同位素稀释液相色谱串联质谱法测量血清未结合雌三醇的协作研究

目的用同位素稀释液相色谱串联质谱(ID-LC/MS/MS)候选参考方法进行实验室间血清未结合雌三醇(u E3)协作研究,通过该研究进一步确认方法的性能特征(正确度和精密度),并将该方法在国内参考实验室推广,推动u E3标准化进程。方法依据ISO/WD 15725-1和GB/T 6379标准,拟定我国血清u E3协作研究方案。协作研究分为两个阶段:初步试验和正式试验,共9家参考实验室参加。按要求收集5个浓度水平样品并进行均匀性评价,分发到9家参考实验室,要求每个样品每日重复测量5次,连续测量3 d。计算各实验室测量结果的偏移和精密度,用格拉布斯(Grubbs)检验和柯克伦(Cochran)检验识别离群值和离群实验室。根据合格实验室的结果计算靶值,并向离群实验室和偏出允许范围的实验室提供整改建议。结果样品均匀性评价:所有样品测量结果计算F值均小于F0.05(9,20)临界值,样品中u E3是均匀的。初步试验:Grubbs检验,1个实验室测量结果为离群值;Cochran检验,2个实验室检验结果为离群值。剔除离群值后计算靶值;2017E301为(22.08±0.24)nmol/L;2017E302为(33.46±1.67)nmol/L。2个实验室结果超出允许范围。正式试验:Grubbs检验,所有实验室测量结果均未检出离群值;Cochran检验,3个实验室数据出现离群值。剔除离群值后计算靶值,2017E303为(10.36±0.35)nmol/L,2017E304为(15.47±0.26)nmol/L,2017E305为(46.97±1.19)nmol/L;各实验室间测量结果不精密度分别为1.14%~2.21%、0.79%~1.93%、0.60%~2.09%,测量结果偏移分别为-6.18%~4.83%、-2.26%~2.39%、-4.19%~4.07%。结论通过组织开展协作研究,确认各参考实验室通过协作研究方案能够快速建立并运行u E3候选参考方法,除个别实验室外各实验室的检测性能满足预定要求(不精密度3.0%,偏移7.5%),进一步确认了该方法的性能和各实验室运行参考方法的能力。通过初步研究和正式研究对研究方案的各实验环节进行了验证和完善;推动了u E3项目参考方法在我国参考实验室的运行,为后续厂商试剂主校准品联合定值或标准物质研制搭建了平台,促进u E3项目的标准化。     

磁共振在单心室心功能检测中的应用

目的利用磁共振成像（MRI）对53例单心室进行心功能检测研究,测得数值与心血管造影（CAG）结果作对照。方法53例单心室,男31例,女22例,平均年龄（4.49±3.95）岁;术前43例,术后10例;分别通过核磁共振的电影序列扫描和CAG计算单心室舒张末、收缩末容量,心每搏量和射血分数,利用均数及t检验进行数据统计分析。结果53例单心室MR心功能测量值：舒张末容量均值（101.61±33.61）ml/m^2,收缩末容量均值（40.31±16.56）ml/m^2,每搏量均值为（62.59±22.66）ml/m^2,射血分数为（61.23±8.45）%;CAG测得单心室舒张末容量均值（137.59±53.59）ml/m^2,收缩末容量均值（61.81±22.13）ml/m^2,每搏量均值为（76.05±35.46）ml/m^2,射血分数均值为（53.61±9.68）%,经统计学分析,两组参数间差异无统计学意义。结论利用MR对单心室的心功能检测是目前非创伤性检查及术后随访中最为可靠的方法。     

鼻声反射在鼻中隔矫正术围术期的临床应用

目的探讨鼻声反射（acoustic rhinometry,AR）技术在鼻中隔偏曲矫正术围术期的临床应用价值。方法应用HOOD实验室生产的ECCOVISION型AR测量系统,对40例鼻中隔偏曲矫正术围术期测量偏向侧及对侧鼻腔容积（NCV）、鼻阻力（NR）、鼻腔最小横截面积（NMCA）及最小横截面距前鼻孔的距离（DCAN）,并进行统计分析。结果偏向侧NCV和NMCA术前分别为（6.46±1.28）cm^3、（0.42±0.11）cm^3,术后为（9.24±1.24）cm^3、（0.61±0.08）cm^3,差异有统计学意义（P〈0.01） 偏向侧NR术前为（3.74±1.38）cmH2O/（L·min）,术后为（2.13±0.63）cmH2O/（L·min）,差异有统计学意义（P〈0.01）,对侧及偏向侧DCAN术前分别为（1.96±0.09）cm、（1.98±0.08）cm,术后为（1.98±0.06）cm、（1.96±0.06）cm,差异无统计学意义（P〉0.05）。结论AR技术能客观评价鼻中隔矫正前后鼻腔通气功能。     

