Unresolved laboratory issues of the heterozygous state of ß-thalassemia: a literature review.

Although considered a mild clinical condition, many laboratory issues of the carrier state of ß-thalassemia remain unresolved. Accurate laboratory screening of ß-thalassemia traits is crucial for preventing the birth of a ß-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 ß 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.

Enhancing thalassemia gene carrier identification in non-anemic populations using artificial intelligence erythrocyte morphology analysis and machine learning.

BACKGROUND: Non-anemic thalassemia trait (TT) accounted for a high proportion of TT cases in South China. OBJECTIVE: To use artificial intelligence (AI) analysis of erythrocyte morphology and machine learning (ML) to identify TT gene carriers in a non-anemic population. METHODS: Digital morphological data from 76 TT gene carriers and 97 controls were collected. The AI technology-based Mindray MC-100i was used to quantitatively analyze the percentage of abnormal erythrocytes. Further, ML was used to construct a prediction model. RESULTS: Non-anemic TT carriers accounted for over 60% of the TT cases. Random Forest was selected as the prediction model and named TT@Normal. The TT@Normal algorithm showed outstanding performance in the training, validation, and external validation sets and could efficiently identify TT carriers in the non-anemic population. The top three weights in the TT@Normal model were the target cells, microcytes, and teardrop cells. Elevated percentages of abnormal erythrocytes should raise a strong suspicion of being a TT gene carrier. TT@Normal could be promoted and used as a visualization and sharing tool. It is accessible through a URL link and can be used by medical staff online to predict the possibility of TT gene carriage in a non-anemic population. CONCLUSIONS: The ML-based model TT@Normal could efficiently identify TT carriers in non-anemic people. Elevated percentages of target cells, microcytes, and teardrop cells should raise a strong suspicion of being a TT gene carrier.

Reliability of hemoglobin A2 value as measured by the Premier Resolution system for screening of ß-thalassemia carriers.

OBJECTIVES: Accurate quantification of hemoglobin (Hb) A2 is vital for diagnosing ß-thalassemia carriers. This study aimed to assess the precision and diagnostic utility of HbA2 measurements using the new high-performance liquid chromatography (HPLC) method, Premier Resolution, in comparison to capillary electrophoresis (CE). METHODS: We analyzed 418 samples, previously identified as A2A by CE, using Premier Resolution-HPLC. We compared the results, established correlations, and determined an optimal HbA2 cutoff value for ß-thalassemia screening. Additionally, we prospectively evaluated the chosen cutoff value in 632 samples. Mutations in the ß- and α-globin genes were identified using polymerase chain reaction (PCR) techniques and DNA sequencing. RESULTS: HbA2 levels were consistently higher with Premier Resolution, yet there was a significant correlation with CE in all samples (bias, -0.33; r, 0.991), ß-thalassemia (bias, -0.27; r, 0.927), and non-ß-thalassemia carriers (bias, -0.36; r, 0.928). An HbA2 cutoff value of ≥4.0 % for ß-thalassemia screening achieved 100 % sensitivity and 99.6 % specificity. Further validation yielded sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 97.3 , 99.8, 97.3, 99.8, and 99.7 %, respectively. We also identified a rare ß-Hb variant, Hb La Desirade [HBB:c.389C>T], associated with ß-thalassemia and co-inherited with a single α-globin gene. CONCLUSIONS: The Premier Resolution HPLC is a reliable and accurate method for routine ß-thalassemia carrier screening, aligning with existing CE methods.

New Born Screening of Hemoglobinopathies in a Center Tunisian Population.

Sickle cell diseases, ß-thalassemia, and other hemoglobinopathies are common in Africa. Their distribution differs from one region to another. There are higher frequencies in Western and Northern Africa. Their clinical complications presented a real public health problem in each country. For this, early treatment can improve the severity of these diseases. Hemoglobinopathies targeted by screening are associated with SCD, ß, and α thalassemia. Our study aim is to report our experience with newborn screening for hemoglobinopathy in Tunis. The 156 newborn's cord blood was collected at the time of childbirth in the center region (Farhat Hached Hôspital). We opted for hemoglobin exploration to achieve maximum efficiency and effectiveness in screening. After that, all patients suspected to have hemoglobinopathies are affected by molecular investigation. Our findings showed the presence of some hemoglobinopathies such as ß-thalassemia and α-thalassemia with the following frequencies: 12% and 0.33%. The molecular results show the presence of HBB: c.93-21G>A, IVS-I-110G>A, HBBc. -106G>A -56G>C, HBBc.404T>C, Hb Yaounde described for the first time in Tunisia and α 3,7 . In conclusion, newborn screening diagnoses neonates with different examples of hemoglobinopathies, which will be beneficial not only for the care of the child but also for genetic counseling of the potential risk's parents.

