血清胆红素水平对HBV DNA检测的影响及其去除胆红素影响的方法

目的探讨血清胆红素水平对乙肝病毒脱氧核糖核酸(HBV DNA)定量检测的影响及其去除胆红素影响的方法。方法采用实时荧光定量聚合酶链反应检测含不同水平胆红素标本中HBV DNA病毒载量,再采用倍比稀释法比较用生理盐水和正常血清作为稀释介质对HBV DNA定量检测的影响。结果血清胆红素水平对HBV DNA定量检测结果有明显影响,但其影响与血清胆红素水平及HBV DNA载量有关。当HBV DNA载量≥1×106 U/mL时,血清胆红素对HBV DNA定量检测结果无影响;当HBV DNA载量1×106U/mL时,其检测结果与胆红素水平相关。当胆红素水平≥250μmol/L时,对HBV DNA定量检测结果有影响;当胆红素水平250μmol/L时,对HBV DNA检测结果无影响。采用倍比稀释法可去除胆红素对HBV DNA定量检测结果的影响,且生理盐水介质明显优于正常血清介质。结论血清胆红素对HBV DNA定量检测结果的影响与胆红素水平和HBV DNA载量相关,采用倍比稀释法可去除胆红素对HBV DNA定量检测结果的影响。     

磁珠分离纯化技术在实时荧光定量PCR检测HBV DNA中的应用

目的 评价磁珠分离纯化技术(简称磁珠法)提取核酸的重复性、提取效率一致性、线性范围、灵敏度及抗干扰性,初步探讨其在实时荧光定量PCR检测HBV DNA中的应用价值.方法 采用磁珠法提取HBV DNA log值分别为4.16,5.23和7.04的质控血清进行重复性试验,HBV DNA阳性血清10倍梯度稀释的系列标本评价其提取效率一致性,HBV DNA强阳性血清作10倍系列稀释后检测其线性范围,HBV DNA标准血清与含干扰成分(溶血、黄疸和脂血)的HBV DNA 阴性血清,按4∶0,3∶1,2∶2,1∶3和0∶4比例分别混合成5个不同浓度标本用于干扰试验.以提取方法为沉淀煮沸法的PCR 荧光诊断试剂作对照,应用磁珠法及配套扩增试剂检测288份乙型肝炎患者血清HBV DNA含量,并比较测定结果.结果 磁珠法批内和批间平均变异系数分别为3.66%和4.75%;HBV DNA含量不同的血清提取效率均在98%以上;在1.28×102IU/L～1.28×109IU/L之间有良好的线性关系;最低检出限为60.5 IU/L;不同浓度的黄疸和脂血对扩增曲线和实验结果无明显影响;血清中血红蛋白浓度至50 g/L时,HBV DNA结果相差约0.5～1.0个数量级.磁珠法和沉淀煮沸法的阳性率分别为88.19%和82.64%.当沉淀煮沸法检测结果log值处在2.699～4.000之间时,两法检测相应标本结果差异有统计学意义.结论 磁珠法高效、快速和易于自动化,应用于HBV DNA检测对乙型肝炎患者的临床诊断和疗效监测将有十分重要的意义.     

