[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 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 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.

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.

[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.

Increase in plasma thrombomodulin and decrease in plasma von Willebrand factor after regular radiotherapy of patients with cancer.

In this study, we investigated plasma levels of thrombomodulin (TM) and von Willebrand factor (vWF) in 51 patients suffering from cancer or tumor undergoing 60 cobalt radiotherapy. Plasma TM and vWF antigen were measured by immunoradiometric assay and ELISA, respectively. During radiotherapy, an increase in plasma TM in patients was observed, which was radiation-dose dependent and there was a positive correlation between plasma TM level and radiation doses. However, the level of plasma vWF in the patients was decreased during radiotherapy and there was an inverse correlation between the amount of plasma vWF and radiation doses. Our data indicate that plasma TM is an useful molecular marker for early detection of radiation injury to endothelial cells in patients undergoing radiotherapy.

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.

Thrombus imaging in dogs with a monoclonal antibody (SZ-51) specific for activated human platelets.

To specifically detect the localization of thrombus in vivo, we prepared a monoclonal antibody (McAb) SZ-51 specific for an alpha-granule membrane protein (GMP-140) on the surface of activated human platelets. The thrombus binding rate in vitro was 80 +/- 5% for 131I-SZ-51 and 4.4 +/- 0.5% for 131I-nonimmune IgG. Owing to the crossreaction of McAb SZ-51 with the activated platelets of dogs, thrombus in the femoral artery and vein of dogs was formed and imaged with single photon emission computerized tomography (SPECT). The ratio of thrombus to blood radioactivity (T/B) was 2.1, 4.8, 14.0 and 18.0 at 1, 2, 3 and 4 hours after injection of 131I-SZ-51 (0.6 mCi, 50 micrograms) into the arterial thrombus respectively, while the T/B ratio was 1.7, 2.2, 5.3 and 8.0 for venous one. The injection of 131I-nonimmune IgG at the same doses yielded a T/B ratio of 1.2 +/- 0.3 at each time period for both arterial and venous thrombi. These findings were in good agreement with the counting of removed thrombi 24 hours after the injection of radiotracer. The ratio was 21.7 and 2.6 for the 131I-SZ-51 in the arterial and venous thrombus, respectively. However, there were only 1.48 and 1.6 for the 131I-nonimmune IgG. These results indicate the great potentiality of McAb SZ-51 in application to detection of thrombi in vivo.

Antifibrin monoclonal antibodies for the detection of venous thrombosis.

In order to specifically detect the localization of thrombus in vivo, we have recently developed two monoclonal antibodies (SZ-58, SZ-63) which can specifically bind to cross-linked fibrin. The binding rates of the two monoclonal antibodies (MoAbs) to human plasma clots in vitro were 46.4 +/- 2.3% for 125I-SZ-58, 50.1 +/- 1.7% for 125I-SZ-63 and 3.4 +/- 1.6% for 125I-SZ-53 (control, MoAb against TM). It was shown that both SZ-58 and SZ-63 possess properties of inhibiting the polymerization of fibrin, and SZ-58 could also inhibit the aggregation of platelets induced by ADP. These characteristics make the two MoAbs suitable in the detection of thrombus in vivo. According to the cross reaction tests, thrombi in the jugular veins and carotid arteries in rabbits were made. After injection of the 125I-labeled MoAbs (100,000 cpm/ml of blood), the thrombi and the blood were taken and weighed at various time intervals and radioactivities were measured by an autogamma counter. The ratios of thrombus to blood radioactivity (T/B) of thrombi in jugular veins were 3.0, 5.6 and 3.0 for 125I-SZ-58, 1.5, 3.0 and 5.2 for 125I-SZ-63 and 1.2, 1.0 and 0.7 for control (125I-SZ-53) at the 3rd, 12th and 24th hour after the injection of the radiolabled MoAbs, while the radioactivities of arterial thrombi were almost the same as that in blood after the injection of the two radiotracers. Therefore, it can be concluded that both SZ-58 and SZ-63 can be used in venous thrombus imaging in vivo and the optimal times of imaging are at the 12th hour for SZ-58, 24th hour for SZ-63 after the injection of the radiolabled MoAbs.

[Molecular pathology of inherited Glanzmann's thrombasthenia. Report of 11 cases].

Glycoprotein IIb-IIIa (GPIIb-IIIa) concentration was studied in 11 patients with Glanzmann's thrombasthenia (GT) with sensitive Western blotting technique. 7 patients with severe GPIIb-IIIa deficiency (less than 10% of the normal) were designated as type I (64% of patients), 2 patients with moderate GPIIb-IIIa deficiency (10-25% of the normal) as type II (18%) and 2 patients with GPIIb-IIIa 40-100% of the normal as variants (18%). Southern Blotting was used to analyze the GPIIb and GPIIIa genes in the 11 patients. The results showed that there were no major deletions or insertions in either the GPIIb or GPIIIa genes. However, a small change in GPIIb gene was demonstrated in two sibling patients and the abnormality of GPIIIa gene was found in another two patients. These observations combined with those from literature provide a basis for discussing the molecular pathology of Glanzmann's thrombasthenia.

Hybridization to an anti-activated platelet monoclonal antibody enhances fibrinolytic potency on urokinase.

The B-chain of urokinase (UK) was covalently linked by disulfide bond to the Fab fragment of an anti-human activated platelet monoclonal antibody (SZ-51). The UK-SZ-51 conjugate retained the original binding specificity of its parent antibody, and produced about a 5-fold enhancement in clot lysis in plasma over that of the urokinase in vitro. Whereas UK significantly decreased the concentration of fibrinogen in plasma clot assay supernatants, UK-SZ-51 did not.