SUPT5H mutations associated with elevation of Hb A2 level: Identification of two novel variants and literature review.

ß-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 A2 level, generally specified as greater than 3.5 %, is commonly used as a high efficiency index for screening of ß-thalassemia carriers. However, mutations in other genes such as GATA1 and KLF1, could also result in increased Hb A2 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 A2 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 A2 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 ß-thalassemia.

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.

Label-free quantification of host cell protein impurity in recombinant hemoglobin materials.

Quantitative analysis relies on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host cell proteins (HCPs) in bioengineered proteins which are intended for use as protein calibration standards. In this study a purified hemoglobin-A2 (HbA2) protein, obtained through its overexpression in E. coli, was used. Two different materials were produced: natural and U15N-labeled HbA2. For the quantification of impurities, precursor ion (MS1-) intensities were integrated over all E. coli proteins identified and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for an E. coli cell lysate, which had been spiked at known mass ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of just nine proteins, and a minimum of five proteins was estimated to be sufficient to keep the sampling error below 15%. For the studied materials, HbA2 mass fractions (or purities) of 923 and 928 mg(HbA2)/g(total protein) were found with expanded uncertainties (U) of 2.8 and 1.3%, resp. Value assignment by LFQ thus contributes up to about 3% of the overall uncertainty of HbA2 quantification when these materials are used as calibrators. Further purification of the natural HbA2 yielded a mass fraction of 999.1 mg/g, with a negligible uncertainty (U = 0.02%), though at a significant loss of material. If an overall uncertainty of 5% is acceptable for protein quantification, working with the original materials would therefore definitely be viable, circumventing the need of further purification.

Molecular Characterization of Haemoglobin E.

OBJECTIVE: To detect mutation in cases having haemoglobin A2 level >7% on high performance liquid chromatography. METHODS: The cross-sectional, descriptive study was conducted from July 2017 to December 2018 at the Department of Haematology and Human Genetics and Molecular Biology, University of Health Sciences, Lahore, Pakistan, and comprised patients of either gender with haemoglobin A2 ≥7%. The samples were collected from different cities of Punjab in collaboration with the Punjab Thalassemia Prevention Programme, Lahore. The samples were subjected to complete blood count and high performance liquid chromatography using automated haematology analysers and variant-II beta thalassemia short programme, respectively. To analyse haemoglobin E mutations at the molecular level, polymerase chain reaction-restriction fragment length polymorphism (PCR_RFLP) was performed using a type IIS restriction endonuclease known as Mnl1 (derived from Moraxella nonliquefaciens) to cleave DNA at specific sites and the results were further confirmed on randomly selected samples using Sanger sequencing. Data was analysed using SPSS 25. RESULTS: Of the 39 patients, 15(38.5%) were males and 24(61.5%) were females. The overall median age was 14 (23) years. There were 29 (74.4%) patients with thalassemia family history, and 22(56.4%) had a positive family history of transfusion related to thalassemia, while no patient had a family history of iron therapy. The median haemoglobin A, haemoglobin A2 and haemoglobin F levels were 72.2 (65.2-79.1) %, 26.6 (19.1-34.0) % and 0.9 (-0.8-2.6) %, respectively. After molecular investigation, HbAE mutation was found in 23(59%) patients, while wild type HbAA genotype was found in 16(41%). The heterozygous HbE mutation was present in 23(59%) patients. CONCLUSIONS: Frequently missed/undiagnosed cases of haemoglobin E that co-elute with haemoglobin A2 in the same high performance liquid chromatography window were detected among those with haemoglobin A2 ≥7%.

Prospective screening for δ-hemoglobinopathies associated with decreased hemoglobin A2 levels or hemoglobin A2 variants: A single center experience.

