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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

LABORATORY TECHNIQUES - URINALYSIS The analysis of urine is an underrated and possibly under-utilised resource. Its collection is often non-invasive and its examination within the scope of any practice laboratory. In fact, its a positive advantage to examine a urine sample fresh in the practice as changes occur on storage that may mislead or confuse diagnosis. There are many possible indications for urinalysis including polydipsia/ polyuria, haematuria, urinary tenesmus, increased frequency of micturition, renal disease and pyrexia of unknown origin. Urine collection Animals should not be sampled after meals, vigorous exercise or excitement. These can cause transient increases in urine protein, haemoglobin, blood, myoglobin and glucose. Also the choice of collecting method can affect the interpretation of results and must always be taken into account. Naturally voided urine is the most frequently collected sample. The best sample to collect is midstream with occasional exceptions (e.g. end stream for prostatic disease and bladder sediment). Urine should be collected into clean and preferably sterile containers. Manual bladder expression can also be performed with care. Cats present an obvious problem. Urine can be collected from an empty litter tray that has been previously scalded in boiling water. Samples may alternatively be collected by catheterisation. This can cause trauma or iatrogenic infection but is conveniently performed alongside radiography using contrast media. Cystocentesis involves the passage of a needle through the abdominal wall into the bladder. It produces the best urine samples for examination and bacterial culture although there can be mild haemorrhage into the urine. Urine storage Urine should be examined within a few hours of collection wherever possible. It may be refrigerated for a short time but should never be frozen. The characteristics of urine may change with storage, particularly at room temperature.

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

e.g. contaminating bacteria can proliferate causing: increased pH false positive protein readings false positive blood reactions decreased solubility of struvite crystals

Strategies have been suggested for combating this including the use of mineral oil overlays. Urine treated this way is very difficult to examine due to the lipid droplets. In one study using urine from normal cats we found no difference between samples with a thymol oil overlay and plain urine samples. Fresh plain urine is the sample of choice for examination, except when urine is required for bacterial cultures in which case a sterile boric acid container should be used to collect the sample. Physical Characteristics Colour: Dilute urine is pale whilst concentrated urine is dark yellow to orange. Pale urine is common in animals that are polydipsic and polyuric whilst concentrated urine is more common in animals with fever, vomiting and diarrhoea or water deprivation. Bilirubin produces orange urine which turns green on standing Haematuria or haemoglobinuria produce a pink to red colouration that may turn brown on standing.

Turbidity: Carnivore urine is usually clear. Urine may appear white due to crystalluria, buff-coloured due to urate crystals or casts, or cloudy due to bacteria or semen. Odour: A sharp ammonia smell suggests urinary tract infection in freshly voided samples. In old samples ammonia develops due to the breakdown of urea. Pear drops (acetone) may be smelled in urine from uncontrolled diabetic dogs and cats. Specific gravity: Measured by refractometer that has a calibrated scale based on refractive index. It is a measure of renal concentrating ability; animals in renal failure are unable to dilute or concentrate their urine and will usually have a specific gravity in the range 1.008-1.012.

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

Dip-stick testing The directions for the time interval between wetting the stick and reading each parameter must be followed carefully. Not all test parameters are valid in cats and dogs: Glucose: no known limitations Bilirubin: no known limitations Ketones: no known limitations Specific gravity: NOT RELIABLE Blood: no known limitations pH: no known limitations Protein: no known limitations Urobilinogen: NOT RELIABLE Nitrite: NOT RELIABLE Leucocytes: cat: NOT RELIABLE, gives false positives, dog: less sensitive (From: Clinical Urine Analysis for the Veterinary Surgeon. Bayer Diagnostics Education Services). Other factors may interfere with the performance of urine dipsticks. Drug therapy can cause transient changes in the urine e.g. xylazine causes a transient glucosuria. Drugs and chemicals can also cause false readings on urine dipsticks e.g. ascorbic acid or chlorine can give false positive glucose results. These effects are not common but some thought should be given to drug therapy when an animal is on when tested. Finally, some companies make available quality control material (artificial urine samples) to test the reactivity of the urine dipsticks. These may be considered if urine dipsticks tend to hang around the practice getting close to their expiry date or if lids are left off the containers for prolonged periods. Normal urine Specific gravity pH Protein Bilirubin Dog Cat Dog Cat Dog Male dog 1.015 1.045 (1.001-1.065) 1.035 1.060 (1.001-1.080) 5.5 7.0 6.0 7.0 trace trace

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

Deposit Microscopy Deposit microscopy is a surprisingly powerful tool in assessing urine. Mix a plain urine sample carefully and centrifuge at least 5ml of urine at 2,000 to 3,000 rpm for 5 minutes. (If you use special urine tubes in the Vet Test centrifuge then fill two tubes and combine the deposit. Use the micro centrifuge setting for urine deposits). Carefully tip away the supernatant and re-suspend the deposit by flicking the tube. Pipette the deposit onto a clean glass slide and add a coverslip. Examine under x400 magnification and report numbers per high power field.

