[Analysis of clinical phenotype and gene mutation characteristics of MYH9-related disorder].

Objective: To investigate the clinical phenotype and gene mutation characteristics of MYH9-related disorder (MYH9-RD). Methods: The clinical data of 66 patients with MYH9-RD in the First Affiliated Hospital of Soochow University from January 2010 to December 2022 were retrospectively analyzed. According to the bleeding symptom, the patients were divided into bleeding and non-bleeding group, and according to the mutation sites, the patients were divided into non-muscle myosin heavy chain ⅡA head region (MD) and tail region (TD) mutation group. Statistical analysis was made to explore the clinical features in different groups such as platelet counts, bleeding, renal function, cataracts and hearing as well as MYH9 gene mutations. Results: A total of 66 MYH9-RD patients were included, with 28 males and 38 females, diagnosis age of 1-63(26±2) years. And 41% (27/66) of the patients had no family history. All patients presented with macrothrombocytopenia and normal platelet aggregation(10/10), 92% (54/59) of the patients had visible blue inclusion bodies in neutrophils, 30% (20/66) had bleeding symptoms, 45% (22/49) had proteinuria or glomerulonephropathy, 20% (8/41) had bilateral hearing impairment, and 10% (4/42) had bilateral cataracts. 18 mutation sites were identified in total, including 15 missense, 1 splicing and 2 termination mutations. Among them, p.Asp1424Asn, p.Arg1933* and p.Arg702His/Cys mutations were identified in 56% (29/52) of the patients, and p.Ser96Leu, Arg1165Cys and p.Glu1841Lys mutations were recurrent mutations, while p.Ala44Thr, p.Asp1447Ala and c.3838-2A>G mutations were novel mutations. The average platelet count of patients in bleeding group was (19±3)×109/L, which was significantly less than (36±3)×109/L in non-bleeding group (P<0.001). Compared with TD mutation group, patients of MD mutation group were presented with lower platelet count and higher risk of bleeding, as well as more severe clinical presentations including renal and hearing impairment and cataracts (all P<0.05). Conclusion: Mutations of p.Asp1424Asn, p.Arg1933* and p.Arg702His/Cys in MYH9 gene are hotspot mutations for MYH9-RD patients, Compared with TD mutation group, patients of MD mutation group were presented with lower platelet count and higher risk of bleeding, as well as more severe clinical presentations including renal and hearing impairment and cataracts.

[Clinical diagnosis and treatment of hereditary thrombocytopenia and purpura: a report of five cases and literature review].

Objective: To report the clinical manifestations and laboratory features of five patients with congenital thrombotic thrombocytopenic purpura (cTTP) and explore its standardized clinical diagnosis and treatment along with a review of literature. Methods: Clinical data of patients, such as age of onset, disease manifestation, personal history, family history, and misdiagnosed disease, were collected. Treatment outcomes, therapeutic effects of plasma infusion, and organ function evaluation were observed. The relationship among the clinical manifestations, treatment outcomes, and ADAMTS13 gene mutation of patients with cTTP was analyzed. Additionally, detection of ADAMTS13 activity and analysis of ADAMTS13 gene mutation were explored. Results: The age of onset of cTTP was either in childhood or adulthood except in one case, which was at the age of 1. The primary manifestations were obvious thrombocytopenia, anemia, and different degrees of nervous system involvement. Most of the patients were initially suspected of having immune thrombocytopenia. Acute cTTP was induced by pregnancy and infection in two and one case, respectively. ADAMTS13 gene mutation was detected in all cases, and there was an inherent relationship between the mutation site, clinical manifestations, and degree of organ injury. Therapeutic or prophylactic plasma transfusion was effective for treating cTTP. Conclusions: The clinical manifestations of cTTP vary among individuals, resulting in frequent misdiagnosis that delays treatment. ADAMTS13 activity detection in plasma and ADAMTS13 gene mutation analysis are important bases to diagnose cTTP. Prophylactic plasma transfusion is vital to prevent the onset of the disease.

[Clinical features of 20 patients with phytosterolemia causing hematologic abnormalities].

