Distribution of serum neuron‐specific enolase and the establishment of a population reference interval in healthy adults

Background Neuron‐specific enolase (NSE) is an important tumor marker in the serum of patients with lung cancer. Elevated serum NSE levels are also associated with many other diseases. However, there is no unified population reference interval for serum NSE. This study aimed to investigate the distribution of serum NSE in healthy Chinese adults aged 20‐79 years and to establish its reference interval in Chinese population. Methods A total of 10 575 healthy subjects were in line with the requirements of this study. The concentration of serum NSE was detected by a fully automated Cobas e602 analyzer with matching reagents. The population reference interval for serum NSE was established using the unilateral 95th percentile (P 95) according to standard guidelines. Results The distributions of serum NSE were not significantly different between males and females (P > 0.05) and also did not differ by age (P > 0.05). Therefore, the population reference interval for serum NSE was established as upper limit 25.4 ng/mL (90% confidence interval: 24.5‐26.2 ng/mL). Conclusions We established the first population reference interval for serum NSE in a large healthy Chinese adult cohort, which was higher than that recommended by Roche Diagnostics GmbH. This new reference interval is more practical and applicable in Chinese adults.

Background: Neuron-specific enolase (NSE) is an important tumor marker in the serum of patients with lung cancer. Elevated serum NSE levels are also associated with many other diseases. However, there is no unified population reference interval for serum NSE. This study aimed to investigate the distribution of serum NSE in healthy Chinese adults aged 20-79 years and to establish its reference interval in Chinese population.
Methods: A total of 10 575 healthy subjects were in line with the requirements of this study. The concentration of serum NSE was detected by a fully automated Cobas e602 analyzer with matching reagents. The population reference interval for serum NSE was established using the unilateral 95th percentile (P 95 ) according to standard guidelines.

Results:
The distributions of serum NSE were not significantly different between males and females (P > 0.05) and also did not differ by age (P > 0.05). Therefore, the population reference interval for serum NSE was established as upper limit 25.4 ng/ mL (90% confidence interval: 24.5-26.2 ng/mL).

Conclusions:
We established the first population reference interval for serum NSE in a large healthy Chinese adult cohort, which was higher than that recommended by Roche Diagnostics GmbH. This new reference interval is more practical and applicable in Chinese adults.

K E Y W O R D S
neuron-specific enolase, population reference interval, serum

| INTRODUC TI ON
Lung cancer is the most commonly diagnosed cancer worldwide (11.6% of all cancer cases) and the leading cause of cancer death (18.4% of all cancer deaths). 1 Lung cancer can be divided into two main types: non-small-cell lung cancer and small-cell lung cancer.
About 80%-85% of lung cancers are non-small-cell lung cancer. 2 Neuron-specific enolase (NSE) is an important tumor marker in the serum of patients with lung cancer. [3][4][5] Elevated serum NSE levels are also associated with neuroblastoma, neuroendocrine neoplasms, renal cell carcinoma, multiple myeloma, brain trauma, Guillain-Barre syndrome, and other diseases. [6][7][8][9] Therefore, measurement of serum NSE can be used as an auxiliary marker for the diagnosis of several diseases. Currently, however, there is no unified population reference interval for serum NSE. Independent clinical laboratories often refer to the reagent manufacturer's instructions for the population reference interval for serum NSE. This interval has not been validated, however, and therefore, the utility of serum NSE is limited in clinical applications. 10 Moreover, the tested level of serum NSE will also be affected by detection system, genotype, geographical location, lifestyle, and many other factors. 11 Thus, the population reference interval of serum NSE could be variable in different population.
However, most Chinese laboratories use the NSE population ref-

| Specimen collection
Five milliliters of fasting venous blood was collected in the morning.
The samples were centrifuged within 2 hours, and packed serum was stored at −80°C until testing. Ten milliliters of fresh morning urine also was collected. Routine urine measurements were completed within 2 hours of collection.

| Instruments and reagents
To minimize analytical errors, the same lots of reagents, standards, and quality control materials (means to check system with traditional QC runs which helps detect both bias shifts and imprecision) were used throughout the analyses. Serum NSE levels were analyzed via electrochemiluminescence immunoassay

| Outlier test
According to the Dixon method recommended in the CLSI C28-A3 guidelines, 12 the detection result is sorted by size to calculate the range, R. The distribution then is used to calculate the difference (D) between the maximum value and the minimum value and its adjacent value. If D/R ≥ 1/3, then the maximum or minimum value is treated as an outlier. This process is repeated on the remaining data until all outliers are eliminated.

| Identification of biological reference interval groups
We referred to the recommended methods in CLSI C28-A3 and WS/T 402-2012 ("Establishment of reference interval for clinical laboratory test items"), 12,13 and applied the Z test to determine whether the data from two comparison groups can be combined.
The formula for the Z test is: where x 1 and x 2 represent the mean values from the two groups, s 1 and s 2 represent the standard deviations from the two groups, and n 1 and n 2 represent the sample sizes of the two groups. If Z > Z*, then the difference between the reference intervals is statistically significant (P < 0.05) and the reference intervals for the two groups should

| Statistical analysis
The data were analyzed in IBM SPSS Statistics version 20 (Armonk, NY, USA). The Kolmogorov-Smirnov test was applied to test the normality of the data. Non-normal data were expressed using the median and interquartile range (IQR), and the Kruskal-Wallis H test was used to compare across multiple groups. Non-parametric (ranked) method was used to establish reference interval by CLSI C28-A3. A P-value <0.05 was considered statistically significant.

| RE SULTS
A total of 10 575 individuals were ultimately retained for inclusion in this study, including 5056 males and 5519 females. Kolmogorov-Smirnov test showed a skewed distribution for serum NSE level (P < 0.05, Figure 2). The distribution of serum NSE levels was not significantly different between males and females, which indicated that the levels of NSE were not correlated with gender (Z = 4.89, Z* = 19.91, P > 0.05; Table 2). Furthermore, no significant age-related differences in serum NSE levels were observed, which showed that the levels of NSE were not correlated with age (P > 0.05; Table 3).
In accordance with non-parametric method recommended by CLSI C28-A3 and WS/T 402-2012, the upper reference limit is important, which was set at the 95th percentile value. Accordingly, the population reference interval for serum NSE was established as upper limit 25.4 ng/mL by using the method of ECLIA in this study.

| D ISCUSS I ON
As an important tumor marker, serum NSE has been widely clinically used in the world. 14,15 Just like other markers, a measurement result of serum NSE by itself without any appropriate reference interval or medical decision limit is of little value. The reference interval is essential for the interpretation of clinical laboratory results and patient TA B L E 2 Distribution of serum neuron-specific enolase (NSE) by sex In summary, we established the reference interval for serum NSE using ECLIA in large-scale healthy Chinese population. Furthermore, the findings from this study suggest that every laboratory should establish a serum NSE population reference interval that accounts for the regional population, factors in the regional environment, and detection system characteristics.

ACK N OWLED G M ENTS
This study was supported in part by grants from Health Scientific