BACKGROUND: δ-hemoglobinopathies may lead to misdiagnosis of several thalassemia syndromes especially ß-thalassaemia carrier, it is important to evaluate the δ-globin gene defects in areas with high prevalence of globin gene disorders. We describe a prospective screening for δ-hemoglobinopathies in a routine setting in Thailand. METHODS: Study was done on a cohort of 8,471 subjects referred for thalassemia screening, 317 (3.7%) were suspected of having δ-globin gene defects due to reduced hemoglobin (Hb) A2 levels and/or appearance of Hb A2-variants on hemoglobin analysis. Hematologic and DNA analysis by PCR and related assays were carried out. RESULTS: DNA analysis of δ-globin gene identified seven different δ-globin mutations in 24 of 317 subjects (7.6%). Both known mutations; δ-77(T>C) (n = 3), δ-68(C>T) (n = 1), δ-44(G>A) (n = 8), Hb A2-Melbourne (n = 5), δIVSII-897(A>C) (n = 5), and Hb A2-Troodos (n = 1) and a novel mutation; the Hb A2-Roi-Et (n = 1) were identified. This Hb A2-Roi-Et, results from a double mutations in-cis, δCD82(AAG>AAT) and δCD133(GTG>ATG), was interestingly found in combination with an in trans, 12.6 kb deletional δß0-thalassemia in an adult Thai woman who had no Hb A2 and elevated Hb F. A multiplex-allele-specific PCR was developed to detect these novel δ-globin gene defects. CONCLUSIONS: The result confirms a diverse heterogeneity of δ-hemoglobinopathies in Thailand which should prove useful in a prevention and control program of thalassemia in the region.

[Causes of Abnormal Hemoglobin Electrophoresis].

OBJECTIVE: To investigate the possible causes of abnormal hemoglobin electrophoresis results. METHODS: The hemoglobin electrophoresis results of 5 696 patients in the First Affiliated Hospital of Chengdu Medical College from September 2018 to July 2021 were collected, and the abnormal results and clinical significance were analyzed. RESULTS: The results of 486 patients (accounting for 8.53%) were abnormal, of which 300 cases had increased HbA2, 135 cases had decreased HbA2, 44 cases had increased F alone, and 7 cases had abnormal hemoglobin bands. Among the 486 patients, 246 patients were thalassemia gene positive (the positive rate was 50.62%), including 29 cases of α thalassemia, 208 cases of ß thalassemia and 9 cases of αß thalassemia. Among the patients with elevated HbA2, 68.67% were detected ß thalassemia, 3.00% αß thalassemia, 9.33% were suspected to be caused by macrocytosis, 6.33% by thyroid dysfunction, and 12.67% by uncertainty of the method. Among the patients with reduced HbA2, 21.48% were detected α thalassemia, 60.00% iron deficiency anemia, 8.15% were suspected to be caused by thyroid dysfunction, and 10.37% by uncertainty of the method. Among the patients with elevated F alone, the results of thalassemia gene detection were negative, 40.91% of them were suspected to be caused by macrocytosis, 27.27% by hereditary persistence of fetal hemoglobin, 29.55% by special physiological condition of pregnant women, and 2.27% by hyperthyroidism. Abnormal hemoglobin bands were detected in 7 patients, including 4 cases of hemoglobin D, 2 cases of hemoglobin E, and 1 case of hemoglobin J. CONCLUSION: Thalassemia, iron deficiency anemia, macrocytosis such as megaloblastic anemia and non-severe aplastic anemia, thyroid dysfunction, hereditary persistence of fetal hemoglobin, abnormal hemoglobin diseases, the uncertainty of the method are all important causes of abnormal hemoglobin electrophoresis results. In clinical work, the patient's indicators should be comprehensively analyzed to determine the possible cause.

Factors affecting the evaluation and use of a hemoglobin A2 method - Lot-to-lot variation, long-term deviation and carry-over.

