Urine Examination&
Analysis
Assigned by: Dr.Javeria Khan
Presented by: Dr.Noor-ul-Ain Sarwar
Contents
1. Collection of sample and preservation.
2. Gross Examination.
3. Determination of specific gravity.
4. Biochemical Analysis.
5. Microscopic Examination.
6. Disease Interpretations
Introduction
• Urinalysis a very useful tool to evaluate healthy and diseased animals. It provides valuable information about the urinary system. There are so many examples of diseases in which specific urine picture can be seen. for example:
A. Kidney diseases: Abnormal specific gravity Proteinurea. Cast Leukocyte Erythrocytes
B. Bladder infection: Proteinuria Leukocytes Bacteria
C. Neoplasia: Exfoliated neoplastic cells Hematuria
D. Liver diseases: Billirubinuria Altered urobilinogen Bilirubin crystals
E. Hemolysis: Post parturient hemoglobinuria Bovine bacillary hemoglobinuria Anthrax Increased urobilinogen
F. Diabetes mellitus: Glycosuria Increased volume, increased specific gravity Ketonurea
G. Diabetes insipidus: Decreased specific gravity
H. AcidosisI. Alkalosis
Collection of Urine
1. Collection of urine sampleCollection can be done either by clean catch
method,catheterization or through cystocentesis.
Sample size: 15- 20mL
Best time for analysis:
A fresh urine sample is preferred for analysis.Ideally urinalysis should be performed within 30 min of sample collection.If delay of examination then following changes takes place:Urea is converted into ammonia that makes the
sample alkaline.Formed elements(cells,casts)are dissolved.
Precautions while collection of urine: Morning samples are most likely to contain
constituents of diagnostic significance.Fluid consumption during the day dilutes the urine
resulting in decreased specific gravity.Collect mid stream urine.In case of diabetes mellitus, sample should be
collected 2 hours after feeding and fasting.For nephritis,use only morning samples.Direct collection is the preferable method in large
animals.although catheterization and cystocentesis
provide high quality of uncontaminated sample but are associated with tissue trauma of varying degree.
Preservation: Store samples in refrigerator at 8 degree C.(warm
at room sample before analysis). Precautions regarding refrigeration:
Maximum upto 12 hoursIt slightly increases specific gravity.It also interferes with tests using enzymes for
reaction.
Chemical preservationCertain chemicals are used with limitations.
a. Toluene:
Quantity: 2ml/100ml of urine for 24 hours.
Only cover urine surface, don't dissolve in urine.
Limitation :Interferes with ketone bodies determination.
b. Thymol:
Quantity: a small lump can preserve for several days.
Limitation: it gives false positive protein reaction.
c. Formaline:
Quantity: 1drop of 40% formalin for 30ml urine for 24 hours.
Limitation: It interferes with glucose reaction.
c. Metaphosphoric acid:
When ascorbic acid is to be determined from the urine. It is added with a ratio of 1:5 ( 1 part 10% aqueous solution of metaphosphoric acid and 5 parts of urine sample.
Collection
2. Gross Examination
a) Volume:
species
Normal values
in liters
Horse 4.7
Cattle 14.2
Sheep, Goat and Dog
0.9
Interpretation
i. Increased volume-Polyurea:
Physiological: Increased water consumption Diuretics Parenteral fluid therapy
Pathological: Chronic progressive renal failure Diabetes mellitus Diabetes insipidus Chronic pylonephritis
Pyometra
ii. Decreased volume-oligouria:Physiological:
Less water intake. High environmental temperature. Panting Dehydration
Pathological: Acute renal disease Urolithiases fever Shock Severe nephritis Edema
b. Color:
Colour of the urine is due to the concentration
of urochromes.Always consider color in association of volume and specific gravity of urine.
Normal Colour:
Freshly voided urine is clear and may range in color from light pale yellow to amber(gold)or straw Colour except horses which have turbid color urine due the presence of calcium carbonate crystals and mucin.
