ObjectiveTo discover and analyze single or several correlative key amino acid sites that influence the host tropism during the influenza A virus （IAV） infection based on complete internal protein gene segments of IAV strains， and to provide evidence for the study of human host-adaptive mutations of IAV. MethodsThe full-length nucleotide sequences of 43 671 IAV strains containing 6 complete internal gene segments were downloaded from the GISAID EpiFlu^TM database， and 698 human-tropic （HU） and 1 266 avian-tropic （AV） representative strains were included. The consensus coding sequences of the representative strains from the amphitropic category were compared by R script， and the differential amino acid sites and their polymorphisms were then obtained. The multi-site combination analysis of differential sites was conducted with R script. ResultsA total of 49 and 57 conserved differential sites were obtained from the consensus sequence comparison between AV and H1N1 （subtype from HU）， and comparison between AV and H3N2 （another subtype from HU）， separately. 79 and 65 multi-site combinations were found between HU and AV strains through 3 and 4 sites combination analysis， respectively， and a total of 11 conserved sites were involved： site 271 and 684 in PB2； site 336， 486， 581 and 621 in PB1； site 204 and 356 in PA； site 33， 305 and 357 in NP. No eligible differential sites were found in M1 and NS1. ConclusionSeveral conserved amino acid differential sites， between HU and AV strains of IAV， are found in PB2， PB1， PA and NP proteins. Instead of working as single units， these sites may have interactions， forming specific amino acid combinations that determine the host tropism of IAV collectively.

Objective:To evaluate the effects of smart stethoscope on the monitoring childhood asthma exacerbation, so as to assist family management in childhood asthma.Methods:A prospective randomized controlled study was carried out.A total of 80 children with asthma who were treated at Department of Pediatric Respiratory, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine and Shanghai Tonxin Pediatric Clinic from November 2020 to May 2021 were enrolled and randomly divided into a test group of 40 cases (used the smart stethoscope) and a control group of 40 cases(not used the smart stethoscope). Medical history data were collected.The control group received monthly routine follow-up, while the test group was followed up both routinely and by smart stethoscope.In the test group, hearing wheezing sound was regarded as asthma exacerbation, and in the control group, the asthma exacerbation was reported by the parents themselves.The frequency of asthma exacerbation, asthma control level and quality of life were compared between the two groups.The recognition, diagnosis, treatment and outcomes of acute asthma exacerbation in two groups of children were described and analyzed.Measurement data were analyzed by t test or Mann- Whitney U test.Numeration data were analyzed by χ2 test. Results:Respiratory sounds collected by smart stethoscope in the test group were assessed by 3 specialist physicians.There were 12 wheezing rales (42.86%), 1 moist rale (3.57%) and 1 rhonchi rale (3.57%). Besides, 12 files (42.86%) were difficult to distinguish, and 2 files (7.14%) induced inconsistent identification.The number of asthma exacerbation was 12 in the test group and 5 in the control group.In the test group, 12 were recognized by the smart stethoscope, and only 6 were recognized by the parents.Comparing the diagnosis and treatment measures between two groups, it was found that there were more children in the test group (38.1%) receiving home treatment through telemedicine than those in the control group (20.0%). Besides, there were less children (61.9%) in the test group receiving unplanned hospital treatment (including unplanned outpatient, emergency and hospitalization) than those in the control group (80.0%). There was no statistically significant difference between the two groups of children during acute asthma exacerbation ( χ2=4.67, P=0.097). Parents were satisfied with the common functions, convenience and stability of smart stethoscope. Conclusions:Smart stethoscope can acquire the respiratory sounds of children with asthma in real time, achieving timely detection, diagnosis and treatment of asthma exacerbation in children.What′s more, smart stethoscope reduces the incidence of unplanned hospital diagnosis and treatment, and assists parents with better family management of children asthma.

Objective:To compare the efficacy of haplotype hematopoietic stem cell transplantation (HIDT) and sibling matched hematopoietic stem cell transplantation (MSDT) in the treatment of complete remission (CR) acute T-lymphoblastic leukemia (T-ALL) .Methods:We retrospectively analyzed the clinical characteristics and outcomes of 98 patients who underwent HSCT in Hebei Yanda Ludaopei hospital with HID ( n=81) or ISD ( n=17) between May 2012 and May 2016. Results:The incidence of grades 2-4 and 3-4 acute-versus-host disease 100 days after HSCT were 51.9% (95% Confidence interval [ CI] 42.0%-64.0%) vs 29.4% (95% CI 14.1%-61.4%) ( P=0.072) and 9.8% (95% CI 5.1%-19.1%) vs 11.8% (95% CI 3.2%-43.3%) ( P=1.000) for HIDT and MSDT. The 100-day cumulative incidences of CMV and EBV viremia were 53.1% (95% CI 43.3%-65.2%) vs 29.4% (95% CI 14.1%-61.4%) ( P=0.115) and 35.8% (95% CI 26.8%-47.9%) vs11.8% (95% CI 3.2%-43.3%) ( P=0.048) . The 5-year overall survival, leukemia-free survival, cumulative incidences of relapse, and no-relapse mortality were 60.5% (95% CI 5.4%-49.0%) vs 68.8% (95% CI 11.8%-40.0%) ( P=0.315) , 58.0% (95% CI 5.5%-46.5%) vs 68.8% (95% CI 11.8%-40.0%) ( P=0.258) , 16.1% (95% CI 9.8%-26.4%) vs 11.8% (95% CI 3.2%-43.3%) ( P=0.643) , 25.9% (95% CI 17.9%-37.5%) vs 19.4% (95% CI 6.9%-54.4%) ( P=0.386) for HIDT and MSDT, respectively. Conclusion:HID could be a valid alternative donor for patients with T-ALL in CR lacking an identical donor.

Objective:To study the clinical efficacy of chimeric antigen receptor T-cell (CART) treatment followed by a second allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with B-cell acute lymphoblastic leukemia (ALL) who relapsed following the first HSCT.Methods:Retrospective analysis of the clinical characteristics and prognosis of 41 patients with B-cell ALL who received a second allo-HSCT from October 2015 to June 2020 in Hebei Yanda Lu Daopei Hospital. After the first HSCT, all patients received CD19-CART, or CD22-CART treatment following a relapse of bone marrow morphology or extramedullary leukemia.Results:A total of 41 patients (male, 21; female, 20) were included in this study. The median age at the second HSCT was 16 (3-46) years. There were 31 cases of bone marrow recurrence (75.6%) , 5 cases of extramedullary recurrence (12.2%) , and 5 cases of bone marrow and extramedullary recurrences (12.2%) . After relapse, 35 patients (85.4%) received CD19-CART treatment, 2 patients received CD22-CART treatment (4.9%) , and 4 patients received CD19-CART and CD22-CART treatments (9.8%) . The expected 3-year overall survival (OS) , leukemia-free survival, cumulative relapse incidence, and non-relapse mortality (NRM) of patients after the second HSCT were 48.9% (95% CI 23.0%-70.6%) , 41.8% (95% CI 17.3%-64.9%) , 8.8% (95% CI 2.9%-26.4%) , and 51.1% (95% CI 31.2%-83.6%) , respectively. The 1-year OS of patients who relapsed ≤6 months and >6 months after the first HSCT were 45.0% (95% CI 12.7%-73.5%) and 75.0% (95% CI 51.4% -88.8%) ( P=0.017) , respectively. Conclusion:CART bridging in the second HSCT enables some B-cell ALL patients who relapsed after the first HSCT to achieve long-term survival. However, because of the high NRM, further modifications could help improve the outcome.

Objective:To study the clinical results and prognostic factors for allo-HSCT of Philadelphia chromosome-positive (Ph +) acute lymphoblastic leukemia (ALL) in complete remission (CR) in the era of tyrosine kinase inhibitors (TKI) . Methods:We performed a retrospective analysis of the clinical characteristics of 116 patients with Ph +ALL who underwent allo-HSCT while in CR. Results:The study population included 72 men and 44 women. The median patient age was 20 years (4-64 years) . The patients received sibling-identical donor ( n=21) , haplo ( n=77) , and unrelated donor ( n=18) HSCT. The overall survival (OS) rate at 5 years was 73.2% (95% CI 63.8% -80.5% ) . In particular, the 5-year OS can reach 87.5% when the time from diagnosis to transplant is <180 days. The 5-years DFS was 61.4% (95% CI 51.8% -69.7% ) , the 5-year molecular and morphology cumulative relapse incidence was 18.5% (95% CI 12.6% -27.3% ) , and the 5-year TRM was 19.9% (95% CI 13.8% -28.7% ) . A multivariate analysis showed that an age range of 15-39 years ( HR=2.730, P=0.044) , time from diagnosis to HSCT ≥ 180 days ( HR=4.534, P=0.010) , and Ⅲ-Ⅳgrade aGVHD ( HR=7.558, P=0.000) were significantly associated with an inferior overall survival. Limited cGVHD subgroup had better OS ( HR=0.300, P=0.034) . Sex, WBC count at diagnosis, type of BCR-ABL fusion genes, somatic gene mutations, CR 1 or >CR 1, MRD negative or positive, conditioning regimen based on TBI or Bu, conditioning intensity, donor source, GVHD prophylactic proposal using cyclosporine or tacrolimus, presence/absence of CMV viremia, and presence/absence of EBV viremia were not significantly different in terms of the OS and DFS. Conclusion:Factors influencing the overall survival of Ph + ALL patients who underwent allo-HSCT in CR in the TKI era include age, time form diagnosis to HSCT, and aGVHD severity.

