Objective:To explore the clinical application value of rapid next-generation sequencing (NGS) strategy based on targeted amplicon sequencing in the diagnosis of myeloid neoplasms.Methods:In this observational study, both rapid NGS and conventional NGS on the bone marrow or peripheral blood samples of 682 patients were prospectively performed from February 2021 to August 2022 in First Affiliated Hospital of Soochow University. The sequencing results were analyzed using the local Ion Reporter software and our lab′s self-built bioinformatics platform, respectively. The timeliness of the two sequencing platforms was compared, and the Kappa consistency test was used to evaluate the consistency between the two sequencing platforms. Patients aged between 18 and 59 years with newly diagnosed acute myeloid leukemia (AML) underwent screening by rapid NGS combining multiplex RT-PCR and in situ fluorescence hybridization technique within 72 hours, from whom high-risk patients according to European LeukemiaNet (ELN) 2017 were screened for individualized induction therapy.Results:In terms of timeliness, the median time from sample receipt to report issuance were 3 (2, 4) days and 13 (11, 15) days under rapid NGS and conventional NGS testing, respectively, with a statistically significant difference ( Z=?22.636, P<0.001). Among 682 specimens with a total of 1 507 variants, rapid NGS detected a total of 1 499 variants, with a detection rate of 99.5% and 674 cases were accurate, with an accuracy rate of 98.8%; the conventional NGS detected 1 506 variants, with a detection rate of 99.9% and 681 cases were accurate, with an accuracy rate of 99.9%. In 682 specimens, there were 181 negative and 501 positive, in which 8 cases were missed under rapid NGS, and 1 case was missed under conventional NGS. The kappa value was 0.967 by Kappa consistency test, and P<0.001, suggesting good consistency and consistency between the two NGS platforms. From February 2021 to July 2022, 286 patients who were rapidly diagnosed of AML contained 78 patients screened as the ELN 2017 adverse-risk category, including 42 patients enrolled, with age 39 (33, 52) years old. After one cycle of venetoclax combined with decitabine induction therapy, 78.6%(38/42) of the patients achieved composite complete remission. Among the rest 104 additional myeloid neoplasms, rapid NGS detected mutations in 80 patients, with a detection rate of 76.9%, among which 89.0%(215/242) of the variants could serve as the basis for the diagnostic classification, prognostic evaluation, and target therapy of myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Conclusion:The rapid NGS based on targeted amplicon sequencing is in good consistency with conventional NGS, and shorters the diagnostic time, whose sensitivity and detection range meets the need for diagnostic classification, prognostic stratification, and target therapy of myeloid neoplasms.

[Objective]To summarize and analyze the educational quality and problems of Traditional Chinese Medicine students in Zhejiang Chinese Medical University,and provide reference for improving the teaching quality and students'clinical knowledge and skills.[Methods]Taking the students of Traditional Chinese Medicine who participated in the step-1 medical licensing examination empirical research in Zhejiang Chinese Medical University from 2018 to 2020 as the research objects,the data was analyzed by variance analysis and χ2 test.[Results]Staged examination scores could reflect students'academic performance,the average passing rate of theoretical examination was 68.64%,and the average passing rate of skill assessment was 95.27%.The average total mastery rate of theoretical examination was 65.06%,including basic medicine(55.57%)and clinical medicine(52.12%),whose comparison with the total mastery rate of theoretical examination was statistically significant(P<0.05).The total mastery rate of skill assessment was 76.57%,and the average mastery rates of stations 3～4(physical examination)were 71.24%and 72.24%,whose comparison with the total mastery rate of skill assessment was statistically significant(P<0.05).It proposed improvement measures and strategies such as establishing exam incentive mechanisms to enhance students'internal motivation in exams,strengthening clinical medicine course training to improve staged examination pass rates of theoretical part,and repeatedly training clinical skills to fill the gaps in skill assessment.[Conclusion]Traditional Chinese medicine universities can improve the clinical knowledge and skills of students by establishing examination incentive mechanisms,strengthening clinical medicine course training and repeating clinical skill training,so as to improve the quality of training for students majoring in Traditional Chinese Medicine.

