Clinical Chemistry 59: (2013)

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1 Clinical Chemistry 59: (2013) General Clinical Chemistry Cystatin C and Creatinine-Based Estimates of Renal Function and Their Value for Risk Prediction in Patients with Acute Coronary Syndrome: Results from the PLATelet Inhibition and Patient Outcomes (PLATO) Study Axel Åkerblom, 1,2* Lars Wallentin, 1,2 Anders Larsson, 3 Agneta Siegbahn, 4 Richard C. Becker, 5 Andrzej Budaj, 6 Anders Himmelmann, 7 Jay Horrow, 8 Steen Husted, 9 Robert F. Storey, 10 Nils Åsenblad, 2 and Stefan K. James 1,2 for the PLATO Investigators BACKGROUND: The estimated glomerular filtration rate (egfr) independently predicts cardiovascular death or myocardial infarction (MI) and can be estimated by creatinine and cystatin C concentrations. We evaluated 2 different cystatin C assays, alone or combined with creatinine, in patients with acute coronary syndrome. METHODS: We analyzed plasma cystatin C, measured with assays from Gentian and Roche, and serum creatinine in patients from the PLATelet Inhibition and Patient Outcomes (PLATO) trial. We evaluated Pearson correlation and agreement (Bland Altman) between methods, as well as prognostic value in relation to cardiovascular death or MI during 1 year of follow up by multivariable logistic regression analysis including clinical variables, biomarkers, c-statistics, and relative integrated discrimination improvement (IDI). RESULTS: Median cystatin C concentrations (interquartile intervals) were 0.83 ( ) mg/l (Gentian) and 0.94 ( ) mg/l (Roche). Overall correlation was 0.86 (95% CI ). The level of agreement was within 0.39 mg/l (2 SD) (n ). The areas under the curve (AUCs) in the multivariable risk prediction model with cystatin C (Gentian, Roche) or Chronic Kidney Disease Epidemiology Collaboration egfr (CKD-EPI) added were , , and Corresponding relative IDI values were 2.96%, 3.86%, and 4.68% (n ). Addition of egfr by the combined creatinine cystatin C equation yielded AUCs of (Gentian) and (Roche) with relative IDI values of 3.54% and 3.24%. CONCLUSIONS: Despite differences in cystatin C concentrations, overall correlation between the Gentian and Roche assays was good, while agreement was moderate. The combined creatinine cystatin C equation did not outperform risk prediction by CKD-EPI American Association for Clinical Chemistry The low-molecular-weight cysteine protease inhibitor cystatin C is synthesized in all nucleated cells at a constant rate and exhibits favorable properties for a renal biomarker such as free filtration by the glomerulus with no reabsorption into the blood (1 3). Compared to creatinine, cystatin C is less influenced by diet, muscle mass, or body constitution, making it a reliable marker of the glomerular filtration rate (GFR) 11 and hence renal function (1 6). Cystatin C concentrations measured at admission in patients with acute coronary syndrome (ACS) also independently correlate with cardiovascular risk, including cardiovascular death and myocardial infarction (MI) (7 10). Despite these favorable properties, the clinical use of cystatin C remains limited, and creatinine-based equations such as 1 Department of Medical Sciences, Cardiology; 2 Uppsala Clinical Research Center; 3 Department of Medical Sciences, Clinical Chemistry; and 4 Department of Medical Sciences, Center of Excellence Inflammation, Uppsala University, Uppsala Sweden; 5 Duke Clinical Research Institute, Durham, NC; 6 Postgraduate Medical School, Grochowski Hospital, Warsaw, Poland; 7 AstraZeneca Research and Development, Mölndal, Sweden; 8 AstraZeneca Research and Development, Wilmington, DE; 9 Department of Cardiology, Århus University Hospital, Århus, Denmark; 10 Department of Cardiovascular Science, University of Sheffield, Sheffield, UK. * Address correspondence to this author at: Uppsala Clinical Research Center, UCR/MTC, Science Park, Dag Hammarskjölds väg 14B, 1tr, Uppsala, Sweden. Fax 46-(0) ; axel.akerblom@ucr.uu.se. Received December 18, 2012; accepted April 22, Previously published online at DOI: /clinchem Nonstandard abbreviations: GFR, glomerular filtration rate; ACS, acute coronary syndrome; MI, myocardial infarction; MDRD, Modification of Diet in Renal Disease; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; LOD, limit of detection; PLATO, PLATelet Inhibition and Patient Outcomes; ID-MS, isotope-dilution mass spectrometry; NT-proBNP, N-terminal pro B-type natriuretic peptide; IDI, integrated discrimination improvement; AUC, area under the curve. 1369

