血清肌酐及cystatinc和尿白蛋白测定和报告中需要强调的问题ppt课件.ppt

血清肌酐及CystatinC和尿白蛋白测定和报告中需要强调的问题 徐国宾 北京大学第一医院检验科,临床检验测量结果的一致可比，对于循证医学和专业团体制定的指南中特定标志物诊断、治疗干预决定水平的有效应用具有重要的作用。,以CKD诊疗指南为例,Outline,Serum/plasma creatinine Serum/plasma cystatin C Urine albumin Urine protein,肾损害 3个月，肾损害指肾脏结构或功能异常，伴/不伴GFR降低，表现为下列之一： 病理异常 有肾损害指标：主要指血或尿成分异常，或影像学检查异常 GFR 60ml/min/1.73m2 3个月，有或无肾损害,K/DOQI（慢性肾脏病临床实践指南）,Early Treatment Makes a Difference,Brenner, et al., 2001,血清肌酐为溯源到同位素稀释质谱的酶法,New equations to estimate GFR in children with CKD GFR ml/(min1.73 m2)=39.1Height(m)/Scr (mg/dl)0.5161.8/cystatin C (mg/L)0.29430/BUN (mg/dl)0.1691.099maleHeight(m)/1.40.188 J Am Soc Nephrol. 2009 Mar;20(3):629-37.,Creatinine Standardization,Improve the precision of creatinine methods to meet the total error goal for serum creatinine measurement described in the paper published in Clinical Chemistry 2006;52(1):5-18.,Imprecision: Standard Deviation 0.08 mg/dL (7.1 mol/L) Bias to IDMS reference method: Bias 0.05 mg/dL (4.4 mol/L),图1 Hitachi 检测系统与Beckman封闭检测系统测定结果的偏差,图2 酶法检测系统与Beckman封闭检测系统测定结果的偏差,实验材料： NIST SRM967 两水平 冰冻人血清 具互通性,常规系统血肌酐检测真实度调查,实验仪器：,实验方法： 实验前，对仪器进行清洗保养和校准，精密度应达到操作手册中的要求； NIST SRM967于室温复融30min，颠倒混匀，每台仪器测量两次，取平均值参与计算。,实验结果一：各系统的检测偏差,实验结果一：各系统的检测偏差,实验结果二：肌酐检测的偏差对eGFR的影响,实验结果二：肌酐检测的偏差对eGFR的影响,Creatinine standardization,By mid 2010, all creatinine methods will have calibration traceable to isotope dilution mass spectrometry (IDMS) reference measurement procedures Some exceptions with minor influence,From a survey of global IVD manufacturers (June 2009),eGFR reporting: CAP Survey of approximately 4000 participants,eGFR reporting: CAP Survey of approximately 4000 participants,60 90 120 All,Specificity of creatinine methods,Calibration traceability to IDMS does not change the influence of interfering substances Drugs Endogenous substances, e.g. Ketoacidosis Bilirubin Hemoglobin Protein,No consensus recommendations for method specificity requirements,Both enzymatic and Jaffe (alkaline picrate) methods are influenced by interfering substances Enzymatic methods have fewer interfering substance influences than Jaffe IFCC and NKDEP are collaborating to compare results for a panel of 389 patient sera and 40 spiked sera containing a wide range of potentially interfering substances,Specificity of creatinine methods,Preliminary data from IFCC/NKDEP evaluation of sera from subjects with interfering substances Three Jaffe and four enzymatic methods vs. IDMS reference method Both Jaffe and enzymatic methods have influence from interfering substances The magnitude of influence for a given substance is different among Jaffe vs. enzymatic methods The same substance interfered with some methods (Jaffe or enzymatic) but not others,Outline,Serum/plasma creatinine Serum/plasma cystatin C Urine albumin Urine protein,Molecular mass 13 kDa 120 amino acid residues Non-glycosylated protein Isoelectric point of 9.3 Positively charged in virtually all body fluids The gene encoding cystatin C is of the “house-keeping” type Produced in all investigated nucleated cells Stable production rate（产生率恒定） Circumstances that impact production Very large doses of glucocorticoids,理想的反映GFR的内源性标志物,Randers E. Clin Chem Lab Med .1999,37(4),First discovered in 1961 as an alkaline protein in normal cerebrospinal fluid (CSF) Amino acid sequence was determined in 1981 Cysteine protease inhibitor belonging to the human cystatin family The distribution in body fluids of the different cystatins is remarkably different,Clin Chem 2001;47:2055-9 Kidney Int 2003;63:1944-7,Blood plasma: 0.96 mg/L CSF: 5.8 mg/L (3.2 12.5) Urine: 0.095 mg/L (0.033 0.29) Saliva : 1.8 mg/L Seminal plasma:51.0mg/L Amniotic fluid: 1.0 mg/L Tears : 2.4 mg/L Milk : 3.4 mg/L,Clin BioChem 2005;38:1-8 J Biol Chem 1986;261(24):11282-9,Clinical application of Cystatin C,Reference Ranges Children/Infants Adults Marker for Glomerular Filtration Rate (GFR) Estimation of GFR,Acute Kidney Injury Kidney Transplants Cancer,ARUP Laborotory , Salt Lake City ,UT,与国外相同方法的报告相似，提示受人种影响小,Reference Ranges,Cystatin C and Creatinine in relation to age in a population of 258 children,Pediatr Nephrol 1998;12(2):125-9,1-60岁之间水平恒定,受肌肉量影响小 在儿童和老年人群中应用较Scr更有特色,Xu guo bing, et al . J Clin Lab Ana . 