Abstract:

Objective To compare the distribution and differences of phenylalanine（Phe） concentration on dry blood spot by fluorescence assay and tandem mass spectrometry,and to provide a methodological reference for newborn hyperphenylalaninemia（HPA）screening program. Methods A total of 62 510 dry blood spot samples were collected for metabolic disease screening,and the Phe concentration was determined by fluorescence assay and tandem mass spectrometry. Tandem mass spectrometry was used to determine tyrosine（Tyr） concentration. The differences and correlation between the 2 methods were analyzed by paired Wilcoxon test and rank correlation analysis,and the consistency was compared by Bland-Altman analysis. Results Both fluorescence assay and tandem mass spectrometry determined 3 cases of HPA in 62 510 dry blood spot samples,and the sensitivities were 100%. The positive predictive value of fluorescence assay was 33.3%. The positive predictive value of tandem mass spectrometry was 18.8%,and it can be up to 100% combined with Phe/Tyr ratio. The distribution of Phe concentrations of normal newborn samples was skewed,and the results of fluorescence assay and tandem mass spectrometry as <60 μmol/L accounted for 96.31% and 95.08%,respectively,and the results as ≥120 μmol/Laccounted for only 0.01% and 0.03%. The concentration by fluorescence assay was lower than that by tandem mass spectrometry. To the 97% percentile （Phe≈63 μmol/L）,the concentration went to flat,then gradually was higher than that by tandem mass spectrometry. The 2 methods were related positively （r=0.43,P<0.01）. For Bland-Altman analysis,the higher the concentration of Phe was,the smaller the bias of the 2 methods was. When Phe was >120 μmol/L,19 sample points all fell within the 95% limit of consistency,the 2 methods were in good consistency,and the results can be replaceable. Conclusions There are differences by fluorescence assay and tandem mass spectrometry to determine Phe with low concentrations,while the 2 methods are in good consistency for that with high concentrations. The 2 methods had no effect on clinical judgment for HPA,and they can be used in newborn screening. Tandem mass spectrometry can simultaneously determine Phe and Tyr concentrations,and the positive predictive value for HPA can be up to 100% combined with Phe/Tyr ratio.

Key words: Phenylalanine, Tandem mass spectrometry, Fluorescence assay, Comparative study, Dry blood spot