多形性胶质母细胞瘤MR弥散张量成像应用研究

目的本研究旨在探讨平均扩散系数（ADC）和各向异性指数（FA）在鉴别多形性胶质母细胞瘤肿瘤组织、水肿及正常脑组织,以及探讨ADC值及FA值在对肿瘤浸润范围中的作用。方法14例多形性胶质母细胞瘤在治疗前行常规MRI、增强扫描及弥散张量成像（DTI）,在T1WI增强、T2WI上确定肿瘤组织、水肿及正常脑组织,在ADC图和FA图上测量这些区域的FA值及ADC值,用方差分析评定值之间的差异。结果肿瘤囊变区的ADC值（2.07±0.631）×10^-3mm^2/s最高,其次为水肿区（1.39±0.164）×10^-3mm^2/s、肿瘤强化中心（1.13±0.187）×10^-3mm^2/s、肿瘤强化边缘（1.04±0.254）×10^-3mm^2/s、瘤周正常白质区（0.779±0.088）×10^-3mm^2/s、对侧正常白质（0.748±0.082）×10^-3mm^2/s。对侧正常白质FA值最高（0.538±0.084）×10^-3mm^2/s,肿瘤囊变区最低（0.09±0.028）×10^-3mm^2/s。肿瘤强化边缘与囊变坏死区、水肿区、瘤周正常白质以及对侧正常白质ADC值差别均具有显著性意义（P〈0.05）,瘤周正常白质区与对侧正常白质区ADC值无显著性意义（P〉0.05）。肿瘤强化边缘与囊变坏死区、瘤周正常白质区、对侧正常白质区FA值差别均具有显著性意义（P〈0.05）,瘤周正常白质区与对侧正常白质区FA值有显著性意义（P〈0.05）,肿瘤强化边缘FA值与水肿区FA值差异无显著性意义（P〉0.05）。结论ADC值可用于区分多形性胶质母细胞瘤正常脑白质、水肿和肿瘤强化边缘,FA值对于组织学鉴别无明显意义,FA值对肿瘤浸润范围有重要的意义。     

中国成人肝微粒体P450酶活性研究

目的观察中国成人肝微粒体细胞色素P450（CYP450）、细胞色素b5（CYb5）的含量及常见几种药物代谢酶活性水平及在性别、民族间的分布差异。方法肝组织匀浆10000×g离心去除沉淀,再以100000×g超速离心60min,取沉淀加PBS甘油缓冲液,充分溶解制成微粒体悬液,用Lowry法测定蛋白含量;用双光道紫外分光光度计测定CYP450和CYb5的含量及P450酶活性。结果48例成人肝微粒体CYP450和CYb5含量分别为（0．46±0．07）nmol／mg和（0．44±0．04）nmol／mg;男性组CYP450和CYb5含量分别为0．49±0．05）nmol／mg和（0．46±0．06）nmol／mg,均高于女性组的（O．41±0．08）nmol／mg、（O．42±0．05）nmol／mg,P〈0．05;汉族、回族和壮族3个民族P450和CYb5含量作q检验进行两两比较,各组间差异均无统计学意义（P〉0．05）。药物代谢酶NADPH细胞色素C还原酶（NR）、乙基吗啡N-脱甲基酶（EDM）、氨基比林N-脱甲基酶（ADM）、苯并芘羟化酶（BPH）和戊巴比妥侧链羟化酶（PSCH）等酶活性水平分别为（0．71±0．13）nmol／（mg·min）,（0．89±0．18）nmol／（mg·min）,（0．99±0．17）nmol／（mg·min）,（0．07±0．01）nmol／（mg·min）,（4．15土0．65）μmol／（mg·min）,其中EDM活性水平女性组高于男性组（P〈0．05）,PSCH活性水平男性组高于女性组（P〈0．05）,其他各种酶活性男女之间无显著性差异;上述5种酶活性水平经q检验,汉族、回族和壮族3个民族之间差异无统计学意义（P〉0．05）。结论测定了中国成人肝微粒体CYP450、CYb5含量及NR,EDM,ADM,BPH,PSCH等酶活性水平,男性组CYP450、CYb5含量及PSCH活性高于女性组,而EDM活性水平女性组高于男性组,上述7种指标在汉族、回族和壮族之间差异均无统计学意义。     