Objective: To investigate the clinical and laboratory features of Phytosterolemia with hematological abnormalities. Methods: A retrospective study was performed on 20 patients with phytosterolemia admitted to the hematology department of the First Affiliated Hospital of Suzhou University during 2004-2017. History of patients was collected and the platelet counts, lipidomic analysis of plasma and osmotic fragility of erythrocytes were carried out. The erythrocyte and platelet morphology was examined by light microscope. Phytosterol levels in serum were measured by high performance liquid chromatography method. All of ABCG5/8 exons and intron-exon boundaries were amplified by PCR and directly sequenced to identify mutations. Results: All patients had been misdiagnosed as immune thrombocytopenia (ITP), or Evans syndrome with a mean delay of 21 years between symptom onset and accuracy diagnosis. The clinical manifestations of the patients were variable, but most of them presented with thrombocytopenia, anemia, splenomegaly from early ages, and xanthomas. Other major features were also observed, such as impaired liver functions (9 cases), premature atherosclerosis (5 cases) and/or arthritis (4 cases). Interestingly, all patients displayed an increased osmotic fragility of red cells and unique blood film features: large unequal platelets surrounded by a circle of vacuoles and various abnormal erythrocyte shapes, especially stomatocyte. Serum levels of the sitosterol and stigmasterol in the patients were remarkably elevated up to 331.05(276.00, 670.20)mg/L and 244.60(193.78,399.40)mg/L, about 10 and 24 times higher than those of normal subjects. There were 14 mutations in ABCG5/8 genes found in the patients. Among them, 2/3 of the mutations were in ABCG5 gene, including p.(E22X), p.(R446X),g.ISV7+3G>A, p.(R446X), p.(R419H), g.ISV7+3G>A, p.(G90E), p.(R389H) and g.7+2G>A), and 1/3 in ABCG8 gene involving p.(M614-K628del), p.(E25X), p.(L86P fs X185), p.(R263Q), p.(E500D fs X604) and p.(G674R) mutation. The ABCG5 p.(R446X) mutation was found in 3 separate families. Conclusions: The phenomena of thrombocytopenia/ stomatocyte/splenomegaly represents a special clinical manifestations of phytosterolemia, and distinct changes of blood cell morphology are the typical characters. Plasma plant sterols and ABCG5/ABCG8 genes should be analyzed when such hematologic abnormalities are unexplained.

[Clinical observation of desmopressin in the treatment of 15 patients with DDAVP].

Objective: To assess the significance of DDAVP use in the diagnosis and treatment of VWD. Methods: An analysis of 15 VWD cases who referred to Hematology Division of First affiliated Hospital of Soochow University and treated with DDAVP from March 2016 to August 2018 was conducted. Efficacy and treatment response of DDAVP were monitored by observations of changes in factor Ⅷ procoagulant (FⅧ∶C) and von Willebrand Factor (VWF) related indicators before and 2 h after DDAVP injection. Results: Of 15 cases with VWD, 7 males and 8 females with a median age of 23 (6-46) years, 7 of 9 type I VWD patients achieved complete response (CR) , 1 type 2A VWD case CR, 5 type 3 VWD ones no response (NR) . The VWF multimer analysis in 5 patients combined with other plasma VWF values were in accordance with the known diagnosis. Conclusions: DDAVP was effective in most type 1 patients, and ineffective in some type 2 and almost all type 3 cases. It was helpful for diagnosis and subsequent treatment planning.

[The diagnostic value of PLASMIC score in the discrimination between thrombotic thrombocytopenic purpura and disseminated intravascular coagulation].

Objective: PLASMIC score was evaluated its value in differential diagnosis between the patients with thrombotic thrombocytopenic purpura (TTP) and those with disseminated intravascular coagulation (DIC) . Method: Twenty-four patients with TTP and 41 cases with DIC were retrospectively analyzed in this study. The platelet count, average red blood cell volume, indirect bilirubin, creatinine and prothrombin time international normalised ratio were collected, and then PLASMIC scores were calculated. Results: According to the risk classification of PLASMIC score, three (12.5%) TTP patients had moderate risk, and the rest 21 (87.5%) cases had high risk. In DIC patients, 92.7% cases were in low risk group, 4.9% at moderate risk, and only one case had high risk. Of these 65 patients, the sensitivity and the specificity to TTP of the high risk of the scoring system were 87.5% and 97.6%, respectively. Conclusion: The patients with high risk of PLASMIC score correlated well with clinical TTP diagnosis. The scoring system showed to be an excellent diagnostic model to distinguish TTP patients from those with DIC.