InterpretationVarious color could be:
i. Less to pale yellow: End stage renal disease Increased uptake of water Diabetes insipidus Hyperadrenocorticism
ii. Dark yellow to yellow brown: Acute nephritis Dehydration Bilirubin
iii. Yellowish brown; In birds ,yellowish green urates indicates
hemolysis or liver disease. Bilirubin
iv. Red: Hematuria Hemoglobinuria
v. Brown to brownish black: Hemoglobin(hemoglobinuria,post parturient
hemoglobinuria,bacillary hemoglobinuria) Myoglobin(Monday morning disease) Melanin
vi. Green: Biliverdin Phenol poisoning
c. Odor Interpretation Ammonia-like :Ammonia-like : Urea-splitting bacteriaUrea-splitting bacteriaFoul, offensive :Foul, offensive : Old specimen, pus or Old specimen, pus or
inflammation inflammation Sweet : Sweet : GlucoseGlucose Fruity : Fruity : KetonesKetones Maple syrup-like: Maple syrup-like: Maple Syrup Urine DiseaseMaple Syrup Urine Disease
d.d. Color:Color:
Colorless Colorless Diluted urineDiluted urine Deep YellowDeep Yellow Conc. Urine, Riboflavin.Conc. Urine, Riboflavin. Yellow-GreenYellow-Green Bilirubin / BiliverdinBilirubin / Biliverdin RedRed Blood / HemoglobinBlood / Hemoglobin Brownish-redBrownish-red Acidified Blood (Actute Acidified Blood (Actute
GN)GN) Brownish-blackBrownish-black Homogentisic acid Homogentisic acid
(Melanin)(Melanin)
3. Specific Gravity
The ability of kidneys to concentrate the urine.Determination: It can be determined by the use of refractometer
or urinometer.the steps are as under: Temperature of the urine must be 20-25°C. cylinder used for floatation of urinometer
should be large enough in diameter so that urinometer can flow in it.
.
Place the urinometer in cylinder containing the urine .Rotate it to prevent its touching to the sides.
Read the scale on the bottom of urinometer
and record it in decimals.
Urinometer
Refractometer
Refractometer
Species Specific Gravity
Horse 1.020 – 1.050
Cattle 1.025 – 1.045
Sheep & Goat 1.015 – 1.024
Dog 1.015 – 1.045
Birds 1.005 – 1.020
Interpretation
i. Increased specific gravity: Acute interstitial nephritis Cystitis Liver failure Diabetes mellitus Glomerulonephritis
ii. Decreased specific gravity: Chronic interstitial nephritis Diabetes insipidus Pylonephritis uremia
Chemical AnalysisChemical Analysis
3. Biochemical Analysis
For biochemical analysis urine must be uncentrifuged.
a. pH:
i. Acidic pH Interpretation: Normal in carnivores. Nursing calves & foals. Excessive diet in protein. Hypokalemia.
ii. Alkaline pH Interpretation:
Normal in herbivores Stale urine sample becomes alkaline. Cystitis.
Normal pH
Species pH
Horses 8
Cattle 7.4-8.4
Sheep & Goat 7-8.2
Dog & Cat 5.5-7
Birds 6-8
b. Protein Determination:
For Protein determination:
a) Reagent strips(dip sticks)
b) Acid prepitation Tests:
i. Nitric Acid Precipitation Test
OR Robert’s Tests:
Principle:
Precipitation of protein occur by concentrated acid
Procedure:
Take 2ml of Robert’s reagent in a test tube.Place 2ml of urine.Wait for few minutes.
Result:
A positive test is indicated by a white ring at the zone of contact of 2 fluids.
Interpretation of Protein determination
Hemoglobinuria.Myoglobinnuria.Pylonephritis.Cystitis UrolithiasesInflammation,Hemorrhage, Glomerular disease.
c. Glycosuria determination
Now a days strips and glucometers are available. Chemical method is Benedict’s test.
Benedict’s test:
Principle:
It depends upon the reducing sugars present in the urine to react with copper sulphate to reduce cupric ions to cuprous oxide giving color.
Composition of Benedict’s Reagent
Copper sulphate 17.3 g
Sodium citrate 173 g
Sodium carbonate 100 g
Distilled water
(To make volume)
1000 mL
Procedure:
Take 5mL of Benedict’s reagent in a test tube.Add 8 drops of urine to the reagent.Mix the 2 fluids.Heat it with constant shaking till boiling.• Result:
Positive Blue color
Negative Orange to brick red or brown
Interpretation
HyperglycemiaAfter general anesthesiaChronic liver diseasesEnterotoxaemia in sheep.
d. Ketonurea Determination
Ross test:Principle:It is based on that the sodium nitroprusside is
decomposed to:Sodium ferrocyanideSodium nitrateFerric hydroxideResults:Purple coloration
Procedure:
Place half inch layer of powdered reagent in test tube.
