酶放大免疫法测定他克莫司血药浓度的临床应用

目的 探讨酶放大免疫法测定他克莫司（FK506）血药浓度的方法学评价和临床应用的可行性.方法 对酶放大免疫法测定他克莫司血药浓度的回收率、精密度、线性范围等方法学指标进行测定.并对1 255例次肝肾移植术后进行他克莫司血药谷浓度测定,进行相关的统计学分析.结果 方法回收率为98.2%~101.03%,批内精密度的平均CV为5.07%,日间精密度的平均CV为6.79%,线性范围2.0~30.0 ng/mL.1 255例次治疗抗移植排斥反应FK506的血药浓度为3.0~12.9 mg/mL,占测定数的91.16%.结论 酶放大免疫法测定他克莫司血药浓度具有准确、稳定、简便、快速、自动化等优点,适用临床他克莫司血药浓度监测.在患者临床状况良好下,他克莫司浓度为3.0~12.9 ng/mL为适合的治疗血药浓度.     

液体芯片技术定量测定人体血清CEA、AFP、NSE和tPSA

目的 应用液体芯片技术,联合定量测定人体血清癌胚抗原（CEA）、甲胎蛋白（AFP）、神经元特异性烯醇化酶（NSE）和前列腺特异性抗原（tPSA）,并对其临床应用进行评价.方法 制备交联微球以及生物素标记抗体,利用双抗体夹心法对60个血清样本进行测定,并将其结果与化学发光免疫分析法（CLIA）作比较.结果 同时检测CEA、AFP、tPSA、NSE的线性范围分别为0.078～200 ng/mL、0.030～30.3 ng/mL、0.007～7.5 ng/mL、0.146～75 ng/mL;最低检测限为26.0 pg/mL、19.7 pg/mL、4.9 pg/mL、73.2 pg/mL;批内精密度CV〈9.0%,批间精密度CV〈13.2%;检测结果与CLIA测值的相关系数r分别为0.986、0.979、0.964、0.958（P〈0.001）.结论 液体芯片技术是一种具有极大优势的新型检测技术.该技术打破传统测定技术每次只能测定一个指标的限制,并且具有高通量、高灵敏度、检测时间短、样本用量少等特点.     

克隆酶供体免疫测定法在环孢菌素A浓度监测中的应用

目的 评价用自动生化分析仪测定全血环孢菌素A（cyclosporine A,CsA)浓度的克隆酶供体免疫,测定（CEDIA）方法。方法 分析测定CEDIA方法的精密度,灵敏度,线性,准确性（回收实验）以及与荧光偏振免疫分析法（FPIA／TDx)和酶扩大免疫测定法（EMIT）之间的相关性。结果 CEDIA测定的批内CV分别为1．8％（455．7μg/L）,2．29％（270．5μg/L）和7．0％（73．8μg/L）;最低检出限为7．0μg/L;测定线性范围可达1800μg/L;平均回收率为99．4％;CEDIA法与FPIA／TDx和EMIT法进行对比,相关系数分别为0．931和0．945。结论 CEDIA方法测定CsA血液浓度具有操作简便,标本不需繁琐的前处理,精密度好和较宽的分析范围等特点,更便于临床实验室应用。     

不同抗凝剂对肝移植受者FK506血药浓度测定的影响及临床意义

目的探讨不同抗凝剂对肝移植受者他克莫司（FK506）血药浓度测定的影响及临床意义。方法采集肝移植受者静脉全血34份,使用乙二胺四乙酸二钾（EDTA-K2）、枸橼酸钠和肝素锂抗凝剂分别抗凝同一份血样本,在IMx型免疫分析仪上用微粒子酶免疫分析法（MEIA）测定FK506血药浓度。结果肝素锂组与EDTA组差异无统计学意义（P=0.660）,呈正相关（r=0.982 8）。枸橼酸钠组与EDTA组差异有统计学意义（P=0.000）,呈正相关（r=0.961 3）。枸橼酸钠组与肝素锂组差异有统计学意义（P=0.000）,呈正相关（r=0.939 8）。结论肝素锂组与EDTA组几乎无偏差,但是枸橼酸钠组分别与EDTA组和肝素锂组存在一定程度的负偏差。枸橼酸钠对MEIA测定FK506血药浓度有一定的影响,应优先考虑EDTA或肝素锂抗凝剂采集全血样本。     