[α1-antitrypsin Pittsburg mutations: report of two cases in the same family].

Objective: To analyze the clinical and laboratory abnormalities of two patients with α1-antitrypsin (α1-AT) Pittsburgh in a family and review the literatures. Methods: Both plasma clotting time and factor activities were performed using clotting or substrate methods. Platelet aggregation was evaluated using an optical aggregometer. The serum protein electrophoresis was performed on Sebia HYDRASYS by using Agarose gel. The exons of α1-AT were amplified by using polymerase chain reaction (PCR) and then sequenced and compared with NCBI GenBank records. Results: The proband had several ruptures of corpus luteum and bleeding after operation, while her daughter had no bleeding history. Both of them showed prolonged coagulation tests which could not be corrected by mixing with the normal plasma. They also showed low levels of plasma coagulation factors, undetected protein C and S activity and abnormal bands of α1-globulin. The results of gene sequencing demonstrated that they were heterozygous for g.T17132G (p.Met358Arg) mutation of α1-antitrypsin gene (NG_008290.1) . Conclusions: Comparing with the data of previously reported cases, our results confirmed the obvious abnormality of coagulation test and the discrepancy of bleeding tendency of α1-antitrypsin Pittsburgh patients, and suggested that the rupture of corpus luteum would be a specific characteristic in women of child-bearing age.

[A Study on the establishment of immune thrombocytopenia model induced by anti-platelet GPâ bα antibodies].

Objective: To establish primary immune thrombocytopenia (ITP) animal model induced by anti-platelet membrane glycoprotein GPⅠbα antibodies AN51 and R300. Methods: Twenty guinea pigs (6-8 week) were divided into 4 groups. Five guinea pigs in each group were intravenously injected with different doses of AN51 (0.05, 0.1, 0.2 µg/g) and 0.2 µg/g IgG as control. The whole blood was collected from inner angular venous plexus. Platelets number was determined by an automated cell counter and Swiss-Jim method. Then, the similar protocol was used to establish ITP nude mice model by intraperitoneal injection of different concentrations of anti-platelet GPⅠbα antibody R300, respectively. Results: ①Five minutes after intravenous injection of AN51 at 0.05, 0.1 and 0.2 µg/g, the platelet counts of guinea pigs reduced about 0-5%, 50%-60% and 70%-80% compared to the control group, respectively. The difference was statistically significant (P<0.01) . ②Six hours after intraperitoneal injection of R300 at 0.05, 0.1, 0.2 µg/g, the platelet counts of nude mice decreased about 20%-30%, 60%-70% and 80%-90% compared to the control group, respectively. The difference was statistically significant (P<0.01) . The nude mice, injected 0.2 µg/g R300 once a day for 2 weeks, showed typical ITP clinical manifestations including large number of petechiaes or ecchymoses on limbs, head and abdomen. Conclusion: AN51 at 0.2 µg/g and R300 at 0.2 µg/g could establish stable ITP model in guinea pigs and nude mice respectively.

[Detection and analysis of plasma VWF levels in healthy Chinese by automatic standardized assay].

