ORIGINAL STUDIES

Assessment of a Veterinary Dipstick for Determination of Urine Protein/Creatinine Ratio in Canines Christina Mamone, DVM*, Mark Mitchell, MS, PhD, DVM, Hugues Beaufrere, PhD, Dr.med.vet.y, Mark Acierno, DVM, DACVIM (Small Animal Internal Medicine)

ABSTRACT The urine protein/creatinine ratio (UPC) is a widely used screening test of glomerular barrier function. It usually requires sending urine samples to a distant laboratory, which is costly and time consuming. A point-of-care urine dipstick that could accurately measure UPC would increase test utility and utilization. The objective of this study was to determine if there was good correlation and agreement between UPC as measured by a dipstick and a laboratory analyzer. Thirty-one canines undergoing UPC measurement as part of a diagnostic workup were included in the study. A total of 2 mL of urine was collected via cystocentesis. Urine was applied to the dipstick then presented to two blinded reviewers to analyze. The remaining urine was submitted to a clinical pathology laboratory for analysis. UPC data from the dipsticks and the analyzer was converted into an ordinal scale. Correlation and agreement between the individual reviews and the analyzer was calculated. Agreement between reviewers was substantial; however, correlation and agreement between the individual reviews and the analyzer was low. On the basis of this information, use of this veterinary urinary dipstick to determine UPC cannot be recommended at this time. (J Am Anim Hosp Assoc 2014; 50:e11–e14. DOI 10.5326/JAAHA-MS-6128)

Introduction

protein and creatinine can be determined. Early detection of

Testing for the presence of proteinuria is commonly performed to

glomerular protein loss is important because studies have dem-

screen for glomerular disease. Historically, measuring glomerular

onstrated that elevations in the UPC are associated with significant

protein loss involved collecting urine for 24 hr and quantifying the

morbidity and mortality; however, treatment can help slow the

amount of protein excreted. That process was necessary to avoid

progression of renal disease and prolong survival.3–5 In addition,

1

the confounding effects of urine concentration on measurement.

because glomerular protein loss can be secondary to an under-

More recently, it has been shown that the urine protein/creatinine

lying disease process, an elevated UPC can prompt the clinician to

ratio (UPC) provides an accurate and convenient assessment of

search for a compatible condition.6

glomerular protein loss.2

An in-house urine dipstick that could accurately detect

Urine dipsticks can provide an effective in-office initial

proteinuria as well as the UPC would greatly benefit practitioners

screening procedure for proteinuria. Once protein is detected,

as well as patients. A previous study has indicated that dipsticks

urine can be sent to an outside reference laboratory for micro-

intended for measuring UPC in people are not highly accurate

scopic examination so that WBCs, epithelial cells, bacteria, and

in canines.7 Recently, a veterinary urine dipsticka that measures

casts can be eliminated as possible protein sources. Then actual

urine protein and creatinine has been introduced. The goal of this

From the Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Baton Rouge, LA (C.M., H.B., M.A.), and Department of Companion and Zoological Animal Health, Small Animal Clinic, Urbana, IL (M.M.).

UPC, urine protein/creatinine ratio

Correspondence: [email protected] (M.A.)

ª 2014 by American Animal Hospital Association

*C. Mamone’s present affiliation is Tampa Bay Veterinary Emergency Service, Tampa, FL. †

H. Beaufrere’s present affiliation is Health Sciences Centre, University of

Guelph, Ontario Veterinary College, Ontario, Canada.

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study was to determine if that dipstick could provide a reasonable

the negatively charged protein amino groups to bind to and

estimation of urine protein and creatinine measurements compared

change the color of acid-base indicators. This reaction is highly

with a veterinary reference laboratory. It is the authors’ hypothesis

sensitive for albumin; however, it is relatively insensitive to glob-

that the urine protein and creatinine determined from the dipsticks

ulins and Bence-Jones proteins.13

will be highly correlated and in good agreement with those ob-

Statistical Analysis

tained at a diagnostic laboratory.

Values of the UPC obtained with the urinary test strip and the

Materials and Methods

laboratory analyzer were converted to an ordinal scale (range, 1–4).

Dogs that presented to the Louisiana State University School

A score of 1 was assigned if UPC # 0.5, a score of 2 was assigned

of Veterinary Medicine Teaching Hospital between August 2010

if 0.5 , UPC # 1, a score of 3 was assigned if 1 , UPC # 2, and

and June 2011 and were determined by a clinician to require

a score of 4 was assigned if UPC . 2. The UPC was determined

a UPC because of proteinuria detected on urinalysis were can-

by dividing the dipstick urine protein by the creatinine. The cor-

didates to be included in the study. No attempt was made to

relation between the urinary test strip of both reviewers and the

screen participants on the basis of sex, age, breed, or degree of

laboratory measure was measured using a Kendall’s rank coeffi-

illness. Exclusion criteria were dogs with urine containing WBCs,

cient. The agreement between the two reviewers and each reviewer

epithelial cells, bacteria, casts, or significant blood contamination

and the laboratory was measured using a weighted k coefficient

Signed per-

using linear weights. k values were interpreted as follows: #0, poor

mission was obtained from all owners prior to enrollment in this

agreement; 0.01–0.2, slight agreement; 0.21–0.4, fair agreement;

study and the Louisiana State University clinical protocol and

0.41–0.6, moderate agreement; 0.61–0.8, substantial agreement;

Institutional Animal Care and Use Committees approved this

and 0.81–1, almost perfect agreement.14

because those can artificially increase the UPC.

8,9

research.