流式荧光免疫法检测多肿瘤标志物的方法学评价

目的评价流式荧光免疫法在多肿瘤标志物定量测定中的可靠性。方法用流式荧光免疫法对试剂盒精密度、线性、携带污染率、抗干扰性等指标进行评价,同时将Luminex^TM-100与Bio-Ekon SerozymeⅠ的结果进行比较。结果本方法化学性能的评价表明,批内变异系数（CV）均〈10%,批间CV均〈14%。线性良好（r〉0.99）。携带污染率均〈0.5%。血红蛋白（Hb）、胆红素（Bil）、三酰甘油（TG）对测定结果无显著干扰（P〉0.05）。与Bio-Ekon SerozymeⅠ内分泌定量分析仪及配套试剂相关性良好（r〉0.975）。对506名年龄在24-84岁健康体检人员标本用本方法进行多肿瘤标志物测定,正常参考值分别为甲胎蛋白（AFP）〈15 ng/mL、糖类抗原125（CA125）〈35 U/mL、癌胚抗原（CEA）〈5 ng/mL、糖类抗原199（CA199）〈35 U/mL、前列腺特异抗原（PSA）〈4 ng/mL。结论本方法测定结果准确、可靠且操作简便、快速,适宜于临床检测推广。     

EMIT法和HPLC法监测苯妥英和苯巴比妥血药浓度比较研究

目的比较酶增强免疫法（EMIT）和高效液相色谱法（HPLC）监测苯妥英（PHT）和苯巴比妥（PB）血浆药物浓度的相关性。方法收集癫痫患者服药后的稳态谷浓度血样,分别用EMIT法和HPLC法进行测定,考察2种测定方法的相关程度。结果以HPLC法测定结果（X）与EMIT法测定结果（Y）所作线性回归方程如下：YPHT=0.505＋1.200X（r=0.991）;YPB=0.103＋1.115X（r=0.970）;EMIT法测定血浆中PHT浓度较HPLC法高3.0μg/mL,EMIT法测定血浆中PB浓度较HPLC法高1.7μg/mL,2种方法测定PHT和PB血浆药物浓度结果差异有统计学意义（P〈0.05）。结论EMIT法和HPLC法测定2种抗癫痫药血浆浓度结果差异具有统计学意义,在PHT和PB治疗药物监测中应予以关注并作相应调整。     

两种磁分离酶联免疫法检测血清甲胎蛋白

目的改进磁分离酶联免疫法(MEIA)。方法采取预加微量标本、将磁铁置于微孔条侧壁和扣除上清液A值法,改良MEIA并建立了"差异磁分离酶联免疫法"(differential magnetic enzyme immunoassay,DMEIA)。求DMEIA法的线性范围、回归方程、变异系数(CV),比较了MEIA和DMEIA配对检测100份血清AFP结果差异。结果 DMEIA法检测AFP的回归方程Y=3.94X+16.45,r=0.992,线性范围5～500ng/mL,批内CV=7.7%,批间CV=8.5%;DMEIA法和MEIA法测定血清AFP值差异无统计学意义(t检验,P0.05),直线回归方程Y=3.418X+70,r=0.981,两法相关良好;检测原倍高浓度AFP时DMEIA法未出现(MEIA法出现)hook效应。结论 DMEIA法可克服hook效应,可用普通微孔板和酶标仪比色,可省去洗涤步骤,检测AFP结果(500ng/mL时)与MEIA法无明显差异。     