BACKGROUND: We compared two Hb A2 methods, high pressure liquid chromatography and capillary zone electrophoresis, at two occasions. In 2014 the methods showed good agreement, while in 2020 HPLC results were clearly higher than CE results. This finding prompted us to investigate the external quality assessment (EQA) outcome and our total patient results obtained by high pressure liquid chromatography over several years. METHODS: The methods compared were Bio-Rad Variant II Beta-Thal Short Program (HPLC) and Sebia Capilllarys Flex Piercing (CE). RESULTS: Our annual patient results obtained by HPLC increased significantly from 2014 to 2020. A similar trend was also seen in our EQA results. When patient results were grouped according to different reagent lots it became evident that method comparisons might be severely affected by lot-to-lot variation. We also noticed that samples analyzed with the HPLC method following a sample containing a high proportion of Hb E where prone to give falsely increased Hb A2 results. CONCLUSION: Standardization of the measurement of Hb A2 is urgently needed. Furthermore, the lot-to-lot variation must be minimized. While waiting for these improvements each laboratory ought to repeatedly evaluate their distribution of patient Hb A2 results.

Effect of megaloblastic anemia on hemoglobin A2 and diagnosis of ß-thalassemia trait.

Context: ß-thalassemia trait is usually diagnosed by raised hemoglobin A2 (HbA2). The presence of megaloblastic anemia can cause an increase in HbA2 and create a diagnostic dilemma. Here, we have analyzed the effect of vitamin B12 and folic acid supplementation on HbA2 and diagnosis of ß-thalassemia trait in cases of megaloblastic anemia with raised HbA2. Materials and Methods: Cases of megaloblastic anemia with raised HbA2 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 HbA2 value, the cases were diagnosed as normal, borderline raised HbA2, or ß-thalassemia trait. Pre- and post-treatment values of red cell parameters and HbA2 were analyzed. Results: There was a significant decrease in HbA2 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 HbA2% showed a significant difference between the thalassemic and normal groups. Conclusions: Megaloblastic anemia can lead to false-positive diagnosis of ß-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 HbA2. Red cell parameters are not helpful to suspect ß-thalassemia trait in presence of megaloblastic anemia. However, HbA2% on HPLC can be a useful parameter to suspect or exclude ß-thalassemia trait in cases of megaloblastic anemia.

Work-up of Patients with Decreased Hemoglobin A2 Identified by Capillary Zone Electrophoresis: A North American Institutional Experience.

OBJECTIVE: Isolated low hemoglobin A2 (HbA2) is rarely encountered in our clinical practice using capillary zone electrophoresis. The study goal was to characterize the work-up at our institution of patients with low HbA2. METHODS: Patients with low HbA2 and a control cohort with normal capillary zone electrophoresis were identified and relevant information extracted from the medical record. RESULTS: Of 44 patients with isolated decreased HbA2, 28 (64%) had corresponding complete blood count/ferritin values. Compared to control patients, patients with low HbA2 were more likely to have iron deficiency and demonstrated a more microcytic, hypochromic blood picture. However, 46% (13/28) of patients with low HbA2 and ferritin for evaluation did not have iron deficiency. Only 2 patients had genetic testing. CONCLUSION: This study redemonstrates the association between low HbA2 and iron deficiency and reinforces the need for iron indices to interpret capillary zone electrophoresis results. Our study population showed incomplete or absent iron studies in most cases.

A stepwise haematological screening and whole-exome sequencing reveal multiple mutations from SUPT5H causing an elevation of Hb A2 from a cohort of 47336 individuals.

INTRODUCTION: Though an increase in Hb A2 is one of the most key markers of ß-thal carriers, a few independent cases are reported to show elevated Hb A2 levels caused by mutations in other genes beyond ß-globin gene. METHODS: We reviewed the haematological indices of 47336 individuals to analyse the phenotype-genotype correlation and identified 1439 individuals (3.04%) positive in the elevation of Hb A2 . Globin and KLF1 genes analysis was performed, and further whole-exome sequencing was carried to dissect the genetic causes of those positive samples without ß-thalassemic or KLF1 mutations. RESULTS: Of these 1439 individuals with elevated Hb A2 , 1381 had a molecular defect in globin genes, and most were ß-thalassemic mutation; 10 had a molecular defect in KLF1 gene. Finally, among the 38 individuals without ß-thalassemic or KLF1 mutations, 7 were identified to carried a loss-of-function mutation in SUPT5H. CONCLUSION: This study has provided a mutation spectrum of SUPT5H in a cohort screening leading to the elevation of Hb A2 . According to the previous observations that individuals with a combination of ß-thal mutation and a SUPT5H variant might present moderate ß-thaelassemia, these findings emphasized the importance of comprehensive molecular diagnosis to prevent birth defects of ß-thaelassemia caused by rare mutations from modifier genes.

