Objective:To explore the clinical features and genetic mutation sites of 28 patients with Congenital fibrinogen disorders (CFDs).Methods:A total of 28 unrelated CFDs patients admitted to Wenzhou People′s Hospital from June 2018 to April 2023 were enrolled into this research. A total of 2.7 mL of peripheral blood was collected from each patient for coagulation function tests, which included thrombin time (TT), fibrinogen activity (Fg: C), fibrinogen antigen (Fg: Ag), and gene detection. The Sanger sequencing method was employed to verify variations in the fibrinogen (Fg) protein-coding gene across 28 patients. Bioinformatics analyses, including harmfulness analysis, conservation analysis across different species, and spatial simulation predictions of variant proteins, were conducted by PolyPhen-2, PROVEAN, SnapGene, and Pymol softwares on the variant sites of these patients. Pathogenicity ratings for the detected variant sites were performed in accordance with the Standards and Guidelines for the Interpretation of Sequence variants by the American College of Medical Genetics and Genomics (ACMG) (hereafter referred to as the ACMG Guidelines). This study received approval from the Ethics Committee of Wenzhou People′s Hospital (Approval No. KY-2023-269), and informed consent was obtained from all participants before enrollment.Results:The clinical and genetic characteristics of 28 patients with CFDs in this study were as follows. ①Clinical data: Among the 28 patients, 2 cases were diagnosed with type I CFDs, while 26 cases were diagnosed with type II CFDs. And 50.0% (14/28) of the patients exhibited no clinical manifestations, while 28.6% (8/28) presented with bleeding manifestations, and 7.1% (2/28) exhibited thrombus manifestations, 3.6% (1/28) experienced both bleeding and thrombosis. Among female patients, 13.0% (3/23) exhibited a history of habitual abortion. All patients demonstrated TT and a significant decrease in Fg: C. ②Sanger sequencing revealed a total of 10 types of heterozygous variations in the FGA, FGB, and FGG genes across 28 patients, distributed among 9 loci. The variation at the γ c. 902G>A/c.901C>T accounted for the highest proportion (35.7%, 10/28), followed by the Bβ c. 569 A>G (28.6%, 8/28). ③Biological informatics analysis: the Aα c. 180+ 1G>T mutation was predicted to be highly deleterious. And the Aα c. 104G>A, Bβ c. 425T>G, Bβ c. 586C>T, and γ c. 902G>A/c.901C>T variations were also predicted to be harmful. Conservation analysis indicates that the 9 variant sites were highly conserved among homo sapiens, musculus, ovis aries, scrofa, and rattus. Spatial conformation analysis revealed that some variations lead to an increase or decrease in the number of hydrogen bonds. ④ACMG guideline rating analysis: Among the ten variations in the Fg protein-coding genes FGA, FGB, and FGG identified in 28 patients, 9 variations (Aα c. 104G>A, Aα c. 180+ 1G>T, Bβ c. 425T>G, Bβ c.569A>G, Bβ c. 586C>T, Bβ c. 643G>A, γ c. 901C>T, γ c. 902G>A, γ c. 1001A>C) were classified as pathogenic, while one variation (γ c. 908C>G) was classified as likely pathogenic. Conclusion:In this study, the majority of CFDs patients are diagnosed with type II CFDs, with 50% presenting clinical symptoms predominantly manifesting as bleeding, thrombosis, and recurrent miscarriage. The mutation hotspots are mainly located in exon 2 of FGA, exon 4 of FGB, and exon 8 of FGG.

