Cystatin C A Paradigm of Evidence Based Laboratory Medicine

Review Article Cystatin C A Paradigm of Evidence Based Laboratory Medicine Janice SC Chew, 1 Mohammed Saleem, 2 *Christopher M Florkowski, 2 Peter M George 2 1 Department of Biochemistry, Medlab South Ltd, 137 Kilmore Street, PO Box 25091, Christchurch 8013, 2 Clinical Biochemistry Unit, Canterbury Health Laboratories, Corner of Tuam Street and Hagley Avenue, PO Box 151, Christchurch 8011, New Zealand. *For correspondence: Assoc Prof Chris Florkowski email: Chris.Florkowski@cdhb.govt.nz Abstract Cystatin C is a 13-kDa protein, of the cysteine proteinase inhibitor superfamily, produced by all nucleated cells. Its production rate is constant throughout the ages of 1 to 50 years. It is freely filtered at the glomerulus and then resorbed and fully catabolised by proximal renal tubules, making it an ideal marker of glomerular filtration rate (GFR). Serum creatinine, the most established marker of renal function, is affected by age, gender, muscle mass, nutritional status and analytical interference. The abbreviated Modification of Diet in Renal Diseases (MDRD) equation has recently been introduced in an attempt to overcome these shortcomings, but still has many limitations. Cystatin C is not affected by gender, muscle mass, malignancy, its production rate is usually constant and its plasma concentration therefore is dependent only on GFR. Cystatin C has been demonstrated to be more accurate than serum creatinine in the detection of early renal impairment and in specific populations may allow for early detection of renal disease. Cystatin C has also been found to be a strong predictor of long-term clinical outcomes in patients with cardiovascular diseases. Although cystatin C may have advantages in detection of early renal impairment there is a paucity of evidence that it significantly improves clinical decision making over creatinine. This coupled with assay cost may be the reason why cystatin C, although well recognised, has not been introduced into routine operational use, although that may eventuate with emerging evidence. Introduction Cystatin C is a 122-amino acid, 13-kDa protein that is a member of the family of cysteine proteinase inhibitors. 1 It is encoded by the housekeeping type CST3 gene, and produced by all nucleated cells at a constant rate. 2 It is freely filtered by the glomerulus and is largely resorbed and catabolised in the proximal tubules. 3 Although its clearance cannot be measured because of this catabolism, its plasma or serum concentration is a good measure of GFR, with possible advantages over more established markers such as serum creatinine. 4 Early studies have suggested no apparent tubular secretion of cystatin C. Newer evidence however has found tubular secretion in a defined group of hypertensive patients, therefore casting doubts on its usefulness as a glomerular filtration marker. 5 This observation, however could be attributed to assay imprecision. 6 The history of cystatin C began in 1961 when Jorgen Clausen described the occurrence in human cerebrospinal fluid (CSF) of a cerebrospinal fluid-specific protein, which he named γ- CSF. 7 Cystatin C has since been found in urine, human plasma, ascitic and pleural fluid. Known also as γ-trace, the complete amino acid sequence of human cystatin C was determined in 1981 by Grubb and Lofberg. 8 The prevalence of chronic kidney disease (CKD) and endstage renal disease (ESRD) is increasing worldwide with ageing of the world population and a global epidemic of type 2 diabetes mellitus. 9 GFR is the most important measure of the renal efficiency for clearing various substances from the blood. A decrease in GFR precedes end-stage renal failure in all forms of progressive kidney disease and knowledge of GFR is therefore critical in the prevention and management CKD. Accurate determination of GFR requires the use of invasive protocols based on injected exogenous substances such as Inulin, 125- Iothalamate, Iohexol, 51 Cr-EDTA or 99m Tc- DTPA. Such procedures are labour intensive, costly, and not entirely free of potential for harm. A 24-hour urine creatinine clearance has been more commonly employed for the detection of renal dysfunction but is inaccurate and inconvenient for the patient. As a substantial Clin Biochem Rev Vol 29 May 2008 I 47

Chew JSC et al. fraction of urinary creatinine is derived from renal tubular secretion, healthy individuals can have a creatinine clearance regularly exceeding their Inulin clearance by 10 to 40%. 10 In practice, serum and plasma creatinine are the most widely used endogenous markers of GFR. Creatinine levels can be estimated by the colorimetric Jaffe reaction as well as by enzymatic methods, high-performance liquid chromatography (HPLC) and the gold standard isotope dilution mass spectrometry (IDMS). Despite known assay interferences such as bilirubin, ketones and protein, the Jaffe method remains the most commonly used method due to convenience and cost-effectiveness. The presence of interfering substances can further lead to the overestimation of serum creatinine by as much as 15-25% by various Jaffe based assays. 11 To help correct for some of these interferences, manufacturers have adjusted the calibration of these assays, producing compensated versions. Serum creatinine is also influenced by muscle mass, sex, age, diet and race. The sensitivity of serum creatinine in the detection of CKD is consequently poor and it will fail to identify half of the patients with crucial stage 3 CKD (GFR of 30-59 ml/min/1.73m 2 ). 