[Preparation and Application of Monoclonal Antibody Against Human von Willebrand Factor Propeptide].

OBJECTIVE: To develop monoclonal antibodies that can specifically recognize human von Willebrand factor (VWF) propeptide (VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody. METHODS: The recombinant human VWFpp (D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma. The levels of VWFpp antigen in plasma of 12 leukemia patients who underwent bone marrow transplantation were dynamically detected. RESULTS: Two hybridoma cell lines that can be subcultured continuously and secrete monoclonal antibodies against VWFpp were obtained and named SZ175 and SZ176 respectively. Identified by ELISA and Western blot, the antibodies could both specifically recognize VWFpp but couldn't recognize mature VWF (without propeptide). Based on the principle of double-antibody sandwich ELISA, monoclonal antibodies SZ175 and SZ176 were successfully made into a kit for detecting VWFpp antigen. The plasma VWFpp levels of leukemia patients before and after bone marrow transplantation were dynamically detected. The results showed that the plasma VWFpp levels of the patients after transplantation were significantly higher than those before transplantation. CONCLUSION: Two monoclonal antibodies against VWFpp were successfully prepared, and a double-antibody sandwich ELISA detection kit for VWFpp antigen was constructed, which provides a powerful tool for further study on the biological function of VWFpp, the clinical diagnosis and classification of von Willebrand disease (VWD), and the prognostic monitoring of endothelial injury-related diseases.

[Application of Bionano-DNA Full-Length Optical Mapping Technology in Genetic Diagnosis of Hemophilia A Carriers].

OBJECTIVE: To apply Bionano Saphyr visual full-length DNA optical mapping technology to the precise genetic diagnosis of hemophilia A carriers. METHODS: For 2 suspected F8 gene deficiency female carriers who could not be diagnosed by conventional next-generation sequencing technology, the full-length DNA optical mapping technology was used to detect and scan the sample X chromosome full-length visual haplotype characteristic map, which was compared with the normal haplotype. The gene structure variation information of the samples was obtained by compare with DNA atlas library. RESULTS: The average fluorescent marker length of the X chromosome DNA molecular where the F8 gene was located in the two samples was greater than 2.5 Mbp, and the average copy number was greater than 20×. After comparative analysis, one of the samples was a proximal inversion of intron 22 of the F8 gene, and another was an inversion of intron 22 accompanied by multiple deletions of large fragments. CONCLUSIONS: Bionano technology has a good detection rate for gene defects with large length and complex variation. In the absence of a proband or accurate genetic diagnosis results of the proband, the application of this technology to detect the heterozygous complex variant of the F8 gene is of great significance for the prenatal diagnosis and pre-pregnancy diagnosis of hemophilia carriers.

[Recent Advances in SARS-CoV-2-Induced Immune Thrombocytopenia --Review].

SARS-CoV-2-induced immune thrombocytopenia (SARS-CoV-2-induced ITP) is an autoimmune disease secondary to virus infections. Its diagnosis is often based on exclusion of other possible causes of thrombocytopenia in COVID-19 patients. Common laboratory examinations include coagulation function, thrombopoietin and drug-dependent antibodies. Since both bleeding and thrombosis risks are seen in SARS-CoV-2-induced ITP patients, individual remedy is essential for the treatment of this disease. Because thrombopoietin receptor agonist(TPO-RA) has the side effect of accelerating thrombosis and may aggravate the pulmonary embolism symptoms of patients, it should be used for refractory SARS-CoV-2-induced ITP patients only. This review briefly summarizes the recent research progress in the pathogenesis, diagnosis and treatment of SARS-CoV-2-induced ITP.

[The Effect of VWF Propeptide on VWF Mutant in D1 Domain].

