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INTRODUCTION
Cystatin C (cysC) is a 13-kDa, low-molecular-weight protein with a high isoelectric point [1]. CysC can be freely filtered through the glomerular membrane and synthesized at a relatively constant rate [2]. Owing to its characteristics, cysC is used as an alternative biomarker for predicting the glomerular filtration rate (GFR) [3], a parameter that reflects kidney function. CysC is less affected by age, race, and muscle mass than creatinine [4], which is the most widely applied marker for GFR estimation. Therefore, the 2012 Kidney Disease: Improving Global Outcomes (KDIGO) clinical practice guidelines suggest measuring cysC in patients with chronic kidney disease (CKD) confirmed solely by a creatinine-based estimated GFR (eGFR) of 45–60 mL/min/1.73 m2 [5]. Furthermore, cysC can have advantages over creatinine for the rapid detection of an abrupt decrease in eGFR in critically ill adults and children [6, 7].
There are two important issues related to measuring cysC in clinical settings. Systematic measurement errors can result from variability in the measuring systems such as reagents, instruments, calibrators, and control materials [8-12]. More than five manufacturers have produced cysC assay kits using different measurement principles such as immunoassay, nephelometric, turbidimetric, or spectrophotometric methodologies, resulting in various reagents [10, 11]. Uncertainties attributable to imprecision, bias, and drift have been reported [13-16], and the need for cysC measurement standardization has increased. Various equations are also used for eGFR. In 2012, the 2012 cystatin C-based Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPIcys) was developed [8]. Subsequently, the Caucasian, Asian, pediatric, and adult equations (CAPAeq) [17] and full age spectrum equation (FASeq) [18] were developed. Recently, the 2023 cystatin C-based European Kidney Function Consortium equation (EKFCcys) [19], which had a scaling factor for cysC that did not differ according to race or sex, was reported to have better accuracy than CKD-EPIcys, CAPAeq, or FASeq.
These two issues for the measurement of cysC have not been resolved, although manufacturers have altered their cysC measurement procedures using the ERM-DA471/IFCC traceable calibrator. Numerous combinations of measuring systems and novel equations have been generated, depending on the laboratory setting and equation selection. Studies on the effect of cysC reagents and equation combinations for eGFR after cysC standardization are limited [12, 15, 20]. We conducted a simulation analysis of cysC measured using two ERM-DA471/IFCC-standardized cysC reagents and analyzed the correlations among eight eGFR combination methods comprising two reagents and four equations as follows: CKD-EPIcys, CAPAeq, FASeq, and EKFCcys. We aimed to elucidate the effect of the cysC reagent and eGFR equation combinations for GFR estimation.
METHODS
Study design and participants
The equation for the Gentian cystatin C immunoassay (Gentiancys; GentianAS, Moss, Norway; Getianeq) was used to estimate the GFR with a single factor of the Gentiancys value [21]. Using a combination of Gentiancys and Getianeq, each eGFR value was categorized as follows: ≥90 mL/min/1.73 m2 (G1), 60–89 mL/min/1.73 m2 (G2), 30–59 mL/min/1.73 m2 (G3), 15–29 mL/min/1.73 m2 (G4), or <15 mL/min/1.73 m2 (G5). Gentiancys values of 1.98 and 1.22 mg/L produced eGFR results of approximately 30 mL/min/1.73 m2 and 60 mL/min/1.73 m2, respectively.
We reviewed the records of 413 participants who were referred for serum Gentiancys measurements at Samsung Medical Center Seoul, Korea, during a 7-day period in July 2021. Of these, 16 participants younger than 18 years of age were excluded; of the remaining 397 participants, 37 had a recorded Gentiancys of >1.98 mg/L and were enrolled (Q1). We selected 37 participants with recorded Gentiancys values ranging between 1.22 and 1.97 mg/L (Q2). As CKD-EPIcys has an inflection point at 0.8 mg/L of cysC, we additionally selected 37 participants with Gentiancys values ranging between 0.80 and 1.21 mg/L (Q3) and 37 participants with Gentiancys values <0.80 mg/L (Q4). Each selected group was matched by age and sex to the group with recorded Gentiancys values >1.98 mg/L. In total, 148 residual samples were analyzed in this study. The participants were categorized as G1 (N=44), G2 (N=36), G3 (N=31), G4 (N=20), and G5 (N=17), according to the eGFR for combined Gentiancys and Getianeq. The study was approved by the Institutional Review Board of Samsung Medical Center Hospital (IRB file no. 2021–06–180). A waiver of consent was obtained given the retrospective nature of the study.