12 In an attempt to overcome the limitations of serum creatinine, the National Kidney Foundation (NKF) and Kidney Disease Outcomes Quality Initiative (KDOQI) and the Australian Creatinine Consensus Working Group recently published recommendations to use estimated GFR (egfr) based on serum creatinine and also including age, racial origin and gender. 13,14 The abbreviated 175 MDRD equation is the recommended equation for use with IDMS aligned assays for laboratories in New Zealand and Australia in the adult population. 15-17 The Cockcroft and Gault (CG) equation is also well recognised and the Schwartz and the Counahan Barratt equations are recommended for estimating GFR in children. 18-20 Although egfr formulae are increasingly used in daily clinical practice, their accuracy is debatable. The MDRD equation was originally validated in CKD patients but its use is unclear for healthy individuals, or in patients with egfr >90 ml/min/1.73m 2. 21-25 There are also many situations in which these equations have not been validated; such as in patients with diabetes, extreme body sizes and in certain races. At higher levels of kidney function, the bias and precision of the MDRD estimate is poorer, tending to underestimate true GFR in most studies. 26-28 Due to the many problems encountered with measurements of creatinine and its use as a GFR estimate, cystatin C has been proposed as an alternative marker of renal function. The potential utility of serum cystatin C in the laboratory lies in its capability to detect early renal failure, i.e. at stage 2 CKD (i.e. GFR level of 60 to 90 ml/min/1.73 m 2 ). We have therefore reviewed the evidence base for cystatin C and its potential clinical utility as a marker of renal function. Diagnostic Accuracy and Performance of Cystatin C Many studies have compared cystatin C levels or cystatin C derived equations with gold standard methods using Bland- Altman and regression analysis to test for agreement. Most studies have found cystatin C, or the reciprocal of cystatin C, to be superior or at least equivalent to serum creatinine for the detection of decreased GFR. Newman et al. concluded that, in addition to being a better estimator of GFR than serum creatinine, cystatin C was a more sensitive marker of small changes in GFR. 29 A meta-analysis based on 46 studies, using ROC curve analysis showed that cystatin C is superior to creatinine for detection of reduced GFR. 30 Roos et al. recently reported a systematic review by comparing the diagnostic accuracy of cystatin C with serum creatinine which included studies that assessed accuracy of cystatin C for all grades of renal function. 31 This review reported that not only is cystatin C a reliable marker of GFR in patients with mildly to moderately impaired kidney function, cystatin C had a higher chance of detecting true renal impairment, in comparison with serum creatinine. Larson et al. also reported cystatin C as having better discriminatory capabilities than Iohexol clearance in the GFR range of 10-20 ml/min, which corresponded to cystatin C values of 2.5 to 4.4 mg/l. 32 Commercially Available Assays for Cystatin C Pergande and Jung developed a sandwich enzyme immunoassay in 1993 for determining cystatin C in serum using rabbit antibodies and a lyophilised urinary protein as a standard. 33 Cystatin C measurements evolved with the introduction of the first fully automated immunoassay, a particle-enhanced turbidimetric immunoassay (PETIA) by Kyhse-Anderson et al. 34 However, in 1995, an assay using the same antibodies and calibrator, but with different particles was described by Newman et al. 29 The PETIA marketed by DAKO has been adapted to a variety of automated analysers that use fixed-interval turbidimetric measurements, and together with its shorter analytical time, this has allowed its introduction into routine laboratory use. 7 In 1997, the particle-enhanced nephelometric immunoassay (PENIA) was introduced by Dade Behring GmBH (Marburg) and subsequently evaluated. 35-37 The PETIA protocol was then modified in order to reduce its imprecision at concentrations near the reference interval and to decrease intra-assay imprecision. 38 Later, the measurement of cystatin C using an adaptation of the PETIA reagents for a nephelometric immunoassay using the kinetic rate was introduced. 39 48 I Clin Biochem Rev Vol 29 May 2008

Cystatic C - A Paradigm of EBLM The advantages of using the PETIA and PENIA include faster turnaround time, lack of interferences by other substances and higher precision. Both assays were reported to be less likely to be influenced by substances that affect serum creatinine measurements, including haemolysis, lipaemia, and icteria. However, it was reported that high concentrations of rheumatoid factor could falsely elevate cystatin C. 40 Newman suggested however, that this interference may be due either to the reagents per se or to the instrument for which the reagents have been adapted. 41 It has been reported that the PENIA method is more sensitive than the PETIA method and should be considered as the method of choice when measuring cystatin C. 7 This is because nephelometry tends to detect smaller immunoaggregates than turbidimetry and the nephelometer monitors an increase in light intensity against a low background signal which accounts for non-specific background scatter, therefore better specificity for the protein. Furthermore, the Dade-Behring (now Siemens) N-Latex cystatin C assay is the only one having FDA approval. 42 Recently, an immunoassay by Gentian (Gentian, Moss, Norway) has also been introduced and evaluated on an Architect ci8200 instrument (Abbott Laboratories, Abbott Park, Ill., USA). 