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1、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 。
2、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
3、 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
4、 ,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.
5、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
6、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
7、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
8、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
9、+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 ,
10、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
11、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
12、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
13、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
14、 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
15、。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 . Alte
16、rnans 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 durin
17、g 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 Can
18、cer/ 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.
19、 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 diseas
20、e 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%. Th
21、e 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 an
22、d 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
23、/ 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 t
24、he 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 pos
25、itive 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 ob
26、viously . 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; Bilirub
27、in 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 ob
28、serve 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 pat
29、ients 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 d
30、ivided 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 lev
31、els 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 Thro
32、mbus 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
33、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 parti
34、al 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) , re
35、duced 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; Coagulat
36、ion 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 i
37、ts 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 myocardial ischemic. ECG was recorded 3 h later, the activities of nitric oxide synthase( NOS) , s
38、uperoxide dismutase( SOD) , the content of malondi- adehyde( MDA) in the myocardium and creatine kinase( CK) activity of serum were examined. Results:Danshen reduced changes in J spot in ECG , in- creased activity of NOS and SOD, inhibited on MAD content and CK activa- tion in the myocareium induced
39、 by pituitrin and nitroglycerin. Conclusion: These results suggested the Danshen can protect myocardium from ischemic. The mechanism may be related to increase activity of NOS and SOD , the re- duction of MAD content and CK activation. Key words:Danshen zhusheye; Myocardial ischemia; NOS; SOD; MAD P
40、age 42/ The Effect of Bood Flow in Nasal Mucosa of Chronic Rhinitis TreatedbyUltrasonic Atomization Inhaling / Zhang Pin, Qiu Ming, Jiang Guangming/Department of Otolaryngology,People s Hospital of Luohu District, Shenzhen 518001 Objective: To investigate the influence of bood flow in nasal mucosa of chronic rhinitis treated by ultrasonic atomization inhaling . Method:60 pa- tients were treated by Danshen injection and Yuxingcao injection, made from medicinal herbs, by mean of ultrasonic, to last three courses. In the mean