OBJECTIVE: To explore the clinical features and genetic mutation sites of 28 patients with Congenital fibrinogen disorders (CFDs). METHODS: A total of 28 unrelated CFDs patients admitted to Wenzhou People's Hospital from June 2018 to April 2023 were enrolled into this research. A total of 2.7 mL of peripheral blood was collected from each patient for coagulation function tests, which included thrombin time (TT), fibrinogen activity (Fg:C), fibrinogen antigen (Fg:Ag), and gene detection. The Sanger sequencing method was employed to verify variations in the fibrinogen (Fg) protein-coding gene across 28 patients. Bioinformatics analyses, including harmfulness analysis, conservation analysis across different species, and spatial simulation predictions of variant proteins, were conducted byPolyPhen-2, PROVEAN, SnapGene, and Pymol softwares on the variant sites of these patients. Pathogenicity ratings for the detected variant sites were performed in accordance with the Standards and Guidelines for the Interpretation of Sequence variants by the American College of Medical Genetics and Genomics (ACMG) (hereafter referred to as the ACMG Guidelines). This study received approval from the Ethics Committee of Wenzhou People's Hospital (Approval No. KY-2023-269), and informed consent was obtained from all participants before enrollment. RESULTS: The clinical and genetic characteristics of 28 patients with CFDs in this study were as follows. CLINICAL DATA: Among the 28 patients, 2 cases were diagnosed with type I CFDs, while 26 cases were diagnosed with type II CFDs. And 50.0% (14/28) of the patients exhibited no clinical manifestations, while 28.6% (8/28) presented with bleeding manifestations, and 7.1% (2/28) exhibited thrombus manifestations, 3.6% (1/28) experienced both bleeding and thrombosis. Among female patients, 13.0% (3/23) exhibited a history of habitual abortion. All patients demonstrated TT and a significant decrease in Fg:C. Sanger sequencing revealed a total of 10 types of heterozygous variations in the FGA, FGB, and FGG genes across 28 patients, distributed among 9 loci. The variation at the γ c.902G>A/c.901C>T accounted for the highest proportion (35.7%, 10/28), followed by the Bβ c.569 A>G (28.6%, 8/28). Biological informatics analysis: the Aα c.180+1G>T mutation was predicted to be highly deleterious. And the Aα c.104G>A, Bβ c.425T>G, Bβ c.586C>T, and γ c.902G>A/c.901C>T variations were also predicted to be harmful. Conservation analysis indicates that the 9 variant sites were highly conserved among homo sapiens, musculus, ovis aries, scrofa, and rattus. Spatial conformation analysis revealed that some variations lead to an increase or decrease in the number of hydrogen bonds. ACMG guideline rating analysis: Among the ten variations in the Fg protein-coding genes FGA, FGB, and FGG identified in 28 patients, 9 variations (Aα c.104G>A, Aα c.180+1G>T, Bβ c.425T>G, Bβ c.569A>G, Bβ c.586C>T, Bβ c.643G>A, γ c.901C>T, γ c.902G>A, γ c.1001A>C) were classified as pathogenic, while one variation (γ c.908C>G) was classified as likely pathogenic. CONCLUSION In this study, the majority of CFDs patients are diagnosed with type II CFDs, with 50% presenting clinical symptoms predominantly manifesting as bleeding, thrombosis, and recurrent miscarriage. The mutation hotspots are mainly located in exon 2 of FGA, exon 4 of FGB, and exon 8 of FGG.