43,44 The main limitation of cystatin C immunoassay determination lies in the standardisation of platforms which will require an international reference standard preparation to allow for valid comparisons. With the emergence of newly developed assays, the best solution may lie in the development of a full reference system to allow traceability of different methods. A complete reference system should include all the following: agreed definition of the measurand, primary reference method, primary and secondary reference materials, and an external quality assessment scheme for cystatin C to evaluate comparability of results and performance. Despite these requirements, cystatin C is currently not listed on the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database. 45 Reference Ranges Cystatin C reference ranges have been published for both adults and children. Most studies show only a slight difference between serum cystatin C concentrations in men and women, suggesting gender-specific reference ranges are not needed. 4,46,47 Cystatin C however, is found to be higher during the first year of life and decreases thereafter. 1,48,49 In children older than one year, the adult reference range can be used. 1 In full term newborns, there is general agreement that there is a significant and progressive decrease of cystatin C over the first week of life, and less significantly, over the first month. 7 The general consensus is to have a common reference range between the ages of 1 to 50 years. Higher cystatin C concentrations are also reported in the elderly population, relating to decreased kidney function in this group. 50 The majority of clinical studies investigating the significance of serum cystatin C as a marker of changes in GFR have measured this protein by using either the PENIA or the PETIA method. The absence of a standardised calibrator, and differences in antibodies and technologies, has led to both manufacturers reporting slightly different reference ranges (Table 1). For example, the PETIA method generally produces reference values that are 20-30% higher than those from PENIA methods. 4 Like creatinine, serum cystatin C is affected by biological variation which has implications for clinical interpretation of results. 57 Intra-individual variation (CVi) describes how tightly repeated measurements over time are distributed around a homeostatic set-point for an individual and inter-individual variation (CVg) reflects differences between individuals within a group. 58 Serum creatinine has a CVi of 4.7% thus tightly distributed results for an individual compared with a CVi of 13.3% for cystatin C. 57 The ratio of CVi to CVg is termed the index of individuality, values less than 0.6 being considered to be highly individual or tightly distributed around a set-point. For creatinine this index is 0.47 which is therefore highly individual compared with 1.64 for cystatin C which is less individual. The corollary of this is that an abnormal result is more likely to fall outside the reference interval for cystatin C, whereas an individual may show a significant change in serum creatinine yet still fall within the reference interval. It has thus been proposed that cystatin C may be more robust for initial diagnosis of renal impairment and creatinine may be more appropriate for serial monitoring of renal function. Although, early data was derived from the old DAKO method, more recent studies have suggested that the intra-individual variability of cystatin C and creatinine are similar. 59 Cystatin C in Specific Populations Paediatric Population Accurate GFR prediction is crucial in the paediatric population to avoid the long-term effects on the kidney by delayed treatment. In this population definitive measurement of GFR is very difficult due to risks of radiation exposure, and timed urine collections are difficult and inaccurate. Despite well-known substantial overestimation of GFR in paediatric patients (especially when GFR <20 ml/min/1.73 m 2 ), the Schwartz equation remains the most widely used surrogate marker of GFR in the paediatric age group. 7,60 Clin Biochem Rev Vol 29 May 2008 I 49

Chew JSC et al. Table 1. Reference ranges for plasma/serum cystatin C in children and adults. Reference Method Age (range) Sample size Reference range (mg/l) Cataldi et al. 51 PETIA 24 hours n = 78 1.40 2.92 72 hours n = 78 1.05 2.47 120 hours n = 78 0.97 2.19 Bökemkamp et al. 49 PETIA 1 3 days n = 23 2.16 * 3 30 days n = 14 2.02 * 2 4 months n = 13 1.59 * 4 12 months n = 8 1.34 * 1 18 years n = 200 0.70 1.38 Finney et al. 48 PENIA 24 36 weeks n = 30 0.43 2.77 0 1 year n = 79 0.59 1.97 1-17 years n = 182 0.50 1.27 Harmoinen et al. 52 PETIA 25 37 weeks n = 58 1.34 2.57 38 40 weeks n = 50 1.36 2.23 9 days 1 year n = 65 0.75 1.87 1 3 years n = 72 0.68 1.60 3 16 years n = 162 0.51 1.31 Norlund et al. 53 PETIA 20 50 years n = 242 0.70 1.21 > 50 years 0.84 1.55 Finney et al. 54 PENIA 60 79 years n = 398 0.93 2.68 > 80 years 1.07 3.35 Finney et al. 46 PENIA 19-49 years n = 258 0.53 0.92 50 67 years n = 51 0.58 1.02 Dade Behring 55 PENIA 1 78 years n = 413 0.53 0.95 (Marburg) DAKO 56 PETIA 1 50 years not specified 0.55 1.15 (Glostrup) > 50 years not specified 0.64 1.44 * 50th percentile Serum creatinine concentrations are considerably lower and highly variable with growth in children. Several studies have shown cystatin C to have higher diagnostic accuracy than serum creatinine in this population. Cystatin C based GFR estimates were also found to be less biased, more sensitive and precise in estimating GFR in children. 