Test Dipstick for Determination of ...

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Original Articles
Test Dipstick for Determination of Urinary Protein, Creatinine and
Protein/Creatinine Ratio
Jiunn-Min Wang
1,2
, Chi-Yu Lin
1
, Feng-An Tsai
1
, Jing-Yi Chen
1
, Yu-Chen Koa
1
1
Department of Medical Laboratory, Taichung Veterans General Hospital,
Taichung 40705, Taiwan
2
Department of Medical Technology, Central Taiwan University of Science and Technology,
Taichung 40601, Taiwan
A slight increase in urinary albumin level could be an advanced indicator of early stage nephropa-
thy. The aim of this study was to detect the presence of albumin adjusted by creatinine using a dip-
stick to test urine samples from 301 patients with nephropathy. The dipstick method can also be
used to calculate the protein-to-creatinine ratio (P/C ratio) as a valuable index for eliminating false
positive or false negative protein reports, which are often caused by abnormal sample concentration
variation. The results showed the test data for the dipstick show good agreement with the quantita-
tive measurements of protein, creatinine and P/C ratio. The P/C ratio obtained by the dipstick
showed a sensitivity of 98% compared with the protein-only results of 80% in diagnosis of clinical
proteinuria/albuminuria. In conclusion, the dipstick is a reliable and practical screening tool for
diagnosing the early stage of nephropathy.
Key words: Creatinine, protein/creatinine ratio, Clinitek Atlas PRO 12 Reagent Pak
Introduction
samples. One of the main factors in variation lies in the
concentrated or diluted samples that lead to urine over-
estimation or underestimation. In order to solve this
problem, 24-hour samples can be used to reduce the
variation in the urine concentration [7]. However, in-
convenience is encountered and re-testing increases the
cost.
It has recently been shown that even small increases in
urinary protein or albumin excretion are early predictors
of kidney failure and end-stage renal disease(ESRD)
[1,2]. Early intervention has been demonstrated to retard
the development or rate of progression of renal disease.
Patients with diabetes and/or hypertension are the pri-
mary risk groups [3-5]. Proteinuria is a major symptom
of renal diseases and the excretive amounts of protein in
the urine could be an index for evaluating the pathologi-
cal stage of renal diseases [6]. Current urinary protein
testing is performed by using a urine dipstick in routine
urine examinations. The principle behind the reaction is
the “protein error of indicators”. Under a fixed pH, indi-
cators will change color when protein exists, and the
concentration of urinary protein can be calculated based
on the difference of color. However, the test results of
urinary protein are often affected by variation in urine
Previous studies had revealed urinary creatinine can
be used to correct the variation occurring in concentration
of random urine samples because the daily excretion of
creatinine is consistent [8-11]. The protein-to-creatinine
ratio (P/C ratio) can be used to correct the false positive
or false negative protein results caused by the variation
in urine concentration. Calculation of the P/C ratio may
be a more objective method for estimating urinary pro-
tein excretion. However, most previous studies used
quantitative methods to calculate the P/C ratio. The
novel urine dipstick, Clinitek Atlas PRO12 Reagent Pak
(PRO12), has the added features of protein-low and
creatinine test pads. The protein-low test pad has im-
proved specificity to albumin and the sensitivity has
Received: December 9, 2008 Revised: March 9, 2009 Accepted: April 6, 2009
Address for correspondence: Jiunn-Min Wang, Department of Medical Laboratory, Taichung Veterans General Hospital,
No. 160, Sec. 3, Chung-Kang Rd., Taichung 40705 Taiwan, R.O.C.
TEL:+886423592525, FAX:+886423741267, E-mail: jmwang@vghtc.gov.tw
J Biomed Lab Sci 2009 Vol 21 No 1
23
 Test Dipstick for Urinary Protein, Creatinine and P/C Ratio
been increased from 15mg/dL to 8mg/dL, which also
leads to the display of “trace” results which are replaced
by the indication “15mg/dL”. Creatinine can also be
used to calculate the P/C ratio.