2 the Modification of Diet in Renal Disease (MDRD), Cockcroft Gault, or Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) are more common for GFR estimation and risk prediction in clinical practice (11 14). Until recently, the CKD-EPI equation was considered the best GFR estimate in a general population, as the equation was developed from a large population and validated in a pooled data set derived from several different cohorts reflecting diverse clinical conditions however, primarily with patients exhibiting normal or mildly impaired kidney function (11).In 2012, an equation combining creatinine and cystatin C concentrations was presented, providing an even more accurate estimate of GFR (15). Contributing factors for the sparse use of cystatin C have been the lack of a standardized method for analyzing cystatin C, uncertainty about what reference values should be recommended, and the relatively higher cost for the reagents compared with assays for creatinine (16). Today, 2 commercial latex-enhanced immunoturbidimetric assays from manufacturers Gentian and Roche are available for implementation on ordinary chemistry analyzers, but the cost of analyzing the cystatin C concentration remains greater than the cost of creatinine analysis (3, 16, 17). Comparisons between different cystatin C assays are few, and limited information has been provided with regard to important subgroups, including ACS patients and those with diabetes. Furthermore, to our knowledge, no previous study has evaluated the possible incremental value in long-term risk prediction by the combined creatinine cystatin C based estimates of GFR (15). Methods STUDY POPULATION The PLATelet Inhibition and Patient Outcomes (PLATO) trial (NCT ) randomized patients with ACS to receive either ticagrelor or clopidogrel treatment in addition to optimal medical therapy and optional revascularization strategies (18, 19). In patients, baseline blood samples with cystatin C (Gentian) were available, and patients had a valid cystatin C result measured by both Gentian and Roche assays, whereas patients also had CKD- EPI calculations. Blood samples were collected just before the administration of study medication at a median of 11.4 h (interquartile range h) after the start of chest pain (n ). The details of the study have been previously published (18, 19). LABORATORY ANALYSIS Venous blood samples were obtained via direct venipuncture and anticoagulated in EDTA. After centrifugation, plasma samples were frozen and sent for central laboratory analysis. We analyzed plasma cystatin C concentrations at baseline with the Gentian assay on the Abbott Architect CI-8200 (Abbott Laboratories). The total analytical CV for cystatin C with the Gentian assay was 1.94% at the mean of 0.87 mg/l and 2.49% at 2.91 mg/l. After analysis of the baseline samples, the samples were again frozen and stored. They were later thawed and again analyzed, in a second set, with the assay from Roche Diagnostics on the Cobas c501 with the following total imprecision: 2.8% at 1.1 mg/l and 2.09% at 4.35 mg/l. The lower limit of detection (LOD) with the Roche assay was 0.41 mg/l and 0.30 mg/l with the Gentian assay. Patients with cystatin C concentrations lower than the LOD were assigned the LOD value. Serum creatinine was measured in a core laboratory using a rate-blanked and compensated modified Jaffe method on the Roche BMD instrument. The method was standardized to isotope-dilution mass spectrometry (ID-MS) with a total CV of 2.3% at 1.09 mg/dl (96 mol/l) and 1.7% at 3.70 mg/dl (327 mol/l) (Precipath U). N-terminal pro B-type natriuretic peptide (NT-proBNP) was analyzed in central core laboratories (Quintiles Laboratories) by the Immulite 2000 assay on the Cobas c601 instrument. Cardiac troponin I was analyzed with the Access AccuTnI assay (Beckman Coulter), in which the 0.08 g/l cutoff concentration corresponds to the 99th percentile. STATISTICAL ANALYSIS Correlation of cystatin C with 2 different assays. All patients with baseline cystatin C values (from both assays) were evaluated by Pearson correlation analysis. The analysis considered all clinically relevant subgroups, including sex, myocardial damage (cardiac troponin I 0.08 g/l), and diabetes. We performed the correlation study stratified by renal function as a crosstabulation of cystatin C quartiles by Gentian (x axis) and Roche (y axis), and -statistics (standard and weighted) were similarly calculated. In addition, the linear regression equation was estimated for cystatin C Gentian as a function of cystatin C Roche. Agreement of the 2 cystatin C assays. We evaluated the agreement between the 2 assays by plotting the difference against the mean of both methods by the Bland Altman method (20), which investigates a possible relationship between the measurement error and the true value. With the true value unknown, the mean of the 2 measurements provides the best estimate (20). The level of agreement was visualized as the degree of the bias, estimated by the mean difference d, the SD of those differences, and the limits of agreement, d 2SD and d 2SD (20) Clinical Chemistry 59:9 (2013)