Objective: To explore the normal range of plasma VWF levels of healthy Chinese and to analyze the influencing factors to VWF level. Methods: To detect the levels of von Willebrand factor antigen (VWF∶Ag) , von Willebrand factor ristocetin cofactor activity (VWF∶Rco) , von Willebrand factor collagen binding activity (VWF∶CB) , and the factor Ⅷ coagulation activity (FⅧ∶C) by using fully automatic and standardized testing instruments and matching reagent in 70 healthy Chinese. The effects of age, ABO blood type, gender and region were also analyzed. Meanwhile, 8 standard plasma samples (2 normal subjects, 6 cases of type 2 VWD) confirmed by NIBSC were tested for VWF values. Results: ① In 70 cases of healthy Chinese, the mean value of plasma VWF∶Ag, VWF∶Rco and VWF∶CB were (95.4±44.9) %, (105.9±35.4) % and (89.8±28.4) %, respectively; the ratio of VWF∶Rco/VWF∶Ag and VWF∶CB/VWF∶Ag was 1.18±0.25 and 1.03±0.29, respectively. ②There was no statistical significance in plasma VWF values between the age ≥30 years and <30 years group (P>0.05) . ③The VWF∶Rco, VWF∶CB of type O blood group were lower than that of non-O group (t=2.074, P=0.042; t=3.949, P=0.001) , but there was no statistical significance in VWF∶Ag, VWF∶Rco/VWF∶Ag, VWF∶CB/VWF∶Ag between the two groups (P>0.05) . ④There was no significant difference in VWF values between male and female groups (P>0.05) . ⑤The VWF∶Ag, VWF∶CB of the northern population (North area of Huaihe River) group were higher than that of southern population (Suzhou area) group (t=4.525, P=0.001; t=3.214, P=0.002) , but VWF∶Rco/VWF∶Ag, VWF∶CB/VWF∶Ag were lower than that of southern population group (t=6.373, P=0.001; t=2.902, P=0.005) , and there was no significant difference in VWF∶Rco between the two groups (t=1.598, P=0.115) . ⑥The VWF values of 8 standard plasma samples were in accordance with the known diagnosis. Conclusions: A more integrate plasma VWF levels of healthy Chinese people were obtained for the first time by using fully automatic and standardized testing instruments. It was also found that ABO blood group and region had a significant impact on the level of VWF, while the age and gender had no significant effect.

[Acquired von Willebrand syndrome in three patients and literature review].

OBJECTIVE: To deepen the understanding of acquired von Willebrand syndrome (AVWS). METHODS: The clinical data of 3 patients were analyzed and related literature were reviewed. RESULTS: ① Case 1, a 70- year- old male, diagnosed as Waldenstrom macroglobulinemia and AVWS, was presented with spontaneous epitaxis and bruising. The VWF∶Ag level was 16%. Treatment was initiated with VWF concentrates. Two cycles of chemotherapy with Bortezomib, thalidomide and Dexamethasone were followed. Partial remission was achieved. Half- year' follow- up showed no sign of spontaneous hemorrhage. ② Case 2, a 48- year- old female, diagnosed as monoclonal gammopathy of undetermined significance and AVWS, was presented with repeated epitaxis. The VWF∶Ag level was 7%. Because the bleeding was slight and self-relieved, no specific treatment was addressed. She was followed up for one and a half year. ③ Case 3, a 50- year- old man, diagnosed as monoclonal gammopathy of undetermined significance and AVWS, was referred to our hospital for presentation with significant hematomas. VWF∶ Ag was reduced to 12%. VWF- containing cryoprecipitate, plasma, intravenous immunoglobulin and rituximab were used to control his bleeding symptom. During the follow-up, spontaneous hemorrhage still occurred occasionally. CONCLUSIONS: Acquired von Willebrand syndrome presented with heterogeneous symptoms. The level of VWF∶Ag and VWF∶Rco for patients with bleeding disorder should be performed. Abnormal bleeding symptoms in elderly patients without personal or family history of bleeding should prompt consideration of the underlying disorders. Treatment included controlling acute bleeding, curing the underlying diseases and preventing bleeding in high- risk situations. The prognosis of acquired von Willebrand syndrome is mainly related to the underlying diseases.

Automedicação em acadêmicos do curso de medicina / Self-medication in academics from medical school

Objetivo: Avaliar a automedicação em estudantes do curso de Medicina da Universidade de RibeirãoPreto (UNAERP) no ano de 2008. Métodos: Estudo transversal quantitativo, realizado no ano de 2008, no qual 200 alunos do curso de Medicina da UNAERP responderam a um questionário autoaplicado, contendo questões de múltipla escolha sobre a prática da automedicação, no intuito de conhecer aspectos do consumo de medicamentos. Para análise estatística foi empregado o teste de correlação de Pearson. Considerou-se como base o intervalo de confiança de 95%. O estudo foi aprovado pelo Comitê de Ética da UNAERP. Resultados: Observaram-se 58,5% de participantes do sexo feminino. A média de idade foi de 21,5 anos. A prevalência da automedicação foi de 92,0%. As prevalências entre os alunos das diferentes etapas do curso não foram significativamente diferentes: 86,6%, 90,0%, 93,3%,94,4% e 97,1%, respectivamente, entre os alunos do 1º ao 4º ano e internato. A aquisição de medicamentos controlados, sem apresentação da prescrição, ocorreu entre 6,5% dos alunos e 69,5% responderam já terem indicado medicamentos para outras pessoas...

