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Technical Briefs |
1
Washington University School of Medicine, Department of Pathology and Immunology, 660 S. Euclid Ave., Box 8118, St. Louis, MO 63110
2
Dade Behring Inc., Newark, DE 19714
aauthor for correspondence: fax 314-362-1461, e-mail mscott{at}labmed.wustl.edu
Cystatin C (cysC) is a 132-amino acid, 13-kDa cysteine protease inhibitor produced by all nucleated cells and whose function is thought to be modulation of the intracellular catabolism of proteins (1). It is formed at a constant rate, freely filtered by the renal glomeruli, and completely reabsorbed and catabolized by the proximal tubular cells (1)(2)(3)(4)(5). Plasma cysC values are reported to be unaffected by age, body weight, diet, medications, or pathologies such as inflammation and cancer. On the basis of these favorable characteristics, cysC has been proposed as a marker of glomerular filtration rate (3)(4)(5)(6). Serum creatinine (creatinine) is widely used for estimation of the glomerular filtration rate, but its tubular secretion, dependence on muscle mass, alteration in some inflammatory diseases, and analytical interferences can limit its utility (7)(8)(9)(10). Thus, cysC has been proposed as an alternative marker in some settings (3)(4)(9)(11)(12).
The purpose of this study was to evaluate the analytical and clinical performance characteristics of a new cysC assay and to establish adult reference intervals for cysC. One hundred thirty-nine presumably healthy adult volunteers participated in the study (age >18 years; 78 females and 61 males) by donating a plasma specimen for creatinine and cysC measurements. Volunteers were recruited with posted signs around our medical center and were offered $25.00 compensation. All volunteers filled out a health questionnaire and were excluded if they had a history of renal disease or chronic health problems. cysC values were also determined in excess residual plasma from 110 patients whose plasma creatinine was measured as part of their clinical management at Barnes-Jewish Hospital. These samples were selected based on their serum creatinine values so that 
60% would be expected to have increased values for cysC. Laboratory and primary diagnoses of these patients were reviewed for the presence of, or a history of renal disease. The Washington University Human Studies Committee approved the study.
cysC was measured by the N Latex Cystatin C assay on the Dade Behring BNTM II analyzer. In this assay, polystyrene beads (13) coated with rabbit antibodies to cysC agglutinate when mixed with samples containing cysC. The intensity of the scattered light in the nephelometer depends on the concentration of cysC (antigen) in the sample. This concentration is determined by comparison with dilutions of a calibrator. Plasma creatinine values were determined by the Jaffe method on the Roche/Hitachi 747 analyzer. All plasma samples (lithium heparin) were analyzed within 3 days or stored frozen until analysis.
Control samples (normal and abnormal) were analyzed in quadruplicate twice a day for 5 days to establish imprecision and to define quality-control ranges. Total imprecision was determined by nested ANOVA. Reference intervals were estimated using a nonparametric procedure (14), and the central 95% interval was determined for each sex and ethnic group.
Total imprecision of the cysC assay was 2.6% at 1.52 mg/L and 4.4% at 0.80 mg/L (n = 40). Creatinine values for the 139 healthy volunteers (ages, 19–69 years) were 8–16 mg/L. The central 95% interval was 8–14 mg/L, which is consistent with the reference intervals at our institution of 6–14 and 7–15 mg/L for females and males, respectively. For these same 139 adult volunteers, cysC values were 0.40–1.03 mg/L, with the central 95% interval being 0.51–0.92 mg/L (mean, 0.67 mg/L; SD, 0.1 mg/L). For the 78 presumably healthy females, the central 95% interval of cysC values was 0.51–0.94 mg/L (range, 0.51–0.97 mg/L; mean, 0.65 mg/L; SD, 0.1 mg/L). For the 61 healthy males, the central 95% interval of cysC values was 0.48–0.98 mg/L (range, 0.4–1.03 mg/L; mean, 0.70 mg/L; SD, 0.1 mg/L). These values are comparable to, albeit 10% lower than previously reported reference ranges for an earlier version of the Dade Behring cysC assay that was performed on a different, batch-mode nephelometer and used a different standard preparation (5). However, our study examined four times as many presumably healthy adults as the previous study and produced reference intervals identical to another recent large study using this method (15). Because gender and race differences can affect reference intervals, 95% confidence intervals for the upper reference value for cysC were calculated across various subgroups (Table 1
). Because of the considerable overlap in the central ranges and in the confidence intervals for the upper reference values for these groups, there was no clinically significant difference among them. However, no estimates were calculated for the Asian ethnic group because of the small sample size.
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The correlation between cysC and creatinine was examined in samples from all patients and volunteers as well as in subpopulations. Considering samples from all 110 patients (including 11 patients with creatinine and/or cysC values determined by sample dilution) and the 139 volunteers, the r value was 0.88. Among the 139 presumably healthy volunteers, the r value was only 0.36, which is not surprising considering the narrow range of both cysC and creatinine values in this population. Within only the 110 patient samples, the correlation of cysC to creatinine produced r values of 0.84, 0.78, and 0.87, respectively, for all 110 patients, the 60 patients with a documented history of renal disease, and the 50 patient samples for whom we could not document a history of renal disease.
Of the values from the 110 patients presented in Fig. 1
, 60 were from patients with a documented history of renal disease by chart review (creatinine range, 7–70 mg/L). Of these 60, 57 had cysC values >1 mg/L (cysC range, 0.56–9.86 mg/L), whereas only 52 had creatinine values >14 mg/L. Two of the three with normal cysC values also had normal creatinine values. Of the 139 healthy volunteers and 50 patients with no documented history of renal disease (creatinine range, 5–58 mg/L), 169 (89%) had cysC values <1 mg/L (range, 0.4–5.89 mg/L) and 178 (94%) had creatinine values <14 mg/L. Nine of the 11 patients in this group with an increased creatinine also had an increased cysC, which strongly suggests the presence of undocumented renal disease.
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These studies indicate that this nephelometric assay for cysC correlates well with creatinine in the patients examined here and that the reference interval we determined is consistent with a previous large study (15). We also confirm that a single reference interval can be used for males, females, and ethnic groups. cysC determined by this method exhibited good diagnostic agreement with creatinine in patients with confirmed renal failure when the central 95% of the cysC values that we observed were used as a reference interval, but cysC may be more sensitive. For example, five patients with confirmed renal failure had normal creatinine but abnormal cysC. Potential advantages of cysC as a renal marker include the lack of differences in values between sexes and reports of it being more sensitive and specific than creatinine in certain clinical settings, including pediatric populations (4)(16), renal transplant patients (17), patients on chemotherapy (18), and patients with hepatic cirrhosis (10)(19).
References
The following articles in journals at HighWire Press have cited this article:
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  B. C. Astor, A. S. Levey, L. A. Stevens, F. Van Lente, E. Selvin, and J. Coresh Method of Glomerular Filtration Rate Estimation Affects Prediction of Mortality Risk J. Am. Soc. Nephrol., October 1, 2009; 20(10): 2214 - 2222. [Abstract] [Full Text] [PDF]
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 O. F. Laterza, C. P. Price, and M. G. Scott Cystatin C: An Improved Estimator of Glomerular Filtration Rate? Clin. Chem., May 1, 2002; 48(5): 699 - 707. [Abstract] [Full Text] [PDF]
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, samples from patients without documented history of renal failure; 
, samples from patients with documented history of renal failure. Dotted lines indicate upper reference limits.