Objective To establish the topotecan(TPT)-resistant cell line of human oophoroma (SKOV3/TPT), and to investigate its biological features. Methods To simulate the condition in clinical chemotherapy, drug resistant SKOV3/TPT cell line of human oophoroma was established by challenging SKOV3 with a large dose of TPT (2160ng/ml). Drug sensitivity was assessed by MTT assay. Light microscopy, electronic microscopy, trypan blue staining rejection, and cell counting were employed to determine the biological features of the cell line. Flow cytometry (FCM) was used to study the cell cycle distribution, the accumulation of intracellular drug, and the expression of P glucoprotein (Pgp). Results SKOV3/TPT cells were established successfully, and the resistance index (RI) was 10.67. They showed an obvious cross-resistance to CPT-11, MX and VCR, but still sensitive to taxol, cis-platin, VP16, MTX and 5-Fluorouracil. Comparing with the parent cells, their morphology was unchanged, but multiplication time was prolonged. The apoptosis rate and the proportion of G2+M phase cells of resistant cells were significantly higher than that of their parent cells (P

Objective: To investigate the relationship between the eosin-5′-maleimide (EMA) binding test and the clinical severity of hereditary spherocytosis (HS). Methods: A total of 258 un-splenectomize HS patients were consecutively enrolled. Correlation of hemoglobin concentration, hemolytic parameters, compensating erythropoiesis and the EMA binding test were evaluated. Results: 258 (128 male and 130 female) patients were included in this study, including 91 compensatory hemolysis patients, 53 patients with mild anemia, 78 patients with moderate anemia and 36 patients with severe anemia. The median age at diagnosis was 23 (2-70) years. The median decreased fluorescence intensity of EMA binding test was 29.97% (16.09%-47.34%) and the average intensity was (29.70±6.28) % of 258 HS patients. The decreased EMA binding fluorescence intensity correlated with MCV (r=-0.343, P<0.001) and MCHC (r=0.223, P<0.001). There was no relationship between EMA fluorescence intensity and absolute reticulocyte count (r=0.080, P=0.198) , reticulocyte percentile (r=-0.015, P=0.813) , IBIL levels (r=-0.009, P=0.902) , HGB levels (r=-0.067, P=0.280). Evaluated as a quartile variable, EMA fluorescence intensity was not correlated with anemia severity (C=0.150, P=0.746). Conclusion EMA binding test does not related to anemia levels and has no major clinical implications for disease severity in HS.

Objective: To evaluate the impact of the targeted next-generation sequencing (NGS) assay for difficult congenital anemias. Methods: Blood Disease Hospital Anemia Panel 2014 (BDHAP-2014) including 217 known genes of congenital anemias was developed. NGS and parental verification were performed for patients who were suspected diagnosed with congenital anaemia from August 2014 to July 2017. Results: A total of 46 patients were enrolled in this study, the clinical suspection were 11 cases Fanconi anemia (FA), 8 cases congenital dyserythropoietic anemia (CDA), 6 cases congenital sideroblast anemia (CSA), 12 cases congenital hemolytic anemia (CHA), 1 case dyskeratosis congenital (DC), 4 cases iron-refractory iron deficiency anemia and 4 cases unexplained cytopenia (Uc), respectively. 28 (60.9%) of 46 patients became confirmed cases after targeted NGS, corresponding to 44 mutations of which 33 were new. 26(56.5%) patients with results of the assay matching to clinical suspection, including FA (5/11, 45.5%), CSA (6/6, 100.0%), CDA (3/8, 37.5%) and CHA (12/12, 100.0%). 2 (4.3%) cases not matching to clinical suspection, including dyskeratosis congenital (DC) was made in 1(2.2%) patients with suspected FA and familial hemophagocytic lymphohistiocytosis (FHL) was made in 1(2.2%) patients with suspected unexplained cytopenia (Uc). In 12 CHA patients, the hemolytic type was further clarified by the NGS. The remaining 18 cases were not clearly diagnosed. Conclusion Targeted NGS assay is of major impact on congenital anemias. The assay should be used routinely in congenital anemias.