Objective: Evaluate the self-medication by students of University of Medicine of Ribeirão Preto (UNAERP) in 2008. Methods: A quantitative cross-sectional study conducted throughout 2008 in with 200 medicine students, who answered a self-administered questionnaire containing multiple choice questions regarding self-medication and consumption of medicines. Statistical analysis was employed to test Pearson correlation. The confidence interval considered was of 95%. The study was approved by UNAERP Ethics Committee. Results: There were 58.5% of female participants. The age average was 21.5 years old. The prevalence of self-medication was 92.0%. The prevalence among students from different stages of the course was not significantly different: 86.6%, 90.0%, 93.3%, 94.4% and 97.1%, among the students from, respectively, the 1st to 4th year and from the internship...

Layer-by-layer assembly of chitosan and platelet monoclonal antibody to improve biocompatibility and release character of PLLA coated stent.

The aim of this study is to construct a biocompatible coating of a drug-eluting stent through the incorporation of chitosan with monoclonal antibody (mAb) to a platelet glycoprotein (GP) IIIa receptor, by electrostatic layer-by-layer (LBL) adsorption of oppositely charged polyelectrolytes and proteins. The platelet maximum aggregation rate and aggregation inhibition rate tests confirm the bioactivity of mAb in different pH assembly environments. The fluorescence spectra test and confocal laser scanning microscopy observation were used to monitor the LBL assembly process of the mAb/chitosan multilayer on the surface of the aminolyzed Poly-L-lactic acid (PLLA) membrane, when using Rhodamine B isothiocyanate-labeled mAb and Fluorescein isothiocyanate-labeled chitosan. The in vitro platelet adhesion experiment demonstrated the amicable blood compatibility of the mAb/chitosan multilayer. The endothelial cell adhesion and migration test revealed that the multilayer could improve the cytocompatibility of the PLLA matrix in terms of cell attachment, proliferation, and migration. An in vitro perfusion circuit was designed to evaluate the release rates measured by a radioisotope technique with ¹²5I-labeled GP IIIa mAb. The different eluting curves of the mAb/chitosan-assembled stent and mAb physically absorbed stent showed the improvement of mAb's release character when using LBL self-assembly technology. Our method to prepare a biocompatible stent surface with mAb/chitosan multilayers has proved to be favorable and effective in vitro, thus justifying further evaluation to improve the biocompatibility in an animal model test.

Ultrasonic atomization and subsequent desolvation for monoclonal antibody (mAb) to the glycoprotein (GP) IIIa receptor into drug eluting stent.

An eluting-stent system with mAb dispersed in the PLLA (poly (L-lactic acid)) was validated in vitro. Specifically designed spray equipment based on the principle of ultrasonic atomization was used to produce a thin continuous PLLA (poly (L-lactic acid)) polymer coating incorporating monoclonal antibody (mAb). This PLLA coating was observed in light microscopy (LM) and scanning electron microscopy (SEM). The concentration of the monoclonal antibody (mAb) to the platelet glycoprotein (GP) IIIa receptor and the eluting rate were then measured by a radioisotope technique with (125)I-labelled GP IIIa mAb. An in vitro perfusion circuit was designed to evaluate the release rates at different velocities (10 or 20 ml min(-1)). The PLLA coating was thin and transparent, uniformly distributed on the surface of the stent. Three factors influenced its thickness: PLLA concentration, duration and gas pressure. The concentration of mAb was influenced by the duration of absorption and the concentration of the mAb solution; the maximum was 1662.23 + or - 38.83 ng. The eluting rate was fast for the first 2 h, then decreased slowly and attained 80% after 2 weeks. This ultrasonic atomization spray equipment and technological process to prepare protein eluting-stents were proved to be effective and reliable.