Objective:To explore the clinical features and treatment strategies of the transsyndesmotic ankle fracture dislocation.Methods:Data of 26 patients of transsyndesmotic ankle fracture dislocation who were treated in our hospital from December 2013 to November 2020 were retrospectively analyzed. There were 16 men and 10 women with an average age of 49.54±12.81 years (range, 26-68 years). Open injuries in 17 cases, of which the Gustilo-Anderson II type in 6 cases, IIIA type in 11 cases, closed injuries in 9 cases. According to the AO/OTA fracture classification, 44B type in 4 cases, 44C type in 22 cases. According to the Lauge-Hansen classification, there were 16 cases of pronation-abduction, 10 cases of pronation-external rotation, including 4 cases of Maisonneuve fractures, and of the 4 cases of Maisonneuve fractures, there were 3 cases of double Maisonneuve fracture. The talar dislocation was anterior, neutral, and posterior within the distal tibiofibular joint in 10 cases, 7 cases, and 9 cases. Fibular fractures in 26 cases, medial malleolar fractures in 24 cases, deltoid ligament rupture in 2 cases, posterior malleolar fractures in 13 cases, and anterior malleolar fractures in 8 cases. All closed injuries were closed reduction and plaster fixation and all open injuries were emergently debridement and reduced under the tibial plafond in the emergency department. Surgical treatment was taken until the soft tissue conditions to be allowed. The reduction quality was assessed by postoperative radiography according to the criteria proposed by Burwell-Charnley. The function of the ankle joint was assessed by the ankle-hindfoot rating system of the American Orthopaedic Foot and Ankle Society (AOFAS), and the posttraumatic arthritis and objectively quantified was assessed using the Kellgren-Lawrence grading scale.Results:There were 4 cases were unreduced due to the tibial posterior tendon to flip through the ankle joint and dislocate anterior to the tibia through the interosseous membrane. Stabilization of fibular fractures were achieved with plate in 25 cases. There were 24 cases of medial malleolar fractures, and the fixation were achieved with cannulated screws in 23 cases and with K-wire fixation in 1 case. There were 12 cases of posterior malleolar fractures treated with open reduction and internal fixation including cannulated screws in 9 cases and antiglide plates in 3 cases. There were 7 cases of anterior malleolar fractures treated with open reduction and internal fixation including suture anchors in 1 case and cannulated screws in 6 cases. Stabilization of syndesmosis was achieved with syndesmotic screws in 14 cases and with TightRope in 2 cases. All patients were followed up for 20.23±9.70 months (range, 12-60 months). According to the Burwell-Charnley criteria of reduction quality, anatomic reduction was obtained in 22 cases, and satisfactory reduction was gained in 4 cases. All fractures healed in 16.31±3.64 weeks (range, 10-24 weeks). Functional examination of the ankle joint (angle measurement method): dorsiflexion average angle 10.38°±6.66°, plantarflexion average angle 34.04°±7.20°. At latest follow up, the AOFAS score was 83.30±13.94 (range, 24-100). Ten (38%) of 26 patients had radiographic evidence of posttraumatic ankle arthritis. According to the Kellgren-Lawrence grading scale criteria, there were grade I in 5 cases, II in 2 cases, III in 2 cases, and IV in 1 case. 2 cases of wound dishence were recovered through changing dressing and 2 cases of skin necrosis were recovered by skin graft and flap transposition respectively. There were no significant complications such as infection, nonunion, or implant failure.Conclusion:The transsyndesmotic ankle fracture dislocation, represents an exceptional pattern of high-energy fractures with significant syndesmotic disruption, and potential soft tissue compromise. Careful attention to radiographic findings can identify unique fracture characteristics relative to operative decision-making. Tibialis posterior tendon dislocation, a rare complication in the transsyndesmotic ankle fracture dislocation injuries, can impede anatomical reduction of the ankle mortise. The open reduction and internal fixation may be an optimal approach to treat transsyndesmotic ankle fracture dislocation injuries. However, the rate of posttraumatic arthritis is relatively high.

