Test Dipstick for Determination of Urinary Protein, Creatinine.pdf

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

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

*: Roche Urisys 2400 with the dipsticks (mg/dL); Neg, negative result.

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)

8-29.9

30-64.9

65-199.9

≥

200

Neg*

15*

30*

100*

≥

121

*: 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*

≥

*: 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*

107

*: 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

*: 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

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-

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.

3. Sarafidis PA, Lasaridis AN, Nilsson PM, et al: The effect

of rosiglitazone on urine albumin excretion in patients

with type 2 diabetes mellitus and hypertension. Am J

Hypertens 2005;18:227-34.

4. Couchoud C, Pozet N, Labeeuw M, et al: Screening

early renal failure:cut-off values for serum creatinine as an

indicator of renal impairment. Kidney Int 1999;55:1878-84.

5. Leung YY, Szeto CC, Tam LS, et al: Urine protein-to-

creatine ratio in an untimed urine collection is a reliable

measure of proteinuria in Lupus nephritis. Rheumatol-

ogy 2007;46(4):649-52.

6. Artunc FH, Fischer IU, Risler T, et al: Improved estima-

tion of GFR by serum cystatin C in patients undergoing

cardiac catheterization. Int J Cardiol 2005;102(2):173-8.

7. Wann S and Kuo TS: The Correlation Between 24 - Hour

- Urine Protein and Spot Urine Protein/Creatinine Ration

and the Inconstancy of Daily Urine Protein and Creatinine

Excretion. TZU-Chi Med J 1992;4:100-5.

8. Al RA, Baykal C, Karacay O, et al: Random Urine Pro-

tein-Creatinine Ratio to Predict Proteinuria in New-Onset

Mild Hypertension in Late Pregnancy. Obstet Gynecol

2004;104:367-71.

9. Waugh J, Bell SC, Kilby MD, et al: Urinary microalbu-

min/creatinine ratios: reference range in uncomplicated

pregnancy. Clin Sci 2003;104(2):103-7.

10. Wallace JF, Pugia MJ, Lott JA, et al: Mutisite Evaluation

of a New Dipstick for Albumin, Protein, and Creatinine. J

Clin Lab Anal 2001;15:231-5.

11. Pugia MJ, Wallace JF, Lott JA, et al: Albuminuria and

Proteinuria in Hospitalized Patients as Measuued by

Quantitative and Dispstick Methods. J Clin Lab Anal

2001;15:295-300.

12. Watanabe M, Funabiki K, Tsuge T, et al: Using Protein/

Creatinine Ratios in Random Urine. J Clin Lab Anal

2005;19:160-6.

13. Shihabi ZK, Sheehan M, Hinsdale M, et al: Clinical

Evaluation of a New Test for Proteinuria on the Clinitek

Atlas R System. AACC Poster No. 511, presented at

AACC/CSCC 2001 National Meting, Chicago, IL, August

2,2001.

References

1. Birmingham DJ, Rovin BH, Shidham G, et al: Spot urine

protein/creatinine ratios are unreliable estimates nephri-

tis flares. Kidney Int 2007;72(7):865-70.

2. Ruggenenti P, Perna A, Mosconi L, et al:Urinary protein

excretion rate is the best independent predictor of ESRF

in non-duabetic proteinuric chronic nephropathies. Kid-

ney Int 1998;53:1209-16.

J Biomed Lab Sci 2009 Vol 21 No 1

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika: