脂质、血红蛋白、胆红素标本对乙肝病毒DNA荧光定量测定的影响.pdf

微循环学杂 志 2006 年第16 卷第 1期 临床研究 作者单位 湖北省黄冈市第一医院检验科, 邮政编码 黄冈 438000 本文 2005-07-01 收到, 2005-10-12 修回,2005-11-14 接受 脂质 、 血红蛋白 、 胆红素标本对乙肝病毒 dna 荧光定量测定的影响 郭华国 姚正国 中图分类号 r446. 11 文献标识码 a 文章编号 1005-1740( 2006) 01-0032-02 乙 肝病毒 dna( hbvdna) 荧光定量测定在临 床上已得到广泛应用。然而 ,有关临床因素 , 诸如高血脂、溶血 、 高胆红素标本对该项实验 结果有无影响尚未见系统研究 。本文旨在探讨溶血 样本、高血脂样本 、高胆红素样本对 hbvdna 荧光 定量测定结果的干扰 。 1 资料与方法 1. 1 高脂血清及对照血清 收集本院门诊乙肝大三阳志愿者血液 9 份, 每 人分两次抽取血液。一次空腹抽血 ,作为正常血脂 水平血清; 一次嘱其食用高脂肪食物后 2 h 左右抽 血,制备高血脂血清 ,从中选取外观呈乳糜样混浊者 测其血清甘油三脂含量( 酶法) , 两管分别作好标记 待用 。 1. 2 溶血血清及对照血清 收集本院门诊乙肝大三阳( 经 elisa 法证实) 志愿者血液 12 份,每人份血液分装 2 管 。一管人工 制备溶血血清( 用冻融法作不完全溶血) , 并在血液 分析仪上( 氰化高铁法) 测其血红蛋白( hb) 含量; 一 管直接分离血清 。两管分别作好标记待用。 1. 3 黄疸血清 收集本院住院病人的高胆红素血清 9 份, 然后 作乙肝三系检测、hbvdna 定量检测, 二项试验连 续检测三次均为阴性则留取血清并测其胆红素含量 ( 钒酸盐法) ,作好标记待用。 1. 4 正常血清 收集门诊健康体检者血清 9 份, 处理方法同高 胆红素血清,留取胆红素正常者血清备用。 1. 5 检测试剂和仪器 实时荧光定量 pcr检测系统( 杭州博日科技有 限公司生产, 型号 : fqd-33 a) ; hbvdna 荧光定量 试剂盒( 上海复星实业股份有限公司生产 ,试剂盒最 低检测限 1 000 拷贝/ml) 。 1. 6 检测方法 将每位乙肝大三阳志愿者的溶血血清和未溶血 血清以及高血脂血清和正常血脂血清制成模板后, 加入 pcr 反应液 , 同时上机作 hbvdna 定量测 定 。将每份高胆红素血清制成模板, 取 4 l 与乙肝 大三阳血清模板 4 l 混合后 , 加入 pcr 反应液, 上 机作hbvdna 的荧光定量测定 。同时用正常血清 模板 4 l 加对应的乙肝大三阳血清模板 4 l 作对 照测定。( 常规操作只加入模板 4 l, 这里考虑到稀 释效应, 所以加入模板量为 8 l。 ) 所有标本模板的 制备均按试剂盒说明书进行 。 1. 7 统计学处理 将配对的溶血血清和未溶血血清、高脂血清和 正常血脂血清以及高胆红素血清和正常胆红素血清 三组检验结果分别进行配对 t 检验 1。 2 结 果 9 份乙肝大三阳志愿者高脂血清与正常血脂血 清 hbvdna 定量结果, 12 份乙肝大三阳志愿者未 溶血血清和溶血血清 hbvdna 定量结果以及 9 份 黄疸血清及正常血清模板分别与 9 份乙肝大三阳血 清模板混合后的 hbvdna 定量结果分别见表 1、 表 2、表 3。 3 讨 论 由表 1 可知 ,同一乙肝大三阳志愿者的高血脂 血清和正常血脂血清 hbvdna 的测定结果存在明 显差异 ,其中1、 2、 4、 5、 7、 9号高血脂血清的hbvdna 微循环学杂 志, 2006, 16 ( 1 ): 32 33 c 2006 chinese journalof microcirculation 表 1 乙肝大三阳志愿者高脂血清与正常血脂 血清 hbvdna 定量结果 标本编号 非脂血血清 ( 拷贝/ml) 高血脂血清 ( 拷贝/ml) 甘油三脂含量 ( mmol/l) 17. 35e+706. 50 23. 21e+605. 27 33. 01e+81. 90e+42. 77 44. 98e+704. 98 56. 25e+603. 89 64. 78e+72. 01e+33. 01 79. 61e+704. 39 85. 80e+72. 20e+42. 80 93. 77e+803. 90 注: 非脂血血清的甘油三脂含量均小于 1. 70 mmol/l ; 甘油三脂含量 指高血脂标本; hbvdna 含量在 1 000拷贝/ml 以下结果为 0 表 2 乙肝大三阳志愿者未溶血血清和 溶血血清 hbvdna 定量结果 标本编号 溶血血清 ( 拷贝/ml) 未溶血血清 ( 拷贝/ml) hb 含量 ( g/l) 15. 57e+74. 98e+76 21. 90e+44. 09e+426 31. 14e+89. 01e+713 42. 01e+71. 73e+718 53. 59e+66. 71e+619 61. 89e+82. 30e+723 73. 36e+72. 80e+79 84. 59e+57. 90e+514 93. 32e+95. 10e+927 106. 89e+74. 90e+713 114. 32e+82. 09e+810 123. 58e+75. 61e+78 注: 未溶血血清 hb 含量均低于 2 g/l ; hb含量指溶血标本 表 3 黄疸血清及正常血清模板分别与乙肝大三阳 血清模板混合后的 hbvdna 定量结果 标本编号 正常血清 ( 拷贝/ml) 黄疸血清 ( 拷贝/ml) 总胆红素 ( mol/l) 14. 50e+73. 98e+736. 10 24. 59e+77. 51e+645. 10 37. 55e+89. 70e+730. 90 44. 32e+62. 87e+628. 80 58. 77e+66. 63e+639. 00 63. 05e+78. 44e+642. 10 73. 99e+72. 70e+734. 00 87. 71e+54. 82e+629. 30 91. 19e+72. 80e+733. 40 注: 正常血清总胆红素含量均低于 10 mol/l; 总胆红素含量指黄疸 血清中的总胆红素 定量结果为 0, 而来自相同志愿者的正常血脂血清 的结果分别为 : 7. 35e +7 拷贝/ml ,3 . 21e+6 拷贝/ ml ,4. 98e + 7 拷贝/ml, 6. 25e+6 拷贝/ml ,9. 61e+ 7 拷贝/ml , 3. 77e +8 拷贝/ml ; 3、6、8 号标本的脂 血和非脂血结果分别为: 1. 90e +4 和 3. 01e +8 拷 贝/ml ,2. 01e + 3 拷贝/ml 和 4. 78e + 7 拷贝/ml ,2. 20e+4 拷贝/ml 和 5. 80e + 7 拷贝/ml, 差异具有显 著性( p 0 . 05) ,因此认为溶血血清( hb 27 g/l 范围 之内) 对 hbvdna 定量测定不存在干扰作用。这 一点与以往文献报道 hb 的衍生物是 taq 酶的抑制 物 ,会严重干扰 pcr结果 2 4不一致。我们认为标 本中 hb 经 100煮沸 10 min 后已经变性沉淀, 再 经过多次离心后, 待测上清液中将不会有 hb 的存 在 ,只会是 hb 的衍生物 。从表 3 结果可看出 1 、 3、 4、 5 、7、9 号黄疸血清与无黄疸血清与对应的 hb- vdna 定量结果都在同一个方次之内 ; 2、6、8 号二 者相差亦不到一个数量级( p 0 . 05) ,均在允许误 差范围之内 5。因此 ,可以认为血清总胆红素在 45 mo l/l 以下时, 对 hbvdna 定量测定无干扰作 用 。其原因可能是胆红素对试验的干扰主要在于它 本身的光吸收作用, 而经过标本处理过程中的丢失 和多次稀释后,它的这种干扰作用就变得很小。 本文第一作者简介: 郭华国( 1960 ) , 男, 汉族, 主管技师 微循环学杂 志 2006 年第16 卷第 1期 临床研究 参考文献 1 李金明, 王露楠. 样本处理对 pcr 反应检测乙型肝炎病毒核酸的 影响. 中华检验医学杂志, 2000, 23( 6) : 337 339. 2 程正江, 付文荣. 荧光定量聚合酶链反应直接检测血清中乙型肝 炎病毒核酸方法的建立及评价. 中华检验医学杂志, 2004, 27( 2) : 102 103. 3 程正江, 曾平凡. 用统计学方法解释和处理定量聚合酶链反应试 验结果, 中华检验医学杂志, 2003, 26( 3) : 158. 4 brechtbehl, whaley sa , dusheiko gm , et al.a rapid real time quantitative polymerase chain reaction for hepertitis b virus. j virol methods, 2001, 93( 1 -2) : 105 113. 5 李金明. 聚合酶链反应临床应用的优越性和局限性. 中华医学检 验杂志, 2005, 28( 3) : 225 227. 33 脂质、血红蛋白、胆红素标本对乙肝病毒 dna 荧光定量测定的影响 本 期 论 文 导 读文摘页1 微循环学杂 志第 16 卷, 第 1 期 第 11 页/低剂量 mptp 致雌鼠黑质听觉 p50 诱发电位动态变化的实验 研究 罗海芸, 何 敏, 孟金兰, 等/昆明医学院生理教研室, 昆明 650031 目的: 功能学角度了解低剂量 mptp 对雌鼠黑质听觉 p50 诱发电位 的动态影响及帕金森病动物模型旋转行为的出现与黑质听觉 p50 诱发 电位的关系。方法: 45 只大鼠随机分为三组: ( 1) 正常对照组; ( 2) mptp 致帕金森病模型组; ( 3) 生理盐水对照组。动态监测术后第 7 至 14 天 m pt p 型帕金森病动物模型黑质听觉 p50 诱发电位。结果: mptp 组的 p50 t/c 值在第 11 14 天均较正常组及生理盐水溶剂对照组( int - saline 组) 降低, 且有统计学意义( p 1. 9 v and was consistently during exercise with heart rate in the specific threthold. results:of 23 patients with ischemic heart disease and vt , t wave alternans was present in 14 cases (60. 7% ) during exercise . alternans was found in 3 of 30 ( 10% ) non -vt ischemic heart disease patients and 0 of 39 control subjects( 0% ) , and the alternans voltage were 2. 31. 1 v , 1. 10. 6 v and 0. 80. 3 v respectively in three groups ( p 0. 05 and p 0. 01 respectively) . conclusion: microvolt- level t wave alternans during exercise may be one of a useful marker for ven - tricular arrythmic risk in patients with ischemic heart disease . key words:t wave alternans; ischemic heart disease; ventricular tachycardia page 29/ significance of detection of p16 gene methylation and cyfra21-1 in serum for diagnosis of lung cancer/ yao qunfeng, ning yong, zhang liping , et al/ department of laboratory medicine, hubei college of traditional chinese medicine, wuhan 430064 objective : to evaluate the feasibility of combined detection of p16 gene methylation and cyfra21 -1 in serum as an approach for diagnosis of lung cancer. method: serum dna was extracted from 53 patients with lung can - cer. the methylation of p16 gene was detected by nested methylation specific polymerase chain reaction. serum content of cyfra21 -1 was determined by radioimmunoassay . in addition, the results from 18 patients with benign lung disease and 25 healthy individuals were also analyzed as control. results: aberrant promoter methylation of the p16 gene was detected in 38 of 53( 71. 