Using the Urine Examination to Its Full Potential
World Small Animal Veterinary Association World Congress Proceedings, 2014
Liesel van der Merwe, BVSc, MMedVet (Med)(Small Animals)
South Africa

The urinalysis provides vital information in many disease conditions. The range of diagnostic tests which can be run on it are expanding. This talk is divided into in-house urinalysis, basic laboratory test requests and the more advanced diagnostic requests.

Before we go to the actual testing though, it is vital to take into account certain factors associated with the patient and technique before we try and interpret the results. The method of urine collection impacts especially the cytology - free flow collections and even catheter urine collection will have increased cellular elements and risk of contamination. Cystocentesis should be sterile, but there may be iatrogenic bleeding. The hydration status of a patient is vital in interpreting SG. A note must be made on the urinalysis sheet if the patient is dehydrated or on fluid therapy. Concurrent medications will also sometimes affect the urine: glucose in the IV fluids, glucocorticoid or diuretic therapy.

I prefer taking a cystocentesis sample. The procedure is relatively a-traumatic, and with experience it becomes very simple. Always check for adequate platelets and that there is no obvious risk of a concurrent coagulopathy, such as petechiae or sever liver disease, before taking a cystocentesis sample. In some situations you may want to take both a cystocentesis sample and a free flow sample - for example, to localise haematuria

In-House Urine Analysis

The analysis should be performed soon after collection, as values may change. Urine can be stored in the fridge for up to 12 h but should be warmed up before analysis.

Macroscopic Evaluation

Urine should be a clear light to darker yellow to orange fluid. Turbidity indicates increased cellular component. Darker yellow-orange discolouration is from bilirubinuria and red from haemoglobinuria or bleeding.

Specific Gravity (SG)

All practices should have a refractometer. SG measures the amount of solute in the urine. Substances must be dissolved in order to affect the SG. SG must be measured and interpreted in relation to the hydration status of the patient. Once fluids have been administered, this chance is gone - so always try to do this first. Basically, if the patient is dehydrated and the SG is not increased > 1.035, then renal concentration ability is compromised. Additionally, a normal SG does not automatically exclude a patient having PU/PD; glucosuria will increase the SG into the normal range in a diabetic with PU/PD.

Urine Dipstick

The leukocyte patch, nitrate patch and SG patch are not at all reliable and should not be read. Heavy discolouration of the urine will make this test ineffective.

 Protein - This semi-quantitative test is most sensitive for albumin and will not detect globulins or Bence-Jones proteins. The result is read as 1–4+ and measures proteins in the 30–2,000mg/dL range (0.3–2g/dl). False increases will occur if the urine is very alkaline (pH> 8). Interpretation of the significance of the proteinuria is affected by the SG, with a low SG increasing the significance of a positive result.

 Glucose - Normally glucose is filtered through the glomerulus and reabsorbed in the proximal convoluted tubule (PCT). Due to a high blood glucose, the proportion filtered may exceed the maximum, which can be reabsorbed. This threshold is > 180mg/dl (10 mmol/L) in dogs and > 280 mg/dl (15 mmol/L) in cats. Primary renal tubular disease can also cause glucosuria.

 Ketones - Ketones are freely filtered by the glomerulus and should be completely resorbed in the PCT. Ketonuria precedes detectable ketonaemia.

 Bilirubin - conjugated bilirubin can easily pass through the glomerulus. Unconjugated bilirubin is bound to albumin and should not normally filter through. A small amount of bilirubin is normal in dogs, especially males, but is always abnormal in cats.

 UBG - The presence of UBG indicates that the bile duct is patent, because it is formed in the small intestine.

 Blood - Only unbound haemoglobin is filtered through the glomerulus. The strip will pick up haemoglobin (solid colour) as well as red blood cells - patchy colouration.

Wet/Sediment Preparation

Urine is placed into a small test tube and is centrifuged. Mucous (Tamm-Horsfall protein) may float in the supernatant and show as threads in the sediment. The supernatant is tipped away and the pellet of concentrated cells will remain. The cells are resuspended in the small amount of remaining fluid. A drop of Sternheimer-Malbin stain may be added. A drop of the urine is placed on a slide and a cover slip placed. Examination is at 10x and 40x magnification. Light intensity should be adjusted downwards. Due to the varying osmolality of the urine and duration in the urine, the morphology of cells is often affected - red blood cells are crenated in concentrated urine and are swollen in dilute urine.

