Measurement agreement of 25-hydroxyvitamin D results derived from Roche immunoassay and liquid chromatography tandem mass spectrometry / 中华检验医学杂志

Objective:To evaluate the measurement agreement of Roche 25(OH)D immunoassay(evaluation method) with LC-MS/MS (reference method).Methods:A total of 909 residual serum samples from routine health check participants were collected from May to June in 2019. 25(OH)D concentrations were measured by evaluation method and LC-MS/MS, respectively. Passing-bablok regression, intraclass correlation coefficient (ICC), Bland Altman plots and Kappa test were used to analyze the consistency and bias on the results derived from the two measurement methods.Results:The 25(OH)D concentration derived from evaluation method was significantly different from those from LC-MS/MS method ( P<0.001). Slope of regression for evaluation method and LC-MS/MS was 0.962(95% CI 0.919-1.007), while intercept was -0.185 (95% CI -1.191-0.745). The ICC was 0.765 (95% CI 0.735-0.792). Altman plot showed that the average deviation between evaluation method and LC-MS/MS was -0.902 ng/ml (0.300%). The coincidence rate of evaluation method′s judgment of vitamin D sufficiency, insufficiency and deficiency with LC-MS/MS was 83.39%, and the weighted Kappa values was 0.790. Conclusion:Roche automatic 25(OH)D immunoassay shows acceptable correlation and agreement with LC-MS/MS, however, it is to note that the deviation between immunoassay and LC-MS/MS may lead to wrong judgment of vitamin D nutritional status. It is recommended that each laboratory should establish own corresponding reference values for 25(OH)D concentrations derived from these two methods.

Clonal Relatedness and Antimicrobial Susceptibility Profiling of Acinetobacter Baumannii Isolates from Senile Patients / 现代检验医学杂志

Objective To investigate the clonal relatedness of A.baumannii isolates from senile patients by conducting cross-sectional and longitudinal studies on antimicrobial susceptibility profiling and genomic diversity.Methods Cross-sectional study was done among the 170 non-repetitive A.baumannii isolates which were collected from senile patients during two years.Longitudinal study was conducted among 77 A.baumannii collected from 8 senile patients within longtime hospitalization.Results 75.3 ％ of the 170 isolates were non-susceptible to carbapenems,and the phenotype were XDR or MDR which spread evenly all over the senile wards.The isolates belonged to 36 pulsotypes determined by PFGE.Groups Ⅰ (contain119 isolates) were major epidemic strains,which were CRAB with XDR phenotype.In longitudinal study,comparison of pulsotypes was performed for each patient and all isolates were clustered into group Ⅰ except one isolate.All the 77 isolates were XDR.Conclusion Extensive drug-resistance of A.baumannii was a serious problem in the gerontal wards.Clone dissemina tion was the most important style of XDR strains spread.Horizontal infection control measures to interrupt person-to-person transmission should be reinforced to reduce the further spread of XDR A.baumannii.

Investigation of detection methods for aggregation function of platelets / 国际检验医学杂志

Objective To adopt 5 kinds of platelets aggregation function analyzer to explore and study the reliable method for detecting the platelets aggregation function and accurate parameters .Methods The platelets aggregation function in the control group and the single clopidogrel group was simultaneously detected by utilizing the ADP induced light turbidimetric platelet aggre‐gation analyzer (LTA) test ,flow cytometry for PAC‐1(receptor of Fg ,Ca2+ ,GPⅡb/Ⅲa forming complex) and CD62p(P selectin) activation percentage detection test ,Innova PL‐11 for platelets analysis test ,VerifyNow anti‐platelet therapy monitoring system and thrombelastogram(TEG) .Results (1)The 6‐parameter differences of ADP% ,PAC‐1 and CD62p receptor activation percentage ac‐tivated by ADP ,MAR% ,INHI% ,ADP induced MA value(mm) detected by TEG had statistical differences between the control group and the case group(P0 .05) .(2)ADP% was positively correlated with (100‐INHI)% ,MAR% ,ADP activated CD62p and PAC‐1 receptor activation percentage(r=0 .565 ,0 .939 ,0 .769 ,0 .583 ,P<0 .05 );while which had no correlation with ADP induced platelets aggregation value(% Agg) detected by TEG(r=0 .794 ,0 .715 ,0 .889) .Conclusion (1)Clopidogre has the anti‐platelets effect .(2)LTA is easily operating ,cheap and stable in detection results ,has high popularizing rate in hospitals and is the first option method for clinically monitoring the platelets aggregation function .

