新生儿黏多糖贮积症的尿滤纸干片法酶联显色法定量检测:改良 DMB 法的初步结果。

Neonatal screening for mucopolysaccharidoses by determination of glycosaminoglycans in the eluate of urine-impregnated paper: preliminary results of an improved DMB-based procedure.

机构信息

Neonatal Screening Laboratory, Department of Paediatrics, University Hospital Complex and University of Santiago de Compostela, Spain.

出版信息

J Clin Lab Anal. 2010;24(3):149-53. doi: 10.1002/jcla.20375.

DOI:10.1002/jcla.20375

PMID:20486194

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6647676/

Abstract

BACKGROUND

The fact that mucopolysaccharidoses (MPSes) are now treatable, and that the earlier treatment is initiated the better, is an indication for neonatal screening. The most efficient approach seems likely to be a multi-tier procedure in which screening for urinary glycosaminoglycan (GAG) is followed by enzyme determinations in heelprick blood of newborns screening positive. Hitherto the method of choice for the determination of GAG has been the measurement of absorbance by a complex of GAG and 1,9-dimethylmethylene blue (DMB).

METHOD

We evaluated a DMB method in which absorbance by DMB is measured following its addition to the eluate obtained from paper-borne newborn urine samples and is normalized relative to urinary creatinine. Calibration is performed with chondroitin-6-sulfate (Ch-6-S).

RESULTS

The limits of detection and quantification of GAG were 1.98 and 5.94 mg/dl, respectively. The within-run coefficients of variation (CVs) of the GAG/creatinine ratio for 25, 31, and 70 mg/dl solutions of Ch-6-S in urine were 21.8, 16.4, and 10.5%, respectively, and the corresponding between-run CVs were 25.0, 13.5, and 10.1%. Recovery from the urine spiked with 31 mg Ch-6-S/dl was 94.8%. Accuracy was also acceptable for all other GAGs except hyaluronic acid. For neonatal screening, the diagnostic threshold was tentatively established as 800 mg GAG/g creatinine, the 95th centile of samples from 903 infants aged 3-28 days, but the value of the GAG/creatinine ratio was negatively correlated with age. Application of the new method to samples from older individuals with and without MPS achieved 100% sensitivity and specificity when used with an age-dependent threshold taken from the literature on the original DMB method.

CONCLUSION

If used in the first tier of a multi-tier screening protocol, the proposed method would allow the detection of abnormal levels of all GAGs except hyaluronic acid.

摘要

背景

黏多糖贮积症（MPS）现在可以治疗，且治疗越早效果越好，这表明需要进行新生儿筛查。最有效的方法似乎是一种多步骤程序，即先对尿糖胺聚糖（GAG）进行筛查，然后对筛查呈阳性的新生儿足跟血进行酶测定。迄今为止，用于测定 GAG 的方法是通过 GAG 与 1,9-二甲基亚甲蓝（DMB）复合物的吸光度来测量。

方法

我们评估了一种 DMB 方法，即在将 DMB 添加到从纸质新生儿尿液样本中洗脱得到的洗脱液后测量 DMB 的吸光度，并相对于尿肌酐进行归一化。用硫酸软骨素 6-硫酸盐（Ch-6-S）进行校准。

结果

GAG 的检测限和定量限分别为 1.98 和 5.94mg/dl。在 25、31 和 70mg/dl 的 Ch-6-S 尿液溶液中，GAG/肌酐比值的日内变异系数（CV）分别为 21.8%、16.4%和 10.5%，相应的日间 CV 分别为 25.0%、13.5%和 10.1%。从 31mg Ch-6-S/dl 的尿液中回收的 GAG 为 94.8%。除透明质酸外，其他 GAG 的准确性也可以接受。对于新生儿筛查，暂定诊断阈值为 800mg GAG/g 肌酐，这是 903 名 3-28 天大婴儿样本第 95 百分位数，但 GAG/肌酐比值与年龄呈负相关。将新方法应用于年龄较大的有或没有 MPS 的个体的样本中，当与原始 DMB 方法文献中的年龄相关阈值一起使用时，达到了 100%的灵敏度和特异性。