Hemoglobin A1c (HbA1c) has a unique structure that makes monoclonal antibody (mAb) preparation challenging. This study aims to develop a method for preparing HbA1c mAbs and establish a fluorescent immunochromatographic assay (FICA) for rapid detection of HbA1c. Three glycosylated peptides were synthesized and used to prepare complete antigens, which were identified by dot enzyme-linked immunosorbent assay (Dot-ELISA) and ultraviolet absorption spectroscopy. The complete antigens and natural HbA1c were used for cross-immunization of mice, and the optimal complete antigen was selected. The mouse with the highest serum titer was chosen for mAb preparation. The purity and specificity of the mAbs were verified, and a FICA method was developed. The optimal complete antigen, with a titer of 1:512 000, was successfully prepared and selected. Fusion with splenocytes resulted in four specific HbA1c antibodies (purity > 90%). The best antibody exhibited a binding constant (Ka) of 1.67×10¹⁰ L/mol with the antigen. Based on this antibody, a FICA method was successfully established, capable of producing results within 15 min. The method demonstrated a good linear range (3%-13% HbA1c, y=0.071 3x+0.005 6, R²=0.993 7), recovery rates of 98%-102%, precision < 10.00%, and no nonspecific reactions. Clinical testing of 210 samples showed positive agreement of 96.36%, negative agreement of 97.00%, and overall agreement of 96.68%. The receiver operating characteristic (ROC) curve analysis yielded an area under curve (AUC) of 0.980 9 [95% confidence interval (CI): 0.961 0-1.000 0], with high consistency verified in multicenter studies. We successfully developed a key technique for preparing HbA1c monoclonal antibodies and established a FICA method for rapid detection of HbA1c. It will provide an efficient and convenient detection method for the early diagnosis and long-term management of diabetes and its complications.
Antibodies, Monoclonal/biosynthesis* ; Animals ; Mice ; Glycated Hemoglobin/immunology* ; Mice, Inbred BALB C ; Humans ; Antibody Specificity ; Chromatography, Affinity/methods* ; Enzyme-Linked Immunosorbent Assay/methods* ; Female

Humans ; Hemoglobinopathies ; Hemoglobins, Abnormal/genetics* ; Oxygen

OBJECTIVE: To explore the genetic basis for a child with Hemoglobin Santa Ana (Hb Santa Ana). METHODS: The child was admitted to the Children's Hospital of Chongqing Medical University on August 4, 2013 due to anemia, splenomegaly and deepening urine color for 5 years. His clinical data were collected. Peripheral blood samples of the child and his parents were collected for routine blood test. The erythrocyte parameters of the child and his parents were determined with an automatic hemocyte analyzer. The hemoglobin components of the child and his parents were detected by high-performance liquid chromatography (HPLC). Common mutations associated with thalassemia were detected by Gap-PCR and PCR-reverse dot blotting (PCR-RDB). Rare variants of the globin genes were detected by next generation sequencing (NGS), and the result was verified by Sanger sequencing. RESULTS: The child has shown mild to moderate hemolytic anemia. Routine blood test showed that he had lower hemoglobin (90 g/L) and mean corpuscular hemoglobin concentration (267 g/L) but a higher reticulocyte ratio (0.141), which indicated hypopigmented hyperplastic anemia. Analysis of hemoglobin component showed that his hemoglobin F was elevated to 10.7%, which indicated abnormal synthesis of β globin peptide chain. HPLC analysis showed an abnormal peak accounting for 4.5% of the total area. Neither of his parents was found to have abnormal results for routine blood test and hemoglobin component analysis. No common globin gene variant was detected in the child. Gene sequencing revealed that the child has harbored a heterozygous variant of HBB: c.266T>C, which was de novo in origin. Based on the guidelines of American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic. CONCLUSION The heterozygous HBB: c.266T>C variant probably underlay the hemolytic anemia associated with Hb Santa Ana in this patient.
Child ; Humans ; Male ; Chromatography, High Pressure Liquid ; Hemoglobins, Abnormal/genetics*