CD62pUsing Light Transmission Aggregometry and Flow Cytometry to Examine the Effect of Clopidogrel / 现代检验医学杂志

Objective To detect clopidogrel effect with light transmission aggregometry (LTA)and flow cytometry (FC). Methods ①Venous blood samples were taken from 71 inpatient with acute corotary syndrome (ACS)in PLA General Hos-pital,including unstable anqina,ST segment elevation myocardial infarction and non ST segment elevation myocardial infarc-tion (46 males,25 females)by random number table from June 2011 to March 2012,whose average age was 69(57~92).②All of them were served 160 mg aspirin and 300 mg clopidogrel after they were in hospital in the beginning,and then served with 75 mg/d clopidogrel for 6 months.On some day,firstly,they were required withdrawing drug for 10 days,and then ve-nous blood samples were separately taken from them before their served-clopidogrel again and their severd-clopidogrel 2 hours later.③The samples were assayed with LTA and FC simultaneously and the platelet aggregation rates before served-clopidogrel (ADPLTA-before serving ),platelet aggregation rates after served-clopidogrel (ADPLTA-after serving ),inhibition rates (ADPLTA-INDU ),PAC-1 activity percentage before served-clopidogrel (PAC-1 before serving ),PAC-1 activity percentage after served-clopidogrel (PAC-1 after serving ),inhibition rates (PAC-1 INHI ),CD62p activity percentage before served-clopidogrel (CD62pbefore serving ),CD62pactivity percentage after served-clopidogrel (CD62pafter serving ),inhibition rates (CD62pINHI )weregotten.All volunteers were signed informed consents and the experiment was approved by the hospital ethics committee.Re-sults ①The paired samples t-test was (t=-2.082,P =0.041)between ADPLTA-before serving (0%~97%)and ADPLTA-after serving (12%~97%),the paired samples t-test was (t = 3.663,P < 0.01)between PAC-1 before serving (15.1% ~ 78.9%)and PAC-1 after serving (14.5% ~ 78.3%);the paired samples t-test was (t = 2.082 and P = 0.041)between CD62pbefore serving (1.5% ~80.8%)and CD62pafter serving (1.4%~41.4%).②The pearson coeffcient correlation results were:ADPLTA-INDU (0%~28.2%) and PAC-1 INHI (0.6%~ 9.1%)(r = 0.297,P = 0.012);ADPLTA-INDU (0% ~ 28.2%)and CD62pINHI (0.1% ~ 48.5%)(r =0.220,P =0.065);PAC-1 INHI (0.6%~9.1%)and CD62pINHI (0.1%~48.5%)(r=0.736,P <0.001).Conclusion Because the correlation was bad between the inhibition rates of clopidogrel detected by FC and them by LTA,FC didn’t apply to clin-ical routine examination of the platelet aggregation.But it could be used to scientific researchs and auxiliary confirmation of routine examination results.

