液相色谱-串联质谱法检测血浆醛固酮和血管紧张素1筛查原发性醛固酮增多症的应用评价

目的 评价液相色谱-串联质谱法（LC-MS/MS）检测血浆醛固酮和血管紧张素1用于原发性醛固酮增多症（PA）筛查的应用效果。方法 建立LC-MS/MS法检测血浆醛固酮和血管紧张素1的检测体系,并进行性能验证。收集疑似PA患者共58例,分别取卧位和立位进行采血检测血浆醛固酮和血管紧张素1,比较LC-MS/MS法与免疫分析法（CLIA）在卧位和立位条件下筛查PA的效能,并进行一致性评价。结果 LC-MS/MS法在性能验证中表现出良好的线性、精密度、准确度和定量下限;LC-MS/MS法与CLIA法的结果呈中等程度正相关性;筛查PA时LC-MS/MS法在卧位和立位条件下的曲线下面积均优于CLIA法,特别是在卧位条件下,LC-MS/MS法的AUC达到了0.85,具有相对较高的准确性。结论 LC-MS/MS法在筛查PA方面可能具有更高的诊断准确性和临床预测价值,但在实际推广中仍需考虑其设备和操作技术的局限性。     

醛固酮肾素定量比值筛查原发性醛固酮增多症的探讨

目的探讨醛固酮肾素定量比值(PAC/PRC以下简称AARR)筛查原发性醛固酮增多症(以下简称为原醛症)的价值。方法使用化学发光方法检测32例原醛症和88例原发性高血压患者立、卧位醛固酮和肾素浓度,计算醛固酮肾素浓度比值(AARR),构建AARR对原醛症的ROC曲线,确定AARR筛查原醛症的最佳切点。结果原醛症患者组立位肾素浓度为4.55(15.67)pg/ml,卧位为2.85(5.34)pg/ml,立位醛固酮浓度为213.70(237.38)pg/ml,卧位为207.52(137.90)pg/ml, 立位AARR为61.53(182.84),卧位为100.69(254.03)。原发性高血压患者组立位肾素浓度为6.80(11.90)pg/ml,卧位为4.79(8.36)pg/ml,立位醛固酮浓度为121.20(31.94)pg/ml,卧位为112.47(23.99)pg/ml,立位AARR为17.49(28.57),卧位为22.67(37.43)。立位AARR筛查原醛症的ROC曲线AUC为0.802,Youden’s指数提示最佳切点为54.40 pg/ml,灵敏度为0.719,特异度为0.852;卧位AARR筛查原醛症的ROC曲线AUC为0.848,最佳切点为64.18 pg/ml,灵敏度为0.750,特异度为0.818。卡方检验提示立、卧位AARR筛查原醛症的诊断效果差异无统计学意义(P>0.05)。结论临床上采用醛固酮肾素定量比值对原发性醛固酮增多症进行筛查有一定的应用价值,且立、卧位AARR诊断效果相当。     

原发性醛固酮增多症合并亚临床型皮质醇增多症临床特点及其发病机制探讨

目的探讨原发性醛固酮增多症合并亚临床型皮质醇增多症（即醛固酮/皮质醇共分泌瘤）的临床特点及其可能的发病机制,提高醛固酮/皮质醇共分泌瘤的诊治水平。方法回顾分析13例临床诊断为原发性醛固酮增多症（其中单纯性醛固酮腺瘤8例,合并亚临床型皮质醇增多症5例）的临床资料。通过免疫组化及RT-PCR检测腺瘤及瘤旁组织类固醇合成限速酶编码基因（CYP11B1和CYP11B2）的表达情况。结果醛固酮/皮质醇共分泌瘤的最大直径[（3.48±1.51）cm]大于单纯醛固酮腺瘤[（1.61±0.36）cm],差异具有统计学意义（t=-3.412,P=0.006）;醛固酮/皮质醇共分泌瘤患者血钾水平[（4.00±0.29）mmol/L]高于单纯醛固酮腺瘤[（3.15±0.72）mmol/L],差异具有统计学意义（t=-2.460,P=0.032）。结论原发性醛固酮增多症合并亚临床型皮质醇增多症并不少见,其具有独特的临床特点。     

