0.98, P

Objeetive To explore change of platelet glycoproteins Ⅵ(GPⅥ)in patients with type 2 diabetes mellitus and its clinical significance.Methods The surface expression of platelet glycoprotein Ⅵ was determined by flow cytometry in 56 patients with type 2 diabetes mellitus and 61 normal individuals.Plasma GP Ⅵ concentration was measured by ELISA in 30 patients with type 2 diabetes mellitus.Platelet surface CD62P was analyzed by flow cytometry and HbA1c was determined in Datients with type 2 diabetes mellitus.Results The geometric mean fluorescence of platelet surface GPⅥ in Datients with type 2 diabetes mellitus was 93.66±35.24,which was significantly enhanced compared with normal subjects (62.83±24.2)(t=-4.927,P＜0.05).Nine patients were positive in plasma GPⅥ among 30 Datients with type 2 diabetes mellitus.Plasma GP Ⅵ concentrations in 9 positive patients were conversely correlated with platelet surface GPⅥ expression(r=-0.633,P＜0.05).However,there was no correlation between platelet Surface CD62P and plasma HbA1c concentrations, and the correlation coemcient is -0.333 and -0.417,respectively(P＞0.05).There was no correlation between platelet surface GP Ⅵ expression with C062P and HbA1c in patients with type 2 diabetes mellitus and the conrrelation coefficient is -0.009 and -0.217,respectively(P＞0.05).Conclusions GPⅥ expression on platelet surface is elevated in Datients with type 2 diabetes mellitus and the determination of platelet surface GPⅥ and plasma GPⅥ concennlation may helP to prognosticate the risk of thrombotic events and may play an important role in evaluating platelet activation in patients with type 2 diabetes mellitus.

Objective To establish a new method for monitoring aspirin response by platelet activated-release experiment.Methods The platelets in whole blood were activated by ARA,and the MPC was measured by hematology analyzer.Blood samples were drawn from five healthy volunteers for measuring MPC,LTAARA and platelet membrane CD62P expression.Blood samples were mixed thoroughly right after venipuncture. The concentration of ARA (0,0. 5,1.0,1.5,5.0 and 10. 0 mmol/L) and the time for platelet activation (5,10,20,30,40,50,60,70,80 and 90 min in 37℃ water bathe) were optimized to evaluate the stability (0,1,2 and 3 h after venipuncture) and reproducibility (MPC, LTAARA and platelet membrane CD62p were measured ten times and the CV was calculated). Platelets were mixed with acetylsalicylic acid at different concentrations in vitro in order to verify the validity for monitoring aspirin response. The percentage of CD62p positive platelets after activated by ARA was measured using flow cytometer with CD61-PerCP and CD62p-PE antibodies. The correlation between MPC and CD62P was determined. 100 patients without taking or stop taking aspirin more than 7 days and 93 patients who took aspirin at least 7 days were enrolled. Duplicate measurements of platelet function were performed using the change of MPC (ARA 0. 5 mmol/L) and LTA (ARA 0. 5 mmol/L) among two patient groups to evaluate the accuracy of the new method. Results Platelcts were completely activated by ARA at final concentration of 0. 5 mmol/L. MPC negatively correlated with platelet membrane CD62p(r=-0. 755,P<0. O1 ). MPC was stable for 30 minutes in 37 ℃ water bathe after ARA activation. The result of MPC was consistent at room temperature within 3 hours after blood collection. This method had good reproducibility. CV in batch using fresh whole blood was 1.35% and CV between batches using commercial control whole blood were 0. 71% and 0. 74%. With the concentration of acetylsalicylic acid increased (0-6. 95 μmol/L), MPC increased as CD62P decreased, which showed negative correlation (r=-0. 765 ,P <0. 01 ). The difference of MPC before and after ARA activation (ΔMPC) and MPC variance ratio of the group taking aspirin were ( 8. 2±8. 6) g/L and ( 3.4±3.6) %, and they were (37.4±10. 3 ) g/L and ( 15.7±4.0) % in control group, respectively.ΔMPC and MPC variance ratio showed significant difference between the two groups ( t=21. 522, 22. 409, all P < 0. 01 ). Area under the ROC curve for MPC variance ratio was 0. 992 with sensitivity of 96. 8% and specificity of 99.0% for monitoring the aspirin response using the cut-off of 8. 7%. MPC variance ratio had good correlation with LTAARA (r = 0. 720, P < 0. O1 ). Conclusions A new method for monitoring aspirin response by platelet activated-release experiment is established. It may replace LTAARA for routine clinical examination.