Investigation of the Antiplatelet Function of Clopidogrel by Thrombelastography and Light Transmission Aggregometry / 现代检验医学杂志

Objective To detect the antiplatelet function of clopidogel by Thrombelastography (TEG)and light transmmi-tance aggregometry (LTA)and analyze efficiency of two methods.Methods ①Venous blood samples were taken from 48 outparients and inpatient with acute corotary syndrome (ACS)in PLA General Hospital,including unstable anqina,ST seg-ment elevation myocardial infarction and non ST segment elevation myocardial infarction (32 males,16 females)by random number table from March to July 2011,whose average age was 73 (62~90).②All of them were served 160 mg aspirin and 300 mg clopidogrel after they were in hospital at first time,and then served with 75 mg/d clopidogrel for 9 months.After that,venous blood samples were drew from them who were served clopidogrel 2 hours later.③Platelet aggregation function was assayed with LTA and TEG.All volunteers were signed informed consent and the experiment was approved by the hos-pital ethics committee.Results The platelet aggregation rate detected with LTA induced by adenosine diphosphate (ADPL-TA-INDU)was 40~80% in 41 individuals (85.4%),>80% in 7 individuals (14.6%),coefficient of variation (CV)was 0.19.The inhibition ration of platelet function (INHI)detected with TEG (ADPTEG-INHI)was 10~60% in 40 individu-als (83.3%),60 in 6 individuals (12.5%),CV was 0.54.The sum of ADPLTA-INDU and ADPTEG-INHI (ADPLTA-INDU+ADPTEG-INHI)tended to a constant (Mean=98%,SD=12.1).There was no significant indifference between every (ADPLTA-INDU+ADPTEG-INHI)and their Mean (98%)by paired t-test.The ADPLTA-INDU was negative correlated with ADPTEG-INHI (r=-0.75,P<0.01).Conclusion The effect of clopidogrel was mild and the efficiency of TEG and LTA was similar in detecting the antiplatelet function of clopidogrel,but the preci-sion of TEG was lower than LTA.

Evaluation of PL-11 in short-term antiplatelet therapy monitoring / 中华检验医学杂志

Objective To evaluate a new platelet function analyzer PL-11 with three major platelet function methods.Methods A randomized controlled trial was adopted.Totally 20 healthy young students from People's Liberation Army Medical School were enrolled in the study during July and August in 2013.Subjects were treated with aspirin or clopidogrel and the platelet function was measured by using of PL-11,as well as light transmittance aggregometry (LTA),VerifyNow and thromboelastography (TEG).Results When monitor aspirin response,correlations between arachidonic acid (AA) induced PL-11 and other methods were:LTA,0.614; VerifyNow,0.829; TEG,0.697,respectively.Biases between AA induced PL-1 1 and LTA were 1.94％ at baseline and 24.02％ during aspirin treatment.Cut-off value of aspirin response tested with AA induced PL-11 was 33.3％.When monitor clopidogrel response,correlations between adenosine piphosphate (ADP) induced PL-11 and other methods were:LTA,0.767; VerifyNow,0.851; TEG,0.608.Biases between ADP induced PL-11 and LTA were 3.01％ at baseline and 6.56％ during clopidogrel therapy.Cut-off value of clopidogrel response tested with ADP induced PL-1 1 was 66％.Conclusion Results of different platelet function testing methods were not equally effective.PL-11 has a high capability when monitoring short aspirin or clopidogrel response.

Clinical and cytomorphological features of bone marrow metastasis of non-hematological malignant carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze the clinical manifestations and hematologic parameters and observe the cytomorphological features of metastatic tumors in the bone marrow originating from different primary sites.</p><p><b>METHODS</b>The clinical data of 77 patients with bone marrow metasta tumors admitted between 2009 and 2014 between 2009 and 2014 in General Hospital of PLA were studied retrospectively to analyze the indications of laboratory examinations (hematological laboratory tests, tumor markers, peripheral blood films, and bone marrow aspirates).</p><p><b>RESULTS</b>Of the 77 patients analyzed, 64.9% were over 50 years of age. The most common clinical characteristics were bone pain (65%), anemia with thrombocytopenia (63.6%) and leukoerythroblastic reaction (61%). The hematological abnormalities included elevation of ESR, ALP, LDH, tumor markers, and hypoproteinemia. Cytological examination of bone marrow aspiration samples revealed different morphological characteristics of the metastatic cells from different primary sites; in most of the cases, scattered or clustered metastatic cells and degenerative tumor cells were found on the edge of the bone marrow smears.</p><p><b>CONCLUSION</b>Detection of the primary tumor site is difficult by cytological examination of bone marrow aspiration samples, but the cytological findings can be of value in the diagnosis of neuroblastoma, small cell lung cancer and gastric cancer (signet ring cell carcinoma). A definite diagnosis of bone marrow metastatic tumor relies on a combined evaluation of the disease history, clinical symptoms and laboratory findings.</p>