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.
Humans ; Female ; Pregnancy ; beta-Thalassemia/genetics* ; Anemia, Iron-Deficiency ; Fetal Hemoglobin/analysis* ; alpha-Thalassemia ; Blood Protein Electrophoresis ; Hemoglobin A2/analysis* ; Hemoglobins, Abnormal/analysis*

Humans ; Hemoglobinopathies ; Hemoglobins, Abnormal/genetics*

Humans ; Hematopoietic Stem Cell Transplantation ; Hemoglobinopathies/therapy* ; Hemoglobins, Abnormal

OBJECTIVE: To identify one case of rare Hb Lepore-BW associated with IVS-II-654 heterozygous mutation in Sichuan area. METHODS: The blood routine examination and hemoglobin electrophoresis methods were used to analyze the blood routine parameters, HbA₂ and HbF in the samples of peripheral blood in proband and his parents, as well as the cord blood of pregnant woman. The detection of thalassemia gene and Sanger sequencing methods were used to detect the hemoglobin mutations. RESULTS: The result showed that the Hb Lepore-BW heterozygous mutation was detected in the father of the proband, while a rare Hb Lepore-BW with IVS-II-654 heterozygous mutation was detected in the proband, as well as his mother and cord blood were both detected as IVS-II-654 heterozygous mutation. CONCLUSION The study identified a rare Hb Lepore-BW with IVS-II-654 heterozygous mutation, which was characterized by intermediate β-thalassemia. It is necessary to hemoglobin electrophoresis combined with routine blood testing in prenatal screening.
Female ; Hemoglobins, Abnormal/genetics* ; Heterozygote ; Humans ; Infant, Newborn ; Male ; Mutation ; Pregnancy ; Prenatal Diagnosis ; beta-Thalassemia/genetics*

Hemoglobins, Abnormal ; Humans ; Singapore/epidemiology* ; alpha-Thalassemia

OBJECTIVE: To analyze the hematological phenotypes of Hb J-Bangkok and concomitant thalassemia. METHODS: In total 72 397 samples were screened by using capillary electrophoresis. Samples with Hb J-Bangkok were identified by DNA sequencing and analysis of red blood cell parameters. Gap-PCR and PCR-reverse dot blotting (PCR-RDB) were used for analyzing the thalassemia genes. RESULTS: Thirty one cases of Hb J-Bangkok were identified, all of which were heterozygotes. The hematological phenotype index (Hb, mean corpuscular volume, mean corpuscular hemoglobin, Hb J-Bangkok, Hb A CONCLUSION Hb J-Bangkok heterozygotes have normal hematological phenotypes, though they may show different hematological characteristics when concomitant with different types of thalassemia, for which genetic counseling should be provided accordingly.
Female ; Hemoglobins, Abnormal/genetics* ; Heterozygote ; Humans ; Male ; Phenotype ; Thailand ; beta-Thalassemia/genetics*

OBJECTIVE: To understand the carrying rate, gene mutation frequency and composition ratio of thalassemia in pregnant women in Suxian and Beihu districts of Chenzhou, Hunan Province. METHODS: Thalassemia gene in 11 212 samples was analyzed by using Next-Generation Sequencing. RESULTS: Among the 11 212 samples, 938 were diagnosed as thalassemia, in which 618 (5.51%) were diagnosed as α-thalassemia, 268 (2.39%) as β-thalassemia, 29（0.26%）as abnormal hemoglobin and 23 (0.21%) as αβ-thalassemia. The gene mutations of --SEA /αα(40.29%) and -α3.7/αα(37.7%) in α-thalassemia were the most common, while for β- thalassemia, the most commonly gene mutation were β41-42M/βN(24.26%) and β654M/βN(23.88%). The detection rate of rare type α,β-thalassemia gene was 0.19%(21/11 212）, 0.53%（59/11 212）, respectively. CONCLUSION The carrying rate of thalassemia in pregnant women is 8.37% in Suxian and Beihu districts of Chenzhou city, and the genotypes are complex. Next-Generation Sequencing can detect rare thalassemia genes and new gene mutations effectively.
China ; Female ; Genotype ; Hemoglobins, Abnormal ; High-Throughput Nucleotide Sequencing ; Humans ; Mutation ; Pregnancy ; Pregnant Women ; alpha-Thalassemia/genetics* ; beta-Thalassemia/genetics*