Sample collection and storage
Peripheral venous blood samples were collected via venipuncture into a BD Vacutainer SST tube (BD, Plymouth, UK) and centrifuged for 10 minutes at 2,270×g. Separated serum aliquots were stored in an Eppendorf tube (Eppendorf, Hamburg, Germany) at 4°C until analysis.
cysC measurement
The cysC value of each aliquot was simultaneously measured using the Gentiancys and Tina-quant Gen.2 assays (Rochecys; Roche, Mannheim, Germany), both on a Cobas c702 automatic analyzer (Roche). Each instrument was set based on the manufacturer’s recommendations and application notes. The Gentiancys calibrator (REF 1012, GentianAS) and Rochecys calibrator (C.f.a.s. Cystatin C, Roche) were standardized against the ERM-DA471/IFCC. The details of the cysC assay conditions are presented in Supplemental Data Table S1.
Equations for cysC-based eGFR
We calculated and compared eGFRs with Gentiancys combined with Gentianeq and each cysC-based eGFR using the two reagents combined with the four equations of CKD-EPIcys, CAPAeq, FASeq, and EKFCcys [16-19, 21] (Table 1). The population-normalized cysC (Qcys) of FASeq was referenced from the FASeq study, which analyzed 6,132 participants from 11 cohorts [18], and the Qcys of EKFCcys was referenced from the EKFCcys study, which analyzed 227,643 participants [19]. For the Qcys of FASeq, values of 0.82 mg/L for ages <70 years and 0.95 mg/L for ages ≥70 years were used, and for the Qcys of EKFCcys, 0.83 mg/L for ages <50 years and 0.83+0.005×(age−50) mg/L for ages ≥50 years were used.
Statistical analysis
The Reed–Dixon analysis was performed to check for the absence of outliers. As an acceptable bias limit, ±7.61% of the total allowable error of the serum cysC measurements was used [22]. Bland–Altman plots were constructed to compare the concordance of each cysC-based eGFR equation and the measuring system. Passing–Bablok regression was performed to obtain a regression line between the Gentiancys and Rochecys values. To compare Getianeq with a single Gentiancys value and CKD-EPIcys, scatter plots and regression lines were constructed. Lin’s concordance correlation coefficient (ρc) [23] was used to assess the concordance between the eGFR calculations. Statistical Package for the Social Sciences version 25.0 (IBM Corporation, Armonk, NY, USA) and MedCalc (version 11.5.1.0; MedCalc Software, Mariakerke, Belgium) were used for statistical analyses and graph construction. Statistically, P<0.05 was considered significant.
RESULTS
The characteristics of the 148 enrolled participants are presented in Table 2. The mean age of the participants was 60.5 years (SD±14.5 years), and 43% were female. The mean cysC was 1.72±1.44 mg/L for Gentiancys and 1.71±1.35 mg/L for Rochecys. There was no significant difference in the mean cysC between Gentiancys and Rochecys.
The arithmetic mean of the percent difference was −2.74%±6.62%, and the lower and upper limits of the 95% confidence interval (CI) were −15.72% and 10.23%, respectively (Fig. 1A). Using a ±7.61% difference as the limit of concordance, the calculated cysC values exceeded the limits. The percent difference plot showed that the bias might have affected the measurements at low concentrations. The percentage difference converged to zero for higher concentrations, although it again widened for extremely high values >5 mg/L.
The obtained regression equation between Gentiancys and Rochecys was Rochecys=0.13 (95% CI, 0.12–0.15)+0.91 (0.90 to 0.93)×Gentiancys (Fig. 1B). According to the regression equation, the limit of the maximum acceptable difference, which satisfied a ±7.61% difference, was 0.85–4.40 mg/L based on Gentiancys. Of the 148 participants, 38 (25.7%) had values <0.85 mg/L and 12 (8.1%) had values ≥4.40 mg/L.
The end point for the linear portion of the regression line was approximately 60 mL/min/1.73 m2 based on a visual assessment (Fig. 2A). Comparing the eGFR of Gentiancys combined with Getianeq and that of Rochecys combined with CKD-EPIcys, the end point for the linear portion of the regression line was 60 mL/min/1.73 m2, after which the distance to the ideal line was greater than that of the combination of Gentiancys and CKD-EPIcys (Fig. 2B). Bland–Altman analysis results showed that differences between Getianeq and CKD-EPIcys increased by approximately 60 mL/min/1.73 m2 (Fig. 2C and D).