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

Objective To investigate the monitoring significance of WT1 gene level in the prognosis of acute myeloid leukemia (AML) patients with normal karyotype after hematopoietic stem cell transplantation (HSCT). Methods The clinical data of 115 AML patients with normal karyotype who were treated with HSCT from July 2009 to March 2017 in the First Affiliated Hospital of Soochow University were retrospectively analyzed. The dynamic detection of bone marrow WT1 gene was carried out by using reverse transcription_polymerase chain reaction (RT_PCR). According to the relative expression level median of WT1 gene before transplantation, the whole patients were divided into the two groups (

Objective: To investigate the immune reconstruct regularity profile of KIR2DL1 and KIR3DL1 in unrelated-donor allogeneic hematopoietic stem cell transplantation (allo-HSCT) with KIR-AA genotype. Method: 75 donor-recipient pairs were performed by KIR genotying using PCR-SSP, and all donors were identified with KIR-AA genotype. Dynamic detections (including unrelated-donor on the day of transplantation and the recipient each month post allo-HSCT) of the expression of KIR2DL1/3DL1 on NK cell and mRNA level were performed in 291 cases using flow cytometry (FCM) and real-time fluorescent quantitation PCR (RT-qPCR) . Result: ①The median expression of KIR2DL1 in unrelated-donor on transplant’s day was 21.60%, the median expression of KIR2DL1 in recipient 1M, 2M, 3M and 3-6M after transplantation were 7.40%, 12.00%, 16.92%, 17.64% respectively. The median expression of KIR2DL1 in unrelated-donor on transplant’s day was 265.14 copies/10 000abl copies, the median expression of KIR2DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 332.17, 438.31, 723.25, 414.17, 180.76 and 234.67 copies/10 000abl copies respectively. The median expression of KIR2DL1 on NK cells and mRNA level gradually increased at all time points after transplantation, and reached the highest expression at 3 months after transplantation. But mRNA expression levels increased earlier than NK cell membrane proteins. ②The median expression of KIR3DL1 in unrelated-donors on transplant’s day was 18.56%, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M after transplantation were 23.83%, 22.57%, 23.02%, 21.60% respectively. The median expression of KIR3DL1 in unrelated-donor on transplant’s day was 572.29 copies/10 000abl copies, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 1 233.74, 1 140.42, 876.73, 1 057.07, 739.02 and 514.43 copies/10 000abl copies respectively. The median expression of KIR3DL1 on NK cells and mRNA level were higher than donors at 1 month after transplantation, and stable expression at all time points after transplantation, so mRNA and NK cell membrane proteins expression increased at the same time. Conclusion The immune reconstruct regularity of KIR2DL1 and KIR3DL1 gene were different, which provided an experimental basis for selecting the best time to detect the expressions of KIR2DL1 and 3DL1 after transplantation.

Objective: To investigate the characteristics of the essential thrombocythemia (ET) cases transformed to the acute myeloid leukemia (AML) and the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of this disease. Methods: The clinical and laboratory characteristics of 3 ET cases before and after transformation and after allo-HSCT were retrospectively analyzed, meanwhile the related literatures were reviewed and discussed. Results: Case 1 was a male patient of 44 years old, whose PLT was 500×10⁹/L when firstly diagnosed ET. After 3 years the disease progressed into myelodysplastic syndrome (MDS) while WT1 expression increased from 77 (first visit) to 13 171 copies/10 000 ABL copies, at the same time chromosome changed dramatically. During the period of decitabine treatment the disease processed into AML. Case 2 was a male of 58 years old whose PLT was 2 100×10⁹/L when firstly diagnosed ET. The disease progressed to AML after 9 years, whose WT1 expression increased from 130 (first visit) to 3 222 copies/10 000 ABL copies, and he relapsed shortly after intensive chemotherapy. Case 3 was a male of 60 years old whose PLT was 900×10⁹/L when firstly diagnosed ET. The disease progressed to AML after 5 years, whose WT1 increased from 56 (first visit) to3 696 copies/10 000 ABL copies. Moreover leukemia spread to central nervous system (CNS) during chemotherapy. Before allo-HSCT, cases 1 did not achieve remission; case 2 relapsed after a short time of remission and case 3 transferred to CNS leukemia. All of the 3 cases underwent allo-HSCT successfully, and they all achieved completely remission, whose chromosome and gene mutation recovered negative. At the same time, CNS leukemia of case 3 disappeared. The median WT1 decreased to 50 copies/10 000 ABL copies. There was no severe complication during the median time of 5 months after allo-HSCT. Conclusions The patients transformed to AML had poor prognosis, allo-HSCT was the only method that can cure the disease now.