Research on the clinical factors of cardiac iron deposition in children with beta-thalassemia major.

Magnetic resonance imaging (MRI) T2* is the gold standard for detecting iron deposition in cardiac tissue, but the technique has limitations and cannot be fully performed in paediatric thalassemia patients. The aim of this study was to analyse clinical data to identify other predictors of cardiac iron deposition. A retrospective analysis was performed on 370 children with ß-TM. According to the cardiac MRI results, patients were allocated to a cardiac deposition group and noncardiac deposition group. Multivariate analysis revealed that genotype and corrected QT interval were associated with cardiac iron deposition, indicating that the-ß0/ß0 genotype conferred greater susceptibility to cardiac iron deposition. Receiver operating characteristic curve (ROC) analysis was performed, and the area under the curve (AUC) of genotype was 0.651. The AUC for the corrected QT interval was 0.711, at a cut-off value of 418.5 ms. ROC analysis of the combined genotype and corrected QT interval showed an AUC of 0.762 with 81.3% sensitivity and 64.7% specificity. Compared to patients with the ß+/ß+ and ß0ß+ genotypes, ß0ß0 children with ß-TM were more likely to have cardiac iron deposition.  Conclusion: The genotype and QTc interval can be used to predict cardiac iron deposition in children with ß-TM who are unable to undergo MRI T2 testing.

Peripheral blood circular RNA circ-0008102 may serve as a novel clinical biomarker in beta-thalassemia patients.

Circular RNA circ-0008102 has previously been found dysregulated in ß-thalassemia (ß-thal) in circRNAs microarray (GSE196682 and GSE241141). Our study is aimed at identifying whether circ-0008102 could be a novel biomarker in ß-thal. The peripheral blood of pediatric ß-thal patients with (n = 39) or without (n = 20) blood transfusion and healthy controls (n = 30) was selected. qRT-PCR, ROC curve analysis, Spearman correlation analysis, and FISH were used to analyze clinical value of circ-0008102. qRT-PCR confirmed that circ-0008102 expression in pediatric ß-thal patients without blood transfusion was significantly higher. ROC curves analysis showed that the AUC of circ-0008102 for differentiating patients without blood transfusion from patients with blood transfusion and healthy controls with an AUC of 0.733 and 0.711. Furthermore, circ-0008102 expression was positively correlated with the levels of RBC, HbF, ß-globin, and γ-globin mRNA, but was negatively corrected with the levels of HbA and Cr. circ-0008102 was mainly located in the cytoplasm. circ-0008102 could induce the activation of γ-globin and negatively regulate the expression of the five highest-ranking candidate miRNAs (miR-372-3p, miR-329-5p, miR-198, miR-152-5p, and miR-627-3p) in K562 cells. CONCLUSION: We demonstrate that peripheral blood upregulated circ-0008102 may serve as a novel clinical biomarker for pediatric ß-thal without blood transfusion. WHAT IS KNOWN: • CircRNAs are known to be involved in various human diseases, and several circRNAs are regarded as a class of promising blood-based biomarkers for detection of ß-thal. • CircRNAs exert biological functions by epigenetic modification and gene expression regulation, and dysregulated circRNAs in ß-thal might be involved in the induction of HbF in ß-thal. WHAT IS NEW: • Peripheral blood circ-0008102 maybe serve as a novel clinical biomarker for detection of pediatric ß-thal without blood transfusion. • Circ-0008102 participates in the pathogenesis of ß-thal through regulating γ-globin expression, and negatively regulates the expression of miR-372-3p, miR-329-5p, miR-198, miR-152-5p and miR-627-3p.

Graves' Disease: Acquired Cause of a Moderate Increase in Hemoglobin A2 Level.

BACKGROUND: Hyperthyroidism can lead to diverse hematological disorders, such as microcytosis and a mild increase in hemoglobin A2 fraction. METHODS: This study reported a 31-year-old woman of Moroccan origin recently diagnosed with Graves' disease. Her blood tests revealed microcytosis, hypochromia, and a normal ferritin level. A phenotypic analysis of hemo-globin was performed using two techniques: capillary electrophoresis and reversed-phase high performance liquid chromatography. RESULTS: Both techniques indicated a slight increase in hemoglobin A2 level. These results initially suggested het-erozygous beta-thalassemia, eventually correlating with the concurrent presence of Graves' disease, as evidenced by the normalization of hemoglobin A2 level following treatment. CONCLUSIONS: This case highlights the importance of having clinical, biological, and therapeutic data for a relevant interpretation of a phenotypic hemoglobin study.

