OBJECTIVES: To investigate the application value of thromboelastography (TEG) in assessing coagulation function in children with severe hemophilia A (HA) after emicizumab (EMI) therapy. METHODS: A retrospective analysis was performed on the activated partial thromboplastin time (APTT) and TEG testing results of 17 children with severe HA before and after EMI treatment at Shenzhen Children's Hospital from January 2023 to July 2024. Correlation analysis was conducted between coagulation factor VIII (FVIII) equivalent activity and reaction time (R value) measured by TEG. RESULTS: After EMI treatment, the mean bleeding rate for children with severe HA was 1.6 events per year, with 15 children (88%) without spontaneous bleeding or joint bleeding. The children with severe HA showed a significant reduction in APTT after EMI treatment (P<0.05), with a significantly shorter APTT than the normal control group (P<0.05). There was no correlation between APTT and FVIII equivalent activity after treatment (P>0.05). After EMI treatment, TEG parameters, including R value, kinetic time, alpha angle (α), maximum amplitude, clot strength, and coagulation index, shifted from a hypocoagulable state before treatment to a nearly normal state after treatment (P<0.05). The R value demonstrated a strong negative correlation with FVIII equivalent activity (r=-0.758, P<0.05). CONCLUSIONS The bleeding condition of children with severe HA can be effectively controlled after EMI treatment. Routine APTT testing cannot reflect true coagulation function, whereas TEG testing is clinically valuable in assessing the coagulation function of children with severe HA undergoing EMI treatment.
Humans ; Thrombelastography ; Hemophilia A/physiopathology* ; Male ; Child ; Antibodies, Bispecific/therapeutic use* ; Antibodies, Monoclonal, Humanized/therapeutic use* ; Blood Coagulation/drug effects* ; Child, Preschool ; Retrospective Studies ; Female ; Partial Thromboplastin Time ; Adolescent ; Infant

OBJECTIVE: To investigate the molecular alterations of splenocytes associated with anti-factor Ⅷ (FⅧ) immune response and the underlying mechanisms based on hemophilia A (HA) murine model via RNA sequencing (RNA-seq) technology. METHODS: Severe HA mice were immunized with recombinant human factor Ⅷ (rhF8) weekly for 4 weeks to establish an FⅧ inhibitor model. High quality raw data were obtained by using bulk RNA-seq and CASAVA base identification technology, and the differentially expressed genes (DEGs) were identified. The DEGs were statistically classified by gene ontology (GO) annotation to obtain information on the major signaling pathways and biological processes involved in anti-FⅧ immune response in HA mouse splenocytes. The cell clusters, genes, and signaling pathway datasets were comprehensively analyzed by GO, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and single cell RNA-seq (ScRNA-seq) analysis, respectively. Flow cytometry analysis was used to verify the changes in T follicular helper cells (Tfh) and regulatory T cells (Treg). RESULTS: A total of 3731 DEGs was identified, including 2275 genes with up-regulated expression and 1456 genes with down-regulated expression. The DEGs were enriched in helper T cell differentiation, cytokine receptor, T cell receptor signaling pathway, ferroptosis, etc. Uniform Manifold Approximation and Project (UMAP) downscaling and visualization analysis yielded a total number of 11 T/NK cell subsets, visualizing the overall expression distribution of C-X-C chemokine-specific receptor gene cxcr5 among these T/NK cell subsets. Higher expression of cxcr5 was found in activated Tfh from FⅧ inhibitor mice, in comparison to the control group. The visualization using Upset plot R language showed a close interaction between Tfh and Treg. Moreover, the increased frequencies of Tfh and the decreased frequencies of Treg in inhibitor mouse splenocytes were further verified by flow cytometry analysis. CONCLUSION Multiple immune cell subsets, signaling pathways, and characteristic genes may be involved in the process of anti-FⅧ immune response in HA mouse splenocytes. The molecules involved in the regulation of Tfh/Treg may play key roles, which provide potential biological targets and therapeutic strategies for HA patients with inhibitors in the future.
Animals ; Hemophilia A/genetics* ; Mice ; Sequence Analysis, RNA ; Disease Models, Animal ; Spleen/cytology* ; T-Lymphocytes, Regulatory/immunology* ; Humans ; Signal Transduction ; Factor VIII/immunology* ; T-Lymphocytes, Helper-Inducer/immunology*

Acquired hemophilia A (AHA) is a rare autoimmune bleeding disorder. Its occurrence secondary to hepatobiliary malignancies is even rarer, and without timely diagnosis and treatment, the mortality rate is extremely high. There is a need to raise awareness of this disease. This report describes a case of a 70-year-old female patient diagnosed with AHA 2 months after surgery for cholangiocarcinoma, admitted to the Second Affiliated Hospital of Bengbu Medical College in October 2022. The patient presented with subcutaneous hematoma in both lower limbs. Coagulation function tests showed a markedly prolonged activated partial thromboplastin time (APTT) of 74.5 seconds, with no correction in the APTT mixing test. Coagulation factor assays revealed a severely reduced coagulation factor VIII activity (FVIII:C) of 0.3%, and an inhibitor titer of 25.6 BU/mL was detected. After ruling out other potential causes, the patient was diagnosed with cholangiocarcinoma-associated AHA. With chemotherapy to control the primary tumor, alongside hemostatic and immunosuppressive therapy for inhibitor eradication, AHA was brought under control. The patient had no further coagulation abnormalities or bleeding, enabling timely and full-course chemotherapy for cholangiocarcinoma and significantly improving survival and quality of life. Therefore, in patients with malignancies who present with spontaneous bleeding or unusual bleeding following surgery, trauma, or invasive procedures, clinicians should be alert to the possibility of secondary AHA. Timely diagnosis and treatment can significantly improve prognosis.
Humans ; Cholangiocarcinoma/surgery* ; Female ; Hemophilia A/drug therapy* ; Aged ; Bile Duct Neoplasms/surgery* ; Factor VIII

OBJECTIVE: To calculate the pharmacokinetic parameters of recombinant human coagulation factor Ⅷ using myPKFiT in patients with severe hemophilia A, and provide an individualized treatment plan for patients. METHODS: A total of 42 patients with severe hemophilia A who were treated with recombinant human coagulation factor Ⅷ were included from January 2021 to December 2021. myPKFiT was used to calculate the pharmacokinetic parameters of FⅧ, and the individualized treatment plan for hemophilia A patients was formulated. RESULTS: The median age of 42 patients with severe hemophilia A was 31（16-50） years old, the average weight was 54.0±9.9 kg, the half-life of FⅧ was 12.05±1.6 h, the time to more than 1% of the baseline was 62.3±15.3 h, and the 0 bleeding rate after the guidance of myPKFiT was significantly increased from 39% to 49%, the Annual bleeding rate was reduced from 3.6±2.5 to 2.1±2.0, and the Annual joint bleeding rate was reduced from 3.2±2.2 to 1.9±0.9, all of which were statistically different (P<0.05). CONCLUSION Individualized therapy in patients with severe hemophilia A who were guided by myPKFiT assay of pharmacokinetics parameters can significantly reduce the annual bleeding rate and annual joint bleeding rate of patients.
Adult ; Humans ; Middle Aged ; Blood Coagulation Factors ; Factor VIII/pharmacokinetics* ; Hemophilia A ; Hemorrhage ; Recombinant Proteins/pharmacokinetics* ; Adolescent ; Young Adult