胆红素对HBV DNA定量检测结果的影响

目的探讨高胆红素血清对实时荧光定量聚合酶链反应(PCR)检测乙型肝炎病毒核酸(HBV DNA)的影响,并选择有效减低胆红素对HBV DNA测定干扰的方法。方法将不同浓度的HBV DNA标准品(S1-S4:3.0×10~7、3.0×10~6、3.0×10~5、3.0×10~4IU/ml)按相同比例分别添加到胆红素正常血清和高胆红素血清中,按不同浓度胆红素进行分组来考察不同浓度胆红素血症对HBV DNA测定结果的影响程度大小,然后再分别测定HBV DNA的结果,每个标本重复检测三次,同时观察扩增曲线是否存在差异。对影响测定的血清标本作10、100、1000倍稀释后,进行检测,并收集5例临床高胆红素的乙肝血清标本进行验证。结果血清胆红素浓度介于401~500μmol/L、501~600μmol/L、600μmol/L三个组,HBV DNA定量检测结果高于胆红素正常血清组,差异有统计学意义(P0.05),血清胆红素浓度浓度介于20.6~100μmol/L、101~200μmol/L、201~300μmol/L和301~400μmol/L四个组,与胆红素正常血清组比较,差异无统计学意义(P0.05);血清胆红素浓度高于400μmol/L血清扩增时,扩增曲线先缓慢升高,其基线高于标准品,其阈值线处于拐点之下非S扩增区,之后升高呈现S型曲线;10、100、1000倍稀释后,扩增曲线平行且等间距,结果一致性较好。5例高胆红素血清有4例在定阈值线时,其阈值线处于拐点之下非S扩增区,定值偏高,结果高于稀释后的标本,差异有统计学意义(P0.05);有1例阈值线处于拐点处,其斜率低于标准品,结果低于稀释后的标本,差异有统计学意义(P0.05)。结论总胆红素高于400μmol/L对荧光定量PCR检测HBV DNA结果存在影响,通过稀释一般就可以降低胆红素因素干扰。     

南昌地区献血者HBV感染隐匿风险与基因型调查分析

目的：探讨南昌地区献血者乙型肝炎病毒（Hepatitis B Virus,HBV）感染隐匿风险与HBV感染人群中 HBV基因分型,为血液筛查策略、地区流行病学提供研究数据。方法（1）对2012年1月1日至2012年12月31日64400份献血者标本采用ELISA法进行HBsAg筛检;（2）对HBsAg筛检阴性标本进行HBV/HCV/HIV 3项联合病毒核酸检测（Nucleic Acid Ampli-fication Testing ,NAT）,再对NAT阳性标本进行HBV DNA定量及血清乙肝五项标志物检测;（3）选取HBsAg筛查阳性标本200份和HBsAg筛查阴性而HBV DNA阳性标本30份,对所有HBV DNA阳性标本进行病毒核酸提取、扩增及测序分型,检测HBV基因型。结果（1）64400份献血者标本中共检出HBsAg阳性862份和阴性63538份;HBsAg检测阴性标本通过NAT检测,共检出HBV DNA 阳性84份,献血者HBV感染率为1.47%,HBV输血感染隐匿风险为0.13%;（2）献血者HBV感染以隐匿型为主（75.0%,63/84）,其病毒载量多数＜20/ml（70.2%,59/84）;3）选取的200份HBsAg阳性标本中共检出HBV DNA阳性118份,148份HBV DNA阳性标本有66份样本分型成功,共检出B基因型为47份（71.2%）,C基因型为19份（28.8%）,未检出其它基因型。结论 ELISA法筛查HBsAg阴性的献血者中HBV感染隐匿风险依然存在,且多以低病毒载量的隐匿性感染为主,应对献血者标本常规开展NAT检测;南昌地区献血者中HBV感染基因型以B型为主,C型次之,未见其它基因型。     

不同标本对荧光实时定量PCR法测定HBV—DNA结果的影响

目的 利用沉淀煮沸裂解法提取HBV核酸，探讨不同的临床标本和贮存条件对荧光定量PCR法测定乙型肝炎病毒核酸（HBV－NDA）结果的影响，为临床标本的收集和贮存提供依据。方法 按不同要求收集特定的临床标本，用本实验室使用的HBV－DNA提取方法提取DNA模板，再用荧光实时定量PCR法检测HBV－DNA的含量。结果 经EDTA、中等含量肝素、枸橼酸钠抗凝血浆标本与相应血清标本测定HBV－DNA的含量无显著性差别均（P＞0．05），但高浓度肝素（1000U／ml)抗凝管结果显著降低（F值为25．96；P＜0．05）；溶血、高血脂标本、标本反复冻融、血浆（清）标本在室温下短期贮存（48h内）对HBV－DNA含量均无明显影响（P＞0．05）。结论 用本实验室使用的HBV－DNA提取方法，可使抗凝剂及其它PCR反应抑制物质对HBV－DNA测定结果影响降到最低，从而使临床无污染标本一般均可接受。     