Very low serum IGF-1 levels are associated with vertebral fractures in adult males with beta-thalassemia major.

PURPOSE: Patients with beta-thalassemia major (BTM) often develop several endocrine disorders due to chronic iron overload. They are also prone to osteoporosis and vertebral fractures. Plasmatic insulin-like growth factor-1 (IGF-1) levels are often low in subjects with BTM, which origin is multifactorial. The aim of this study was to evaluate a possible relationship between serum IGF-1 levels and the presence of osteoporosis and/or vertebral fractures. METHODS: We retrospectively evaluated the occurrence of vertebral fractures in 30 adult male patients affected by BTM (mean age 43.3 ± 7.9 years) with low serum IGF-1 (median value 52.4 ng/ml, 38.5-83.4). Only 6 of them (20.0%) were diagnosed with GH deficiency (GHD) after GHRH/arginine stimulation test, while 23 (76.7%) had osteoporosis and 12 (40.0%) had known vertebral fractures. All patients except one also showed at least one endocrine disorder. RESULTS: Serum IGF-1 was significantly lower in BTM patients with vertebral fractures compared to patients without vertebral fractures (U = 41.0, p = 0.005) while it was not significantly different between patients with low bone mass compared to patients without low bone mass. The diagnosis of GHD was significantly associated with lower serum IGF-1 (p = 0.001) and vertebral fractures (p = 0.002) but not with low bone mass. After ROC analysis, we found that very low IGF-1 (≤ 50.0 ng/dl) was associated with vertebral fractures (sensitivity 83.3%, specificity 75.0%) and was also predictive of GHD (sensitivity 75.0%, specificity 100.0%). CONCLUSION: Our study shows that, in male patients with BTM, serum IGF-1 ≤ 50.0 ng/dl is a marker of vertebral fractures and it is predictive of a diagnosis of GHD.

Multi-criteria decision making to validate performance of RBC-based formulae to screen [Formula: see text]-thalassemia trait in heterogeneous haemoglobinopathies.

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.

Newborn Screening for ß-Thalassemia Identifies a Complex Genotype Involving a Novel ß-Globin Gene Mutation (HBB:c.336dup).

Newborn screening identified a Chinese-Canadian infant who was positive for possible ß-thalassemia (ß-thal). Detailed family studies demonstrated that the proband was a compound heterozygote for the Chinese Gγ(Aγδß)0-thal deletion and a novel frameshift mutation within exon 3 (HBB:c.336dup), and heterozygous for the Southeast Asian α-thal deletion (--SEA/αα). This case illustrates the importance of follow-up molecular testing of positive newborn screening results to confirm the diagnosis and define risks for future pregnancies.

ß0-Thalassemia Caused by a Novel Nonsense Mutation [HBB:c.199A > T].

We report two hemoglobinopathy cases involving a novel ß-thalassemia (ß-thal) nonsense mutation, HBB:c.199A > T. One patient had Hb S/ß-thal, and a second unrelated patient had Hb D-Punjab/ß-thal. The HBB:c.199A > T mutation introduces a premature termination codon at amino acid codon 66 (AAA→TAA) in exon 2, resulting in typical high Hb A2 ß0-thal.

First Report of Filipino ß0-Thalassemia/ß-Thalassemia in a Chinese Family.

A pregnant woman living in Fujian Province, southeastern China, presented due to a risk of having a baby with ß-thalassemia major, during her second pregnancy, since she and her husband were suspected as ß-thalassemia carriers and their affected daughter was a transfusion-dependent patient. Using the common α-thalassemia and ß-thalassemia genotypes test, the pregnant woman was diagnosed as a ß-thalassemia carrier with ßIVS-2 - 654 (C→T)/ßN genotype and her daughter had a homozygosity for IVS - 2 - 654 (C→T) mutation, however, no abnormalities were detected in her husband. SMRT identified a Filipino ß0-deletion in her husband, and MLPA also revealed an unknown deletion in the HBB gene. Electrophoresis showed approximately 350 bp of the PCR product, and the ß-Filipino genotype presented novel fracture fragments ranging from 5,112,884 to 5,231,358 bp, and lacked a 118,475 bp fragment relative to the wild-type sequence. The daughter was therefore diagnosed with the ßIVS-2 - 654 (C→T)/ßFilipino genotype. Prenatal diagnosis with umbilical cord blood at 27th week of gestation showed heteroztgosity for IVS - 2 - 654 (C→T) mutation in the fetus and continued pregnancy was recommended. In conclusion, we identified the Filipino ß0-deletion in a Chinese family, from Fujian area, for the first time, during prenatal screening.