The technology of morphological analysis using blood cell analyzer and microscopic screening examination / 中华检验医学杂志

In this paper,the technology development process of morphological analysis was reviewed based on the blood cell analyzers in recent 40 years.The clinical values,advantages and limitations using blood cell analyzer to classify the white blood cell in different development stages were illuminated.It was evident that the manual microscopic examination is still a necessary way for clinical diagnosis,which is not necessarily replaced by the automated clinical analyzers,although some high-precision or high-intelligence machines are helpful for detecting the disease better on various levels.Therefore,it should be adopted in the rational ways for clinical examination based on the evidence-based medicine,and the appropriate diagnosis instruments should be chosen which not only satisfy the needs of clinical diagnosis,but also conform to the national benefit-for-people policies and requirements of healthcare reform.Herein,valuing the morphological examination in clinical diagnosis and strengthening the basic skills trainings should be advocated to improve the qualities and academic levels of clinic examination technicians.

Several hot topics and thinking about routine examination of domestic coagulation tests / 中华检验医学杂志

The progress on methodology evaluation and quality control of laboratory diagnosis of venous thrombosis and hemophilia and that of platelet function assessment were summarized.Methods to promote the development of routine test of hemorrhagic and thrombotic disorders and to improve the quality of laboratory testing were suggested.

The development trends of laboratory medicine under the new situation / 中华检验医学杂志

In order to implement the spirit of the document awarded by the three ministries,promote the medical reform process of public hospital,promote the heahhy development of the laboratory medicine,this paper expounds the significance of the study and implementation of the file,and puts forward several ideas.

Moving forward with times, improving the cytological diagnosis levels / 中华检验医学杂志

In this paper,we reviewed the recent developments of blood cell analyzer,summarized the problems during the analyzer-operation,presented our viewpoints on how to optimize the methods and innovate the technologies in the field of cytological diagnosis.Based on the actual demands of cytological diagnosis in China,in order to solve the emerging problems better in the process of updating technologies and operation models of blood analyzer,we advocate to comply with the core principles,including abiding by the “best-convictive-evidence oriented” rule,valuing the “combination-test model” using multiple methods,raising the levels on clinic diagnosis and prognosis evaluation,improving the qualities and capabilities of diagnosis technicians,to finally achieve the aims of narrowing the gaps and advancing the overall diagnosis levels.Moreover,we also strongly suggest making the realistic policies to support the developments of morphological examination,reasonably increasing the charging standard in some specific morphologicalexamination-items characterized by high-technology,time-costing and working-costing to faithfully reflect their values.

Pay attention to quality management of post-analytic phase / 中华检验医学杂志

Based on the connotation of ISO15189 and philosophy of current laboratory medicine,this article discussed about the important significance and role of quality management in diagnosing and curing of patients during post-analytic phase.This article also expounded the attention to these questions of examination results verification,procedure of reviewing and reporting examination results,establishment of principle for reserving samples after examination,range of consulting service and methods of handling complaint,the relationship between the whole quality management and information exchange of laboratory and clinical department.