Direct oral anticoagulants,include direct thrombin inhibitor and direct factor Xa inhibitor.As the pharmacokinetics and pharmacodynamics of these drugs are known,their plasma concentration is not food-effective,and theoretically it is not necessary to monitor routinely.However, clinical practice in recent years has shown that anticoagulant effect of DOACs is required to evaluate in patients with thrombosis, major bleeding, emergency surgery, hepatic/renal dysfunction and other special situations.Recent studies have shown that routine coagulation assays such as activated partial thromboplastin time(APTT) and thrombin time(TT) can be used as laboratory screening tests for direct thrombin inhibitor dabigatran and prothrombin time(PT) can be used as laboratory screening test for direct factor Xa inhibitor rivaroxaban.DOAC′s quantitative measurements include dilute thrombin time(dTT),hemoclot thrombin inhibitor (HTI),ecarin clotting time (ECT) and ecarin chromogenic assay (ECA) for direct thrombin inhibitor and anti-FXa assay(rivaroxaban calibration) for rivaroxaban.Laboratories should establish their own monitoring range when performing these assays.

Manual microscopic differential of white blood cell has been challenged by multiparameter flow cytometry,using monoclonal antibodies to define the different leukocyte types.Compared with manual differential,flow cytometry method is more sensitive,specific,objective and has good repeatability.Recent studies demonstrated flow cytometric differential correlates well with manual microscopic method and has good clinical performance for blast and immature granulocyte.Meanwhile more leukocyte populations can be identified with flow cytometric method,such as lymphocyte subset and CD 16 + monocyte,thus helping in monitoring blast in acute leukemia,B lymphocyte proliferative disorder differential diagnosis and minimal residual disease.With the development and improvement of flow cytometric differential,it might be a candidate reference method of leukocyte differential,gradually applied in the routine work.

Objective To establish a method of competitive polymerase chain reaction combined with restrictive endonucleases to measure the methylation quantitatively in MDR1 promoter region.Methods One sense primer and two anti-sense primers were designed to amplify a fragment of MDR1 gene promoter region, which was located between -102 and +186 bp containing a methylation site. The internal reference DNA fragment, witch was less 30bp than target fragment was made by three times of polymerase chain reaction(PCR)using different anti-sense primer and then subcloned into the Pbluescriptsk+ plasmid. The genomic DNA digested by Hpa, a methylation-sensitive restrictive endonuclease and competitive internal reference DNA were competitively amplified for MDR1 promoter in the same tube by PCR. The PCR products were electrophoresed on agarose gel, stained by ethidium bromide and subjected to image analysis scanner. The amount of target fragment was calculated as following, the optical density ratio of target fragment to competitive internal reference fragment multiplied the amount of competitive internal reference DNA. The ratio of PCR products amplified from HpaⅡ digested DNA and undigested DNA was named the methylation rate.Results The genomic DNA serially diluted with optimal amount of competitive internal reference DNA were co-amplified for MDR1 promoter. The significant positive correlation between the ratio of two products and the amount of genomic DNA was demonstrated. The correlation coefficient was 0.992, P