山莨菪碱对兔心室肌细胞离子通道电流的影响

目的研究山莨菪碱对兔正常离体心室彤睫田胞离子通道电流的影响,探讨其抗心律失常的细胞学离子机制。方法以酶解的方法分离兔心室肌外膜单个心室肌细胞,采用全细胞膜片钳技术,研究不同浓度山莨菪碱对兔正常心室肌细胞跨膜钠离子通道电流（INa）、L-钙通道电流（ICa-L）及瞬间外向钾通道电流（Ito）的影响。结果山莨菪碱浓度为10nmol／L时,对INa无明显影响（P〉0．05）。山莨菪碱浓度为100nmol／L时,INa电流密度减少到（-33．25±4．46）pA／pF,1000nmol／L时INa电流密度减少到（-29．32±3．55）pA／pF,抑制率分别为22．3％和31．5％,与基础值比较,差异均有统计学意义（P〈0．01）。山莨菪碱浓度为10nmol／L时,对ICa-L无明显影响（P〉0．05）。山莨菪碱浓度为100nmol／L时,ICa-L．电流密度减少到（-2．15±1．02）pA／pF,1000nmol／L时ICa-L电流密度减少到（-1．82±0．86）pA／pF,抑制率分别为31．3％和41．8％,与基础值比较,差异均有统计学意义（P〈0．01）。山莨菪碱浓度为10nmol／L时,Ito电流密度由（17．41±3．13）pA／pF减少到（16．13±2．93）pA／pF,100nmol／L时减少到（15．11±2．88）pA／pF,1000nmol／L时减少到（14．96±2．82）pA／pF,抑制率分别为7．3％、13．2％和14．1％,均无统计学差异（P〉0．05）。结论山莨若碱对离体正常单个心室肌细胞INa和ICa-L具有剂量依赖性抑制作用。     

前臂加压和上臂加压对高频超声检测肱动脉内皮依赖性舒张功能的影响

目的探讨不同加压部位对高频超声检测肱动脉内皮依赖性舒张功能（FMD）结果的影响。方法对65例正常人,应用高频超声分别检测上臂加压后和前臂加压后血流介导的肱动脉峰值FMD值并做对比分析。结果（1）上臂加压后肱动脉的最大扩张值明显大于前臂加压后[（3.99±0.60）mm和（3.88±0.66）mm,P〈0.01];（2）上臂加压后肱动脉的峰值流速明显大于前臂加压[（185.69±56.03）cm/s和（156.88±40.36）cm/s,P〈0.05];（3）上臂加压后的峰值FMD明显大于前臂加压[（11.70±3.44）%和（8.90±2.02）%,P〈0.01]。结论加压阻断血流的部位是FMD值的重要决定因素之一,上臂加压后的肱动脉FMD大于前臂加压,导致其差别的主要原因在于上臂加压后高速血流引起的切应力较大。     

血清降钙素原及C反应蛋白在社区获得性肺炎中的临床意义

目的 探讨血清降钙素原（PCT）及C-反应蛋白（CRP）,WBC在社区获得性肺炎实验诊断中的价值.方法 采用酶免荧光定量法,测定116例细菌感染性肺炎及36例非细菌感染性肺炎患者、80例正常对照的PCT,以 PCT＞0.25 μg/L作为社区获得性肺炎细菌感染的阳性界值,上述三组同时测定了PCT,CRP和WBC.结果 细菌感染性肺炎患者血清PCT（2.08±1.13）μg/L,CRP（67.12±23.54）mg/L和WBC（10.09±2.89）×109/L均比非细菌感染性肺炎患者的PCT（0.53±0.67）μg/L,CRP（46.18±31.42）mg/L,WBC（7.36±3.24）×109/L升高（P＜0.05）,但PCT的灵敏度及特异度更高（P＜0.01）,分别为96.6%,93.8%,而CRP,WBC灵敏度和特异度在细菌感染性肺炎和非细菌感染性肺炎患者中都没有差异（P＞0.05）.结论 在社区获得性肺炎中,PCT是一个较好的诊断或鉴别诊断的指标,比CRP更有临床价值.     

