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.

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.

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.

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.

OBJECTIVE:To optimize the preparation technology of Sinomenine topical paste. METHODS:Using“initial vis-cous force”,“holding viscous force”and“peeling strength”as index,heating and stirring time (A,h),heating temperature (B,℃) and the sequence of adding composition (softening agent,blank matrix and sinomenine)(C) as influential factors,the preparation technology of Sinomenine topical paste was optimized by orthogonal test and verified. At the same time,the content of sinomenine was determined by HPLC method. RESULTS:The optimal preparation technology of Sinomenine topical pasta was as follows as adding blank matrix and sinomenine,and then adding softening agent,heating at 80 ℃,stirring for 1 h. In verification test,RSD of comprehensive score for 3 batches of samples were 2.09%(n=3);average contents of samples were 6.7 mg/g, which was in line with the requirement of ≥6.0 mg/g. CONCLUSIONS:The optimal preparation technology of Sinomenine topical pasta is reasonable,stable and feasible. The paste shows good adhesiveness and is qualified in content.

Objective To evaluate the clinical performance of AQT90 FLEX,a novel time-resolved fluorescence based point-of-care test (POCT) for quantification of D-dimer in elderly patients.Methods The method from Quantitative D-dimer assay (WS/T 477-2015) for testing equipment performance was used as a reference to evaluate the clinical performance of AQT90 FLEX.The correlation was compared between testing results of D-dimer using the AQT90 immune-assay analyzer and those using the ACL TOP coagulation analyzer.Results At high concentration of D-dimer,the within-run precision coefficient of variation (CV)was 2.619％,and at low concentration of D-dimer,the within-run precision CV was 2.767％.The pollution-carrying rate was 0.12％.The measured data from AQT90 and ACL TOP had a correlation coefficient of r =0.9491 (P ＜ 0.01).The equation of the line of best fit for D-dimer with which all AQT90 results can be adapted to the ACL TOP was:AQT90 =2.52 ACL TOP + 0.15.The number from the equation was slightly greater in female than that in male,and it was also increased in elderly.Conclusions The AQT90 FLEX had rational precision and linearity in determination of concentration.There was a high agreement between the testing results from AQT90 and those from ACL TOP.It was recommended to use a slope of 2.52 and an intercept of 0.15 to adjust the D-dimer values of the ACL TOP to the AQT90 FLEX assay systems.POCT for D-dimer by AQT90 FLEX raises feasibility for use in elderly patients.

Objective To compare the consistency of thrombelastography (TEG) and light transmittance aggregometry (LTA) in monitoring the antiplatelet therapy of the patients with coronary heart disease (CHD) after percutaneous coronary intervention (PCI),and observe the changes of mean platelet volume (MPV) of the patients treated with aspirin and clopidogrel after PCI.Methods A total of 177 patients undergoing PCI and the treatment of aspirin and clopidogrel in Peking University First Hospital during March 2014 and May 2015 were enrolled in the study.Their adenosine diphosphate (ADP) or arachidonic acid (AA) induced platelet inhibition rates determined by TEG,MPV before and after antiplatelet therapy,and the maximum platelet aggregation rates measured by LTA from 99 patients were retrospectively analyzed.Results There was no any correlation between the maximum aggregation rates measured by LTA and the platelet inhibition rates determined by TEG regardless of using ADP or AA as agonist (all P ＞ 0.05).The detection rates of clopidogrel hyporesponsiveness determined by LTA and TEG were 30.3％ and 45.5％,respectively,while those of aspirin hyporesponsiveness were 19.2％ and 31.3％,respectively.The detection rate of hyporesponsiveness determined by LTA was significant lower than that by TEG (P ＜ 0.05).The MPVs after antiplatelet therapy were significant lower than that before treatment (all P ＜ 0.01) regardless of clopidogrel hyporesponsive or sensitive and aspirin hyporesponsive or sensitive.The MPVs in clopidogrel hyporesponsive group before and after treatment were significantly lower than that in clopidogrel sensitive group (all P ＜ 0.05).The PLT counts in clopidogrel or aspirin hyporesponsive groups after treatment were significantly higher than that before treatment (all P ＜ 0.05).Conclusion There is poor correlation between LTA and TEG.It should be noted that the incidence rate of antiplatelet drug hyporesponsiveness is high in clinical practice.The MPVs of the patients significantly decrease after antiplatelet therapy.The patients with a significant increase of PLT after antiplatelet therapy are more likely to become drug hyporesponsiveness,while the patients with lower MPV are more likely to have clopidogrel hyporesponsiveness.