3 Cystatin C vs Creatinine-Based Estimates Risk prediction with cystatin C alone or in combination with creatinine. We analyzed the incremental predictive value of cystatin C with the 2 different assays at baseline regarding outcome with logistic regression models, c-statistics, and the relative integrated discrimination improvement (IDI) index (21). Similarly, we assessed the combined creatinine cystatin C equation (with both assays separately analyzed) and the CKD-EPI. The IDI is a measure to assess the added predictive ability of a marker for a binary outcome (21). More specifically, the IDI can be viewed as the difference in the discrimination slopes between a new model (e.g., with cystatin C) and the old model (e.g., without cystatin C), where the discrimination slope is defined as the difference of mean predicted probabilities of event and nonevents. In the multivariable analysis, clinically relevant risk factors (age, sex, body mass index, randomized treatment, type of ACS, habitual smoking, and history of heart failure, diabetes, hypertension, nonhemorrhagic stroke, myocardial infarction, peripheral artery disease, coronary artery bypass grafting, or percutaneous coronary intervention) and the renal variable (e.g., cystatin C) were included and constituted a first model (model 1). In an expanded multivariable analysis, biomarkers log(ntprobnp) and cardiac troponin I dichotomized at 0.08 g/l were included (model 2). All analyses were done with SAS software (version 9.2, SAS Institute). The CKD-EPI equation (2009) was defined as 141 min(creatinine/, 1) max(creatinine/, 1) age (if female) (if black), where is 0.7 for females and 0.9 for males, is for females and for males, min indicates the minimum of (creatinine/ ) or 1, and max indicates the maximum of (creatinine/ )or1(11). The combined creatinine cystatin C equation (2012) can be expressed as a single equation: 135 min(scr/, 1) max(scr/, 1) min(scys/0.8, 1) max(scys/0.8, 1) age (if female) 1.08 (if black), where Scr is serum creatinine, Scys is serum cystatin C, is 0.7 for females and 0.9 for males, is for females and for males, min indicates the minimum of Scr/ or 1, and max indicates the maximum of Scr/ or 1 (15). Estimations of the GFR based on cystatin C concentrations were derived from equations provided by the manufacturers: Gentian, GFR cystatin C (mg/ L) , and Roche, GFR cystatin C (mg/ L) In the risk-prediction analysis, the cystatin C concentrations and not the estimated GFR were used. Results PATIENT POPULATION Regarding baseline characteristics, the study population with both Gentian and Roche cystatin C concentrations (n ) was comparable to the previously reported biomarker population with cystatin C Gentian only (n ) (data not shown) (9). CYSTATIN C CONCENTRATIONS AND GFR ESTIMATES The median cystatin C concentrations (interquartile intervals) were 0.83 ( ) and 0.94 ( ) mg/l by Gentian and Roche assays, respectively. The mean cystatin C concentrations were 0.89 (SD 0.35) mg/l for Gentian compared to 1.02 (0.37) mg/l for Roche (n ). The calculated median GFRs derived by the Gentian and Roche assays alone were 104 and 94 ml min 1 (1.73 m 2 ) 1, respectively, whereas calculated GFRs by the combined creatinine cystatin C equation by Gentian and Roche samples were 89.9 and 82.7 ml min 1 (1.73 m 2 ) 1. CKD-EPI was calculated for comparison and exhibited a median GFR of 82.6 ml min 1 (1.73 m 2 ) 1 (n ). CORRELATION AND AGREEMENT OF THE 2 CYSTATIN C ASSAYS The correlation coefficient (r) between the Gentian and Roche assays of cystatin C in the entire population was 0.86 (95% CI ) (n ) and was consistent in important subgroups: women, patients with increased troponin I ( 0.08 g/l), and patients with diabetes (Table 1). Overall values by cystatin C quartiles were (95% CI ) (standard) and ( ) (weighted). The regression equation was as follows: cystatin C Gentian (mg/l) cystatin C Roche (mg/l). The agreement evaluated by the Bland Altman plot is presented in Fig. 1, where 3.1% of observations fall outside the limits of agreement of 0.52 mg/l and 0.26 mg/l (20). Overall cystatin C by the Roche method was more variable, with SD 0.37 mg/l compared to 0.35 mg/l by Gentian, and produced more outliers visually (Fig. 1). RISK PREDICTION WITH DIFFERENT CYSTATIN C ASSAYS AND THE COMBINED CREATININE CYSTATIN C EQUATION We evaluated both cystatin C assays as well as the combined creatinine cystatin C equation (with both Gentian and Roche values) and the CKD-EPI as predictors of the composite endpoint of cardiovascular death and MI in a multivariable model (with adjustment for clinical risk factors and biomarkers) during 1 year of follow up (n ). The total number of myocardial infarctions in the cohort was 806 (6.2%); 550 (4.2%) individuals died from cardiovascular causes. The c-statistics [areas under the curve (AUCs)] after adding the renal variable to the risk prediction model with clinical variables are presented in Table 2. The addition of the biomarkers NT-proBNP and cardiac troponin I and the renal variable further increased the AUC (model 2), but the incremental separate value of the renal variable, reflected by the relative Clinical Chemistry 59:9 (2013) 1371

