Light transmittance aggregometry with non-adjusted PRP is more suitable for clinical laboratory =monitoring antiplatelet therapy / 中华检验医学杂志

Objective In this study,maximum platelet aggregation rate of Light transmittance aggregometry (LTA) for coronary heart disease(CHD) patients taking antiplatelet drug and patients without antiplatelet therapy was measured in non-adjusted and platelet count-adjusted platelet-rich plasma (PRP).The aim of this study is to compare which method is superior in evaluation of antiplatelet drug effect.Methods This is a methodology comparative research.560 CHD outpatients and inpatients that visited Beijing Anzhen Hospital of the Capital University of Medical Sciences from May to June,2012 were chosen,who were treated with aspirin monotherapy,or patients on combination therapy with aspirin and clopidogrel,as well as patients without antiplatelet therapy.LTA was performed in non-adjusted (improved method) and platelet count (200 × 109/L)-adjusted PRP (original method),using 6 μmol/L adenosine diphosphate (ADP) and 0.5 mmol/L arachidonic acid (ARA) as agonists.The maximum aggregation rates in 5 min were detected,and consistency and differences of the two methods were compared.Results There is no statistically significant correlation between maximum aggregation rate and platelet count in PRP with 6 μmol/L ADP or 0.5 mmol/L ARA as agonists in all subgroups including aspirin monotherapy,combination therapy with aspirin and clopidogrel and patients without antiplatelet therapy (-0.21 ≤ r ≤0.111,P ＞ 0.05).The maximum aggregation rate using ADP as agonists in original method is decreased compared with improved method,there is statistically significant difference in all subgroups including patients without antiplatelet therapy,aspirin monotherapy,combination therapy with aspirin and clopidogrel less than one week and more than one week.The variability of platelet aggregation rate using ADP as agonists with improved method is lower than that with original method in all subgroups.Yet the maximum aggregation rates using ADP as agonists with improved method and original method correlate well with each other in all subgroups (r =0.78,0.73,0.40,0.71,P ＜0.01).In the subgroup of subjects without antiplatelet therapy using ARA as agonist,platelet aggregation rate is decreased in original method compared with improved method,there is statistically significant difference,and the variability of the aggregation rate with improved method is also lower than that with original method,ranging from 62％-98％ relative to 5％-89％.The decrease of aggregation rate using ARA as agonist for patients taking antiplatelet drug compared with patients without antiplatelet therapy can be detected both with improved method and original method.Conclusion Non-adjusted PRP in LTA is more convenient and time-saving,and it also means less effects on platelet in vitro.Therefore,non-adjusted PRP is more suitable for monitoring efficacy of antiplatelet therapy in clinical laboratory.

A new platelet activated-release test for monitoring aspirin response / 中华检验医学杂志

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.

Risk factors for deep vein thrombosis in lower extremity after hip or knee arthroplasty and hip fracture internal fixation / 中华麻醉学杂志

Objective To determine the factors contributing to the development of deep vein thrombosis (DVT) in the lower extremity in patients after hip or knee arthroplasty and hip fracture internal fixation.Methods One hundred and forty-seven consecutive patients receiving hip or knee arthroplasty and hip fracture internal fixation from 2004 to 2005 were included in this study. Their age ranged from 33 to 92 years. Duplex color ultrasonic inspection was performed on veins of the bilateral lower extremities before operation and 2 weeks after operation for detection of DVT. The patients were divided into a DVT group and a DVT-free group based on the development of DVT after operation. Detailed perioperative clinical information about the patients, surgery and anesthesia was collected.Results Lower extremity DVT was found in 42.2% of the patients after operation, while the incidence of proximal DVT was 2.7%. Compared with the DVT-free group, the usage rate and dosage of ephedrine increased significantly, the duration of anesthesia was significantly longer, and the white blood cell count (WBC) on the 1st postoperative day and the highest WBC count were significantly higher in the DVT group(P＜0.05). Logistic regression analysis indicated that the above factors were closely related to DVT.Conclusion Duration of anesthesia ＞ 3 h, ephedrine administration and a marked increase in WBC count after operation are the risk factors for DVT in the lower extremities in patients after hip or knee arthroplasty and hip fracture internal fixation.

