Cystatin C Den svenske erfaringen.

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1 Cystatin C Den svenske erfaringen. Cystatin C some basic facts the new IFCC cystatin C calibrator assay harmonization between different companies cystatin C not just a new marker for renal function! the combined use of cystatin C and creatinine Or Why some of us in Sweden love cystatin C Lars-Olof Hansson Associate professor Clinical Chemistry Karolinska University Hospital Stockholm, Sweden lasse.hansson@me.com

2 Some background facts on cystatin C

3 γ-trace = cystatin C

4 Cystatin C First published as γ-trace in the early 70th 13,3 kda, 120 amino acid residues Non-glycosylated protein Isoelectric point of 9.3 Cystein C is a cystein protease inhibitor A Grubb

5 Cystatin C The Cystatin C gene is a so called housekeeping gene Mostly stable production in all nucleated cells Production is independent of gender, age and muscle-mass The first correlation between GFR and 1/Cystatin C was 1985 The synthesis is significantly increased by hyperthyroidism and by high doses of cortico-steroids.

6 S-Creatinine during childhood A Grubb

7 S-Cystatin C in 258 children A Grubb

8 Cystatin C in the United States Serum cystatin C percentiles (5th, 50th, and 95th) by age and (A) sex and (B) race/ethnicity Anna Köttgen et al. American Journal of Kidney Diseases, Vol 51, No 3, 2008:

9 Does systemic inflammation influence cystatin C? Persons (n=20) were subjected to elective surgery and the levels of inflammatory markers and cystatin C and creatinine were followed for 7 postoperative days. Grubb A et al. Scand J Clin Lab Invest 71: (2011)

10 Does systemic inflammation influence cystatin C? A Grubb

11 Cystatin C in relation to CRP

12 Characteristics of an ideal GFR marker and Early detection of KD

13 Characteristics of an ideal GFR marker Demonstrates the early and potentially reversible decrease of GFR Independent of diet and hormonal or inflammatory changes No tubular secretion Low influence by muscle mass, gender and race (African American) Independent of age for children above 1 year and adults Demonstrates the decrease of GFR in old persons Mirrors the diurnal GFR variation Elucidates filtration quality and life expectancy

14 Normal glomeruli 40 Å Glomeruli pore Cystatin C Creatinine 30 Å 5 Å

15 Diseased glomeruli No passage Passage

16 Correlation between Iohexol GFR and cystatin C or creatinine

17 The IFCC cystatin C standardization efforts

18 IFCC-EU Working Group for Standardization of Cystatin C

19 The new ERM-DA471/IFCC Cystatin C calibrator was released June 2010

20 IFCC-EU Working Group for Standardization of Cystatin C GOALS 1. To produce an international cystatin C-calibrator. (Done June 2010: ERM-DA471/IFCC) 2. To produce an assay-independent cystatin C-based GFRprediction equation in collaboration with major diagnostic companies. (Ongoing)

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23 Problem 1 How to achieve minimal batch-to-batch variation and stable egfr results Company X and Y Year 2006 very similar results were achieved both for cystatin C in mg/l and for egfr in ml/min/1.73 m 2. However year 2010 discrepant results were delivered using these two assays. New calibrator needed! The very tight fit around the line indicates that both assays have superior precisions!

24 Bias % Reagens Kalibrator (8,25) 8,00 7,00 6,00 5,00 Cystatin C Architect, Gentian y = 1,0474x - 0,0852 R 2 0,9998 = Solution to Problem 1 Very strict batch-to-batch control of reagents and calibrators. This will give stable egfr results. 4,00 3,00 2,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 Pool Cystatin C Architect, Gentian 10,00 5,00 0,00-5,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00-10,00 mg/l

25 Problem 2 How to achieve proper assay set-up Assay setup: Compa ny Z Rate 548 nm Conc. Sample buffer antisera Total volume Percent antisera 0, ,6 1, ,4 2, ,8 5, ,4 9, ,6 Company Z calibration curve with the stated volumes. The blue dots: manually dilutions of Company Z calibrator.

26 Solution to Problem 2 Company Z calibration with modified (corrected) calibrator volumes Assay setup: Compa ny Z Rate 548 nm Conc. Sample buffer antisera Total volume Percent antisera 0, ,6 1, ,6 2, ,5 5, ,4 9, ,6 Instrument dilution of Company Z calibrator Manual dilution of Company Z calibrator Company Z calibration curve with modified volumes. The blue dots: manually dilutions of Company Z calibrator.

