31 August 2016

Urinalysis - a review Bradley Galgut, BVSc (Hons), DACVP Specialist Veterinary Clinical Pathologist

UrinalysisA Review

Bradley Galgut, BVSc (Hons), DACVPSpecialist Veterinary Clinical Pathologist

ASAP Laboratory, VIC

Functions of the Urinary System• Kidneys:

• Eliminates liquid waste (i.e. metabolic by-products such as urea and creatinine, drugs, toxins etc) in the form of urine

• Regulates electrolytes (potassium, sodium, chloride, calcium etc)

• Regulates acid/base balance• Controls water balance• Kidneys help regulate blood pressure• Produce erythropoietin, which controls

red blood cell production in the bone marrow

• Key role in Vitamin D metabolismhttp://www.theveterinaryexpert.com/urinary-incontinence/urinary-incontinence-in-the-female-dog-part-1/

Urinary Tract Anatomy

http://www.theveterinaryexpert.com/urinary-incontinence/urinary-incontinence-in-the-female-dog-part-1/ http://www.umich.edu/~elements/fogler&gurmen/html/web_mod/viper/

kidney_function.htm

~1 million nephrons in each kidney

Urinalysis• Vital component of routine diagnostic evaluation along with complete

blood count (CBC) and biochemistry

• “Liquid gold”

• Often overlooked/ignored/forgotten

• Detects disease of the urinary system and some metabolic disturbances• As urine is a filtrate of blood, we can assess urine for drug by-products,

toxins/venoms etc

“Recommend obtaining a urine sample”

Urine Composition• Determined by 3 major factors:

• Blood/plasma presented to the kidneys• Renal functions

• Filtration• Resorption• Secretion

• Added during flow• In the kidneys• After the kidneys (bladder, urethra, skin)

• Always consider method of collection• Voided, cystocentesis, catheter, off floor• Important to note on lab submission form

Urine Specimen Storage• Urine must be collected into clean (preferably sterile)

container (usually yellow-top urine containers)

• Ideally urine should be analysed immediately after collection• pH may change over time• Cells may deteriorate• Crystals may dissolve / precipitate• Casts may break up• Bacteria may proliferate with time

• If analysis will be delayed, refrigerate immediately

• Allow sample to return to room temp before analysis

Urinalysis Procedure1) Physical evaluation

• Colour• Clarity• Urine specific gravity (USG)

2) Chemical evaluation• Dipstick (reagent pad) analysis

3) Microscopic examination• Sediment evaluation

Physical Evaluation - Colour• Normal yellow colour due to

urochromes• Poorly defined group of pigments

• Generally urine concentration can be predicted by colour• Pale yellow = dilute• Dark yellow = concentrated

Physical Evaluation - Colour• Pigmenturia (abnormal urine colour)

• Red, clear Haemoglobinuria, myoglobinuria• Red, cloudy Haematuria • Orange-brown Bilirubinuria• Coffee-brown Myoglobinuria

Colour –Red/Brown UrineHaematuria • Red, cloudy urine; supernatant is

clear or only slightly red• RBC’s in sediment• Positive blood and protein on

dipstick• Normal plasma colour (clear)• RBC’s may lyse if urine pH > 7.5

Myoglobinuria• Occurs due to muscle injury/necrosis • Red-brown supernatant; few or no

RBCs in sediment• Positive blood and protein on dipstick • Plasma normal colour (clear)• Increased CK and AST (snake bite,

trauma, ‘tying up’ in horses)

Haemoglobinuria• Occurs due to intravascular

haemolysis• Red, clear supernatant• Few / no RBC’s in sediment• Positive blood and protein on dipstick• Red plasma (haemoglobinaemia)• Haemolytic anaemia (CBC)

Clarity• Definition: Clearness / transparency

• Clear • Expected in all healthy dogs and cats

• Hazy or cloudy/turbid• Reflects the presence of suspended particles • Crystals, cells, mucus, casts, spermatozoa, lipid, other

material• Equine urine is often cloudy due to mucoproteins

• Dissolved ions and molecules in urine• Electrolytes and metabolic products (urea, creatinine etc)• Concentrations modified by tubular resorption or secretion of solutes

and by resorption of H2O (i.e. normal kidney functions)

• Analytical concepts• Osmolality (what we’re most interested in!)

