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Issue number 29.2 Other Scientific

Urinalysis: what can go wrong?

Published 03/10/2019

Written by Paola Scarpa

Also available in Français , Deutsch , Italiano , Español and ภาษาไทย

Although urinalysis is a routine and everyday test for all small animal practices, there are various potential pitfalls that can markedly affect the reliability of the results obtained, as Paola Scarpa explains.

Urinalysis: what can go wrong?

Key Points

Dipsticks are inexpensive and – assuming correct use – offer a simple qualitative and semi-quantitative test for urinalysis.


A urine protein to creatinine ratio may be indicated if a sample is proteinuric, although care must be taken when interpreting the results.


Introduction

Urinalysis is one of the most common and easiest tests performed in small animal practice, but a surprising number of factors can lead to potential inaccuracies in the results. This short paper identifies some common pitfalls and offers tips on how to achieve best practice for urinalysis.

Urine sample collection 

Cystocentesis, preferably via ultrasound, allows an uncontaminated urine sample to be obtained.
Figure 1. Cystocentesis, preferably via ultrasound, allows an uncontaminated urine sample to be obtained. © Paola Scarpa

Urine collection must be carried out by an appropriate method. Cystocentesis, preferably guided by ultrasound (Figure 1), is necessary whenever urine culture is required, as it can allow an uncontaminated sample to be obtained. The needle is inserted into the ventral or ventrolateral wall of the bladder at a 45° angle; this allows the muscle fibers of the bladder to rapidly close the hole caused by withdrawal of the needle. The patient must be immobile and calm, usually in lateral or dorsal recumbency, and the sampling area is generally shaved and disinfected prior to inserting the needle. Iatrogenic microhematuria often occurs following the procedure. Cystocentesis is contraindicated if the bladder is empty, if the patient is non-compliant, or if pyoderma is present. Note that metastatic dissemination along the needle has been reported in a case of transitional cell carcinoma of the bladder 1.

 
Spontaneous urination is the least traumatic method of collection, although it is not always easy, and samples must be collected in suitably sterile containers.
Figure 2. Spontaneous urination is the least traumatic method of collection, although it is not always easy, and samples must be collected in suitably sterile containers. © Paola Scarpa

Spontaneous urination (Figure 2) is the least traumatic method of collection, although it is not always easy (e.g., in small dogs) or even possible (with cats). Only samples collected in suitable sterile containers should be accepted for analysis. Detergents and disinfectants used to clean containers and litter trays may alter the results of the dipstick test.

A spontaneous urination sample is generally suitable for a “standard” (i.e., first screening) urine test, although note that the sample may be contaminated by prostatic fluid, sperm or debris from the urethra and prepuce. With this caveat, the determination of dipstick proteinuria and UPC ratio is not influenced by the method of collection, so analysis of this type of sample may be satisfactory for diagnosing and monitoring a protein-losing nephropathy. Furthermore, the UPC in cats is not affected by the sampling method (e.g., cystocentesis or by manual compression of the bladder).

Proteinuria 

Urinary dipsticks are inexpensive and offer a simple qualitative and semi-quantitative test.
Figure 3. Urinary dipsticks are inexpensive and offer a simple qualitative and semi-quantitative test. © Ewan McNeill

An initial evaluation of proteinuria can be made with a dipstick (Figure 3). The protein pad is impregnated with indicators (e.g., tetrabromophenol blue) that interact with the amine groups of urinary proteins; the resulting color change (from yellow to green and finally to blue) allows a numerical interpretation of the degree of proteinuria, usually assessed from 0-4. This method is sensitive for detection of albumin, but less sensitive for globulins or protein fractions (e.g., Bence-Jones proteins), which typically have lower levels of amine groups.

Various factors, including an alkaline pH, presence of hemoglobinuria, pyuria or bacteriuria, or the use of quaternary ammonium or chlorhexidine disinfectants when collecting samples, can result in false positives.