3种化学发光免疫分析法检测系统与同位素稀释液相色谱串联质谱法检测总甲状腺素的一致性评价

目的比较3种化学发光免疫分析法检测系统与同位素稀释液相色谱串联质谱法(ID-LC/MS/MS)检测总甲状腺素(tT4)结果的一致性。方法参考美国临床和实验室标准化协会(CLSI)EP-15A方案评价3种检测系统(Antolumo A2000、CL-1000i为国产,ADVIA Centaur~为进口)检测不同浓度水平tT4的不精密度。选择北京协和医院2015年10月至2016年1月144例剩余血清样品,浓度覆盖tT4的测量范围。以ID-LC/MS/MS候选参考方法为参比方法,分别评价Antolumo A2000、CL-1000i及ADVIA Centaur~检测系统tT4检测结果与参比方法的一致性。以Passing&Bablok回归分析2种方法之间的相关性,绘制Bland-Altaman图并比较医学决定水平处的偏倚,以一致性相关系数(CCC)评估方法间的一致性。结果 3种化学发光免疫分析法检测系统的实验室内不精密度均小于说明书标示值,但在低浓度水平的不精密度(以变异系数CV表示)均大于来源于生物学变异的允许不精密度(3.7%),各化学发光法检测系统tT4结果与ID-LC/MS/MS结果相关性良好(r均>0.975),但仅有Antolumo A2000与ID-LC-MS/MS相比的斜率的95%CI包含1,且3种方法的截距的95%CI均不包含0;与ID-LC-MS/MS比较,平均偏差超过最低偏倚要求(4.5%),且在医学决定水平处各法均未能全部满足最低偏倚要求。一致性分析显示,Antolumo A2000和CL-1000i与ID-LC/MS/MS均具有较好的一致性(CCC:0.95～0.99),ADVIA Centaur~与ID-LC/MS/MS具有中度一致性(CCC:0.90～0.94)。结论 2种国产和1种进口tT4检测系统与ID-LC-MS/MS方法一致性良好,但在精密度、偏倚等方面均仍需进一步提高。     

血浆醛固酮浓度/血浆肾素活性比值对原发性醛固酮增多症的诊断价值

目的探讨血浆醛固酮浓度/血浆肾素活性比值(ARR)对原发性醛固酮增多症的鉴别诊断价值。方法随机选取2017年1月至2018年12月85例原发性醛固酮增多症患者作为研究组,另选取我院同期收治的85例原发性高血压患者作为对照组,两组患者均予以血浆醛固酮浓度、血浆肾素活性检测,记录两组上述指标检测结果、计算各组血浆醛固酮浓度/血浆肾素活性比值。结果研究组血浆醛固酮浓度、血浆醛固酮浓度/血浆肾素活性比值显著高于对照组,研究组血浆肾素活性显著低于对照组,差异有统计学意义(P0.05)。结论应用血浆醛固酮浓度/血浆肾素活性比值可为鉴别诊断原发性醛固酮增多症、原发性高血压提供有力依据,提高原发性醛固酮增多症诊断准确率。     

原发性醛固酮增多症诊治分析

目的 探讨原发性醛固酮增多症的诊断及治疗.方法 回顾性分析68例原发性醛固酮增多症患者的临床资料.结果 全部患者均有不同程度的低血钾,卧、立位血浆醛固酮平均值分别为(682±332)pmol/L、(881±406)pmol/L,29例患者尿醛固酮值升高,其中48例立位的醛固酮/肾素活性比值≥50.通过影像学检查62例为原发性醛固酮增多症腺瘤型,其余6例为双侧肾上腺增生;32例予以手术治疗,24例予以口服安体舒通治疗,经治疗后56例患者血压及血钾均有不同程度的恢复.结论 原发性醛固酮增多症主要通过影像学检查定位,血、尿醛固酮测定定性诊断.肾上腺腺瘤切除仍是当前的首选治疗方法,安体舒通也是重要的治疗手段.     