• Solute concentration in a liquid • Can’t measure this easily (specialised testing)

• Specific gravity • Ratio of solution’s weight (density) to that of an equal volume of water• ↑ solute concentration → density → ↑ SG• Rarely performed

• Refractive index – determined by refractometer• Estimate of the specific gravity of the solution• Light waves slow down and bend (refract) when they enter the solution• Degree of refraction ↑’s proportionately to the solute concentration

Solute Concentration Urine Specific Gravity (USGref)• Refractive index is an estimation of USG• Dependent on:

• Solute concentration• Chemical composition of the solute• Temperature

• Refrigerated / cold urine → false increase in USG• Suspended, non-dissolved particles (cells, casts, crystals

etc) do NOT refract light and do NOT alter refractive index

• Do NOT use dipstick USG pad – poor accuracy

• Refractometers generally calibrated for human urine

• Veterinary refractometers available• Canine/large animals, feline • Calibration scale for dogs is slightly

different from calibration scale for people

• Calibration scale for cats is moderately different from calibration scale for people

Urine Specific Gravity (USGref)• The higher the number, the more concentrated the sample.• The lower the number, the more dilute the sample.

• Maximal urine dilution ~1.001 (jargon = 10,01)

• Maximal urine concentration• Cats: > 1.080 Dogs: ~1.060

• Usual USG values in health when water intake is adequate• Cats: 1.035 – 1.065 Dogs: 1.015 – 1.045

Urine Specific Gravity (USGref)

Urine Specific Gravity (USGref)Isosthenuria• Urine osmolality ≈ plasma osmolality

• Kidneys have not concentrated nor diluted the tubular fluid

• USG 1.007 – 1.013

Hypersthenuria• Urine osmolality > plasma osmolality

• Implies that the kidneys have concentrated the tubular fluid

• USG > 1.013

Hyposthenuria• Urine osmolality < plasma osmolality

• Implies that the kidneys have actively diluted the tubular fluid

• i.e. NOT renal failure• USG ≤ 1.006

Chemical Evaluation

• Semiquantitative

• Chemical properties may differ before and after centrifugation• E.g. If RBCs are present protein and blood/haem reaction

may differ• Best to perform dipstick analysis before centrifuging

Dipstick evaluation Dipstick EvaluationCorrect technique

• Urine should be at room temperature (enzymatic reactions)• Prevent ‘spill-over’ from one pad to the next

Dipstick EvaluationCorrect technique

• Urine should be at room temperature (enzymatic reactions)• Prevent ‘spill-over’ from one pad to the next

XX

Gloves should always be worn!!

Dipstick evaluationCorrect technique

• Read at appropriate times

Dipstick Evaluation• Reliable

• Protein• Glucose• Ketones• pH• Bilirubin• Blood/Haem

• Unreliable (Do not use)• Urobilinogen

• Product of bacterial degradation of bilirubin in gut; used in people not dogs/cats

• Nitrite• Screen for bacteriuria in people;

not reliable in dogs/cats

• Leukocyte esterase• Screen for inflammatory cells in

people; not reliable in dogs/cats• False negatives in dogs• False positives in cats

• Specific gravity

USG and Dipstick resultsProtein Neg. Trace 1+

30 mg/dL2+

100 mg/dL3+

300 mg/dL

100 mL300 mg/dLUSG 1.045

3+Protein = 300 mg

200 mLwater

+ =

300 mL100 mg/dLUSG 1.015

2+Protein = 300 mg

300 mL100 mg/dLUSG 1.015

2+Protein = 300 mg

600 mLwater+ =

900 mL33 mg/dLUSG 1.005

1+Protein = 300 mg

Same amount of protein in each containerDegree of proteinuria3+ protein @ 1.045 Æ same as Æ 1+ protein @ 1.005

Significance of a positive reaction needs to be determined in light of the

reaction strength and the USG

Dipstick Evaluation - pH• Effected by

• Diet (carnivores usually have acidic urine pH 5.5 – 7.0)

• Systemic acid-base balance

• Delayed analysis / exposure to air

• Aciduria (pH <7)

• Metabolic / respiratory acidoses

• Urinary acidifying agents

• Protein catabolic states

• Paradoxical aciduria (severe vomiting)

• Alkalinuria (pH >7)

• Some bacterial infections (e.g. Staph, Proteus)

• Alkalosis

• Following a meal (alkaline tide)