Prognostic relevance of immunophenotyping in 131 patients with acute myeloid leukemia / 中华检验医学杂志

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.

Flow cytometric immnno-bead array assay for detection of platelet-specific autoantibodies / 中华检验医学杂志

Objective To establish a new flow cytometric immuno-bead array assay (FCIBA)to detect several platelet-specific autoantibodies simultaneously in a single serum sample.Methods A series of beads of different red fluorescent intensity were used for coating with different anti-platelet membrane protein monoclonal antibodies(anti-GP Ⅱb/a,anti-GP Ⅰ a/Ⅱ a,anti-GP Ⅳ,anti-GP Ⅰ b/Ⅸ and anti-HLAABC)to detect five platelet-specific autoantibodies in serum. The beads captured platelet antigenautoantiboay complex.Subsequently,different platelet-specific autoantibodies in a patient serum can be detected simultaneously by the flow cytometry.In addition,we evaluated the new FCIBA,and compared its results with modified antigen capture ELISA method(MACE).The new FCIBA was used to detect five platelet-specific autoantibodies in serums of autoimmune thrombocytopenic purpura(AITP)and nonautoimmune thromboeytopenic purpura patients.Results The new FCIBA can be used to detect five plateletspecific autoantibodies simultaneously(Anti-GP Ⅱ b/Ⅲ a,Anti-GP Ⅰ a/Ⅱ a,Anti-GP Ⅳ,Anti-GP Ⅰ b/Ⅸand Anti-HLA-ABC).The coefficient of variation(CV)of intra-repetition are 4.82%,6.09%,5.04%,5.73%and 5.30%,respectively.The dilution test results are in good logarithm linearity which are 0.997 2,0.996 6,0.998 8,0.996 5 and 0.998 2,respectively. The resuhs of the new FCIBA are highly correlated with those with MACE method,and the coefficient correlation were 0.928 9,0.922 4,0.889 4,0.910 0 and 0.913 4,respectively(P＜0.01).51.69%samples of AITP patients show positive for platelet-specific autoantibodies as detected by the new FCIBA.Among the AITP patients,the positivity of specific autoantibodies for anti-GPⅡb/ Ⅲ a,anti-GP Ⅰ a/Ⅱ a,anti-GP Ⅳ,anti-GP Ⅰb/Ⅸ and anti-HLA-ABC were 40.82%,24.45%,19.39%,32.65%and 17.35%.Among the 40 non-autoimmune thrombocytopenic purpura patients,none of platelet-specific autoantibodies in serum samples can be detected.Conclusion A new FCIBA is established successfully to detect five platelet-specific autoantibodies coefficient correlation.

Screening of some vegetables that inhibit platelet aggregation and their mechanism:a preliminary study / 中华检验医学杂志

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.

Research on whole blood control materials for lymphocyte subset counting by flow cytometry / 中华检验医学杂志

Objective To research on a whole blood control material for lymphocyte subset counting by flow cytometry(FCM).Methods To detect lymphocyte subset in whole blood with different preservers by flow cytometric multi-color analysis.Results whole blood control material for lymphocyte subset counting by FCM was prepared.In 2-8℃ refrigerator, the light scatter and CD45 of leukocytes of whole blood control were stable in 72 days. The cluster of lymphocyte, monocyte, neutrophil in plot were separated easily from debris. The lymphocyte subset of whole blood control can be counted by FSC/SSC or CD45/SSC gating. The variation of lymphocyte subset count was less in different preserving day. The coefficient of variation (CV) of lymphocyte subset count was less than 6.5% in our laboratory and less than 13% in external quality assessment among 56 laboratories in China.Conclusion The whole blood control prepared by us can be used for internal quality control and external quality assessment in lymphocyte subset counting by FCM, it is very important signification to ensure the quality and accuracy of lymphocyte subset count.