4 Table 1. Correlation of plasma cystatin C between Gentian and Roche assays. n Correlation (95% CI) Whole population ( ) Sex Male ( ) Female ( ) GFR 60 ml min 1 (1.73 m 2 ) ( ) 60 ml min 1 (1.73 m 2 ) ( ) Myocardial damage Cardiac troponin I 0.08 g/l ( ) Cardiac troponin I 0.08 g/l ( ) Diabetes Yes ( ) No ( ) IDI, was lower in the context of the model that included both clinical variables and biomarkers, as expected (Table 2). The AUCs for the multivariable model including cystatin C by Gentian and Roche alone were and , respectively, compared with in the multivariable model without a renal marker (Table 2 and Supplemental Fig. 1, which accompanies the online version of this article at content/vol59/issue9). The relative IDIs were 2.96% and 3.86%, respectively (Table 2). The combined creatinine cystatin C equation yielded c-statistic AUCs of (Gentian) and (Roche), with respective relative IDIs of 3.54% and 3.24% (see Table 2 and online Supplemental Fig. 1). The CKD-EPI equation (11), used for comparison, exhibited an AUC of and relative IDI of 4.68% (n ). Discussion Our results demonstrate that the baseline cystatin C concentrations measured with two different assays yielded different median and mean concentrations. This could be due to several factors including calibration errors, different antibodies in the assays and although unlikely, we cannot rule out that the extra cycle of freeze and thaw may have influenced the results. Nonetheless, the correlation between results for the two assays was good and cystatin C values measured at baseline had incremental predictive capability for clinical outcomes in ACS patients receiving current recommended antiplatelet treatment irrespective of assay used (19, 22). The correlation was consistent throughout important subgroups such as women, patients with increase in cardiac troponin I or with diabetes mellitus. The current study complements a previous study by Shlipak et al., where cystatin C concentrations measured by assays from the manufacturers Siemens, Gentian and Roche were well correlated in patients with suspected or verified coronary artery disease (n 948) (16). That study included more men and older patients, with worse renal function (median cystatin C of 1.12 mg/l), as compared with our study (16). Our study also evaluated the level of agreement between the two assays, and although a good correlation was present, the level of agreement was only moderate and a discrepancy between the two methods was obvious. The level of agreement has greater clinical relevance as it provides information about comparison of the actual measurements, not just the direction. The outer borders of the 2 SD range of differences between assays, encompassing roughly 95% of observations, were 0.26 mg/l and 0.52 mg/l. The importance of these differences depends on the underlying GFR but in some cases is large enough to possibly change the staging in the CKD classification of individual patients. The discrepancy between the two assays was more pronounced at higher cystatin C concentrations, more often with Roche as the outlier. The clinical significance of the outliers is of unknown importance and the prognostic value of either cystatin C assay in a risk prediction model was of similar incremental value. Inker and colleagues have proposed a new combined creatinine cystatin C equation to provide improved accuracy in estimating the GFR (15). We hypothesized that both Gentian and Roche values, when added to the equation, might improve the AUC for risk 1372 Clinical Chemistry 59:9 (2013)

5 Cystatin C vs Creatinine-Based Estimates Fig. 1. (A), Difference versus means plot for the cystatin C concentrations by Gentian and Roche. The difference in mg/l between the two methods was calculated by subtracting Gentian values from Roche values. (B), Scatter plot of cystatin C concentrations in mg/l by Gentian and Roche, with linear regression (n 16279). prediction compared to the cystatin C values alone. Importantly the equation by Inker was derived by cystatin C values from Dade Behring and Siemens AG. However, in a comparison of cystatin C results from the Gentian and Dade Behring assays, based upon 200 individuals and performed at the Department of Clinical Chemistry in Uppsala University Hospital, the correlation between the two assays was R and slope 1.05 (Anders Larsson, personal communication March 2013). Furthermore, the calibration of the Clinical Chemistry 59:9 (2013) 1373