On low magnification, the slide is evaluated for casts and crystals. Casts are formed in the distal tubules and are composed of cellular components as well as a protein matrix in varying proportions. They generally face in different directions. If the coverslip is moved, the sediment may "roll," resembling casts - but these all face the same direction. A variety of crystals may be found. Microscopic crystalluria does not always imply clinically significant disease - precipitation increases as soon as the urine starts cooling.

At 40/50x magnification, the cellular component is evaluated. The cells can be graded large, medium and small. Large cells are epithelial cells and transitional cells. Renal tubular cells, red blood cells and white blood cells are smaller, and bacteria are the smallest.

Epithelial cells are large flat angular cells and are from contamination.

RTE cells are small, round and slightly larger than neutrophils with a granular cytoplasm and an eccentric nucleus.

Degenerated and dead neutrophils stain red with Sternheimer; live cells are light purple. Neutrophilia may not always be accompanied by bacteriuria and vice versa.

Bacilli (singly or chains) are relatively easy to identify, whereas a relatively large number of cocci need to be present before easily identified. Be careful not to misinterpret Brownian motion or stain deposit as bacteria.

The retrograde movement of sperm into the urine in intact dogs is normal.

Stained Smear

Kwik-Diff. The sample is often very acellular relative to blood, so a very thick smear, or blob of urine should be dried and stained. It may be difficult to focus initially and will be impossible if the slide is upside down. Label them! Mucosal epithelial cells respond to irritation by hyper- and metaplasia, changes which may appear similar to neoplastic changes. As a general rule - if a cause for inflammation is present (uroliths, bacterial cystitis), then reassess the cytology once this is resolved. Abnormal cytology in the absence of inflammatory changes is more significant.

Laboratory Tests

Urine Protein Creatinine Ratio (UPC)

The UPC is the most reliable way to measure urine protein and as it is a ratio, the effect of urine concentration is eliminated. UPC can only be run once postrenal causes of proteinuria have been excluded - urine sediment must be evaluated. UPC has high variability and 2–3 serial samples, a week or so apart, need to be evaluated before a baseline can be established. Two to three urine samples, collected a week apart, can also be refrigerated and equal portions pooled to determine a baseline.

A significant change in UPC is about 80% in a UPC of about 3–5 and about 35% in a UPC of about 10. In cats, the UPC should be < 0.4 but is usually < 0.1, and in dogs a UPC of < 0.5 is considered normal. Glomerular disease is likely if the UPC is ≥ 3 and tubulointerstitial disease normally causes low range proteinuria.

Culture and Antibiogram

Only urine collected by cystocentesis should be sent for culture. If this is not possible, an aseptically placed catheter sample can be submitted.

Urolith Analysis

This must be by diffraction analysis - where the layering of the stone is analysed to determine what the initial component was - because that is what needs to be managed. Uroliths can also be submitted for culture of the nidus.

Extended Laboratory Tests - What Is on the Horizon and Available

1.  Amino acid excretion in the urine - enzyme deficiencies, storage diseases

2.  Bence jones proteinuria

3.  Neutrophil gelatinase-associated lipocalin (NGAL) - a protein indicating real-time renal injury

4.  Urinary biomarkers for acute renal disease3 - filtered proteins: retinol binding protein, cystatin C, tubular enzymes; N-acetyl-β-D-glucosaminidase (NAG) and alkaline phosphatase (AP) are lysosomal enzymes and GGT and alanine aminopeptidase (AAP) are brush border enzymes. Increases are related to tubular injury.

References

1.  Latimer K, Mahaffey E. Duncan and Prasse's Veterinary Laboratory Medicine: Clinical Pathology. 4th Ed. Iowa: Blackwell Publishing; 1994.

2.  Consensus recommendations for the diagnostic investigation of dogs with suspected glomerular disease. J Vet Intern Med. 2013;27:S19–S26.

3.  De Loor J, Daminet S, Smets P, et al. Urinary biomarkers for acute kidney injury in dogs. J Vet Intern Med. 2013;27:998–1010.

  

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Liesel van der Merwe, BVSc, MMedVet (Med)
South Africa


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