OBJECTIVE: To investigate whether co-transfection of wild-type VWFpp with VWF mutant in D1 region is able to correct VWF defects in biosynthesis and secretion. METHODS: Four VWF mutant plasmids were single transfected into HEK 293 cells, or co-transfected into HEK 293 cells with the wild type VWFpp plasmids. The VWF in supernatant and lysate of transfected cells were analyzed by ELISA, vertical VWF multimer electrophoresis. The retention of VWF in endoplasmic reticulum of transfected cells were detected by immunofluorescence confocal microscope. RESULTS: In the vertical VWF multimer analysis, with co-expressing VWF mutant and VWFpp, the VWF multimer bands disappeared, and the VWF antigen in both supernatant and lysate of cells decreased, compared with the single expression of VWF mutant. Although the intracellular levels of VWF antigens decreased after co-expression, the retention rate of VWF mutant decreased in endoplasmic reticulum. CONCLUSION: VWFpp can reduce the retention of VWF in endoplasmic reticulum, assists the transport of VWF between subcellular organelles. However, VWFpp inhibits the biosynthesis and secretion of VWF about the mutant in D1 domain.

Bifunctional effect of the inflammatory cytokine tumor necrosis factor α on megakaryopoiesis and platelet production.

BACKGROUND AND OBJECTIVES: Platelets are affected by many factors, such as infectious or aseptic inflammation, and different inflammatory states may induce either thrombocytopenia or thrombocytosis. Tumor necrosis factor α (TNFα) is an important inflammatory cytokine that has been shown to affect the activity of hematopoietic stem cells. However, its role in megakaryocyte (MK) development and platelet production remains largely unknown. This study aimed to investigate the effects of TNFα on MK and platelet generation. METHODS AND RESULTS: The ex vivo study with human CD34+ cells demonstrated that TNFα differentially modulated commitment toward the MK lineage. Specifically, a low concentration of 0.5 ng/ml TNFα promoted MK maturation, proplatelet formation, and platelet production, whereas a high concentration of 10 ng/ml or more TNFα exhibited a substantial inhibitory effect on MK and platelet production. The distinct effect of TNFα on MKs was mainly dependent on TNFα receptor 1. TNFα differentially regulated the MAPK-ERK1/2 signaling pathway and the cytoskeletal proteins cofilin and MLC2. The in vivo study with Balb/c mice indicated that low-dose or high-dose TNFα administration differentially affected short-term platelet recovery after bone marrow transplantation. CONCLUSIONS: Our study revealed distinct roles for TNFα in megakaryopoiesis and thrombopoiesis and may provide new insights regarding the treatment for platelet disorders.

[Clinical Significance of Pretransplant Serum Ferritin Level in Patients with Prolonged Thrombocytopenia after Allogeneic Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To investigate the correlation between pretransplant serum ferritin (SF) level and prolonged or prolonged isolated thrombocytopenia (PT) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: The clinical data of 35 patients with PT after allo-HSCT were retrospectively analyzed, and 35 patients were matched according to age and sex as a controls from 424 allo-HSCT patients with normal platelet count. The serum ferritin level before the transplantation was analyzed. The potential risk factors were analyzed by chi-square test and Fisher's exact test as well as univariate and multivariate logistic regression. The survival curve was estimated by the Kaplan-Meier model to explore its clinical significance. In addition, ROC curve was used to verify the predictive power of SF. RESULTS: Compared with control group, the SF level in the PT group before transplantation significantly increased (P=0.001). Multivariate analysis results showed that SF level before transplantation was a risk factor for prolonged thrombocytopenia after HSCT, and patients with SF≥1000 ng / ml showed a higher risk of death (P=0.014). ROC curve showed that SF level could be used as a predictor of prolonged thrombocytopenia after allo-HSCT. CONCLUSION: The SF level before allo-HSCT relates with occurrence and prognosis of PT in patients after allo-HSCT. Detection of SF level can provide guidance for the intervention of prolonged thrombocytopenia after HSCT.

[Reaserch Advances in the Treatment of Primary Immune Thrombocytopenia--Review].

Primary immune thrombocytopenia (ITP) is a blood system disease mediated by autoimmune mechanism. Currently, the goal of treatment for primary ITP is to keep patients' peripheral platelet count at a safe level to prevent severe bleeding. Recently, avatrombopag and fostamatinib have been approved by the FDA for the treatment of primary ITP in adults, while new drugs such as rozanolixizumab, efgartigimod, PRTX-100, decitabine and atorvastatin have shown efficacy in early clinical trials. This review summarizes the current accepted therapies for the clinical treatment of primary ITP in adults, and briefly discuss the progress of new therapies.

Clinical significance of FLT3-ITD/CEBPA mutations and minimal residual disease in cytogenetically normal acute myeloid leukemia after hematopoietic stem cell transplantation.

PURPOSE: Genetic changes have prognostic significance in cytogenetically normal acute myeloid leukemia (CN-AML). We set out to evaluate the prognostic of 6 gene mutations in CN-AML. METHODS: We performed a mutational analysis and evaluated prognostic findings of six genes (NPM1, CEBPA, DNMT3A, FLT3-ITD, FLT3-TKD, and C-KIT) in 428 CN-AML patients at our center over 10 years. RESULTS: A total of 282 patients (65.9%) had at least one gene mutation, and the mutation frequencies were as follows: 29.7% (NPM1), 24.1% (CEBPA), 20.1% (FLT3-ITD), 4.0% (FLT3-TKD), 11.9% (DNMT3A), and 4.7% (C-KIT). Multivariate analysis indicated that FLT3-ITDmut and CEBPAwt were independent risk factors correlated with poor overall survival (OS) and disease-free survival (DFS) of CN-AML. Compared with patients who received chemotherapy as consolidation, hematopoietic stem cell transplantation (HSCT) significantly improved OS of CN-AML patients. For standard/high risk patients, HSCT improved both OS and DFS. Combined analysis showed that patients with CEBPAmut/FLT3-ITDwt had the best prognosis, and patients with CEBPAwt/FLT3-ITDmut had the worst OS, with 3-year OS of only 44%. In 212 patients who received HSCT, FLT3-ITD/CEBPA mutations and minimal residual disease (MRD) were correlated with OS and DFS in univariate analysis. CONCLUSIONS: We found that HSCT significantly improves the prognosis of standard/high risk CN-AML patients with superior OS and DFS. Molecular marker analyses, especially combined analysis of the FLT3-ITD and CEBPA status revealed a correlation with the prognosis of CN-AML. For patients who have received HSCT, MRD before transplantation was a strong prognostic marker predicting patient outcome.

[Efficacy of Haploidentical Hematopoietic Stem Cell Transplantation in Treatment of Intermediate Risk Acute Myeloid Leukemia with Negative for FLT3-ITD, NPM1 or Biallelic CEBPA Mutation].

OBJECTIVE: To compare the efficacy of haploidentical hematopoietic stem cell transplantation (hi-HSCT) HLA-matched sibling donor hematopoietic stem cell transplantation (MSD-HSCT) and post-remission chemotherapy (PR-CT) in treatment of intermediate risk acute myeloid leukemia with negative for FLT3-ITD, NPM1 or biallelic CEBPA mutation. METHODS: The clinical data of patients with intermediate risk NPM1wt/non-CEBPAdm/FLT3-ITDneg AML from October 2009 to May 2016 were retrospectively analyzed. RESULTS: The overall survival rate of the patients treated with PR-CT, MSD-HSCT or hi-HSCT was 63.7%, 71.7%, 75.5%, respectively (P＜0.05); the disease-free survival (DFS) rate was 52.8%, 67.1%, 71.3% respectively (P＜0.001); the cumulative incidence of relapse was 24.7%, 16.9%, 14.4% respectively (P＜0.05); the non-relapse mortality was 26.2%, 17.3%, 14.4% reapectively (P＞0.05). The analysis of transplantation, related adverse events showed that II-IV grade of aGVHD in the MSD-HSCT group and hi-HSCT group was 48.9% and 45.6% respectively (P＞0.05); the extensive cGVHD event was 21.6% and 8.8% (P＜0.05) respectively. CONCLUSION: The efficiency of hi-HSCT and MSD-HSCT is superior to that of PR-CT for treatment of patients with intermediate risk NPM1wt/non-CEBPAdm/FLT3-ITDneg AML after CR1, there is no statistically significant difference in the efficiency of consolidatorg treatment and the transplantation-related mortality between hi-HSCT and MSD-HSCT.

[Effect of Protein Kinase A Activation on Aggregation Function of Platelets].

OBJECTIVE: To investigate the effect of protein kinase A (PKA) activation on aggregation funetion of platelets in vitro. METHODS: The peripheral blood of healthy adults were collected, and the washed platelets were gained from collected peripheral blood. The washed platelets were treated with PKA activator Forskolin, then the platelet aggregation was induced by using Ristocetin, Thrombin, Collagen and ADP respectively, the platelet aggregation level was detected by the platelet aggregator. RESULTS: Compared with the controls, 5 µmol/L forskolin significantly inhibited ADP and collagen-induced platelet aggregation (P＜0.001), and showed mild inhibiting effect on Thrombin-induced platelet aggregation (P＜0.05). 2.5-10 µmol/L forskolin significantly inhibited ADP and Collagen -induced platelet aggregation (P＜0.001); but not showed significantly inhibitory effects on Ristocetin-induced platelet aggregation (P＞0.05). CONCLUSION: PKA activation inhibits agonists-induced platelet aggregation.

[Establishment and Clinical Application of Flow Cytometric Immunobead Array in Detecting Plasma von Willebrand Factor Antigen].

OBJECTIVE: To establish a novel flow cytometric immunobead array (FCIA) for detecting plasma von Willebrand factor antigen (vWF:Ag), and to analyze the clinical value of FCIA in predicting the prognosis of patients with ischemic stroke (IS). METHODS: Anti-human vWF monoclonal antibody SZ29 IgG was coated on microspheres overnight, the diluted plasma was added after blocking, then incubated with FITC-conjugated sheep-anti-human vWF IgG polyclonal antibody, and finally detected by flow cytometry. The plasma vWF in 21 case of von Willebrand disease (vWD) and 105 controls (CTL) were detected by FCIA and ELISA, so as to carry out methodological assessment. Plasma vWF:Ag of 61 IS patients was detected by FCIA and the data of prognosis followed-up for 2-year were collected. RESULTS: The linear fitting of FCIA was good (R2=0.99) without significant difference between FCIA and ELISA. The Bland-Altman bias was 1.12% with 95% limits of agreement that spanned from -45.06% to 47.30%, and the slope of the linear regression was 0.97 (r=0.86, P＜0.01). Importantly, the FCIA method was faster than ELISA, and superior to the ELISA in the detection of low levels of vWF:Ag. The levels of vWF:Ag, vWF:GPIbR and vWF:CB in IS patients were significantly higher than those in healthy controls (Z=8.36, 8.71, 6.22, respectively, P＜0.01). CONCLUSION: The FCIA for detecting plasma vWF:Ag is not only an effective supplement to ELISA, but also the efficiency is faster and more sensitive, thus improves the diagnosis of type 3 vWD. Elevated levels of vWF: Ag in IS patients indicate the poor recovery of daily activities and prognosis.

Antiplatelet Effect of a Pulaimab [Anti-GPIIb/IIIa F(ab)2 Injection] Evaluated by a Population Pharmacokinetic-pharmacodynamic Model.

BACKGROUND: Cardiovascular disease has one of the highest mortality rates among all the diseases. Platelets play an important role in the pathogenesis of cardiovascular diseases. Platelet membrane glycoprotein GPIIb/IIIa antagonists are the most effective antiplatelet drugs, and pulaimab is one of these. The study aims to promote individual medication of pulaimab [anti-GPIIb/IIIa F(ab)2 injection] by discovering the pharmacological relationship among the dose, concentration, and effects. The goal of this study is to establish a population pharmacokineticpharmacodynamic model to evaluate the antiplatelet effect of intravenous pulaimab injection. METHODS: Data were collected from 59 healthy subjects who participated in a Phase-I clinical trial. Plasma concentration was used as the pharmacokinetic index, and platelet aggregation inhibition rate was used as the pharmacodynamic index. The basic pharmacokinetics model was a two-compartment model, whereas the basic pharmacodynamics model was a sigmoid-EMAX model with a direct effect. The covariable model was established by a stepwise method. The final model was verified by a goodness-of-fit method, and predictive performance was assessed by a Bootstrap (BS) method. RESULTS: In the final model, typical population values of the parameters were as follows: central distribution Volume (V1), 183 L; peripheral distribution Volume (V2), 349 L; Central Clearance (CL), 31 L/h; peripheral clearance(Q), 204 L/h; effect compartment concentration reaching half of the maximum effect (EC50), 0.252 mg/L; maximum effect value (EMAX), 54.0%; and shape factor (γ), 0.42. In the covariable model, thrombin time had significant effects on CL and EMAX. Verification by the goodness-of-fit and BS methods showed that the final model was stable and reliable. CONCLUSION: A model was successfully established to evaluate the antiplatelet effect of intravenous pulaimab injection that could provide support for the clinical therapeutic regimen.

[Effect of Recombinant Protein in the Spacer Domain on ADAMTS13 Activity].

OBJECTIVE: To obtain the recombinant protein of spacer domain in von Willebrand factor cleaving protease (ADAMTS13), and further study its biological function in ADAMTS13. METHODS: The prokaryotic expression vector was constructed by using the template of plasmid with full-length ADAMTS13, and then transfected into E coli., following the induction of IPTG with the low temperature (30 ℃). The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blot respectively. By Adding the recombinant protein to the plasma of immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients, the activity of ADAMTS13 was tested. RESULTS: The prokaryotic expression vector was successfully constructed and the protein of spacer domain with the high purity was obtained. Western blot showed that the recombinant fragment could both react with monoclonal antibody against 6×His and polyclonal sheep IgG against ADAMTS13 (Gln34-Trp688). The protein formed a main lane at the position of 15 kDa with SDS-PAGE. It was demonstrated that the recombinant protein could efficiently elevate the ADAMTS13 activity in plasma of iTTP patients to reach normal level by functional experiment. CONCLUSION: The recombinant protein has high purity and immune activity, which provides the experimental basis for further research on mechanism of iTTP involved in spacer domain.

[Establishment of Flow Cytometric Immunobead Array for Detecting Plasma Von Willebrand Factor Activity and Its Clinical Application in the Prognosis of Ischemic Stroke].

OBJECTIVE: To establish a novel flow cytometric immunobead array (FCIA) for detecting plasma von Willebrand factor activity (vWF:GPIbR) and apply it in ischemic stroke (IS). METHODS: Microspheres coated with anti-human platelet glycoprotein Ibα (GPIbα) monoclonal antibody SZ151 IgG, were incubated with recombinant fragment of GPIbα, then added ristocetin and plasma, finally incubated with FITC-conjugated sheep-anti-human vWF IgG polyclonal antibody, and detected by flow cytometry. vWF antigen (vWF:Ag), vWF:GPIbR, and vWF collagen binding assay (vWF:CB) were also included for evaluating vWF levels in IS patients. RESULTS: The intra-assay coefficient variations (CVs) and inter-assay CVs of FCIA were 7.7% and 13.5%, respectively. The slope of the linear regression was 0.9739 (r=0.855, P<0.001), and the Bland-Altman bias was 9.95%, indicating a good correlation between FCIA and ELISA. The FCIA had better sensitivity, specificity and accuracy as compared with those by ELISA (P<0.05). The levels of vWF:Ag, vWF:GPIbR and vWF:CB in IS patients were significantly higher in comparison with those in healthy controls (H=7.8, 6.4, 6.2, respectively, P<0.01), the level of vWF:GPIbR in IS patients positively correlated with levels of vWF:Ag, high-sensitivity C-reactive protein, Autar score and hospitalization time. CONCLUSION: The FCIA for detecting plasma vWF:GPIbR is more specific and accurate than ELISA. The vWF:GPIbR is involved in the paroxysm of IS, which could be used to evaluate the risk of thrombosis in IS patients.

Incidence, risk factors, and clinical significance of Epstein-Barr virus reactivation in myelodysplastic syndrome after allogeneic haematopoietic stem cell transplantation.