他克莫司血药浓度检测方法比较与差异研究

目的：回顾性分析肾移植患者临床检测他克莫司血药浓度由于实验室采用方法的不同而对结果造成的差异。方法：应用意大利DiaSorin公司酶联免疫吸附试验（ELISA）法和美国Abbott公司微粒子酶联免疫分析（MEIA）法分别对200份肾移植术后患者标本进行检测,比较两种方法对结果造成的差异。结果：两种方法在保证各自实验最佳条件下进行,t检验结果P〈0.05,差异有显著性,通过线性回归法分析得知,在浓度小于9.489 6 ng/mL的范围内,ELISA法检测值低于MEIA法检测值;在大于9.489 6 ng/mL范围内,ELISA法检测值高于MEIA法检测值。结论：两者方法学上的差异不会超出患者药物治疗的影响限度,两种方法具有很好的线性关系,相关性系数很高,两者的检测结果可以通过回归方程来互相推算。     

肾移植术后患者西罗莫司血药浓度的测定

目的建立肾移植术后患者西罗莫司血药浓度的液相色谱-质谱联用(HPLC-MS)测定方法。方法选取服用FK506/Cs A+西罗莫司+糖皮质激素三联免疫治疗的肾移植术后患者20例,采集西罗莫司谷浓度全血样本,以达那唑为内标,经沉淀蛋白及液液萃取处理后,经液相色谱分离,采用ESI离子源,以SIR正离子方式进行检测,检测离子为m/z 936.76(西罗莫司),m/z 338.26(内标)。同时将所建立的HPLC-MS法和微粒子酶免疫法(MEIA)分别测定肾移植患者全血西罗莫司浓度,评价两种分析方法测定结果的一致性。结果西罗莫司在0.2~100μg/L范围内线性关系良好(r=0.9992),最低定量限为0.2μg/L。日内、日间精密度均小于6%,方法学回收率为92.93%~102.98%。两种方法测得的数据相关系数为0.979(P0.05),两组数据间存在相关性(n=20)。当西罗莫司浓度在1.9~11.1μg/L时,两种方法测定的平均偏差为1.4μg/L。结论本文建立的HPLC-MS测定方法快速、准确、灵敏、选择性高,适用于临床对西罗莫司的血药浓度监测;HPLC-MS法和MEIA法测定西罗莫司全血浓度存在相关性,同一份样本用MEIA测定的浓度均值比HPLC-MS法测定的值高。     

酶联免疫吸附试验检测乙型肝炎病毒表面抗原临界样本复检范围的探讨

目的探讨酶联免疫吸附试验（ELISA）检测乙型肝炎病毒表面抗原（HBsAg）临界样本的复检范围。方法收集用ELISA初检HBsAg吸光度（A）值在0.07～2.00范围内的样本,用ELISA复检;初、复检结果不符和2次结果均在临界的样本,用微粒子酶免疫法（MEIA）进行复核,并用中和法做确认试验。统计分析结果,选择一个合适的样本复检范围。结果样本初检结果A值在0.070～0.104、0.105～0.300范围内,ELISA复检符合率分别为85.2%、56.9%;MEIA与ELISA复检结果的符合率分别为90.1%、93.9%。A值在0.301～0.500、0.501～1.000、1.001～1.500、1.501～2.000范围内,ELISA复检符合率依次为88.3%、93.8%、99.1%、99.2%,MEIA与ELISA复检结果的符合率为100%。确认试验和MEIA复检结果的符合率样本测定值（S）/阴性对照值（N）在1.50～1.99、2.00～5.00,分别为50.0%和92.7%,其他组均在97.1%以上。结论在操作规范化的实验室内,ELISA初检HBsAg复检范围宜选择样本A值在0.070～0.300范围内;大批量体检时,复检范围应放宽至0.070～0.500;乙肝标志物模式异常的样本也需复检。规定适宜的复检范围既能确保检测结果的准确性,又能避免人力和试剂等物品的浪费。     

Therapeutic monitoring of Tacrolimus: Aberrant results by an immunoassay with automated pretreatment