60-63 Neither gender nor the presence of dystrophy had any effect on the accuracy of cystatin C. 63 Contradicting results were seen in some studies reporting no apparent advantage over serum creatinine, while others concluded that GFR is better estimated by considering both serum cystatin and creatinine levels. 64,65 Analytical interferences of bilirubin and haemoglobin seen with the measurements of serum creatinine are important in this population, because of the problems of neonatal jaundice and the in vitro haemolysis that occurs with collection of small paediatric samples. 7 Cystatin C measurements have been reported to be less affected by bilirubin and haemoglobin interference than serum creatinine. 35,36 Elderly Population As elderly patients often have low muscle mass and poor nutrition, serum creatinine may remain in the normal blind range despite decreasing GFR and therefore renal impairment may go unrecognised. Ageing is also associated with decreasing GFR, at a yearly rate of approximately 1 ml/ min/1.73 m 2 in those over the age of 40 years. 54 Moreover, failure to appreciate renal insufficiency commonly results 50 I Clin Biochem Rev Vol 29 May 2008

Cystatic C - A Paradigm of EBLM in drug dosage errors in older people. 66 Thus an accurate reflection of GFR is crucial in the elderly. Although the MDRD and CG formulae have been developed to overcome these problems, cystatin C has been shown to be superior for the detection of mild renal impairment in this population. 50,54,66 Diabetic Population Diabetic nephropathy is the single most frequent cause of ESRD and is predominantly due to type 2 diabetes. Although measurements of albumin excretion rate are useful in clinical practice, there is a 40% day to day variability in albumin excretion rate. 67 Numerous studies have investigated the use of cystatin C in the diabetic population. 68-71 Hoek et al. showed that not only was cystatin C a better indicator of GFR than creatinine, it was the parameter which had the best correlation (r = 0.66) with changes in GFR over two years, making it a useful analyte for follow up of patients with diabetes. 69 A four year follow-up study in Pima Indians demonstrated close correlation (Spearman r = 0.77) between longitudinal trends in Iothalamate clearance and the trends in renal function estimated from serum cystatin C. 72 In contrast, the trends for commonly used creatinine-based estimates of GFR, such as serum creatinine, the CG formula, and the MDRD formula compared poorly with trends in Iothalamate clearance (Spearman r < 0.35). The annual percentage change in GFR based on Iothalamate clearance and cystatin C were also found to be similar, again emphasising the accuracy of cystatin C in monitoring small changes of GFR. The clinical reliability of cystatin C as a measure of GFR has also been documented in type 1 diabetes. 73,74 In their recent study, Pucci et al. reported that levels of cystatin C could discriminate degrees of renal impairment in a population of type 1 and 2 diabetic patients. 71 Cystatin C was found to correlate more strongly with Iohexol clearance (r = 0.857) than creatinine (r = 0.772), CG egfr (r = 0.750) and MDRD egfr (r = 0.806). Cystatin C was also found to be able to predict progression to pre-diabetes stages in normoglycaemic subjects. The Western New York Health study found that elevated baseline cystatin C levels was associated with a three fold risk of progression from normoglycaemia to pre-diabetes over a six year period. 75 Renal Transplant Patients In renal transplant patients, early detection of impaired kidney function is critical so that efforts to prevent further deterioration of graft function or rejection can be instituted. Because serum creatinine has a smaller intra-individual variability than cystatin C, it is postulated that serum creatinine will more reliably detect small changes in GFR and therefore will be better able to identify those at risk of acute transplant rejection. From their preliminary study of renal transplant patients, Le Bricon et al. however found that cystatin C was more sensitive than serum creatinine for detecting decreases in GFR and delayed graft function, with the potential for more timely intervention. 76 Follow-up studies have found GFR over-estimation of 30% and 40% by plasma creatinine and 24- hour creatinine clearance respectively. 77 Even though cystatin C underestimated GFR by 14% it was still more sensitive in detecting impaired renal function (GFR cut-off <80 ml/ min/1.73 m 2 ) than serum creatinine and creatinine clearance, with no false-negative results. 77 Glucocorticoid therapy in adult renal transplant patients is known to be associated in a dose-dependent manner with increased cystatin C, leading to systematic underestimation of GFR. This however does not preclude the use of cystatin C in detecting impaired renal function in renal transplant patients on glucocorticoids, given that many studies have shown cystatin C to be still significantly more accurate in detecting impaired renal function in this patient group. 78-80 Plasma cystatin C also appears to be superior to creatinine and 24- hour creatinine clearance for evaluation of GFR in the postoperative follow up of adult kidney transplant patients. 78,81 In addition to detecting post-transplant renal damage earlier than serum creatinine, cystatin C has been suggested to predict renal function recovery earlier than serum creatinine. The mean time to spontaneous decrease in cystatin C occurred at 14.8 days post-transplant compared to 18.8 days for the decrease in serum creatinine (p<0.002). 