间接免疫荧光试验检测抗核抗体的前带效应分析

Prozone effect on indirect immunofluorescence assay for anti-nuclear antibody testing

目的探讨间接免疫荧光试验(IIFA)检测抗核抗体(ANA)中的前带效应对ANA荧光滴度的影响。方法将880例血清样本以1∶100稀释进行手工检测,筛选ANA荧光滴度≥1∶1 000的样本分别以1∶100、1∶1 000、1∶10 000稀释检测,统计有前带效应(1∶1 000稀释比1∶100稀释荧光亮)的样本数;将前带效应的样本采用欧蒙Sprinter XL全自动荧光加样仪和EUROPattern全自动荧光分析仪检测ANA(1∶100稀释),与手工法检测ANA(1∶100稀释)进行比较,检测前带效应样本的特异性自身抗体,分析其荧光模型、荧光滴度及阳性特异性自身抗体的分布特点。结果有明显前带效应的样本数为34例,占总样本数的3.86%,占高滴度(≥1∶1 000)ANA样本数的29.57%。通过手工法检测或通过Sprinter XL全自动荧光加样仪检测均发现前带效应,其荧光模型及荧光滴度相似,值得注意的是,EUROPattern只能选取图片中间区域判读。在有前带效应的血清样本中,以荧光滴度≥1∶10 000最常见(74.42%)。在荧光模型方面,以斑点型最为多见(46.51%)。在特异性自身抗体的分布方面,以抗核糖核蛋白(RNP)抗体最多见(62.79%),其次为抗双链DNA抗体(51.16%)和抗SSA抗体(51.16%)。结论 IIFA检测高滴度ANA样本在低稀释度时容易产生前带效应,从而导致错误的ANA荧光滴度判断,临床检验实验室应引起重视。

ObjectiveTo explore the influence of prozone effect on anti-nuclear antibodies(ANA)testing by indirect immunofluorescence assay(IIFA).MethodsThe samples with high titer of ANA(≥1∶1 000)were selected from 880 fresh serum samples,and were subsequently diluted in 1∶100,1∶1 000 and 1∶10 000 ratio.Prozone effect was defined as fluorescence intensity from 1∶1 000 dilution was stronger than that from 1∶100 dilution.The samples with prozone effect were determined manually or by Sprinter XL and EUROPattern.The samples with prozone effect were further characterized by combinations of fluorescence patterns,fluorescence intensities and autoantibody specificities.ResultsA total of 880 samples were tested.Importantly,34 samples displayed prozone effect(3.86%in total and 29.57%in samples with ANA≥1∶1 000).Interestingly,prozone effect was identified by manual detection as well as by Sprinter XL with similar fluorescence patterns and fluorescence intensities.Notably,EUROPattern can only select the central area for identification.Among all samples with prozone effect,74.42%samples exhibited fluorescence intensities of≥1∶10 000.Speckled pattern was the most prevalent fluorescence patterns in samples with prozone effect(46.51%).In addition,anti-RNP antibodies(62.79%)were the most popular autoantibodies in samples with prozone effect,followed by anti-dsDNA antibodies(51.16%)and anti-SSA antibodies(51.16%).ConclusionProzone effect was present in ANA testing,especially in samples with high titers,resulting in underestimating the titers.The study highlighted that special attention should be paid to the prozone effect in clinical practice.