Each component of the urine deposit should be reported. Component Red Blood Cells White Blood Cells Epithelial Cells Red, White & Epithelial Cells Crystals Casts Bacteria / Yeasts Spermatozoa Amorphous debris Report as number per high power field (h.p.f.) if more than 5. number per h.p.f. if more than 5. number per h.p.f. if more than 5. Occasional if less than 5. Occasional / Moderate / Large numbers of [type of crystal] Occasional / Moderate / Large numbers of [type of cast] Occasional / Moderate / Large numbers of Occasional / Moderate / Large numbers of Small / Moderate / Large amounts of

Red, white and epithelial cells Any number of red blood cells is abnormal in a urine deposit but the method of collection should always be considered. Greater than 5 white blood cells per x400 microscope field are taken as a rule of thumb to indicate urinary tract inflammation/ infection. Epithelial cells may be present in large numbers in catheterised samples, in urinary tract infections, associated with urolithiasis or associated with urinary tract neoplasia. Crystalluria Some crystals (especially struvite and calcium oxalate) are commonly found in small numbers in normal dog and cat urine, but their presence in large numbers, depending on the urine pH, concentration and solubility of the individual salts, may be significant.

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

Effect of urine pH on commonly occurring urine crystals Type Struvite Calcium oxalate Calcium phosphate Ammonium urate Acidic +/+ +/+ pH Neutral + + + + Alkaline + +/+ +

Urologists refer to the habit of a mineral. This means the characteristic shape that the mineral can assume in crystalline form. Crystal shape should only however be taken as a tentative identification of the mineral type. Struvite crystalluria Typically 3 to 6 sided, colourless coffin-like prisms. They can occasionally aggregate into fern-like structures. These crystals are common in dogs and cats. They occur: in clinically normal animals in animals with struvite uroliths associated with infection in animals with sterile struvite uroliths in cats with urethral obstruction in animals with non-struvite or mixed uroliths in animals with urinary tract infection and no uroliths

Calcium oxalate crystalluria There are two types of calcium oxalate crystals. The more common is calcium oxalate dihydrate (weddellite). These crystals are typically colourless with an octahedral or envelope shape. Calcium oxalate monohydrate (whewellite) crystals are typically spindle, oval hemp seed or dumbbell shaped. Calcium oxalate dihydrate crystals are found: in clinically normal animals in animals with calcium oxalate uroliths

Calcium oxalate monohydrate crystals are found: in ethylene glycol poisoning associated with excess calcium in the urine (e.g. hypercalcaemia)

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

Calcium phosphate crystalluria Calcium phosphate crystals take on many different forms. They are most commonly amorphous and need to be distinguished by solubility tests from amorphous urates. Calcium phosphates also form long, thin colourless prisms. Calcium phosphate crystals are seen: in clinically normal dogs in dogs with persistent alkaline urine in dogs with calcium oxalate uroliths in dogs with mixed calcium phosphate/ calcium oxalate uroliths in small numbers in infection associated struvite crystalluria

Urate crystalluria Ammonium urate (ammonium biurate) crystals are yellow/brown and often form spherical bodies with long irregular protrusions (thorn-apple forms). Amorphous urates are sodium, potassium, magnesium or calcium urate salts. As they grow they can become yellow/brown. Urates are seen: in clinically normal cats and dogs in dogs with portosystemic shunts in Dalmatians and English Bulldogs

Cystine crystalluria Cystine crystals are colourless, flat and hexagonal with equal or unequal sides. They are most common in concentrated acidic urine. Cystine crystals are found: In dogs and cats with inherited cystinuria

Drug associated crystalluria Sulphonamide drugs typically appear in urine as sheaves of clear or brown crystals. They may also form spheroids or amorphous crystals. Other drugs do not commonly produce crystals but radiopaque dyes, ampicillin, primidone and ciprofloxacin all produce crystalluria in man.

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

Less common crystallurias include: Tyrosine and leucine crystals may be associated with severe liver disease. Bilirubin crystals may occur in concentrated urine from normal male dogs or in cases of marked bilirubinaemia. Cholesterol crystals may be seen in clinically normal dogs Uric acid crystals may be found along with urate crystals Xanthine crystals may be seen with excessive administration of allopurinol

Crystal morphology is not always distinct enough to allow for confident identification. Solubility tests can also be applied to help identification. Crystal type Phosphates of Ca and Mg Calcium oxalate Uric acid and urates Cystine Cholesterol Sulphonamides Casts Casts are an uncommon finding in urine deposits. Low numbers may be found in clinically normal cats and dogs. Large numbers indicate renal tubule pathology and are often associated with renal failure. Hyaline (mucoprotein) casts: are clear and difficult to see. Small numbers may be seen with transient proteinuria. Most common in pre-renal and glomerular cases of proteinuria. Alkali Insoluble Insoluble Soluble Soluble Insoluble ? Solubility Dilute HCl Acetic acid Soluble Soluble Soluble Insoluble Soluble Insoluble ? Insoluble Insoluble Insoluble Insoluble ?

Acetone Insoluble Insoluble Insoluble Insoluble Soluble Soluble

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

Red cell casts: indicate haematuria of renal origin

White cell casts: form due to tubulointerstitial inflammation

Epithelial cell casts: contain desquamated tubular epithelial cells and may be associated with ischaemic tubular necrosis or toxic tubular insults. Lipid may accumulate to give fatty casts. The cells may degenerate to form waxy casts. Waxy and fatty casts are more common in cat than dog urine.

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

Granular (degenerative cellular) casts: contain fine granular material thought to result from degenerating white or red cell casts.

Bacteriology Isolation, identification and antibacterial sensitivity testing of organisms from urine is best undertaken in a specialist laboratory. Fresh urine in a sterile boric acid preservative should be submitted (boric acid fixes urine pH and acts as a bacteriostat). Other procedures Other specialised procedures performed at commercial diagnostic laboratories include: Qualitative or quantitative urolith analysis Urine protein: creatinine ratio Urine cortisol: creatinine ratio Urine protein electrophoresis

Tim Jagger BVM&S MSc DipRCPath MRCVS Leeds Veterinary Laboratories 2002

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