Objective: To observe the correlation between Nε-carboxymethyl-Lysine (CML), the main component of advanced glycation end products and the calcification of the anterior tibial artery plaque in patients with diabetic foot post foot amputation. Methods: Sixty patients hospitalized for foot amputation operation due to diabetic foot from June 2012 to June 2016 in the Department of Orthopedics, Affiliated Hospital of Jiangsu University were prospectively recruited.The patients were categorized into mild stenosis (0n=20), moderate stenosis (50%≤stenosis<70%, n=20) and severe stenosis (70%≤stenosis≤100%, n=20) based on the color Doppler ultrasound assessed severity of anterior tibial artery stenosis.The baseline clinical data of patients were collected and anterior tibial artery was isolated.Then, HE staining, O-Cresolphthalein Complexone method, enzymic method and ELASA analysis were then performed to detect the evolution of calcification, arterial calcium content, alkaline phosphatase activity and serum CML concentration, respectively. Results: The results from both color Doppler ultrasound scan before amputation and HE staining after amputation showed that echo intensity as well as spotty blue calcium particles of anterior tibial artery plaque increased significantly in proportion to degree of stenosis and destructed elastic plate of the arterial wall was evidenced in patients with severest stenosis.The content of calcium ((2.3±0.9), (3.9±1.3), (6.6±1.7) μmol/mg, respectively, P<0.001), ALP activity ((102.4±39.4), (202.3±73.4), (483.7±117.9) U/mg, respectively, P<0.001) and serum CML level ((28.9±4.4), (37.9±5.3), (57.3±7.1)μg/L, respectively, P<0.001) increased significantly in proportion to stenosis severity.Pearson correlation analysis showed that serum CML level was positively correlated with the content of calcium (r=0.749, P<0.001) and ALP activity (r=0.923, P<0.001), respectively. Conclusions Serum CML level is positively correlated with the calcification of anterior tibial arterial plaque in patients with diabetic foot and could be used to evaluate the calcification of anterior tibial arterial plaque and stenosis degree of anterior tibial arterial in these patients.

Klebsiella pneumoniae carbapenemases-producing Klebsiella pneumoniae (KPC-Kp) has caused a global public health crisis, and the severity of its infection is associated with high mortality in hospitalized patients. Therefore, the KPC-Kp prevention methods and the corresponding treatment strategy exploration are imminent. The risk factors and the treatment progress of KPC-Kp colonization or infection are reviewed in this paper to explore corresponding preventive measures and treatment strategies for clinical prevention and treatment.

OBJECTIVETo report clinical and laboratory features of 4 cases of myeloid neoplasm with t (5;12) (q33;p13).

METHODSCytogenetic examination of bone marrow cells obtained from patients was performed by 24 h culture method. R banding technical was used for karyotype analysis. PDGFRβ gene rearrangement was detected by FISH using dual color break apart PDGFRβ probe. ETV6-PDGFRβ fusion genes were detected by multiple-reverse transcription polymerase chain reaction (RT-PCR). Direct sequencing analysis was performed on the PCR products in case 1. Immunophenotype analysis was carried out by flow cytometry. Four cases were treated with imatinib (IM) and followed up.

RESULTSThe diagnoses included 3 MPN and 1 AML-M2. The t (5;12) (q33;p13) was a primary abnormality in 3 cases of MPN and a secondary abnormality in 1 case of AML-M2. PDGFRβ gene rearrangement and ETV6-PDGFRβ fusion genes were detected by FISH and multiple-RT-PCR in 4 cases, respectively. The immunophenotypical analysis of leukemia cells showed positive for CD13, CD33 and CD34. Two cases obtained MMR after the treatment of IM, one case complete hematologic and complete cytogenetic response. ETV6-PDGFRβ was negative detected by multiple-RT-PCR after the treatment of IM, but relapsed and died soon in case 4.

CONCLUSIONSThe t (5;12) myeloid neoplasm was a subtype with unique features. The t (5;12) maybe a primary chromosome abnormality in MPN and a secondary in AML. MPN with t (5;12) could benefit from IM, but not for AML. Dual-FISH was a reliable tool for detecting PDGFRβ rearrangement.

Chromosome Banding ; Gene Rearrangement ; Hematologic Neoplasms ; genetics ; Humans ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; Karyotyping ; Myeloproliferative Disorders ; genetics ; Polymerase Chain Reaction ; Proto-Oncogene Proteins c-ets ; genetics ; Receptor, Platelet-Derived Growth Factor beta ; genetics ; Remission Induction ; Repressor Proteins ; genetics ; Translocation, Genetic