2000,分布,1201例健康成年人，男529人，女672人，年龄20-84岁 Cystatin C在人群中基本呈正态分布，均值约0.73mg/L,参考范围,Cystatin C、Scr 参考范围,Cystatin C在三个水平的参考值上限男、女均没有显著差异 推荐以99%的双侧范围作为参考-其上限为1.00mg/L Scr受肌肉量影响，男、女性别间具有显著差异 Scr的参考值上限也显著低于苦味酸终点法的参考上限-133 mol/L,Reference Intervals,Individuals 1-60 years: 0.5-1.1 mg/L Individuals 60 years: 0.6-1.4 mg/L (Represent 99% of the population),测定方法 金标准：99mTc-DTPA清除率 GFR=Dln (P1/P2)/(T2-T1)exp(T1lnP2)-(T2lnP1)/(T2-T1) 研究对象 265例CKD患者 (平均年龄5316岁,男/女: 147/118),CKD人群Cys、Scr与金标准比较,相关性比较,Cystatin C、Scr与金标准呈双曲线相关(r= -0.660 vs r= -0.510) GFR4080时，Cystatin C水平显著的升高, Scr 水平无显著变化 GFR80组，Cystatin C有38例高于正常上限；Scr仅有12例；,ROC曲线分析,CystatinC:AUC=0.907 ; Scr: AUC=0.862 ( p0.05) Cys ROC曲线拐点处界值为1.17mg/L, 此时诊断灵敏度和特异性分别为83.6%、84.4% Scr ROC曲线拐点处界值为107 mol/L ，诊断灵敏度和特异性分别为74.6%、90.0%,Estimation of GFR,Clinicians need fast estimate of renal funtion to calculate correct amount of antibiotics or cytotoxic drugs Before initiating a treatment For subsequent monitoring of response prevalence Early indication of renal damage allows for adjustment of dosage,Estimation of GFR,Prediction equation from serum cystatin C Transformation of cystatin C in mg/L to GFR in mL/min/1.73 m2 Estimation of GFR from cystatin C provides a more precise and accurate estimate of GFR than other available formulas based on creatinine,Scand J Clin Lab Invest 2005;65:153-62 Clin Chem 2005;51:1420-31,Study performed by Prof. Anders Grubb in Lund, Sweden 536 patients Age range from 4 months old to 93 years old Determination of iohexol clearance and serum creatinine,Clin Chem 2005;51:1420-31,Relationship between cystatin C and GFR,Clin Chem 2005;51:1420-31,Equation for estimating GFR GFR (mL/min/1.73 m2) = 84.69 x (cystatin C (mg/L)-1.680* * If the patient is 14 years of age, the result has to be multiplied with a prepubertal factor of 1.384,Clin Chem 2005;51:1420-31,Estimation of GFR Table,Clin Chem 2005;51:1420-31,Current limitation in using cystatin C,Results do not agree among methods eGFR equations have been proposed but: Limited to the method used to develop the equation Not validated in large populations,Cystatin C检测中应该注意的问题 *方法多 *试剂不同测定值有差异/（方法/校准） *测定结果，特别是高值时结果有差异 *参考值要多中心验证 *方法间的比较，要做多点验证 *不同方法间测定值的可比性，是一个问题 *eGFR方程具试剂特异性 *国际标准正在研发中 *测定的干扰因素：类风湿因子/嗜异抗体/色原 *质量控制，特别是室间质评要跟上 *炎症、激素治疗、肿瘤、白血病对测定的影响报告少,Standardization of cystatin C,IFCC work group (chair: A. Grubb) Primary reference preparation (PRP) Pure recombinant human Cystatin C Secondary reference preparation (SRP) PRP added to delipidated, stabilized human serum pool Characterization and value assignment complete Commutability validation underway To be available in 2010 from Institute for Reference Methods and Materials (IRMM - EU) as ERM-DA 471/IFCC,Commutable reference material,Commutable means a standard reference material has a numeric relationship between two, or more, methods equivalent to that observed for clinical samples. Tracing calibration to a non-commutable RM will cause mis-calibration for patient samples.,Numeric relationship for patients,0,2,4,6,8,10,0,2,4,6,8,10,Method A (RMP if available),Method B,Clinical Specimen,95% prediction interval,Commutable if same as patients,0,2,4,6,8,10,0,2,4,6,8,10,Method A (RMP if available),Method B,Clinical Specimen RM Commutable,95% prediction interval,Not-commutable if different than patients,0,2,4,6,8,10,0,2,4,6,8,10,Method A (RMP