Objective To establish and validate a droplet digital PCR(ddPCR) method for the detection of template DNA residues in SARS-CoV-2 mRNA vaccine, thereby providing technical support for the quality control of SARS-CoV-2 mRNA vaccine.Methods With the replicon of the common DNA sequence of different plasmids as the target gene, specific primers and probe were designed to establish a ddPCR method for detecting the template DNA residues in SARS-CoV-2 mRNA vaccine. The established method was validated for linear range, repeatability, accuracy, intermediate precision, robustness,limit of detection(LOD), limit of quantification(LOQ), and specificity. The validated ddPCR method was subsequently applied to detect template DNA residues in SARS-CoV-2 mRNA vaccines targeting various variants from different manufacturers.Results The optimal annealing temperature for the established ddPCR was determined to be 58 ℃. The method demonstrated good linearity within a copy number range of 19 to 4 729 copies/μL, with a correlation coefficient(R2) of 0. 999 9.The repeatability coefficient of variation(CV) was less than 10%, and the accuracy recovery rate ranged from 99% to 121%,with a CV of less than 10%. Both CVs of intermediate precision and robustness exhibited less than 10%. The LOD and LOQ of the method were 9 copies/μL and 26 copies/μL, respectively, with good specificity. When applied to detect template DNA residues in SARS-CoV-2 mRNA vaccines from four different manufacturers targeting various variants, the CVs were all not more than 15%, and the recovery rates ranged from 79% to 138%.Conclusion The established ddPCR method for detecting template DNA residues in SARS-CoV-2 mRNA vaccine demonstrates high sensitivity, excellent stability, strong specificity, and high accuracy, making it a suitable approach for the detection of template DNA residues in SARS-CoV-2mRNA vaccine.

Objective:Induced pluripotent stem cells (iPSCs) cell lines were established using peripheral blood mononuclear cells (PBMCs) from a patient suffering from neonatal respiratory distress syndrome (NRDS) who carried Adenosine triphosphate-binding cassette transporter A3 ( ABCA3) compound heterozygous mutations. Methods:Cell experimental research.Peripheral venous blood was collected and PBMCs were isolated and cultured in vitro. PBMCs were transfected with non-integrated Sendai vector carrying reprogramming factors.The chromosome karyotypes of the established iPSCs were analyzed.Immunofluorescence and flow cytometry were used to detect pluripotency markers of stem cells and verify their differentiation potential.Sanger sequencing was performed to analyze gene mutations.In addition, short tandem repeat (STR) analysis was performed, polymerase chain reaction(PCR) and agarose gel electrophoresis were used to detect virus residual. Results:Karyotype analysis of established iPSCs cell lines showed normal diploid 46, XY karyotype.Immunofluorescence showed positive staining of stem cell pluripotency markers OCT4, SSEA4, Nanog and Sox2.Flow cytometry was used to detected stem cell pluripotency markers and showed expression of TRA-1-60, SSEA-4 and OCT4.After differentiation into all three germ layers, immunofluorescence was performed to detect ectoderm (Pax-6), mesoderm (Brachyury) and endoderm alpha-fetoprotein markers, and the results showed positive staining, which confirmed that the iPSCs had the potential to differentiate.Sanger sequencing showed c. 3997_3998del and c. 3137C>T compound heterozygous mutations.STR analysis showed they originate from PBMCs, and no Sendai virus residual was detected by PCR and agarose gel electrophoresis.Conclusions:In this study, PBMCs from patient carrying ABCA3 compound heterozygous mutations was used to establish iPSCs cell lines.The research lays a foundation for the study of pathogenesis, therapeutic drug screening and cell therapy of NRDS caused by ABCA3 gene mutations.