• Exposure of sample to air for extended periods of time

Proteinuria – Chemical Methods

• Reagent strip method

• Negatively charged proteins bind dye → colour change• Detects albumin better than globulins

• False positive in alkaline urine (pH > 7)

Proteinuria• Large proteins do not pass through the glomerulus filtration

barrier in health• Small proteins may pass through the glomerular filtration barrier

→ almost completed resorbed by proximal tubules• Interpret in light of USG• Healthy dogs may have small amount of protein (trace or 1+) in

concentrated urine in the absence of evidence of urinary tract disease

• Proteinuria in cats is always pathologic• False positives in alkaline urine (pH > 7)

1) Prerenal (overflow) proteinuria• Excessive filtration of small proteins → exceeds tubular

resorptive capacity• Haemoglobinuria (haemoglobinaemia), myoglobinuria

2) Glomerular proteinuria• Leakage of plasma proteins through

permeable/damaged glomeruli• Most severe proteinurias• +/- hypoproteinaemia• Glomerulonephritis, amyloidosis

Proteinuria – 4 main causes3) Tubular proteinuria

• Defective/damaged proximal tubular cells• Congenital (e.g. Fanconi syndrome)• Tubular injury (toxicosis and hypoxia)

• Small filtered proteins lost in the urine

4) Haemorrhagic or inflammatory proteinuria• Confirm with urine sediment examination• Voided sample → consider reproductive tract

sources

Proteinuria – 4 main causes

Glucosuria• Glucose is filtered at the glomerulus and then reabsorbed in the proximal

tubule in health

• Hyperglycaemic glucosuria• Filtered glucose exceeds renal threshold

• Dog: ~ 10 mmol/L Cat: ~ 16 mmol/L• Stress (cats), diabetes mellitus, pheochromocytoma

• Renal glucosuria• Glucosuria but normal blood glucose• Proximal tubular disease/transport defect (filtered glucose not resorbed)

• Congenital • Fanconi syndrome (Basenji) • Primary renal glucosuria (Basenji, Norwegian Elkhound, Scottish

Terriers)• Acquired (toxic/hypoxic)

Glucosuria – Chemical Methods

• Glucose oxidase reaction• False positives - hydrogen peroxide (H2O2)

- bleach (sodium hypochlorite)• False negatives in cold urine

Ketonuria • Ketones = acetoacetate, acetone, β-hydroxybutyrate• Ketones are alternative fuels for the body that are made

when glucose is in short supply (i.e cells are starved of glucose for energy). They are made in the liver from the breakdown of fats.

• Excess ketone production → ketonaemia → ketonuria

• Causes• Diabetes mellitus• Starvation/prolonged fasting• Carbohydrate-poor diet• Persistent hypoglycaemia

Ketonuria – Chemical Methods

• Reagent strip method

• Acetoacetate and to a lesser extent acetone react with nitroprusside → colour change

• β-hydroxybutyrate does not react

• Acetone is highly volatile (rapidly lost)

Haem in Urine (blood)• Reagent strip method:

• Peroxidase activity of haem → enzymatic colour change• Detects haemoglobin and myoglobin• Intact RBCs → speckling• Free haem → diffuse colour change

• Differentiate haematuria, haemoglobinuria and myoglobinuria• Examine urine sediment for RBCs (haematuria)• CBC/biochem for evidence of haemolytic anaemia

(haemoglobinuria)• Biochem (CK) for evidence of muscle damage

(myoglobinuria)

Bilirubinuria• Physiology

• Haem degradation → processed in the liver → excreted via bile• If excessive bilirubin is present in the blood → bilirubinuria• Dogs

• Low renal threshold for bilirubin• Small (+) or moderate (++) reactions in concentrated canine

urine (USG >1.025) in health• Bilirubinuria may occur before hyperbilirubinaemia

• Causes of pathologic bilirubinuria• Haemolytic disease• Impaired excretion of bilirubin from the liver (cholestasis, bile duct

obstruction etc)

Bilirubinuria – Chemical Methods• Reagent strip method

• Bilirubin + diazonium salt → colour change• May be difficult to interpret colour change in highly concentrated or

discoloured urine

• Ictotest (tablet) method• Based on same principle• Lower detection limit• Confirmatory test

• Exposure to UV light → bilirubin degradation → false negative

Microscopic Sediment Examination• Perform on fresh urine (ideally <2 hours old)