In this study, we examined urine samples from the
outpatient department and evaluated the comparability of
protein, creatinine, and P/C ratio data obtained from the
PRO12 dipstick using quantitative chemical methods.
The results showed that the novel dipstick can eliminate
false negative and false positive reports and provide
more accurate and objective data for clinical physicians
in screening and diagnosis of renal disease.
is automatically calculated by the analyzer based on the
light absorbent readings of the protein and creatinine test
pads.
Results were analyzed by computing the percentage
of same level agreement,
＋
/
－
one-level agreement, sen-
sitivity, specificity and Cohen
’
s Kappa coefficient.
Results
The correlation of the protein test results between the
dipsticks of the Roche Urisys 2400 and the quantitative
methods was shown in Table 1. The rate of complete
coincidence was 77.7%. Rates of 77.42%, 94.92%, and
87.71% were observed for the agreement rate with the
cutoff point between negative and positive results being
“trace” for the dipsticks and “25 mg/dl” for the quantita-
tive methods.
The correlation of the protein test results between
the PRO12 dipsticks and the quantitative methods was
shown in Table 2. The rate of complete coincidence was
69.44%. Rates of 93.88%, 78.57%, and 86.05% were
observed for the agreement rate with the cutoff point
between negative and positive results being “15 mg/dL”
for the Pro12 test strips and “8 mg/dL” for the quantita-
tive methods. The sensitivity of the PRO12 dipsticks, but
the specificity showed a minor decline.
The correlation of the creatinine test results between
the PRO12 dipsticks and the quantitative methods was
shown in Table 3. The rate of complete coincidence was
52.16%. Agreement was 95.68%, if we extended the
comparison range to +/- one level.
Table 4 shows the correlation of the P/C ratio results
between the PRO12 dipsticks and the quantitative
methods. Only 286 samples were compared with the P/C
ratio results of the quantitative methods. (There were 39
over-diluted samples, which were re-collected.) The rate
of complete coincidence was 67.44%. Rates of 98.64%,
76.98%, and 88.11% were observed for the agreement
Materials and Methods
We collected 301 random urine samples from the outpa-
tient department of Taichung Veterans General Hospital,
Taichung, Taiwan. All samples were examined by two
models of automatic urine chemistry analyzers (Roche
Urisys 2400 with the dipsticks,Urisys 2400 cassette and
Siemens/Bayer Clinitek Atlas with the dipsticks, Clinitek
Atlas PRO12 Reagent Pak). The following quantitative
methods were also performed by a Hitachi chemistry
analyzer for each urinary sample: total protein concen-
tration in urine using the total protein dye-binding assay
(pyrogallol red), urinary albumin concentration using the
immuno-turbidimetric method, and urinary creatinine
concentration using Jaffe’s method.
The Clinitek Atlas PRO12 Reagent Pak is enhanced
with a new protein-low pad which uses bis(3’,3”-diiodo-
4’,4”-dihydroxy-5’,5”-dinitrophenyl)-3,4,5,6-tetrabromo
-
sulfonephthalein (DIDNTB) dye for the detection of
albumin at
>
8 mg/dL. Also present on the dipstick is an
original pad (protein-high pad) that is based on the pro-
tein error of indicator method (tetrabromophenol blue
(TBPB)) for detection of protein at
>
30 mg/dl, and a
pad for detection of creatinine that uses the peroxidase
activity of the copper-creatinine complex. The P/C ratio
Table 1.
Correlation between the results of the quantitative method and the protein test portion of the Roche Urisys
2400 with the dipsticks.
Total protein concentration in urine (mg/dL)
<25
25-49.9
50-112.4
112.5-325
>=325
Neg*
25*
75*
150*
500*
168
9
0
0
0
23
29
4
0
0
4
9
32
3
0
1
2
5
3
4
0
0
1
2
2
*: Roche Urisys 2400 with the dipsticks (mg/dL); Neg, negative result.
24
J Biomed Lab Sci 2009 Vol 21 No 1
Table 2.
Correlation between the results of the quantitative methods and the protein test portion of the PRO12
dipsticks.