Incidence of deep venous thrombosis (DVT) in patients undergoing thoracotomy and changes of hemostasis / 北京大学学报(医学版)

Objective:To survey the incidence of deep venous thrombosis (DVT) in high risk patients undergoing thoracotomy and observe the changes of hemostatic activity. Methods: Fifty-two consecutive patients (ages that ranged from 35 to 79, 34 men and 18 women) with lung or esophagus cancer were enrolled into this prospective trial. The patients included underwent major thoracic surgery from February 2003 to April 2003. Bilateral lower extremity duplex ultrasonography for DVT screening was performed 3-10 days post surgery in all 52 patients and 57 matched clinic normal controls. Venous blood was collected to determine fibrinogen(FIB), D-dimer(D-D), plasminogen activator inhibitor (PAI), antithrombin (AT), thrombin antithrombin complex (TAT), prothrombin time (PT),international normalized ratio(INR), and activated partial thromboplastin time (APTT) immediately before surgery, the third and tenth days postoperatively. No patient had a prior thromboembotic history. Risk factors for DVT were evaluated. Results: Of the 52 patients, 28 (53.8%) had an acute postoperative DVT detected in the calf. One patient died of suspected pulmonary embolism postoperatively. Plasma levels of FIB and D-D increased significantly up to 7 d after operation. AT level decreased significantly 3 d after operation and returned to normal 7 d latter. TAT increased significantly 3 d post operation and decreased to normal on day 7. PAI level was lowered 3 d after surgery, but increased significantly on day 7 compared with that on day 3. With the addition of risk factors related to thrombosis, the incidence of DVT was increased correspondingly. Conclusion: Of the patients undergoing major thoracic surgery,53.8% of them had a postoperative DVT by postoperative screening duplex ultrasound. In Chinese population, incidence of DVT appears to be high without prophylaxis, which is similar to other reports of westerners. These patients had a number of risk factors for the development of DVT, which include older age, overweight, hypertension, diabetes, and history of thromboembolism, etc. Prophylactic measures should be taken against postoperative venous thromboembolism in major thoracic surgery with high risk, including early mobilization, anticoagulant therapy with heparins, and intermittent pneumatic compression (IPC).

Helping to early diagnosis of SARS by flow cytometric three color absolute count of T lymphocyte subsets in blood / 中华检验医学杂志

Objective To investigate the changes of T lymphocyte subsets of severe acute respiratory syndrome(SARS) patients during acute phase and recovery stage and study its clinical significance in early diagnosis of SARS.Methods T lymphocyte subsets of 40 patients of SARS and 22 cases of SARS recovery stage were detected by flow cytometry.Results Compared with normal group, T lymphocyte, Th and Ts cell counts of SARS patients decreased obviously( P =0.013, P 0.05). Conclusion The cellular immunity of SARS patients were badly damaged during acute phase. Their T lymphocyte, Th and Ts cell counts decreased obviously. This could be a guideline of early diagnosis of SARS. The counts of T lymphocyte subsets of SARS patients can return to normal after they were cured.

Laboratory diagnosis of leukocyte myeloperoxidase deficiency / 中华检验医学杂志

Objective To study the pathological characteristics of leukocyte myeloperoxidase (MPO) deficiency and clinical laboratory diagnostic strategy for this disease. Methods The Bayer ADVIA 120 blood cell analyzer differentiate leukocyte and detect myeloperoxidase index (MPXI) . The leukocyte morphology and differentiating count on blood smear by Wright’s stain were finished. The peroxidase positive cell percentage and score were counted by peroxidase stain. The expressive levels of leukocyte MPO antigen were measured by flow cytometric monoclonal antibodies method.Results The prevalence of mild neutrophil MPO deficiencies was 2.61%, and partial neutrophil MPO deficiencies was 0.39% in 5 761 in-patients. The significant changes in the dot plot from ADVIA 120 blood cell analyzer were seen in 3 patients with MPO deficiencies, and MPXI,MPO activities and MPO antigen levels of the patient’s neutrophils and monocytes decteased remarkably,but there were normal levels in eosinophils.Conclusion Neutrophil MPO deficiencies were not a rare disorder,but the mild deficiency cases were seen usually.The low activity of MPO is an important evidence for MPO deficiencies diagnosis.The Bayer ADVIA 120 blood cell analyzer can be used simply and fleetly for screening MPO deficiencies in routine hematology laboratory.The peroxidase stain of blood smear is an improtant diagnostic method for MPO deficiencies.