实时荧光PCR与COBAS Amplicor PCR-ELISA 定量检测血清HBV DNA的比较

目的研究实时荧光聚合酶链反应（PCR）与COBAS Amplicor PCR-酶联免疫吸附试验（ELISA）定量检测血清标本中乙型肝炎病毒（HBV）DNA水平的相关性。方法采用实时荧光定量PCR与COBAS AmplicorPCR-ELISA平行检测110例HBV携带者和40名健康体检者的血清标本,比较2种方法检测结果的相关性和差异程度。结果实时荧光PCR与COBAS Amplicor PCR-ELISA定量检测血清HBV DNA相关性较好,相关系数为0.944,对高浓度标本检测结果的差异性更小。结论我们采用的实时荧光PCR检测血清标本HBV DNA与COBAS Amplicor PCR-ELISA具有较好的相关性,可以为临床提供可靠的结果。     

不同核酸提取方法对母乳HBV DNA定量检测结果的影响

目的评价不同核酸提取方法对母乳乙型肝炎病毒(HBV)DNA定量检测结果的影响。方法采用聚乙二醇(PEG)病毒沉淀浓缩法(PEG法)和碱液直接裂解法提取HBV DNA阳性产妇乳汁HBV DNA,并进行荧光定量聚合酶链反应检测,以酚-氯仿提纯法作为核酸提取对照方法。结果当母乳HBV DNA≥104 copy/mL时,PEG法、碱性直接裂解法提取标本的检测阳性率均为100.00%(21/21);当母乳HBV DNA水平为103～<104 cop-y/mL时,PEG法提取标本检测阳性率为93.75%(30/32),高于碱液直接裂解法提取标本的检测阳性率71.88%(23/32)(P<0.05)。结论 PEG法提取母乳HBV DNA能提高母乳HBV DNA检测阳性率,能够为科学、安全的母乳喂养提供可靠的实验室依据。     

多种一步法快速提取DNA对肺炎支原体PCR检测的影响

目的 对比不同一步法提取DNA在肺炎支原体PCR检测中的效果及应用范围,为临床标本中肺炎支原体的检测提供依据。方法 以两种经典的DNA提取一步法（ROSE和Chelex-100法）及其优化方案和商品化DNA提取试剂盒,提取肺炎支原体纯培养菌株及30份肺炎支原体阳性临床咽拭子标本DNA,分别进行普通PCR和荧光PCR检测,对比不同方法提取的DNA对PCR检测结果的影响。结果 ROSE法和Chelex-100一步法中的SDS严重影响Taq酶活性,提取物稀释100倍方可进行荧光PCR扩增,稀释10倍可用于普通PCR扩增。改良的Chelex-100一步法提取肺炎支原体纯菌DNA产量最高,且PCR扩增效果最好。对于30份临床标本来说,试剂盒法、改良Chelex-100法和水煮法的阳性率分别为100.00%（30/30）、80.00%（24/30）和43.33%（13/30）,且对相同肺炎支原体阳性标本提取的DNA,试剂盒法检测的循环阈值（Ct值）比上述两种方法分别小1.95和2.38。结论 经典一步法提取的DNA必须经稀释后方可作为扩增模板。改良的Chelex-100法操作简便,可用于纯菌培养的DNA提取。临床标本中肺炎支原体DNA提取以试剂盒法最优,改良的Chelex-100法提取的DNA也可用于临床标本中肺炎支原体的检测,但不可用于定量分析。     

超敏HBV DNA定量检测系统的临床应用研究

目的:比较超敏HBV DNA检测系统与传统荧光定量PCR外标法定量检测血清乙肝病毒DNA,探讨其临床应用价值。方法:采用超敏PCR检测系统和传统荧光定量PCR外标法同时检测80例HBsAg阳性患者的血清HBV DNA浓度。结果:超敏法阳性检出率(92.5%)高于传统外标法(50.0%)(P0.01)。超敏法阳性定量结果中位值为2.95×10~5IU/mL,高于传统外标法(5.63×10~4IU/mL)(P0.01),两种方法对高载量病毒(≥10~4IU/mL)血清标本检测结果的相关性有统计学意义(R~2=0.89,P0.01)。超敏法对26例HBeAg阴性慢性乙肝患者的阳性检出率(100.0%)高于传统外标法(11.5%)(P0.01)。结论:超敏HBV DNA定量检测系统检测灵敏度高,更适用于临床低载量病毒(10~4IU/mL)血清标本的检测。     