OBJECTIVE: To analyze the results of activated partial thromboplastin time (APTT) mixing test in coagulation factor Ⅷ inhibitor-positive hemophilia patients, so as to increase the value of APTT mixing test in the screen of factor Ⅷ inhibitor. METHODS: Eighty plasmas samples with different titers of coagulation factor Ⅷ inhibitors had been collected and diluted for routine immediate APTT mixing test and at 37 ℃ 2 hours incubation APTT mixing test. Fifteen samples were selected for immediate and normal temperature incubation for 15 min, 30min, 1 hour, 2 hours and 37 ℃ for 30 min, 1 hour, 2 hours APTT mixing test. RESULTS: The results of APTT mixing test were significantly correlated with the titers of coagulation factor Ⅷ inhibitors. The ROC curve result showed that the best diagnostic cut-off value for 2 hours incubation APTT mixing test at 37 ℃ to determine the presence or absence of coagulation factor Ⅷ inhibitors was 43.8 s (sensitivity and specificity was 85.90% and 100%, respectively), while the best diagnostic cut-off value for distinguishing high-titer and low-titer Ⅷ inhibitors was 52.4 s (sensitivity and specificity was 98.18% and 95.65%, respectively). The critical coagulation factor Ⅷ inhibitor titer that could not be corrected by immediate APTT was 5.14 BU/ml, while that could not be corrected by 37 ℃ 2 hours incubation APTT was 1.31 BU/ml. Paired samples t -test was performed on the APTT mixing test results at different times and temperatures, and the differences were statistically significant (P < 0.05). CONCLUSIONS The APTT mixing test can be used as a screening index for coagulation factor Ⅷ inhibitors. APTT mixing test result shows a significant time-temperature dependence with lower titers of coagulation factor Ⅷ inhibitor. Patients with hemophilia who cannot be corrected by immediate APTT mixing test should be alert to the possibility of high titer of coagulation factor Ⅷ.
Humans ; Factor VIII ; Hemophilia A/diagnosis* ; Blood Coagulation Tests/methods* ; Partial Thromboplastin Time ; Blood Coagulation Factors

Hemophilia A(HA) is an X-linked recessive bleeding disorder caused by mutations in coagulation factor VIII. Nowadays, exogenous coagulation factor replacement therapy is the main treatment. With the continuous development of gene therapy, new research directions have been provided for the treatment of hemophilia A. CRISPR-Cas9 technology was applied to select suitable target sites, and mediate the targeted knock-in and efficient expression of exogenous B-domain-deleted FⅧ variant gene through corresponding vectors for the treatment of hemophilia A.CRISPR-Cas9 technology is an emerging gene editing tool with great efficiency, safety and effectiveness, and has been widely used in hemophilia gene therapy research. This paper reviews the vector selection, construction of therapeutic genes, gene editing technology and selection of expression target sites for hemophilia A gene therapy at this stage.
Humans ; Hemophilia A/therapy* ; CRISPR-Cas Systems ; Hemophilia B/therapy* ; Gene Editing ; Genetic Therapy ; Genetic Vectors

OBJECTIVE: To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia. METHODS: Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene. RESULTS: The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups. CONCLUSION After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.
Humans ; Transduction, Genetic ; Genetic Vectors ; Hemophilia A/genetics* ; Transfection ; Blood Coagulation Factors/genetics* ; Lentivirus/genetics*

Systemic lupus erythematosus (SLE) complicated with acquired hemophilia A (AHA) is a rare condition with frequently delayed diagnosis and a high mortality rate, so it is necessary to strengthen the understanding of this disease. In this study, the characteristics and treatment in 1 case of SLE complicated by AHA is reported and analyzed, and a literature review is conducted. The patient was a 29-year-old young female with a 10-year history of SLE, the main clinical manifestation was severe abdominal bleeding. Laboratory tests revealed that the activated partial thromboplastin time (APTT) was notably prolonged (118.20 s), and the coagulation factor VIII activity (FVIII꞉C) was extremely decreased (0.20%) with high-titer of factor VIII (FVIII) inhibitor (31.2 BU/mL). After treating with high-dose glucocorticoid, immunoglobulin, cyclophosphamide, rituximab, blood transfusion, and intravenous infusion of human coagulation FVIII, the coagulation function and coagulation FVIII꞉C were improved, and FVIII inhibitor was negative without serious adverse reactions. During the next 5-year follow-up, the patient's condition was stable and no bleeding occurred. In the case of coagulation dysfunction in SLE, especially with isolated APTT prolongation, AHA should be screened. When the therapeutic effects of glucocorticoid combined with immunosuppressants are not desirable, rituximab could be introduced.
Female ; Humans ; Adult ; Hemophilia A/therapy* ; Rituximab ; Glucocorticoids ; Factor VIII ; Lupus Erythematosus, Systemic/complications* ; Hemorrhage/complications*