Humans ; Female ; Male ; Afibrinogenemia/congenital* ; Fibrinogen/metabolism* ; Mutation ; Genotype ; Adult ; Child ; Adolescent ; Child, Preschool ; Infant

Objective:To explore the clinical features and genetic mutation sites of 28 patients with Congenital fibrinogen disorders (CFDs).Methods:A total of 28 unrelated CFDs patients admitted to Wenzhou People′s Hospital from June 2018 to April 2023 were enrolled into this research. A total of 2.7 mL of peripheral blood was collected from each patient for coagulation function tests, which included thrombin time (TT), fibrinogen activity (Fg: C), fibrinogen antigen (Fg: Ag), and gene detection. The Sanger sequencing method was employed to verify variations in the fibrinogen (Fg) protein-coding gene across 28 patients. Bioinformatics analyses, including harmfulness analysis, conservation analysis across different species, and spatial simulation predictions of variant proteins, were conducted by PolyPhen-2, PROVEAN, SnapGene, and Pymol softwares on the variant sites of these patients. Pathogenicity ratings for the detected variant sites were performed in accordance with the Standards and Guidelines for the Interpretation of Sequence variants by the American College of Medical Genetics and Genomics (ACMG) (hereafter referred to as the ACMG Guidelines). This study received approval from the Ethics Committee of Wenzhou People′s Hospital (Approval No. KY-2023-269), and informed consent was obtained from all participants before enrollment.Results:The clinical and genetic characteristics of 28 patients with CFDs in this study were as follows. ①Clinical data: Among the 28 patients, 2 cases were diagnosed with type I CFDs, while 26 cases were diagnosed with type II CFDs. And 50.0% (14/28) of the patients exhibited no clinical manifestations, while 28.6% (8/28) presented with bleeding manifestations, and 7.1% (2/28) exhibited thrombus manifestations, 3.6% (1/28) experienced both bleeding and thrombosis. Among female patients, 13.0% (3/23) exhibited a history of habitual abortion. All patients demonstrated TT and a significant decrease in Fg: C. ②Sanger sequencing revealed a total of 10 types of heterozygous variations in the FGA, FGB, and FGG genes across 28 patients, distributed among 9 loci. The variation at the γ c. 902G>A/c.901C>T accounted for the highest proportion (35.7%, 10/28), followed by the Bβ c. 569 A>G (28.6%, 8/28). ③Biological informatics analysis: the Aα c. 180+ 1G>T mutation was predicted to be highly deleterious. And the Aα c. 104G>A, Bβ c. 425T>G, Bβ c. 586C>T, and γ c. 902G>A/c.901C>T variations were also predicted to be harmful. Conservation analysis indicates that the 9 variant sites were highly conserved among homo sapiens, musculus, ovis aries, scrofa, and rattus. Spatial conformation analysis revealed that some variations lead to an increase or decrease in the number of hydrogen bonds. ④ACMG guideline rating analysis: Among the ten variations in the Fg protein-coding genes FGA, FGB, and FGG identified in 28 patients, 9 variations (Aα c. 104G>A, Aα c. 180+ 1G>T, Bβ c. 425T>G, Bβ c.569A>G, Bβ c. 586C>T, Bβ c. 643G>A, γ c. 901C>T, γ c. 902G>A, γ c. 1001A>C) were classified as pathogenic, while one variation (γ c. 908C>G) was classified as likely pathogenic. Conclusion:In this study, the majority of CFDs patients are diagnosed with type II CFDs, with 50% presenting clinical symptoms predominantly manifesting as bleeding, thrombosis, and recurrent miscarriage. The mutation hotspots are mainly located in exon 2 of FGA, exon 4 of FGB, and exon 8 of FGG.

Background: Clinical chemistry tests are most widely used in clinical laboratories, and diverse measurement systems for these analyses are available in China. We evaluated the imprecision of clinical chemistry measurement systems based on internal QC (IQC) data. Methods: IQC data for 27 general chemistry analytes were collected in February each year from 2013 to 2022. Four performance specifications were used to calculate pass rates for CVs of IQC data in 2022. Boxplots were drawn to analyze trends of CVs, and differences in CVs among different groups were assessed using the Mann–Whitney U-test or Kruskal– Wallis test. Results: The number of participating laboratories increased significantly from 1,777 in 2013 to 5,425 in 2022. CVs significantly decreased for all 27 analytes, except creatine kinase and lipase. Triglycerides, total bilirubin, direct bilirubin, iron, and γ-glutamyl transferase achieved pass rates > 80% for all goals. Nine analytes with pass rates < 80% based on 1/3 allowable total error were further analyzed; the results indicated that closed systems exhibited lower CVs than open systems for all analytes, except total protein. For all nine analytes, differences were significant between tertiary hospitals and non-tertiary hospitals and between accredited and non-accredited laboratories. Conclusions The CVs of IQC data for clinical chemistry have seen a continuous overall improvement in China. However, there is ample room for imprecision improvement for several analytes, with stricter performance specifications.