Objective To express and purify recombinant human platelet glycoprotein Ⅵ extracellular domain and prepare the polyclonal antibody against it.Methods Human platelet glycoprotein Ⅵ extracellular domain fragment (123~268 residues) was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-3x.The recombinant plasmid was constructed and expressed in E.coli after induction with isopropyl-?-D-thiogalactopyranoside (IPTG).The fusion protein was identified by Western blot analysis after purification by affinity chromatography.Rabbits were immunized with the purified fusion protein, and the collected rabbit antiserum was evaluated by sandwich ELISA, Western blot and flow cytometry.Results The coding sequence of GPVI extracellular domain was successfully inserted into pGEX-3x.Sequencing result showed that the cloned gene was identical as reported.After induction, a Mr 42kd fusion protein was expressed and confirmed by Western blot, which was identical to that expected.The titers of the antisera were up to 1∶[KG-*2]128. Sandwich ELISA result demonstrated that the prepared antibody recognized GPVI in human platelet. Western blot and flow cytometry revealed that the prepared antibody reacted with GPVI of platelet lysate and the native GPVI on human platelet surface.Conclusions Using purified prokaryotic expressed the fusion protein as antigen, the specific antibody was elicited in the immunized animals. The prepared polyclonal antibodies react specially with GPVI on human platelet surface and can be used for further studies of GPVI.

Objective To establish and evaluate the new method of 8-color flow cytometry (8c-FCM) with two tube detecting bone marrow minimal residual disease (MRD) in B lineage acute lymphoblastic leukemia (B-ALL).MethodsThe MRD cells were analyzed by using two combinations of 8c-FCM antibody panels,gating with CD19/Side scatter(SSC),CD45/CD10 and CD34(or cTdT).Nalm-6 cell of B-ALL was mixed into normal marrow cells,with proportion of 10.00％,1.00％,0.10％,0.01％and 0.005％,and recovery test and reproducibility test were carried with 8c-FCM established to value its accuracy and precision of.Fluorescence intensity was detected on different time points after marked Nalm-6 by antibodies to evaluate the fluorescent stability of the antibodies.The immunophenotyping was analyzed in 39 bone marrow specimens,including 9 cases of normal control,9 cases of B-ALL primary or recurrent,21 cases of complete remission (CR) after chemotherapy or bone marrow transplantation ( BMT),to evaluate the detection of normal B lymphocyte lineage,leukemia cells of B-ALL,MRD cells of B-ALL using the 8c-FCM and compare it with 4c-FCM in being.ResultsThe method of two tube 8c-FCM with main antibodies of CD19,CD45 and CD10 to detect MRD of B-ALL was founded; CV ＜ 2.5％,average recovery rate was 95.81％,when the actual percent of Nalm-6 mixed into normal bone marrow≥0.10％,and the percent of Nalm-6 detected and actual was linear dependent ( r =0.99,P ＜ 0.05 ) ranged from 10.00％ - 0.01 ％,when 106 cells were acquired ; the sensitivity of the method established could reach 0.01％.The fluorescent intensity decreased along with the time after Nalm-6 cell marked,but less than 10％ in 24 hours.Using the antibody combinations and analyze strategy,the immunophenotye of B lymphocyte in normal bone marrow presented four sequential stages:Stage Ⅰ CD45low/CD10stro/CD20 -/CD38stro/CD34 + or cTdT +,Stage ⅡCD45 +/CD10 + / CD20 -/CD38stro/CD34+ or cTdT +,Stage Ⅲ CD45 +/CD10 +/CD20 -/CD38stro/CD34 -or cTdT-,Stage Ⅳ CD45stro/CD10 -/CD20 +/CD38low/CD34or cTdT.Antigen expressions of leukeamic cells of B-ALL primary or recurrent were different compared with control team; there were 5 cases with MRD positive in CR team,and the main antigen expression was consistent with the results from 4c-FCM.The range of the percent of MRD cells was 0.02％ - 5.42％ of the 5 cases of MRD positive.ConclusionsThe new method of two tube 8c-FCM established shows good reproducibility and high accuracy,and can identify normal B lymphocyte populations in bone marrow and regenerated B-precursors in CR cases with MRD cells;compared with 4c-FCM,the new method of two tube 8c-FCM with the fewer specimen is faster and efficient to diagnose MRD of B-ALL.