Causes of Abnormal Hemoglobin Electrophoresis / 中国实验血液学杂志

OBJECTIVE@#To investigate the possible causes of abnormal hemoglobin electrophoresis results.@*METHODS@#The hemoglobin electrophoresis results of 5 696 patients in the First Affiliated Hospital of Chengdu Medical College from September 2018 to July 2021 were collected, and the abnormal results and clinical significance were analyzed.@*RESULTS@#The results of 486 patients (accounting for 8.53%) were abnormal, of which 300 cases had increased HbA2, 135 cases had decreased HbA2, 44 cases had increased F alone, and 7 cases had abnormal hemoglobin bands. Among the 486 patients, 246 patients were thalassemia gene positive (the positive rate was 50.62%), including 29 cases of α thalassemia, 208 cases of β thalassemia and 9 cases of αβ thalassemia. Among the patients with elevated HbA2, 68.67% were detected β thalassemia, 3.00% αβ thalassemia, 9.33% were suspected to be caused by macrocytosis, 6.33% by thyroid dysfunction, and 12.67% by uncertainty of the method. Among the patients with reduced HbA2, 21.48% were detected α thalassemia, 60.00% iron deficiency anemia, 8.15% were suspected to be caused by thyroid dysfunction, and 10.37% by uncertainty of the method. Among the patients with elevated F alone, the results of thalassemia gene detection were negative, 40.91% of them were suspected to be caused by macrocytosis, 27.27% by hereditary persistence of fetal hemoglobin, 29.55% by special physiological condition of pregnant women, and 2.27% by hyperthyroidism. Abnormal hemoglobin bands were detected in 7 patients, including 4 cases of hemoglobin D, 2 cases of hemoglobin E, and 1 case of hemoglobin J.@*CONCLUSION@#Thalassemia, iron deficiency anemia, macrocytosis such as megaloblastic anemia and non-severe aplastic anemia, thyroid dysfunction, hereditary persistence of fetal hemoglobin, abnormal hemoglobin diseases, the uncertainty of the method are all important causes of abnormal hemoglobin electrophoresis results. In clinical work, the patient's indicators should be comprehensively analyzed to determine the possible cause.

RS12574989 and haplotype associated with α/ß-chain imbalance and population HbA2 reduction.

Determining the associated relationship of genotype and phenomenon would benefit the understanding of disease and renew disease intervention means. 14,518 patients who underwent haemoglobin electrophoresis from June 2020 to December 2020 were enrolled in our study, and additional data including sex, age and routine blood examination results were collected. We focused on individuals with normal red blood cell indices and no common thalassemia pathogenic mutation and selected three groups for the following study: the control group (2.5% ≤ HbA2 ≤ 3.5%), the HbA2 under 2.5 group (HbA2 < 2.5%) and the HbA2 under 2.4 group (HbA2 < 2.4%). Four regions of ß-globin regulation were sequenced. Statistical analysis was conducted to compare the collected information of the three groups and the genotype distributions in the control group and sequenced group. The HbA2 under 2.5 group was characterized by a majority of females and lower red blood cell counts and haemoglobin compared with the control group. There were genotypes associated with the grouping as the T of rs12574989 and TTTAGC of the haplotype were significantly increased in the HbA2 under 2.4 group and CTTAGC was significantly decreased in the HbA2 under 2.4 group. This study demonstrated that the genotypes of the population associated with HbA2 were reduced in southern China.

