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Background: Hemoglobinopathies are among the most prevalent inherited disorders globally, with carrier prevalence varying significantly across regions. In Saudi Arabia, high rates of consanguineous marriages amplify the risk of these disorders., Aim: This study aims to assess the burden of hemoglobinopathies by evaluating the prevalence and regional distribution of beta-hemoglobin variants, including rare variants, among couples participating in the national premarital screening program., Methods: Data were collected from the premarital genetic screening program and entered into the SEHA platform, covering the 13 administrative regions of Saudi Arabia. Blood samples underwent various screening tests for infectious and genetic diseases. Hemoglobin electrophoresis samples were analyzed using capillary electrophoresis, High-Performance Liquid Chromatography (HPLC), or a combination of both methods., Results: From 2011 to 2018, 1,871,184 individuals were included in the study, with 49.8% male and 50.2% female. The average age was 30.2 years. Hemoglobin S (HbS) was identified in 88,431 individuals (4.7% of the tested population and 78.5% of abnormal screening results), primarily as a sickle cell trait. β-thalassemia was the second most common disorder, identified in 22,420 individuals (1.2% of the population and 19.9% of hemoglobin disorders). HbC and HbD were each detected in 0.04% of cases, while HbO-Arab was identified in 0.007% and HbG in 0.006%. Hemoglobin E and hemoglobin Lepore were found to be extremely rare., Conclusion: The study demonstrates regional variation in the prevalence of hemoglobin genetic variants in Saudi Arabia. To effectively mitigate this risk, it is imperative to strengthen public education and awareness, particularly focusing on genetic screening and counseling., (© 2024. The Author(s).)

Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype-phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and β-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a β-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with β 0 -thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.

Context.—: Recently, new technologies, such as next-generation sequencing and third-generation sequencing, have been used in carrier screening of thalassemia. However, there is no direct comparison between the 2 methods in carrier screening of thalassemia., Objective.—: To compare the clinical performance of third-generation sequencing with next-generation sequencing in carrier screening of thalassemia., Design.—: Next-generation sequencing and third-generation sequencing were simultaneously conducted for 1122 individuals in Hainan Province., Results.—: Among 1122 genetic results, 1105 (98.48%) were concordant and 17 (1.52%) were discordant between the 2 methods. Among the 17 discordant results, 4 were common thalassemia variants, 9 were rare thalassemia variants, and 4 were variations with unknown pathogenicity. Sanger sequencing and polymerase chain reaction for discordant samples confirmed all the results of third-generation sequencing. Among the 685 individuals with common and rare thalassemia variants detected by third-generation sequencing, 512 (74.74%) were carriers of α-thalassemia, 110 (16.06%) were carriers of β-thalassemia, and 63 (9.20%) had coinheritance of α-thalassemia and β-thalassemia. Three thalassemia variants were reported for the first time in Hainan Province, including -THAI, -α2.4, and ααααanti3.7. Eleven variants with potential pathogenicity were identified in 36 patients with positive hemoglobin test results. Among 52 individuals with negative hemoglobin test results, 17 were identified with thalassemia variants. In total, third-generation sequencing and next-generation sequencing correctly detected 763 and 746 individuals with variants, respectively. Third-generation sequencing yielded a 2.28% (17 of 746) increment compared with next-generation sequencing., Conclusions.—: Third-generation sequencing was demonstrated to be a more accurate and reliable approach in carrier screening of thalassemia compared with next-generation sequencing., Competing Interests: Liu, Mao, R. Xu, and Chen are employees of Berry Genomics Corporation. The authors have no other relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for β-thalassemia major in children. However, it often induces graft-versus-host-disease (GVHD), which is associated with complications. In the present study, we used cyclophosphamide (Cy) to treat a thalassemia patient post-HSCT to reduce the adverse effects of GVHD. We monitored the numbers and phenotype of granulocytes. In this case study, an 11-year-old female patient, diagnosed with β-thalassemia major (Pesaro class II), was treated with Cy before and after HSCT with mobilized CD34+ cells. Both the relative and absolute granulocyte counts, as well as CD33+CD11b+ cell counts, increased significantly after HSCT until day 56. However, they suddenly began to decrease after day 56, accompanied by severe diarrhea, skin rash, and a decrease in bilirubin levels compared to day -12. Furthermore, compared to day -12, IL-22 levels increased until day 56, and then decreased, while IDO levels continued to rise after day 56. Our data suggest the potential use of IL-22 and IDO as biomarkers for GVHD assessment. It also indicates that Cy promotes HSCT reconstitution by increasing CD33+CD11b+ cells, which may play a crucial role in reducing GVHD risks. However, further studies are needed to elucidate the mechanism behind GVHD recurrence.

Aim: To develop a non-invasive prenatal test for beta-hemoglobinopathies based on analyzing maternal plasma by using next generation sequencing., Methods: We applied next generation sequencing (NGS) of maternal plasma to the non-invasive prenatal testing (NIPT) of autosomal recessive diseases, sickle cell disease and beta-thalassemia. Using the Illumina MiSeq, we sequenced plasma libraries obtained via a Twist Bioscience probe capture panel covering 4 Kb of chromosome 11, including the beta-globin (HBB) gene and >450 genomic single-nucleotide polymorphisms (SNPs) used to estimate the fetal fraction (FF). The FF is estimated by counting paternally transmitted allelic sequence reads present in the plasma but absent in the mother. We inferred fetal beta-globin genotypes by comparing the observed mutation (Mut) and reference (Ref) read ratios to those expected for the three possible fetal genotypes (Mut/Mut; Mut/Ref; Ref/Ref), based on the FF., Results: We bioinformatically enriched the FF by excluding reads over a specified length via in-silico size selection (ISS), favoring the shorter fetal reads, which increased fetal genotype prediction accuracy. Finally, we determined the parental HBB haplotypes, which allowed us to use the read ratios observed at linked SNPs to help predict the fetal genotype at the mutation site(s). We determined HBB haplotypes via Oxford Nanopore MinION sequencing of a 2.2 kb amplicon and aligned these sequences using Soft Genetics' NextGENe LR software., Conclusion: The combined use of ISS and HBB haplotypes enabled us to correctly predict fetal genotypes in cases where the prediction based on variant read ratios alone was incorrect.

The blood counts of α thalassemia carriers (α-thal) are similar to those of β thalassemia carriers, except for Hemoglobin A 2 (Hb A 2 ), which is not elevated. The objective of this study was to determine whether mathematical formulas are effective for detecting suspected α-thal. The data were obtained from the database of the prevention program for detecting couples at risk for having a child with hemoglobinopathy. Red Blood Cells (RBC) indices were analyzed using mathematical formulas, and the sensitivity and negative predictive value (NPV) were calculated. Among 1334 blood counts suspected of α-thal analyzed, only the Shine and Lal and the Support Vector Machine formulas revealed high sensitivity and NPV. Sensitivity was 85.54 and 99.33%, and NPV was 98.93 and 99.93%, respectively. Molecular defects were found in 291, and 81 had normal α genes. Molecular analysis was not performed in 962 of the samples. Based on these results, mathematical formulas incorporating one of these reliable formulas for detecting suspected α or β thalassemia carriers in the program of the automatic analyzers can flag these results, increase the awareness of the primary physicians about the carrier risk, and send an alert with a recommendation for further testing.

Hematopoietic stem cell transplantation is a curative treatment for many malignant and nonmalignant diseases in children and adults. It is performed with peripheral blood stem cells, bone marrow, and umbilical cord blood. Anaphylaxis may occur during hematopoietic stem cell transplantation, similar to that shown with blood transfusions. In children, although a few cases of anaphylaxis have been reported with cord blood transplantation, no cases of anaphylaxis have been reported with other hematopoietic stem cell transplantations. In this case report, we present the cases of 2 children, one diagnosed with thalassemia major and the other with aplastic anemia, both of whom developed anaphylaxis associated with bone marrow transplantation products cryopreserved with dimethyl sulfoxide and hydroxyethyl starch. Hematopoietic stem cell transplantation-induced anaphylaxis could be associated with cryoprotective agents, especially dimethyl sulfoxide, and alloantigens. In both anaphy-lactic reactions, dimethyl sulfoxide was thought to be the trigger, but it could not be excluded that it was related to stem cell components, plasma, or hydroxyethyl starch.