实时荧光定量PCR检测布鲁氏菌核酸DNA的应用评价

目的探讨实时荧光定量PCR检测布鲁氏菌核酸DNA浓度的检测范围和检测健康人全血中核酸DNA的Ct本底值。方法采用试剂盒提取纯菌、健康人和临床患者血液中的核酸DNA,应用实时荧光定量PCR进行检测。结果经检测,40份健康人群血液标本Ct值平均值为37.0,标准差为1.9,Ct值95%置信区间为33.0 ~ 38.8。 将布鲁氏菌核酸DNA标准品倍比稀释（56.2 ng ~ 0.026 5 fg）,实时荧光定量PCR检测灵敏度为6.7 fg。结论实时荧光定量PCR检测纯菌核酸DNA的灵敏度相当于2个基因组DNA拷贝数,但用于检测血液标本尚待进一步研究。     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

俯卧位通气对高海拔地区肺复张治疗无效急性呼吸窘迫综合征患者氧合的影响

目的 探讨俯卧位通气对高海拔地区肺复张术(RM)治疗无效急性呼吸窘迫综合征(ARDS)患者的治疗作用.方法 从海拔2260m的地区医院筛选RM治疗无效的41例ARDS患者[平均氧合指数( PaO2/FiO2)较RM前升高＜20％视为RM无效],依不同病因分为肺内源性ARDS组(ARDSp组)和肺外源性ARDS组(ARDSexp组),每组再按信封法随机分为俯卧位组和仰卧位组,即ARDSp俯卧位组(11例)、ARDSp仰卧位组(9例)、ARDSexp俯卧位组(10例)、ARDSexp仰卧位组(11例).在通气前及通气1、2、3、4h监测动脉血氧分压( PaO2)、PaO2/FiO2、静态顺应性(Cst)、气道阻力(Raw)的变化.结果 通气lh时,ARDSexp俯卧位组PaO2/FiO2( mm Hg,l mm Hg=0.133 kPa)即较通气前显著升高(157.4±40.6比129.3±48.7,P＜0.05),并随通气时间延长呈持续增高趋势,4h达峰值(219.1 ±41.1);且ARDSexp俯卧位组通气3h内PaO2/FiO2较其他3组显著增高,另3组间则差异无统计学意义.ARDSp俯卧位组、ARDSexp俯卧位组通气4h时PaO2/FiO2均较相应仰卧位组显著增高(208.8±39.7比127.4±47.1,219.1±41.1比124.9±50.8,均P＜0.05).4组通气前后Cst无显著改变,各组间差异也无统计学意义.ARDSp俯卧位组通气4h时Raw(cmH2O·L-1·s-1)较通气前显著降低(6.8±1.7比10.7±1.8,P＜0.05),且明显低于其他3组;其他3组各时间点Raw组内及组间比较差异均无统计学意义.结论 俯卧位通气作为ARDS机械通气重要策略之一,可以改善RM无效高原ARDS患者的氧合,为抢救患者赢得宝贵的时间.     

