia. The entire plant, especially the leaves, is used in herbal medicine for several therapeutic purposes. Acute intravascular haemolysis and methaemoglobinaemia have been reported in patients who consume this herb. We present a case of a previously healthy middle-aged man who ingested boiled leaves of A. indica The patient developed clinical symptoms and signs of intravascular haemolysis 7 days after ingestion. Peripheral blood smear showed typical findings of glucose-6-phosphate dehydrogenase (G6PD) deficiency with acute haemolysis. The G6PD activity was low during active haemolysis. The G6PD level, however, returned to normal after 4 months of follow-up. The patient was further tested for common G6PD gene mutations in Southeast Asia and was negative. Ingestion of A. indica may induce transient G6PD deficiency, which in this patient led to acute haemolysis and methaemoglobinaemia.

The serum ferritin levels and liver iron concentrations in patients with alpha-thalassemia: is there a good correlation?

INTRODUCTION: Liver iron overload is common in patients with thalassemia. In patients with beta-thalassemia, the correlation between serum ferritin and liver iron concentration is well established. The correlation between serum ferritin levels and liver iron concentrations in patients with alpha-thalassemia remains limited. METHODS: This is a cross-sectional study in patients with alpha-thalassemia aged ≥ 18 years old at Srinagarind Hospital, Khon Kaen University, Thailand. Liver iron concentration (LIC) was evaluated by the MRI-T2* technique. Linear logistic regression analysis was used to determine the correlation between serum ferritin levels and liver iron concentrations. RESULTS: One hundred and thirty-one of the MRI-T2* measurements from 65 patients with alpha-thalassemia were evaluated. Patients with non-deletional alpha-thalassemia had higher LIC compared to patients with deletional alpha-thalassemia. The serum ferritin levels were relatively low at the same levels of LIC in patients with non-deletional alpha-thalassemia compared to deletional alpha-thalassemia. CONCLUSIONS: The correlation of serum ferritin levels and LIC was modest and different among alpha-thalassemia genotypes. A different serum ferritin threshold is needed to guide iron chelation therapy in patients with alpha-thalassemia. Evaluation of liver iron concentration is necessary for patients with alpha-thalassemia, especially in patients with non-deletional alpha-thalassemia.

Clinical factors predictive of mortality in acute leukemia patients with febrile neutropenia.

BACKGROUND: Acute leukemia is mainly treated with chemotherapy leading to febrile neutropenia (FN). There is limited data on clinical factors predictive of mortality in adults with acute leukemia and FN. METHODS: This was a retrospective cohort study and enrolled adult patients, diagnosed as acute leukemia, and developed FN. The eligible patients were admitted and followed up with mortality as the primary outcome. A stepwise, multivariate logistic regression analysis was used to find predictors for mortality. RESULTS: There were 203 patients met the study criteria. Of those, 14 patients died (6.89%). AML was the most common type of acute leukemia with FN (64.04%). There were five remaining factors in the final model: AML, FN at admission, prolong broad spectrum antibiotics, lower respiratory tract infection, and Aspergillosis. Only lower respiratory tract infection was significant with adjusted odds ratio of 7.794 (95% CI of 1.549, 39.212). The Hosmer-Lemeshow Chi square was 2.74 (p value 0.907). The lower respiratory tract infection group had higher proportions of Gram negative and fungus than the non-lower respiratory tract infection group; specifically E. coli (p 0.003), and Aspergillus (P < 0.001). CONCLUSIONS: There were two independent predictors of mortality in acute leukemia patients with FN: septic shock and lower respiratory tract infection regardless of leukemia type or pathogen. E. coli and Aspergillus were more common in those with lower respiratory tract infection than those without. No specific pathogens were found in cases of septic shock.

The Trabecular Bone Score as a Predictor for Thalassemia-Induced Vertebral Fractures in Northeastern Thailand.

INTRODUCTION: Thalassemia bone disease is one of the disease-related complications in patients with thalassemia. Prevalence of fractures and the role of a trabecular bone score (TBS) as a predictive factor for fractures were evaluated in patients with thalassemia. METHODS: A cross-sectional study was conducted in patients with thalassemia aged ≥18 years at Srinagarind Hospital, Khon Kaen University, Thailand. A lateral thoracolumbar radiograph and bone mineral density (BMD) at the lumbar spine and hip, as well as the TBS measured by dual-energy X-ray absorptiometry (DXA), were evaluated in all patients. RESULTS: Among 86 patients, 14 patients were found to have radiographic vertebral fracture yielding a prevalence of 16.3%. All patients who had fractures were ß-thalassemia/Hb E. Combined low BMD and TBS at lumbar spines and a presence of endocrinopathies were significantly associated with vertebral fractures. CONCLUSIONS: The prevalence of vertebral fractures in patients with thalassemia was not uncommon. A combined low BMD and TBS and a presence of endocrinopathies were associated with vertebral fractures. These findings suggested that BMD testing and TBS measurement have a clinical implication as a screening tool for evaluating the risk of vertebral fractures in thalassemic patients, particularly in ß-thalassemia/Hb E who have endocrinopathies.

Massive pleural effusion due to IgG-Kappa subtype multiple myeloma: A case report.

Multiple myeloma (MM) is a hematologic malignancy of plasma cell origin. Incidence of pleural effusion in multiple myeloma patients is approximately 6%. Myelomatous pleural effusions (MPE) are rare and occur in less than 1% of all MM cases. MPE is associated with advanced diseases, decreased survival time, and poor treatment response. In our case report, we describe a 59-year old man who presented with MPE at the initial diagnosis of MM. A diagnosis of MPE was reach through pleural fluid cytology and pleural tissue histology. The MPE had good response to initial dexamethasone without local therapy.