β-thalassemia is one of the most common monogenic disorders in areas of the tropics and subtropics, which represents a major familial and social burden to local people. The elevated Hb A 2 level, generally specified as greater than 3.5 %, is commonly used as a high efficiency index for screening of β-thalassemia carriers. However, mutations in other genes such as GATA1 and KLF1, could also result in increased Hb A 2 level. In this study, we identified two novel variants in the SUPT5H gene: a frameshift mutation (SUPT5H: c.3032_3033delTG, p.M1011Mfs*9) and a nonsense mutation (SUPT5H: c.397C > T, p.Arg133*) in two Chinese individuals. Utilizing a combination of phenotype analysis, bioinformatics analysis, and functional analysis, we deduced that these two variants modified the SUPT5H protein's structure, thereby impacting its function and consequently leading to the heightened Hb A 2 level phenotype found in the carriers. Furthermore, through a comprehensive literature review, a mutation spectrum was consolidated for SUPT5H, an investigation into the genotype-phenotype correlation was conducted, and factors known to influence Hb A 2 levels were identified. Based on this in-depth understanding, clinicians are better equipped to carry out large scale screenings in regions with high prevalence of β-thalassemia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Celocentesis is a new sampling tool for prenatal diagnosis available from 7 weeks in case of couples at risk for genetic diseases. In this study, we reported the feasibility of earlier prenatal diagnosis by celocentesis in four cases of cystic fibrosis and one case of cystic fibrosis and β-thalassemia co-inherited in the same fetus. Celomic fluids were aspired from the celomic cavity between 8 +2 and 9 +3 weeks of gestation and fetal cells were picked up by micromanipulator. Maternal DNA contamination was tested and target regions of fetal DNA containing parental pathogenetic variants of CFTR and HBB genes were amplified and sequenced. Four of the five fetuses resulted as being affected by cystic fibrosis and, in all cases, the women decided to interrupt the pregnancy. In the other case, the fetus presented a healthy carrier of cystic fibrosis. The results were confirmed in three cases on placental tissue. In one case, no abortive tissue was obtained. In the last case, the woman refused the prenatal diagnosis to confirm the celocentesis data; the pregnancy is ongoing without complications. This procedure provides prenatal diagnosis of monogenic diseases at least four weeks earlier than traditional procedures, reducing the anxiety of patients and providing the option for medical termination of the affected fetus at 8-10 weeks of gestation, which is less traumatic and safer than surgical termination in the second trimester.

Hemoglobin (Hb) Lepore is a rare deletional δβ-thalassemia caused by the fusion between delta-beta genes, and cannot be identified by traditional thaltassemia gene testing technology. The aim of this study was to conduct molecular diagnosis and clinical analysis of Hb Lepore in four unrelated Chinese families using third generation sequencing. Decreased levels of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and an abnormal Hb band were observed in the probands of the four families. However, no common α and β-thalassemia variants were detected in the enrolled families using polymerase chain reaction-reverse dot blot hybridization based traditional thalassemia gene testing. Further third-generation sequencing revealed similar Hb Lepore-Boston-Washington variants in all the patients, which were resulted from partial coverage of the HBB and HBD globin genes, leading to the formation of a delta-beta fusion gene. Specific gap-PCR and Sanger sequencing confirmed that all the patients carried a similar Hb Lepore-Boston-Washington heterozygote. In addition, decreased levels of MCH and Hb A2 were observed in the proband's wife of family 2, an extremely rare variant of Hb Nanchang (GGT > AGT) (HBA2:c.46G > A) was identified by third-generation sequencing and further confirmed by Sanger sequencing. This present study was the first to report the similar Hb Lepore-Boston-Washington in Chinese population. By combining the utilization of Hb capillary electrophoresis and third-generation sequencing, the screening and diagnosis of Hb Lepore can be effectively enhanced., (© 2024. The Author(s).)

Rationale: Compound heterozygotes for deletional β-thalassemia can be difficult to diagnose due to its diverse clinical presentations and no routine screenings. This can lead to disease progression and delay in treatment., Patient Concerns: We reported pedigree analysis and genetic research in a family with rare β-thalassemia., Diagnosis: Pedigree analysis and genetic research demonstrated that the patient was a compound heterozygote for β-thalassemia CD17/Southeast Asian hereditary persistence of fetal hemoglobin deletion, inherited from the parents. Magnetic resonance imaging T2* examination revealed severe iron deposition in the liver. Echocardiography revealed endocardial cushion defect., Interventions: The patient was treated with Deferasirox after receiving the final molecular genetic diagnosis. The initial once-daily dose of Deferasirox was 20 mg/kg/d., Outcomes: The patient discontinued the medication three months after the first visit. Two years later, the patient visited the Department of Hepatobiliary and Pancreatic Diseases. He was recommended to undergo splenectomy after surgical repair of the congenital heart disease. However, the patient refused surgical treatment because of the economic burden., Lessons: We report that fetal hemoglobin is a sensitive indicator for screening large deletions of the β-globin gene, which can be effectively confirmed by the multiplex ligation-dependent probe amplification assay. In non-transfusion-dependent thalassemia patients, iron status assessment should be regularly performed, and iron chelation treatment should be initiated early. This case will provide insights for the diagnosis of rare genotypes of β-thalassemia and has important implications for genetic counseling., Competing Interests: The authors have no other conflicts of interest to declare., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Context.—: Thalassemia is the most widely distributed monogenic autosomal recessive disorder in the world. Accurate genetic analysis of thalassemia is crucial for thalassemia prevention., Objective.—: To compare the clinical utility of a third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles with routine polymerase chain reaction (PCR) in genetic analysis of thalassemia and explore the molecular spectrum of thalassemia in Hunan Province., Design.—: Subjects in Hunan Province were recruited, and hematologic testing was performed. Five hundred four subjects positive on hemoglobin testing were then used as the cohort, and third-generation sequencing and routine PCR were used for genetic analysis., Results.—: Of the 504 subjects, 462 (91.67%) had the same results, whereas 42 (8.33%) exhibited discordant results between the 2 methods. Sanger sequencing and PCR testing confirmed the results of third-generation sequencing. In total, third-generation sequencing correctly detected 247 subjects with variants, whereas PCR identified 205, which showed an increase in detection of 20.49%. Moreover, α triplications were identified in 1.98% (10 of 504) hemoglobin testing-positive subjects in Hunan Province. Seven hemoglobin variants with potential pathogenicity were detected in 9 hemoglobin testing-positive subjects., Conclusions.—: Third-generation sequencing is a more comprehensive, reliable, and efficient approach for genetic analysis of thalassemia than PCR, and allowed for a characterization of the thalassemia spectrum in Hunan Province., (© 2024 College of American Pathologists.)

Hemoglobin (Hb) disorders are among the most common monogenic diseases affecting nearly 7% of the world population. Among various Hb disorders, approximately 1.5% of the world population carries β -thalassemia ( β -Thal), affecting 40,000 newborns every year. Early screening and a timely diagnosis are essential for β -thalassemia patients for the prevention and management of later clinical complications. However, in Africa, Southern Europe, the Middle East, and Southeast Asia, where β -thalassemia is most prevalent, the diagnosis and screening for β -thalassemia are still challenging due to the cost and logistical burden of laboratory diagnostic tests. Here, we present Gazelle, which is a paper-based microchip electrophoresis platform that enables the first point-of-care diagnostic test for β -thalassemia. We evaluated the accuracy of Gazelle for the β -Thal screening across 372 subjects in the age range of 4-63 years at Apple Diagnostics lab in Mumbai, India. Additionally, 30 blood samples were prepared to mimic β -Thal intermediate and β -Thal major samples. Gazelle-detected levels of Hb A, Hb F, and Hb A 2 demonstrated high levels of correlation with the results reported through laboratory gold standard high-performance liquid chromatography (HPLC), yielding a Pearson correlation coefficient = 0.99. This ability to obtain rapid and accurate results suggests that Gazelle may be suitable for the large-scale screening and diagnosis of β -Thal.