Objective:The allowable total error ( TEa),allowable imprecision ( CV)and allowable bias( Bias)were recommended for 34 routine chemistry analytes in China. Methods:According to the performance specification setting mode newly determined at the Milan conference in Italy,the performance specification was derived based on components biological variation (BV)and current state of the art mode. The data(including EQA data and IQC data)of laboratories participating in the routine chemistry and lipids and lipoproteins EQA activities of the national center for clinical laboratories from 2019 to 2021 was collected through clinet-EQA. For the analytes with biological variation(BV)data,compared the'percentage difference′ of EQA data and the'in-control coefficient of variation of the month′ of IQC data of each research analyte with the three levels evaluation criteria derived based on BV,and calculated the percentage difference passing rate and CV passing rate of all batches in each year. When the passing rate reaches 80%,the performance specifications of this level met the requirements of the recommended performance specifications of the analyte. For the analytes without BV data or analytes whose performance specifications at three levels derived based on BV could not be used as recommended standards,the recommended performance specifications are derived based on the current state of the art. After obtaining the recommended TEa and allowable CV for each analyte,used the formula | Bias|≤ TEa-z? CV to derive the recommended allowable bias. Results:The results of TEa ( CV)% recommended by 34 analytes are as follows:K4.7(2),Na4(1.5),Cl4(1.4),Ca5(2),P9.6(3.9),Glu6.4(2.5),Urea8(3),UA12(4.1),Cre11(3.3),TP5(2),Alb5.2(2.4),TC8.6(2.7),TG13.5(5),HDL-C16.5(4.3),LDL-C20.5(6.2),ApoAⅠ16(5.3),ApoB 17.1(5.5),Lp(a) 24.1(10.4),TBil 12.4(5),DBil 20(7.3),ALT16(5),AST13.5(4.8),ALP17.5(4.8),AMY13.1(3.3),CK11.3(3.8),LDH11(3.9),CHE13.4(5.3),LIP20(6.9),Fe13.3(5.2),Mg14(4.5),Cu17.9(6.8),Zn15.1(6.4),γ-GGT10(3.3),α-HBDH18(5.8).The formula | Bias|≤ TEa-z? CV is used to derive the allowable bias of 34 analytes. Conclusions:For 34 clinical routine chemistry quantitative analytes,the allowable total error,allowable imprecision and allowable bias that meet the current state of the art of Chinese laboratories are recommended.

The role of glial scar after intracerebral hemorrhage(ICH)remains unclear.This study aimed to inves-tigate whether microglia-astrocyte interaction affects glial scar formation and explore the specific function of glial scar.We used a pharmacologic approach to induce microglial depletion during different ICH stages and examine how ablating microglia affects astrocytic scar formation.Spatial transcriptomics(ST)analysis was performed to explore the potential ligand-receptor pair in the modulation of microglia-astrocyte interaction and to verify the functional changes of astrocytic scars at different periods.During the early stage,sustained microglial depletion induced disorganized astrocytic scar,enhanced neutrophil infiltration,and impaired tissue repair.ST analysis indicated that microglia-derived insulin like growth factor 1(IGF1)modulated astrocytic scar formation via mechanistic target of rapamycin(mTOR)signaling activation.Moreover,repopulating microglia(RM)more strongly activated mTOR signaling,facilitating a more protective scar formation.The combination of IGF1 and osteopontin(OPN)was necessary and sufficient for RM function,rather than IGF1 or OPN alone.At the chronic stage of ICH,the overall net effect of astrocytic scar changed from protective to destructive and delayed microglial depletion could partly reverse this.The vital insight gleaned from our data is that sustained microglial depletion may not be a reasonable treatment strategy for early-stage ICH.Inversely,early-stage IGF1/OPN treatment combined with late-stage PLX3397 treatment is a promising therapeutic strategy.This prompts us to consider the complex temporal dynamics and overall net effect of microglia and astrocytes,and develop elaborate treatment strategies at precise time points after ICH.