if available),Method B,Clinical Specimen RM Commutable RM Not-Commutable,95% prediction interval,Cystatin C eGFR equation,IFCC work group Plans to perform a multi-site evaluation of a new equation for eGFR using standardized methods,Outline,Serum/plasma creatinine Serum/plasma cystatin C Urine albumin Urine protein,NKDEP/IFCC conference held in March 2007 Clinical Chemistry 2009; 55: 24-38.,Standardization of urine albumin and creatinine measurement and reporting,2010年 5月 18日 Lancet 发表荟萃分析 eGFR75ml/min,与死亡呈线性相关 尿蛋白（试纸定性，微量白蛋白定量）与死亡呈线性相关 似乎蛋白测定具有更大意义,【据JAMA2010年2月】 肾功能、蛋白尿和不良临床结局间的关系（作者Hemmelgarn BR.等） 约有89.1%研究对象eGFR60 mL/min/1.73m2。 在eGFR60 mL/min/1.73m2 轻度 vs 重度蛋白尿 全因死亡 急性心肌梗死住院 肾脏终末期疾病 随访结束时血肌酐较基线水平升高2倍以上 发生风险增高,重度蛋白尿且eGFR60 mL/min/1.73m2时，全因死亡率 蛋白正常且eGFR 4559.9 mL/min/1.73m2两倍多。 尿试纸或ACR检测结果对临床不良结局风险的评估具有近似相同的价值。 与无重度蛋白尿者相比， 若ACR2000mg/g时 患者不良临床结局发生率显著升高。也就是说，患有严重蛋白尿但eGFR未明显异常者较eGFR中度减少但蛋白尿正常者的临床结局更差。,72,72,Albumin,MW 66 kDa Non-glycoprotein Membrane stabilizer Carrier protein Maintenance of osmotic pressure Glomerular marker, tubular marker (not-to-be-missed) Diagnostic and prognostic marker for diabetic nephropathy, other renal diseases including CKD Risk factor for cardiovascular disease, cerebrovascular disease,73,73,The difference is caused by many factors,Preanalytical factors Sampling and preservation Analytical Heterogeneity of molecular form in calibrator Heterogeneity of albumin in urine under different pathophysiologic conditions including degraded or antibody-unreactive albumin Poor assay performance Other known and unknown factors Tackling in the logic of quality assurance system,74,74,Nonspsecific Binding of Albumin on Tubes,Courtesy by Professor Hara, Gunma Univ.,75,Adsorption of albumin on plastic tubes,Albumin value,Hydrophilic Hydrophobic Super-hydrophilic,76,Effects of Freeze and Thawing Using super hydrophilic tube,% decrease,% decrease,cycles,cycles,78,78,Marker,HSA,A B C,Marker,199,131,75,41.7,Heterogeneity of Product Calibrator,Routine Calibrator (A - C) was analyzed on SDS-PAGE,Assigned value in each calibrator has been much varied by undefined value assignment,Albumin in urine is heterogeneous,Large and small fragments exist in plasma and urine C- and N-terminal truncation occurs Tubular uptake is receptor mediated influences enrichment of modified plasma forms in urine (e.g. glycated) Many ligands are concentrated in urine and bind to albumin Proteolytic degradation and chemical modifications may occur in tubules, bladder and urine after collection,80,80,Presence of Degraded Albumin in Urine,NU：Normal urine NS：Normal serum,Albumin measurement procedures,Immunoassays Primarily nephelometric and turbidimetric procedures Influenced by: Epitope(s) recognized by the antibodies Ab reactivity with modified forms of albumin Polyclonal assays are reactive with some modified albumin forms,Difference in Urine Albumin Value between Immunoassay Systems in Japan,Four urine with different concentration were measured by 20 assays.,Immunoassay precision,CAP Survey, pooled human urine supplemented with albumin, within method comparison,Immunoassay vs LC-MS,Seegmiller et al. Clin Chem 2009;55: epub,Polyclonal Ab,Monoclonal Ab,Average difference = 24% (N = 92 patient urines),Albumin measurement procedures,HPLC assays (size exclusion) Does not resolve albumin from other co-eluting urine proteins causing overestimation Hypothesis of “non-immunoreactive albumin” likely related to non-specificity of HPLC,Immunoassay and HPLC vs LC-MS,Shaikh et al. Clin Chem 2008; 54: 1504-1510,Immunoassay,HPLC,Immunoassay and HPLC for predicting cardiovascular events,McQueen et al. Am J Kidney Dis 2006 Dec;48:889-96,113,113,Difference in the value is more or less inevitable outcome,Preparation of well-defined reference