27 Problem 3 How shall the calibrator-function be set? Upper and lower calibrator points? How many points? Which curve fitting model is to be used? Original calibration for both methods Company A and B Original calibration for both assays from Company A and B on the left graph. On the right graph the assay from Company A has been calibrated using the same distribution of calibrator points as used in the assay from Company B.

28 Problem 4 At which concentration level shall the assay performance be optimal?

29 Result after optimal assay set-up for Company A and B Both assays from Company A and B has been re-calibrated and their assay-set up has been optimally harmonized Iohexol samples from Lund

30 Mathematical models of equations with cystatin C as single variable 1. CYS simple: A x CysC -1 - k ( k might be interpreted as a constant extrarenal elimination of cystatin C) 2. CYS 1: A x CysC -k 3. CYS knots: If CYSC<Q, A x CysC -k1 If CYSC>/=Q, B x CysC -k2 4.?

31 egfr ml/min/1.73m Effect of different egfr equations Uppsala CysC 1 CysC <1.6 and CysC Sample no

32 The IFCC cystatin C standardization efforts Next steps: Finalize the development of a common equation for egfr from cystatin C Validation of the assay-independent cystatin C-based equation Characterize if, and how much, the P30-value can be improved by adding further variables, e.g. age, sex, BMI, race/ethnicity, to the prediction equation. Determine how much egfr will be improved by combining and comparing optimal cystatin C- and creatinine-based egfrs according to Grubb et al. SJCLI 2012, 72:73-77: Improved estimation of GFR by comparison of egfr cystatin C and egfr creatinine.

33 Examples on the clinical usefulness of cystatin C for detection of progression of loss of kidney function and progression of diseases. Or The importance of early detection of kidney disease

34 Factors involved in the rate of progression of chronic diseases Pre-eclampsia Increasing Age Atherosclerosis Metabolic syndrome Hypertension Genetics Diabetes Pre-chronic kidney disease Epigenetics Chronic kidney disease Smoking Renal failure Stress Alzheimer Dementia Overweight CVD Cancer Food Lipids Inflammation Pre-timely aging Pre-timely loss of physical function Pre-timely death

35 Inflammatory stimuli Trauma Irradiation Heat Cold Tumor Neuro-stress Parasites Fat tissue Thromboembolism Inflammation Fungi Viruses Anoxia Enzymes Immuncomplexes Toxins Bacteria Different inflammatory stimuli inducing release of proinflammatory cytokines (IL-6, IL-8 and tumor necrosis factor alfa (TNF-a) It is the sum of inflammation during life-time that matters!

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37 Percent of Obese (BMI > 30) in U.S. Adults

38 Percent of Obese (BMI > 30) in U.S. Adults

39 Percent of Obese (BMI > 30) in U.S. Adults

40 Percent of Obese (BMI > 30) in U.S. Adults

41 Percent of Obese (BMI > 30) in U.S. Adults

42 Global projections for the diabetes epidemic:

43 Incidence of Kidney Failure in per million inhabitants in U.S 1990

44 Incidence of Kidney Failure in per million inhabitants in U.S 2000

45 Serum cystatin C as a marker for early detection of chronic kidney disease and grade 2 nephropathy in Japanese patients with type 2 diabetes Yoshitake Suzuki1, Kazuyuki Matsushita1,2, Masanori Seimiya1,2, Toshihiko Yoshida1, Yuji Sawabe1, Makoto Ogawa3 and Fumio Nomura1,2 1 Division of Laboratory Medicine, Chiba University Hospital, Chiba, Japan 2 Department of Molecular Diagnosis and 3 Nephrology, Chiba University Graduate School of Medicine, Chiba, Japan Yoshitake Suzuki et al. Clinical Chemistry and Laboratory Medicine 2012

46 Serum cystatin C as a marker for early detection AUC scysc =0.864 (95% CI, ) AUC scr=0.603 (95% CI, ) AUC sβ2m=0.816 (95% CI, ) Control patients (n = 88) vs. grades 2 and 3 diabetes nephropathy (n=151) Yoshitake Suzuki et al. Clinical Chemistry and Laboratory Medicine 2012

47 Serum cystatin C as a marker for early detection grade 1 DN (ACR <30 mg/g) grade 2 DN (ACR mg/g) grade 3 DN (ACR >300 mg/g) Yoshitake Suzuki et al. Clinical Chemistry and Laboratory Medicine 2012