Objective:To investigate the application value of antegrade splenic superior region dissection first in laparoscopic total gastrectomy of obesity gastric cancer.Methods:The retrospective and descriptive study was conducted. The clinicopathological data of 21 obesity patients with gastric cancer who underwent laparoscopic total gastrectomy in Renji Hospital of Shanghai Jiaotong University School of Medicine from July 2018 to October 2023 were collected. There were 16 males and 5 females, aged (58±13)years. All 21 patients underwent laparoscopic total gastrec-tomy with antegrade splenic superior region dissection first. Observation indicators: operation time, volume of intraoperative blood loss, laparotomy conversion, intraoperative splenic hemorrhage or gastric hemorrhage, lymph node dissection, time to postoperative first flatus, time to postoperative initial liquid food intake, duration of postoperative hospital stay, postoperative complication. Measure-ment data with normal distribution were represented as Mean± SD, and count data were expressed as absolute numbers. Results:All 21 patients underwent laparoscopic total gastrectomy success-fully, with the operation time of (283±47)minutes, time for splenogastric ligament and vascular manage-ment of (34±12)minutes, volume of intraoperative blood loss of (143±86)mL, and no laparotomy conversion. There was no intraoperative splenic hemorrhage or gastric haemorrhage. The total number of lymph node dissected in 21 patients was 375, with the number of lymph node dissected as (21±9)per case. Time to postoperative first flatus, time to postoperative initial liquid food intake and duration of postoperative hospital stay in 21 patients were (3.1±0.7)days, (4.0±0.8)days and (10.1±3.0)days, respectively. There were 2 patients with postoperative complications, including 1 case of incision infection and 1 case of lung infection. The 2 patients with postoperative com-plications were recovered and discharged after conservative treatment. There was no death during the postoperative 30 days.Conclusion:The application of antegrade splenic superior region dissec-tion first in laparoscopic total gastrectomy is safe and feasible, which can reduce surgical difficulty.

Purpose: This study examined the diagnostic value of high-frequency ultrasound (HFUS) features in differentiating between benign and malignant skin lesions. Methods: A total of 1,392 patients with 1,422 skin lesions who underwent HFUS examinations were included in an initial dataset (cohort 1) to identify features indicative of malignancy. Qualitative clinical and HFUS characteristics were recorded for all lesions. To determine which HFUS and clinical features were suggestive of malignancy, univariable and multivariable logistic regression analyses were employed. The diagnostic performance of HFUS features combined with clinical information was evaluated. This assessment was validated using internal data (cohort 2) and multicenter external data (cohort 3). Results: Features significantly associated with malignancy included age above 60 years; lesion location in the head, face, and neck or genital regions; changes in macroscopic appearance; crawling or irregular growth pattern; convex or irregular base; punctate hyperechogenicity; blood flow signals; and feeding arteries. The area under the receiver operating characteristic curve, sensitivity, and specificity of HFUS features combined with clinical information were 0.946, 92.5%, and 86.9% in cohort 1; 0.870, 93.1%, and 80.8% in cohort 2 (610 lesions); and 0.864, 86.2%, and 86.6% in cohort 3 (170 lesions), respectively. However, HFUS is not suitable for evaluating lesions less than 0.1 mm in thickness or lesions exhibiting surface hyperkeratosis. Conclusion In a clinical setting, the integration of HFUS with clinical information exhibited good diagnostic performance in differentiating malignant and benign skin lesions. However, its utility was limited in evaluating extremely thin lesions and those exhibiting hyperkeratosis.

