血样低温放置时间对黏多糖贮积症酶学检测的影响

Effects of storage time of whole blood samples on enzyme activity assay for diagnosis of mucopolysaccharidosis at low temperature

目的探讨4℃条件下血样放置时间对黏多糖贮积症(MPS)酶学检测的影响。方法选择2020年1月5日至9日,采集自5例健康志愿者(男性为2例,女性为3例,年龄为24~30岁)的血样各6管为研究对象。将其依次命名为血样1、2、3、4、5(研究组)。将采集的5×6管血样,置于4℃条件保存,于不同放置时间点(0、24、48、72、96、120 h)对其进行MPS酶学检测。同时收集本院医学遗传科20例健康受试者进行外周血细胞染色体检查后的剩余血样,将其混合与研究组同批次检测作为MPS酶活性正常值(对照组)。①观察研究组血样放置不同时间点,血浆颜色变化,并进行血浆游离血红蛋白(f-Hb)检测,评估血样溶血情况。②进行白细胞分离,并检测放置不同时间点白细胞中α-硫酸艾杜糖醛酸酶(IDUA)、α-N-乙酰葡萄糖胺酶(NAGLU)和β-葡萄糖醛酸酶(GUSB)活性水平,比较各种酶活性水平在不同检测时间点的差异。所有受试者均签署临床研究知情同意书,本研究遵循的程序符合四川大学华西第二医院伦理委员会制定的伦理学批准,并得到该委员会批准(审批文号:20180094)。结果①血样在放置72 h时血浆颜色开始逐渐变红,放置时间越长,变红的程度越深;血浆f-Hb水平提示放置72 h呈增高趋势,并且随着放置时间延长,溶血程度也越来越严重。②血样1~5放置24、48、72、96、120 h时,IDUA、NAGLU活性水平分别与自身未放置血样比较,差异均无统计学意义(P>0.05)。③血样1~5在放置24、48、72、96 h的GUSB活性水平分别与未放置血样比较,差异亦均无统计学意义(P>0.05);但是,放置120 h时GUSB活性水平明显低于未放置血样,2组比较,差异有统计学意义(t=4.601、P=0.002)。结论综合溶血情况和酶活性水平等多种因素,建议进行MPS酶学检测的外周血血样于采集后72 h(3 d)内送达检测实验室,72~120 h(3~5 d)送达实验室的血样,需取决于血样自身质量和待检测溶酶体酶类型,于120 h(5 d)时送达的检测失败可能性极大。

Objective To evaluate the impact of different storage time of enzymatic detection of whole blood samples on enzyme activity assays for the diagnosis of mucopolysaccharidosis(MPS)at low temperature(4℃),and provide experimental basis for controlling the storage time of MPS enzymatic samples before detection and the delivery time of samples.Methods From January 5 to 9,2020,five healthy volunteers,two males and three females,aged 24 to 30 years,were enrolled.Six tubes of blood samples were collected from each volunteer and placed for testing at different time points(0,24,48,72,96,120 h)after collection and named in order as samples 1,2,3,4,5.The remaining samples from 20 cases after peripheral blood cell chromosome examination at the Department of Medical Genetics&Prenatal Diagnosis Center of same hospital were also collected and mixed as the source of normal values of enzyme activity in this study.①The impact of sample placement time on hemolysis was evaluated by visual observation and quantitative determination of free hemoglobin(f-Hb).②Leukocytes were isolated and the activity ofα-L-iduronidase(IDUA),α-N-acetylglucosaminidase(NAGLU),andβ-glucuronidase(GUSB)were analyzed.The differences of enzyme activity at each time points mentioned above were compared.All subjects signed the clinical study informed consent forms,and the procedures followed in this study were in accordance with the ethical approval established by the Ethics Committee of West China Second University Hospital,Sichuan University,and approved by this committee(approval No.20180094).Results①By visual observation,the longer time duration was,the deeper the color of the samples was.The color of plasmas began to turn red slightly at 72 h and became redder with the prolonged storage time.The results of quantitative analysis also showed that the level of f-Hb increased significantly at 72 h.With prolonged storage time,the degree of hemolysis became more deeper.②There were no statistically significant differences in the levels of IDUA and NAGLU activity at 24,48,72,96 and 120 h of sample 1-5 placement compared to 0 h,respectively(P>0.05).③The difference between the GUSB activity levels of samples 1-5 at 24,48,72,and 96 h of placement compared with these at 0 h was also not statistically significant(P>0.05);however,the GUSB activity level was significantly lower at 120 h of placement than that at 0 h.The difference was statistically significant between them(t=4.601,P=0.002).Conclusions Considering various factors such as hemolysis and enzyme activity levels,it is recommended that peripheral blood samples for MPS enzymatic testing be delivered to the laboratory within 72 h(3 d)after collection,and that samples delivered between 72 and 120 h(3-5 d)depend on the quality of the sample and the type of lysosomal enzyme to be tested,with a high probability of test failure if delivered after 120 h(5 d).