摘要目的 建立干血滤纸片和门细胞酸性α-葡萄糖苷酶(GAA)活性测定方法 及正常参考值,用于糖原累积病Ⅱ型(GSD Ⅱ)患者的酶学诊断.方法 利用GAA特异性水解荧光底物4-MUG的原理,采用阿卡波糖抑制其同工酶,建立干血滤纸片(DBS)和白细胞测定GAA活性方法 .取700例DBS样本和100例白细胞样本作为正常对照建立DBS和白细胞测定GAA活性正常参考值,并对临床疑诊4例患者及其父母进行GAA活性测定.结果 DBS法具有良好的精密度,室温或低于室温保存至少4周时DBS GAA活性无明显影响.正常新生儿和儿童-成人DBS的GAA参考范围分别为8.92～60.03 pmol/(punch·h)和8.00～37.43 pmol/(punch·h);正常人白细胞GAA参考范围:12.56～50.26 nmol/(mg蛋白·h),共检出4例GSD Ⅱ型患者.结论 DBS法测定GAA活性具有敏感、快速、高通量等特点,适于GSD Ⅱ型高危人群筛查和诊断;白细胞法测定GAA活性也具有快速、特异性高等特点,适于患者的确诊.

abstractsObjective Glycogen storage disease type Ⅱ(GSD Ⅱ,Pompe disease)is caused by the deficiency of acid α-glucosidase(GAA)that leads to lysosomal glycogen accumulation.Early diagnosis and treatment of GSD Ⅱ are considered to be critical for maximum efficacy of the enzyme replacement therapy.The aim of this study was to introduce two reliable methods and to generate the reference range of GAA activitv.Method The assay of GAA activity was performed in dried blood spots(DBS)and mixed leukocytes with acarbose to eliminate isoenzyme interference and to generate the reference range.GAA activitv was assayed in 700 specimens for DBS from normal subjects and 100 specimens for mixed leukocytes from normal subjects to set up reference range.GAA activity in the samples of 4 patients who were clinically suspected of GSD Ⅱ and their parents were also assayed.Result The intra-run and inter-run precision of the DBS method wag less than 10%.GAA activity tested by DBS was stable for 28 days between room temperature and-80 ℃.The reference range of newborns and children-adults in DBS samples was 8.92-60.03 pmol(punch·h) and 8.00-37.43 pmol/(punch·h),respectively.The reference range in mixed leukocytes samples was 12.56-50.26nmol/(mg protein·h).Four patients were diagnosed as GSDⅡ with the above-mentioned two methods.Conclusion The determination of GAA activity in DBS is sensitive and time-saving,and is suitable for high throughput analysis and newborn screening for GSD Ⅱ.The assav of GAA activity in mixed leukocytes is accurate,fast and specific,and is suitable for final diagnosis of GSD Ⅱ.