人工肝和肝移植术后的乙肝三系统定量观察

目的观察人工肝技术和肝移植手术对重症乙肝患者乙肝病毒标志物的影响。方法乙肝三系统定量采用时间分辨免疫荧光技术,乙肝病毒DNA定量采用实时荧光定量PCR法。结果28例实施人工肝技术的重型肝炎患者治疗前后的乙肝三系统定量和HBVDNA定量结果为：HBsAg171．19±32．10ng／mL,HBeAg0．03±0．029NCU／mL,抗-HBe3．97±4．61NCU／mL,抗-HBc5．98±3．31NCU／mL,HBVDNA（1．1×10^7）±（6．81×10^6）copy／mL和HBsAg168．14±39．40ng／mL,HBeAg0．02±0．023NCU／mL,抗-HBe3．95±4．34NCU／mL,抗-HBc6．41±3．13NCU／mL,HBVDNA（1．1×10^7）±（6．23×10^6）copy／mL。P值均大于0．05,差异无统计学意义。26例施行肝移植手术的患者治疗前后的乙肝三系统定量和HBVDNA定量结果分别为HBsAg144．65±77．00ng／mL,HBeAg0．02±0．028NCU／mL,抗-HBe4．32±6．43NCU／mL,抗-HBc6．04±4．88NCU／mL,HBV DNA（1．0×10^7）±（6．89×10^6）copy／mL和HBsAg6．54±3．32ng／mL,HBeAg0．02±0．016NCU／mL,抗-HBe4．79±6．44NCU／mL,抗-HBc5．97±4．64NCU／mL,HBV DNA（1．04×10^2）±（3．40×10^2）copy／mL。除抗-HBe和抗-HBc,P值大于0．05外,其他项目P值均小于0．05,差异有统计学意义。结论肝移植手术可有效清除乙肝患者体内的乙肝病毒,人工肝支持系统虽不能有效清除乙肝患者体内的乙肝病毒,但可改善重型肝炎患者体内严重紊乱的内环境,为肝移植手术创造合适的条件和机会,赢得宝贵时间。     

溃疡性结肠炎患者血浆和淋巴细胞内亲环素A测定的临床意义

目的 探讨血浆和淋巴细胞内亲环素A（CyP A）水平与溃疡性结肠炎（ulcerative colitis,UC）的关系.方法 用酶联免疫吸附试验（ELISA）同时检测116例UC患者（其中活动期67例,缓解期49例）和50例正常人血浆和淋巴细胞内的CyP A水平.结果 活动期UC患者血浆和淋巴细胞内CyP A含量分别为（380±145）μg/L和（2 780±500）μg/g蛋白,均显著高于正常对照组的（160±78）μg/L （u值为10.54,P〈0.01）和（2 280±375）μg/g蛋白（u值为6.18,P〈0.01）,也显著高于缓解期UC患者（u值分别为8.19和5.37,P均〈0.01）.结论 血浆和淋巴细胞内CyP A升高可作为UC活动期诊断的辅助参考指标.     

LC–MS/MS progress in newborn screening

Newborn screening programs detect treatable disorders in infants before they become symptomatic. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) has greatly increased the screening possibilities by monitoring levels of amino acids and acylcarnitines. After the initial screening step, LC–MS/MS can also be used in screening positive samples as a second tier test to differentiate between true and false positive samples.As the list of disorders screened for by LC-tandem MS increases, questions arise about screening for untreatable disorders, such as some lysosomal storage diseases (LSDs). For LSDs screening methods are being developed and tested more quickly than treatments are becoming available. This goes against one of the main tenets of newborn screening which requires that a treatment be available.LC–MS/MS can detect several disorders with a single injection, which is important in high throughput laboratories. Measuring different amino acids and acylcarnitines can be used to detect up to 45 different inherited disorders depending on how diseases are counted. The LSD assays are designed in a similar way to detect multiple disorders with common sample preparation and a single injection. The clinical implications of applying this technology to NBS on a large scale in many jurisdictions across the world are discussed.     

串联质谱和高效液相色谱-串联质谱二次筛查联合应用于新生儿MMA筛查

目的探讨串联质谱和高效液相色谱-串联质谱二次筛查联合应用在甲基丙二酸血症(MMA)中的筛查价值。方法收集新生儿串联质谱初筛结果中C3、C3/C2、C3/C0单一或多个指标异常的新生儿干血滤纸片标本,用高效液相色谱-串联质谱的方法定量检测原始血片中甲基丙二酸、甲基枸橼酸和高半胱氨酸,对二次筛查后疑似阳性的新生儿进行召回复查,并进行尿气相色谱/质谱检测。临床诊断患儿进一步予以基因检测进行确诊。结果共收集423例C3、C3/C2、C3/C0单一或多个指标异常的新生儿筛查标本,初筛阳性率约为1%,行联合筛查检测结果发现8例标本中甲基丙二酸和同型半胱氨酸表达水平明显升高,召回复查尿气相色谱质谱提示甲基丙二酸轻度升高。结论串联质谱和高效液相色谱-串联质谱联合应用可以提高新生儿MMA筛查的阳性预测值、降低假阳性率,在新生儿遗传代谢病筛查中具有重要价值。     

