全自动时间分辨荧光免疫分析检测新生儿滤纸血斑甲状腺素(T4)改良竞争法的建立与评价OA

Objective To establish and evaluate a fully automatic time-resolved fluoroimmunoassay(TRFIA)based on a modified competitive method for detecting thyroxine(T4)in neonatal dried blood spots(DBS).Methods Microplates coated with T4 competitive-method paired monoclonal antibody(capture antibody)were used as the solid phase.Europium(Eu)-labeled T4 monoclonal antibody(anti-T4)was used as the tracer.The fully automatic TRFIA system was applied to quantitatively detect the T4 concentration in the blood spots on the filter paper fixed onto plate needles.The analytical performance indicators,including sensitivity,accuracy,linearity,precision,specificity,chromatographic effect,matrix effect,recovery test,volume effect,hematocrit effect,and stability,were evaluated.120 paired serum and DBS samples were selected for the comparison test between the self-developed method and the T4 chemiluminescence method.Residual DBS samples from 312 newborns were used for combined T4 and thyroid-stimulating hormone(TSH)detection.Results The limit of blank was 0.44 μg/dl serum,the limit of detection was 0.81μg/dl serum,and the limit of quantification was 1.61 μg/dl serum.The relative deviations for certified reference materials ranged from-5.33％to 1.83％.Excellent linearity was observed within the range of 1.61～50.00 µg/dl serum(r=0.999 6).The intra-assay,inter-assay,repeatability,laboratory and inter-laboratory coefficients of variation(CV)were all within 6.00％.The cross-reactivity rates for nine high-concentration cross-reaction substances ranged from 0.021％to 1.980％,and the cross-reactivity rate for 20µg/dl D-T4 DBS sample was 43.900％.The interference rates of nine high-concentration interfering substances on T4 measurements were all within±10％.Blood spots punched from the edge yielded higher results than those punched from the center or from the midpoint between the central and edge(t=4.44,5.26,both P<0.05).The relative deviations between measured and theoretical values for DBS samples prepared with three different batches of 903 filter paper were all within the acceptable range(±15.00％).Recovery rates ranged from 91.64％to 104.07％.Compared with DBS samples prepared at a volume of 50 μL/drop,those prepared at other volumes showed statistically significant differences(all P<0.05).The relative deviations for DBS samples prepared at hematocrit levels of 35％to 60％compared with those at 50％were all within±15％.The Eu-marker could withstand up to four freeze-thaw cycles at-20℃,and the reagent could be stored at 2 to 8℃for 14 months and at 37±1℃for 9 days.Within the range of 2.07～36.11 µg/dl serum,the results of the established method showed a strong correlation with those of the comparator method(r=0.995 5,tr=112.73,P<0.05),with no statistically significant difference in detection rates between the two methods(χ2=0.50 and 0.50,both P>0.05).The average concentration of T4 in neonatal DBS samples was 18.90 µg/dl serum.The detection rates of T4 and TSH differed significantly(χ2=11.56 and 16.52,all P<0.05).Conclusions The established method demonstrates high sensitivity,good accuracy,wide linear range,excellent precision and high specificity.It shows strong correlation and consistency with the comparative method,and meets the requirements for routine clinical testing.