7% )of the serum samples. the positive rates of cyfra21 -1 were 64. 2% ( 34/53) . parallel combined test increased the diagnostic sensitivity to 89. 8%. the positive rates of p16 gene methylation and cyfra21 -1 of the group bld were 22. 2%and 11. 1%, respectively . no aberrant rmethylation of the p16 gene was found in 25 healthy individuals. conclusion: combined detection of p16 gene methylation and cyfra21-1 in serum could increase the sensitivity and specificity in diagnosing lung cancer. key words:lung cancer; p16 gene; cyfra21-1; nested methyla- tion specific polymerase chain reaction( nmsp) page 32/ investigation on effect of hemolytic blood, serum lipids and serum bilirubin on real -time flourimetry pcr of hbvdna /guo huaguo , yao zhengguo/ the first hospital of huanggang city , huanggang , 438000 objective : t o investigate the effect of the high lipid, high bilirubin and hemolytic blood specimens on real time fluorimetry pcr of hbvdna . method: the serums from the case that were hbsag +, hbeag +and anti- hbc+, and the serums from the same case but those were high lipids or hemolytic blood were testedby real -time fluorimetry pcr of hbvdna paral - lel; collecting serums from the case that hbvdna was negative but with high bilirubin and serum that hbvdna was negative but with nomorl level of bilirubin,mixed them with a same positive serum of hbvdna respective- ly , and then tested by real time flurimetry pcr of hbvdna parallel. re- sults: 12 sample of the hemolytic blood and 9 high serum bilirubin have no siginificant change compared with the contrast in hbvdna ; 9 sample of the high lipids were lower than the contrast obviously . conclusion: t he hemolyt- ic blood and the serum with high bilirubin have no affecting to the real time fluorimetry pcr of hbvdna . however, the sample of high lipids will affect on it seriously . key words:real-time fluorimetrypcr of hbvdna ; sample; serum lipids; hemolytic blood; bilirubin page 34/ the predictive value of serum level of n -terminal pro - brain natriuretic peptide in heart function after acute myocardialinfarction /hu xiaozhou, zhangjie, cui liyan / department of clinical laboratory medicine, the third hospital of peking university , beijing 100083 objective : to observe the correlation between the levels of serum n-ter - minal pro-brain natriuretic peptide ( nt-probnp)on the 3rd day after acute myocardial