胶乳增强免疫比浊法测定血浆和尿液中性粒细胞明胶酶相关脂质运载蛋白的方法学评价

Evaluation of the latex particle-enhanced turbidimetric immunoassay for the measurement of plasma and urinary neutrophil gelatinase-associated lipocalin

目的 对胶乳增强免疫比浊法测定血浆和尿液中性粒细胞明胶酶相关脂质运载蛋白（NGAL）进行方法学评价.方法 按照美国临床和实验室标准协会（Clinical and Laboratory Standards Institute,CLSI）评价方案和相关文件的要求进行验证性研究,对胶乳增强免疫比浊法的不精密度、检测限、线性范围、正确度和抗干扰性等进行评价.同时,测定20例急性肾损伤患者（AKI）、166例慢性肾病患者（CKD）以及100名健康对照者血浆和尿液中NGAL水平.结果 胶乳增强免疫比浊法测得NGAL低值和高值的重复性估计值为1.90％（低值）和1.22％（高值）,实验室内不精密度为4.06％（低值）和2.57％（高值）;空白限（limit of blank,LoB）为3.1μg/L,血浆NGAL（sNGAL）和尿液NGAL（uNGAL）的检出限（limit of detection,LoD）分别为12.5和21 μg/L.用胶乳增强免疫比浊法测定血浆和尿液NGAL在25～ 4250 μg/L的线性良好;sNGAL和uNGAL平均回收率分别为98.89％、98.28％（低值）和102.61％、95.44％（高值）;与国外同类试剂盒相比,测定结果基本一致.血红蛋白≤7 g/L,胆红素≤0.6 g/L,维生素C≤0.6 g/L,肝素钠≤5g/L,内脂≤1％时,对sNGAL测定结果无显著性干扰,血红蛋白≤10 g/L,胆红素≤0.6 g/L,维生素C≤0.6 g/L,uNGAL测定结果在可接受范围内.AKI组、CKD组血浆和尿液NGAL水平与健康对照组比较差异均有统计学意义;AKI组较CKD组显著升高.sNGAL、uNGAL的水平均与肾小球滤过率估算值（eGFR）呈负相关（r=-0.758、-0.692,P均＜0.01）.结论 胶乳增强免疫比浊法测定血浆和尿液NGAL的不精密度符合要求,线性范围宽,具有自动化程度高、检测速度快等优点.

Objective To evaluate the performance of the latex particle-enhanced turbidimetric immunoassay for the determination of plasma and urinary neutrophil gelatinase-associated lipocalin.Methods Latex particle-enhanced turbidimetric immunoassay was used to measure the concentration of NGAL in plasma and urinary samples for confirmatory study.The assay imprecision,limit of blank （LOB）,limit of detection （LOD）,linearity,accuracy and interference factors were assessed according to CLSI protocols.Twenty samples of patients with acute kidney injury （AKI）,166 samples of patients with chronic kidney disease（CKD） and 100 healthy adult samples were tested.Results Repeatability estimate of the NGAL was 1.90％ （Level 1） ＆ 1.22％ （Level 2）,and the within-Laboratory imprecision was 4.06％（Level 1） ＆ 2.57％ （Level 2） respectively.The LOB was 3.1 μg/L and the LOD of plasma NGAL （sNGAL） and urinary NGAL （uNGAL） were 12.5 μg/L,21 μg/L respectively.The linear coverage was from 25 to 4250 μg/L.The accuracy of sNGAL and uNGAL was 98.89％,98.28％ （Level 1） ＆102.61％,95.44％ （Level 2）.The results were basically identical with similar foreign kit measurement results.No interference in plasma was observed with hemoglobin ≤7 g/L,bilirubin ≤0.6 g/L,vitamin C ≤0.6 g/L,sodium heparin ≤ 5 g/L and introlipid ≤ 1％.The concentration of uNGAL was in the acceptable range with the hemoglobin ≤10 g/L,bilirubin ≤0.6 g/L and vitamin C ≤0.6 g/L.In general,the measured NGAL levels in AKI group and CKD group were higher than control group; AKI group was significantly higher than CKD group.The evaluated glomerular filter rate （eGFR） was significantly inverse correlated with sNGAL and uNGAL （r =-0.758 ＆-0.692,P ＜ 0.01）.Conclusions The data show that excellent imprecision and linearity are achieved using the particle-enhanced turbidimetric immunoassay for testing plasma and urinary NGAL concentrations.The assay of NGAL has advantages including high automation and fast speed detection which are suited for clinical application and laboratory automation.