Urine Kathleen Tennant BVet Med Cert SAM Cert

Urine Kathleen Tennant BVet. Med Cert SAM Cert VC FRCPath MRCVS Clinical Lead Diagnostic Laboratories Langford Veterinary Services

Requirements • Microscope with a 100 x and 400 x magnification capacity • Centrifuge capable of approx 1500 rpm • Conical centrifuge tube (preferably capped) • Sediment stain • Standard graticule grid • Cytological stain (e. g. Modified Wright’s)

Requirements

Storage of samples • Analyse within 1 hour of collection • Refrigeration preserves physical and chemical properties of urine, slows bacterial overgrowth and maintains cellular elements, but…

Albasan et al (2003), JAVMA vol 222, no. 2 pp 176 - 179 • 31 dogs and 8 cats • Urine analysed within 1 hour, or refrigerated for 6 or 24 hours • Crystals formed in vitro in 11 of 39 samples (calcium oxalate and magnesium ammonium phosphate) • Refrigeration increased the numbers of crystals present (significantly for Ca. Ox)

Gross appearance • “Record appearance including turbidity, colour and any odour” • Really? Deliberately sniffing potentially pathogen laden urine? • (P. S. Are you wearing gloves? )

Barlough et al (1981) JAVMA 178 (1): 61 -63 • 1000 dog urines, 1000 cat urines • Compared subjective abnormalities in appearance vs microscopic findings • 16. 5% of canine urines appearing normal had abnormalities (mostly pyuria and bacteriuria) on microscopy, 5. 7 % of cats (haematuria/ bacteriuria) • 50% with subjective abnormal appearances had abnormal microscopic findings

Specific gravity • Measure by refractometer • Quality control: measure specific gravity of distilled water (should be 0. 0), 5% Na. Cl should be 1. 022) • Should be measured at room temperature

Van Vonderen et al (1995) Tijdschr Diergeneskd 120 (13) 400 - 402 • Compared urine strip, refractometry (s. g. ) and osmolality in 80 canine urine samples • Good correlation refractometry and osmolality (r = 0. 98) • Poor between strip and refractometry (r =0. 39), strip and osmolality (r = 0. 36) • 77. 5% had a difference of >0. 005 • Does it matter? Threshold decisions

Dipsticks • Manual reading or automated systems to read the sticks • Paquinon et al (1993) Lab Anim 27 (3) evaluated Clinitek 200 in dog and rat, found good correlation between visual reading and automated, reported over and under - estimation of protein at low concentrations, overestimation at high concentrations • Published and in – house data available for other makes

14 y. o. DSH with polyuria • Positive result on leucocyte panel • Is this evidence of a urinary tract infection?

Dipstick interpretation • Leucocyte esterase panel: designed for human use (where there approx 0. 9% false negatives). • Holan et al (1997) Vet Clin Path 26 (3): 126 – 131 evaluated a test strip vs microscopy in cats, found sensitivity of 77% and a specificity of 34%

Dipstick - nitrites • Generally more specific than sensitive – many non- nitrite producing urinary tract infections exist

p. H • Rapidly affected by storage of the sample • Heuter et al (1998) JAVMA 213 (7): 996 998 compared p. H meter and dipstick : found only 40% of dipstick results were within 0. 25 p. H units Picture, W. Millard

Urinary protein measurement • Dipstick and sulphosalicylic acid techniques predominantly measure albumin • Dipsticks may give false positives in alkaline urine or in the presence of detergents

Protein: creatinine ratio • Ratio removes the influence of concentration • A point of care immunoassay (E. R. D. -Screen Urine Test, Heska Corp) has become available : one canine study showed 90% of samples negative for microalbuminemia (semiquantative) correlating to a UPr: Cr of<0. 5, 92% with high and very high microalbuminaemia correlating to UPr: Cr of >0. 5 (Garner and Wiedmeyer, 2007)

Protein: creatinine ratio • Influence of cells on protein levels • Vaden et al (2004) found 70 samples with pyuria had a normal UPr: Cr • Whole blood was added and the UPr: Cr did not exceed 0. 4 at the point where the sample was visibly pink • If it’s pink, don’t trust the UPr: Cr – get more measures

Other panels • Good correlation of other panels in spiking studies • No ability to distinguish intact red cells, haemoglobin and myoglobin: microscopy required to see if intact red cells, other clinical finding for myoglobinuria • Dipsticks should be checked between batches or in doubt

E. g. Chek – stix (Bayer) Picture, W Millard

Sediment examination • Mix sample • Transfer standard volume (5 ml) - to allow sample to sample comparison - to a conical tipped capped centrifuge tube • Centrifuge at 1500 rpm for 5 minutes • Decant supernatant with a pipette leaving a small amount (0. 5 ml) for resuspension

Sediment examination • Add stain if desired, usually at equal volume with sediment – remember will alter quantification. Consider doing both stained and unstained. • Resuspend by flicking the pot until well mixed • The sample can then be examined in either a commercial graticule or a single drop under a standard square coverslip to allow quantification

Sample examination • Examine under reduced light : drop the condenser or reduce iris size • 100 x scanning overview and to allow initial quantification of elements • 400 x to allow identification of morphology and bacteria • Numerical scores either per high power field (40 x objective) or subjective low, medium, high for bacteria, crystals, sperm, parasites

What is this?

