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.

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. ① The 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 of α-L-iduronidase (IDUA), α-N-acetylglucosaminidase (NAGLU), and β-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).

① By 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. ② There 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).

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).