Objective: To reveal the genetic characteristics of erythrocyte membrane protein in hereditary spherocytosis (HS) in China. Methods: Next-generation sequencing technology was used to detect mutations in genes of erythrocyte membrane proteins in 51 clinically diagnosed HS patients. The relationship between gene mutations and clinical phenotypes was analyzed. Results: Mutations in erythrocyte membrane protein genes were detected in 37 patients, including 17 with ANK1 mutations (17/37, 45.9%), 14 with SPTB mutations (14/37, 37.8%), and 5 with SLC4A1 mutations (5/37, 13.5%). One patient carried both heterozygous ANK1 mutation and SPTB mutation (1/37, 2.7%). SPTA1 and EPB42 mutation was not fou nd in any patient. Nonsense mutations (36.8%) and missense mutations (31.6%) were most common. Of the 38 mutations detected, 34 were novel mutations and have not been reported elsewhere (89.5%). Sixteen HS patients underwent parental genetic validation, 6 patients (37.5%) inherited gene mutation from parents and 10 (62.5%) were de novo. The peripheral blood cell parameters of HS patients were not related to the mutant genes and gene mutation types. However, it seems that HS patients with mild clinical status are prone to carry SPTB mutations while more patients with severe clinical status have ANK1 mutations. Conclusions ANK1 and SPTB are the most common mutant genes in Chinese HS patients, mainly with missense mutations and nonsense mutations. There was no significant correlation between the mutation of HS related genes and the severity of HS.

Clinical data of 19 patients with congenital pyruvate kinase deficiency were analyzed. Insufficient pyruvate kinase confirmed the diagnosis. Laboratory parameters of hemolysis were summarized. In cases of neonatal hyperbilirubinemia and unexplained hemolytic anemia, pyruvate kinase activity and next generation sequencing test may help the early diagnosis.

Objective:To investigate the clinical characteristics of patients with acquired aplastic anemia (AA) accompanied by abnormal antinuclear antibody (ANA) and autoantibodies and their effects on the efficacy of immunosuppressive therapy (IST).Method:A retrospective case-control study was conducted, analyzing the clinical data of 291 patients with AA who underwent IST and were screened for autoantibodies at initial diagnosis between January 2018 and December 2019 at Blood Diseases Hospital, Chinese Academy of Medical Sciences. According to the titer of ANA at the initial diagnosis, extracted nuclear antigen antibodies (ENAs) abnormality and the change of ANA titer after treatment, the treatment responses of 3 months and 6 months after IST were compared. The correlation between clinical features and ANA abnormality was analyzed by univariate and multivariate logistic regression analysis. The parameters of univariate analysis P<0.1 were included in multivariate analysis, stepwise regression analysis and subgroup analysis. Results:A total of 291 patients were included in the study, of which 145 (49.83%) were male. Among all patients, 147 (50.52%) tested positive for ANA at initial diagnosis, with titers of 1∶100, 1∶320, and 1∶1 000 observed in 94, 47, and 6 cases, respectively. Female gender, older age, presence of paroxysmal nocturnal hemoglobinuria (PNH) clone, and higher levels of IgG, IgA, and thyroid hormone were significantly associated with ANA positivity at initial diagnosis, while white cell counts, reticulocytes, and free triiodothyronine were significantly lower than that of ANA-negatively patients (all P<0.05). Furthermore, logistic regression analyses revealed that female gender ( OR=1.980, 95% CI 1.206-3.277), older age ( OR=1.017, 95% CI 1.003-1.032), and presence of PNH clone ( OR=1.875, 95% CI 1.049-3.408) were independent risk factors for ANA positivity at initial diagnosis. Subgroup analysis indicated that the risk of ANA positivity at initial diagnosis was even higher in PNH clone-positive patients in the subgroups of females ( OR=1.24, 95% CI 1.02-1.51), severe AA ( OR=1.26, 95% CI 1.07-1.47), and age≥40 years ( OR=1.26, 95% CI 1.05-1.52) (all P<0.05). However, ANA titers at initial diagnosis, presence of other abnormal ENAs, and changes in ANA titers after treatment with IST were not correlated with treatment response (all P>0.05). Conclusions:Approximately 50% of patients with AA had abnormal ANA, and their presence was significantly associated with female gender, older age, and presence of PNH clone at initial diagnosis. However, the presence of abnormal ANA and changes in ANA titers after treatment did not affect the efficacy of IST in patients with AA.

Objective:Short-term efficacy and safety of afatrombopag conversion therapy in patients with aplastic anemia (AA) who were previously ineffectively treated with intense immunosuppressive therapy (IST) combined with TPO receptor Agonist (TPO-RA) or who were unable to tolerate the side effects of TPO-RA.Methods:Analysis of patients with severe aplastic anemia (SAA) treated in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College from January 2021 to December 2021 who received IST combined with TPO-RA (eltrombopag/hatrombopag) for at least 6 months, but was ineffective for at least 3 months or patients who cannot continue to use TPO-RA due to side effects, and switched from TPO-RA to avatrombopag (APAG) , and treated for at least 6 months. This study analyzed its short-term efficacy and evaluated its safety.Results:The median age was 54 (14-68) years old among the 16 patients with AA (8 male and eight female) . A total of ten patients (refractory group) who did not respond to IST combined with TPO-RA were converted to APAG median therapy for 6 (6-10) months. Only seven patients (70% ) obtained trilineage HR [four cases of complete treatment response (CTR) , one case of good treatment response (GPR) , and two cases of partial treatment response (PR) ], all of which began to take effect at 3 months of APAG treatment. There were six patients with TPO-RA intolerance, and APAG was treated for 6 (2 to 8) months. About four patients (67% ) received HR (three GPR cases and one PR case) , of which two patients received PR at 3 months and four patients received HR at 6 months of APAG treatment. No APAG-related grade 2 or more adverse events occurred during the APAG therapy, no thrombotic events occurred, no fibrologic tissue hyperplasia was found in the bone marrow pathology reexamination at 6 months of treatment, and none of the patients discontinued the drug due to adverse events.Conclusion:APAG may be a better switching treatment option for patients with AA who are refractory or are intolerant to TPO-RA.