Epstein-Barr virus (EBV) reactivation is a life-threatening complication after allogeneic haematopoietic stem cell transplantation (allo-HSCT). In this study, we investigated the characteristics of EBV reactivation in 186 consecutive myelodysplastic (MDS) patients who underwent allo-HSCT in our centre. In 35 patients (18.8%) who experienced EBV reactivation after allo-HSCT, the median onset was 53 days (range 4-381 days). The cumulative incidence of EBV reactivation at the first, sixth, and twelfth month after allo-HSCT was 10.7%, 15.1%, and 17.9%, respectively. Twenty-five patients (71.4%) received pre-emptive rituximab therapy, and no patients developed post-transplant lymphoproliferative disorders. Stem cell source was proven to be a risk factor correlated with EBV reactivation. The cumulative incidence of relapse in the EBV-positive group was 11.4%, 25.2%, and 31.0% at the first, second, and third year after transplantation, respectively, being significantly higher than the corresponding 6.8%, 10.2%, and 10.2%, in the EBV-negative group (P = 0.014). Prognostic analysis showed that EBV reactivation was an independent risk factor for relapse-free survival (RFS). Patients in the EBV-positive group showed obviously shorter RFS than those in the EBV-negative group, with 3-year RFS of 62% and 85%, respectively (P = 0.017).

FLT3-ITD and CEBPA Mutations Predict Prognosis in Acute Myelogenous Leukemia Irrespective of Hematopoietic Stem Cell Transplantation.

Cytogenetic and genetic changes have prognostic significance in acute myelogenous leukemia (AML). In our study, we compared the cytogenetic changes and gene mutations (NPM1, CEBPA, DNMT3A, FLT3-ITD, FLT3-TKD, and C-KIT) with clinical outcomes in 1132 patients with AML enrolled at our center over a 10-year period. A total of 977 patients provided gene mutation data. There were subsets of patients who exhibited mutations in NPM1 (17.9%), CEBPA (16.4%), FLT3-ITD (18.5%), FLT3-TKD (3.9%), DNMT3A (8.6%), and C-KIT (8.8%). A total of 557 patients (49.2%) underwent hematopoietic stem cell transplantation (HSCT) as consolidation therapy. Multivariate analysis identified an adverse karyotype (hazard ratio [HR], 1.48; P = .001), the presence of FLT3-ITD (HR, 1.90; P < .001), and receipt of nonstandard first-line induction chemotherapy (HR, 1.45; P = .003) as significant risk factors for poor overall survival (OS), and the presence of CEBPAmut (HR, .42; P < .001) and receipt of HSCT (HR, .35; P < .001) as prognostic factors for favorable OS. In addition, the presence of FLT3-ITDmut (HR, 2.11; P < .001) was identified as an independent risk factor for poor disease-free survival (DFS), and receipt of HSCT was correlated with improved DFS (HR, .74; P = .046). Compared with chemotherapy as consolidation therapy, HSCT improved the prognosis and overcame the prognostic effect of karyotype from the initial diagnosis; however, the presence of FLT3-ITD or CEBPA mutation can predict prognosis in AML irrespective of HSCT.

[Research Advances of Acquired Thrombotic Thrombocytopenic Purpura--Review].

Thrombotic thrombocytopenic purpura (TTP) is an acute, potentially life-threatening thrombotic microangiopathies (TMA). TTP has mainly been diagnosed by clinical findings, such as thrombocytopenia and microangiopathic haemolytic anemia. In addition, the reduced activity of von Willebrand factor-cleaving metalloproteinase ADAMTS13 below 10% has been accepted internationally as a diagnostic criterion for TTP. In clinic, the accurate diagnosis and early initiation of therapy can significantly improve the survival rate of patients. Therapy should be focused on increasing ADAMTS13 activity and eliminating or inhibiting ADAMTS13 antibody by the infusion or exchange of therapeutic plasma and corticosteroids. Both the administration of recombinant ADAMTS13 and reducing the release of human neutrophil peptides (HNPs) would be novel promising strategies for the prevention of platelet-vWFinteraction. This review briefly summarizes the recent advances in terms of pathogenesis, diagnosis and treatment of TTP.

Pyrosequencing quantified methylation level of miR-124 predicts shorter survival for patients with myelodysplastic syndrome.