48 Early nephropathy in type I diabetes the importance of early renal function decline ml/min/1.73 m2 Early renal function decline can be diagnosed using serial measurement of serum cystatin C but not by MDRD Serial determinations of glomerular filtration rate by measurement of iothalamate clearance, 100/cystatin C, and the MDRD equation from a representative example in the diabetic renal disease study Perkins B A, Krolewski A S. Curr Opin Nephrol Hypertens 2009; 18:

49 Elevated Cystatin C Concentration and Progression to Pre-Diabetes 6 years follow-up Model 1 was adjusted for age. Model 2 was adjusted for age, BMI, family history of diabetes, smoking, lifetime pack-years, fasting glucose, current drinker, baseline hypertension, HOMA-IR, and egfr (MDRD). Our findings provide an important new avenue for future research: Mild renal impairment may occur early in the natural history of diabetes. Donahue R H et al. Diabetes Care 30: , 2007

50 Progression to CKD after 4 Year Follow-Up (Cardiovascular Health Study cohort) Incidence (%) Unadjusted OR (95%CI) Adjusted OR* (95%CI) Normal (egfr>60) 7.5% Preclinical Kidney Disease (cystatin C>1.0) 27% 4.53 ( ) 4.18 ( ) *Adjusted for age, gender, race, current smoking, LDL, HDL, CRP, diabetes, and hypertension Shlipak et al. Ann Intern Med 2006

51 Cystatin C and Measures of Physical Function in Elderly Adults The Health, Aging, and Body Composition Study. M Schlipak Mild decrements in kidney function, as measured by cystatin C concentration, were associated with poorer physical function. Not completing a 400-m walk 10.9-second longer 400-m walk time 0.11-point reduction in lower extremity performance score 1.12-kg lower grip strength 4.7-newtonmeter lower knee extension strength In contrast, when kidney function was measured by MDRD, the association of kidney function with physical function was only evident below 60 ml/minute/1.73 m2. MC Odden, MG Shlipak IB Tager J Gerontology 2009:64A;

52 Creatinine, MDRD, and cystatin C and risk for death in participants without chronic kidney disease Creatinine Ann Intern Med. 2006;145:

53 Cystatin C, Albuminuria, and Mortality Among Older Adults With Diabetes A single cystatin C measurements carry important prognostic information even without prior context! Ian H. De Boer et al. Diabetes Care 32: , 2009

54 Cystatin C and Aging Success MJ. Sarnak, MG Shlipak et al. Arch Intern Med. 2008;168(2):

55 Incidence Rates per Year(%) Kidney Function and Heart Failure Incidence *Significant adjusted hazard ratio 6 5 * 4 3 * * Cystatin C egfr MDRD Quintiles Sarnak MJ et al. Ann Intern Med. 2005;142:

56 Mortality from All Causes According to Quintile of Measures of Renal Function Shlipak M et al. N Engl J Med 2005;352:

57 GFR = 120 ml/min/1.73m 2 P-Creatinine 52 µmol/l P-Cystatin C = 0.75 mg/l GFR = 120 ml/min/1.73m 2 P-Creatinine 102 µmol/l P-Cystatin C = 0.75 mg/l A Grubb

58 Suggestion for optimal diagnostic use of egfr Run both cystatin C and creatinine and calculate egfr (cystatin C) and egfr (creatinine) at the initial patient contact. If they agree: GFR is correct, no invasive clearance determination is required. If they do not agree: Try to find a biomedical explanation (low muscle mass, high dose of glucocorticoid). If an obvious explanation is found no invasive clearance determination is required. The non-affected egfr is used. P/S-Creatinine can be used to follow a correct egfr as defined above. If the two egfrs do not agree and no obvious explanation for the difference can be found: An invasive clearance determination is justified (iohexol clearance). Site: egfr.se

59 Creatinine-based egfr equations MDRD : x [S- creat ( mol/l)/88.4] x age x (if female) x (if African American) Only for adults Schwartz: 0.55 x height (cm) x [P- creat ( mol/l)/88.4] -1 Only for children Counahan Barratt : 0.43 x height (cm) x [P- creat ( mol/l)/88.4] -1 Only for children Lund-Malmö : e x creat x ln(age) x age (if female) For children and adults

60 Age-related GFR-predictions at a constant creatinine level of 80 µmol/l for 3 prediction equations MDRD Lund-Malmö Counahan Barratt

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64 The light of cystatin C is rising in the west Cystatin C and Aging Success Thank you all! MJ. Sarnak, MG Shlipak et al. Arch Intern Med. 2008;168(2):