原发性醛固酮增多症患者肾丢钾的标准

原发性醛固酮增多症是少数几个可以治愈的高血压疾患之一,临床表现:高血压、低血钾、肾功能正常的肾丢钾、低肾素高醛固酮血症。当血清钾<3.5mmol/L时,肾丢钾的标准在权威书刊各不相同,有>25mmol/d,>25mmol/L,>30mmol/L,>40mmol/d,给临床应用带来困惑。本文收集近年来我院经手术证实的16例原发性醛固酮增多症患者尿钾排泄量,结合文献提出原发性醛固酮增多症肾丢钾的标准。 1 临床资料 住院患者16例,年龄24～45岁,病程2个月～20年。血清钾及尿钾测定用自动生化仪(Beckman,ELISE)。患者均正常饮食,未用任何影响血钾及尿钾排泄     

血清醛固酮候选参考测量程序的建立及性能评估

目的建立基于同位素稀释液相色谱-串联质谱(ID-LC-MS/MS)的血清醛固酮候选参考方法。方法以甲基叔丁基醚为萃取剂,采用液液萃取方式进行样本前处理。以醛固酮-2H7为内标,采用电喷雾离子源(ESI)负离子模式建立检测血清醛固酮的ID-LC-MS/MS方法,并对方法的准确度、精密度、灵敏度、线性等基本分析性能进行验证。结果 ID-LC-MS/MS测定血清醛固酮的线性范围为0.08～3.55 nmol/L,检测限和定量限(LOQ)分别为0.012 nmol/L和0.045 nmol/L。批内和批间变异系数(CV)分别为5.0%和4.1%。平均加标回收率为98.52%～100.14%。测定国际临床化学和检验医学联合会参考实验室外部质量评价计划(RELA)样本的结果偏移1.6%。结论建立的测定血清醛固酮的ID-LC-MS/MS方法准确、精密,有望作为测定血清醛固酮的参考方法。     

原发性醛固酮增多症误诊一例报告

甲状腺功能亢进(甲亢)合并原发性醛固酮增多症的报道较少,该文报道了1例原发性醛固酮增多症被误诊为甲亢性低血钾麻痹的患者,患者以反复下肢无力2年,再发并加重3 d为主诉入院,入院后血压升高,低血钾症状改善不明显,从而怀疑原发性醛固酮增多症,通过药物试验性治疗、辅助检查及实验室检查,明确诊断为原发性醛固酮增多症.     

血清肌酐参考方法的建立及其在正确度调查中的应用

目的建立同位素稀释液相色谱串联质谱(ID-LC/MS/MS)测定人血清肌酐的参考方法,并运用于临床实验室正确度调查新鲜冰冻血清样本靶值的确立。方法按照国际检验医学溯源联合委员会(JCTLM)推荐方法,建立本实验室肌酐参考方法,利用参考物质SRM909c及RELA比对样本考察所建方法的精密度与准确度,并将建立的参考方法用于上海地区小分子正确度调查新鲜冰冻血清肌酐靶值的确立。结果参考方法测量参考物质SRM909c相对偏移为0.69%,测量不精密度2%,初步验证了方法的准确度和精密度。正确度调查样本201411、201412、201421、201422酶法检测结果与参考方法测定值的相对偏移分别为14.97%、-2.77%、-1.37%、-1.92%;苦味酸法检测结果与参考方法测定值的相对偏移分别为21.39%、2.10%、6.22%、2.38%。除201411样本(为低浓度样本)外,其余样本不同常规分析系统测定的血清肌酐浓度与参考方法赋值结果的相对偏移均不超过8%。结论建立的ID-LC/MS/MS测定血清肌酐的方法精密度、准确度均较好,靶值的确定对实验室检测结果分析有重要意义,该参考方法的建立有望在上海地区临床实验室正确度计划中发挥一定作用,并建议临床实验室重视低浓度下肌酐测量的准确度和一致性。     

The analytical performance of six urine drug screens on cobas 6000 and ARCHITECT i2000 compared to LC-MS/MS gold standard