Urinary albumin conc. (mg/dL)
Urinary total protein conc. (mg/dL)
<8
8-29.9
30-64.9
65-199.9
≥
200
Neg*
15*
30*
100*
≥
121
24
9
0
0
9
10
18
0
0
0
0
40
24
0
0
0
1
31
4
0
0
0
3
7
*: PRO12 test pad (mg/dL); Neg, negative result.
Table 3.
Correlation between the results obtained from the quantitative methods and the creatinine test portion of
the test pad.
Urinary creatinine concentration (mg/dL)
<25
25-74.9
75-149.9
150-249.9
≥
250
Neg*
50*
100*
200*
≥
9
1
1
0
1
27
39
7
1
0
3
30
63
12
6
0
1
31
38
5
0
0
0
18
8
*: PRO12 test pad, protein test for creatinine (mg/dL).
T
able 4.
Correlation of the P/C ratio between the results from the quantitative method and the PRO12 dipsticks.
P:C(quantification) (mg/gCr)
Normal dilution
<80
80-299.9
300-499.9
≥
500
Normal dilution*
Normal*
150*
300*
>500*
0
14
107
26
6
0
1
2
28
20
4
0
0
4
12
13
0
0
0
8
56
*: Roche Urisys 2400 with the dipsticks (mg/dL); Neg, negative result.
Table 5.
Comparison of protein test results from the conventional dipsticks and the P/C ratio from the quantitative
methods.
Negative (<80)
Positive (
≥
80)
Negative * (-)
Positive * (
≥
25)
156
11
40
94
*: Roche Urisys 2400 with the dipsticks (mg/dL); Neg, negative result.
rate with the cutoff point between negative and positive
results being “150 mg/gCr” for the Pro12 dipsticks and
“80 mg/gCr” for the quantitative methods.
Table 5 shows the correlation between the protein
test results obtained with the conventional dipsticks and
the P/C ratio results obtained with the quantitative
methods. Rates of 70.15% and 93.41% were observed
for the agreement rate with the cutoff point between
negative and positive results being
“>
25 mg/dL” for the
Roche test strips and “80 mg/gCr” for the quantitative
methods.
J Biomed Lab Sci 2009 Vol 21 No 1
25
300*
300*
Test Dipstick for Urinary Protein, Creatinine and P/C Ratio
Discussion
all correlation showed that the new creatinine test pad is
reliable.
In comparison with the conventional dipsticks, the
PRO12 dipsticks can provide both more sensitive protein
and P/C ratio results for evaluating clinical proteinuria.
Furthermore, the P/C ratio can act as an indicator to de-
tect over-diluted samples which have less than 10 mg/dL
of urinary creatinine. When negative protein results are
found in over-diluted samples, the samples should be
re-collected and re-examined to ensure accurate results.
About 10% of routine urine screening samples were di-
luted samples. That also means without the P/C ratio, the
diagnosis of proteinuria will be underestimated because
of false negative results. In this study, we ruled out the
diluted samples and only compared the P/C ratio data of
the other 286 samples between the PRO12 dipsticks and
the quantitative methods.
Microalbuminuria has been defined as a urinary al-
bumin-to-creatinine ratio (A/C ratio) in the range of
30~300 mg/gCr. Many studies have revealed that micro-
albuminuria can be an early warning sign of renal dis-
eases. The high-risk population
includes hypertensive
and diabetic patients, so we suggest regular monitoring
of urinary microalbumin by using the A/C ratio data. In
this study, we used 80 mg/gCr as the cutoff point for
comparisons with the quantitative results [13]. The re-
sults showed that the coincidence rate was 88.11%. The
false negative rate was only 1.3%, which indicated the
P/C ratio could decrease the chance of false negatives.
We also compared the results with those obtained with
the Roche Urisys 2400 conventional dipsticks, the
original method we used for routine urine tests. The sen-
sitivity was 98.64% and specificity was 76.98%, which
means 30% of early nephropathy cases could potentially
be misdiagnosed due to negative protein reports. The
data shows the P/C ratio of PRO12 could be an effective
diagnostic tool for the monitoring of early stage renal
diseases, especially for diabetes, hypertension and eld-
erly health screening.