Add 5mL of urine.Agitate the 2 components in the test tube.’Overlay 1-2 ml of ammonium hydroxide over
the mixture.Wait for 4-5 minutes.Development of purple color indicates the
presence of ketone bodies in urine.
Interpretations
Diabetes mellitusHigh fat dietStarvationImpaired liver functionsAfter ether chloroform anesthesiaMilk fever
e. Hematuria detection
Benzidine test:Take 2mL of glacial acetic acid in a test tube.Add small amount of Benzidine reagent.Add 1 ml of urineAdd 1 mL of fresh hydrogen per oxide.Wait for 5 minutes.Result:• Green or blue color development.
Interpretation• Acute nephritis
• Urolithiases
• Cystitis
• Tumor of the urethra
• Severe infections like, anthrax, leptospirosis,infectious canine hepatitis.
• Chemicals like copper, mercury or phenol poisoning.
• Parasites like Dicroflaria immitus,Dictophyma renale,Capillaria plica.
f. Billirubinuria determination
Foam test:
Procedure:Take 1-2 mL of urine in a test tube.Shake it vigorously.
Result:
• Appearance of yellow, greenish yellow or brown colour foam above the surface of urine indicates presence of Bilirubin.
Interpretation:
Infectious canine hepatitis,leptospirosis.NeoplasiaObstruction of bile duct.Jaundice
Calcium Determination
Sulkowitch test:
Calcium present in urine reacts with sulkowitch reagent ,ppt in the form of calcium oxalate.
Procedure:Take 5mL distilled water & add 5mL urine in
1 test tube as control.In another test tube,mix equal amount of urine
& sulkowitch reagent.
Result:Compare the 2 test tubes in light after 2-10 min.
Interpretation:Increased:After Ca administration.HyperthyroidismHypervitaminosis
Decreased: In bovines, it is not reliable. In canines,pre-renal tetany.hypothyroidism
5) Microscopic examination
Purpose:Recognition of cells for urinary tract
infections.Exfoliative cytology of tumors.
Procedure; Centrifuge sample @ 1500 rpm for 2-3 min. Pour off the supernatant. Place a drop of sediment on slide and cover it
with a cover slip. Observe the slide @ 10x and 40x.Result variations:i. Voided sample: more cellular, bacterial
contamination.ii. Catheterized sample: increased transitional cell
content, iatrogenic hemorrhage.iii. Cystocentesis: least extraneous contamination,
more specific for changes in the tract,
Interpretations:• Epithelial cells in neoplasia diagnosis.
• more than 5RBCs/HPF indicate Hematuria.
• Leukocytes indicate infection(pyouria).
• More than 5/HPF.
• Elongated structures like casts indicate presence of Urolithiases.
• 10,000 bacterial rods/ml and >100,000 bacterial cocci/mL of urine are required to consistently find bacteria in a urine sample using light microscopy. and readings are normally below this.
Some of the drugs that excreted in urine also appear in crystals.e.g:
Sulfonamide crystals spherical with spikes.Ampicillin crystals form long needle lik
arrays.Calcium oxalate crystals are like colorless
squares indicate: UrolithiasesEthylene glycol toxicosis
Cytological ExaminationCytological Examination
• Staining:Staining:
– PapanicolauPapanicolau– Wright’sWright’s– ImmunoperoxidaseImmunoperoxidase– ImmunofluorescenceImmunofluorescence
Staining:
WRIGHT STAIN PROCEDURE:Make a air dried smear.Fix it in methanol for 30 sec.Take a disposable pipette and flood the Wright Stain
on the appropriately labeled slides.Wait for 3 min. Place 1ml oxidizing Wright Stain and Wright Stain
Buffer Mixture on Wright stained slides laying on slide rack (Displacing the Wright Stain off the slides with the pipette filled with Wright Stain/buffer mixture and viewing a metallic sheen on the top of slides.)____
Staining:
Wait for 6 min. Place slides in Wright Stain Buffer for 1.5
minutes.wait for 1.5 minutes. Rinse, dry and examine under oil immersion
lens,100x.
Parasites
Capillaria plica
Dioctophyme renale.
Trichuris
Casts
RBCs Cast - HistologyRBCs Cast - Histology
RBCs CastRBCs Cast
WBCs CastWBCs Cast
Tubular Epith. CastTubular Epith. Cast
Tubular Epith. CastTubular Epith. Cast
Granular CastGranular Cast
Hyaline CastHyaline Cast
Waxy CastWaxy Cast
Fatty CastFatty Cast
Crystals
Calcium Oxalate CrystalsCalcium Oxalate Crystals
Calcium Oxalate CrystalsCalcium Oxalate Crystals
Triple Phosphate CrystalsTriple Phosphate Crystals
Urate CrystalsUrate Crystals
Leucine CrystalsLeucine Crystals
Cystine CrystalsCystine Crystals
Bilirubin
Ammonium Biurate CrystalsAmmonium Biurate Crystals
Cholesterol CrystalsCholesterol Crystals
Cytology
carcinoma
Cytology: Polyoma (Decoy Cell)Cytology: Polyoma (Decoy Cell)
Cytology: Squamous Cell Ca.Cytology: Squamous Cell Ca.
Cytology: Renal Cell Ca.Cytology: Renal Cell Ca.
Cytology: Prostatic CarcinomaCytology: Prostatic Carcinoma
Cytology
Cytology: NormalCytology: Normal
Cytology: NormalCytology: Normal
Cytology: ReactiveCytology: Reactive
Cytology: ReactiveCytology: Reactive
Tubular Epithelial CellsTubular Epithelial Cells
WBCs
RBCs
Cocci
Transitional CellsTransitional Cells
Oval Fat BodyOval Fat Body
Transitional CellsTransitional Cells
LE CellLE Cell
Squamous cell
CytomegalovirusCytomegalovirus
YeastsYeasts
YeastsYeasts
BacteriaBacteria
Amorphous Substance
Bacilli
Mucous
Interpretations of Urine Analysis
ProteinuriaCasts & cellsHematuriaHemoglobinuriaMyoglobinuriaPyuriaBacteriuriaCrystalluriaGlycosuriaKetonuriaParasites
6.Interpretation Of
Diseases of
Urinary System
Common Findings in:Common Findings in:
Acute Tubular NecrosisAcute Tubular NecrosisMicroscopic:Microscopic:Renal tubularRenal tubular epithelial cellsepithelial cellsPathological casts.Pathological casts.
Microscopic:Microscopic:Renal tubularRenal tubular epithelial cellsepithelial cellsPathological casts.Pathological casts.
GlucoseGlucose
BilirubinBilirubin
KetonesKetones
S.G.S.G.
BloodBlood
pHpH
ProteinProtein
UrobilinogenUrobilinogen
DecreasedDecreased
+ / -+ / -
+ / -+ / -
Common Findings inCommon Findings in::
Acute GlomerulonephritisAcute Glomerulonephritis
Microscopic:Microscopic:Erythrocytes (dysmorphic)Erythrocytes (dysmorphic)Erythrocyte castsErythrocyte castsMixed cellular castsMixed cellular casts
Microscopic:Microscopic:Erythrocytes (dysmorphic)Erythrocytes (dysmorphic)Erythrocyte castsErythrocyte castsMixed cellular castsMixed cellular casts
GlucoseGlucoseGlucoseGlucose
BilirubinBilirubinBilirubinBilirubin
KetonesKetonesKetonesKetones
Specific GravitySpecific GravitySpecific GravitySpecific Gravity
BloodBloodBloodBlood
pHpHpHpH
ProteinProteinProteinProtein
UrobilinogenUrobilinogenUrobilinogenUrobilinogen
NitriteNitriteNitriteNitrite
Leukocyte EsteraseLeukocyte EsteraseLeukocyte EsteraseLeukocyte Esterase
IncreasedIncreased
IncreasedIncreased
Common Findings inCommon Findings in::Chronic GlomerulonephritisChronic GlomerulonephritisMicroscopic:Microscopic:Pathological castsPathological casts
(broad waxy casts, RBCs)(broad waxy casts, RBCs)
Microscopic:Microscopic:Pathological castsPathological casts
(broad waxy casts, RBCs)(broad waxy casts, RBCs)