Background: India has the most significant number of children with thalassemia major worldwide, and about 10,000-15,000 children with the disease are born yearly. Scaling up e-health initiatives in rural areas using a cost-effective digital tool to provide healthcare access for all sections of people remains a challenge for government or semi-governmental institutions and agencies., Methods: We compared the performance of a recently developed formula SCS[Formula: see text] and its web application SUSOKA with 42 discrimination formulae presently available in the literature. 6,388 samples were collected from the Postgraduate Institute of Medical Education and Research, Chandigarh, in North-Western India. Performances of the formulae were evaluated by eight different measures: sensitivity, specificity, Youden's Index, AUC-ROC, accuracy, positive predictive value, negative predictive value, and false omission rate. Three multi-criteria decision-making (MCDM) methods, TOPSIS, COPRAS, and SECA, were implemented to rank formulae by ensuring a trade-off among the eight measures., Results: MCDM methods revealed that the Shine & Lal and SCS[Formula: see text] were the best-performing formulae. Further, a modification of the SCS[Formula: see text] formula was proposed, and validation was conducted with a data set containing 939 samples collected from Nil Ratan Sircar (NRS) Medical College and Hospital, Kolkata, in Eastern India. Our two-step approach emphasized the necessity of a molecular diagnosis for a lower number of the population. SCS[Formula: see text] along with the condition MCV[Formula: see text] 80 fl was recommended for a higher heterogeneous population set. It was found that SCS[Formula: see text] can classify all BTT samples with 100% sensitivity when MCV[Formula: see text] 80 fl., Conclusions: We addressed the issue of how to integrate the higher-ranked formulae in mass screening to ensure higher performance through the MCDM approach. In real-life practice, it is sufficient for a screening algorithm to flag a particular sample as requiring or not requiring further specific confirmatory testing. Implementing discriminate functions in routine screening programs allows early identification; consequently, the cost will decrease, and the turnaround time in everyday workflows will also increase. Our proposed two-step procedure expedites such a process. It is concluded that for mass screening of BTT in a heterogeneous set of data, SCS[Formula: see text] and its web application SUSOKA can provide 100% sensitivity when MCV[Formula: see text] 80 fl., (© 2023. The Author(s).)

Background: Thalassemia is a common genetic disorder in southwestern China, and an increasing number of cases from eastern China have been recently reported. Here, we developed a rapid, convenient, and accurate assay to evaluate the mutation spectrum of thalassemia in eastern China., Methods: A carrier screening assay for 61 hotspot variants among HBA1/HBA2 and HBB (OMIM: 141800, 141850, and 141900) genes was developed by SNaPshot/high-throughput ligation-dependent probe amplification (HLPA) technology. We used this assay to detect the mutation spectrum of thalassemia in individuals from eastern China and compared with the data collected from literatures focused on southern and northern China for variant distribution., Results: Among 4276 tested individuals, 2.62% (112/4276) were α-thalassemia carriers, with 90 carrying one deletion or mutation and 22 carrying two deletions. 0.40% (17/4276) were β-thalassemia carriers, and the most common variant of β-thalassemia was c.126_129delCTTT (29.41%) followed by c.316-197C>T (23.53%). The genotype distribution in our study was similar to those from southern China populations., Conclusion: The Chinese population from different regions presented comparable mutation spectrum of thalassemia, and the SNaPshot/HLPA technique may serve as a capable assay for a routine genetic test in clinical practice with its accurate, rapid, and inexpensive advantage., (© 2023 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Although considered a mild clinical condition, many laboratory issues of the carrier state of β-thalassemia remain unresolved. Accurate laboratory screening of β-thalassemia traits is crucial for preventing the birth of a β-thalassemia major child. Identification of carriers in the laboratory is affected by factors that influence red cell indices and HbA2 quantification. Silent mutations and co-inheriting genetic and non-genetic factors affect red cell indices which decreases the effectiveness of the conventional approach. Similarly, the type of β mutation, co-inheriting genetic and non-genetic factors, and technical aspects, including the analytical method used and variations in the HbA2 cut-off values, affect the HbA2 results, leading to further confusion. However, the combination of mean corpuscular volume, mean corpuscular hemoglobin, and hemoglobin analysis increases the diagnostic accuracy. Diagnostic problems arising from non-genetic factors can be eliminated by carefully screening the patient's clinical history. However, issues due to certain genetic factors, such as Krüppel-like factor 1 gene mutations and α triplication still remain unresolved. Each laboratory should determine the population-specific reference ranges and be wary of machine-related variations of HbA2 levels, the prevalence of silent mutations in the community.

Background: Beta thalassemia, related to HBB mutation and associated with elevated hemoglobin A2 (HbA2), is an important genetic hemoglobinopathy with high incidences of disease and carrier rates in Singapore. Carrier screening is essential to facilitate prenatal counseling and testing. However, when individuals with elevated HbA2 do not have an identifiable HBB disease-associated variant, there is ambiguity on risk to their offspring., Methods: We describe a case report of a proband with elevated HbA2, no identifiable HBB disease-associated variant, whose partner was a beta thalassemia carrier. Through clinical HBB gene sequencing, multiplex ligation-dependent probe amplification (MLPA) analysis, as well as targeted Nanopore long read sequencing of selected genes, we performed a complete analysis of HBB including the promoter region, 5'UTR and coding gene sequence, as well as evaluation for potential modifier variants and other rare structural variants., Results: This process identified that the proband was heterozygous for KLF1:c.544T>C (p.Phe182Leu), a potential functional polymorphism previously known to be associated with benign elevated HbA2 levels. The presence of disease variants in the HBB locus was excluded., Conclusion: This finding provided clarity and enabled family planning for the proband and her family., (© 2023 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Objective: The capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) methods were compared in terms of HbA2 measurement for the assessment of hemoglobinopathies., Materials and Methods: CZE was compared with HPLC for the evaluation of patients without hemoglobinopathy (n=321), with β-thalassemia trait (n=113), and with common (HbD-Punjab, E, C, S/A, and S/S) and rare (HbS/D, O-Arap, Lepore, G-Coushata, Setif, Hamadan, Q-Iran, and H) variants (n=21). The reference range for HbA2 was determined by CZE., Results: Among patients without hemoglobinopathy, the median (2.5 th -97.5 th percentiles) values were 97.4% (97.0-98.0%) and 97.5% (96.6-98.4%) for HbA (p=0.060) and 2.4% (1.6-3.0%) and 2.5% (1.6-3.1%) for HbA2 (p<0.001) by HPLC and CZE, respectively. The reference range for HbA2 was 1.6-3.1% by CZE. In the comparison of methods for HbA2, there was a constant error of 0.255 (confidence interval: 0.062-0.448) and bias of 0.10% (limit of agreement: 0.33-0.53), and higher values were obtained with CZE. A strong correlation was observed between the methods (r=0.782). Interrater agreement was almost perfect for clinical diagnosis (ϰ=0.911). The two methods detected and identified the common variants similarly. All rare variants, except HbH by HPLC and HbS/D by CZE, were detected as separate peaks by both methods., Conclusion: The two methods were in agreement regarding the preliminary identification of β-thalassemia patients. Different Hb variants were detected by both methods but with possible methodological interference for HbA2 measurements. CZE is a reliable and simple alternative for the evaluation of hemoglobinopathies. The standardization of HbA2 measurements should be prioritized as more techniques become available in routine laboratory practice., Competing Interests: Conflict of Interest: No conflict of interest was declared by the authors., (©Copyright 2023 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House.)