Objective:To investigate the effect of on-demand glucocorticoid strategy on the occurrence and outcome of porcine anti-lymphocyte globulin (p-ALG) -associated serum sickness in aplastic anemia (AA) .Methods:The data of AA patients who received in the Anemia Diagnosis and Treatment Center of Haematology Hospital, CAMS & PUMC from January 2019 to January 2022 were collected. Among them, 35 patients were enrolled in the on-demand group, with the glucocorticoid strategy adjusted based on the occurrence and severity of serum sickness; 105 patients were recruited in the usual group by matching the age and disease diagnosis according to 1∶3 ratio in patients who received a conventional glucocorticoid strategy in the same period. The incidences, clinical manifestations, treatment outcomes of serum sickness, and glucocorticoid dosage between the two groups were analyzed.Results:The incidences of serum sickness in the on-demand group and the usual group were 65.7% and 54.3% ( P=0.237) , respectively. The median onset of serum sickness was the same [12 (9, 13) d vs the 12 (10, 13) d, P=0.552], and clinical symptoms and signs, primarily joint, and/or muscle pain, fever, and rash were similar. Severity grades were both dominated by Grades 1-2 (62.8% vs 51.4%) , with only a few Grade 3 (2.9% vs 2.9%) , and no Grades 4-5. No significant difference in the serum sickness distribution ( P=0.530) . The median duration of serum sickness was the same [5 (3, 7) d vs 5 (3, 6) d, P=0.529], and all patients were completely cured after glucocorticoid therapy. In patients without serum sickness, the average dosage of prophylactic glucocorticoid per patient in the usual group was (469.48 ±193.57) mg (0 in the on-demand group) . When compared to the usual group, the average therapeutic glucocorticoid dosage per patient in the on-demand group was significantly lower [ (125.91±77.70) mg vs (653.90±285.56) mg, P<0.001]. Conclusions:In comparison to the usual glucocorticoid strategy, the on-demand treatment strategy could significantly reduce glucocorticoid dosage without increasing the incidence of serum sickness; in addition, the duration of serum sickness and the incidence of above Grade 2-serum sickness were similar.

Objective:To analyze the diagnostic value of cell-free plasma metagenomic next-generation sequencing (mNGS) pathogen identification for severe aplastic anemia (SAA) bloodstream infection.Methods:From February 2021 to February 2022, mNGS and conventional detection methods (blood culture, etc.) were used to detect 33 samples from 29 consecutive AA patients admitted to the Anemia Diagnosis and Treatment Center of the Hematology Hospital of the Chinese Academy of Medical Sciences to assess the diagnostic consistency of mNGS and conventional detection, as well as the impact on clinical treatment benefits and clinical accuracy.Results:①Among the 33 samples evaluated by mNGS and conventional detection methods, 25 cases (75.76%) carried potential pathogenic microorganisms. A total of 72 pathogenic microorganisms were identified from all cases, of which 65 (90.28%) were detected only by mNGS. ②All 33 cases were evaluated for diagnostic consistency, of which 2 cases (6.06%) were Composite, 18 cases (54.55%) were mNGS only, 2 cases (6.06%) were Conventional method only, 1 case (3.03%) was both common compliances (mNGS/Conventional testing) , and 10 cases (30.3%) were completely non-conforming (None) . ③All 33 cases were evaluated for clinical treatment benefit. Among them, 8 cases (24.24%) received Initiation of targeted treatment, 1 case (3.03%) received Treatment de-escalation, 13 cases (39.39%) received Confirmation, and the remaining 11 cases (33.33%) received No clinical benefit. ④ The sensitivity of 80.77%, specificity of 70.00%, positive predictive value of 63.64%, negative predictive value of 84.85%, positive likelihood ratio of 2.692, and negative likelihood ratio of 0.275 distinguished mNGS from conventional detection methods (21/12 vs 5/28, P<0.001) . Conclusion:mNGS can not only contribute to accurately diagnosing bloodstream infection in patients with aplastic anemia, but can also help to guide accurate anti-infection treatment, and the clinical accuracy is high.