Assessment of a Veterinary Dipstick for Determination of Urine Protein/Creatinine Ratio in Canines

Introduction

Testing for the presence of proteinuria is commonly performed to screen for glomerular disease. Historically, measuring glomerular protein loss involved collecting urine for 24 hr and quantifying the amount of protein excreted. That process was necessary to avoid the confounding effects of urine concentration on measurement.¹ More recently, it has been shown that the urine protein/creatinine ratio (UPC) provides an accurate and convenient assessment of glomerular protein loss.²

Urine dipsticks can provide an effective in-office initial screening procedure for proteinuria. Once protein is detected, urine can be sent to an outside reference laboratory for microscopic examination so that WBCs, epithelial cells, bacteria, and casts can be eliminated as possible protein sources. Then actual protein and creatinine can be determined. Early detection of glomerular protein loss is important because studies have demonstrated that elevations in the UPC are associated with significant morbidity and mortality; however, treatment can help slow the progression of renal disease and prolong survival.^3–5 In addition, because glomerular protein loss can be secondary to an underlying disease process, an elevated UPC can prompt the clinician to search for a compatible condition.⁶

An in-house urine dipstick that could accurately detect proteinuria as well as the UPC would greatly benefit practitioners as well as patients. A previous study has indicated that dipsticks intended for measuring UPC in people are not highly accurate in canines.⁷ Recently, a veterinary urine dipstick^a that measures urine protein and creatinine has been introduced. The goal of this study was to determine if that dipstick could provide a reasonable estimation of urine protein and creatinine measurements compared with a veterinary reference laboratory. It is the authors’ hypothesis that the urine protein and creatinine determined from the dipsticks will be highly correlated and in good agreement with those obtained at a diagnostic laboratory.

Materials and Methods

Dogs that presented to the Louisiana State University School of Veterinary Medicine Teaching Hospital between August 2010 and June 2011 and were determined by a clinician to require a UPC because of proteinuria detected on urinalysis were candidates to be included in the study. No attempt was made to screen participants on the basis of sex, age, breed, or degree of illness. Exclusion criteria were dogs with urine containing WBCs, epithelial cells, bacteria, casts, or significant blood contamination because those can artificially increase the UPC.^8,9 Signed permission was obtained from all owners prior to enrollment in this study and the Louisiana State University clinical protocol and Institutional Animal Care and Use Committees approved this research.

Either a veterinarian or a qualified technician obtained the urine samples by cystocentesis. Urine was collected using a 6 mL syringe^b and a 22 gauge 1.5 inch needle^c. Once urine was collected, 1 mL was placed in a red top tube^d and immediately submitted to a veterinary clinical pathology laboratory^e for analysis, while the remainder was used for dipstick testing. Urine was applied to the dipsticks that were then handed to the two reviewers who recorded their observations. Each reviewer was blinded to the other’s interpretation.

An automated chemistry analyzer^f was used to measure all urinary creatinine and protein concentrations. The analyzer determined urine creatinine based on a modified Jaffe procedure in which creatinine reacts with alkaline picrate resulting in the formation of a red color, which is measured at 505 nm.¹⁰ Urine protein was measured using a colorimetric method in which pyrogallol red is combined with molybdate and the color changes are directly proportional to the protein concentration in the urine.¹¹

The UPC dipstick utilizes the Benedict Behr reaction to measure urine creatinine. In that test, creatinine reacts with 3,5-dinitrobenzene and is enzymatically degraded. That process liberates hydrogen peroxide, which subsequently reacts with a color indicator.¹² As the quantity of creatinine in the urine increases, so does the amount of hydrogen peroxide liberated to drive the color change. Measurement of urine protein is based on the protein error of pH indicator test. That test is dependent on the ability of the negatively charged protein amino groups to bind to and change the color of acid-base indicators. This reaction is highly sensitive for albumin; however, it is relatively insensitive to globulins and Bence-Jones proteins.¹³

Results

Thirty-one dogs were included in the study, including 14 males. Ages ranged from 2 to 15 yr, with a median age of 10 yr. There were six Labrador retrievers, three golden retrievers, three Shetland sheepdogs, five mixed-breed dogs, and one of each of the following breeds: beagle, boxer, Cavalier King Charles spaniel, Chinese crested, dachshund, Jack Russell terrier, miniature schnauzer, Pekapoo, Pit bull terrier, rat terrier, Siberian husky, West Highland white terrier, Welsh terrier, and a Yorkshire terrier.

A statistically significant, but low correlation, was found between each reviewer’s dipstick interpretation and chemistry analyzer’s measurement (τ = 0.37 and 0.31, respectively; P < .05). A statically significant but slight agreement was found between each reviewer’s dipstick interpretation and chemistry analyzer’s measurement (κ = 0.35 and 0.26, respectively; P < .05). The agreement between the two reviewers was substantial (κ = 0.77; P < .01); however, in three cases, there was significant disagreement between the reviewers.

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

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

*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%.

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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 was independent of the degree of proteinuria despite substantial 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 for accurate determination the UPC. Larger studies should be performed before this test is clinically used.