This study described the distribution and dynamic changes of biochemistry analytes from healthy term infants during the first 3 days of life in northeast China. There was heterogeneity in analytes with different biological sources of variations, however, day age showed a predominant correlation with the concentration for most analytes. Premature and term infants were prone to different diseases, and the demand for clinical evaluation and nursing intervention varied . Therefore, the local RIs were derived for ensuring a high accuracy of clinical management.
Age-dependent RIs for 26 common biochemical analytes
In reviewing the literature, gestational age and birthweight were regarded as key factors for establishing RIs for preterm infants, but little difference of the two would be found in healthy term newborns. Although the guidance documents for establishing RIs only set the lowest limit for the number of subgroup participants, there was no single, specific definition for age intervals. If the age intervals were too wide, the rapid changes in a short time might be otherwise overlooked. Therefore, this study statistically depended upon SDR-dm, SDR-sex and SDR-age to analyze differences between subgroups, and then judged whether it was appropriate for clinical practice to partition RIs according to delivery mode, sex and postnatal age. Regarding the test results of SDR-dm and SDR-sex, RIs of all items did not require specific partitions (SDR <0.40) except for K, P and BUN. It was suggested that the combination of scatter plots and SDR values decide whether to partition RIs for test results comparing across different groups. This helps avoid exaggerated SDR for the actual narrow range of RIs or masked differences caused by the interaction between the factors.[2, 21] In effect, no sex difference for all analytes was detected , which was consistent with data in different regions [8, 17, 22].
However, there were some considerable differences in postnatal age. This might be due to the rapid physiological adaptation to extrauterine life for newborns. A possible explanation for this might be that perinatal data were mainly affected by maternal and neonatal physiological factors during the first days of life. The concentration of each analyte reflected the balance between the production, metabolism and clearance. First, some easy-to-understand mechanisms may cause short-term fluctuations of most analytes, such as proteins, electrolytes, lipids  and renal function markers , listed as follows: 1) the exchange of substances based on placental transfer between the fetus and mother ceased abruptly after delivery; 2) developmental and maturational changes occurred in the organs during the perinatal period, especially liver and kidney；3) the intake of external nutrients directly and indirectly affected the concentration of metabolites. Furthermore, the stress response during labour can also have an impact. Enzyme levels evaluated in our study were consistent with Lackmann et al. who found that the cytoplasmic and mitochondrial enzymes presented similar activity curves, whereas the membrane-bound enzymes showed the opposite. The differences in enzymes were considered the result of minor cell damage caused by physiological hypoxia during labour . However, the changes in our study may be not obvious enough because the release of enzymes was also affected by uterine contractions and physical stress through the birth canal, while the small number of natural delivery were included . The regulation of substances by hormones was equally important after birth, which can be demonstrated by bone metabolism status in neonates. In addition to maternal vitamin D during pregnancy , serum Ca, Mg and P homeostasis was also regulated by foetal parathyroid hormone (PTH), calcitriol, calcitonin, calcium sensing receptor (CaSR) and fibroblast growth factor-23 (FGF-23), which affecting bone physiology, intestinal absorption and renal excretion .
Other variables that related to RIs of biochemical analytes
Generally the main considerations for sources of variations of each tests were sex, age and BMI in adults and children, besides races and regions. In the case of neonates, even more factors came into play, including maternal and infant health, delivery mode, gestational age and weight at birth. The comparison between the MLA model and the nested ANOVA method indicated that the results of delivery mode and sex were almost the same in present study, and postnatal age was the main influence on the concentration of most analytes. Birthweight and gestational age were widely used in evaluating neonatal clinical outcomes, while both had limited effects on a few items. A possible explanation for this might be that the premature or overdue babies and the low birthweight or fetal macrosmia were not included in this study.
In this study, there was poor negative correlation between birthweight and ALT and ALP, but positive correlation with P and TIBC although in term healthy newborns. Surprisingly, the small but consistent inverse associations of birthweight with ALT and ALP remained in late adolescent  and adult . In fact, an elevated level of ALT within the normal range was proven to predict the hepatic steatosis of the metabolic syndrome [30, 31]. It may be assumed that birthweight is a proxy for exposures such as intrauterine nutrition or genetic factors that directly affect the liver. In addition, the opposite correlation of birthweight with ALP and P may reflect the inverse association between birthweight and bone strength. For another, the observed increasing levels in CK, TP and ALT with GA could be attributed to the rate of the babies' muscular mass, or to the development of their metabolism in advanced pregnancy [13, 32]. It is noteworthy that the expression of LC3-II and p62 in cord blood was associated with serum total protein in infants, perhaps suggesting that the autophagy reaction introduced by postnatal starvation played a crucial part in the maintenance of protein or amino acid metabolism during the perinatal period .
Comparison to Other Studies
RIs in our study were compared with those of kit inserts and other studies, which covered different experimental types and designs, assay systems, characteristics of the studied population and specimen types. The manufacturer RIs were less reliable in the evaluation of clinical applicability before use in Chinese laboratories, because they were generally based on Caucasian people and lacked data on children. The RIs of Cl, K and Na were similar to those from reagent inserts and previous findings for children and adolescents . Therefore, it seemed that the three electrolytes remained relatively stable in one’s life. The differences of RIs for Ca, Mg, P and lipids between this study and a study in Korea, to some extent, might be explained by the fact that umbilical cord contained maternal substances for fetal development. Compared to another two studies in other parts of China, there might be some factors that contributed to the differences between RIs, including actual differences between cities, laboratories, analytical systems and statistical calculation methods reported in these studies. Moreover, Liu et al . used an indirect method for determination of RIs, which based on the stricter exclusion criteria and wider age intervals, and then little data about the rapid changes was presented during the first days.
As mentioned in the Caliper’s report about transferred RIs from Abbott to Ortho assays , Ca, CO2, Mg and LDH did not meet transference or verification criteria. Except these, the RIs of most corresponding analytes showed higher values, which might be attributed to racial and diet differences. This finding further support that laboratories should verify transferred RIs for local population and analytical platform—that covers as many partitioned RIs as possible—according to CLSI guidelines.
Unfortunately, several limitations existed. First, being limited to recruiting healthy newborns, this study just determined the RIs in newborns aged 0-3 days, and new data on infant period should be supplemented in the future. Second, once the RI was partitioned, the small sample size would be hard to assure a highly accurate RI with narrow confidence intervals. Finally, all the analytes should be evaluated the validity of routine use in clinics and further investigated in preterm infants. The percentile charts provided might be integrated into the hospital and laboratory information system to implement new strategies for result display.