6 Table 2. c-statistics for risk prediction models and relative IDI for the renal variable regarding cardiovascular death or MI. a Renal estimate variable c-statistic (AUC) Relative IDI, % P IDI Model 1 Baseline model with clinical variables, but no cystatin C or creatinine NA b NA Cystatin C Gentian Cystatin C Roche CKD-EPI Combined creatinine cystatin C Gentian Combined creatinine cystatin C Roche Model 2 Baseline model with clinical variables and biomarkers but not NA NA cystatin C or creatinine Cystatin C Gentian Cystatin C Roche CKD-EPI Combined creatinine cystatin C Gentian Combined creatinine cystatin C Roche a The first multivariable analysis (model 1) included only clinical variables (n ). Model 2 includes both clinical variables and biomarkers NT-proBNP and cardiac troponin I (n ). b NA, not applicable. Gentian assay did not change in the new International Federation of Clinical Chemistry universal cystatin C calibration, (Anders Larsson, personal communication March 2013, Bård Sundrehagen, personal communication March 2013). Despite the similarity between Gentian and Dade Behring cystatin C assays and the concordance to the IFCC calibration, the incremental prognostic value in risk prediction was not improved with this combined creatinine cystatin C equation. Rather the highest AUC was seen with the CKD-EPI equation (11), similarly as previously reported in this population (9). Worth noticing is also that the incremental value of a renal biomarker was diminished when other biomarkers like NT-proBNP and Troponin I were added to the risk prediction model reflected by a fall in the relative IDI. Limitations of our study include that, de facto, no gold standard measurement of GFR was provided; therefore it was not possible to observe a calibration error in either or both of the assays. The analysis of the samples also differed in time as the Roche samples were analyzed several months later than the Gentian samples. Furthermore, the combined creatinine cystatin C equation by Inker et al. used factorized cystatin C values by Dade Behring, not completely agreeing with the values by Gentian or Roche methods. The investigated cohort of ACS patients did not include patients requiring dialysis and the results are not necessarily generalizable to other populations. In conclusion, despite differences in baseline cystatin C estimations evaluated in a large cohort of ACS patients including subgroups not previously evaluated, the overall correlation of two clinically available assays was good. Nonetheless, the moderate level of agreement between the two cystatin C assays may lead to clinically important differences in how renal dysfunction is classified in individual patients. Risk prediction with cystatin C alone or in combination with creatinine, irrespective of assay, adds important information on the composite endpoint of cardiovascular death and MI during one year of follow up. However the solely creatinine-based CKD-EPI equation exhibited the highest predictive power. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: A. Himmelmann, AstraZeneca; J. Horrow, AstraZeneca Clinical Chemistry 59:9 (2013)

7 Cystatin C vs Creatinine-Based Estimates Consultant or Advisory Role: R.C. Becker, Bayer, Boehringer Ingelheim, Merck, and Daiichi Sankyo; A.J. Budaj, Eli Lilly, GlaxoSmith- Kline, AstraZeneca, Bristol Myers Squibb/Pfizer, Sanofi-Aventis, and Novartis; S.E. Husted, AstraZeneca; R.F. Storey, AstraZeneca, Daiichi Sankyo/Eli Lilly, Merck, Roche, Sanofi Aventis/Regeneron, Bristol Myers Squibb, Novartis, Eisai, and Accumetrics; S.K. James, AstraZeneca, Eli Lilly, and MSD. Stock Ownership: None declared. Honoraria: A. Åkerblom, AstraZeneca; R.F. Storey, AstraZeneca, Merck, Accumetrics, Iroko, Eli Lilly/Daiichi Sankyo, Novartis, and Medscape; S.K. James, Eli Lilly, AstraZeneca, MSD, and Iroko. Research Funding: The PLATO study was funded by AstraZeneca. Support for the analysis and interpretation of results and preparation References of the manuscript was provided through funds to the Uppsala Clinical Research Center and Duke Clinical Research Institute as part of the Clinical Study Agreement. The cost for the Gentian assay was provided by AstraZeneca, and Roche provided their assays free of charge. R.C. Becker, Johnson & Johnson. Expert Testimony: None declared. Role of Sponsor: The executive and operations committee, consisting of both academic members and representatives of the sponsor, AstraZeneca, designed and oversaw the conduct of the main trial. The sponsor played no role in the design of the present study, interpretation of data, or preparation of drafts but reviewed the final manuscript before submission. 1. Grubb AO. Cystatin C: properties and use as diagnostic marker. Adv Clin Chem 2000;35: Laterza OF, Price CP, Scott MG. Cystatin C: an improved estimator of glomerular filtration rate? 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