82 The relative imprecision of creatinine and cystatin C measurement is also an important factor for detecting very small significant changes in concentration that may suggest rejection of a transplant. Initial evaluation of cystatin C assays reported imprecision at <4.5% (between-batch) for the nephelometric method and <4.0% (between-batch) for the improved turbidimetric method. 35,38 The broader variability of cystatin C could be attributable to the fact that it reflects small temporary changes of GFR more accurately than creatinine. Moreover, coefficients of variation of cystatin C and creatinine tend to equalise with increasing concentrations of cystatin C, suggesting that creatinine and cystatin C have the same variability. 83 With a low GFR, both markers accurately reflect renal function, while in mild renal impairment, creatinine is not sensitive enough to assess transient changes, thus perhaps incorrectly indicating a low variability. Serum cystatin C has also been reported to be a determinant of fasting total homocysteine levels in renal transplant recipients Clin Biochem Rev Vol 29 May 2008 I 51

Chew JSC et al. with normal serum creatinine. Here it was reported that serum cystatin C levels may reflect subtle decreases in renal function that independently predict fasting total homocysteine levels. 84,85 Cancer Patients During treatment with nephrotoxic chemotherapeutic agents, early recognition of alterations in renal function is crucial too because it may lead to impaired metabolism and accumulation of chemotherapeutic agents and their metabolites. Studies investigating the use of cystatin C as a renal marker in malignancy are currently scarce. Initially it was thought that the extent of malignancy may influence the cystatin C concentration due to tumour mass. 86 Later studies have revealed no correlation between serum cystatin C concentration and tumour burden. 87-90 Cystatin C has also been found to be superior to creatinine in monitoring renal function before and during chemotherapy. 87 One study investigating the effects of cisplastin chemotherapy on cystatin C found that changes in cystatin C serum concentrations correlated well with GFR decrease as measured by Inulin clearance. 91 Another study reported that cystatin C was at least as good as serum creatinine as a marker for the handling of carboplatin. 92 Patients with Liver Disease Cirrhosis of the liver is often accompanied by functional renal failure and it is known that serum creatinine overestimates GFR in patients with liver disease, especially cirrhosis. 93 This is postulated to be due to reduced muscle mass, reduced creatinine synthesis and increased tubular secretion. 94 Cystatin C has been shown to have significantly higher sensitivity for detecting reduced GFR in cirrhotic patients. 95,96 In an evaluation of the diagnostic value of cystatin C in hepatorenal syndrome, cystatin C was suggested to replace or at least be added to serum creatinine in the evaluation of renal function in patients with cirrhosis of the liver. 97 Evaluation of cystatin C levels for assessing renal function in a large cohort of paediatric patients with chronic liver disease before and after liver transplantation proved cystatin C as highly sensitive in detecting reduced GFR. 98 Cystatin C was also assessed in an adult population immediately after liver transplantation, and later in the post-transplant phase. 99-101 In all these studies, cystatin C was again shown to be a reliable marker of renal function. In Pregnancy During uncomplicated pregnancy, the renal plasma flow progressively increases, leading to above 40% higher GFR than in non-pregnant women. 102 GFR increases in pregnancy are without substantial alterations in the production of creatinine and urea. 102 Preeclampsia is a condition associated with substantial foetal and maternal morbidity and mortality. Altered renal function is an essential component of the pathophysiological process in preeclampsia and close monitoring of renal function is essential to ascertain the optimal time for delivery to avoid renal damage. Cystatin C does not cross the placental barrier and no correlation was found between maternal and neonatal serum cystatin C values, suggesting neonatal serum cystatin C originate almost exclusively in the neonate. 51,103 Furthermore serum cystatin C levels were not found to be decreased in term pregnancy compared with other low molecular mass, substances which are known to increase by at least 40% by the third trimester. 104 In an attempt to determine whether serum cystatin C is a reliable GFR marker in pregnant women, a study by Strevens et al. found serum cystatin C to reflect GFR (measured by Iohexol) reliably in both non-pregnant and pregnant, healthy and hypertensive women. 104 Cystatin C was also shown to be superior to traditional markers, such as serum creatinine and urea used commonly in assessment of preeclamptic women. 105 Increasing serum cystatin C levels also have a highly significant linear trend and correlations with structural renal changes as shown by renal biopsies in normal, hypertensive and preeclamptic patients. 106 Equations Estimating GFR based on Cystatin C Equations based on creatinine that take into account age, gender, and race, have been developed to improve its accuracy in the prediction of GFR. However, equations that are routinely used currently, such as the MDRD equation and CG equation, have been derived from hospitalised populations with CKD. Therefore, the generalisability of these equations in normal healthy individuals has been questioned. The reciprocal of cystatin C levels has been found to correlate well with measured GFR and many equations based on cystatin C in specific populations have also been developed. However, cystatin C based equations were generated and validated in fewer subjects using different gold-standard measurements than creatinine based equations. 