1. Ron T. Gansevoort Coordinator PREVEND Study Department of Nephrology University Medical Center Groningen The Netherlands Ron T. Gansevoort Coordinator PREVEND Study Department of Nephrology University Medical Center Groningen The Netherlands Assessing albuminuria Methodological considerations with clinical impact

2. Introduction Albuminuria is a urinary biomarker that has been shown to be a predictor of renal and CV events. As such albuminuria has a place in clinical practice: kDOQI stages 1 and 2 are defined by presence of micro-albuminuria. There is strong lobby for standardisation of measurement serum creatinine (Cleveland Clinic / IDMS traceable) to obtain the most reliable GFR estimate. Untill recently little attention has been paid to standardisation of albuminuria (exception Miller et al, Clin Chem 2009;55:24-38)

3. Micro-albuminuria - Definition and classification - Spot urines (first morning void, or random) 17 - 170 25 - 250 < 17 < 25 Alb/creat ratio (mg/gram) > 200> 300> 200Macro-albuminuria 20 – 20030 – 300MFMF 20 – 200Micro-albuminuria < 20< 30MFMF < 20Normal Albumin Excretion (  g/min) Albumin Excretion (mg/24h) Albumin Concentration (mg/l) Overnight (timed) 24h urine > 170 > 250 MFMF

4. Assessment of albuminuria Questions to address 1.What assay to use? (answer: immunochemistry polyclonal) 2.What urine sample to use? 24hr urine collection, first morning void or a spot sample? 3.Which albuminuria measure to use: urinary albumin concentration, albumin/creatinine ratio, or 24hr albumin excretion? 4.Does it matter whether we use fresh urine samples or stored samples? 5.If we are going to use frozen urine samples, what is important? pre-storage handling, storage temperature, sample handling

5. Monomer Bakker, Gansevoort et al, Curr Hypert Rep 2009;11:111-7 Dimerization Polymerization Fragmentation Loss of immunoreactivity ?? Type of assay Monoclonal AB Polyclonal AB Colorimetric test strips Immunochemistry based Size exclusion (HPLC) Assessment of albuminuria Which assay to use?

6. 24-hourFMVSpot (morning) Albumin concentration (mg/L) 0 20 40 60 80 Median 24-hour[IQ-range] 7.6 [4.8-12.7] Median Overnight[IQ-range] 7.2 [4.5-12.0] Median Spot (morning) [IQ-range] 11.9 [7.8-25.8] P = < 0.01 P = 0.43 N=250 Witte et al, JASN 2009;20:436-43 What urine samples to use ? Median urinary albumin concentration PREVEND

7. Urinary Albumin Concentration 24-hourFMVSpot (morning) Intra-subject coefficient of Variation (%) 0 20 40 60 80 100 P = < 0.01 P = 0.08 PREVEND What urine samples to use ? Coefficient of variation N=250 Witte et al, JASN 09;20:436-43

8. 24-hourFMVSpot (morning) Albumine:creatinine ratio (mg/mmol) 0 2 4 6 8 10 Median 24-hour[IQ-range]1.00 [0.65-1.54] Median Overnight[IQ-range] 0.67 [0.50-1.17] Median Spot (morning) [IQ-range] 1.21 [0.68-2.37] P = 0.023 P = < 0.001 P < 0.001 Witte et al, JASN 09;20:436-43 What urine samples to use ? Median albumin:creatinine ratio PREVEND N=250

9. PREVEND What urine samples to use ? Coefficient of variation Albumin:creatinine ratio 24-hourFMVSpot (morning) 0 20 40 60 80 100 P = < 0.01 P = 0.58 N=250 Intra-subject coefficient of Variation (%) Witte et al, JASN 09;20:436-43

10. 0 10 20 30 Overall Male Female Prevalence of microalbuminuria (%) # * # * # * # * # * # * 24hr UAE UAC ACR 0 10 20 30 Overall Male Female Prevalence of microalbuminuria (%) First Morning Void # * # * # * # * # * # * PREVEND Spot Morning Urine Sample Witte et al, JASN 09;20:436-43 UACACR What urine samples to use ? Prevalence of microalbuminuria

11. PREVEND Which albuminuria measure to use? Predicting CV outcome 24 hr urineFirst morning void UAE (mg/24hr)UAC (mmol/LACR (mg/mmol) Overall 0.650.620.66* Subgroups Male 0.640.620.68* Female 0.660.59 # 0.66* <47 yr 0.580.52 >47yr 0.650.64 * p < 0.05 vs UAC, # p < 0.05 vs UAE AUC ROC curve N=3432 Random sample of the general population Lambers-Heerspink et al, Am J Epidemiol 08;168:897-905

12. Lambers Heerspink et al, submitted N=701 RENAAL: DM2 nephropathy Which albuminuria measure to use? Predicting renal outcome 100806040200 0 40 60 80 100 Sensitivity (%) Specificity (%) UAE 24hr; AUC = 0.78 UPE 24hr; AUC = 0.78 p<0.001 ACR FMV; AUC = 0.82

13. First morning void -60 -40 -20 0 20 40 60 80 3545556575 Age (years) Difference (%) UAC UCrC ACR 24-hour urine collection -60 -40 -20 0 20 40 60 80 3545556575 Age (years) Difference (%) UAE UCrE ACR Lambers-Heerspink, Gansevoort et al, Am J Epidemiol 2008;168:897-905 PREVEND Which albuminuria measure to use? ACR “incorporates” the influence of age

14. PREVEND Frozen storage (-20 C) of urine samples Influence of duration of storage and sample handling 3 to 5 5 to 8 8 to 12 12 to 18 18 to 24 months * Not significantly different from zero Hand-inversion ** Percentage change in UAC, % Vortex mixing No sample handling -80 -60 -40 -20 0 20 40 Brinkman et al, Clin Chem 2005;51:2181-3

15. Predictive value of albuminuria Does it matter when urine has been stored frozen ? Brinkman et al, Clin Chem 2007;53:153-4 PREVEND P<0.01 Predictive value of UAE for CV endpoints

16. PREVEND Frozen storage of urine samples Does urinary pH matter ? -100 -75 -50 -25 0 50 75 Change in urinary albumin concentration (%) -20 °C- 20  C pH8 -80  C pH8 Storage condition - - - - - Lambers Heerspink et al, Diabetic Med 2009;26:556-9

17. Screening for albuminuria The past (1892) Gansevoort and Ritz, Nephrol Dial Transplant 2008

18. Conclusions When assessing the clinical impact of urinary biomarkers it is essential to take into consideration methodological issues 1.Which assay was used? Polyclonal? Intra- and interassay CV? 2.What urine samples were used? Preferably 24hr collections or first morning voids 3.In case first morning void samples are used, normalise for creatine concentration 4.Fresh or frozen? Preferably use fresh urine samples. 5.If frozen, what were storage conditions and how was sample handling? Frozen at -80 0 Celsius, pH adjustment (or protease inhibitors?), vortexing?