Background and aimsThe therapeutic monitoring of Tacrolimus (FK506) is necessary since low doses may cause graft rejection while overdosage is linked to nephrotoxicity, neurotoxicity, and many other adverse effects. Occasional notices of elevated values recorded in patients under maintenance regimen have prompted us to record all results exceeding the therapeutic range (>15 ng/mL) with no clinical signs or explanation and to compare the routine method (Siemens-Dade Dimension XPand) with other assays.MethodsEighty-four whole blood samples from 8 patients have been assayed by Dimension and by one or more of three other commercial assays (CMIA and MEIA, Abbott; EMIT, Siemens-Dade). As a reference, an automated LC-MS/MS method has been performed.ResultsIn all cases the raised Tacrolimus values were observed only by ACMIA, while the correlation (r²) of the other assays with LC-MS/MS was excellent for CMIA (0.97) and good for MEIA (0.88) and EMIT (0.83). The aberrant results were often recorded over a span of several weeks or months and could not be ascribed to a common cause.DiscussionAbnormally high Tacrolimus results by the ACMIA method have been observed in 1% of the patients currently followed up in our Center. Since these results may lead to erroneous adjustments of the drug dosage, we suggest checking any elevated or clinically unexplained Tacrolimus result by the ACMIA assay with other method(s) requiring an external pretreatment.     

β-人绒毛膜促性腺激素光激化学发光免疫分析方法的建立

目的建立一种检测人血清β-人绒毛膜促性腺激素（β-HCG）浓度的光激化学发光免疫分析方法。方法采用2株针对不同抗原表位的抗β-HCG抗体,其中一株抗体用来包被发光微粒,另一株抗体标记生物素,以双抗体夹心法检测人血清中的β-HCG浓度,并与Beckman-Coulter公司试剂（化学发光法）进行比较。结果发光微粒浓度为100μg/mL、生物素标记抗体浓度为7μg/mL时,检测范围为0~1 000 mIU/mL,灵敏度为0.16 mIU/mL,平均回收率为100.3%,批内变异系数（CV）为0.80%~3.99%,批间CV为2.25%,与TSH、FSH和LH的交叉反应率低,稳定性较好,与化学发光法的符合率好（r=0.99）。结论光激化学发光免疫分析方法测定β-HCG具有较高灵敏度、精密度和准确性,与化学发光法的符合率较好,适用于临床测定。     

Analytical performance and clinical results of a fully automated MEIA system for brain natriuretic peptide assay: comparison with a point of care testing method.

The aim of this study was to evaluate the analytical performance of a recently available immunoassay for brain natriuretic peptide (BNP), based on microparticle enzyme immunoassay (MEIA, AxSYM System, Abbott Laboratories), whose analytical characteristics and clinical results were compared with those of a point of care testing (POCT) method (TRIAGE system, Biosite Diagnostics). The within-run and total imprecision of the MEIA system were 18.4% and 19.8% at 21 ng/l, 8.0% and 14.8% at 183 ng/l, and 5.7% and 14.0% at 319 ng/l, respectively. The detection limit of the MEIA system was tested by repeatedly measuring (n=20) the 0 calibrator in four different runs; a mean +3 SD value of 5.6+/-4.8 ng/l (range 1.8-12.6 ng/l) was obtained. A close linear relationship (MEIA= -22.5+/-1.71 POCT method, R=0.950, n=296) was found (BNP concentration: 5-5500 ng/l), with a significant bias (mean difference: 164.8 ng/l, p<0.0001). Mean BNP concentration measured in 94 reference subjects (57 women and 37 men; mean age 43.5+/-14.0 years) was higher with MEIA than POCT, (25.9+/-32.7 ng/l vs. 11.7+/-8.9 ng/l, p<0.0001). The same trend was observed also in 202 cardiac patients (620.6+/-1082.2 ng/l vs. 386.1+/-594.5 ng/l, p<0.0001). Our data suggest that MEIA and POCT have quite similar analytical performance but different clinical results. Then, different reference values, as well as cut-off values, should be taken into account for the clinical use of these two immunoassays.     