107 Table 2 shows equations that have been published for both adult and children using different cystatin C methods. Comparison of cystatin C based equations with creatinine based equations such as the Schwartz, MDRD, and CG formulae have been rigorously assessed. Many found cystatin C based equations to be superior to creatinine based estimates of GFR. 107 Estimated GFR using cystatin C has been reported to have better correlation with gold-standard GFR than equations based on creatinine. 60,62,69,110,111,114,115 Studies have also evaluated the generalisability of cystatin C equations obtained 52 I Clin Biochem Rev Vol 29 May 2008

Cystatic C - A Paradigm of EBLM by other studies in different settings by using these equations in their own study population. 114,115 For example, in cirrhotic patients and transplant patients, cystatin C equations were found to correlate better and were more accurate at predicting GFR than traditional equations based on creatinine. 114,115 Cystatin C and Clinical Outcomes CKD is well recognised as a risk factor for cardiovascular diseases (CVD) and several studies have shown an independent and graded relationship between the degree of kidney function and risk for CVD. 116-121 Several studies have also shown that cystatin C has a stronger association with CVD outcomes than creatinine concentration or egfr. 122-128 Analysis of the Cardiovascular Health Study cohort of community-dwelling elderly patients, showed that higher levels of cystatin C were associated with increased mortality and CVD outcomes. 122 Compared with the two lowest cystatin C quintiles combined (cystatin C level 0.99mg/L), the highest quintile of cystatin C ( 1.29mg/L) was associated with elevated risk of death from cardiovascular causes (hazard ratio 2.27[1.73 to 2.97]), myocardial infarction (hazard ratio 1.48[1.08 to 2.02]) and stroke (hazard ratio 1.47[1.09 to 1.96]) after multivariate adjustment. In contrast, creatinine concentration had a J-shaped relationship with mortality with no independent relationship with CVD outcomes. The lowest values of estimated egfr, using the MDRD equation, were associated with increased risk for death but not associated with CVD outcomes. Sequential quintiles of cystatin C concentration were also associated with stepwise increased risk for heart failure whereas quintiles of serum creatinine were not. 123 Furthermore, participants without CKD but with elevated cystatin C concentrations ( 1.0 mg/l) had a fourfold risk of progressing to CKD after four years of follow-up compared with those with normal cystatin C concentrations. 124 In this setting, cystatin C seems to identify a preclinical state of kidney dysfunction that is not detected with serum creatinine or estimated GFR. In a subgroup of subjects with heart failure cystatin C was a stronger predictor of mortality than creatinine. 125 An evaluation of 825 study participants in the MDRD study with stage 3 or 4 non-diabetic CKD, the association of cystatin C level with all cause mortality was as strong or stronger than that of Iothalamate GFR. 126 Data from the Heart and Soul study, a prospective cohort study designed to investigate the influence of psychosocial factors on coronary heart disease (CHD) progression, high cystatin Table 2a. Cystatin C based equations for the estimation of GFR using the PENIA method. Reference Equation Sample Size Population Equations for adults Le Bricon et al. 77 egfr = 78/cystatin C [mg/l] + 4 (n = 25) Renal Transplant Hoek et al. 69 egfr = 80.35 /cystatin C [mg/l] - 4.32 (n = 123) CKD patients Larsson et al. 32 egfr = 77.239 x cystatin C [mg/l] -1.2623 (n = 100) Adults MacIsaac et al. 108 egfr = 86.7 / cystatin C [mg/l] 4.2 (n = 251) Diabetics Rule et al. 109 egfr = 66.8 x cystatin C [mg/l] -1..30 (n = 357) CKD patients egfr = 76.6 x cystatin C [mg/l] -1.16 (n = 103) Renal Transplant Equations for children Filler et al. 60 log egfr = 1.962 + [1.123 (n = 536) CKD patients x log (1 / cystatin C [mg/l])] Zappitelli et al. 110 egfr = 75.94 / cystatin C [mg/l] -1.17 (n = 103) Children x 1.2 (if renal transplant) Clin Biochem Rev Vol 29 May 2008 I 53

Chew JSC et al. Table 2b. Cystatin C based equations for the estimation of GFR using the PETIA method. Reference Equation Sample Size Population Equations for adults Tan et al. 74 egfr = 87.1 / cystatin C [mg/l] 6.87 (n = 40) Adults (n = 29) Diabetics Larsson et al. 32 egfr = 99.434 x cystatin C [mg/l] -1.5837 (n = 100) Adults Grubb et al. 111 egfr = 99.19 x cystatin C [mg/l] -1.713 (n = 451) Adults x 0.823 (if female) Grubb et al. 62 egfr = 87.62 x cystatin C [mg/l] -1.693 (n = 451) Adults x 0.94 ( if female) Sjöström et al. 112 egfr = 124 / cystatin C [mg/l] 22.3 (n = 381) Adults Equations for children Bökenkamp et al. 63 egfr = 162 / cystatin c [mg/l] 30 (n = 184) CKD patients Grubb et al. 62 egfr = 87.62 x cystatin C [mg/l] -1.693 (n = 85) Children x 1.376 (if < 14 years) x 0.94 (if female) Table 2c. Cystatin C based equation for the estimation of GFR using the Gentian method. Reference Equation Sample Size Population Equation for adult Jonsson et al. 113 egfr = 79.901 x cystatin C [mg/l] -1.4389 ( n = 94) Patients with reduced GFR C levels predicted substantial increased risks of all-cause mortality, cardiovascular events, and incident heart failure among ambulatory persons with CHD. 127 A study investigating risk of secondary cardiovascular events in a large cohort of patient with CHD showed that patients in the top quintile of cystatin C distribution at baseline had a statistically significant increased risk of secondary CVD events even after adjustment for classic risk factors, (hazard ratio 2.27 [95% CI: 1.05 to 4.91]). 