Objective To explore the diagnosis of clinically suspicious von Willebrand disease(vWD)in a family and its pathogene-sis.Methods The pedigree information and the biological specimen were collected from the clinically suspected VWD patient and her family members(4 persons in total)in Peking University First Hospital.The levels of platelet count(PLT),activated partial thrombo-plastin time(APTT),vWF antigen(vWF:Ag),vWF activity(vWF:Ac)and FⅧ activity(FⅧ:C)were detected,and vWF risto-cetin cofactor(vWF:RCo)assay,ristocetin-induced platelet aggregation assay(RIPA)and vWF collagen binding(vWF:CB)assay were performed for phenotype diagnosis.The peripheral blood genomic DNAs were extracted from the proband and her family members to perform whole-exome sequencing for identifying the mutation of vWF gene,The mutation site was analyzed by using bioinformation tools to explore the pathogenesis of the proband.Results The APTT of proband(m 1)was slightly prolonged and her vWF:Ag,vWF:Ac,vWF:RCo and vWF:CB were significantly decreased.There was no obvious aggregation in RIPA assay(1.0 mg/mL and 1.25 mg/mL).In her father(Ⅱ3),APTT,FⅧ:C,vWF:Ag,vWF:Ac and vWF:CB were normal,but vWF:RCo was slightly decreased.In her mother(Ⅱ4),APTT,FⅧ:C,vWF:Ag,vWF:RCo and vWF:CB were all normal,but vWF:Ac significantly decreased.In her brother(Ⅲ2),APTT and FⅧ:C were normal,but vWF:Ag,vWF:Ac,vWF:RCo and vWF:CB were reduced to varying degrees.In all the family members(father,mother and brpther),no apparent aggregation in RIPA(1.0 mg/mL)was shown.Genetic analysis showed that the proband(Ⅲ1)carried a compound heterozygous mutation of vWF gene c.7288-9T＞G and c.1117C＞T,her father(Ⅱ3)carried vWF gene c.7288-9T＞G heterozygous mutation,and vWF gene c.1117C＞T heterozygous mutation was presented in both mother(Ⅱ4)and brother(Ⅲ2).Conclusion According to the results of laboratory tests,the proband was diagnosed as type 2A vWD.The hetero-zygous mutation in vWF gene c.1117C＞T and c.7288-9T＞G may be the molecular mechanism leading to type 2A vWD in the proband.

Objective:To discuss the effect of downregulating of high mobility group box protein 2(HMGB2)expression on the biological behavior of the liver cancer cells and the epithelial-mesenchymal transition(EMT)process,and to clarify its mechanism.Methods:The human liver cancer LM3 cells at logarithmic growth phase were divided into negative control group and HMGB2 RNA interference group(HMGB2 siRNA group);the cells in two groups were transfected with RNA oligonucleotides(RNA oligos)with irrelevant sequences and RNA oligos designed to knock down HMGB2,and the Lipofectamine 2000 was regarded as the vector.The expression levels of HMGB2 mRNA and protein in the cells in two groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods;cell scratch assay and Transwell chamber assay were used to detect the migration and invasion abilities of the cells in two groups;the expression levels of E-cadherin,N-cadherin,and Vimentin proteins and protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway related proteins in the cells in two groups were detected by Western blotting method.Results:Compared with negative control group,the expression levels of HMGB2 mRNA and protein in the cells in HMGB2 siRNA group were significantly decreased(P<0.05),the cell scratch healing rate was significantly decreased(P<0.01),the number of invasion cells was significantly decreased(P<0.01),and the expression level of E-cadherin protein in the cells was significantly increased(P<0.01),while the expression levels of N-cadherin,Vimentin,mTOR,AKT,and phosphorylated AKT(p-AKT)proteins in the cells were significantly decreased(P<0.05 or P<0.01).Conclusion:Downregulating the expression of HMGB2 can reduce the migration and invasion abilities of the liver cancer LM3 cells and inhibit the EMT,and its mechanism may be related to regulating the expression of the AKT/mTOR pathway related proteins.