A model approach was also used to further investigate the

Either a veterinarian or a qualified technician obtained the

agreement. The absolute bias was calculated as the absolute value of

urine samples by cystocentesis. Urine was collected using a 6 mL

the laboratory value minus the dipstick value. This bias was

syringeb and a 22 gauge 1.5 inch needlec. Once urine was collected,

modeled using an intercept only ordinal logit model. The labo-

d

1 mL was placed in a red top tube and immediately submitted to

ratory value was added to the model to test whether it influenced

a veterinary clinical pathology laboratorye for analysis, while the

the magnitude of the bias. Rg was used for statistical analysis and

remainder was used for dipstick testing. Urine was applied to the

the R-package “VGAM” for ordinal logit model.15,16 An a of 0.05

dipsticks that were then handed to the two reviewers who

was used for statistical significance.

recorded their observations. Each reviewer was blinded to the

Results

other’s interpretation. f

An automated chemistry analyzer was used to measure all

Thirty-one dogs were included in the study, including 14 males.

urinary creatinine and protein concentrations. The analyzer de-

Ages ranged from 2 to 15 yr, with a median age of 10 yr. There were

termined urine creatinine based on a modified Jaffe procedure in

six Labrador retrievers, three golden retrievers, three Shetland

which creatinine reacts with alkaline picrate resulting in the

sheepdogs, five mixed-breed dogs, and one of each of the following

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formation of a red color, which is measured at 505 nm. Urine

breeds: beagle, boxer, Cavalier King Charles spaniel, Chinese crested,

protein was measured using a colorimetric method in which

dachshund, Jack Russell terrier, miniature schnauzer, Pekapoo, Pit

pyrogallol red is combined with molybdate and the color

bull terrier, rat terrier, Siberian husky, West Highland white terrier,

changes are directly proportional to the protein concentration in

Welsh terrier, and a Yorkshire terrier.

the urine.

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A statistically significant, but low correlation, was found

The UPC dipstick utilizes the Benedict Behr reaction to

between each reviewer’s dipstick interpretation and chemistry ana-

measure urine creatinine. In that test, creatinine reacts with 3,5-

lyzer’s measurement (t ¼ 0.37 and 0.31, respectively; P , .05). A

dinitrobenzene and is enzymatically degraded. That process lib-

statically significant but slight agreement was found between each

erates hydrogen peroxide, which subsequently reacts with a color

reviewer’s dipstick interpretation and chemistry analyzer’s mea-

indicator. As the quantity of creatinine in the urine increases, so

surement (k ¼ 0.35 and 0.26, respectively; P , .05). The agreement

does the amount of hydrogen peroxide liberated to drive the color

between the two reviewers was substantial (k ¼ 0.77; P , .01);

change. Measurement of urine protein is based on the protein

however, in three cases, there was significant disagreement between

error of pH indicator test. That test is dependent on the ability of

the reviewers.

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Determination of Urine Protein/Creatinine Ratio via Dipstick

was independent of the degree of proteinuria despite substantial

TABLE 1 Probability of Observing Bias when Using the Urinary Strip Test* Probability

95% Confidence Interval

No. of biases Bias of at least 1

0.44 0.56

0.32–0.56 0.44–0.68

Bias of at least 2

0.26

0.17–0.38

Bias of at least 3

0.15

0.08–0.26

*Bias was defined as the difference between the reviewers’ observations after conversion to an ordinal scale; therefore, the probability of the reviewers’ observations differing by $2 units on the scale is 26%.

agreement between the two reviewers.

Conclusion Early detection of glomerular protein loss has been shown to be important, as studies have demonstrated that elevations in UPC are associated with significant morbidity and mortality.3 An in-house urine dipstick that could accurately measure proteinuria as well as the UPC would greatly benefit practitioners as well as patients; nevertheless, on the basis of results presented in this study, use of this newly developed veterinary dipstick cannot be recommended

Using the ordinal logit model, the probability of having different degrees of bias was computed (Table 1). There was no

for accurate determination the UPC. Larger studies should be performed before this test is clinically used.

observable trend in the direction of the bias (negative or positive). That was further confirmed by fitting a quasi-symmetry log-linear model to the data. The laboratory value of the UPC did not significantly influence the degree of bias.

Discussion A urine dipstick that accurately measures urine protein and creatinine could provide an effective in-office diagnostic test for proteinuria. In this study, two reviewers used a dipstick to determine UPC in canine patients and compared the results to the UPCs measured by a veterinary clinical pathology laboratory. Furthermore, there was a high probability of having a large and clinically significant bias when using the urinary dipstick. Only dogs diagnosed with relatively high UPC ratios on the urinary dipstick (UPC . 4) would have a high probability of having a truly abnormal UPC (Table 1). With any colorimetric reaction, human visual perception and interpretation could be a significant source of variation. For this reason, automated readers exist for most commercially available urine dipsticks; however, no such reader exists for this product. Nevertheless, in this study, statistical analysis demonstrated that there was substantial agreement between the two blinded reviewers. In addition, the goal of this study was to examine the performance of this urine dipstick in a clinical setting. One shortcoming of this study is that the authors attempted to compare continuous UPC data provided by the laboratory to the relatively limited ordinal data obtained by the dipstick. However, because the urinary dipstick is marketed to estimate the UPC in dogs, the study authors suggest that the study methodology is still justified. To compensate for this, all UPC ratios from both methods were converted into four ordinal data groups as described in the methods section. Even with that limited scale, correlation and agreement failed to achieve moderate levels of agreement or correlation. The lack of agreement and correlation

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