Recent Advances in SARS-CoV-2-Induced Immune Thrombocytopenia --Review / 中国实验血液学杂志

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.

Clinical diagnosis and treatment of hereditary thrombocytopenia and purpura: a report of five cases and literature review / 中华血液学杂志

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.

Reaserch Advances in the Treatment of Primary Immune Thrombocytopenia--Review / 中国实验血液学杂志

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 Pretransplant Serum Ferritin Level in Patients with Prolonged Thrombocytopenia after Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

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.

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 / 中国实验血液学杂志

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 NPM1/non-CEBPA/FLT3-ITD 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 NPM1/non-CEBPA/FLT3-ITD 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 / 中国实验血液学杂志

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 / 中国实验血液学杂志

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.

Establishment of Flow Cytometric Immunobead Array for Detecting Plasma Von Willebrand Factor Activity and Its Clinical Application in the Prognosis of Ischemic Stroke / 中国实验血液学杂志

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.

Effect of Recombinant Protein in the Spacer Domain on ADAMTS13 Activity / 中国实验血液学杂志

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.

Research Advances of Acquired Thrombotic Thrombocytopenic Purpura--Review / 中国实验血液学杂志

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.

Establishment of Double Antibody Sandwich ELISA for the Determination of Human Soluble VE-Cadherin in Human Plasma and Its Application / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human soluble VE-cadherin in human plasma and to investigate its value in clinical use.</p><p><b>METHODS</b>The monoclonal antibodies against human VE-cadherin were prepared from BALB/c mice immunized with prokaryotic expression recombinant proteins, and the best combination of double antibodies was selected by checkerboard titration method. Double antibody sandwich ELISA for the determination of human VE-cadherin was established by using HRP-labeled McAb as a detection antibody and a capture antibody. The methodology performance was evaluated. The plasma concentrations of VE-cadherin in 28 healthy subjects and 60 patients with cancer were determined.</p><p><b>RESULTS</b>The double antibody sandwich ELISA for the determination of human VE-cadherin was established by selecting the combination of double antibodies. The detection limit was 24.7 pg/ml, the coefficients of variation for inner-batch and inter-batch were 4.1%-7.7% and 8.7%-10.8% respectively. The average recovery was 96.7%. The plasma level of soluble VE-cadherin in normal controls was 262.1±11.75 pg/ml. The plasma level of soluble VE-cadherin was 173.9±17.98 pg/ml in 24 patients with leukemia, 311.7±25.24 pg/ml in 14 patients with stomach cancer, 206.8±25.01 pg/ml in 11 patients with lung cancer, and 310.7±11.82 pg/ml in others patients(9 patients with breast cancer, 1 patients with gliomas, 1 patients with liver cancer).</p><p><b>CONCLUSION</b>The developed ELISA kit has better sensitivity and specificity, and can be used in detection of human soluble VE-cadherin in human plasma, therefore, it can provide a new mathod for diagnosis of cancer patients.</p>

Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies against ADAMTS13 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Influence of HSCT Preconditioning on ADAMTS-13 Activity and vWF level and Its Clinical Significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the influence of precondintioning on ADAMTS-13 activity and vWF level, and its clinical significance by measuring the alterations of ADAMTS-13 activity and vWF antigen levels before and after preconditioning of hematopoietic stem cell transplantation (HSCT) in the patients.</p><p><b>METHOD</b>A total of 113 patients received HSCT in the First Hospital affiliated to Soochow University were investigated, 20 healthy volunteers were used as the control. The ADAMTS-13 activity and vWF antigen level were measured by FRETS-vWF73 and ELISA respectively. Modified BU/CY, TBI/CY or BEAM were used as preconditioning regimen.</p><p><b>RESULTS</b>(1) out of all the patients enrolled in this study, 8 patients were diagnosed as thrombotic disorders, and 49 patients were with occurence of acute graft-versus-host disease (aGVHD); (2) comparing with the control group, the ADAMTS-13 activity were lower and vWF antigen was higher in the patients after preconditioning (P < 0.01). Among all the patients, 69 (59.3%) cases showed the decrease of ADAMTS-13 activity after preconditioning, including 9 patients with more than 60% (9/113, 8.0%) decrease, in the meantime, the average plasma vWF antigen level of these 69 patients was significantly increased after preconditioning (P < 0.05); (3) the plasma ADAMTS-13 activity in 8 patients with thrombotic complications decreased after preconditioning, and there was significant difference in comparision with that of patients without thrombotic complication (P < 0.01). The patients with decrease of ADAMTS-13 activity more than 60% accounted for 37.5% (3/8), at the same time, the the level of vWF antigen increased (P < 0.01); (4) The medium level of ADAMTS-13 activity was dropped in 49 patients with aGVHD after preconditoning, but there was no abvious difference in comparision with that of patients without aGVHD. Among them 25 patients were with significant drop actvity of ADAMTS-13 at that time when aGVHD occurred (P < 0.01). Out of them, the patients with drop more than 60% before preconditioning accounted for 60% (2/35). The logistic regression analyse showed that the drop of ADAMTS-13 activity more than 60% before preconditioning was the risk factor for occurence of thrombosis at the later stage (P < 0.01), but the drop of ADAMTS-13 activity after preconditioning did not was the risk factor for occurence of aGVHD.</p><p><b>CONCLUSION</b>The decrease of ADAMTS-13 activity after preconditioning of HSCT confirmed to be lower than that befor preconditioning of HSCT, and the level of vWF antigen after preconditioning of HSCT could be higher than that before precondifioning, especially in patients with thrombosis. The decrease of ADAMTS-13 activity after preconditioning has been found to be mone than 60%, then this decrease can be considered as an independent risk factor for later thrombogenesis, but the decrease of ADAMTS-13 after precoditioning activity did not associate with occurence of aGVHD, so the decrease of ADAMTS-13 activity can be used as an important predictor of thrombosis after HSCT.</p>

Identification of Monoclonal Antibodies against von Willebrand Factor Cleaving Protease (ADAMTS13) and Their Function / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To construct and identify the monoclonal antibodies against von willebrand factor cleaving protease(ADAMTS13), and to study their biological function.</p><p><b>METHODS</b>BALB/c mice were immunized by purified recombinant ADAMTS13 truncated eukaryotic protein (ADAMTS13-T7). Murine anti-human ADAMTS13 monoclonal antibodies (McAbs) were constructed by standard hybridoma technology and identified by ELISA. The recognition of McAbs with full-length recombinant ADAMTS13 protein was identified by Western blot. In function assay, the influence of McAbs on the proteolysis of vWF by ADAMTS13 was observed.</p><p><b>RESULTS</b>A group of 6 murine anti-ADAMTS13 McAbs was obtained with the clone number 1G11, 2F11, 6G3, 9E1, 10A8 and 10B4. In ELISA, the highest titers of 1G11 and 2F11 were observed, both of which showed a higher affinity to ADAMTS13-T7 than full-length ADAMTS13. The Western blot demonstrated that the 6 McAbs all could recognize ADAMTS13, among which 1G11 and 2F11 showed stronger reaction with ADAMTS13. In addition, under the denatured conditions, 1G11 and 2F11 could inhibit hydrolysis of vWF by ADAMTS13, and that was stronger with the increasing of McAbs concentration.</p><p><b>CONCLUSION</b>McAbs against ADAMTS13 have been gained, two of which are inhibitory antibodies.</p>

Changes of ADAMTS13 activity and vWF antigen level in patients with acute myelogenous leukemia and their significance / 中国实验血液学杂志