Purpose: This study examined the diagnostic value of high-frequency ultrasound (HFUS) features in differentiating between benign and malignant skin lesions. Methods: A total of 1,392 patients with 1,422 skin lesions who underwent HFUS examinations were included in an initial dataset (cohort 1) to identify features indicative of malignancy. Qualitative clinical and HFUS characteristics were recorded for all lesions. To determine which HFUS and clinical features were suggestive of malignancy, univariable and multivariable logistic regression analyses were employed. The diagnostic performance of HFUS features combined with clinical information was evaluated. This assessment was validated using internal data (cohort 2) and multicenter external data (cohort 3). Results: Features significantly associated with malignancy included age above 60 years; lesion location in the head, face, and neck or genital regions; changes in macroscopic appearance; crawling or irregular growth pattern; convex or irregular base; punctate hyperechogenicity; blood flow signals; and feeding arteries. The area under the receiver operating characteristic curve, sensitivity, and specificity of HFUS features combined with clinical information were 0.946, 92.5%, and 86.9% in cohort 1; 0.870, 93.1%, and 80.8% in cohort 2 (610 lesions); and 0.864, 86.2%, and 86.6% in cohort 3 (170 lesions), respectively. However, HFUS is not suitable for evaluating lesions less than 0.1 mm in thickness or lesions exhibiting surface hyperkeratosis. Conclusion In a clinical setting, the integration of HFUS with clinical information exhibited good diagnostic performance in differentiating malignant and benign skin lesions. However, its utility was limited in evaluating extremely thin lesions and those exhibiting hyperkeratosis.

Purpose: This study examined the diagnostic value of high-frequency ultrasound (HFUS) features in differentiating between benign and malignant skin lesions. Methods: A total of 1,392 patients with 1,422 skin lesions who underwent HFUS examinations were included in an initial dataset (cohort 1) to identify features indicative of malignancy. Qualitative clinical and HFUS characteristics were recorded for all lesions. To determine which HFUS and clinical features were suggestive of malignancy, univariable and multivariable logistic regression analyses were employed. The diagnostic performance of HFUS features combined with clinical information was evaluated. This assessment was validated using internal data (cohort 2) and multicenter external data (cohort 3). Results: Features significantly associated with malignancy included age above 60 years; lesion location in the head, face, and neck or genital regions; changes in macroscopic appearance; crawling or irregular growth pattern; convex or irregular base; punctate hyperechogenicity; blood flow signals; and feeding arteries. The area under the receiver operating characteristic curve, sensitivity, and specificity of HFUS features combined with clinical information were 0.946, 92.5%, and 86.9% in cohort 1; 0.870, 93.1%, and 80.8% in cohort 2 (610 lesions); and 0.864, 86.2%, and 86.6% in cohort 3 (170 lesions), respectively. However, HFUS is not suitable for evaluating lesions less than 0.1 mm in thickness or lesions exhibiting surface hyperkeratosis. Conclusion In a clinical setting, the integration of HFUS with clinical information exhibited good diagnostic performance in differentiating malignant and benign skin lesions. However, its utility was limited in evaluating extremely thin lesions and those exhibiting hyperkeratosis.

Purpose: This study examined the diagnostic value of high-frequency ultrasound (HFUS) features in differentiating between benign and malignant skin lesions. Methods: A total of 1,392 patients with 1,422 skin lesions who underwent HFUS examinations were included in an initial dataset (cohort 1) to identify features indicative of malignancy. Qualitative clinical and HFUS characteristics were recorded for all lesions. To determine which HFUS and clinical features were suggestive of malignancy, univariable and multivariable logistic regression analyses were employed. The diagnostic performance of HFUS features combined with clinical information was evaluated. This assessment was validated using internal data (cohort 2) and multicenter external data (cohort 3). Results: Features significantly associated with malignancy included age above 60 years; lesion location in the head, face, and neck or genital regions; changes in macroscopic appearance; crawling or irregular growth pattern; convex or irregular base; punctate hyperechogenicity; blood flow signals; and feeding arteries. The area under the receiver operating characteristic curve, sensitivity, and specificity of HFUS features combined with clinical information were 0.946, 92.5%, and 86.9% in cohort 1; 0.870, 93.1%, and 80.8% in cohort 2 (610 lesions); and 0.864, 86.2%, and 86.6% in cohort 3 (170 lesions), respectively. However, HFUS is not suitable for evaluating lesions less than 0.1 mm in thickness or lesions exhibiting surface hyperkeratosis. Conclusion In a clinical setting, the integration of HFUS with clinical information exhibited good diagnostic performance in differentiating malignant and benign skin lesions. However, its utility was limited in evaluating extremely thin lesions and those exhibiting hyperkeratosis.