Background: In China, conventional genetic testing methods can only detect common thalassemia variants. Accurate detection of rare thalassemia is crucial for clinical diagnosis, especially for children that need long-term blood transfusion. This study aims to explore the application value of third-generation sequencing (TGS) in the diagnosis of rare thalassemia in children with anemia., Methods: We enrolled 20 children with anemia, excluding from iron deficiency anemia (IDA). TGS was employed to identify both known and novel thalassemia genotypes, while sanger sequencing was used to confirm the novel mutation detected., Results: Among the 20 samples, we identified 5 cases of rare thalassemia. These included β -4.9 (hg38,Chr11:5226187-5231089) at HBB gene, α -91 ( HBA2 :c.*91delT), α CD30 ( HBA2 :c.91-93delGAG), Chinese G γ + ( A γδβ) 0 (NG&#95;000007.3: g .48795-127698 del 78904) and delta - 77(T > C) ( HBD :c.-127T>C). Notably, the - SEA /α -91 α genotype associated with severe non-deletional hemoglobin H disease (HbH disease) has not been previously reported. Patients with genotypes β 654 /β -4.9 and - SEA /α -91 α necessitate long-term blood transfusions, and those with the - SEA /α CD30 α, Chinese G γ + ( A γδβ) 0 and delta thalassemia demonstrate mild anemia., Conclusions: TGS demonstrates promising potential as a diagnostic tool for suspected cases of rare thalassemia in children, especially those suspected to have transfusion-dependent thalassemia (TDT).

Objectives: Clinical manifestations of patients with Hemoglobin E/beta-thalassemia vary from mild to severe phenotypes despite exhibiting the same genotype. Studies have partially identified genetic modifiers. We aimed to study the association between rare variants in protein-coding regions and clinical severity in Thai patients., Methods: From April to November 2018, a case-control study was conducted based on clinical information and DNA samples collected from Thai patients with hemoglobin E/beta-thalassemia over the age of four years. Cases were patients with severe symptoms, while patients with mild symptoms acted as controls. Whole exome sequencing and rare variant association study were used to analyze the data., Results: All 338 unrelated patients were classified into 165 severe and 173 mild cases. Genotypes comprised 81.4% of hemoglobin E/beta-thalassemia, 2.7% of homozygous or compound heterozygous beta-thalassemia, and 0.3% of (δβ) 0 thalassemia Hb E while 15.7% of samples were not classified as beta-thalassemia. A novel cis heterozygotes of IVS I-7 (A > T) and codon 26 (G > A) was identified. Six genes ( COL4A3 , DLK1 , FAM186A , PZP , THPO , and TRIM51 ) showed the strongest associations with severity (observed p -values of <0.05; significance lost after correction for multiplicity). Among known modifiers, KLF1 variants were found in four mild patients and one severe patient., Conclusion: No rare variants were identified as contributors to the clinical heterogeneity of hemoglobin E/beta-thalassemia. KLF1 mutations are potential genetic modifiers. Studies to identify genetic factors are still important and helpful for predicting severity and developing targeted therapy.

Thalassaemia is a typically monogenic disease caused by mutations or deletions in the globin gene and has a high prevalence in southern China. Prenatal screening for thalassaemia can be effective in reducing the incidence of thalassaemia. Haematologic parameters of pregnant thalassaemia carriers are diverse and potentially valuable for identifying different types of genotypes. By comparing and evaluating haematological parameters, formulas in the literature, we tried to reveal differences between pregnant women carrying different types of thalassaemia genes. The Mentzer formula (MCV/RBC) showed a strong ability to differentiate thalassaemia genotypes in pregnant women. In addition, combined with haemoglobin electrophoresis HbA2 can further distinguish the -α/αα, α T α/αα, -/αα, β + /N and β 0 /N groups. HbA2 divides them into two groups. Based on the Mentzer formula, we can further decide which type of thalassaemia to screen (α/β and the subgroups) for genotyping. Therefore, this simpler and more cost-effective workflow has great potential for application in screening pregnant women for thalassaemia carriers.Impact Statement What is already known on this subject? Currently, it is known that thalassaemia gene carriers have abnormal blood indicators. Many findings describe their important values in distinguishing thalassaemia and other blood diseases. They combined different metrics as an algorithm to distinguish thalassaemia and iron deficiency anaemia. Prenatal screening is an effective method to reduce the incidence of thalassaemia. The current main method is PCR. Due to technical and financial constraints, many backward places cannot use this technology. The necessity for prenatal screening for thalassaemia has been overlooked. What the results of this study add? Among these algorithms, Mentzer formula revealed differences in haematological parameters during pregnancy between normal individuals and thalassaemia carriers. Combining the HbA2, thalassaemia carriers can be distinguished from normal individuals, including -α/αα, α T α/αα, -/αα, β 0 /N and β + /N. What are the implications of these findings for clinical practice and/or further research? We provide another tool for these hospitals that donot have Hb electrophoresis test and PCR. Then the clinical doctor can get some evidence and suggest women go to another big hospital for essential tests. It is an excellent suggestion. In the future, we will collect more specific gene types and further investigate their potential relationship using these formulas.

Objective: To compare the diagnostic performance of 10 mathematical formulae for identifying thalassemia trait in blood donors., Methods: Compete blood counts were conducted on peripheral blood specimens using the UniCel DxH 800 hematology analyzer. Receiver operating characteristic curves were used to evaluate the diagnostic performance of each mathematical formula., Results: In the 66 donors with thalassemia and 288 subjects with no thalassemia analyzed, donors with thalassemia trait had lower values for mean corpuscular volume and mean corpuscular hemoglobin than subjects without thalassemia donors (77 fL vs 86 fL [P < .001]; 25 pg vs 28 pg [P < .001]). The formula developed by Shine and Lal in 1977 showed the highest area under the curve value, namely, 0.9. At the cutoff value of <1812, this formula had maximum specificity of 82.35% and sensitivity of 89.58%., Conclusions: Our data indicate that the Shine and Lal formula has remarkable diagnostic performance in identifying donors with underlying thalassemia trait., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: To evaluate the value of the real-time PCR-based multicolor melting curve analysis (MMCA) with an automatic analysis system used in a mass thalassemia screening and prenatal diagnosis program., Methods: A total of 18,912 peripheral blood samples from 9456 couples and 1150 prenatal samples were detected by MMCA assay. All prenatal samples were also tested by a conventional method. Samples with unknown melting peaks, unusual peak height ratios between a wild allele and a mutant allele, or a discordant phenotype-genotype match were further studied by using multiplex ligation-dependent probe amplification (MLPA) or Sanger sequencing. All MMCA results were automatically analyzed and manually checked. The consistency between MMCA assay and conventional methods among prenatal samples was investigated., Results: Except for initiation codon (T > G) (HBB:c.2T > G), all genotypes of thalassemia inside the scope of conventional methods were detected by MMCA assay. Additionally, 27 carriers with 10 rare HBB variants, 13 with α fusion gene, 1 with a rare deletion in α globin gene, and 1 with rare HBA variant were detected by using MMCA assay., Conclusion: MMCA can be an alternative approach used in routine thalassemia carrier screening and prenatal diagnosis for its high throughput, sufficient stability, low cost, and easy operation., (© 2023 University College London (UCL) and John Wiley & Sons Ltd.)