Some consensus for urinalysis / 中华检验医学杂志

At present,the urinalysis has many puzzles and problems,this paper is proposed consensus of the key problems including standardization method in the urine formed elements examination,the urinalysis screening test,the results of reference intervals between the different examination methods,quantitative of the visible components,using centrifugal or non-centrifugal urine to do urine formed elements examination and the development direction of automatic urine sediment analyzer.These key problems are discussed and the consensus are put forward,in order to cause concern.( Chin J Lab Med,2012,35:790-791 )

Establishment and evaluation of the review criteria of automatic urine analysis workstations / 中华检验医学杂志

ObjectiveTo establish the proper review rules for the microscopic screening of urine samples tested by automatic urinalysis work station which is composed of LabUMat urine dry chemical analyzer and Urised urine sedimental analyzer.Methods The paired comparison was used to analyze the results tested by microscopy and Urised.A total of 2015 random urine samples were enrolled to establish and validate review rules.All the samples were collected from the inpatients and ontpatients of General Hospital of the People's Liberation Army from May to November 2011 and tested by urinalysis work station.2015 urine samples were firstly tested by urinalysis work station,including both urine dry chemical analyzer and urine sediments analyzer.Then each urine sample was examined microscopically by two technicians-in-charge using double-blind method.The average results from the two technicians were used as review results.Compared with review results,we set up the review rules and evaluated the Irue positive rate,false positive rate,true negative rate,false negative rate (omission diagnostic rate) and review rate.According to different test methods by automatic urinalysis work station,four microscopic review protocols were defined:( 1 ) Protocol 1:based on chemistry results only,microscopy review was performed when any of WBC,RBC,PRO and NIT was positive; (2) Protocol 2:based on urine sedimental analysis only,microscopy review was performed when any of WBC,RBC and CAST count was over upper limit of the reference range ; (3) Protocol 3:if any of BLD vs.RBC,LEU vs.WBC was different between two systems,or quantitative results had two or more than two gradient differences,microscopy review was performed; (4) Protocol 4:if any of BLD vs.RBC,LEU vs.WBC was different between two systems,or CAST was over upper limit of the reference range,or alam appeared,microscopic review was performed.300 randomly selected urine samples were tested to validate the review rules.Omission diagnostic rate and review rate were used to evaluate the rules.Results According to our review rules,the positive samples rate was 41.14％ (829/2015) and the negative rate was 58.86％ ( 1186/2015 ) ; Positive samples were composed of RBC (50.30％),WBC (53.32％) and CAST (3.74％).The review rates of four protocols were 42.93％ (865/2015),39.70％ (810/2015),29.58％(596/2015),18.91％ (381/2015 ),respectively.The false negative rates (omission diagnostic rates) were 6.36％ (128/2015),4.42％ (89/2015),1.34％ (27/2015)and 1.04％ (21/2015)respectively.Protocol 4 was selected as an ideal plan.Additional 300 urine samples were tested using protocol 4 in order to confirm the review rule.The review rate,consistency rate,true positive rate,false positive rate,true negative rate,omission diagnostic rate were 19.67％ (59/300),91.67％ (275/300),35.67％ (107/300),7.67％(23/300),56.00％ (168/300),0.67％ (2/300),respectively.After image review revised,the review rate was 8.67％ (26/300).ConclusionThe review rules established by our research for Urinalysis Work Station can find the abnormal urine samples effectively and exactly and can reduce the workload significantly.(Chin J Lab Med,2012,35:810-814)

Study and application of the combination of urine dry chemistry examination and urine sediment flow cytometry examination on the microscopic screening of the urine formed component / 中华检验医学杂志