Purpose: This study examined the diagnostic value of high-frequency ultrasound (HFUS) features in differentiating between benign and malignant skin lesions. Methods: A total of 1,392 patients with 1,422 skin lesions who underwent HFUS examinations were included in an initial dataset (cohort 1) to identify features indicative of malignancy. Qualitative clinical and HFUS characteristics were recorded for all lesions. To determine which HFUS and clinical features were suggestive of malignancy, univariable and multivariable logistic regression analyses were employed. The diagnostic performance of HFUS features combined with clinical information was evaluated. This assessment was validated using internal data (cohort 2) and multicenter external data (cohort 3). Results: Features significantly associated with malignancy included age above 60 years; lesion location in the head, face, and neck or genital regions; changes in macroscopic appearance; crawling or irregular growth pattern; convex or irregular base; punctate hyperechogenicity; blood flow signals; and feeding arteries. The area under the receiver operating characteristic curve, sensitivity, and specificity of HFUS features combined with clinical information were 0.946, 92.5%, and 86.9% in cohort 1; 0.870, 93.1%, and 80.8% in cohort 2 (610 lesions); and 0.864, 86.2%, and 86.6% in cohort 3 (170 lesions), respectively. However, HFUS is not suitable for evaluating lesions less than 0.1 mm in thickness or lesions exhibiting surface hyperkeratosis. Conclusion In a clinical setting, the integration of HFUS with clinical information exhibited good diagnostic performance in differentiating malignant and benign skin lesions. However, its utility was limited in evaluating extremely thin lesions and those exhibiting hyperkeratosis.

Objective:To construct a droplet digital PCR (ddPCR) for quantification of virus particles in recombinant adenovirus-vectored SARS-CoV-2 vaccine.Methods:The genome of SARS-CoV-2 BA.1 strain was used as the target gene. A set of primer and probe was designed based on the conserved sequence of spike protein, and the ddPCR for quantification of virus particles in recombinant adenovirus-vectored SARS-CoV-2 vaccine was established. The linearity, accuracy, specificity, repeatability and durability of the method were verified.Results:The optimal annealing temperature for ddPCR was 63℃. When the number of virus particles was in the range of 5×10 5-2×10 7 VP/ml, the linearity and the recovery rate of the method were good; the specificity of the probe and primer was good; the coefficient of variation (CV) values of the repeatability and the intermediate precision were within 10%; the CV value of the durability was within 15%. When comparing ddPCR with qPCR, the CV values were all within 10%. Conclusions:The established ddPCR had high sensitivity, good stability and strong specificity, suggesting that it could be used for the quantitative detection of virus particles in recombinant adenovirus-vectored SARS-CoV-2 vaccine.

OBJECTIVE To establish and verify a ddPCR method to determinate the proportion of mRNA components in multivalent mutant COVID-19 mRNA vaccine. METHODS Different types of coronavirus mRNA were used as target genes, and their conserved sequences were selected to design primers and probes. The ddPCR method for measuring the proportion of multivalent mutant COVID-19 mRNA vaccine components was established, and the linear range, repeatability, accuracy, intermediate precision, durability and applicability of the method were verified. RESULTS When the total mRNA concentration ranged from 1 to 150 pg·mL-1, the linearity was good. Repeatability coefficient of variation(CV) was within 10%. The recoveries of accuracy verification were 84%-114%, CV＜10%. The intermediate precision and durability coefficient of variation CV were all＜ 5%, and the CV of the proportion of components in the four different bivalent COVID-19 mRNA vaccines was all≤5%. CONCLUSION The ddPCR method established for detecting the component proportion of multivalent mutant COVID-19 mRNA vaccine has high sensitivity, good stability and strong specificity, which is suitable for detecting the component proportion and content of multivalent COVID-19 mRNA vaccine.