两种免疫法联合肾功能指标测定他克莫司血药浓度的比较

背景：不同免疫法测定的他克莫司血药浓度,对免疫抑制作用及毒性反应的预测能力是否有差异,在肾功能异常时是否能够更加敏感的反映患者血药浓度,是值得研究的问题。目的：联合肾功能指标,分析酶联免疫吸附技术和酶增强免疫技术测定他克莫司血药浓度的相关性。方法：收集133例应用他克莫司治疗的肾移植患者血样,分别用酶联免疫吸附技术、酶增强免疫技术测定血药浓度。分析两种方法测定的相同浓度范围内肾功能异常的发生率;分析不同浓度范围内,两种方法测定结果的相关性。结果与结论：在肾功能异常时,比较两种方法测定的浓度,酶联免疫吸附法测定结果均值较高,且波动较大。酶联免疫吸附法测定他克莫司血药浓度5-20μg/L范围内,肾功能异常发生率较酶增强免疫法少,分析显示两种方法测定结果总体差异无显著性意义（r=0.9045,P＞0.05）。以酶联免疫吸附法为标准,在他克莫司血药浓度＜2.0μg/L时,酶增强免疫法测定结果明显较高（P＜0.01）。在血药浓度为≥2.0μg/L时,两种方法测定结果差异无显著性意义（P＞0.05）。说明酶增强免疫法与酶联免疫吸附法相关性较好,均适合于临床常规测定他克莫司的血药浓度,建议结合肾功能指标,细化区分两种测定方法的参考范围。同时,在患者血药浓度极低（＜2.0μg/L）时,不建议采用酶增强免疫法。     

Performance evaluation of a new chemiluminescent cardiac troponin I assay

Cardiac troponin I is a marker for diagnosis of myocardial damage. Several immunoassays are currently available for determination of concentrations of troponin I in serum. We evaluated a chemiluminescent assay for troponin I using ACS:180 automated analyzer (Bayer Diagnostics). We compared our results with two other immunoassays using the OPUS Magnum (OPUS troponin I assay, Dade Behring) and AxSYM (microparticle enzyme immunoassay, Abbott) analyzers. The within-run and between-run CVs were less than 5% for all three levels of controls. The chemiluminescent assay for troponin I was linear up to a serum troponin I concentration of 50 ng/mL and the detection limit was 0.1 ng/mL of troponin. A good correlation between troponin I concentration measured by the chemiluminescent assay (y axis) and the microparticle enzyme immunoassay (MEIA) (x axis) was observed, although the concentrations of troponin I in individual specimens were approximately four times higher, when measured by the MEIA assay, than those measured by chemiluminescent assay. The correlation coefficient was 0.98 with the regression equation y = 0.22x + 1.125. We also observed a good correlation in troponin I concentrations obtained by the chemiluminescent assay (y axis) and OPUS troponin I assay (x axis). The correlation coefficient was 0.96 and the regression equation was y = 0.79x - 0.52. The correlation coefficient was 0.93 when we compared troponin I concentrations obtained by the OPUS assay (x axis) with the corresponding concentrations obtained by the MEIA assay (y axis). The corresponding regression equation was y = 0.25x + 3.5. We conclude that the chemiluminescent troponin I assay showed good analytical performance.     

肝移植后他克莫司血药浓度对外周血单个核细胞内HBV DNA的影响

背景：肝移植后他克莫司等免疫抑制剂的长期应用导致机体细胞免疫功能降低,并有可能影响机体对乙肝病毒的清除。目的：分析乙肝相关肝移植患者后不同浓度他克莫司对外周血单个核细胞中的HBVDNA含量的影响。方法：纳入乙肝相关终末期肝病肝移植受者23例,根据移植后12周清晨空腹他克莫司血药浓度,分为高浓度组（≥10μg/L）9例和低浓度组（〈10μg/L）14例,同时用荧光标记单克隆抗体结合流式细胞技术检测外周血T细胞亚群的百分比,用实时荧光定量PCR检测外周血单个核细胞内的HBVDNA。结果与结论：用多元线性回归分析外周血单个核细胞内的HBVDNA含量与CD8＋CD152＋呈正相关,与CD8＋CD28＋呈负相关。高血药浓度他克莫司的患者外周血单个核细胞内的HBVDNA高于低浓度组,其改变与反映细胞免疫功能的指标CD8＋CD152＋和CD8＋CD28＋的变化有关。     

Effect of the hematocrit and its correction on the relationship between blood tacrolimus concentrations obtained using the microparticle enzyme immunoassay (MEIA) and enzyme multiplied immunoassay technique (EMIT)

The Abbott microparticle enzyme immunoassay (MEIA) and the Dade Behring enzyme multiplied immunoassay technique (EMIT) are the most frequently used methods in the therapeutic drug monitoring of tacrolimus; however, a hematocrit-dependent interference for the MEIA has been described. In 244 whole blood samples from patients with liver (n=152) and kidney (n=92) transplants, the MEIA/EMIT ratio presented a highly significant negative correlation with the hematocrit (r = -0.482, p < 0.001). On distributing the samples into three groups with a hematocrit of less than 30%, 30-40%, and higher than 40%, different regression equations were found between the results of MEIA and EMIT and demonstrate the different effect of the hematocrit on both immunoassays. Correcting the MEIA results by calculation for a hematocrit of less than 30% and higher than 40% (Hermida et al. Clin Lab 2005; 51: 43-45) led to a regression with EMIT that was similar to that found between MEIA and EMIT for the group of samples with a hematocrit of 30-40%. Furthermore, the corrected MEIA/EMIT ratio had a poor correlation with the hematocrit (r = 0.149, p < 0.05). In 95 samples with a hematocrit of less than 25% (n=73) and higher than 40% (n=22) we also determined the tacrolimus levels using the modified MEIA method to correct hematocrit interference, as proposed by Tomita et al. (Ther Drug Monit 2005; 27: 94-97). In the samples with a hematocrit of less than 25%, correcting the MEIA results by calculation produced results that were similar and had a high correlation coefficient (r = 0.954, p < 0.001) to those of the modified MEIA method, whose application as a routine practice is more expensive and laborious. Calculation of the corrected MEIA values in anemic patients may be useful for the therapeutic monitoring of tacrolimus.     

Increased digitalis-like immunoreactive substances in neonatal plasma measured using fluorescence polarization immunoassay

OBJECTIVE: To better define the reported increased digitalis-like immunoreactive substances (DLIS) in neonatal plasma, we studied the relation among plasma DLIS level, blank intensity (BLK-I) value at FPIA measurement and plasma total bilirubin level. METHODS: The DLIS levels were measured in 10 neonates with or without jaundice and 10 infants in good health, using fluorescence polarization immunoassay (FPIA) and microparticle enzyme immunoassay (MEIA). BLK-I value and plasma total bilirubin level were also measured simultaneously. RESULTS: In neonates with jaundice, DLIS using FPIA, BLK-I and total bilirubin level were 0.58 +/-0.13 ng/mL, 2598 +/- 408, and 17.98 +/- 1.13 mg/dL, respectively, before phototherapy, and 0.33 +/-0.06 ng/mL, 1886 +/- 237, and 15.16 +/- 2.07 mg/dL after phototherapy. Corresponding values in neonates without jaundice were (DLIS: 0.34 +/-0.04 ng/mL; BLK-I: 1,764 +/- 278; total bilirubin: 10.37 +/- 4.54 mg/dL); in healthy infants (0.12 +/-0.06 ng/mL, 400.7 +/- 4.6 and 0.42 +/- 0.13 mg/dL, respectively) and in healthy volunteers (0.10 +/-0.07 ng/mL, 403.1 +/- 8.4, and 0.58 +/- 0.30 mg/dL, respectively). Using MEIA, DLIS was not detected in 10 neonates, 10 infants and 20 healthy volunteers. CONCLUSIONS: A fluorescent compound related to bilirubin increased the BLK-I value in the measurement of neonatal plasma using FPIA. The fluorescence was not the result of endogenous digitalis-like factors.