Misdiagnosis of ß-Thalassemia Major Due to Chinese Gγ+(Aγδß)0-Thalassemia Combined with ß0-Thalassemia.

δß-thalassemia is a rare type of thalassemia characterized by increased Hb F levels, including mainly Chinese Gγ(Aγδß)0-thalassemia, Yunnanese Gγ(Aγδß)0-thalassemia, Cantonese Gγ(Aγδß)0-thalassemia in China. Due to the low rate of δß-thalassemia carriers, there are few reports of δß-thalassemia combined with ß-thalassemia causing ß-thalassemia major. Herein, we described the combination of Chinese Gγ(Aγδß)0-thalassemia and ß-thalassemia leading to ß-thalassemia major in a Chinese patient. Hemoglobin analysis was performed by capillary electrophoresis (CE). Routine genetic analysis was carried out by gap-polymerase chain reaction (Gap-PCR) and PCR and reverse dot blot (PCR-RDB). Multiple ligation-dependent probe amplification (MLPA) was used to detect the large deletion, and Gap-PCR confirmed the deletion. A CE result showed an elevated Hb F level of 98.7% and 11.7% in the proband and her mother, but the proband was diagnosed with ßCD17M/ßCD17M using routine genetic analysis. However, her father was heterozygous for CD17 in ß-globin, and her mother was detected as SEA heterozygous. The further analysis presented that the proband had actually missed the diagnosis of Chinese Gγ(Aγδß)0-thalassemia by MLPA and PCR-RDB. Finally, the genotype of the proband was corrected from ßCD17M/ßCD17M to ßCD17M/ßGγ(Aγδß)0. This is the first report of Chinese Gγ(Aγδß)0-thalassemia combined with ß-thalassemia resulting in ß-thalassemia major in China. Screening for δß-thalassemia by Hb analysis could be an effective method.

Identification of a Novel Variant c.163delG in HBB Gene Resulting in a Beta Null Phenotype in a Proband with Thalassemia Intermedia.

A 21-year-old patient presented with a previous medical history of pallor, mild icterus, increased fatigue, low hemoglobin, and abnormal hemoglobin variant analysis with more than 70 transfusions. He was referred for genetic analysis to identify the pathogenic variations in the ß-globin gene. Sanger's sequencing of the proband and his family revealed the presence of a novel frame shift variant HBB:c.163delG in a compound heterozygous state with hemoglobin E (HbE) (HBB:c.79G > A) variant. The father and the sibling of the patient were found to be normal for the HBB gene. Mother was found to be heterozygous for HbE (HBB:c.79G > A) variant. In silico analysis by Mutalyzer predicted that c.163delG variant generated a premature stop codon after seven codons, leading to a truncated protein. FoldX protein stability analysis showed a positive ΔΔG value of 45.27 kcal/mol suggesting a decrease in protein stability. HBB:c.79G > A is a known variant coding for HbE variant, which results in the reduced synthesis of ß-globin chain and shows mild thalassemia. Combined effect of HBB:c.163delG and HBB:c.79G > A variants in the proband might have led to the reduced synthesis of ß-globin chains resulting in a thalassemia intermedia type of clinical manifestation.

A Population-Oriented Genetic Scoring System to Predict Phenotype: A Pathway to Personalized Medicine in Iraqis With ß-Thalassemia.

