Urinalysis: what can go wrong?

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 

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 (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 

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. 

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). 

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).

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. 

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.

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 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.