ron t. gansevoort coordinator prevend study department of nephrology university medical center...
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Ron T. GansevoortRon T. Gansevoort
Coordinator PREVEND StudyCoordinator PREVEND StudyDepartment of NephrologyDepartment of NephrologyUniversity Medical CenterUniversity Medical Center
GroningenGroningenThe NetherlandsThe Netherlands
Ron T. GansevoortRon T. Gansevoort
Coordinator PREVEND StudyCoordinator PREVEND StudyDepartment of NephrologyDepartment of NephrologyUniversity Medical CenterUniversity Medical Center
GroningenGroningenThe NetherlandsThe Netherlands
Assessing albuminuriaAssessing albuminuria
Methodological considerations with clinical impactMethodological considerations with clinical impact
IntroductionIntroduction
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-albuminuriaMicro-albuminuria- - Definition and classification -Definition and classification -
Spot urines(first morning void, or random)
17 - 17025 - 250
< 17< 25
Alb/creat ratio
(mg/gram)
> 200> 300> 200Macro-albuminuria
20 – 20030 – 300MF
20 – 200Micro-albuminuria
< 20< 30MF
< 20Normal
AlbuminExcretion
(g/min)
AlbuminExcretion(mg/24h)
AlbuminConcentration
(mg/l)
Overnight (timed)24h urine
> 170> 250
MF
Assessment of albuminuriaAssessment of albuminuriaQuestions to addressQuestions 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
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 albuminuriaAssessment of albuminuriaWhich assay to use?Which assay to use?
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 ?What urine samples to use ?Median urinary albumin concentrationMedian urinary albumin concentration
PREVENDPREVEND
Urinary Albumin Concentration
24-hour FMV Spot (morning)
Intr
a-su
bjec
t co
effic
ient
of
Var
iatio
n (%
)
0
20
40
60
80
100P = < 0.01
P = < 0.01
P = 0.08
PREVENDPREVEND
What urine samples to use ?What urine samples to use ?Coefficient of variationCoefficient of variation
N=250
Witte et al, JASN 09;20:436-43
24-hour FMV Spot (morning)
Alb
umin
e:cr
eatin
ine
ratio
(m
g/m
mol
)
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 ?What urine samples to use ?Median albumin:creatinine ratioMedian albumin:creatinine ratio
PREVENDPREVEND
N=250
PREVENDPREVEND
What urine samples to use ?What urine samples to use ?Coefficient of variationCoefficient 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
Intr
a-su
bjec
t co
effic
ient
of
Var
iatio
n (%
)
Witte et al, JASN 09;20:436-43
0
10
20
30
Overall
Male
Female
Pre
vale
nce
of
mic
roa
lbu
min
uri
a (
%)
#*
#*
#*#*
#*
#*
24hr
UAE
UACACR
0
10
20
30
Overall
Male
Female
Pre
vale
nce
of
mic
roa
lbu
min
uri
a (
%)
First Morning Void
#*
#*
#*#*
#*
#*
PREVENDPREVEND
Spot Morning Urine Sample
Witte et al, JASN 09;20:436-43
UACACR
What urine samples to use ?What urine samples to use ?Prevalence of microalbuminuriaPrevalence of microalbuminuria
PREVENDPREVEND
Which albuminuria measure to use? Which albuminuria measure to use? Predicting CV outcomePredicting 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 curveAUC ROC curve
N=3432
Random sample of the general populationLambers-Heerspink et al, Am J Epidemiol 08;168:897-905
Lambers Heerspink et al, submitted
N=701
RENAAL: DM2 nephropathy
Which albuminuria measure to use? Which albuminuria measure to use? Predicting renal outcomePredicting renal outcome
100 80 60 40 20 00
20
40
60
80
100S
ensi
tivi
ty (
%)
Specificity (%)
UAE 24hr; AUC = 0.78
UPE 24hr; AUC = 0.78
p<0.001
ACR FMV; AUC = 0.82
First morning void
-60
-40
-20
0
20
40
60
80
35 45 55 65 75
Age (years)
Diff
ere
nce
(%
)
UAC
UCrC
ACR
24-hour urine collection
-60
-40
-20
0
20
40
60
80
35 45 55 65 75
Age (years)
Diff
ere
nce
(%
)
UAE
UCrE
ACR
Lambers-Heerspink, Gansevoort et al, Am J Epidemiol 2008;168:897-905
PREVENDPREVEND
Which albuminuria measure to use? Which albuminuria measure to use? ACR “incorporates” the influence of ageACR “incorporates” the influence of age
PREVENDPREVEND
Frozen storage (-20 C) of urine samplesFrozen storage (-20 C) of urine samplesInfluence of duration of storage and sample handlingInfluence 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 zeroHand-inversion
* *
Per
cen
tag
e ch
an
ge
in U
AC
, %
Vortex mixing
No sample handling
-80
-60
-40
-20
0
20
40
Brinkman et al, Clin Chem 2005;51:2181-3
Predictive value of albuminuria Predictive value of albuminuria Does it matter when urine has been stored frozen ?Does it matter when urine has been stored frozen ?
Brinkman et al, Clin Chem 2007;53:153-4
PREVENDPREVEND
P<0.01
Predictive value of UAE for CV endpoints
PREVENDPREVEND
Frozen storage of urine samplesFrozen storage of urine samplesDoes urinary pH matter ?Does urinary pH matter ?
-100
-75
-50
-25
0
25
50
75C
han
ge
in u
rin
ary
alb
um
in c
on
cen
trat
ion
(%
)
-20°C - 20CpH8
-80C -80CpH8
Storage condition
-
-
-
-
-
Lambers Heerspink et al, Diabetic Med 2009;26:556-9
Screening for albuminuriaThe past (1892)
Gansevoort and Ritz, Nephrol Dial Transplant 2008
ConclusionsConclusions
When assessing the clinical impact of urinary biomarkers it isessential 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?