To assess the roles of genetic modifiers in Iraqi ß-thalassemia patients, and determine whether a genotype-based scoring system could be used to predict phenotype, a total of 224 Iraqi patients with molecularly characterized homozygous or compound heterozygous ß-thalassemia were further investigated for α-thalassemia deletions as well as five polymorphisms namely: rs7482144 C > T at HBG2, rs1427407 G > T and rs10189857 A > G at BCL11A, and rs28384513 A > C and rs9399137 T > C at HMIP. The enrolled patients had a median age of 14 years, with 96 males and 128 females. They included 144 thalassemia major, and 80 thalassemia intermedia patients. Multivariate logistic regression analysis revealed that a model including sex and four of these genetic modifiers, namely: ß+ alleles, HBG2 rs7482144, α-thalassemia deletions, and BCL11A rs1427407 could significantly predict phenotype (major versus intermedia) with an overall accuracy of 83.9%. Furthermore, a log odds genetic score based on these significant predictors had a highly significant area under curve of 0.917 (95% CI 0.882-0.953). This study underscores the notion that genetic scoring systems should be tailored to populations in question, since genetic modifiers (and/or their relative weight) vary between populations. The population-oriented genetic scoring system created by the current study to predict ß-thalassemia phenotype among Iraqis may pave the way to personalized medicine in this patient's group.

Splice Acceptor Mutation [HBB:c.93-2A > T] in a Patient with Hb S/ß0-Thalassemia.

We report a case of Hb S/ß0-thalassemia (Hb S/ß0-thal) in a patient who is a compound heterozygote for the Hb Sickle mutation (HBB:c.20A > T) and a mutation of the canonical splice acceptor sequence of IVS1 (AG > TG, HBB:c.93-2A > T). This is the fifth mutation involving the AG splice acceptor site of IVS1, all of which prevent normal splicing and cause ß0-thal.

Premarital Screening is Pivotal in Reducing the Births of Babies Affected with Thalassemia Major in Iraq.

Thalassemia major is one of the health problems in Iraq, especially in Kurdistan. Pre-marriage mandatory preventive screening program was established in Kurdistan in 2008, which allowed us to study the prevalence of different hemoglobinopathies among newly married young adults in this region. A total of 1154 subjects (577 couples) attending the Koya district, premarital Health center, were screened using red cell indices. Those who had mean corpuscular volume (MCV)<80 fl and mean corpuscular hemoglobin (MCH)<27 pg had high-performance liquid chromatography and iron studies. Out of 1154 individuals that were evaluated, 183 (11.9%) had low MCV and MCH. Of the former 183 subjects, 69 (5.97%) had ß-thalassemia trait, 10 (0.86%) had δß-thalassemia trait, and no other hemoglobinopathies were recorded in our study. There was second-degree consanguinity in 4.7% of all 577 couples. In two couples, both partners had ß-thalassemia trait and both were consanguineous. Both couples decided to separate after counseling. Based on the current study, the role of the premarital screening program in decreasing the number of new thalassemia major cases among the Kurdish population is laudable. Therefore, mandatory premarital screening is advised in all parts of Iraq.

Non-invasive prenatal testing of beta-hemoglobinopathies using next generation sequencing, in-silico sequence size selection, and haplotyping.

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.

Support Vector Machine-Based Formula for Detecting Suspected α Thalassemia Carriers: A Path toward Universal Screening.

The blood counts of α thalassemia carriers (α-thal) are similar to those of ß thalassemia carriers, except for Hemoglobin A2 (Hb A2), 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 ß 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.

[Study of the types of mutations of Thalassemia in Shanghai area].

OBJECTIVE: To analyze the mutations of globin genes among patients suspected for thalassemia from the Shanghai area. METHODS: A total of 4 644 patients diagnosed at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine between June 2016 and December 2019 were selected as the study subjects. The patients were tested for common mutations associated with thalassemia gene by Gap-PCR and reverse dot blotting (RDB). Patients were suspected to harbor rare mutations based on the inconsistency between hematological phenotypes and results of common mutation detection, and were further analyzed by Gap-PCR and Sanger sequencing. RESULTS: Among the 4 644 patients, 2 194 (47.24%) were found to carry common thalassemia mutations, among which 701 (15.09%) were α-thalassemia, 1 448 (31.18%) were ß-thalassemia, and 45 (0.97%) were both α- and ß-thalassemia. Forty six samples were found to harbor rare mutations, which included 17 α-globin gene and 29 ß-globin gene mutations. CD77(CCC>ACC) (HBA2: c.232C>A) of the α-globin gene, NG_000007.3: g.70567_71015del449, codon 102(-A) (HBB: c.308_308delA) and IVS-Ⅱ-636 (A>G) (HBB: c.316-215A>G) of the ß-globin gene were previously unreported new types of globin gene mutations. CONCLUSION: Among the 4 644 patients, the detection rate for common thalassemia mutations was 47.24%, whilst 46 samples were detected with rare gene mutations. The type of gene mutation types were diverse in the Shanghai area. The study has provided more accurate results for genetic diagnosis and counseling.