128 Neither serum creatinine nor creatinine clearance were associated with the incidence of secondary CVD events. Jernberg et al. evaluated the prognostic value of cystatin C in 726 patients with suspected or confirmed acute coronary syndrome with regard to death and myocardial infarction respectively. 129 In Cox regression models including well-known predictors of outcome, cystatin C levels were independently associated with mortality but not with the risk of subsequent myocardial infarction. In a comparison of plasma cystatin C, plasma creatinine and creatinine clearance, using ROC curve analysis, cystatin C had the best ability to discriminate between survivors and non survivors. Limitations of Cystatin C Limitations exist which may restrict the use of cystatin C in certain clinical situations. 54 I Clin Biochem Rev Vol 29 May 2008

Cystatic C - A Paradigm of EBLM Knight et al. analysed the performance characteristics of serum cystatin C and creatinine levels for estimating measured creatinine clearance utilising data collected in the Prevention of Renal and Vascular End-Stage Disease (PREVEND) cohort, a population-based sample of 8592 individuals. 130 They found that older age, male gender, greater weight, greater height, current cigarette smoking and serum CRP levels were independently associated with higher serum cystatin C levels after adjusting for creatinine clearance. 130 These results are in contrast with other reports that show no association between the above factors and cystatin C. 46,48 However in the study of Knight et al., a creatinine clearance was used instead of a gold standard GFR method which may question the validity of renal assessment used. Thyroid function is also known to effect serum cystatin C and serum creatinine levels independently of renal function. In a prospective case series of 22 consecutively referred patients with thyroid dysfunction, Fricker et al. evaluated the changes in serum cystatin C and creatinine levels during treatment of hypo- and hyperthyroidism. 131 In contrast to creatinine concentrations, cystatin C levels were lower in the hypothyroid and higher in the hyperthyroid state as compared with the euthyroid state. This association of cystatin C levels with thyroid state has also been shown for patients with subclinical hypo- and hyperthydoidism. 132 It is postulated that there is an altered production rate of cystatin C and serum creatinine in the context of a changed cell turnover and/or metabolic rate in thyroid dysfunction and that this overrides the effect of thyroid hormones on GFR. An average change of about 30% in cystatin C levels might be seen with frank thyroid disease and 15% with sub-clinical thyroid disease. 132 This has led to the recommendation by some authors that cystatin C levels should be determined with TSH to exclude primary thyroid disorders, especially in populations with a high incidence of thyroid diseases. It is also well known that corticosteroid treatment significantly induces cystatin C production. Pöge et al. demonstrated that cystatin C levels rose markedly in 22 renal transplant patients three days after routine treatment with corticosteroids post transplantation and that high dose methyl-prednisone application for rejection therapy led to a significantly increased serum cystatin C concentration. 133 In vitro studies have also shown a dose-dependent increase of up to 80% of cystatin C secretion by HeLa cells following dexamethasone exposure. 134 Significantly higher cystatin C concentrations were also found in adult asthmatic patients compared with controls and methyl prednisone decreased serum cystatin C concentrations after one week of therapy. 135 In contrast, Risch et al. have shown that adults on low-dose steroids tend to have somewhat higher cystatin C levels, albeit not significantly. 79 Apart from renal dysfunction, serum cystatin C concentrations may be influenced by underlying malignancy. 86 One study revealed a significant correlation between increased cystatin C and malignant progression in melanoma and colorectal cancer. 86 Average serum cystatin C concentrations in leukaemia patients have also been shown to be significantly higher than that of controls. 136 It has to be noted however, that in some of these studies, creatinine clearance was again used as a measure of GFR and not gold reference GFR methods. Discussion Cystatin C, first introduced in 1985, is now emerging as a potentially better marker of GFR than traditional markers such as serum creatinine. 137 The meta-analysis by Dharnidharka et al. and recent publication by Roos et al. provides evidence that cystatin C has superiority over serum creatinine as a marker of renal impairment. 30,31 Despite this, cystatin C has not been introduced into clinical laboratory practice and remains very much a research tool. Possible reasons include; (i) Clinicians do not like to replace familiar markers with new tests unless proven extensively to influence clinical decision making. (ii) Despite superior diagnostic accuracy, there is little evidence that cystatin C improves clinical decision making over the use of serum creatinine. (iii) The potential confounding effects of steroid therapy and thyroid disease and lack of data on other potential confounding variables such as malignancy. (iv) Different reference ranges have been published for different age groups and in addition, clinical decision points for cystatin C are not well defined. (v) Lack of uniformity and standardisation of available commercial assay formats may be contributing to this limitation. (vi) Contradicting results in the literature although the majority of studies showed superiority or at least equal performance of cystatin C in comparison with serum creatinine for detection of renal impairment. (vii) Cost and turnaround time of cystatin C measurements. For example, a nephelometric cystatin C measurement takes approximately 18 minutes to complete and the cost per test on the Dade Behring nephelometer is approximately 20 times more than a Jaffe creatinine Clin Biochem Rev Vol 29 May 2008 I 55