What is this? • Red cells • Calcium oxalate monohydrate 1. 2.

There are problems with only being able to go to 400 x (dry lens)

Erythrocytes

Erythrocytes • Usually < 5 per 400 x field • Haemolyse in low s. g. urine (<1. 008), but also affected by other factors (time, temperature) • Biconcave discs, may be crenated or swollen • If ruptured, membrane only noted (ghost cell) • Few expected in cysto sample

Leucocytes Usually <5 per 400 x field 1. 5 – 2 x size of erythrocytes Granular cytoplasm, lobed or segmented nucleii Difficult to distinguish from epithelial cells – consider air drying a sample and Giemsa stain • Denote inflammation but may be difficult to tell visually if septic or sterile (e. g. in response to uroliths) • •

Leucocytes

Leucocytes and bacteria

Squamous epithelial cells • • Originate externally or the distal urethra Large, flattened cell, usually anucleated May be folded or rolled to imitate casts If external in origin, may have attached bacteria

Squamous epithelial cells

Transitional epithelial cells • Large cells (up to 40 um) Leucocytes 12 - 14 • Smallest basal layer more columnar than round • Transitional cell carcinoma difficult to diagnose without nuclear staining – to differentiate neoplasia from dysplasia need high cell yield and air dried cytological staining (e. g. Giemsa, rapid stains) • Changes in morphology may also be caused by exposure to urine

Transitional epithelial cells

Is this a neoplastic population or a normal one? • Neoplastic • Normal • Can’t tell 1. 2. 3.

Transitional cells • You need to be able to see internal cell detail to be able to judge if normal, dysplastic or neoplastic. • Make a concentrated sample and allow to air dry, then use a cytologically stain the standard way

Renal epithelial cells • Proximal tubular cells rectangular, some may show microvillous border • Vary widely in size • Difficult to differentiate from other epithelial cells or leucocytes • Normal turnover releases some, higher numbers expected in disease

Renal epithelial cells

What do the crystals we see tell us?

Magnesium ammonium phosphate • Struvite • May also form fern –frond structures • May be seen in normal animals, those with uroliths (any type), or other urinary tract disease including UTI • Dissolve if acidified

Calcium phosphate • Many forms: amorphous; clear prisms, clear elongated type may form rosettes • Normal dogs, those with alkalinised urine, with or without uroliths

Calcium oxalate dihydrate • Squares with intersecting lines from corner • May occur singly or as conglomerate structures • With or without calcium oxalate uroliths • In any p. H urine

Calcium oxalate monohydrate • In hypercalcaemia and excessive oxaluria • Ethylene glycol toxicity • Rhubarb leaf toxicity • Seen alone or with calcium oxalate dihydrate • Polarising light helps to identify – present also in renal histological slides

Ammonium biurate • ‘Thorn apple’ morphology • With liver function compromise, particularly portosystemic shunts • Increased incidence in dalmations

Amorphous urates • Similar circumstances to ammonium biurate • Other amorphous crystals include amorphous phosphates – differentiate with alkali: phosphates dissolve, urates remain

Uric acid • May occur singly or in sheaves/rosettes • Diamond or rhomboid • Significance as for ammonium biurate • Can be grown from amorphous urates by adding acetic acid – helps confirm identity

2 y. o. DSH free catch urine sample Is this…… • Debris from the litter tray? 1. • Rod bacteria 2. • Fungal elements 3. • Bilirubin 4.

Bilirubin • May be normal in concentrated urine in dogs – renal bilirubin conjugation • Abnormal in cats • May see increased amounts in pre-hepatic, hepatic or post hepatic origin icterus

Cystine • Clear hexagons, often stacked • Cystinuria (defect in renal transport of cystine in the tubule) • Many breeds, reported autosomal recessive in some Newfoundlands and Labradors • Can lead to uroliths • In acidic urine

Leucine • Brownish spherical with ‘target’ lamination • Possibly associated with aminoaciduria or severe liver disease

Urinary casts • Tamm – Horsfall mucoprotein secreted from loop of Henle, distal tubules, collecting duct: this alone may become a hyaline cast • Cells may become snared in the protein: possibly degenerate as continue down tubule/duct • Low numbers unremarkable, high numbers consistent with renal insult: hypoxia, ischaemia although absence is not a rule out

Granular/hyaline cast

Cellular cast

Lipid • Normal in cats • Lipid droplets float on a higher plane than much of sediment • Result of normal tubular degeneration • No direct relationship between lipidaemia/lipiduria

Yeast/Fungi • May be pathological especially in immunocompromised animals on chronic antibiotics • Candida or systemic fungal (e. g. Cryptococcus) • If no inflammation, suspect contamination and overgrowth

Parasites • Capillaria plica or Capillaria felis cati – bipolar plugs and lemon shape • Differentiate from faecal contamination with Trichuris vulpis • Also Dioctophyme renale and Dirofiliria microfilaria

Contaminants