• Cells/casts degenerate rapidly at room temperature• Crystals precipitate/dissolve• Bacteria proliferate/die

• Refrigerate immediately if cannot be evaluated in this time• Note urine collection method• Ideally use standardised urine volume for sedimentation

• Semiquantitative, comparison of results• Thoroughly mix sample if pouring off for centrifugation• Standardise speed & duration of centrifugation

• ~450G for 5 mins (~1500 rpm)

Microscopic Sediment Examination• Use of a sediment stain (Sedi-stain™) is optional• Lower microscope condenser to improve contrast

• Low-power field (10x objective)• Casts • Crystals

• High-power field (40x objective) • Erythrocytes (RBCs)• Leukocytes (WBCs)• Epithelial cells • Bacteria

Sediment Exam – Leukocytes (WBCs)• Pyuria (WBCs in urine)= >5 WBCs per HPF

• Usually infectious (UTI)• Can be sterile (neoplasia, necrosis, urolithiasis

etc)• Unstained WBCs appear as round granular cells• 1.5 – 2 times diameter of RBC• Usually neutrophils• Consider collection method when interpreting

• Cystocentesis sample localises inflammation to urinary tract

WBC

RBC

Sediment Exam –Erythrocytes (RBCs)• Haematuria = > 5 RBCs per HPF• RBC morphology depends on urine concentration and time• Consider collection method• Lysis of erythrocytes can occur in poorly-concentrated or alkaline

urine samples.

Crenated RBC

RBC

Sediment exam – Epithelial Cells• Squamous cells

• Contamination from distal urethra, genital tract, skin

• Transitional cells• May slough for inflamed or

hyperplastic mucosa• Transitional cell carcinoma –

cytology best tool• Renal epithelial cells (rare)

• May be difficult to differentiate

Sediment Exam - Bacteria• Voided/catheterised samples may have few bacteria• Cystocentesis samples should have none• Bacteriuria due to infection usually accompanied by

pyuria (but not always!)• Can confirm bacterial presence via urine cytology• Centrifugation of sample does NOT

concentrate bacteria• Detection of bacteria in urine

• Rods > 10,000 / mL• Cocci > 100,000 /mL

Sediment Exam - Casts• Cylindrical molds that form in the tubules

• Tamm-Horsfall mucoprotein• Named and classified according to composition

• Cells/material that is within or adhered to the mucoprotein matrix • Cellular, granular & waxy casts represent different stages of cell

degeneration• How a cast appears in the urine

largely depends upon the length of time it was in the tubulesprior to being shed

• Degenerate in urine with time

• Large numbers of casts indicate renal tubular disease• Usually acute damage

Sediment Exam - Casts• Hyaline casts

• Composed of protein only • Low numbers may be normal• Increased numbers suggest glomerular proteinuria

• Epithelial casts, coarse and finely granular cellular casts, waxy casts • Ischaemic or toxic renal tubular injury

• White cell casts• Suggest pyelonephritis

• Red cell casts• Suggest glomerular or tubular haemorrhage

Sediment Exam - Crystals• Precipitation depends on:

• pH• Ion concentration• Temperature

• Prolonged storage/refrigeration enhances formation of some crystals and dissolution of others

• Crystalluria is a common finding• Insignificant?• Pathologic state causing change in urine pH or ion

concentration?• Risk factor for development of urolithiasis

• Most animals with crystalluria do not have uroliths• Some with uroliths do not have crystalluria

CrystalluriaCrystal Significance Urine pH

pH<7 pH>7

Ammonium biurateAmorphous urates

Common in Dalmation and English Bulldogs, liver dysfunction and portosystemic shunts.

+

Bilirubin Bilirubinuria +

Calcium oxalate dihydrate Common in healthy animals, ethylene glycol toxicity, hypercalcaemia

+

Calcium oxalate monohydrate

Ethylene glycol toxicity, hypercalcaemia +

Cystine Congenital/hereditary cystinuria, liver disease

+

Magnesium ammonium phosphate (struvite)

Common in healthy animals with alkalinuria.May be associated with urease-producing bacteria (UTI).

+

Uric acid As with ammonium biurate +

Struvite Calcium oxalate (dihydrate)

Bilirubin

Ammonium biurate

Cystine

Calcium oxalate (monohydrate)

The End!