Objectives: Allogeneic hematopoietic stem cell transplant is the only curative treatment for patients with transfusion-dependent thalassemia major. In recent years, a number of novel approaches have improved patient outcomes and quality of life by minimizing the toxicity of conditioning regimens. The objective of this study was to compare the role of treosulfan- and busulfan-based conditioning in transfusion-dependent thalassemia., Materials and Methods: Data were collected retrospectively on 121 children with beta thalassemia major who underwent hematopoietic stem cell transplant using treosulfan-based (n = 37) or busulfan-based (n = 84) conditioning regimens between 2012 and 2022., Results: Two-year overall survival was 87.5% in the busulfan-based conditioning group and 91.1% in the treosulfan-based conditioning group.The group given the busulfan regimen compared with treosulfan regimen had significantly increased number of side effects (58.3% vs 21.6%, respectively; P < .001). When the busulfan-based regimen by level was evaluated, we observed no significant differences between the frequency of side effects according to drug serum levels. In addition, no significant differences were shown between the 2 regimen groups for cumulative incidence of acute and chronic graft-versus-host disease., Conclusions: The safety and effectiveness of a treosulfan-based myeloablative conditioning regimen has been confirmed by ourretrospective investigation of pediatric patients with beta thalassemia.

Background: Sepsis and thrombo-embolic disease are well known complications of thalassemia major. Intracardiac thrombi are however rare and can lead to diagnostic dilemmas., Case Presentation: We report the case of a 20-year-old female splenectomised thalassaemia major patient with severe iron overload, who presented with life threatening sepsis associated with a liver abscess. Discovery of a large oscillating intra cardiac lesion on 2D echocardiogram confirmed by Contrast Enhanced Computed Tomography (CECT) chest in the right atrium extending from the left hepatic vein through the inferior vena cava complicated the clinical course. After a prolonged Intensive Care Unit (ICU) stay supported with antibiotics and anticoagulation, she recovered with evidence of resolution of the intra cardiac thrombus., Conclusions: Early recognition and prompt aggressive treatment of sepsis in patients with thalassemia is essential to prevent complications. Intracardiac thrombosis is a potentially treatable cause for an intra cardiac mass in patients with thalassemia major, which should not be missed., (© 2023. The Author(s).)

Background: The B cell CLL/lymphoma 11A (BCL11A) is a key regulator of hemoglobin switching in β-thalassemia (β-thal). Previous study has suggested that dysregulated microRNAs are involved in the regulation of BCL11A expression. The aim of this study was to investigate the clinical value of hsa-miR-190b-5p in β-thal, and to confirm the regulatory effect of hsa-miR-190b-5p on BCL11A expression., Methods: The peripheral blood of 25 pediatric β-thal patients and 25 healthy controls were selected, and qRT-PCR was used to analyze the levels of hsa-miR-190b-5p and BCL11A mRNA. The relationship between hsa-miR-190b-5p expression and hematological parameters was assessed by Pearson's correlation test. The diagnostic power of hsa-miR-190b-5p was evaluated by ROC curves analysis. The direct integration between hsa-miR-190b-5p and BCL11A 3'-UTR was confirmed by luciferase reporter assay., Results: Hsa-miR-190b-5p expression in pediatric β-thal was upregulated, and negatively correlated with the MCH and HbA levels, but positively correlated with the HbF level. Hsa-miR-190b-5p showed a good diagnostic capability for pediatric β-thal equivalent to that of HbA2 (AUC: 0.760 vs. 0.758). Moreover, the levels of BCL11A mRNA in pediatric β-thal were decreased, and hsa-miR-190b-5p had a negative correlation with BCL11A mRNA expression (r = -0.403). BCL11A was a target gene of hsa-miR-190b-5p. The mRNA and protein levels of BCL11A were diminished by introduction of hsa-miR-190b-5p, whereas its expression was upregulated by knockdown of hsa-miR-190b-5p., Conclusions: Hsa-miR-190b-5p expression was upregulated in pediatric β-thal and might be an effective diagnostic biomarker. BCL11A was negatively regulated by hsa-miR-190b-5p, which might provide new target for the treatment of pediatric β-thal., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright: © 2023 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Abstract: A 57-year-old male had abdominal pain and distension for 6-7 months with a palpable swelling in the right lumbar region. Contrast-enhanced computed tomography abdomen showed a large heterogeneous lesion with fat density measuring 22 cm ´ 16.5 cm in the right suprarenal region. Laparotomy was done which showed an encapsulated mass measuring 21 cm ´ 14 cm ´ 5 cm. Cut section revealed yellowish areas admixed with hemorrhage and large areas of necrosis. Microscopy revealed adrenal myelolipoma. The patient was found to have sickling positive. High-performance liquid chromatography showed double heterozygous for HbS and beta-thalassemia trait. The association of giant adrenal myelolipoma with double heterozygous for HbS and beta-thalassemia trait is rare, and as clinically it simulates retroperitoneal sarcoma, awareness of this rare entity is critical for its accurate diagnosis and proper management., (Copyright © 2023 Copyright: © 2023 Journal of Cancer Research and Therapeutics.)

Thalassemia is one of the most prevalent genetic disorders worldwide. The present study aimed to explore the mutational spectrum of all hemoglobin (HB) encoding genes and to identify the potentially damaging and pathogenic variants in the beta (β)-thalassemia major patients and thalassemia minor carriers of Southern Punjab, Pakistan. A total of 49 β-thalassemia major patients and 49 carrier samples were screened for the identification of HBA1, HBA2, HBB, HBD, HBE1, HBG1 and HBG2 variants by NGS. PCR was performed for the amplification of HB encoding genes and the amplified product of 13 patients and 7 carrier samples were processed for the Sanger sequencing. Various bioinformatics tools and databases were employed to reveal the functional impact and pathogenicity potential of the observed variants. Results depicted a total of 20 variants of HB-related genes by NGS and 5 by Sanger sequencing in thalassemia patients. While 20 variants by NGS and 3 by Sanger were detected in carriers. Few known genetic variants of HB-encoding genes are being reported for the first time in Pakistani thalassemia patients and carriers. However, two novel HBB variants c.375A>C (p.P125P) and c.*61T>G and a novel variant of HBE1 (c.37A>T (p.T13S)) were also documented. Pathogenicity analysis predicted the pathogenic potential of HBB variants (c.47G>A (p.W16*), c.27-28insG (p. S10fs), and c.92+5G>C) for β thalassemia. The study of functional impact indicated that these HBB variants result in the premature termination of translation leading to the loss of functional β-globin protein. It is therefore suggested that the pathogenic HBB variants, identified during present study, can be employed for the diagnosis, carrier screening, and planning therapy of thalassemia., (© 2023. Springer Nature Limited.)

Background and Aim: Beta thalassemia major (β-TM) is a genetic blood disorder requiring lifelong blood transfusions.  The resulting iron overload damages multiple organs, particularly the heart and endocrine organs. This study aimed to describe and assess the predictors of survival and complications in Omani patients with β-TM.   Methods: All β-TM patients registered in the day care of Sultan Qaboos University Hospital were included in this retrospective study.   Results: There were 187 patients with β-TM with a median follow-up of 24.9 years.  The median ages at diagnosis and the start of chelation were 0.7 and 4.8 years, respectively. The following complications developed at different time points [Median (age in years), Complication Free Probability at 20 years]: Death (20.0 years;85%), hypogonadism (15.9 years;50%), insulin-dependent or non-insulin dependent diabetes (20.0 years;88%), cardiac complications (20.3 years;91%), osteoporosis (20.7 years;96%), hypothyroidism (25.7 years;97%), liver complications (7.3 years;54%). The number of complications predicted death (P = 0.0038). Those born after 1980 had a lower risk of death (P = 0.005), hypogonadism (P = < 0.0001), and cardiac complications (P = 0.004). Higher serum ferritin at the start of chelation was associated with the development of diabetes (P = < 0. 001).   Conclusions: This long-term study shows complications development at different ages, and the number of complications is associated with survival. Later birth cohorts had a lower risk of death, hypogonadism, and cardiac complications. There was a persistent negative impact of delay in the start of iron chelation that is present even after a long follow-up. (www.actabiomedica.it).