Cryptogenic organic pneumonia (COP) refers to organic pneumonia that has not been identified a clear cause by current medical methods. A small proportion of COP can exhibit severe and progressive characteristics, while severe COP can cause systemic inflammatory storms and can be secondary to hemophilia. This article reported a case of acute severe COP secondary to hemophilia. A 67-year-old male patient was admitted to the hospital due to cough, shortness of breath, and fever. At first, he was misdiagnosed as severe pneumonia, but failed to receive anti infection treatments. Sputum pathogenetic examination and Macrogene testing of alveolar lavage fluid were performed, and no etiology was found to explain the patient's condition. The condition was gradually worsened and hemophilia occurred to explain, suggesting that acute severe COP was relevant. After receiving hormone treatment, the condition gradually relieved and the absorption of lung lesions improved. Hemophilia secondary to COP is rare, and the specific mechanism needs further study.
Male ; Humans ; Aged ; Hemophilia A/complications* ; Pneumonia/diagnosis* ; Bronchoalveolar Lavage Fluid ; Cough ; Dyspnea/etiology*

BACKGROUND: Total knee arthroplasty (TKA) can reduce severe joint pain and improve functional disability in hemophilia. However, the long-term outcomes have rarely been reported in China. Therefore, this study aimed to evaluate the long-term outcomes and complications of TKA in Chinese patients with hemophilic arthropathy. METHODS: We retrospectively reviewed patients with hemophilia who underwent TKA between 2003 and 2020, with at least 10 years of follow-up. The clinical results, patellar scores, patients' overall satisfaction ratings, and radiological findings were evaluated. Revision surgery for implants during the follow-up period was recorded. RESULTS: Twenty-six patients with 36 TKAs were successfully followed up for an average of 12.4 years. Their Hospital for Special Surgery Knee Score improved from an average of 45.8 to 85.9. The average flexion contracture statistically significantly decreased from 18.1° to 4.2°. The range of motion (ROM) improved from 60.6° to 84.8°. All the patients accepted patelloplasty, and the patients' patellar score improved from 7.8 preoperatively to 24.9 at the last follow-up. There was no statistically significant difference in clinical outcomes between the unilateral and bilateral procedures, except for a better ROM at follow-up in the unilateral group. Mild and enduring anterior knee pain was reported in seven knees (19%). The annual bleeding event was 2.7 times/year at the last follow-up. A total of 25 patients with 35 TKAs were satisfied with the procedure (97%). Revision surgery was performed in seven knees, with 10- and 15-year prosthesis survival rates of 85.8% and 75.7%, respectively. CONCLUSIONS TKA is an effective procedure for patients with end-stage hemophilic arthropathy, which relieves pain, improves knee functions, decreases flexion contracture, and provides a high rate of satisfaction after more than ten years of follow-up.
Humans ; Arthroplasty, Replacement, Knee/methods* ; Hemophilia A/surgery* ; Follow-Up Studies ; Retrospective Studies ; Treatment Outcome ; Knee Joint/surgery* ; Range of Motion, Articular ; Arthritis/complications* ; Pain ; Contracture/surgery* ; Surgeons ; Knee Prosthesis