material/ Calibrator is only solution! The precise causes can be elucidated by its introduction, thus promoting and expanding urine albumin measurement standardization Minimizing the difference between assay systems,114,114,Preparation of RM as Working Reference Material,Funded by AIS (National Institute of Advanced Industrial Science and Technology), NEDO (the New Energy and Industrial Technology Development Organization and JCCLS,115,115,Properties of Reference Material,Monomeric human serum albumin with purity of more than 98.9% on HPLC Matrix: 0.15 M NaCl, 1% Sucrose, 0.05% NaN3 (0.5 mg/vial) in 20 mM PB Lyophilized Non-biohazard (all negative: HBs Ag, HBs Ab, HBc Ab, HBe Ab, HCV Ab, HCV Ag, TPHA, HIV Ab) Intervial difference less than 3% in albumin and TP value More stable than material stored 3 years at 5C to 10C demonstrated by prototype II RM Stable 20 hours at 10C after reconstitution with pure water Dry weight: 544 mg Assigned value from CRM470: 226.1 10.6 mg/L reconstituted with 3.0 ml of pure water Can improve standardization for qualitative and quantitative measurement of total protein and albumin in body fluids RM indicates reference material.,116,116,Traceability Chain for Urine Albumin RM,ERM DA470/CRM470 Working Reference Material Product Calibrator Urine Sample,Selected method Master Method Routine Method,Calibration Value transfer,Uncertainty,Traceability,Laboratory Data,Until the preparation of primary reference material, CRM470 is an actual RM of higher hierarchy,117,117,118,118,SDS- PAGE Native- PAGE,Monomeric Albumin on SDS-PAGE and Native PAGE,119,119,1価 proton,2価 proton,Unknown contaminets,No n specific,Monomeric Albumin on TOF MASS,120,120,Turbidemetric Immunoassay (Sysmex Corp) on Hitachi automated analyzer model 7170,Albumin value (mg/L),Hour,100,110,120,130,140,150,0,5,10,15,20,Stability after reconstitution with pure water,121,121,Long-term Stability,Stability for 3 years has been demonstrated at 10C.,122,122,Value Assignment,Assignment from ERM DA470 to the RM (target) by the closed procedure All reconstitutions and dilutions were controlled by weighing RM and CRM470, serially diluted from approximately 200 mg/L in concentration Measuring 3 vials for 5 consecutive times per day Transfer factor calculated from the ration of y-slope between RM and ERM DA470 on conditions that proportionality and linearity are assured,In a similar way as ISO guide 35 on Certification of Reference Material by WHO/IFCC meeting Recommendation, present investigation was made,123,123,1 2 3,1 2 3,1、，： ,Marker,Marker,ERM DA470 on SDS-PAGE and Western Blots,ERM DA470 mainly consists of monomeric albumin,Alb value： 39.7 g/L,124,124,Results,Successful value assignment from ERM DA470 to RM, precisely and accurately Assigned value properly determined Realization of standardization by limited number of assay systems, being tolerable to complex matrix effects of urine demonstrated by commutability study,125,125,Standardization of Urine Albumin Immunoassay realized!,Commutability Study,State of the art: results reporting,A variety of reporting systems: Albumin concentration (e.g. mg/L) Albumin excretion rate (AER, mg/24 h) Albumin/creatinine ratio (ACR) SI (molar) and non-SI units mg/mmol mg/g A variety of decision points with different numbers,Recommendations: implement now,Albumin concentration (mg/L) is difficult to interpret and should not be reported alone Problem for dipsticks Albumin/Creatinine ratio should always be reported “mg/mmol” or “mg/g” should be used uniformly in a country or region,Develop a reference method (LC-MS) Develop reference standard materials Clarify adsorption to containers Clarify biological variability Clarify molecular forms to measure Clarify current immunoassay performance,Recommendati