Background: Hemoglobinopathies are the most common inherited diseases in humans resulting from impaired globin chain synthesis of hemoglobin. The progression of thalassemia rates is prevented with prenatal screening methods., Aims: To evaluate the hematological parameters of α- and β-thalassemia and normal fetuses aged 17-25 weeks of gestation., Study Design: A cross-sectional study., Methods: Pregnant women who underwent cordocentesis in the second trimester because of the risk of having a baby with thalassemia were included in the study. Hematological indices and molecular DNA methods were analyzed from the cord blood samples of 129 women who were 17-25 weeks into pregnancy. The HPLC method was used for Hb fraction analysis. Amplification refractory mutation system, restriction enzyme analysis, multiplex polymerase chain reaction, and sequencing methods were used for the molecular analysis. Maternal contamination was eliminated by the short tandem repeat method., Results: In total, 112 of the fetuses carry α- and β-thalassemia heterozygous or homozygous (α: 37, β: 58, mixed: 17) and 17 fetuses had a normal genotype for thalassemia. Significant differences in adult hemoglobin (HbA), fetal hemoglobin (HbF), Hb Barts, MCV, MCH, and RDW were detected in three groups compared with the normal group (p < 0.001, except for RBC, Hb, HCT, and MCHC). Differences in HbF, Hb Barts, MCV, MCH, and RDW were observed in the α-thalassemia groups compared with the normal group (p < 0.001). Among the five β-thalassemia subgroups, only HbA and RDW were different from the normal group (p < 0.001)., Conclusion: This study could be a good reference for future studies and prenatal diagnostic applications in emphasizing the importance of changes in the blood parameters of fetuses before molecular genotyping. These hematological data give valuable information to clinicians about the fetus to enlighten families in making appropriate decisions during prenatal diagnosis.

Background: Large β-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or β-, δβ-, γδβ-, and ϵγδβ-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for β-thalassemia. Notably, ϵγδβ-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψβ loci with intact LCR, δ-, and β-regions in 2 women and newborn twins., Methods: Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed., Results: NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψβ (HBBP1) loci., Conclusions: This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδβ-thalassemia., (© American Association for Clinical Chemistry 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Microcytosis and hypochromia result from deficient hemoglobin synthesis in red blood cells and are easily detected in a complete blood count test. These conditions are mainly due to iron nutritional deficiency, but may also result from some genetic diseases, such as thalassemia. The aim of this study was to determine the contribution of β- and α-thalassemia to these abnormal hematological phenotypes in a representative sample of adult individuals living in Portugal who participated in the first Portuguese National Health Examination Survey (INSEF)., Material and Methods: Among the 4808 INSEF participants, 204 had microcytosis, hypochromia or both. The corresponding 204 DNAs were screened for changes in the β-globin gene by next-generation sequencing and Sanger sequencing. In addition, α-thalassemia deletions within the α-globin cluster were investigated by Gap-PCR and multiplex ligation-dependent probe amplification., Results: In this selected subgroup of INSEF participants, 54 had α-thalassemia (26%), predominantly caused by the -α3.7kb deletion, and 22 were β-thalassemia carriers (11%) mainly due to point mutations in the β-globin gene previously known in Portugal., Conclusion: Thalassemia trait is a frequent cause of microcytosis or hypochromia in Portugal since this genetic condition was found in 37% of the investigated cases.

Background: Differentiation between thalassemia trait (TT) and iron deficiency anemia (IDA) is challenging and costly. This study aimed to construct and evaluate a model based on red blood cell (RBC) parameters to differentiate TT and IDA in the southern region of Fujian Province, China., Methods: RBC parameters of 364 TT patients and 316 IDA patients were reviewed. RBC parameter-based Logistic-Nomogram model to differentiate between TT and IDA was constructed by multivariate logistic regression analysis plus nomogram, and then compared with 22 previously reported differential indices., Results: The patients were randomly selected to a training cohort (n TT  = 248, n IDA  = 223) and a validation cohort (n TT  = 116, n IDA  = 93). In the training cohort, multivariate logistic regression analysis identified RBC count, mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC) as independent parameters associated with TT susceptibility. A nomogram was plotted based on these parameters, and then the RBC parameter-based Logistic-Nomogram model g (μ y ) = 1.92 × RBC count-0.51 × MCH + 0.14 × MCHC-39.2 was devised. The area under the curve (AUC) (95% CI) was 0.95 (0.93-0.97); sensitivity and specificity at the best cutoff score (120.24) were 0.93 and 0.89, respectively; the accuracy was 0.91. In the validation cohort, the RBC parameter-based Logistic-Nomogram model had AUC (95% CI) of 0.95 (0.91-0.98); sensitivity and specificity were 0.92 and 0.87, respectively; accuracy was 0.90. Moreover, compared with 22 reported differential indices, the RBC parameter-based Logistic-Nomogram model showed numerically higher AUC, net reclassification index, and integrated discrimination index (all p < 0.001)., Conclusion: The RBC parameter-based Logistic-Nomogram model shows high performance in differentiating patients with TT and IDA from the southern region of Fujian Province., (© 2023 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Background: β 0 -thalassemia deletion removing 5´ β -globin promoter usually presents phenotype with high hemoglobin (Hb) A 2 and Hb F levels. We report the molecular characteristics and phenotype-genotype correlation in a large cohort of the β 0 -thalassemia with 3.4 kb deletion., Methods: A total of 148 subjects, including 127 heterozygotes, 20 Hb E- β -thalassemia patients, and a double heterozygote with α -globin gene triplication, were recruited. Hb and DNA analysis were performed to identify thalassemia mutations and four high Hb F single nucleotide polymorphisms (SNPs) including four base pair deletion (-AGCA) at A γ -globin promoter, rs5006884 on OR51B6 gene, -158 G γ - Xmn I, BCL11A binding motifs (TGGTCA) between 3´ A γ -globin gene and 5´ δ -globin gene., Results: It was found that heterozygous β 0 -thalassemia and Hb E- β 0 -thalassemia with 3.4 kb deletion had significantly higher Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and Hb F values as compared with those with other mutations. Co-inheritance of heterozygous β 0 -thalassemia with 3.4 kb deletion and α -thalassemia was associated with even higher MCV and MCH values. The Hb E- β 0 -thalassemia patients carried a non-transfusion-dependent thalassemia phenotype with an average Hb of around 10 g/dL without blood transfusion. A hitherto undescribed double heterozygous β 0 -thalassemia with 3.4 kb deletion and α -globin gene triplication presented as a plain β -thalassemia trait. Most of the subjects had wild-type sequences for the four high Hb F SNPs examined. No significant difference in Hb F was observed between those of subjects with and without these SNPs. Removal of the 5´ β -globin promoter may likely be responsible for this unusual phenotype., Conclusions: The results indicate that β 0 -thalassemia with 3.4 kb deletion is a mild β -thalassemia allele. This information should be provided at genetic counseling and prenatal thalassemia diagnosis., Competing Interests: The authors declare there are no competing interests., (©2023 Soontornpanawet et al.)

