微量连续流动荧光法测定滤纸干血样苯丙氨酸及其在新生儿疾病筛查中的应用

Micro-continuous flow fluorescence for determinating phenylalanine in dry blood specimens of filter paper and its application in newborn screening

目的研究滤纸干血样苯丙氨酸(Phe)微量连续流动荧光法测定实验方法,并评价其在新生儿苯丙酮尿症筛查中的应用价值。方法直径3.2mm的新生儿滤纸干血样经萃取后用微量连续流动荧光分析仪测定Phe,确定苯丙酮尿症的筛查阳性临界值。结果在所建立的标本保存和分析条件下,新生儿滤纸干血样经30～120min萃取后,Phe在0～20mg/dl浓度范围内线性良好,最低检出限为0.2mg/dl。Phe高值和低值质控滤纸干血样的批内变异度分别为4.1%和5.3%,批间变异度分别为5.0%和5.6%,测定平均回收率为98.2%,并与PerkinElmer公司Phe荧光定量法测定结果高度相关。50009例新生儿滤纸干血样Phe呈非正态分布,用百分位数法(95%)确定筛查新生儿苯丙酮尿症的临界值为Phe>3.9mg/dl为阳性。苯丙酮尿症筛查阳性率为64.0/10万,发病率为16.0/10万。结论微量连续流式荧光分析法检测新生儿滤纸干血样Phe具有灵敏、特异、重复性好,为筛查新生儿苯丙酮尿症提供了简便、快速、费用低的实验方法。

Objective To discuss a experimental methods for determinating Phenylalanine （Phe） in dry blood specimens of filter paper with micro-continuous flow fluorescence, and to evaluate its application in newborn screening test for phenylketonuria. Methods A neonatal dry blood specimen of filter paper with 3.2mm was extracted first, then measured its Phenylalanine concentration with micro-continuous flow fluorescence analyzer to determine cut-off value in screening of phenylketonuria. Results Under the condition of specimen preservation and analysis which had established, after 30 - 120min extraction of neonatal dry blood specimens of filter paper, there is a good linear correlation when phenylalanine is in the range of 0-20mg/dl, the lowest detection value is 0.2 mg/dl. For Phenylalanine quality control in dried blood spot of filter paper, the intra-assay variation of high-value and low-value were 4.1% and 5.3% respectively, and the inter-assay variation were 5.0% and 5.6% respectively. The average recovery rate was 98.2%, these results were highly correlated with the determined results by using PerkinElmer＇ s Phe fluorescence quantitative method. There was a non-normal distribution of Phe values among 50,009 dry blood specimens of filter paper. The positive critical value of Phe is more than 3.9 mg/dl which determined by 95% percentile for newborn screening for phenylketonuria. The positive rate of phenylketonuria screening was 64/100 000, and the incidence rate was 16/100 000. Conclusions Micro-continuous flow fluorescence analysis can detect phenylalanine levels in neonatal dry blood specimens of filter paper with good sensitivity, specificity and reproducibility. It would be a simple, fast and low-cost method for newborn phenylketonuria screening.