According to the groups with Gentiancys values, the eGFR results calculated for each combination are shown in Table 3. The mean eGFR range of all combinations was 63.4±38.0–58.8±28.6 mL/min/1.73 m2. Differences in eGFRs between Gentiancys and Rochecys were found mainly in the Q4 (6.8–11.7 mL/min/1.73 m2) and Q3 (2.4–3.4 mL/min/1.73 m2) groups rather than in the Q1 (−0.3 to −0.4 mL/min/1.73 m2) or Q2 (−0.8 to −1.0 mL/min/1.73 m2) groups.
According to the groups with Gentiancys values, comparisons of the ρc between the two reagents are described in Supplemental Data Table S2. In the Q1 and Q2 groups, there was a negligible difference between Gentiancys and Rochecys, whereas the difference between Gentiancys and Rochecys was significant in the Q3 and Q4 groups. Comparisons of the ρc among the four equations with the same reagent are described in Supplemental Data Table S3. In the Q1 and Q2 groups, FASeq and EKFCcys resulted in higher eGFR values than CKD-EPIcys and CAPAeq. In contrast, FASeq, but not EKFCcys, resulted in lower eGFR values in the Q3 and Q4 groups. The largest difference in eGFR was found between Rochecys combined with FASeq and Gentiancys combined with CAPAeq, but this was not the case for Gentiancys combined with FASeq, Rochecys combined with CAPAeq, and Rochecys combined with EKFCcys (Supplemental Data Table S4).
DISCUSSION
Initially, simple equations such as “eGFR=A×cysC–B” were developed for cysC-based GFR calculation [21, 24-27]. Potentially different results obtained with different measurement systems could potentially lead to larger differences in eGFR [8]. Each equation is recommended for use with a specific associated measuring system because of the discrepancy in eGFR among equations despite a fixed cysC value [14, 21, 24, 26, 28]. For the standardization of cysC, a certified reference material, ERM-DA471/IFCC, was produced by the Working Group for the Production of an International Cystatin C Calibrator (WG-SCC) in 2010 [29]. The 2012 cysC-based CKD-EPIcys was developed to include age and sex factors as well as the standardized cysC value [8], and the KDIGO guidelines recommend use of CKD-EPIcys for reporting cysC-based eGFR or the alternative equations shown to improve the accuracy of GFR estimation compared with CKD-EPIcys [5]. Therefore, CKD-EPIcys appears to be the most broadly applicable equation [30], although CAPAeq [17], FASeq [18], and EKFCcys [19] were suggested as their GFR estimation showed improved accuracy compared with that of CKD-EPIcys.
In 2010, ERM-DA471/IFCC was approved for cysC measurements in specific systems with successful calibration and negligible method bias [20, 31, 32]. Using ERM-DA471/IFCC as a calibrator or measurement trueness control material, each manufacturer generated adjusted calibration curves with improved measurement uncertainty [20, 31, 33, 34]. However, the outcome of harmonization and standardization for cysC measurement remains controversial [10, 11], although a few studies have reported improvements in measurement uncertainty for specific measuring systems [20, 31, 34]. Both Gentiancys and Rochecys, which use the particle-enhanced immunoturbidimetric (PETIA) method, have low analytical variability and bias [11, 32]. ERM-DA471/IFCC requires dilution because it is a single-level calibrator with a high cysC concentration of 5.48 mg/L. The results of the College of American Pathologists 2014 CYS Survey, international proficiency testing programs [11], and the 2015 survey involving seven clinical laboratories located in France and Belgium [10] showed limitations in the metrological traceability of commercial measuring systems for cysC. We found significant differences between Gentiancys and Rochecys at concentrations <0.85 mg/L and cysC concentrations ≥4.40 mg/L calculated using Gentiancys. Gentiancys resulted in higher values than Rochecys at very high cysC concentrations, similar to the results of a previous report [10]. In contrast, the Bland–Altman analysis suggested that Rochecys shows higher values than Gentiancys at low concentrations (<0.85 mg/L). These results are in line with a 2015 survey [10], which reported that clinically significant positive bias decreased with higher concentrations of Rochecys on the Cobas c502 system. Based on our results, the equivalence of cysC values at low concentrations (<0.85 mg/L) between the two PETIA-based measuring systems was unsatisfactory.