Background & Objectives: Non-invasive prenatal testing (NIPT) of maternally inherited alleles of β-thalassaemia (MIB) remains to be a challenge. Furthermore, current techniques are not available for use as routine tests. NIPT for β-thalassaemia disease was developed by using a specific droplet digital polymerase chain reaction (ddPCR) assay to analyze the cell-free foetal DNA (cffDNA) derived from maternal plasma., Methods: Pregnant women and their spouses who are at risk of bearing an offspring with β-thalassaemia disease from common MIB mutations (CD 41/42-TCTT, CD17A>T, IVS1-1G>T and CD26G>A) were enrolled. The ddPCR assay sets were constructed for each of the four mutations. All cell-free DNA samples were first screened for the paternally inherited β-thalassaemia (PIB) mutation. The PIB-negative samples were considered as non-disease and were not further analyzed. For PIB-positive samples, DNA fragments of 50-300 base pairs in size were isolated and purified, and further analyzed for MIB mutation. The allelic ratio between the mutant and the wild-type was used to determine the presence of MIB in cffDNA. All cases underwent a prenatal diagnosis by amniocentesis for a definite diagnosis., Results: Forty two couples at risk were enrolled. Twenty two samples were positive for PIBs. Among these 22 samples, there were 10 cases with allelic ratio >1.0 (MIB positive). All foetuses with over-represented mutant alleles were further diagnosed with β-thalassaemia disease; eight with compound heterozygous and two with homozygous mutations. The 20 PIB-negative and 12 MIB-negative foetuses were non-affected., Interpretation & Conclusions: The results of this study suggest that NIPT utilizing the ddPCR assay can be effectively used for the screening and diagnosis of foetal β-thalassaemia in at risk pregnancies., Competing Interests: None

Objective: To determine the frequency and etiology of unnecessary prenatal diagnosis for hemoglobinopathies during 12 years of services at a single university center in Thailand., Methods: We conducted a retrospective cohort analysis of prenatal diagnosis during 2009-2021. A total of 4,932 couples at risk and 4,946 fetal specimens, including fetal blood (5.6%), amniotic fluid (92.3%), and chorionic villus samples (2.2%) were analyzed. Identification of mutations causing hemoglobinopathies was carried out by PCR-based methods. Maternal contamination was monitored by analysis of the D1S80 VNTR locus., Results: Among 4,946 fetal specimens, 12 were excluded because of poor PCR amplification, maternal contamination, non-paternity, and inconsistency of the results of the fetuses and parents. Breakdown of 4,934 fetuses revealed 3,880 (78.6%) at risk for the three severe thalassemia diseases, including β-thalassemia major, Hb E-β-thalassemia, and homozygous α0-thalassemia, 58 (1.2%) at risk for other α-thalassemia diseases, 168 (3.4%) at risk for β+-thalassemia, 109 (2.2%) at risk for high Hb F determinants, 16 (0.3%) at risk for abnormal Hbs, and 294 (6.0%) with no risk of having severe hemoglobinopathies. The parents of 409 (8.3%) fetuses had inadequate data for fetal risk assessment. Overall, we encountered unnecessary prenatal diagnostic requests for 645 (13.1%) fetuses., Conclusions: The frequency of unnecessary prenatal diagnosis was high. This could lead to unnecessary risk of complications associated with fetal specimen collection, psychological impacts to the pregnant women and their families, as well as laboratory expenses and workload., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Singha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The high prevalence of β-thalassemia indicates the severe medical burden in Guangxi province in China. Millions of thousands of prenatal women with healthy or thalassemia-carrying fetuses received an unnecessary prenatal diagnosis. We designed a prospective single-center proof-of-concept study to evaluate the utility of a noninvasive prenatal screening method in the stratification of beta-thalassemia patients before invasive procedures., Methods: Next-generation and optimized pseudo-tetraploid genotyping-based methods were utilized in preceding invasive diagnosis stratification to predict the mater-fetus genotype combinations in cell-free DNA, which is from maternal peripheral blood. Populational linkage disequilibrium information with additional neighboring loci to infer the possible fetal genotype. The concordance of the pseudo-tetraploid genotyping with the gold standard invasive molecular diagnosis was used to evaluate the effectiveness of this method., Results: 127 β-thalassemia carrier parents were consecutively recruited. The total genotype concordance rate is 95.71%. The Kappa value was 0.8248 for genotype combinations and 0.9118 for individual alleles., Conclusion: This study offers a new approach to picking out the health or carrier fetus before invasive procedures. It provides valuable novel insight into patient stratification management on β-thalassemia prenatal diagnosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Jia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Context: β-thalassemia trait is usually diagnosed by raised hemoglobin A 2 (HbA 2 ). The presence of megaloblastic anemia can cause an increase in HbA 2 and create a diagnostic dilemma. Here, we have analyzed the effect of vitamin B12 and folic acid supplementation on HbA 2 and diagnosis of β-thalassemia trait in cases of megaloblastic anemia with raised HbA 2 ., Materials and Methods: Cases of megaloblastic anemia with raised HbA 2 on high-performance liquid chromatography (HPLC) were supplemented with vitamin B12 and folic acid. Post-treatment evaluation was done after 2 months. Cases showing adequate hematological response were subjected to statistical analysis. Based on post-treatment HbA 2 value, the cases were diagnosed as normal, borderline raised HbA 2 , or β-thalassemia trait. Pre- and post-treatment values of red cell parameters and HbA 2 were analyzed., Results: There was a significant decrease in HbA 2 value after vitamin B12 and folic acid supplementation. The diagnosis was changed in 70.97% of the cases after treatment. The chance of inconclusive diagnosis was decreased from more than 50% to less than 10%. Pre-treatment mean corpuscular volume (MCV) and HbA 2 % showed a significant difference between the thalassemic and normal groups., Conclusions: Megaloblastic anemia can lead to false-positive diagnosis of β-thalassemia trait on HPLC. Repeat HPLC should be done after adequate supplementation of vitamin B12 and folic acid in cases of megaloblastic anemia with raised HbA 2 . Red cell parameters are not helpful to suspect β-thalassemia trait in presence of megaloblastic anemia. However, HbA 2 % on HPLC can be a useful parameter to suspect or exclude β-thalassemia trait in cases of megaloblastic anemia., Competing Interests: None

Introduction: The α-globin fusion gene between the HBA2 and HBAP1 genes becomes clinically important in thalassemia screening because this fusion gene can cause severe hemoglobin (Hb) H disease when combining with α 0 -thalassemia (α 0 -thal). Due to its uncommon rearrangement in the α gene cluster without dosage changes, this fusion gene is undetectable by common molecular testing approaches used for α-thal diagnosis., Methods: In this study, we used the single-molecule real-time (SMRT) sequencing technique to detect this fusion gene in 23 carriers identified by next-generation sequencing (NGS) among 16,504 screened individuals. Five primers for α and β thalassemia were utilized., Results: According to the NGS results, the 23 carriers include 14 pure heterozygotes, eight compound heterozygotes with common α-thal alleles, and one homozygote. By using SMRT, the fusion mutant was successfully detected in all 23 carriers. Furthermore, SMRT corrected the diagnosis in two "pure" heterozygotes: one was compound heterozygote with anti-3.7 triplication, and the other was homozygote., Conclusion: Our results indicate that SMRT is a superior method compared to NGS in detecting the α fusion gene, attributing to its efficient, accurate, and one-step properties., (© 2022 John Wiley & Sons Ltd/University College London.)

Background: Early detection of iron overload in transfusion-dependent thalassemia (TDT) patients is critical to prevent complications and improve survival., Objectives: Evaluate the utility of serum ferritin (SF) in the prediction of hepatic and myocardial iron overload (HIO and MIO) compared to T2*-MRI., Design: Retrospective SETTINGS: Governmental hospitals., Patients and Methods: Patients with TDT who had T2*-MRI examinations between January 2016 to October 2019 were included. The predictive value of SF for detection of HIO and MIO was assessed by measuring area under the curve (AUC). A sample size of 123 cases was calculated to detect a correlation of 0.25 with 90% power and a two-sided type I error of 0.05., Main Outcome Measures: The correlation between SF and estimated hepatic iron concentration., Sample Size: 137 TDT patients who required regular blood transfusions., Results: The predictive value of SF was excellent for detection of HIO (AUC=0.83-0.87) but fair for detection of MIO (AUC=0.67). The two independent predictors of MIO were age and SF. The log of (age × SF) enhanced the SF predictive value for MIO (AUC=0.78). SF values of 700 and 1250 mg/L effectively excluded mild and moderate HIO with a sensitivity of 97.8% and 94.2%, respectively (LR-=0.1). While SF values of 1640 and 2150 mg/L accurately diagnosed mild and moderate HIO with a specificity of 95.55% and 96.4%, respectively (LR+>10). A log of (age × SF) cut-off value of 4.15 effectively excluded MIO (LR-=0.1), while a value of 4.65 moderately confirmed MIO (LR+=3.2)., Conclusions: SF is an excellent predictor of hepatic IO in TDT. Age adjustment enhanced its myocardial IO predictive accuracy. Likelihood ratio-based SF cut-off values may help clinicians in risk stratification and treatment decision-making., Limitations: The laboratory data were gathered retrospectively and although the risk of selection bias for T2*-MRI examination is thought to be low, it cannot be ignored., Conflict of Interest: None.