A New Mutation, Hb A2-Canakkale [δ10(A7)Ala→Val; HBD: c.32C>T], and Other Well-Known δ Variants Identified in a Selected Cohort with Low Hb A2 Levels.

Hemoglobinopathies are the most common single-gene disorders, and ß-thalassemia (ß-thal) imposes a tremendous health burden on Turkey. Thus, premarital carrier screening is obligatory in Turkey, as it is in some other countries. As a result of this mandatory procedure, at routine clinical checkups, many individuals who had undergone premarital screening but did not have any clinical symptoms and/or hematological findings, have compulsorily been required to undergo further evaluation due to abnormal levels of hemoglobin (Hb) fractions (Hb A, Hb A2 and Hb F). Many consequences, such as mutations in unrelated gene(s) or someone's nutritional status, have been reported to affect the Hb fractions levels. In the present study, we aimed to determine whether HBD has a molecular causative role in patients with low Hb A2 levels (below 1.8%). The study was conducted with 20 individuals with low Hb A2 levels who had applied to our outpatient clinic. All DNA samples were analyzed for the HBD gene. Nineteen of the 20 subjects were diagnosed to carry a mutation with one of four different δ-globin variants. Three of them had been described previously [Hb A2-Yialousa (HBD: c.82G>T), Hb A2-Bornova (HBD: c.350G>C) and Hb A2-Yokoshima (HBD: c.77G>A)]. The novel [δ10(A7)Ala→Val, HBD: c.32C>T] mutation was defined as a new δ variant and reported to the HbVar database as Hb A2-Canakkale. In conclusion, the molecular characterization of Hb A2 low levels has been suggested to be significant for a definite diagnosis and counseling.

Establishment of pregnant-specific intervals for hemoglobin (Hb) A2, HbF and cut-off points for HbA2 for thalassemia in Chongqing, China.

OBJECTIVES: To analyze pregnant-specific intervals for hemoglobin A2 (HbA2), hemoglobin fetal (HbF), and cut-off points of HbA2 for thalassemia in Chongqing, China. METHODS: Between September 2015 and April 2019, the study recruited 10039 individuals of reproductive age. Of which, 4399 healthy normal individuals were selected to determine reference values for HbA2 and HbF. The remaining 5640 individuals suspected of thalassemia were included to explore the cut-off points of HbA2 for thalassemia. RESULTS: The reference values of HbA2 in males was 2.3-3.2%, in females was 2.1-3.1%, and in pregnant women was 1.9-3.1%. While the reference values of HbF in males was 0.0-0.0%, in females was 0.0-0.9%, and in pregnant women was 0.0-4.3%. Approximately 2.3% cut-off points for pregnant women was determined to be optimal for α-thalassemia screening. In the entire group, 2.5% was best for all α-thalassemia screenings. The cut-off for ß-thalassemia screening using HbA2 was 3.2% for the entire group. CONCLUSION: The reference interval of HbA2 for pregnant females group was significantly lower than other groups. Therefore, we recommend cut-off points of HbA2 for α-thalassemia at 2.3% for pregnant women. While partitioning was not needed due to gender. Gender and pregnancy had little effect on the cut-off points of HbA2 for ß-thalassemia carrier.

[Genetic Analysis of Thalassemia in 22 940 Pregnant Women in Xiangxi Tujia and Miao Autonomous Prefecture].