In dogs, interpretation of the dipstick results along with the urine specific gravity (USG) may indicate when it is necessary to evaluate the UPC ratio (Table 1). Dipstick negative subjects may be considered non-proteinuric, while subjects with a USG < 1.012 and 1+ on the dipstick test should be considered proteinuric. Subjects with 2+ on dipstick are definitely proteinuric. 

USG Protein level = 0 Protein level 1+ Protein level 2+
< 1.012 Non-proteinuric Likely proteinuric
Perform UPC
Proteinuric
Perform UPC
> 1.012 –
< 1.030
Non-proteinuric Non-proteinuric Proteinuric
Perform UPC
> 1.030 Non-proteinuric Non-proteinuric Proteinuric
Perform UPC

Table 1. Assessing the USG and protein dipstick result can help determine if a UPC ratio should be obtained 2

The UPC ratio

Values above 0.4 in cats and 0.5 in dogs are indicative of renal proteinuria. However, in order to correctly interpret the test result it is important to be aware of the possible biological and analytical variables, as follows:

Daily variability

To have a reliable estimate of proteinuria, it is generally necessary to determine the UPC ratio over several consecutive days and to calculate the mean value. Alternatively, the UPC ratio may be determined from a pooled sample taken over three consecutive days. Since the ratio can vary considerably over several days, when assessing UPC on an ongoing basis, two serial samples can only be considered significantly different if the variance is ~80% for low UPC values (i.e., around 0.5) and ~35% for high values (i.e., around 12). One measurement is adequate to reliably estimate the UPC when UPC < 4, but 2-5 determinations are necessary at higher UPC values (Table 2). 

UPC (baseline) UPC definitely decreased UPC definitely increased Number of samples for reliable quantitation of proteinuria
0.5 < 0.1 > 0.9 1
1 < 0.3 > 1.7 1
2 < 0.9 > 3.1 1
4 < 2.1 > 5.9 1
6 < 3.5 > 8.8 2
8 < 4.9 > 11.1 3
10 < 6.3 > 13.7 4
12 < 7.8 > 16.2 5

Table 2. The mean UPC ratio should be calculated using samples collected over several consecutive days. Since the ratio can vary considerably, two serial samples can only be considered significantly different if the variance is ~80% for low UPC values and ~35% for high UPC values 3.

Analytical variability

The coefficients of variation (CV) of the UPC ratio are 10-20% where the UPC = 0.2 and around 10% for UPC = 0.5. This analytical inaccuracy can then lead to a sub-staging error, especially around the extremes of the “borderline” values, i.e., a patient may be wrongly classified as being non-proteinuric when the UPC value is around 0.15-0.25, or wrongly classified as being proteinuric when the UPC is around 0.45 to 0.55 (Figure 4).

Imprecision in the analysis of the UPC ratio can lead to erroneous interpretation of the result. The coefficients of variation of the UPC ratio are approximately 10-20% where the UPC = 0.2 and around 10% when the UPC = 0.5 ( 4 ).

Figure 4. Imprecision in the analysis of the UPC ratio can lead to erroneous interpretation of the result. The coefficients of variation of the UPC ratio are approximately 10-20% where the UPC = 0.2 and around 10% when the UPC = 0.5 4. © Paola Scarpa / redrawn by Sandrine Fontègne

Laboratory method

The UPC ratio may be obtained by different methods (Coomassie Brilliant Blue and Pyrogallol Red) which can lead to differing results (with a mean difference of 0.1-0.2). It is therefore advisable to always refer to the same laboratory to avoid further variation.

Urinary sediment

The presence of contaminants such as blood (macroscopic hematuria) (Figure 5) and/or pyuria (Figure 6) will lead to a significant increase in the UPC ratio. In cats, this increase is also seen with microhematuria. It may be better to avoid performing a UPC on a sample with an “active” sediment, and if urolithiasis, urinary infection or feline idiopathic cystitis is present it is therefore appropriate to determine the UPC ratio after resolution of the disease. 