Objective:To measure the morphological parameters of distal tibiofibular syndesmosis in healthy adults using multi-slice CT (MSCT) so as to provide a reference for the diagnosis of distal tibiofibular syndesmosis injury.Methods:The ankle MSCT imaging data in 110 normal adults were retrieved from the image report database of Cangzhou People′s Hospital from May 2019 to May 2021, including 56 males and 54 females; aged 18-60 years [(38.2±11.0)years]. There were 51 patients with imaging on the right ankle and 59 on the left ankle. Picture archiving and communication system (PACS) was used to measure parameters at 10 mm above the articular surface of the distal tibia on MSCT, including the anterior tibiofibular space (L1), posterior tibiofibular space (L2), middle tibiofibular space (L3), depth of fibula in notch (L4), distance of anterior tibiofibular edge (L5), distance of posterior tibiofibular edge (L6), anterior tibiofibular syndesmosis angle (A1), and fibular rotation angle (A2), and the measurements were compared by sex, age and side. The positive rate of "tibiofibular line" was observed. The morphological classification of distal tibiofibular syndesmosis was performed.Results:There was no significant difference in L1-L6, A1 and A2 among different age and side (all P>0.05). No significant difference was found in L4, L5, A1 and A2 between males and females ( P>0.05), but L1, L2, L3 and L6 were larger in males than in females ( P<0.05 or 0.01). The positive rate of "tibiofibular line" was 80.4% (45/56) in males compared to 74.1% (40/54) in females ( P>0.05), 77.2% (44/57) in the youth compared to 77.4% (41/53) in the middle-aged, and 78.0% (46/59) in the left ankle compared to 76.5% (39/51) in the right ankle (all P>0.05). Morphological classification of distal tibiofibular syndesmosis was crescent in 61 patients (55.5%), trapezoid in 14 (12.7%), I-shaped in 3 (2.7%), M-shaped in 17 (15.5%), V-shaped in 10 (9.1%), Г-shaped in 5 (4.5%). Conclusions:When L1, L2, L3 and L6 are used as references in the diagnosis of adult distal tibiofibular syndesmosis injury, gender factors rather than age or side factors should be considered. Males have wider distal tibiofibular space than females, with the fibula more forward. The "tibiofibular line" has a high positive rate and is not affected by gender, age or sides, providing a new idea for the diagnosis of distal tibiofibular syndesmosis injury and anatomical reduction. There are many variations in the morphology of distal tibiofibular syndesmosis, so it is easy to be misdiagnosed as the separation of distal tibiofibular syndesmosis on X-ray, which should be noted.

Objective:To evaluate the performance of an artificial intelligent (AI)-based automated digital cell morphology analyzer (hereinafter referred as AI morphology analyzer) in detecting peripheral white blood cells (WBCs).Methods:A multi-center study. 1. A total of 3010 venous blood samples were collected from 11 tertiary hospitals nationwide, and 14 types of WBCs were analyzed with the AI morphology analyzers. The pre-classification results were compared with the post-classification results reviewed by senior morphological experts in evaluate the accuracy, sensitivity, specificity, and agreement of the AI morphology analyzers on the WBC pre-classification. 2. 400 blood samples (no less than 50% of the samples with abnormal WBCs after pre-classification and manual review) were selected from 3 010 samples, and the morphologists conducted manual microscopic examinations to differentiate different types of WBCs. The correlation between the post-classification and the manual microscopic examination results was analyzed. 3. Blood samples of patients diagnosed with lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, or myeloproliferative neoplasms were selected from the 3 010 blood samples. The performance of the AI morphology analyzers in these five hematological malignancies was evaluated by comparing the pre-classification and post-classification results. Cohen′s kappa test was used to analyze the consistency of WBC pre-classification and expert audit results, and Passing-Bablock regression analysis was used for comparison test, and accuracy, sensitivity, specificity, and agreement were calculated according to the formula.Results:1. AI morphology analyzers can pre-classify 14 types of WBCs and nucleated red blood cells. Compared with the post-classification results reviewed by senior morphological experts, the pre-classification accuracy of total WBCs reached 97.97%, of which the pre-classification accuracies of normal WBCs and abnormal WBCs were more than 96% and 87%, respectively. 2. The post-classification results reviewed by senior morphological experts correlated well with the manual differential results for all types of WBCs and nucleated red blood cells (neutrophils, lymphocytes, monocytes, eosinophils, basophils, immature granulocytes, blast cells, nucleated erythrocytes and malignant cells r>0.90 respectively, reactive lymphocytes r=0.85). With reference, the positive smear of abnormal cell types defined by The International Consensus Group for Hematology, the AI morphology analyzer has the similar screening ability for abnormal WBC samples as the manual microscopic examination. 3. For the blood samples with malignant hematologic diseases, the AI morphology analyzers showed accuracies higher than 84% on blast cells pre-classification, and the sensitivities were higher than 94%. In acute myeloid leukemia, the sensitivity of abnormal promyelocytes pre-classification exceeded 95%. Conclusion:The AI morphology analyzer showed high pre-classification accuracies and sensitivities on all types of leukocytes in peripheral blood when comparing with the post-classification results reviewed by experts. The post-classification results also showed a good correlation with the manual differential results. The AI morphology analyzer provides an efficient adjunctive white blood cell detection method for screening malignant hematological diseases.

Platelet surface is rich in glycocalyx. It has been found that platelet glycosylation plays an important role in the physiological hemostasis mechanism, regulating the interaction between platelets and receptor proteins, and dynamically reshaping the surface glycosylation through its own glucose metabolism system. Platelet glycosylation also participates in platelet aging and clearance, and regulates platelet counts. Meanwhile, abnormal platelet glycosylation is closely related to primary immune thrombocytopenia, coronary heart disease and other related diseases, being a potential therapeutic target.

To explore the pharmacogenomic markers that affect the platinum-based chemotherapy response in non-small-cell lung carcinoma (NSCLC), we performed a two-cohort of genome-wide association studies (GWAS), including 34 for WES-based and 433 for microarray-based analyses, as well as two independent validation cohorts. After integrating the results of two studies, the genetic variations related to the platinum-based chemotherapy response were further determined by fine-mapping in 838 samples, and their potential functional impact were investigated by eQTL analysis and in vitro cell experiments. We found that a total of 68 variations were significant at P < 1 × 10^-3 in cohort 1 discovery stage, of which 3 SNPs were verified in 262 independent samples. A total of 541 SNPs were significant at P < 1 × 10^-4 in cohort 2 discovery stage, of which 8 SNPs were verified in 347 independent samples. Comparing the validated SNPs in two GWAS, ADCY1 gene was verified in both independent studies. The results of fine-mapping showed that the G allele carriers of ADCY1 rs2280496 and C allele carriers of rs189178649 were more likely to be resistant to platinum-based chemotherapy. In conclusion, our study found that rs2280496 and rs189178649 in ADCY1 gene were associated the sensitivity of platinum-based chemotherapy in NSCLC patients.

Objective:To investigate the role and influencing factors of case design in PBL teaching.Methods:Thirty-two six-year-program undergraduates from the Department of Medicine of Peking University in batch 2014 and batch 2015 were selected as the subjects. PBL teaching was used in the practice class of experimental diagnostics. The feedback effects of four times PBL courses were analyzed by collecting questionnaires for teachers, students, and supervisors. The data obtained from the five-point questionnaire and the question-and-answer questionnaire were analyzed by one-way ANOVA and statistics respectively. Then the problems in case preparation process are discussed and the experience of case design is summarized. SPSS 13.0 was used in this study.Results:The 5-point questionnaire showed that the average score of anemia PBL course was the highest among students' self-evaluation and mutual evaluation of teachers and students (4.84 points, 4.79 points), with statistical significance compared with other courses ( P<0.05). The question-and-answer questionnaire survey showed that 93.75% of the students generally agreed with the teaching model of anemia cases; 78.13% and 59.38% of the students believed that it was difficult to set up cases of infection and coagulation, which affected the classroom effect; and 50% of the supervisors thinked that the students' level should be taken into account in case design and oral expression should be avoided. Conclusion:Case design is the key to PBL teaching. Summarizing the experience of case design can lay a good foundation for the establishment of PBL teaching database.

Objective To explore the molecular pathogenesis of 3 Glanzmann's thrombasthenia pedigree by using bioinformatics software and provide evidence for in vitro experiments. Methods The genetic analysis of 3 pedigree diagnosed as Glanzmann's thrombasthenia was carried out. Clustalx-2.1 win software was used to analyze the conservatism of mutant sites in homologous sequences. Bioinformatics software such as PolyPhen-2, PROVEAN, SIFT and Mutationtaster was used to analyze the biological effect of mutation. SPDBV software constructed the molecular structure model of mutant protein and evaluated the influence of mutation on protein structure. Results The "new mutations" found in 3 Glanzmann's thrombasthenia pedigree were ITGA2B:c. 814G>C (p. Val272Leu), ITGA2B:c. 432G>A (p. Trp144Ter) and ACTN1:c. 2458A>G (p. Ile820Val). All three mutations were highly conserved among homologous species. Mutationtaster software showed that 3 new mutations were likely pathogenic. PolyPhen-2 and PROVEAN software showed ITGA2B p.Val272Leu and ACTN1 p.Ile820Val were benign and SIFT software showed that ITGA2B p. Val272Leu were likely pathogenic, while ACTN1 p. Ile820Val is benign. The result of SPDBV software showed that the Val272 of ITGA2B was transformed to Leu, neutralizing all the original hydrogen bond. The Trp144 of ITGA2B is transformed to Ter, resulting in the truncated proteins with only 113 amino acid residues. All these mutations affected the molecular structure of GPⅡb, resulting in a decrease ofGPⅡb/Ⅲa expression. When the Ile820 of ACTN1 is transformed to Val, onlyretained the hydrogen bond of Ile820 and Asp822, neutralized the rest hydrogen bond, whichaffected the molecular structure and protein function of ACTN1. Conclusion The mutations of ITGA2B:c.814G>C (p.VAL272LEU), ITGA2B:c.432G>A (p.Trp144Ter) and ACTN1:c.2458A>G (p.Ile820Val) are pathogenic.