OBJECTIVE: To investigate the incidence and types of thalassemia in Xiangxi Tujia and Miao Autonomous Prefecture. METHODS: Automatic capillary electrophoresis was used to screen the thalassemia phenotypes of 22 940 blood samples of pregnant women and puerperants collected in our hospital and some other medical institutions in the prefecture during 2017-2019, among which there were 3 356 cases of Tujia ethnicity, 2 821 cases of Miao ethnicity, and 2 233 cases of Han ethnicity included, whose ethnicity were indicated. The samples with positive result would undergo further genetic testing. RESULTS: There were 2 314 cases of suspicious thalassemia were screened from 22 940 cases by the electrophoresis, thus the positive rate was 10.1% (hematological phenotypes from some other institutions were not included). Specifically, there were 1 706 cases with HBA2 less than 2.5%, 255 cases with HBA2 ranged from 2.5% to 3.5%, which displayed abnormal hematology (MCV or/and MCH) or other abnormal bands, and 353 cases with HBA2>3.5%. There were 436 suspected positive patients in 2 314 suspicious samples received further thalassemia gene testing in our hospital, among them 48 cases were diagnosed with α-thalassemia, 85 cases with ß-thalassemia, and 2 cases as compound type. The positive diagnosis rate of α-thalassemia gene test was 11.0%, ß-thalassemia was 19.4%, and positive pregnant women was 31.0%. CONCLUSION: The positive rate of thalassemia screening in Xiangxi Autonomous Prefecture is roughly the same as that in other regions of Hunan. The positive predictive value of ß-thalassemia screening is as high as 86%. Compared with the missed screening data, it is recommended to use hematology (MCV, MCH) method combined with capillary hemoglobin electrophoresis for thalassemia screening.

Importance of Sequencing HBA1, HBA2 and HBB Genes to Confirm the Diagnosis of High Oxygen Affinity Hemoglobin.

High oxygen affinity hemoglobin (HOAH) is the main cause of constitutional erythrocytosis. Mutations in the genes coding the alpha and beta globin chains (HBA1, HBA2 and HBB) strengthen the binding of oxygen to hemoglobin (Hb), bringing about tissue hypoxia and a secondary erythrocytosis. The diagnosis of HOAH is based upon the identification of a mutation in HBA1, HBA2 or HBB in specialized laboratories. Phenotypic studies of Hb are also useful, but electrophoretic analysis can be normal in 1/3 of cases. The establishment of the dissociation curve of Hb can be used as another screening test, a shift to the left indicating an increased affinity for Hb. The direct measurement of venous P50 using a Hemox Analyzer is of great importance, but due to specific analytic conditions, it is only available in a few specialized laboratories. Alternatively, an estimated measurement of the P50 can be obtained in most of the blood gas analyzers on venous blood. The aim of our study was therefore to determine whether a normal venous P50 value could rule out HOAH. We sequenced the HBB, HBA1 and HBA2 genes of 75 patients with idiopathic erythrocytosis. Patients had previously undergone an exhaustive medical check-up after which the venous P50 value was defined as normal. Surprisingly, sequencing detected HOAH in three patients (Hb Olympia in two patients, and Hb St Nazaire in another). A careful retrospective examination of their medical files revealed that (i) one of the P50 samples was arterial; (ii) there was some air in another sample; and (iii) the P50 measurement was not actually done in one of the patients. Our study shows that in real life conditions, due to pre-analytical contingencies, a venous P50 value that is classified as being normal may not be sufficient to rule out a diagnosis of HOAH. Therefore, we recommend the systematic sequencing of the HBB, HBA1 and HBA2 genes in the exploration of idiopathic erythrocytosis.

[The value of combined detection of HbA2 and HbF for the screening of thalassemia among individuals of childbearing ages].

OBJECTIVE: To assess the application value of combined detection of HbA2 and HbF for the screening of thalassemia among a population of childbearing age in Quanzhou, Fujian, and determine the optimal cut-off values for the region. METHODS: Capillary hemoglobin electrophoresis and genetic testing for α and ß globin gene mutations were simultaneously carried out on 11 428 patients with suspected thalassemia. Statistical methods were used to analyze the distribution of various types of thalassemia and compare the performance of HbA2 and HbF measurement for the screening of various types of thalassemia. The optimal cut-off values for HbA2 and HbF were determined with the ROC curves. RESULTS: 4591 patients with α, ß, and αß compound thalassemia were identified by genetic testing. The most common genotypes for α and ß thalassemia included --SEA/αα and ß654/ßN, ß41-42/ßN, and ß17/ßN. The ROC curves were drawn to compare the performance of HbA2 screening for α-, ß-, αß-compound, static α-, mild α-, and intermediate α-thalassemia, and the maximum area under the curves was 0.674, 0.984, 0.936, 0.499, 0.731, 0.956, and the optimal cut-off values for HbA2 were 2.45%, 3.25%, 3.65%, 2.95%, 2.55%, 1.75%, respectively. CONCLUSION: HbA2 is an efficient indicator for identifying intermediate types of α-, ß-, and αß compound thalassemia. The combination of HbA2 and HbF measurement can effectively detect carriers for ß-thalassemia mutations.