Chew JSC et al. measurement and three times the cost of an enzymatic creatinine measurement. At present, the convenience and low cost of serum creatinine assays has allowed this marker to be still widely used at the expense of accuracy. More work needs to be done on the effect of drugs (cyclosporin, chemotherapeutic agents, antibiotics, etc.), and generalisability of cystatin C equations in other populations. More research is also required in other patient groups, such as those with malignancy. Despite their gold standard status, reference GFR measurements such as the use of radio-pharmaceuticals are known to have variability and measurement uncertainty in comparison to the gold standard Inulin GFR clearance. For example, the variability in Iothalamate clearance may arise from both human and technical error during repeated timed urine collections and multiple assays for the calculation of Iothalamate clearances. 138,139 For Iohexol, it is interesting that determining GFR on three difference occasions with a 12 day period for 24 patients with a wide range of renal function can generate GFR results that differ by around 10% for many patients. 140 Furthermore although the usage of radiopharmaceuticals such as 99m Tc-DTPA and 51 Cr-EDTA has simplified the measurement GFR, these are less accurate than Inulin and may give substantial systematic error in GFR of up to 10-15 ml/min. 141,142 Several other low molecular weight proteins such as β2- Microglobulin, β-trace protein and retinol binding protein have also been evaluated as possible replacements for creatinine in the assessment of GFR. These proteins however, are affected by the presence of infections or malignancies and determinations are relatively more difficult. In contrast, cystatin C levels are not influenced by inflammation and levels are easily measured by commercially available latex based immunoassays. In renal transplant recipients where concerns arise due to increased production by glucocorticoid therapy, cystatin C has been found to offer similar and slightly better accuracy than these proteins. 143 More recently, a high profile group published results in their study of 3418 CKD patients pooled from different research studies. 144 Using newly developed equations, percentages of estimated GFR within 30% of measured GFR for equations based on serum cystatin C alone, serum cystatin C, serum creatinine, or both levels with age, sex and race were 81%, 83% and 89%, respectively. This lead to the conclusion that an equation including serum cystatin C level in combination with serum creatinine level, age, sex, and race provides the most accurate estimates of GFR. In conclusion, cystatin C is a more robust marker and reflects GFR more accurately than serum creatinine. Its introduction into laboratory use will depend firstly on analytical issues such as the production of an international standard calibrator. More importantly, it will depend on the establishment of an evidence base that it significantly improves clinical decision making to justify its cost. Despite the superior diagnostic accuracy of cystatin C compared with serum creatinine for detection of early renal impairment and also prediction of long-term outcomes, there is still a paucity of evidence that it actually leverages important clinical decisions more effectively than the use of serum creatinine alone or egfr. Ongoing research may yet provide that evidence and lead to its adoption into operational use. Competing Interests: None declared. References 1. Newman DJ. Cystatin C. Ann Clin Biochem 2002;39: 89-104. 2. Abrahamson M, Olafsson I, Palsdottir A, Ulvsbäck M, Lundwall A, Jensson O, et al. Structure and expression of the human cystatin C gene. Biochem J 1990;268: 287-94. 3. Tenstad O, Roald AB, Grubb A, Aukland K. Renal handling of radiolabelled human cystatin C in the rat. Scand J Clin Lab Invest 1996;56:409-14. 4. Laterza OF, Price CP, Scott MG. Cystatin C: an improved estimator of glomerular filtration rate? Clin Chem 2002;48:699-707. 5. van Rossum LK, Zietse R, Vulto AG, de Rijke YB. Renal extraction of cystatin C vs 125I-iothalamate in hypertensive patients. Nephrol Dial Transplant 2006;21:1253-6. 6. Ix JH. Utility of cystatin C measurement--precision or secretion? Nephrol Dial Transplant 2006;21:3614. 7. Mussap M, Plebani M. Biochemistry and clinical role of human cystatin C. Crit Rev Clin Lab Sci 2004;41: 467-550. 8. Lofberg H, Grubb AO. Quantitation of gamma-trace in human biological fluids: indications for production in the central nervous system. Scand J Clin Lab Invest 1979;39:619-26. 9. Hamer R, El Nahas AM. The burden of chronic kidney disease. BMJ 2006;332:563-4. 10. Perrone RD, Madias NE, Levey AS. Serum creatinine as an index of renal function: new insights into old concepts. Clin Chem 1992;38:1933-53. 56 I Clin Biochem Rev Vol 29 May 2008

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