BACKGROUND: Aberrant CpG island methylation has been increasingly recognized as a common event in myelodysplastic syndrome (MDS). To date, most of the previous studies of miR-124 in MDS have focused on epigenetic changes and little is known about the underlying mechanism through which miR-124 regulates CDK6 expression. RESULTS: In the present study, we employed pyrosequencing analysis to quantify the methylation levels of upstream regions of the miR-124 genes (miR-124-1, miR-124-2 and miR-124-3) in 56 primary MDS patients. We found the three miR-124 genes were methylated in MDS patients. Univariate analysis revealed that the World Health Organization (WHO) classification, marrow blast count, karyotype, International Prognostic Scoring System (IPSS), mean corpuscular volume, as well as high methylation of miR-124-1, miR-124-2 and miR-124-3 were significantly related to overall survival. In leukaemia-free survival, patients who were older and had an advanced WHO classification, high marrow blast counts, high IPSS risk and high methylation of miR-124-1 and miR-124-2 progressed rapidly to acute myeloid leukaemia. Multivariate analysis demonstrated that high methylation of miR-124-3 was an independent factor of overall survival. Median survival of patients with high miR-124-3 methylation was significantly shorter (7.6 months) than patients with low methylation (32.7 months; P = 0.010). A functional study revealed that silencing of miR-124 resulted in upregulation of its target gene, cyclin dependent kinase CDK6, which in turn promoted cell proliferation in the MDS cell line SKM-1. CONCLUSIONS: High methylation of miR-124-3 predicts shorter survival for patients with MDS, which may be a useful prognostic marker in MDS.

[Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies against ADAMTS13].

OBJECTIVE: To establish the hybridoma cell lines secreting anti-ADAMTS13-T7 monoclonal antibodies (MA6) and to construct the MAb directed against different domains of ADAMTS13-T7. METHODS: The trucated type protein ADAMTS13 of eukaryote-expressed recombinant ADAMTS13-T7 was constructed and was purified by using Ni-NTA agarose. Then the purified ADAMTS13 was used to immunize the BALB/c mice; the antiserum titer of ADAMTS13 protein of immunized mice was deteceted by ELISA. The spleen was taken from mice for construcing the single cell suspension, then the single cell suspension was mixed with myeloma cells SP2/0 at 10:1 ratio for cell fusion, and the elution culture of hybridoma cells was carried out; after 2 weeks, the wells in which clones were well grown were selected for detection, then the positive clonal wells were expansively cultured and the detection again was performed. RESULTS: The purified ADAMTS13-T7 protein was gained. The ELISA showed that the antiserum titer in mice immumized by ADAMTS13-T7 protein was 1:20000. The results of fusion culture by hybridoma techmique showed that 30 hyridoma cell lines secreting MAb against recombinant ADAMTS13 were established. CONCLUSION: The hybridoma cell lines secreting MAb against recombinant ADAMTS13 have been successfully established by fusion culture, which provide more powerful tools for further screening the MAb with certain functional activity and studying the structure and function of ADAMTS13.

Recombinant Human Thrombopoietin Promotes Platelet Engraftment and Improves Prognosis of Patients with Myelodysplastic Syndromes and Aplastic Anemia after Allogeneic Hematopoietic Stem Cell Transplantation.

Poor platelet graft function (PPGF) is a significant complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, no optimal treatment has been recommended. This study investigated aspects of platelet recovery after allo-HSCT, including prognostic value and the effect of recombinant human thrombopoietin (rhTPO). We retrospectively analyzed 275 patients who received allo-HSCT in our center. Of them, 135 (49.1%) patients had good platelet graft function (GPGF) and 140 (50.9%) had PPGF. The latter included 59 (21.5%) patients with primary PPGF and 81 (29.4%) with secondary PPGF. Multivariate analysis showed that male gender (P = .024), lower CD34+ cell count (P = .04), and no use of rhTPO (P <.001) were associated with PPGF. The 3-year overall survival rate of patients with PPGF (58%) was significantly less than that of patients with GPGF (82%; P <.001). We further analyzed the effect of rhTPO on prognosis of patients after allo-HSCT. Although no advantage was apparent when analyzing the entire cohort, for patients with myelodysplastic syndromes and aplastic anemia, rhTPO was associated with a significant survival advantage (P = .014).