Study on the action of three kinds of anti-platelet drugs for in vitro inhibiting platelet activation / 中华检验医学杂志

Objective To study the action of aspirin, ticlopidine and cilostazol in inhibiting platelet activation in vitro. Methods Three kinds of anti platelet drugs, which inhibited the expression of fibrinogen receptor (FIB R) and P selectin (CD62P) on activated platelets surface in vitro, were analyzed by tri color flow cytometry. Results Three kinds of anti platelet drugs inhibited the expression of FIB R and CD62P on ADP activating platelet membrane surface differently. Cilostazol could inhibit the expression of FIB R and CD62P of the platelets markedly. Ticlopidine could inhibit the FIB R expression markedly, but CD62P indistinctly. Aspirin could not inhibit the expression of either FIB R and CD62P on ADP activating platelets. Conclusion Cilostazol is a strong anti platelet drug as compared with aspirin and ticlopidine. It could not only inhibit FIB R expression of activated platelets, which can prevent platelet aggregation, but also decrease the release reaction and procoagulative activities of activated platelets.

Effects of graduated compression stocking and intermittent pneumatic compression on the incidence of deep vein thrombosis after major surgery / 中华麻醉学杂志

60 yr, hypertension, diabetes mellitus, smoking, long term inactivity etc), and duration of operation. The incidence of DVT was 49.3% in control group, 30.0% in group CS alone, 15.0% in group CS + IPC1 and 23.3% in group CS + IPC2 (P

Application of Reptilase in the Radical Correction of Malignant Tumor of Urinary System and Its Effects on the Deep Venous Thrombosis of Lower Limbs / 中国药房

0.05).There were no significant differences in thrombus-associated indexes except a slight change of plasminogen activator antigen.CONCLUSIONS:The incidence of DVT after tumor removal of urinary system is mainly associated with malignant tumor itself, operation wound, postoperative immobilization and the risk factors of thrombus,the administration of reptilase does not increase the incidence of DVT,but might promote the fibrinolytic activity of patients.

THE STUDY OF REVERSAL RESISTANCE EFFECT AND ITS MECHANISM OF BERBAMINE IN MCF7/ADR CELLS / 解剖学报

Objective To explore the efficacy of calmodulin antagonist berbamine(BBM)on multidrug resistance(MDR)reversal and its mechanism. Methods Human breast cancer cell line MCF7 and its adriamycin-resistant counterpart MCF7/ADR were used in the study.The cells were cultured with ADR and different concentration of BBM. MTT assay was used to analyze the effect of BBM on cell growth inhibition.According to the MTT assay,the 50% inhibitory concentration(IC 50 ),the multiples of drug resistance and increased sensitivity of ADR were calculated.The concentration of intracellular ADR and expression level of P-glycoprotein(P-gp)were detected by flowcytometry(FCM).The mRNA expression level of mdr1 gene was detected by semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR)with ?-actin as internal reference. Results The IC 50 of ADR in MCF7 and MCF7/ADR cells were(0.98?0.06)?mol/L and(101.20?5.72)?mol/L,respectively.The resistant multiple of MCF/ADR cells to ADR was 103 folds higher than that of MCF7 cells.BBM increased the chemo-sensitivity of ADR in MCF7/ADR cells with dose-dependent relationship,i.e.when 5*!?mol/L ,10*!?mol/L and 20*!?mol/L BBM was added into the culture the chemo-sensitivity of ADR was increased to 2.76,5.88,and 28.26 folds(P