Background: Serum C-peptide results from various routine methods used in China are highly variable, warranting well-performing methods to serve as an accuracy base to improve the harmonization of C-peptide measurements in China. We developed an accurate isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC–MS/MS) method for serum C-peptide measurement and explored its use in harmonization. Methods: After protein precipitation with ZnSO4 solution, C-peptide was extracted from serum samples by anion-exchange solid-phase extraction and quantified by ID-LC–MS/MS in positive ion mode. The precision and analytical recovery of the ID-LC–MS/MS method were assessed. Seventy-six serum samples were analyzed using the ID-LC–MS/MS method and six routine immunoassays. Ordinary linear regression (OLR) and Bland-Altman (BA) analyses were conducted to evaluate the relationship between the ID-LC–MS/MS method and routine immunoassays. Five serum pool samples assigned using the ID-LC–MS/MS method were used to recalibrate the routine assays. OLR and BA analyses were re-conducted after recalibration. Results: The within-run, between-run, and total precision for the ID-LC–MS/MS method at four concentrations were 1.0%–2.1%, 0.6%–1.2%, and 1.3%–2.2%, respectively. The analytical recoveries for the ID-LC–MS/MS method at three concentrations were 100.3%–100.7%, 100.4%–101.0%, and 99.6%–100.7%. The developed method and the immunoassays were strongly correlated, with all R2 >0.98. The comparability among the immunoassays was substantially improved after recalibration. Conclusions The performance of the ID-LC–MS/MS method was carefully validated, and this method can be used to improve the harmonization of serum C-peptide measurements in China.

Background: Using commutable external quality assessment (EQA) materials is important for monitoring successful harmonization efforts. We assessed the commutability of four human serum pool (HSP) preparations to identify candidate EQA materials for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity measurement. Methods: One set each of 85 clinical samples (CSs) was collected for ALT and AST activity measurement. The 15 candidate EQA materials included four types of HSP preparations (A to D): materials A, C, and D contained human original recombinant (HOR) aminotransferases; materials B was mixed leftover samples. The CSs and 15 candidate EQA materials were analyzed using seven routine assays, and the ln-transformed results were analyzed in 21 assay pairs. Commutability was assessed using Deming regression, with a 95% prediction interval (CLSI approach) and the difference in bias with an error component model (International Federation of Clinical Chemistry and Laboratory Medicine [IFCC] approach). Results: For ALT, all materials were commutable for 14–21 assay pairs according to the CLSI and IFCC approaches. For AST, B01-03 showed commutability for 14-21 assay pairs, and C01-03 and D01-03 showed commutability for no less than 10 assay pairs according to the two approaches. A01-06 were commutable for 9-16 assay pairs according to the CLSI approach, but for 6-9 assay pairs according to the IFCC approach. Conclusions Mixed leftover samples showed desirable commutability characteristics as candidate EQA materials for routine aminotransferase activity measurements. Human serum bases supplemented with HOR were commutable for most routine ALT activity measurements.

Objective:To describe the incidence, diagnosis rate, treatment rate and treatment pattern of hyperkalemia, and serum potassium retesting rate among hyperkalemia patients in the emergency department.Methods:Data were derived from Military Data Center for Rational Use of Drugs. Patients who accessed emergency medical services (≥18 years old) with record(s) of serum potassium between 2015 and 2017 were included. The data of laboratory test, diagnosis, and treatment were analyzed. The main outcomes included the incidence of hyperkalemia, the diagnosis rate, the treatment rate, treatment pattern and the 7-day retesting rate.Results:A total of 1 039 245 patients who met the above criteria were included, of whom, 36 615 (3.52%) had at least one hyperkalemia event. Among the emergency patients with chronic kidney disease, heart failure, diabetes mellitus and hypertension, the proportions of patients who experienced hyperkalemia were 47.69%, 29.13%, 21.69% and 10.16%, respectively. The diagnosis rate of emergency hyperkalemia patients was 9.23%. The overall hyperkalemia treatment rate was 42.1%. Insulin + glucose injection was the most commonly used therapy for emergency hyperkalemia patients. The overall serum potassium retesting rate within 7 days was 28.8%.Conclusions:Hyperkaliemia is more common and more severe in patients with chronic kidney disease, heart failure, diabetes and hypertension. The diagnosis rate and retesting rate of hyperkalemia are low, suggesting that the identification and management of hyperkaliemia in emergency patients should be strengthened.