Digital imaging among medical facilities

The Department of Veterans Affairs (VA) in the USA operates a network of 172 medical centres which all utilize a hospital information system (HIS) which has been developed and is currently maintained by the VA. During the past several years, an image management and communication module has been developed, installed and clinically utilized at the Washington DC and Maryland VA Medical Centres. This image management and communication system, referred to as the decentralized hospital computer program (DHCP) imaging system, is fully integrated with a commercial picture archiving and communication system (PACS). The system is utilized to capture, archive, and display all images generated within the hospital including radiology, nuclear medicine, pathology, endoscopy, bronchoscopy, and dermatology, intraoperative photographs, ECG data, and a limited number of paper documents. The ultimate goal of the project is to have all patient text and image data available at any clinical workstation to any authorized user anywhere within the network of medical centres. Clinical requirements for an imaging workstation include ease of use, rapid and reliable access to the complete set of patient information, and images which are of acceptable quality to meet the requirements of the user and the subspecialty. Patient confidentiality and data security must be safeguarded at all times. Integration of the images with the remainder of the patient's database was found to be critical to the success of the project. The experience at the Washington and Maryland facilities suggests that an imaging system that is successfully integrated with a hospital information system can provide substantial clinical and economic benefits both within and among medical centres. Clinical acceptance and utilization of the system has been excellent, particularly in diagnostic radiology where DHCP Imaging has been interfaced to a commercial PAC system. Based upon this initial experience, the VA has begun to deploy the system throughout its large network of medical centres.     

Myocardial elastography at both high temporal and spatial resolution for the detection of infarcts

Myocardial elastography is a novel method for noninvasively assessing regional myocardial function, with the advantages of high spatial and temporal resolution and high signal-to-noise ratio (SNR). In this paper, in-vivo experiments were performed in anesthetized normal and infarcted mice (one day after left anterior descending coronary artery [LAD] ligation) using a high-resolution (30 MHz) ultrasound system (Vevo 770, VisualSonics Inc., Toronto, ON, Canada). Radiofrequency (RF) signals of the left ventricle (LV) in longitudinal (long-axis) view and the associated electrocardiogram (ECG) were simultaneously acquired. Using a retrospective ECG gating technique, 2-D full field-of-view RF frames were acquired at an extremely high frame rate (8 kHz) that resulted in high-quality incremental displacement and strain estimation of the myocardium. The incremental results were further accumulated to obtain the cumulative displacements and strains. Two-dimensional and M-mode displacement images and strain images (elastograms), as well as displacement and strain profiles as a function of time, were compared between normal and infarcted mice. Incremental results clearly depicted cardiac events including LV contraction, LV relaxation and isovolumetric phases in both normal and infarcted mice, and also evidently indicated reduced motion and deformation in the infarcted myocardium. The elastograms indicated that the infarcted regions underwent thinning during systole rather than thickening, as in the normal case. The cumulative elastograms were found to have higher elastographic SNR (SNR(e)) than the incremental elastograms (e.g., 10.6 vs. 4.7 in a normal myocardium, and 6.0 vs. 2.4 in an infarcted myocardium). Finally, preliminary statistical results from nine normal (m = 9) and seven infarcted (n = 7) mice indicated the capability of the cumulative strain in differentiating infracted from normal myocardia. In conclusion, myocardial elastography could provide regional strain information at simultaneously high temporal (>/=0.125 ms) and spatial ( approximately 55 microm) resolution as well as high precision ( approximately 0.05 microm displacement). This technique was thus capable of accurately characterizing normal myocardial function throughout an entire cardiac cycle, at the same high resolution, and detecting and localizing myocardial infarction in vivo.     

Clinical Pharmacokinetics of Morphine

Morphine, the most widely used mu-opioid analgesic for acute and chronic pain, is the standard against which new analgesics are measured. A thorough understanding of the pharmacokinetics of morphine is required in order to safely and effectively use this analgesic in a wide variety of patients with different levels of organ function. A MEDLINE search was conducted to identify literature published between 1966 and January 2002 relevant to the pharmacokinetics of morphine. These publications were reviewed and the literature summarized regarding unique and clinically important elements of morphine disposition relative to its parenteral administration (including intravenous, intramuscular, subcutaneous, epidural and intrathecal administration), absorption profile (immediate release, controlled release, and sublingual/buccal, and rectal administration), distribution, and its metabolism/ excretion. Special populations, including infants, elderly, and those with renal/liver failure, have a unique morphine pharmacokinetic profile that must be taken into account in order to maximize analgesic efficacy and reduce the risk of adverse events.     