LC-MS/MS systematic toxicological analysis: comparison of MS/MS spectra obtained with different instruments and settings

OBJECTIVES: To compare the mass spectra obtained using a linear-ion-trap (LIT) tandem mass spectrometer (QTRAP) operated in the "enhanced product ion scan" (EPI) mode with those obtained in the classical triple-quadrupole product ion scan (PIS) mode run on the same as well as on two other instruments (TSQ-Quantum and Quattro-Micro). DESIGN AND METHODS: After tentative standardization of ion fragmentation and transmission in both polarities using a reference compound (glafenine) on the three instruments, eight test compounds detected in the positive mode and five in the negative mode were systematically infused in different ionization sources and spectral acquisition performed over approximately 5 s. The relative intensity of the ions present in the resulting spectra was quantitatively and statistically compared. Also, the intra-day and inter-day variabilities of these relative intensities, as well as the effect of increasing compound concentration, were studied using QTRAP operated in EPI mode. RESULTS: The EPI and PIS modes operated on a single LIT MS/MS instrument resulted in significant differences in relative ion intensities in both polarities, and so did the other two instruments despite prior standardization with glafenine. Some fragments could be absent in certain spectra, but no unexpected or unique fragments showed up. Intra-day variability was smaller in the LIT EPI than in the regular PIS mode and in the positive than in the negative polarity. In EPI mode, both intra- and inter-day variabilities increased when the relative intensity decreased. The effect of increasing concentration on the relative intensity of major and minor ions was small but significant in both polarities. Finally, contamination and cleansing of the ionization source also had noticeable effects on MS/MS spectra, though the cause is unclear. CONCLUSIONS: MS/MS spectra do not offer the expected inter-instrument reproducibility despite an attempt at standardizing the fragmentation conditions using a reference compound. However, although the inter-instrument differences in ion relative intensity were significant, the spectra obtained looked almost similar. This suggests that in library searching algorithms, higher weight should be assigned for the m/z ratios than for their relative intensity in the spectra.     

Multi-center evaluation of a commercial Kit for tacrolimus determination by LC/MS/MS

ObjectivesA multi-center evaluation (3 sites) of the LC/MS/MS MassTrak? tacrolimus Immunosuppressants Kit (Kit) was undertaken.Design and methodsTen aspects of the analytical performance of the Kit were investigated based on FDA and CLSI guidelines.ResultsThe linear analytical range of the procedure was between 0.68 and 31.7 ng/mL. Within-run and total imprecision were < 6% and < 8% (n = 240), respectively. Recoveries of tacrolimus added to clinical samples that contained between 5 and 10 ng/mL of tacrolimus before addition were 99, 102 and 105% at 5.0, 10 and 20 ng/mL, respectively. Comparison of in-house and Kit procedures in samples from liver (n = 50–58) or kidney (n = 50 or 51) transplant recipients yielded method mean biases between ? 2.0 and + 10.7% at 5 and 15 ng/mL.ConclusionsThis evaluation indicates that the Kit is suitable for the monitoring of tacrolimus in kidney and liver transplant recipients.     

高效液相色谱法及串联质谱法在苯丙酮尿症筛查诊断中的应用

目的考查了高效液相色谱(HPLC)法和串联质谱(MS/MS)法在遗传代谢性疾病苯丙酮尿症(PKU)筛查中的应用及意义。方法利用MS/MS法和HPLC法分别分析了1 860例出生3d至11岁儿童的干血滤纸片及全血标本中苯丙氨酸(Phe)、酪氨酸(Tyr)浓度及其比值。结果 MS/MS法和HPLC法Phe线性范围为26.02~101.11μmol/L和32.04~132.50μmol/L,Tyr线性范围为41.50~253.07μmol/L和32.85~111.50μmol/L,平均回收率Phe为97.36%和98.43%,Tyr为96.71%和98.99%,批内CV Phe为4.31%和3.97%,Tyr为4.09%和4.01%,批间CV Phe为5.73%和4.58%,Tyr为6.01%和5.24%。结论两种方法均能灵敏,特异的测定血中Phe和Tyr浓度,满足对PKU筛查及诊断的需要。