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目的:研究滤纸干血样苯丙氨酸和葡萄糖-6-磷酸脱氢酶微量连续流式荧光法联合测定实验方法,并评价其在新生儿苯丙酮尿症和葡萄糖-6-磷酸脱氢酶缺乏症筛查中的应用价值。方法:直径3.2 mm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同时测定苯丙氨酸和葡萄糖-6-磷酸脱氢酶浓度,确定苯丙酮尿症和葡萄糖-6-磷酸脱氢酶缺乏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30～120 min萃取后,苯丙氨酸在0～20mg/dl、葡萄糖-6-磷酸脱氢酶在0～150μM NADPH浓度范围内线性良好,苯丙氨酸和葡萄糖-6-磷酸脱氢酶最低检出限分别为0.2 mg/dl和2.0μM NADPH。苯丙氨酸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%;葡萄糖-6-磷酸脱氢酶批内变异度分别为4.9%和5.4%,批间变异度分别为5.1%和5.9%,测定平均回收率分别为98.2%和99.2%,并与PerkinElmer公司PHE和G6PD荧光定量法测定结果高度相关。50009例新生儿滤纸干血样PHE和G6PD呈非正态分布,用95%百分位数法确定筛查新生儿苯丙酮尿症的临界值为PHE>3.9 mg/dl为阳性,筛查新生儿G6PD缺乏症的临界值为G6PD<42μM NADPH为阳性,苯丙酮尿症和葡萄糖-6-磷酸脱氢酶缺乏症筛查阳性率分别为0.064%和0.352%,发病率分别为0.016%和0.042%。结论:微量连续流式荧光分析法可同时检测新生儿滤纸干血样苯丙氨酸和葡萄糖-6-磷酸脱氢酶含量,且灵敏度、特异性、重复性好,为筛查新生儿苯丙酮尿症和葡萄糖-6-磷酸脱氢酶缺乏症提供了简便、快速、费用低的方法。
OBJECTIVE: To study the combination of dry-flow phenylalanine and glucose-6-phosphate dehydrogenase in filter paper by micro-continuous flow-cytometry and to evaluate its clinical significance in neonatal phenylketonuria and glucose-6-phosphate dehydrogenase deficiency Applied value of disease screening. Methods: Neonatal filter paper with a diameter of 3.2 mm was extracted from dry blood of filter paper, and the concentrations of phenylalanine and glucose-6-phosphate dehydrogenase were determined simultaneously with a microfluidic continuous flow fluorescence analyzer to determine phenylketonuria and glucose-6-phosphate Screening positive threshold for hydrogenase deficiency. Results: Under the conditions of sample preservation and analysis, the newborn filter paper dried blood samples were extracted from 30 to 120 min with phenylalanine at 0 to 20 mg / dl, glucose-6-phosphate dehydrogenase at 0 to 150 μM NADPH The linearity was good in the concentration range. The detection limits of phenylalanine and glucose-6-phosphate dehydrogenase were 0.2 mg / dl and 2.0 μM NADPH, respectively. The intra-assay variability of phenylalanine high-value and low-value filter paper dry blood samples were 4.1% and 5.3% respectively, and the inter-assay variability was 5.0% and 5.6% respectively. The intra-assay variability of glucose- Degrees were 4.9% and 5.4%, respectively. The variation between batches was 5.1% and 5.9%, respectively. The average recoveries were 98.2% and 99.2%, respectively, and were highly correlated with the results of PHE and G6PD fluorescence quantification tests by PerkinElmer. The plasma PHE and G6PD of 50009 newborn filter paper samples were non-normal distribution. The critical value of 95% percentile screening screening for neonatal phenylketonuria was positive for PHE> 3.9 mg / dl, and the screening of newborns The cut-off value of G6PD deficiency was positive for G6PD <42μM NADPH, positive rates for phenylketonuria and glucose-6-phosphate dehydrogenase deficiency were 0.064% and 0.352%, respectively, with a prevalence of 0.016% and 0.042%, respectively . CONCLUSION: Micro-continuous flow fluorescence spectrometry can simultaneously detect dry blood-like phenylalanine and glucose-6-phosphate dehydrogenase in neonatal filter paper, and has good sensitivity, specificity and repeatability. To screen newborns with phenylketonuria Symptoms and glucose-6-phosphate dehydrogenase deficiency provide a simple, fast, and cost effective method.