Introduction

Serum creatinine is the most widely used marker to predict glomerular filtration rate. However, serum creatinine concentrations are not determined only by glomerular filtration [1], as creatinine production is proportional to muscle mass [2]. In children, muscle mass increases significantly with linear growth. To reflect the renal function, serum creatinine concentrations should be adjusted for body height and body size. In childhood, therefore, serum creatinine levels are dependent on age and muscle mass [3–6].

Cystatin C, a 13 kDa non-glycosylated low molecular weight protein [7], is a proteinase inhibitor involved in the intracellular catabolism of proteins [8]. Unlike creatinine, cystatin C is produced in all investigated nucleated cells at a constant rate, freely filtered in the renal glomeruli, and almost completely reabsorbed and catalyzed in the renal proximal tubular cells [9, 10].

In the existing literature, the proposed ranges for serum cystatin C in pediatric populations are inconsistent, with several small, single-institution, hospital, or clinic-based studies [11, 12]. In addition, the reported cystatin C ranges are affected by use of different cystatin C assays [13]. Furthermore, some previous studies suggested that cystatin C levels were independent of gender, age and body composition [14, 15], whereas others showed differences in serum cystatin C levels according to gender, age and race [16]. The aim of this study was to establish reference ranges for cystatin C levels in Japanese children by using 4 different assays.

Subjects and methods

Serum cystatin C levels were studied in 1128 children (503 boys and 625 girls) aged between 3 months and 16 years visiting the outpatient pediatric clinic, or hospitalized at Aichi Children’s Health and Medical Center, Tokyo Metropolitan Children’s Medical Center, Yokohama City University Medical Center, Niigata University, Seirei Hamamatsu General Hospital, Fussa Hospital, or Tokyo Health Service Association between 2008 and 2009 without clinical evidence of kidney diseases, urogenital diseases, infectious diseases, inflammatory diseases, muscular diseases, malignant diseases, cardiovascular diseases, liver or pancreas diseases, anomaly syndrome, hypertension, dehydration, or pregnancy. None of the subjects had hyperthyroidism or hypothyroidism. The children’s parents provided written informed consent according to the Declaration of Helsinki, and ethics approval was obtained from the institutional review board.

Serum cystatin C was analyzed at SRL Inc (Tokyo, Japan) by using 4 different cystatin C assays—Nescaute GC cystatin C (Alfresa Pharma Corporation, Osaka, Japan), LZ TEST ‘EIKEN’ cystatin C (Eiken Chemical, Tokyo, Japan), and Iatro Cys-C (Mitsubishi Chemical Medience, Tokyo, Japan) on the BioMajesty JCA-BM8020, and the N Latex Cystatin C assay (Siemens Healthcare Diagnostics Inc., Tokyo, Japan) on the Behring Nephelometer II (BNII; Siemens Healthcare Diagnostics Inc., Tokyo, Japan). All assays were programmed and calibrated according to the manufacturer’s instructions.

The central 95 % reference ranges were calculated using the nonparametric method, and the Mann–Whitney U test was used for the analysis. All p values were based on two-sided testing and a significance level of 0.05 was used for the analysis.

Results

Subject characteristics are shown in Table 1. The subjects’ median height and weight were 117.6 cm (range 57.0–184.6), and 21.7 kg (range 5.0–100.8 kg), respectively. The median body mass index (BMI) of the subjects was 16.4 (range 12.2–32.5) and 2 (0.2 %) of 1128 subjects had a BMI of ≥30.

Table 1 Patient characteristics
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The serum concentrations of cystatin C were highest after birth followed by a decrease over the following months in each assay, when normal adult ranges of cystatin C were reached (Fig. 1). After the first 2 years of life, the median serum cystatin C became constant and slightly decreased in children aged 15–16 years. The median serum cystatin C level in children aged 2–11 years was similar in males and females (p ≥ 0.05; all assays). However, the median serum cystatin C level in children aged 12–16 years was significantly higher in males than in females (p < 0.0001; all assays) (Fig. 2).

Fig. 1
figure 1

Serum cystatin C in children aged 3 months to 16 years. The box plot extends from the 25th percentile to the 75th percentile, with the horizontal line at the median, and the whiskers show the central 95 % of the data for Alfresa (a), Eiken (b), Mitsubishi (c), and Siemens assays (d)

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Fig. 2
figure 2

Serum cystatin C in male and female children. The box plot extends from the 25th percentile to the 75th percentile, with the horizontal line at the median, and the whiskers show the central 95 % of the data for Alfresa (a), Eiken (b), Mitsubishi (c), and Siemens assays (d)

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The distribution of serum cystatin C for children by age, gender and assay is shown in Table 2. The reference ranges in children aged 2–11 years were Alfresa, 0.59–1.01 mg/L; Eiken, 0.61–1.04 mg/L; Mitsubishi, 0.50–0.83 mg/L; and Siemens, 0.52–0.88 mg/L. Overall, the serum cystatin C levels measured using the Alfresa and Eiken assays were significantly higher than those measured using the Mitsubishi and Siemens assays (p < 0.0001).

Table 2 The central 95 % reference ranges and median of serum cystatin C (mg/L) measured by the 4 assays in children
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Discussion

Serum cystatin C concentrations were measured in children by using 4 different automated assays and calculated assay-specific cystatin C ranges in this study. The highest serum cystatin C concentration measured by all 4 assays was found after birth, followed by a rapid decrease over the following months, consistent with previously published data [17, 18]. Cataldi et al. [19] reported that serum cystatin C does not cross the placental barrier; therefore, the high values of serum cystatin C after birth probably reflect the degree of maturation of the glomerular filtration capacity.

The concentrations of serum cystatin C were constant in children >2 years, and the nonparametric reference ranges of the Alfresa and Eiken assays were higher than that obtained by the Mitsubishi and Siemens assays. The difference had been explained by the differences in the methods for measurement of a particle-enhanced nephelometric immunoassay in contrast to a particle-enhanced turbidimetric immunoassay [20]. However, since the Eiken assay and the Mitsubishi assay are both particle-enhanced turbidimetric immunoassays, the difference of the reference ranges for cystatin C was not explained by the use of the different methodologies.

This study showed that cystatin C decreased in children aged 15–16 years, and serum cystatin C in children aged 12–16 years was higher in males than in females, and supported the result of a previous study conducted in US adolescents [21]. In addition, assay-specific differences in serum cystatin C levels in children were also observed in this study. There are concerns raised with regard to measuring serum cystatin C levels, as assay-specific differences were observed in levels of serum cystatin C measured.

The Institute for Reference Materials and Measurements (IRMM) announced the availability of the new certificated reference material ERM-DA471/IFCC [22]. The standardized measurement of serum cystatin C using ERM-DA471/IFCC is now being developed.

In conclusion, our study provided age-, gender- and assay-specific ranges of cystatin C for Japanese children. Age-, gender-, race- and assay-specific ranges for serum cystatin C should be used as another tool to assess kidney function in children. The standardized measurement of serum cystatin C will be a reliable marker for the recognition of abnormal renal function compared to serum creatinine.