Transcript Dia 1 - KDIGO

Assessing albuminuria
Methodological considerations with clinical impact
Ron T. Gansevoort
Coordinator PREVEND Study
Department of Nephrology
University Medical Center
Groningen
The Netherlands
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)
Micro-albuminuria
- Definition and classification -
Spot urines
(first morning void, or random)
Albumin
Concentration
(mg/l)
Normal
Alb/creat
ratio
(mg/gram)
Albumin
Excretion
(mg/24h)
Overnight
(timed)
Albumin
Excretion
(g/min)
24h urine
< 20
M
F
< 17
< 25
< 30
< 20
Micro-albuminuria
20 – 200
M
F
17 - 170
25 - 250
30 – 300
20 – 200
Macro-albuminuria
> 200
M
F
> 170
> 250
> 300
> 200
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
Assessment of albuminuria
Which assay to use?
Type of assay
Polymerization
Colorimetric test strips
Immunochemistry based
Monoclonal AB
Dimerization
Polyclonal AB
Monomer
Size exclusion (HPLC)
Fragmentation
Loss of immunoreactivity ??
Bakker , Gansevoort et al, Curr Hypert Rep 2009;11:111-7
What urine samples to use ?
Median urinary albumin concentration
PREVEND
80
Albumin concentration (mg/L)
P = < 0.01
P = < 0.01
60
40
P = 0.43
20
N=250
0
24-hour
Witte et al, JASN 2009;20:436-43
FMV
Spot (morning)
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]
What urine samples to use ?
Coefficient of variation
Intra-subject coefficient of Variation (%)
PREVEND
P = < 0.01
100
P = < 0.01
80
P = 0.08
60
40
N=250
20
0
24-hour
FMV
Spot (morning)
Urinary Albumin Concentration
Witte et al, JASN 09;20:436-43
What urine samples to use ?
Median albumin:creatinine ratio
Albumine:creatinine ratio (mg/mmol)
PREVEND
10
P = 0.023
8
P = < 0.001
6
P < 0.001
4
2
0
Witte et al, JASN 09;20:436-43
N=250
24-hour
FMV
Spot (morning)
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]
What urine samples to use ?
Coefficient of variation
Intra-subject coefficient of Variation (%)
PREVEND
P = < 0.01
100
P = < 0.01
80
P = 0.58
60
40
N=250
20
0
24-hour
FMV
Spot (morning)
Albumin:creatinine ratio
Witte et al, JASN 09;20:436-43
What urine samples to use ?
Prevalence of microalbuminuria
PREVEND
#*
Prevalence of microalbuminuria (%)
30
#*
#*
#*
20
Overall
Male
Female
#*
#*
10
0
First Morning Void
Witte et al, JASN 09;20:436-43
Spot Morning Urine Sample
Which albuminuria measure to use?
Predicting CV outcome
PREVEND
AUC ROC curve
24 hr urine
UAE (mg/24hr)
UAC (mmol/L
ACR (mg/mmol)
0.65
0.62
0.66*
Male
0.64
0.62
0.68*
Female
0.66
0.59#
0.66*
<47 yr
0.58
0.52
0.52
>47yr
0.65
0.64
0.64
Overall
Subgroups
First morning void
* p < 0.05 vs UAC, # p < 0.05 vs UAE
N=3432
Lambers-Heerspink et al, Am J Epidemiol 08;168:897-905
Random sample of the general population
Which albuminuria measure to use?
Predicting renal outcome
100
Sensitivity (%)
80
60
40
ACR FMV; AUC = 0.82
UAE 24hr; AUC = 0.78
20
UPE 24hr; AUC = 0.78
p<0.001
0
100
80
60
40
20
0
Specificity (%)
N=701
Lambers Heerspink et al, submitted
RENAAL: DM2 nephropathy
Which albuminuria measure to use?
ACR “incorporates” the influence of age
PREVEND
24-hour urine collection
First morning void
80
80
ACR
60
ACR
60
UAE
40
20
UAC
0
35
45
55
65
75
-20
UCrC
-40
-60
Age (years)
Difference (%)
Difference (%)
40
20
0
35
45
55
-40
Lambers-Heerspink, Gansevoort et al, Am J Epidemiol 2008;168:897-905
75
UCrE
-20
-60
65
Age (years)
Frozen storage (-20 C) of urine samples
Influence of duration of storage and sample handling
PREVEND
* Not significantly different from zero
Hand-inversion
Vortex mixing
No sample handling
40
Percentage change in UAC, %
* *
20
0
-20
-40
-60
-80
3 to 5
5 to 8
Brinkman et al, Clin Chem 2005;51:2181-3
8 to 12
12 to 18
18 to 24 months
Predictive value of albuminuria
PREVEND
Does it matter when urine has been stored frozen ?
P<0.01
Brinkman et al, Clin Chem 2007;53:153-4
Predictive value of UAE for CV endpoints
Frozen storage of urine samples
Does urinary pH matter ?
PREVEND
Change in urinary
albumin concentration (%)
75
50
25
0
--25
--50
--75
--100
-20 °
C
- 20C
pH8
-80C
Storage condition
Lambers Heerspink et al, Diabetic Med 2009;26:556-9
-80C
pH8
Screening for albuminuria
The past (1892)
Gansevoort and Ritz, Nephrol Dial Transplant 2008
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 0Celsius, pH adjustment (or protease
inhibitors?), vortexing?