assessing albuminuria - kdigo · predicting cv outcome 24 hr urine first morning void uae (mg/24hr)...
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Ron T. Gansevoort
Coordinator PREVEND Study
Department of Nephrology University Medical Center
Groningen The Netherlands
Assessing albuminuria
Methodological considerations with clinical impact
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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)
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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 > 200 Macro-albuminuria
20 – 200 30 – 300 M F
20 – 200 Micro-albuminuria
< 20 < 30 M F
< 20 Normal
Albumin Excretion
(µg/min)
Albumin Excretion (mg/24h)
Albumin Concentration
(mg/l)
Overnight (timed) 24h urine
> 170 > 250
M F
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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
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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?
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24-hour FMV Spot (morning)
Alb
umin
con
cent
ratio
n (m
g/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 = < 0.01
P = 0.43
N=250
Witte et al, JASN 2009;20:436-43
What urine samples to use ? Median urinary albumin concentration PREVEND
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Urinary Albumin Concentration 24-hour FMV Spot (morning)
Intra
-sub
ject
coe
ffici
ent o
f Var
iatio
n (%
)
0
20
40
60
80
100 P = < 0.01
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
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24-hour FMV Spot (morning)
Alb
umin
e:cr
eatin
ine
ratio
(mg/
mm
ol)
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
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PREVEND
What urine samples to use ? Coefficient of variation
Albumin:creatinine ratio
24-hour FMV Spot (morning) 0
20
40
60
80
100 P = < 0.01
P = < 0.01
P = 0.58
N=250
Intra
-sub
ject
coe
ffici
ent o
f Var
iatio
n (%
)
Witte et al, JASN 09;20:436-43
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0
10
20
30 Overall Male Female
Pre
vale
nce
of m
icro
albu
min
uria
(%)
# *
# *
# * # *
# *
# *
0
10
20
30 Overall Male Female
Pre
vale
nce
of m
icro
albu
min
uria
(%)
First Morning Void
# *
# *
# * # *
# *
# *
PREVEND
Spot Morning Urine Sample
Witte et al, JASN 09;20:436-43
What urine samples to use ? Prevalence of microalbuminuria
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PREVEND
Which albuminuria measure to use? Predicting CV outcome
24 hr urine First morning void
UAE (mg/24hr) UAC (mmol/L ACR (mg/mmol)
Overall 0.65 0.62 0.66*
Subgroups 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
* 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
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Lambers Heerspink et al, submitted
N=701
RENAAL: DM2 nephropathy
Which albuminuria measure to use? Predicting renal outcome
100 80 60 40 20 0 0
20
40
60
80
100 Se
nsiti
vity
(%)
Specificity (%)
UAE 24hr; AUC = 0.78
UPE 24hr; AUC = 0.78
p<0.001
ACR FMV; AUC = 0.82
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First morning void
-60
-40
-20
0
20
40
60
80
35 45 55 65 75
Age (years)
Diff
eren
ce (%
)
UAC
UCrC
ACR
24-hour urine collection
-60
-40
-20
0
20
40
60
80
35 45 55 65 75
Age (years)
Diff
eren
ce (%
)
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
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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
* *
Perc
enta
ge c
hang
e in
UA
C, %
Vortex mixing No sample handling
-80
-60
-40
-20
0
20
40
Brinkman et al, Clin Chem 2005;51:2181-3
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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
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PREVEND
Frozen storage of urine samples Does urinary pH matter ?
- 100
- 75
- 50
- 25
0
25
50
75 C
hang
e in
urin
ary
albu
min
con
cent
ratio
n (%
)
-20 ° C - 20°C pH8
-80°C -80°C pH8
Storage condition
-
-
-
-
-
Lambers Heerspink et al, Diabetic Med 2009;26:556-9
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Screening for albuminuria The past (1892)
Gansevoort and Ritz, Nephrol Dial Transplant 2008
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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?