手转胎头术失败的原因与分娩结局临床分析

目的 探讨手转胎头术失败的原因与分娩结局.方法 选择2008年1月至2010年12月于我院住院分娩的持续性枕横位、枕后位产妇198例,根据行手转胎头术后结果分为成功组126例、失败组72例.比较两组分娩结局,对比分析失败原因.结果 失败组胎儿体质量≥3500 g的发生率[76.4％(55/72)]明显高于成功组[31.7％(40/126)],差异有统计学意义(x2=30.177,P=0.001)、失败组宫缩乏力发生率[58.3％(42/72)]高于成功组[38.1％ (48/126)],差异有统计学意义(x2=7.569,P=0.006)、失败组骨盆临界或轻度狭窄发生率[38.9％ (28/72)]高于成功组[23.8％(30/126)],差异有统计学意义(x2 =5.030,P=0.002)、失败组手转胎头时机不当(宫口开大＜6 cm、胎头位于坐骨棘上及宫口开大8～10 cm、胎头位于坐骨棘下≥2 cm)发生率[61.1％(44/72)]高于成功组[38.9％(49/126)],差异有统计学意义(x2=9.084,P=0.003).失败组母儿并发症(产后出血、产褥病率、胎儿窘迫、新生儿窒息)发生率高于成功组(x2 =9.586,P=0.002、x2=9.334,P=0.002、x2=5.910,P=0.015、x2=5.240,P=0.022)、失败组剖宫产发生率[72.2％(52/72)]明显高于成功组[34.1 ％(43/126),x2=26.641,P=0.001)].结论 手转胎头术能使难产变顺产,降低剖宫产率,减少母儿并发症,但须积极预防、处理导致手转胎头术失败的原因,对矫正失败后继续矫正及试产应慎重.     

Evidence-Based Pain Management and Palliative Care in Issue Four for 2009 of The Cochrane Library

ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly. Issue 4 for 2009 contains 4027 complete reviews, 1906 protocols for reviews in production, and 11447 one-page summaries of systematic reviews published in the general medical literature. In addition, there are citations of 600,000 randomized controlled trials, and 12,200 cited papers in the Cochrane methodology register. The health technology assessment database contains over 7500 citations. This edition of the Library contains 90 new reviews, of which 19 have potential relevance for practitioners in pain and palliative medicine.     

Wie zuverlässig sind Angaben zu nichtentzündlichen ausgedehnten Schmerzen?

ZusammenfassungFragestellung Es wurde geprüft, wie sich der Differenziertheitsgrad zweier Schmerzmessmethoden auf Angaben zur Ausgedehntheit klinischer Schmerzen auswirkt. Zugleich wurde der Referenzzeitraum variiert, über den die Patienten berichten sollten.Methode Erfasst wurde der Einfluss zu Lasten der Befragungsdifferenziertheit durch den Vergleich zweier Körperschema-Bildvorlagen. Drei Referenzzeiträume (Schmerz aktuell, letzte Woche, letztes halbes Jahr) wurden vorgegeben.Ergebnisse Patienten mit ausgedehnten Schmerzen gaben bei differenzierter Befragung um so mehr Schmerzen an, je weiter die Schmerzen zurück lagen und je größer der Berichtszeitraum war. Patienten mit gelenknahen Schmerzen gaben bei hoch differenzierter Befragung weniger ausgedehnte Schmerzen in der Vergangenheit an als bei globaler Einschätzung. Patienten mit Rückenschmerzen berichteten bei differenzierter Befragung zum aktuellen Schmerz über weniger ausgedehnte Schmerzen als bei globaler Befragung.Schlussfolgerung Die Angaben zur Schmerzausdehnung variieren vor allem bei Patienten mit ausgedehnten Schmerzen in Abhängigkeit von der Differenziertheit der Befragung. In diesen Fällen ist die Wahrscheinlichkeit erhöht, dass sich die Beschwerdesymptomatik zumindest teilweise erst in der Reaktion auf die situativen Befragungsbedingungen konstituiert und daher nicht auf andere Befragungsbedingungen generalisiert werden kann.