infarction( ami)and left ventricular remodeling to evaluate the predictive levels of nt-probnp for left ventricular remodeling after ami. method: in 106 patients with first mi were examined after three days and 3 months with blood samples and echocardiography . left ventricular remodeling is estimated by the change of left ventricular end -diastolic volume ( lvedv) between the 3rd day and 3 months. based on the change of lvedv , the 106 patients were divided into two groups: left ventricular remodeling group and non- left ventricular remodeling group. results:the correlation coefficients with the change of lvedv were 0. 702 for serum nt-pro bnp . with a cut- off value of 0. 20, the area under the roc curve ( aucs)was 0. 892. con- clusion: the levels of serum of nt-probnp appropriately reflects heart func- tion after ami. key words: n-terminal pro -brain natriuretic peptide( nt -probnp); acute myocardial infarction( ami); left ventricular re - modeling (lvremodeling);echocardiography;roc curve page 37/ effects of qidan tongmai tablet on thrombus formation and blood viscosity in rats / long yin, wang zongren, ma jing, et al/ department of traditional chinese med- cine, xijing hospital, forth military medical university of pla , xi an 710032 objective: to study effects of qidan tongmai tablet ( qdtmt) , a compound preparation made up of pure traditional chinese medicine, on thrombus formation, plasma coagulation time and blood viscosity in wistar rats. method: the suspension of qdtmt was given to wistar rats by gas- trogavage for 7 days and the indexes of thrombus length, wet weight, dry weight, prothrombin time( pt) , kaolin partial thromboplastin time( kptt) , thrombin time( t t)and whole blood viscosity( b)were detected. results: compared with control group, qdtm tmaxand qdtmtmidmarkedly short- ened thrombus length( p 0. 05 0. 01) , wet weight and dry weight of thrombus ( p 0. 01) , extended pt , kptt and tt( p 0. 05) , reduced whole blood mid -shear( bm)and low-shear viscosity ( bl)( p0. 05) . con- clusion: qdtmt can significantly inhibit thrombus formation, extend blood coagulation, reduce blood viscosity and the effects are dosage-dependent. key words:qidan tongmai tablet;herb drugs;thrombus forma - tion; coagulation time; blood viscosity;rats page 40/ protective effect of danshen zhusheye on the ischemia myocardium / jiang haiping, wu yantao, cheng jingang/ the first people s hospital of wuxue, wuxue 435400 objective: to observe the effect of danshen zhusheye on the myocardial ischemia in rat and to study its mechanism. method : rat were randomly di- vied into four groups and supplied with 40% danshen zhusheye . one week later, the rat were injected with pituitrin( 30 u/kg) intraperitoneally to re- model myoc