Objective: To investigate the molecular pathogenesis for a patient with Glanzmann thrombasthenia (GT). Methods: The peripheral blood of a patient with Glanzmann′s thrombasthenia was collected, and the genetic mutations were detected by gene sequencing technology. The mutant plasmids were prepared by PCR site-directed mutagenesis and transfected into CHO-K1 cells of Chinese hamster ovary to construct in vitro eukaryotic expression system. The expressions of αⅡb and β3 protein subunits in CHO-K1 cells were detected by western blot. The expression levels of αⅡb and β3 in cellular membrane and cytoplasm of CHO-K1 cells were detected by flow cytometry. The expression and distribution of αⅡb and β3 in CHO-K1 cells were observed by immunofluorescent labeling under microscope. Results: This patient was diagnosed with type Ⅱ GT. Gene sequencing revealed two mutations in ITGB3 gene which has not been reported in the literature. ITGB3 c.1495 T＞C missense mutation resulted in replacement of cysteine no.499 by arginine (p.C499R). ITGB3 c.1728 delC code shift mutation resulted in a change in the amino acid synthesis initiated by the β3 protein subunit serine no.577 and terminated by the 92nd amino acid following these changes. The results of western blotting showed that the synthesis and expression of primary structures of αⅡb and β3 were detectable in the lysates of mutant CHO-K1 cells. The results of flow cytometry showed that no expression of β3 on the surface and intracellular of mutant CHO-K1 cells was observed. Under fluorescence microscopy no distribution of β3 protein subunit was displayed in mutant CHO-K1 cells. Conclusion The mutation of ITGB3 c.1728 del C or ITGB3 c.1495 T＞C should be relevant to the cause of GT in this patient. The mutation of ITGB3 c.1728 del C and ITGB3 c.1495 T＞C seems not to affect the formation of the primary structure of β3 protein subunit, but did affect the formation of its high-level structure.

Objective: To establish a set of rules for autoverification of blood analysis, in order to provide a way to validate autoverification rules for different analytical systems, which can ensure the accuracy of test results as well as shorten turnaround time (TAT) of test reports. Methods: A total of 34 629 EDTA-K2 anticoagulated blood samples were collected from multicenter cooperative units including the First Hospital of Jinlin University during January 2017 to November 2017. These samples included: 3 478 cases in Autoverification Establishment Group, including 288 cases for Delta check rules; 5 362 cases in Autoverification Validation Group, including 2 494 cases for Delta check; 25 789 cases in Clinical Application Trial Group. All these samples were analyzed for blood routine tests using Sysmex XN series automatic blood analyzers.Blood smears, staining and microscopic examination were done for each sample; then the clinical information, instrument parameters, test results and microscopic results were summarized; screening and determination of autoverification conditions including parameters and cutoff values were done using statistical analysis. The autoverification rules were input into Sysmex Laboman software and undergone stage Ⅰ validation using simulated data, and stage Ⅱ validation for post-analytical samples successively. True negative, false negative, true positive, false positive, autoverification pass rate and passing accuracy were calculated. Autoverification rules were applied to autoverification blood routine results and missed detection rates were validated, and also data of autoverification pass rate and TAT were obtained. Results: (1)The selected autoverification conditions and cutoff values included 43 rules involving WBC, RBC, PLT, Delta check and abnormal characteristics. (2)Validation of 3 190 cases in Autoverification Establishment Group showed the false negative rate was 1.94%(62/3 190)(P<0.001), autoverification pass rate was 76.74%, passing accuracy was 97.47%; Validation of 2 868 cases in Autoverification Validation Group, the false negative rate was 3.38%(97/2 868)(P=0.002), autoverification pass rate was 42.26%, passing accuracy was 92.00%; Validation of Delta check on 288 cases in Autoverification Establishment Group and 2 494 cases in Autoverification Validation Group showed the false negative rates were respectively 1.39% and 2.61%(P<0.001). (3)Three hospitals adopted these rules of autoverification for 25 789 blood routine samples, and found that the average TAT of blood routine test reports were shortened by 24min, 32min and 7min respectively, the rate of samples reported within 30min were elevated by 33%, 53% and 7%. The autoverification pass rates were 72%-74%. Conclusions The application of this set of 43 autoverification rules in blood sample analysis can ensure test quality while shortenTAT and improve work efficiency. It is worth pointing out that for the same analytical systems in this research, validation is necessary before application of this set of rules, and periodic validation is required during application to make necessary adjustment; for different analytical systems, as this research provide a way to establish autoverification rules for blood routine tests.Clinical labs may establish their own suitable autoverification rules on the basis of technological parameters. (Chin J Lab Med, 2018, 41: 601-607)