BackgroundImmunoassays provide a rapid tool for the screening of drugs-of-abuse (DOA). However, results are presumptive and confirmatory testing is warranted. To reduce associated cost and delay, laboratories should employ assays with high positive and negative predictive values (PPVs and NPVs). Here, we compared the results of urine drug screens on cobas 6000 (cobas) and ARCHITECTi2000 (ARCHITECT) platforms for six drugs against LC-MS/MS to assess the analytical performance of these assays.MethodsEighty nine residual urine specimens, which tested positive for amphetamine, THC-COOH, benzoylecgonine, EDDP, opiates and/or oxycodone during routine drug testing, were stored frozen until later confirmation by LC-MS/MS. Immunoassays were performed on cobas and ARCHITECT using a split sample. A third aliquot from these samples was tested by LC-MS/MS to assess the percentage of false positive, false negative, true positive and true negative results and calculate the PPVs and NPVs for each immunoassay.ResultsThe PPVs of THC-COOH and EDDP assays were 100% on both platforms. Suboptimal PPVs were achieved for oxycodone (cobas, 57.1% vs ARCHITECT, 66.7%), amphetamine (77.8 vs. 100%), opiates (80.0 vs. 84.6%) and benzoylecgonine (88.9 vs. 84.2%) assays. The NPV was 100% for cobas and ARCHITECT oxycodone assays. Lower NPVs were achieved for THC-COOH (cobas, 28.6% vs ARCHITECT, 25.0%), EDDP (72.7% for both assays), benzoylecgonine (74.4% vs 73.8%), amphetamine (83.3% vs 82.8%) and opiates (100% vs 85.3%).ConclusionOverall, cobas and ARCHITECT urine drug screens have comparable analytical performance. Confirmatory testing is warranted for positive test results especially for oxycodone, amphetamine, opiates and cocaine. Negative drug screen results must be interpreted with caution especially for THC-COOH, EDDP, benzoylecgonine, amphetamine and opiates.     

Developing an ultra‐performance liquid chromatography‐tandem mass spectrometry for detecting aldosterone in human plasma

BackgroundAccurately measuring plasma aldosterone concentration is difficult but meaningful for primary aldosteronism (PA) diagnosis.MethodsIn this study, we developed an ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) method for plasma aldosterone detection, evaluated its performance according to guidelines issued by CLSI, including detection limit, linearity, precision, and compared it with chemiluminescence immunoassay. Then, a reference range of plasma aldosterone in young people was established by using this method.ResultsThe lower limit of quantitation (LOQ) was 10 pg/ml. The mean recovery rates of analyte added to serum were 100.07–102.05% in different concentrations. The linearity range was 20–2000 pg/ml. Inter‐assay CVs were 2.20–3.97% at aldosterone concentrations of 65.66–854.75 pg/ml. The regression equation of UPLC‐MS/MS (x) and chemiluminescence immunoassay (y) was y = 1.002x + 65.854 (r = 0.9456, n = 237). The reference range of plasma aldosterone detected by UPLC‐MS/MS was 11.30–363.82 pg/ml in young people in South China, and there was no statistically significant difference in plasma aldosterone concentration between two genders.ConclusionIn conclusion, UPLC‐MS/MS can rapidly and accurately detect plasma aldosterone and is appropriate for clinical application.     

Pilot study for the standardization of insulin immunoassays with isotope dilution liquid chromatography/tandem mass spectrometry

BACKGROUND: An international working group convened by the American Diabetes Association (ADA) called for a reference measurement procedure for use in a trueness-based standardization project of insulin immunoassays. In view of this demand, we conducted a pilot study to investigate the feasibility of such a standardization project with our isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/tandem MS) procedure. METHODS: We evaluated the precision, accuracy, and limit of quantification (LoQ) of the ID-LC/tandem MS procedure by use of insulin-free serum supplemented with insulin to give 3 pools with concentrations of 0.0796, 0.769, and 5.56 microg/L. We conducted a pilot method comparison study with 4 immunoassays and 80 samples from fasting and glucose-stimulated patients. RESULTS: The within-run and total imprecision (CV) ranged from 3.2% to 6.3% and from 4.9% to 12.1% (listing sequence from the high to the low pool). The recovery from supplemented insulin-free sera ranged from 101.8% to 104.1%, and the LoQ was 0.07 microg/L (12 pmol/L). Weighted Deming regression and correlation analysis of the method-comparison data showed considerable between-assay variation for the immunoassays but, with the exception of one assay, excellent correlation with ID-LC/tandem MS. Recalibration of the immunoassay results considerably reduced the between-assay variation. Moreover, after recalibration, 3 of the 4 assays fulfilled the total error specification of 32% proposed by the ADA Workgroup. CONCLUSIONS: Recalibration of insulin assays by regression equations established from method comparison with ID-LC/tandem MS can result in successful standardization and fulfillment of the total error criterion proposed by the ADA Workgroup.     