Objective:To investigate the molecular mechanism of proliferation-inhibiting effect of icaritin on hepatoma cells via regulating miRNA-329 (miR-329) and miRNA-1236 (miR-1236).Methods:Hepatoma cell line HepG2 was treated with icaritin at different concentrations (2.5, 5.0, 10.0, 20.0, 40.0 μmol/L), and the control group only added dimethyl sulfoxide (DMSO). The half inhibitory concentration ( IC50) of icaritin on HepG2 cells and cell proliferation rate were detected by CCK-8 after cultured for 36 h. HepG2 cells were treated with 400 μg/L alpha fetoprotein (AFP). After cultured for 0, 12, 24, 36, 48 and 60 h, the effect of AFP on the proliferation of HepG2 cells was detected by CCK-8 method. AFP-3'UTR reporter plasmid pmirGLO-AFP-3'UTR plasmid was constructed, pmirGLO blank vector plasmid, pmirGLO-AFP-3'UTR plasmid, miR-329 or miR-1236 mimics or inhibitors, control plasmid of mimics (NC), control plasmid of inhibitors (INC) were respectively co-transfected with HepG2 cells, and the effect of miR-329 and miR-1236 on the luciferase activity was detected by dual-luciferase reporter assay after cultured for 24 h. Western blot and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to detect the effects of icaritin on the expressions of AFP, miR-329 and miR-1236 in HepG2 cells. HepG2 cells were respectively transfected with mimics and inhibitors of miR-329 and miR-1236 to detect the effects of miR-329 and miR-1236 on the expression of AFP. Results:The cell proliferation rates of 2.5, 5.0, 10.0, 20.0, 40.0 μmol/L icaritin group and control group were (80.4±2.3)%, (73.2±1.6)%, (51.7±3.3)%, (38.2±4.6)%, (29.5±4.3)%, and (94.0±2.9)%, respectively, and the difference was statistically significant ( F = 75.65, P < 0.01); the differences in the proliferation rate of HepG2 cells between different concentrations of icaritin group and control group were statistically significant (all P < 0.01). The IC50 of icaritin on HepG2 cells was 10 μmol/L. The relative expressions of AFP mRNA in 2.5, 5.0, 10.0, 20.0, 40.0 μmol/L icaritin group and control group were 0.83±0.06, 0.69±0.02, 0.53±0.07, 0.45±0.01, 0.33±0.07, and 1.00±0.01, respectively, and the difference was statistically significant ( F = 42.67, P < 0.01); the differences in the relative expressions of AFP mRNA between different concentrations of icaritin group and control group were statistically significant (all P < 0.01). HepG2 cells were treated by 400 μg /L AFP for 0, 12, 24, 36, 48 and 60 h, and the cell proliferation rates were (102±5)%, (138±13)%, (186±24)%, (260±12)%, (311±15)%, and (348±25)%, respectively, and the difference was statistically significant ( F = 27.483, P < 0.01); the differences in the cell proliferation rate between different time of AFP treatment and 0 h were statistically significant (all P < 0.01). Compared with the control group, different concentrations of icaritin can promote the expression of miR-329 and miR-1236 (all P < 0.01). After co-transfection of miR-329 and miR-1236 mimics and AFP-3'UTR, the luciferase activity decreased by about 40%; after co-transfection of miR-329 and miR-1236 inhibitors and AFP-3'UTR, the luciferase activity increased about 1.5 times. Both miR-329 and miR-1236 can reduce the expression levels of AFP protein and mRNA (both P < 0.05). Conclusion:Icaritin can promote the binding of miR-219, miR-1236 and AFP-3'UTR by promoting the expression of miR-329 and miR-1236, inhibit the stability and translation activity of AFP mRNA, inhibit the expression of AFP, and thus inhibit the proliferation of hepatoma cells in vitro.