The presence of blood contamination (macroscopic hematuria) in a urinary sediment leads to a significant increase in the UPC ratio.
Figure 5. The presence of blood contamination (macroscopic hematuria) in a urinary sediment leads to a significant increase in the UPC ratio. © Paola Scarpa
Pyuria in a urinary sediment will also significantly increase the UPC ratio.
Figure 6. Pyuria in a urinary sediment will also significantly increase the UPC ratio. © Paola Scarpa

Gender

Intact male dogs may have a UPC between 0.2 and 0.5, but this can drop to below 0.2 after castration.

Place of collection

The UPC is higher when the urine is collected in a clinical environment rather than in a domestic environment.

Sample handling and storage

Storing a urine sample before testing can potentially cause several problems.

Bilirubin

It is important to note that bilirubin is an unstable compound, easily oxidizing to biliverdin when exposed to light or air. For this reason, bilirubinuria should be determined within 30 minutes of collection when using a dipstick.

 
Paola Scarpa

An initial evaluation of proteinuria can be made with a dipstick, but while this method is sensitive for detection of albumin, it is less sensitive for globulins or protein fractions.

Paola Scarpa

Ketones

The dipstick test for ketones can give a false negative result if the sample to be analyzed has been exposed to air for more than 2 hours, if the pad has been exposed to light, heat or humidity, or if the urine is very acidic.

Temperature 

Refrigeration of a urine sample can cause precipitation of struvite crystals.
Figure 7. Refrigeration of a urine sample can cause precipitation of struvite crystals. © Paola Scarpa

Refrigeration preserves many of the chemical and physical characteristics of urine, but the sample must be brought back to room temperature before performing a dipstick analysis to avoid analytical errors such as inhibition of the glucose reaction. Refrigeration also inhibits bacterial overgrowth, but causes precipitation of calcium oxalate and struvite crystals; these will increase in number and size with time (Figure 7). When performing a UPC ratio, the sample is stable at both room temperature and at +4°C for up to 12 hours post-sampling; after this timepoint the UPC ratio will tend to increase, so it is advisable to freeze the supernatant to reduce any artifacts from long-term conservation.

Technique

The dipstick container must be kept tightly sealed at all times and the expiration date of the test strips verified. Accurate interpretation of dipstick results depends on reading the color change at the specified time for each parameter, and a timer can be used if necessary. Very dark or concentrated urine can also change the dipstick color response, and tests should be repeated as necessary to ensure accurate results.

Whilst urinalysis is a commonplace, invaluable and simple diagnostic tool, errors in sample collection, storage and testing parameters may lead to inaccurate outcomes; in addition, correct interpretation of certain results, especially the UPC ratio, can be problematic. The clinician should bear these factors in mind whenever urine tests are required.

References

  1. Vignoli M, Rossi F, Chierici C, et al. Needle tract implantation after fine needle aspiration biopsy (FNAB) of transitional cell carcinoma of the urinary bladder and adenocarcinoma of the lung. Schweizer Archiv für Tierheilkunde 2007;149;314-318.
  2. Zatelli A, Paltrinieri S, Nizi F, et al. Evaluation of a urine dipstick test for confirmation or exclusion of proteinuria in dogs. Am J Vet Res 2010;236(4):439.
  3. Nabity MB, Boggess MM, Kashtan CE, et al. Day-to-day variation of the urine protein: creatinine ratio in female dogs with stable glomerular proteinuria caused by X-linked hereditary nephropathy. J Vet Intern Med 2007;21(3):425-430.
  4. Rossi G, Giori L, Campagnola S, Zatelli A, et al. Evaluation of factors that affect analytic variability of urine protein-to-creatinine ratio determination in dogs. Am J Vet Res 2012;73:779-788.
Paola Scarpa

Paola Scarpa

Professor Scarpa graduated from the Faculty of Veterinary Medicine in Milan and studied for her PhD at the same university. Read more

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