Feasibility of standardization of serum C-peptide immunoassays with isotope-dilution liquid chromatography-tandem mass spectrometry

BACKGROUND: Serum C-peptide concentrations reflect pancreatic function in different clinical and diagnostic settings; however, the utility of C-peptide testing is limited by the lack of standardized commercial immunoassays. Standardization can best be done by split-sample comparison with a hierarchically higher reference measurement procedure with a set of native sera. For serum peptides, isotope-dilution liquid chromatography-mass spectrometry (ID-LC/MS) is recommended as a reference measurement procedure. METHODS: We evaluated the analytical performance characteristics of an ID-LC/tandem MS procedure for measurement of serum C-peptide after a 2-step solid-phase extraction. To investigate the feasibility of this procedure for use in standardization, we also performed a method comparison with 3 representative commercial assays. RESULTS: The ID-LC/tandem MS procedure showed maximum within-run, between-run, and total CVs on dedicated sera (C-peptide concentrations, 1.6 and 4.0 mug/L) of 2.1%, 2.5%, and 2.9%, respectively; an accuracy of 94.6%-104.1%; a minimum trueness of 98.1% (95% confidence interval, 96.2%-100.0%), and limits of quantification and detection of 0.15 and 0.03 mug/L, respectively. Deming linear regression analysis of the method-comparison data showed that the immunoassays correlated well with ID-MS and were specific, but lacked intercomparability and trueness. We propose that the deficiencies can be resolved by recalibration on the basis of the method comparison. CONCLUSIONS: The ID-LC/tandem MS procedure is suitable for specific and accurate measurement of basal and stimulated serum concentrations of proinsulin C-peptide fragment 33-63 and is suitable for use in standardization of C-peptide immunoassays.     

Proposal of a candidate international conventional reference measurement procedure for free thyroxine in serum.

In the present paper the IFCC WG-STFT recommends and provides the rationale to establish metrological traceability of serum free thyroxine (FT4) measurements to a candidate international conventional reference measurement procedure. It is proposed that this procedure be based on equilibrium dialysis combined with determination of thyroxine in the dialysate with a trueness-based reference measurement procedure. The measurand is thus operationally defined as "thyroxine in the dialysate from equilibrium dialysis of serum prepared under defined conditions". With regard to the trueness-based reference measurement procedure, the WG-STFT recommends use of an isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/tandem MS) procedure for total thyroxine that has been optimized towards measurement at picomolar concentration levels and that is listed in the database of the Joint Committee for Traceability in Laboratory Medicine (JCTLM). For calibration, the purified thyroxine material IRMM-468 (resulting from a project funded by the European Commission and recently submitted to the JCTLM) is proposed. The WG-STFT stresses that according to this recommendation it is a prerequisite to strictly adhere to the defined equilibrium dialysis procedure, whereas it is permissible to introduce variants in the ID-LC/tandem MS procedure.     

A multi-target screening analysis in human plasma using fast liquid chromatography-hybrid tandem mass spectrometry (Part II)

Objectives

Perform a comparison of results obtained with a LC-MS/MS method and a Remedi® instrument on clinical serum samples.

Design and methods

Results obtained on 146 selected plasma samples were compared between the two methods.

Results

On the 336 positive identifications, 89% were obtained using the LC-MS/MS technique and 57% by the LC-DAD. Benzodiazepines were well recognized by LC-MS/MS. For some compounds such as antidepressant agents, sensitivity was improved using LC-MS/MS. Moreover, this method extended the panel of drugs detected in clinical toxicology.