Objective To study the mechanism of some vegetables inhibiting human platelet aggregation function.Methods Some vegetable juice was mixed(tomato juice as positive control)with platelet-rich plasma(PRP),and maximum ratio of platelet aggregation was measured after induction by agonists on the aggregometer.The expression levels of platelet activation marker were detected,including fibrinogen receptor(Fib-R),P-selectin(CD62P),and the combination amount of fibrinogen on the surface of platelet by flow cytometry to validate the inhibitory effect of platelet aggregation by animal tests.Ordinary white rabbits were randomly divided into five groups,each group was fed with saline,cooked tomato juice,blanched garlic leaves juice,Chinese cabbage juice or spinach juice,respectively.And the maximum ratio of rabbit platelet aggregation in different time were observed.Results Cooked vegetable juice couldn't inhibit human platelet aggregation induced by AA,but blanched garlic leaves juice,Chinese cabbage juice and spinach juice could inhibit human platelet aggregation obviously induced by ADP.collagen or epinephrine.The inhibition intensity of platelet aggregation increased markedly along with the increase of the concentration of the cooked vegetables juice.Cooked juice of blanched garlic leaves,Chinese cabbage,spinach could not inhibit the expression of Fib-R and CD62P,Whereas they were able to significantly decrease the amount of Fib-R and fibrinogen binding. ADP-induced rabbit platelet aggregation ratios in the group fed with cooked juice of blanched garlic leaves,Chinese cabbage or spinach were significantly lower than control group after 3,5,8 weeks,respectively.The inhibition ratios of the platelet aggregation in cooked Chinese cabbage juice group were 45.7%,53.6%,48.5% after 3,5,8 weeks,respectively.Cooked juice of blanched garlic leaves group were 10.7%,66.7%,46.3%,respectively. Cooked spinach juice group were 8.7%,21.0%,42.6%,respectively.The collagen-induced rabbit platelet aggregation ratios in the groups fed with cooked juice of blanched garlic leaves or Chinese cabbage were significantly lower than control group after 5 and 8 weeks respectively. The inhibition ratios of the platelet aggregation in cooked Chinese cabbage juice group were 54.9%,56.3%after 5 and 8 weeks,respectively. Cooked juice of blanched garlic leaves group were 28.4%and 86.7%,respectively. Cooked tomato juice could not inhibit rabbit platelet aggregation in 8 weeks. Conclusions After induced by ADP or collagen,cooked juice of blanched garlic leaves,Chinese cabbage and spinach could significantly decrease the amount of Fib-R and fibrinogen binding,and inhibit platelet aggregation function significantly.It may have potential application of therapy or prlevention of thrombotic diseases.

Objective To investigate the correlation of prognosis with the immunophenotype in acute myeloid leukemia(AML)patients.Methods Immunophenotyping was performed in 131 patients with AML by mtdtieolor flow cytometry.Correlation of immunophenotype with other laboratory parameters such as initial white blood cell count(WBC),platelet count(PLT),hemoglobin(Hb),and the complete remission(CR)ratio was analyzed.Results In these AML patients,myeloid antigens CDl3,CD33 and myeloperoxidase(MPO)were more highly expressed than other antigens.Expression of CD34 and HLA-DR were lower in acute promyelocytic leukemia(M3)subtype.The expression of lymphocyte antigen CD19 and CD7 were the highest.CD7 expression was associated with age(t=-2.27,P＜0.05).CD14 was associated with initial WBC(Z=-2.284,P＜0.05).The overall CR ratio was 56.5%among all patients.CD34positive patients had a significantly lower CR rato(45.1%),compared with the CD34 negative patients whose CR ratio was 75.6%(x2=11.524,P=0.001).The CR ratio was significantly lower in cases expressing both CD34 and HLA-DR(74 patients with CR rate 41.9%)than in cases expressing only CD34 or HLA-DR(38 patients with CR rate 78.9%)and both negative(19 patients with CR rate 68.4%)(Z=-3.492,P＜0.01).However,other antigens,including CD7,CD19,CD13,CD33,CD38,CD15,CD64,CD14 and MPO had no significant association with CR ratio.Logistic regression analysis showed that age(60 years or older),initial WBC(more than 50 x 109/L),PLT(more than 30 x 109/L),Hb(less than 60g/L)and CD34 were independent adverse factors for achieving CR.Conclusions In AML patients,age,initial WBC,PLT,Hb and CD34 are associated with CR ratio.Detection of immunophenotype may help to estimate prognosis of patients with AML and guide the treatment of AML.