This study was purposed to investigate the changes of von Willebrand factor cleaving protease (ADAMTS13) activity and vWF antigen level in patients with acute myelogenous leukemia (AML) before and after treatment and evaluate their clinical significance. Seventy-three AML patients were enrolled in this study, the sodium citrate anticoagulated plasma was collected before and after their induction chemotherapy. Fluorescence resonance energy transfer substrate vWF73 (FRETS-vWF73) assay was established to detect the plasma ADAMTS13 activity while vWF antigen level was measured by ELISA. The results showed that the ADAMTS13 activity in newly diagnosed patients with AML before induction therapy was obviously lower than that in normal controls (63.3 ± 25.5)% vs (105.1 ± 37.7)(P < 0.01), while the vWF antigen level was higher than that in normal controls (226.6 ± 127.0)% vs (111.4 ± 39.7)% (P < 0.01). After standard induction chemotherapy, the ADAMTS13 activity of AML patients in complete remission period was higher than that in AML patients before therapy (P < 0.01), and was not significant difference with that in normal controls; the vWF antigen was significantly lower than that in AML patients before therapy (P < 0.01), but it still was higher than that in controls (P < 0.05). The ADAMTS13 activity in newly diagnosed AML patients complicated with infection before therapy was obviously lower than that in AML patients without infection (52.2 ± 20.6)% vs (73.9 ± 24.7)% (P < 0.01), while the vWF antigen level was significantly higher than that in AML patients without infection (262.2 ± 135.7)% vs (193.8 ± 110.2)% (P < 0.05). The ADAMTS13 activity in AML patients with disseminated intravascular coagulation (DIC) was significantly lower than that in AML patients without DIC (42.0 ± 14.5)% vs (73.4 ± 22.7)% (P < 0.01), while the vWF antigen level was obviously higher that in AML patients without DIC (274.2 ± 140.0)% vs (204.7 ± 115.5)% (P < 0.01). It is concluded that the ADAMTS13 activity in newly diagnosed AML patients befor induction therapy has been confiremed to be lower and the vWF antigen level to be higher, especially in AML patients with infection or DIC. The ADAMTS13 and vWF antigen may play a role in the pathogenesis of AML and the formation of infection and DIC.

Isolation, culture and characterization of outgrowth endothelial cells from the human peripheral blood / 中国实验血液学杂志

Compared with endothelial progenitor cells, outgrowth endothelial cells (BOECs) from peripheral blood are rich in protein for blood angiogenesis and cell adhesion, similar to mature endothelial cells in biological characteristics. Moreover, they are now replacing human umbilical vein endothelial cells for the latter's limited life span and drift of phenotype, and might become a new tool for exploring the vascular abnormalities. This study was aimed to establish the protocol of producing BOECs, and then analyze the cell phenotype and function of BOECs. Mononuclear cells were collected from peripheral blood by gradient centrifugation and then seeded on plates and cultivated in EGM-2 medium for 4 weeks. The morphological changes of cells were observed and cell phenotype was examined by flow cytometry. VWF multimers were used to analyse the distribution of vWF multimers in superment of BOECs and the storage of vWF in BOECs, and the secretion of vWF in BOECs under stimulation was detected by confocal fluorescence microscopy. The results showed that after 4-week-culture in vitro, the cell colonies and characteristic cobblestone-like morphology of BOECs were found in plates. For another three weeks of expansion, BOECs expressed CD31, CD34, and EPCR, without the expression of CD14, CD45 and CD133. The vWF from BOECs cell supernatant shared the same multimer pattern as that in normal plasma. By confocal fluorescence microscopy, vWFs were observed in BOECs. The amount of vWF increased in cells, and vWF strings were formed on cell surface by the stimulation of phorbol-12-myristate-13-acetate(PMA). It is concluded that the BOECs are first successfully established, and the phenotype and function of BOECs are analyzed. They are the native cell models for the pathogenesis of von Willebrand diseases (vWD), and may be used as new gene therapy tools for vWD.