Conclusion

The new software platform developed for screening and identification of small molecules (SmileMS) allows an easy and reproducible detection of drugs and toxic compounds in blood for general unknown screening. It offers automated generation of reports, which makes the LC-MS/MS easier to use without having specialised skills in mass spectrometry.This LC-MS/MS screening method will be a reliable alternative to the Remedi® instrument in the global process of screening in emergency clinical toxicology laboratories.     

免疫测定技术的临床应用及应注意的问题

在临床实验室,感染性疾病血清学标志物、肿瘤标志、激素、特定蛋白、自身抗体等均可采用免疫学方法进行检测,而且使用的免疫测定技术也多种多样,既涉及经典的免疫凝集试验、免疫沉淀试验,以及对检测微量抗原和抗体必须使用的标记免疫测定技术,如ELISA、免疫化学发光试验、时间分辨荧光试验等,又有一些新的免疫测定技术,如蛋白芯片、免疫传感器等.对于新技术能否用于临床实际检测,最重要的应通过充分的评价试验来决定.采用国际标准物质对定量免疫测定进行量值溯源,以及对免疫测定试剂、方法和程序进行标准化,是保证不同试剂与方法、不同实验室间结果一致性的前提.而室内质量控制的实施及分析评价,是实验室检测质量持续改善的源泉.     

An isotope dilution LC-MS/MS based candidate reference method for the quantification of cyclosporine A,tacrolimus, sirolimus and everolimus in human whole blood

An isotope dilution LC-MS/MS based candidate reference measurement procedure for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood is presented to be used for evaluation and standardization of routine assays applied for therapeutic drug monitoring. The assay allows baseline separation of the four immunosuppressive drugs within a total runtime of 9 minutes using a C4 reversed phase column. Sample preparation is based on protein precipitation with zinc sulphate followed by purification with solid phase extraction. Reference materials used in this reference measurement procedure were characterized by qNMR and an absolute content of analytes calculated to guarantee traceability to SI units. As internal standards the corresponding deuterated and ¹³C-labelled analytes were used. The method allows the measurement of cyclosporine A in the range of 5 ng/mL to 2100 ng/mL; tacrolimus, sirolimus and everolimus were analysed in the range of 0.25 ng/mL to 50 ng/mL. Imprecision for inter-day measurements were found to be ≤3.5% for cyclosporine A and ≤4.4% for tacrolimus, sirolimus and everolimus. Accuracy was found to be within 101% and 108% for cyclosporine A and between 95% and 104% for the macrolide compounds. The uncertainty was evaluated according to the GUM. Expanded measurement uncertainties were found to be ≤7.2% for cyclosporine A, ≤6.8% for tacrolimus, ≤9.0% for sirolimus and ≤8.9% for everolimus (k = 2).     

临床化学检测的标准化现状

实验室的质量管理和检验结果标准化是临床化学检测项目实现结果互认的2个重要基础。开展标准化工作,建立检验项目的标准化体系可为检验质量提供准确性依据,是确保检验结果准确、具有可比性的最有效措施。实现检验结果准确、可比的重要方法之一是建立和保证检验结果的溯源性。国际组织一般通过建立参考测量程序、参考物质和一致性方案等来解决不同项目的量值溯源性问题。正确度验证计划和能力验证的开展为临床化学检验项目的结果互认提供了依据。文章对临床化学检测标准化的现状进行综述,以期为临床实验室质量管理提供参考。     

临床检验量值溯源与参考系统

医疗卫生工作需要准确可比的检验结果，实现临床检验标准化的有效途径是建立和保证检验结果的计量学溯源性。溯源性通过一系列由不同等级测量程序和校准物实现的比较测量而建立，较低级别程序足够特异和校准物具有互通性是计量学溯源的前提。较高级别的测量程序和校准物称参考系统，参考系统需符合特定计量学要求，为此有关国际组织成立检验医学溯源联合委员会（JCTLM），鉴定现有国际参考测量程序、参考物质和参考实验室。总之，临床检验量值溯源问题已受到广泛重视，发展和应用参考系统将可能成为临床检验领域的重要课题。