Objective:To evaluate the association of TP53 mutations with the clinical outcomes of Ph-negative B-ALL following allogeneic hematopoietic stem cell transplantation (allo-HSCT) .Methods:Total 300 patients with Ph-negative B-ALL who underwent allo-HSCT at the Hebei Yanda Ludaopei Hospital from May 2012 to May 2017 were retrospectively analyzed; their clinical characteristics, TP53 gene mutation type, and association between TP53 mutations and transplantation outcomes, including leukemia-free survival (LFS) , overall survival (OS) , non-relapse mortality (NRM) , relapse, and GVHD, were evaluated.Results:Total 23 patients had TP53 mutations; all the TP53 mutations affected P53’DNA-binding domain. The 5-year-LFS, OS, and RI were 34.8% and 62.3% ( P=0.001) , 41.9% and 65.1% ( P=0.020) , and 47.8% and 14.8% ( P=0.000) , respectively, for TP53 mutations and wild-type TP53 patients. However, there were no significant differences in NRM and GVHD. Multivariate analysis showed that TP53 mutations remained adverse prognostic factors for LFS, OS, and RI after allo-HSCT. Conclusion:Some patients with TP53 mutations can achieve long-term survival with allo-HSCT. TP53 mutations are adverse prognostic factors for Ph-negative B-ALL patients who undergo allo-HSCT.

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 analyze the effect of NCCN (2015) risk stratification on prognosis of patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: Retrospective analysis of 258 patients with AML in CR (186 cases in CR₁, 72 cases in CR₂) who underwent allogeneic HSCT in our hospital between April 2012 and March 2015 according to NCCN (2015) risk stratification. Of them, 63 cases were classified as low risk, 112 cases intermediate risk and 83 cases high risk. Results: ①With the median follow up of 18 (5-41) months, two-year disease free surviva (DFS) in 258 patients was 78.0% (95% CI 60.4%-96.6%) . Two-year DFS in AML after transplantation was 78.6% (95% CI 61.0%-96.2%) in low risk, 76.0% (95% CI 84.0%-93.6%) in intermediate risk and 80.3% (95% CI 62.7%-97.9%) (P=0.886) in high risk groups respectively. ②Univariate analysis showed that DFS has no significant difference in patient age, the median disease course before HSCT, the WBC number at the beginning of the disease, blood routine and chromosomes examination before transplantation, extramedullary disease before transplantation, disease status before transplantation, conditioning regimen, donor type, donor and recipient sex, recipient blood type, transfused MNC number, transfused CD34⁺ cell number and transfused CD3⁺ cell number. DFS was significant lower in primary AML than that in secondary AML (P=0.006) and also lower in MRD positive than that in MRD negative (P=0.003) . The accumulative relapse was significant higher in CR₂ compared to that in CR₁ (P=0.046) . Accumulative non-relapse mortality (NRM) was significanlyt higher in secondary AML compared to that in primary AML (P=0.004) and also higher in MRD positive compared to that in MRD negative (P=0.010) . ③Multivariate analysis showed that MRD positive was the only significant factor in DFS and NRM. Conclusion Allo-HSCT treatment of AML CR patients could achieve a high efficacy, which is similar between CR₁ and CR₂ patients. There is no significant correlation between NCCN (2015) risk stratification and the prognosis of AML patients with allo-HSCT treatment. Pre-conditioning MRD status monitored by multiparameter flow cytometry was the only impact factor on DFS and NRM in allo-HSCT for CR-AML patients.