Congenital afibrinogenemia caused by a novel insertion mutation in the FGB gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the genetic defect and its mechanism in a patient with congenital afibrinogenemia.</p><p><b>METHODS</b>The plasma fibrinogen activity and antigen of the patient was determined using the Clauss method and immuno-nephelometric assay, respectively. Genomic DNA was isolated from peripheral blood of the proband and his related family members. All exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by PCR followed by direct sequencing. Thrombin fibrin aggregation curve were detected in the plasma of the patient. Wild-type and mutation type fibrinogen vectors were constructed, and then transfected into COS-7 cells. The wild-type and mutant proteins from the culture media and cell lysates were tested by Western blot and ELISA.</p><p><b>RESULTS</b>APTT, PT, TT were significantly longer in the proband. Plasma fibrinogen activity and antigen of the patient could not be detected using the Clauss method and immuno-nephelometry, respectively. Gene analysis revealed that a novel homozygous GTTT insertion between nucleotides 2833 and 2834 in FGB exon 2 in the proband. The proband's father, mother, brother and son were heterozygous. The polymerization curves of the patient did not show a lag phase or final turbidity, compared with the normal controls. Western blot analysis showed the lack of complete half-molecules of the fibrinogen molecule and fibrinogen in patient's plasma under non-reducing conditions. It also could not detect the truncated Bβ chain under reducing conditions. Abnormal fibrinogen molecule (molecule weight>340 000) were found in transfected COS-7 cells by Western blot, which indicated that the mutation caused the abnormal intracellular fibrinogen molecule assembly. The fibrinogen band was absent in culture media transfected by the mutation. Fibrinogen levels of mutant fibrinogen were no significant different from those of wild-type fibrinogen in cell lysates by ELISA analysis [(2.47 ± 0.30) μg/ml vs (2.65±0.60) μg/ml, P=0.0889]; However, the levels of the mutant fibrinogen were statistically significant lower than those of wild type fibrinogen in culture media [(0.01 ± 0.01) μg/ml vs (3.80±0.80) μg/ml, P=0.0001].</p><p><b>CONCLUSION</b>Congenital afibrinogenemia was caused by this frameshift mutation in exon 2 of FGB. This novel mutation impaired fibrinogen assembly and secretion.</p>

Clinical and experimental characteristics of 20 patients with acute myeloid leukemia with complex variant of t(8; 21) / 中国实验血液学杂志

This study was aimed to summarize and analyze the morphology, immunophenotype, cytogenetics, molecular biology (MICM), tyrosine kinase (TK) gene mutations and clinical features of acute myeloid leukemia (AML) with complex variant of t(8;21). A retrospective study was performed for 20 AML patients with complex variant of t(8;21) in our hospital from January 1994 to April 2012, including analysis of clinical feature, immunophenotype, chromosome karyotype, treatment regimen, as well as the overall survival (OS) and relapse-free survival (RFS). Mutations of C-KIT, FLT3-ITD, FLT3-TKD and JAK2V617F were detected by genomic DNA PCR and the sequencing was per-formed in 13 AML patients with complex variant of t(8;21). The results showed that (1) the incidence of 20 AML patients with complex variant of t(8; 21) was 2.4% of total t(8; 21) AML patients. In 20 AML patients with complex variant of t(8;21), 1 case was M1, 17 cases were M2, 2 cases were M4; 10 cases were myeloid phenotype and the other 3 were myeloid plus lymphoid phenotype. There were 16 kinds of cytogenetics additional involvement of chromosomal breakpoints: lp22, 1p32, 2q35, 2q14, 3p25, 5q13, 6p22, 7q21, llq11, 1lq13, 12q14, 12q24, 12p12, 14q32, 15p13, 20q12. (2) C-KIT aberrations were detected in 30.8% cases, all mutated in exon 17 (mutkit 17), only 1 case had JAK2V617F mutation. The result of FLT3 mutation screenings in AML patients with complex variant of t(8; 21) was negative. Of 5 patients with gene mutations, 1 patient (20%) achieved complete remission (CR), the median RFS and median OS time were 6.5 months and 8.9 months respectively. Of the 8 patients without gene mutations, 6 patietns (75%) achieved CR; the median RFS and median OS time were 26.6 months and 27.7 months respectively. It is concluded that the AML patients with complex variant of t(8;21) shows typical features of t(8;21) AML, but the existence of the tyrosine kinase-related gene mutation has important implications on remission rate and long-term survival of patients treated by induction chemotherapy.

Gene analysis and pathogenesis in 40 patients with hemophilia B / 中国实验血液学杂志

Hemophilia B (HB) is a recessive X-linked inherited disorder, the pathogenesis of HB is deficiency or functional abnormalities of coagulation factor IX, which is caused by F9 gene mutations. To explore the mechanism of its molecular pathology, 40 patients with HB were studied with polymerase chain reaction (PCR) and direct sequencing. The diagnosis of HB patients were based on clinical manifestation and deficient factor IX activity in plasma. DNA was routinely extracted from peripheral blood cells of the patients and their relatives, all the 8 exons and their flanking boundaries were amplified by PCR, and the PCR products were screened by direct sequencing. Mutations which were found in study need to exclude polymorphism. The results showed that 34 mutations were confirmed in 40 HB patients, including 6 nonsense mutations, 24 missense mutations, 2 splice site mutations and 2 frame mutations for 1 or 2 nucleotide insertion. After retrieved, 4 missense mutations and 1 frameshift mutation were found for the first time. Among the 34 mutations, 2 mutations in signal peptide, 7 mutations in propeptide and gla domain, 7 mutations in epidermal growth factor-like domain, 3 mutations in activation domain, 15 mutations in serine protease or catalytic domain. It is concluded that gene analysis can directly explain molecular mechanism of hemophilia B and also provides the foundation for further studies to the function of coagulation factor IX. There is obvious heterogeneity in F9 gene mutation and missense mutation is still the main way of mutation, which are closely related to clinical features. DNA sequencing and linkage analysis are efficient methods for HB carriers and prenatal gene diagnosis.

Detection of alloantibodies against Factor VIII in plasma of patients with hemophilia A and its relationship with Factor VIIIC domain / 中国实验血液学杂志

This study was purposed to detect the alloantibodies against Factor VIII (FVIII) by ELISA for estimating the incidence of the alloantibodies against Factor VIII (FVIII) in patients with hemophilia A, and to investigate the relationship between factor VIIIC domain and alloantibodies. Total of 140 patients with hemophilia A and 80 normal controls were enrolled in this study, among them plasma FVIII level of 84 patients was less than 1%, plasma FVIII level of 34 patients was between 1% and 5%, and plasma FVIII level of 22 patients was more than 5%. All patients were treated with plasma-derived FVIII concentrate or plasma before. The ELISA plate was coated with McAb (SZ-132) against FVIII prepared in our laboratory, then human recombinant FVIII concentrates were applied. After incubation in room temperature for 2 hours, diluted plasma samples and HRP-conjugated goat anti-human IgG were added successively, finally A490 was recorded. The threshold of alloantibody of patient plasma was set as mean value>3 SD more than control. The plate was coated with antibody against His, then human recombinant FVIII-C1C2 prepared in our laboratory was added. After incubation in room temperature for 2 hours, diluted plasma samples and HRP-conjugated goat anti-human IgG were added successively, finally A490 were recorded. The threshold was set as the mean value>3 SD more than control. The results showed that the alloantibodies against FVIII were found in 56 patients (40%) by ELISA, and 82.1% (46/56) of this kind of alloantibody could interact with the C domain of FVIII. It is concluded that C domain of FVIII is one of the primary binding sites for the alloantibodies against FVIII in Chinese patients with hemophilia A.