An online alpha-thalassemia carrier discrimination model based on random forest and red blood cell parameters for low HbA2 cases.

BACKGROUND: Since screening of α-thalassemia carriers by low HbA2 has a low positive predictive value (PPV), the PPV was as low as 40.97% in our laboratory, other more effective screening methods need to be devised. This study aimed at developing a machine learning model by using red blood cell parameters to identify α-thalassemia carriers from low HbA2 patients. METHODS: Laboratory data of 1213 patients with low HbA2 used for modeling was randomly divided into the training set (849 of 1213, 70%) and the internal validation set (364 of 1213, 30%). In addition, an external data set (n = 399) was used for model validation. Fourteen machine learning methods were applied to construct a discriminant model. Performance was evaluated with accuracy, sensitivity, specificity, etc. and compared with 7 previously published discriminant function formulae. RESULTS: The optimal model was based on random forest with 5 clinical features. The PPV of the model was more than twice the PPV of HbA2, and the model had a high negative predictive value (NPV) at the same time. Compared with seven formulae in screening of α-thalassemia carriers, the model had a better accuracy (0.915), specificity (0.967), NPV (0.901), PPV (0.942) and area under the receiver operating characteristic curve (AUC, 0.948) in the independent test set. CONCLUSION: Use of a random forest-based model enables rapid discrimination of α-thalassemia carriers from low HbA2 cases.

Genetic Analysis of Thalassemia in 22 940 Pregnant Women in Xiangxi Tujia and Miao Autonomous Prefecture / 中国实验血液学杂志

OBJECTIVE@#To investigate the incidence and types of thalassemia in Xiangxi Tujia and Miao Autonomous Prefecture.@*METHODS@#Automatic capillary electrophoresis was used to screen the thalassemia phenotypes of 22 940 blood samples of pregnant women and puerperants collected in our hospital and some other medical institutions in the prefecture during 2017-2019, among which there were 3 356 cases of Tujia ethnicity, 2 821 cases of Miao ethnicity, and 2 233 cases of Han ethnicity included, whose ethnicity were indicated. The samples with positive result would undergo further genetic testing.@*RESULTS@#There were 2 314 cases of suspicious thalassemia were screened from 22 940 cases by the electrophoresis, thus the positive rate was 10.1% (hematological phenotypes from some other institutions were not included). Specifically, there were 1 706 cases with HBA2 less than 2.5%, 255 cases with HBA2 ranged from 2.5% to 3.5%, which displayed abnormal hematology (MCV or/and MCH) or other abnormal bands, and 353 cases with HBA2>3.5%. There were 436 suspected positive patients in 2 314 suspicious samples received further thalassemia gene testing in our hospital, among them 48 cases were diagnosed with α-thalassemia, 85 cases with β-thalassemia, and 2 cases as compound type. The positive diagnosis rate of α-thalassemia gene test was 11.0%, β-thalassemia was 19.4%, and positive pregnant women was 31.0%.@*CONCLUSION@#The positive rate of thalassemia screening in Xiangxi Autonomous Prefecture is roughly the same as that in other regions of Hunan. The positive predictive value of β-thalassemia screening is as high as 86%. Compared with the missed screening data, it is recommended to use hematology (MCV, MCH) method combined with capillary hemoglobin electrophoresis for thalassemia screening.