Background: Thalassemia trait (TT) and iron deficiency anemia (IDA) are the most common conditions of microcytic hypochromic anemia (MHA) in pregnant women. Accurate discrimination between TT and IDA is an important issue, and better methods are urgently needed. Although considerable RBC formulas and indices have been developed since 1973, distinguishing between IDA and TT is still a challenging problem due to the diversity of various anemic populations. To address this problem, we assessed the diagnostic function of 43 different differential formulas in patients with microcytic anemia by using accuracy measures and recommending a new log-based differential formula., Methods: The data of 430 pregnant women (229 with TT and 201 with IDA) were enrolled, and 44 formula performances were evaluated with receiver operating characteristic (ROC) analysis., Results: The newly introduced logarithm-based formula XS-1 performs better than the general discriminant index with sensitivity and specificity of 82.10 and 89.05, which are better than other formulas. In the pregnant population, the Shine and Lal and Roth..SVM. formulas have shown excellent performance, while other formulas showed poorer discriminative abilities in our study than in the original authors., Conclusion: The logarithm-based formula XS-1 can be used to screen thalassemia and iron deficiency anemia during the first trimester. Considering the particularity of pregnancy, medical personnel in different regions should choose a screening formula similar to that of the local region and population when identifying thalassemia in pregnancy. Any formula should be independently verified locally before use. For the convenience of the health care team and experimental scientists, a web-based tool has been established at http://yyy.yiyiy.top/XS-1/ by which users can easily get their desired screening test result without going through the underlying mathematical and computational details., (© 2023. The Author(s).)

Background: Diabetes mellitus is one of the most common diseases worldwide with significant morbidity and mortality. HbA1c remains one of the most important methods for diagnosis and monitoring of the disease. Since HbA1c is a reflection of the glucose attached to red blood cells, factors affecting hemoglobin and red blood cells' half-life can influence HbA1c measurements., Objective: This study aims to evaluate the effect of different types of anemia including iron deficiency anemia, sickle cell anemia, β -thalassemia trait, and megaloblastic anemia on HbA1c levels in a tertiary hospital over the past 6 years (2016-2022)., Method: This is a retrospective chart review study of 324 patients including those with one of the four types of anemia mentioned above and a control group. The control group were healthy adults with normal HbA1c and hemoglobin, who were not known to have diabetes or anemia. Patients with diabetes or prediabetes based on self-reporting or elevated fasting, random blood sugar, or 2 hours post-prandial blood glucose were excluded., Results: The mean HbA1c levels were significantly higher in sickle cell anemia at 5.83% (95% CI = 5.39-6.28) and in iron deficiency anemia at 5.75% (95% CI = 5.68-5.82) when compared to the control group at 5.32% (95% CI = 5.22-5.41). However, the mean HbA1c levels in megaloblastic anemia were 5.38% (95% CI = 5.26-5.5) and 5.45% (95% CI = 5.21-5.69) in beta thalassemia trait, which were not significantly different when compared to the control group. HbA1c significantly decreased from 5.75 to 5.44% after treatment in the iron-deficient group with a p-value of < 0.001. Moreover, lower hemoglobin and higher red cell distribution width correlated with higher HbA1c levels in patients with sickle cell anemia., Conclusion: This study found a significant increase in HbA1c levels in iron deficiency anemia and sickle cell disease in patients not known to have diabetes. However, there was no significant effect in those patients with β-thalassemia trait and megaloblastic anemia. Treatment of iron deficiency anemia significantly decreased the HbA1c level, bringing it back to normal., (© 2023. The Author(s).)

Objective: Single-molecule real-time technology (SMRT) is a sequencing technology using the DNA polymerases and fluorescently tagged nucleotides to accurately sequence DNA strands. The purpose of this study was to evaluate the detection accuracy of SMRT for identification of the Hong Kongαα (HKαα) thalassemia allele., Methods: We conducted a blinded study of 33 samples of known HKαα alleles. These alleles were detected using SMRT to evaluate accuracy., Results: We conducted a blinded study of 33 known HKαα samples and found all HKαα variants detected by SMRT to be concordant with those independently assigned by gap-polymerase chain reaction (gap-PCR), reverse dot blot hybridization, and 2-round nested PCR. In addition, SMRT detected 2 β-thalassemia variants that were missed by conventional techniques., Conclusion: The results demonstrate that SMRT offers a higher detection accuracy of thalassemia rare and new loci. It is an efficient, reliable, and broad-spectrum test that can be widely used for thalassemia screening in the clinic., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society for Clinical Pathology.)

Objective: To determine the safety and outcomes of trans-abdominal chorionic villus sampling technique., Study Design: Observational study., Place and Duration of Study: Departments of Obstetrics and Gynaecology, PNS Shifa Karachi, Pak-Emirates Military Hospital Rawalpindi and CMH Lahore, from 2005-2020.   Methodology: A total of 1530 consecutive chorionic villus samplings (CVS) were performed on pregnant females between 10-20 weeks of gestation using the transabdominal approach. Patients were subjected to integrated, stepwise sequential screening. Analysis of data was based on demographic features, indications for sampling, gestational age, attempts of CVS, needle aspiration time, assessment, placental location, sample yield, complications, pain estimation by visual analogue scale (VAS), CVS culture results and pregnancy outcomes., Results: The most common indication for CVS was couple having thalassemia traits and history of having a thalassemia major child previously (55.2%). Pain was the most common complication (64.1%). Procedure-related pregnancy loss (considered to be till 20 weeks of gestation) was observed in two cases (0.1%) only. The most common abnormal karyotype was found to be β-Thalassemia trait (23.6%) followed by β-Thalassemia major (22.1%) and Trisomy 21(16.8%). No abnormality was detected in 33.5% of the cases. Five hundred and eighty-nine (38.4%) interruptions of pregnancies were done on the basis of CVS results., Conclusion: CVS is a safe and useful technique for sampling in prenatal diagnosis of genetic disorders, markedly affecting the management., Key Words: Chorionic villus sampling, Pre-natal diagnosis, Karyotype.

Context: Z score defines the shift of an observed value from the mean., Aims: By determining the direction of this shift and its absolute value for mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), one can quickly screen the hemogram for any spurious results in RBC parameters and also predict the type of anemia. This is because MCH and MCHC are derived parameters (from Hb, RBC, MCV) and thereby reflect the true as well as false changes in an erythrogram., Materials and Methods: A total of 975 hemograms were studied retrospectively. Basic statistical formulae using mean and standard deviation were applied to calculate z scores for MCH and MCHC. Results obtained were compared with the standard method and validated by an independent cohort of 100 random samples run on a different machine., Results and Statistical Analysis: Z score was found to be statistically significant (p <.001) in diagnosing iron deficiency anemias, megaloblastic anemias, hemolytic anemias, regenerative anemias, anemia of chronic disease and spurious findings. Z score was not significant (p = 0.9) in predicting beta thalassemia trait. The sensitivity was low for the differentials of microcytic hypochromic anemias., Conclusions: Despite this, Z score can be of immense help to the clinicians and pathologists in making quick interpretation of the underlying red cell abnormalities. Also, it can be used as a quality assessment tool in hematology laboratories taking pre analytical and analytical factors into account., Competing Interests: None