Objective To establish the proper review rules for the microscopic screening of urine analyzed by UF-1000i automatic urinalysis work station (composed of UF-1000i urine flow cytometer and AX-4030 urine dry chemical analyzer).Methods A total of 2 839 random urine samples were collected at Chinese People′s Liberation Army General Hospital from September 2009 to February 2010, and were analyzed using UF-1000i urinalysis work station.The parameters obtained from UF-1000i and AX-4030 included RBC, WBC, CAST and ERY, LEU, PRO.After analysis by urinalysis work station, each urine sample was examined microscopically by two technologists using double-blind method.The average results got from the two technologists were regarded as the judging criterion.Based on the criterion, the review rules for the 2 839 urine samples tested by urinalysis work station were created and adjusted, and the true positive rate, false positive rate, true negative rate, false negative rate and review rate of these review rules were calculated.After that, 299 randomly selected urine samples were tested to validate these review rules.Omission diagnostic rate and review rate were used to assess the clinical practicability of the review rules.Results Thirty seven rules for microscopic review and twenty seven rules without further microscopic examination were set up based on six parameters using UriAccess 3.0 Software.The microscopic examination result was taken as the judging criterion, the consistency rate of these rules was 81.11%(2 311/2 839), the true positive rate was 40.51%(1 150/2 839), the false positive rate was 16.17%(459/2 839), the true negative rate was 41.00%(1 164/2 839), the false negative rate(omission diagnostic rate) was 2.43%(69/2 839) and the review rate was 18.28% (519/2 839).Additional 299 urine samples were assayed using UriAccess3.0 software to further verify these review rules.The consistency rate was 82.27%(246/299), the true positive rate was 36.12%(108/299), the false positive rate was 16.39%(49/299), the true negative rate was 46.15%(138/299), the false negative rate(omission diagnostic rate) was 1.34%(4/299), the review rate was 19.06%(57/299). The 4 false negative samples selected by these review rules did not come from the nephropathy department or the urology department.Microscopic results of RBC and WBC form these 4 samples ranged 3-8 cells/HP. Thus, these review rules could avoid the missed diagnosis of those patients with severe renal dysfunction.Conclusion The review rules established from this study for the UF-1000i urinalysis work station can effectively detect abnormal urine samples and improve the efficiency and the quality of urinalysis in routine clinical practice.

Examination of the urine sediment and establishment of the criterion for microscopy review / 中华检验医学杂志

Urine sediment analysis is of importance for the diagnosis, differential diagnosis and prognosis in urinary system diseases.To understand the standardization of sediment analysis and its development, This article analyzes the advantage and disadvantage of automated instrument used in urine sediment analysis, and then developes the criteria for microscopy review following automated urinalysis.

Evaluation of coagulation in vitro with platelet mapping and rapid TEGTM on thrombelastography / 中华检验医学杂志

Objective To evaluate performance of PlateletMapping(R) and RapidTEGTM based on thrombelastography by blocking platelet function with Reopro.Methods PlateletMapping was carried out with whole blood from healthy volunteers mixed with Reopro in vitro in a serial of titration.TEG(R) ACT of heparinized blood was tested with Rapid TEG kits.Linearity, repeatability and validity were calculated for two methods.Results MA activated by Kaolin, AA and ADP decreased with the increase of the concentration of Reopro. Inhibition rates (%)for AA and ADP induced aggregation were repeatable in channels and systems.At the Reopro levels of (1-4) × 10-2 mg, inhibition rate increased statistically( AA:27.99% ± 2.8% vs 63.37% ± 0.0% ,t = 21.9, P ＜ 0.01;ADP: 35.9% ± 0.56% vs 91.42% ± 1.14%,t=58.9,P ＜ 0.01 ) after addition of Reopro.Dose-dependent effect relationship could be seen between TEG(R) ACT and heparin;In Rapid TEG assay, measurement repeatability of K, α angle, MA and TEG(R)ACT were all good ( CV ＜ 5% ) except for R.Conclusions PlateletMapping(R) is sensitive to the inhibition of platelet function with good precision with dose-dependent effect.Moreover, Rapid TEG provides analysis of the overall coagulation function besides monitoring heparin therapy.

