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 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.

Inherited bleeding and thrombotic disorders (BTD) are a group of heterogeneous diseases related to the coagulation system, platelet function and fibrinolytic system. In addition to the common BTD, it is difficult to diagnose such patients by routine laboratory tests, and special laboratory tests are often required to confirm the diagnosis. Therefore, the diagnosis and treatment of patients may be delayed, or even life-threatening. With the increasing use of high-throughput sequencing (HTS) technology in clinical practice, the diagnosis and differential diagnosis of BTD have made great progress.

Central nervous system (CNS) involvement is a serious complication of hematological malignancies. At present, the gold standard to detect CNS involvement is cerebrospinal fluid (CSF) cytology, but it has low sensitivity. The multiparameter flow cytometry (MFC) shows higher sensitivity than CSF cytology. The occult central nervous system involvement with negative conventional cytology but positive MFC result is the same as dominant central nervous system involvement, which is associated with a high risk of recurrence of hematological tumors. However, due to the particularity of cerebrospinal fluid specimens--less number of cells, low activity, and more interfering cells, the application of MFC for detection of cerebrospinal fluid is limited. Therefore, there is an urgent need to standardize the MFC detection of CSF for hematological tumors, including defining specimen transportation, antibody selection and cut-off value, standardizing data analysis and strengthening employee training, which will greatly improve the role of MFC in the diagnosis of CNS with hematological malignancies.

Flow cytometry has been widely used in clinical practice. Due to the powerful user-defined function of flow cytometry, different instrument settings among manufacturers and lack of standard materials, the comparability of results needs to be improved and flow cytometry is facing the great challenge of normalization and standardization. In this paper, the recent progress of normalization and standardization about flow cytometry was discussed form the aspects of standardizing operation protocol, including specimen and centrifugation, standardizingantibody selection and panel combination and ensuring instrument and data analysis consistency.

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 compare the practical value of three statistical methods in establishing reference intervals by the indirect method.Methods:This is a methodological evaluation study. The data of coagulation parameters were obtained from laboratory information system, which were from pregnant women who had done prenatal examination in Peking University First Hospital from January 2017 to December 2019. The test results from 32 401 pregnant women were collected. Those healthy pregnant women were divided into three groups: early pregnancy group(n=11 151), middle pregnancy group(n=4 872) and late pregnancy group(n=16 378). Statistical analysis was performed for the result of PT, APTT, FIB, TT, D-D and FDP, and the necessity of stratification based on age in different periods of pregnancy was analyzed. Stratification based on age was necessary in three pregnancy groups for PT and APTT, in late pregnancy group for FIB and TT, and in early pregnancy group for D-D. The reference intervals of coagulation parameters were calculated by three statistical methods: non-parametric method, Hoffmann method and Q-Q plot method. Forty-two healthy pregnant women from October 2019 to January 2020 were enrolled as reference individuals for the validation of the reference intervals.The proportions of test results outside the reference intervals in the reference population calculated and compared.Results:The levels of the six coagulation assays vary significantly during the three periods of pregnancy, stratification based on age was necessary in three pregnancy groups for PT and APTT, in late pregnancy group for FIB and TT, and in early pregnancy group for D-D. If the number of test results was large, non-parametric and Hoffmann method provided more similar results, while the reference intervals calculated with Q-Q plot method was slightly wider than Hoffmann method. If the number of test results was small, reference intervals calculated with Hoffmann and Q-Q plot method were more reliable. For pregnant women during early pregnancy under the age of 35, the reference intervals of PT, APTT, FIB and TT calculated by this method were (10.44-13.11)s, (25.29-35.88)s, (2.61-4.64)g/L and (11.53-15.58)s.Conclusion:When establishing the reference interval, stratification according to pregnancy period and age was needed. Hoffmann method can be used as an alternative to the direct method.

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 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.