Eluting characteristics of a platelet glycoprotein receptor antibody using a PLLA-coated stent.

Drug-eluting stents (DES) have emerged as a recognized alternative to treat stent restenosis but many questions remain regarding the optimal type and eluting characteristics of both drug and stent. The first component of the study examines the extent of surface coating of PLLA (poly(L-lactic acid)) on a Nitinol stent. The second characterizes the adsorption and elution rates of monoclonal mouse anti-human platelet glycoprotein (GP) IIIa antibody SZ-21 from a PLLA-coated surface. The PLLA coating was examined by fluorescence staining and image analysis using the Image Processing Box of MATLAB. Stents exposed to the monoclonal mouse anti-human platelet GP IIIa antibody were tested for their adsorption characteristics by radioisotope technique with (125)I-labelled SZ-21. The elution rates were then measured in looped circuits at different velocities (10 or 20 ml/min) and durations (30 min up to 312 h). Results showed that the fluorescence staining and image analysis showed a striking difference in the extent of coating between PLLA-coated stents and SZ-21 eluting stents on the gray-scale distribution of Nitinol surfaces. The amount of SZ-21 adsorbed onto the PLLA-coated stents was dependent on the concentration and duration of immersion in the solution. The method of preparation the mAb eluting stent significantly influenced the elution characteristics for a continuous perfusion of more than 300 h. The eluting curve was biphasic with initial rapid elution for the first 24 h followed by a gradual slow elution. These results indicate that the Image Processing Box of MATLAB appears to be a useful method for semi-quantitative analysis of fluorescence images. Furthermore, SZ-21 can be passively adsorbed onto PLLA-coated stents and predictably influenced by the concentration and duration of immersion. These studies may pave the way to developing stent-based delivery of a potent anti-platelet agent.

Comparative study of platelet activation markers in diabetes mellitus patients complicated by cerebrovascular disease.

Cerebrovascular disease and other vascular diseases are common complications of non-insulin-dependent diabetes mellitus (NIDDM) and are associated with its increased morbidity and mortality. Platelet activation plays an important role in the pathomechanisms of these vascular diseases. Although several indices have been used to assess platelet activation, few data are available indicating which are more sensitive or more valuable in this situation. We have measured platelet arachidonic acid metabolites [thromboxane B2 (TXB2) and 11-dehydro-thromboxane B2 (TXB2)] and plasma P-selectin, platelet fibrinogen binding and membrane glycoproteins in 47 well-characterized NIDDM patients with cerebrovascular disease, 38 NIDDM patients without vascular diseases, and 36 age-matched healthy individuals. Our study shows that platelets were remarkably activated in NIDDM patients with cerebrovascular diseases. The measurement of plasma 11-dehydro-TXB2 and the determination of fibrinogen binding to, and P-selectin expression on platelets would reveal a higher diagnostic sensitivity for detecting in vivo platelet activation than other markers.

[Expression and drug resistance of human MGMT gene in hemopoietic cells mediated by bicistronic retroviral vector].

OBJECTIVE: To increase myeloid progenitors resistance to chemotherapy and prevent myelosuppression caused by alkylating agents. METHODS: Total cellular RNA was extracted from human liver and cDNA was synthesized by superscript reverse transcriptase, a polymerase chain reaction(PCR) was conducted. We obtained a full length cDNA fragment encoding human alkyguarine-DNA-alhyltransferase(MGMT). The PCR product was cloned into pGEMT-T vector and further subcloned into G1Na retrovirus expression vector. Then the recombinant plasmid was transduced into the packaging cell lines GP+E86 and PA317 by lipofect AMINE. RESULTS: By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic produced clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 8.6-10 CFU/ml. Human hematopoietic cells were infected repeatedly with this high titer virus under stimulation of hemopoietic growth factors IL-3, IL-6 and SCF. PCR, RT-PCR, Southern blot, Western blot and MTT analyses showed that MGMT gene has integrated into the genomic DNA of human hemopoietic cells and expressed efficiently. CONCLUSIONS: This study provides a foundation for application of gene therapy to tumor clinical trial.