Prevalence and risk factors for aspirin resistance in elderly patients with type 2 diabetes / 中华老年医学杂志

Objective Although aspirin resistance has been recognized to occur in patients with diabetes mellitus, the prevalence and related risk factors for aspirin resistance in elderly patients with diabetes mellitus have not been reported yet. The purpose of the present study was to evaluate the prevalence and potential risk factors for aspirin resistance in elderly patients with type 2 diabetes.Methods The 140 elderly patients [aged from 60 to 92 years, mean age (73.8±8. 0) years] with type 2 diabetes receiving daily aspirin therapy (≥ 75 mg) over one month were recruited. Platelet aggregation was measured by light transmittance aggregometry (LTA) and thrombelastograph (TEG)platelet mapping assay. Results By LTA, 6 patients (4.3%) of the diabetic patients were found to be resistant to aspirin therapy, 44 patients (31.4 %) were semi-responders. By TEG, 31 patients (22. 1%) were aspirin resistant. Among the 31 patients who were aspirin resistant by TEG, 3 were aspirin resistant by LTA. In the multivariate logistic regression analysis, female gender (OR= 5. 54,95%CI: 1.17-27.47, P=0.036) and homocysteine level (OR=1.15, 95%CI: 1.00-1.35, P=0. 043) were statistically significant risk factors for aspirin resistance by TEG. Conclusions The prevalence of aspirin resistance in elderly patients with type 2 diabetes is considerably higher in elderly female patients and in elderly patients with higher serum homocysteine level.

Activating profile of platelet receptors in patients with coronary disease after clopidogrel intake / 中华检验医学杂志

Objective To study the characteristic of inhibition on platelet P2Y12 and short-term change after clopidogrel intake in patients with cardiovascular disease. Methods Thirty-two patients with cardiovascular disease were enrolled. Samples at baseline, 10 h and 36 h after 300 mg loading dose and 75 mg/d maintenance dose of clopidogrel with 100 mg/d Aspirin intake were measured respectively. Platelet aggregation (PAgT) was measured on thromboelastograph(TEG) induced by ADP/AA. INH was detected and calculated activated by Kaolin, AA, ADP and Activator((R)) in the TEG reagent. CD62p and VASP phosphorylation (PRI), platelet activation markers were tested with FACSCalibur Flow Cytometry, and platelet secretion activity and suppression of P2Y12 receptor were detected respectively. The changes of indicators were compared before and after clopidogrel intake, and evaluate their function in platelet receptor activation. Results INHADP at baseline was (11.5 ±9.3)%, and increased to (42.5 ±29.1)% statistically (t =3.155, P<0.05) after taking the P2Y12 at 10 h, but decreased to (20.4±13.1)% at 36 h, non-statistical to baseline (t = 2.078, P > 0. 05) , INHAA increases from baseline level (56. 6 ± 36. 6) % to (83.0 ±27. 3)% at 10h(t=2.086,P>0.05) and (85. 4 ±20. 8)% at 36 h (t= 1. 888, P>0.05), no statistical defferences were found. Inhibition on platelet activativation induced by ADP function well till 36 h after 300 mg loading dose. PAgTADP decrease from (53. 7 ± 14. 1)% at baseline to (49. 2 ±22. 8)% at 10 h non-statistically (t=0.656, P>0.05), and (40.7±12.8)% at 36 h statistically (t=2.418, P<0.05), however PAgTAA decrease at both 10 h and 36 h statiscally, from (34. 3 ± 18. 1) % to (17.4 ± 13. 1) % , (t=3.134, P<0.05) and (14.6±5.1)%, (t=2.532, P<0.05), respectively. Data of PAgT was not corresponding to that from TEG for the difference in sample type partly. PRI in VASP assay was (78. 6 ± 22.3)% before loading dose, and decreased to (70.7 ±9.4)% at 10 h without significance (t = l. 194, P>0.05) and (59.6 ±28.0)% at 36 h (t=1.930,P<0.05) statistically, similarly to INHADP,indicating that within 36 h clopidogrel did not have strong inhibitory effect on the ADP receptor. On the contrary, CD62p changed from (7. 5 ± 1. 4) % at baseline to (4. 2 ± 1. 1) % statistically (10 h, t = 18. 027, P < 0. 05) and ( 4. 3 ± 0. 2 ) % non-statistically (36 h, t = 2. 908, P > 0. 05 ). Inhibition of secretion activity reflected by CD62p was significant. In contrast, it was more obvious inhibition in COX-1 passway, while the inhibition of P2Y12 receptor varied due to assay difference. Conclusions AA-induced platelet activation is significantly decreased in the inhibition of clopidogrel and aspirin, while ADP receptor is significantly inhibited until 36 h after the loading dose of clopidogrel. Platelet function in whole blood reflects total activity of platelet interaction with other components, in which no significant inhibition could be witnessed within 10 h.