A low cysC concentration might be the baseline for evaluating abrupt cysC elevations in critically ill adults and children [35, 36], and the bias at low concentrations might dramatically affect GFR estimations [14, 15]. Recently, a study showed that the predicted concentrations from recalibration against ERM-DA471/IFCC were consistently 17% higher than the previously measured values using calibration with the Siemens nephelometer [31]. These values were 5–6% higher than the previous prediction used for the development of CKD-EPIcys.
In the current study, CKD-EPIcys, with an inflection point at 0.8 mg/L of cysC, resulted in the highest ρc value between the two reagents in the Q3 and Q4 groups (Supplemental Data Table S2). According to CKD-EPIcys, the exponent number of cysC is altered from −1.33 to −0.50 at 0.8 mg/L of cysC, which leads to a reduction in effectiveness for bias at low cysC concentrations. Conversely, CAPAeq and FASeq, without a compensation function at low cysC concentrations, resulted in dynamic ρc values ranging from 0.73 to 0.93 in the Q3 and Q4 groups, depending on the combination of reagents (Supplemental Data Table S4).
Our study showed that differences in eGFR values between Getianeq and CKD-EPIcys within Gentiancys were increased by approximately 60 mL/min/1.73 m2. Although the manufacturer continues to recommend the use of Getianeq for eGFR using Gentiancys values after standardization, CKD-EPIcys and two equations developed after standardization revealed different trends for eGFRs from approximately 60 mL/min/1.73 m2 compared with those of Getianeq. Thus, the initial equation cannot be confidently used in the absence of evidence indicating the superior accuracy of GFR estimations compared with that of CKD-EPIcys [5].
FASeq and EKFCcys tended to represent higher eGFR values for the Q1 group, which mainly consists of G4 and G5 eGFR categorization, compared with those obtained using CKD-EPIcys or CAPAeq. These results might reflect a relatively higher proportion of healthy participants in the FASeq development population [18, 37]. In addition, FASeq resulted in a less sensitive reflection of the eGFR in the Q3 and Q4 groups with low cysC concentrations compared with that obtained using CKD-EPIcys, CAPAeq, or EKFCcys (Table 3 and Supplemental Data Fig. S1). Because Qcys rescaling would require only a single rescaling factor, specifically the spectrum of age [19], EKFCcys would have the advantage of easily reducing bias based on the measured GFR. Compared with FASeq, EKFCcys used a modified Qcys, and each Qcys induced a marked difference in eGFR in the Q3 and Q4 groups.
Furthermore, the cumulative frequency distribution of eGFRs from each combination showed the effect of both reagents and equations on the eGFR results (Supplemental Data Fig. S1). The frequency distribution indicated that the equation prominently affects the eGFR at levels less than approximately 60 mL/min/1.73 m2, whereas the measuring system and equation have either an additive effect or a decreased effect according to each combination at levels greater than approximately 60 mL/min/1.73 m2. In contrast to a previous study [20], our data indicate that the diversity of cysC reagents is a cause of discordance in accuracy in comparison studies of equations.
To overcome the current limitations of the cysC measurement system, methodological improvements are required. First, the most promising candidate, isotope dilution mass spectrometry (IDMS), will be a reference measurement method for cysC [38, 39], and current commercial methods should be calibrated to be traceable with respect to the IDMS method. Second, it is essential to develop novel reference materials for cysC that are assigned clinically useful values and do not require dilution.
Our study has several limitations. First, we could not measure the GFR, which serves as the reference method for the eGFR. Second, we only performed one-point calibration, whereas multi-point calibration based on serial dilutions of a certified reference material or commercial calibrator would help to adjust the calibration factor. Finally, owing to limited resources, we evaluated only a limited number of participants. Due to these limitations, we could not show the accuracy and bias of each cysC value and eGFR equation.
Taken together, our data show that the equivalence of cysC values at low concentrations (<0.85 mg/L) between the two PETIA-based measuring systems is not satisfied after standardization. Results obtained with different measurement systems could lead to larger differences in the eGFR, which vary based on the combination used. Bias and accuracy analyses for cysC-based eGFR equations should be conducted using a standardized measurement system for cysC, as well as measured GFR.
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