Since conventional dipsticks have sensitivity and
specificity limitations, the urinary protein results could
be false positive or false negative. If the false results are
not detected, the consequences can be serious due to
delays in diagnosis and treatment. However, it is costly
to retest false negative data. The results of this study
show that the new urine dipsticks, which have the added
features of the creatinine test and P/C ratio, increase both
the sensitivity and specificity of the protein test. These
improvements could effectively eliminate most false
negative results. We also found that using both the pro-
tein and P/C ratio results to evaluate clinical proteinuria
In Taiwan, there are more than 46,000 dialysis patients
and the number is increasing. Urine protein examination
is the first step in screening for renal diseases. However,
the urine dipstick currently used is semi-quantitative.
Also, compared with the quantitative method the dip-
stick has sensitivity limitations (about 15-30 mg/dL).
The urinary protein results can also be affected by varia-
tions in sample concentrations. Thus, physicians cannot
distinguish between pathological proteinuria and false
positives caused by concentrated samples when the uri-
nary protein report shows “trace”. Many studies have
shown measurement of urinary creatinine could be a
good way to correct the variation in sample concentra-
tion. The protein-to-creatinine ratio (P/C ratio) can be
used to correct the false protein results caused by the
physiological differences in urine concentration. Calcu-
lating the P/C ratio is a more objective method for as-
sessing urinary protein excretion.
In this study, we evaluated the application of the
PRO12 dipstick [12]. The PRO12 is enhanced with a
protein-low test pad, which has higher specificity and
affinity for detecting lower concentrations of albumin.
The protein-low pad has improved specificity to urinary
albumin and the sensitivity has been increased to 8~15
mg/dL. The original protein test pad on the PRO12 dip-
stick is named the “protein-high pad” and it detects the
concentration of total urinary protein. The PRO12 test
strips have an added feature, a creatinine test, that can
detect urinary creatinine and it also acts as a corrective
factor for urine concentration variations. We compared
both the conventional dipsticks and the PRO12 with the
quantitative methods. The sensitivity of the PRO12 for
urinary protein tests was 93.88%, but the sensitivity of
the conventional dipsticks was only 77.42%. Further-
more, the complete coincidence rate of PRO12 urinary
protein tests reached 86.05%.
We also compared urinary creatinine results be-
tween the PRO12 dipsticks and the quantitative methods.
The complete coincidence rate was 52.16%. By extend-
ing the comparison range to one upper or one lower re-
port unit, the overall coincidence rate reached 95.68%.
The principle of the creatinine test in the PRO12 dip-
sticks is based on the peroxidase-like activity of the
copper creatinine complex, and vitamin C or blood cells
in urine samples can interfere with the reaction. This
could be the reason why we did not get a high correla-
tion using the quantitative methods. However, the over-
26
J Biomed Lab Sci 2009 Vol 21 No 1
was better than using the protein-only results. If we use
the routine urine tests to identify patients with potential
early stage renal diseases, treatment could lower the
chances of serious nephropathy. With the current preva-
lence of renal diseases increasing, the PRO12 test strip
could be used as a reliable and effective tool for early
screening.
According to recent data, the population with dia-
betic nephropathy is expanding year by year and is also
the most rapidly growing disease population preceding
end-stage renal disease( ESRD). In Taiwan, the medical
costs for dialysis, which is often required for end-stage
renal disease (ESRD) patients, have reached almost 28
billion New Taiwan Dollars (NT$) per year. The new
dipstick is convenient, rapid, accurate, reliable and eco-
nomical. It can effectively help prevent chronic renal
diseases by early detection and also save medical re-
sources.
In conclusion, we suggest that physicians use the
P/C ratio or A/C ratio to monitor the potential risks of
renal diseases in outpatients, hypertensive, and diabetic
patients. The P/C ratio data, which is easily obtained by
dipsticks, should be included in the routine urine data
and could improve the early diagnosis and monitoring of
renal diseases. Decelerating the prevalence of end-stage
renal disease (ESRD) could effectively relieve the bur-
den on the national health insurance program in Taiwan.
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