Determination of antigen-specific CTL induced by the gene vaccine in vitro with MHC pentamer / 中华检验医学杂志

Objective To determine the antigen-specific CTL in PBMC induced by a fusional family-gene vaccine of the immunoglobulin heavy chain variable gene framework region combined with the sequence of cytokine CM-CSF in vitro with MHC pentamer. Methods Peripheral blood samples were collected from two healthy donors and two patients. One was follicular lymphoma and another was hair cell leukemia. PBMC were isolated by density gradient centrifugalization with Ficoll and then subsequently differentiated into immature DCs (imDCs) induced by recombinant human GM-CSF and recombinant human IL-4. Gene gun was used to deliver the plasmids of the gene vaccine or the control plasmids into the imDCs. RT-PCR and ELISA assay were used to detect IgHVl-GM-CSF mRNA and GM-CSF in order to validate the transfection of the vaccine. After adding the cytokine cocktail, the imDCs became mature DCs. Then the mature DCs were co-cultured with lymphocytes from the blood samples for the induction of the antigen-specific CTL. The cultured cells were classified into vaccine group and control group and harvested at different time points of 0 d,7d, 17 d and 24 d after transfection. The subset of CD3+CD8+ T cells was analyzed by FCM assay. Finally, the CTL levels were detected with fluorescently labeled MHC pentamer antibody targeting vaccine epitopes. Results With the induction of cytokines, the imDCs with typical morphology were generated in PBMC. After delivering, the efficient expressions of the vaccine in the imDCs were determined by RT-PCR. And ELISA results also confirmed that GM-CSF was produced at a level of (28 ±6) ng/106 cells of the imDCs loaded with the vaccine, which was significantly different from that of control group (10 ± 3) ng/106 cells (t = 5. 191, P <0.01). FCM assay result showed that the CD3+ CD8+ T cells increased in a stepwise pattern during the culture. For control group, the levels at 0 d,7d, 17d and 24 d were ( 34. 24 ± 2. 72 )% , (46.06 ± 3.08)%, ( 65. 34 ± 4. 26 )% and (73.86 ±4.85 )% , respectively. For vaccine group, the results were (32. 28 ± 2. 08 ) % , (45. 32 ± 3. 81)% , ( 63. 37 ± 4. 21)% and (75. 01 ±3. 20)%. The differences between each time point had statistical significance (F = 176. 966 ,P <0.01) ,but there was no statistical differences between vaccine group and control group ( F = 0.657,P>0.05). The MHC pentamer analysis showed that the DCs loaded with IgHV1-GM-CSF fusional vaccine could efficiently induce the antigen-specific CTL response and the CTL levels increased gradually with the culture time, with the highest level of 4. 36% in the lymphoma blood and 3. 89% in the hair cell leukemia blood. Conclusions MHC pentamer assay could efficiently determine the antigen-specific CTLs response induced by the gene vaccine of family IgHV frame region in vitro. It could be a useful method for monitoring of anti-tumor cell immunity and evaluating of diagnosis and prognosis of the tumors in clinical application.