Validation of creatinine assays utilizing HPLC and IDMS traceable standards in sera of children

Abstract

The purpose of this study was to validate serum creatinine (SCr) concentrations assayed in the Central Biochemistry Laboratory of the National Institutes of Health (NIH)-funded Chronic Kidney Disease in Children (CKiD) study utilizing an enzymatic assay (Siemens Advia 2400) against a method traceable to reference isotope dilution mass spectroscopy (IDMS) developed by the National Institute of Standards and Technology (NIST). High-performance liquid chromatography (HPLC) measured SCr after external validation utilizing IDMS-based standard reference materials. Sera from the first 201 subjects enrolled in CKiD were analyzed and compared for creatinine concentration by enzymatic and HPLC methods. Fifty "normal" pediatric sera were subsequently analyzed. Finally, a "pediatric" reference standard was prepared and examined for accuracy and precision. Enzymatic SCr concentrations (median 1.4 mg/dl) of CKiD subjects were well correlated with HPLC (r = 0.984) but were slightly higher (+7%; p < 0.001). Agreement was poorer at lower SCr (median 0.4 mg/dl) when using samples from normal children and the "pediatric" reference standard. However, the Roche enzymatic assay was comparable with HPLC in accuracy and precision. Referring physicians should be aware of the accuracy and reproducibility of their laboratory's SCr assay. Our enzymatic assay agreed well with HPLC in CKiD subjects with elevated SCr. We suggest that NIST develop a pediatric SCr standard reference material for use by assay manufacturers to improve accuracy and precision of assays at the low SCr levels observed in most pediatric patients.

Fig. 1

Comparison of Siemens Advia enzymatic assay against high-performance liquid chromatography (HPLC) using sera from the Chronic Kidney Disease in Children (CKiD) subjects. a Histogram of results and comparison of each determination showing a correlation of 0.98. The y axis is in milligrams per deciliter (mg/dl) presented in log scale. b Bland-Altman plot showing mean of Advia and HPLC values on the x axis and ratio on the y axis (difference of logs), with a bias of 7% overestimation by the Advia enzymatic assay. The scatter clearly appears larger at low serum creatinine values

Fig. 2

Comparison of Siemens Advia enzymatic assay against HPLC at low levels of creatinine. The y axis is in milligrams per deciliter (mg/dl) presented in log scale. Histogram of results and comparison of each determination showing a substantial negative bias compared with high-performance liquid chromatography (HPLC) values

Fig. 3

Comparison of Siemens Advia enzymatic assay against high-performance liquid chromatography (HPLC) using 251 samples from the 201 CKiD subjects and the 50 healthy pediatric cases. Histogram of results and comparison of each determination showing a correlation of 0.97. The y axis is in milligrams per deciliter (mg/dl) presented in log scale. There is more scatter in the Advia assay at levels of serum creatinine below 1.0 mg/dl

Fig. 4

Box plots of multiple analyses (20) of diluted pediatric standard by enzymatic and high-performance liquid chromatography (HPLC) assays. The rectangle describes the interquartile range (IQR, 25th and 75th percentiles) and any horizontal line through the rectangle denotes the median. The horizontal portion of the “T” depicts upper and lower adjacent values, respectively, defined as the largest observation that is ≤75th percentile + 1.5 × IQR and the smallest observation that is ≥25th percentile minus 1.5 × IQR. Points above or below the upper and lower adjacent values are outliers. a Box plot of HPLC against Siemens Advia showing that the Siemens assay has a positive bias of 19% and a much larger range of values compared with HPLC. The CV of HPLC was 2% compared with 16% of the Advia. b Box plot of HPLC against the Roche enzymatic analyzer showing close agreement and narrow interquartile ranges. The CVs were 1.6% for HPLC and 2.3% for the Roche