GlucoseGlucoseGlucoseGlucose
BilirubinBilirubinBilirubinBilirubin
KetonesKetonesKetonesKetones
Specific GravitySpecific GravitySpecific GravitySpecific Gravity
BloodBloodBloodBlood
pHpHpHpH
ProteinProteinProteinProtein
UrobilinogenUrobilinogenUrobilinogenUrobilinogen
NitriteNitriteNitriteNitrite
Leukocyte EsteraseLeukocyte EsteraseLeukocyte EsteraseLeukocyte Esterase
DecreasedDecreased
IncreasedIncreased
IncreasedIncreased
Common Findings inCommon Findings in::
Acute PyelonephritisAcute Pyelonephritis
Microscopic:Microscopic:BacteriaBacteriaLeukocytesLeukocytesLeukocyte, granular, andLeukocyte, granular, and
waxy castswaxy castsRenal tubular epithelialRenal tubular epithelial
cell castscell casts
Microscopic:Microscopic:BacteriaBacteriaLeukocytesLeukocytesLeukocyte, granular, andLeukocyte, granular, and
waxy castswaxy castsRenal tubular epithelialRenal tubular epithelial
cell castscell casts
GlucoseGlucoseGlucoseGlucose
BilirubinBilirubinBilirubinBilirubin
KetonesKetonesKetonesKetones
Specific GravitySpecific GravitySpecific GravitySpecific Gravity
BloodBloodBloodBlood
pHpHpHpH
ProteinProteinProteinProtein
UrobilinogenUrobilinogenUrobilinogenUrobilinogen
NitriteNitriteNitriteNitrite
Leukocyte EsteraseLeukocyte EsteraseLeukocyte EsteraseLeukocyte Esterase
TraceTrace
PositivePositive
Common Findings inCommon Findings in::Nephrotic SyndromeNephrotic SyndromeMicroscopic:Microscopic:Oval fat bodiesOval fat bodiesFatty castsFatty castsWaxy castsWaxy casts
Microscopic:Microscopic:Oval fat bodiesOval fat bodiesFatty castsFatty castsWaxy castsWaxy casts
GlucoseGlucoseGlucoseGlucose
BilirubinBilirubinBilirubinBilirubin
KetonesKetonesKetonesKetones
Specific GravitySpecific GravitySpecific GravitySpecific Gravity
BloodBloodBloodBlood
pHpHpHpH
ProteinProteinProteinProtein
UrobilinogenUrobilinogenUrobilinogenUrobilinogen
NitriteNitriteNitriteNitrite
Leukocyte EsteraseLeukocyte EsteraseLeukocyte EsteraseLeukocyte Esterase
++++++++
Common Findings inCommon Findings in::
Eosinophilic CystitisEosinophilic Cystitis
Microscopic:Microscopic: Numerous eosinophilsNumerous eosinophils
(Hansel’s stain)(Hansel’s stain) NO significant castsNO significant casts..
Microscopic:Microscopic: Numerous eosinophilsNumerous eosinophils
(Hansel’s stain)(Hansel’s stain) NO significant castsNO significant casts..
GlucoseGlucoseGlucoseGlucose
BilirubinBilirubinBilirubinBilirubin
KetonesKetonesKetonesKetones
Specific GravitySpecific GravitySpecific GravitySpecific Gravity
BloodBloodBloodBlood
pHpHpHpH
ProteinProteinProteinProtein
UrobilinogenUrobilinogenUrobilinogenUrobilinogen
NitriteNitriteNitriteNitrite
Leukocyte EsteraseLeukocyte EsteraseLeukocyte EsteraseLeukocyte Esterase
++
Common Findings inCommon Findings in::
Urothelial CarcinomaUrothelial CarcinomaMicroscopic:Microscopic: Malignant cells on Malignant cells on urine cytology urine cytology (urine sample should(urine sample should be submitted be submitted separately toseparately to cytology, cytology, void or 24 hrs.)void or 24 hrs.)
Microscopic:Microscopic: Malignant cells on Malignant cells on urine cytology urine cytology (urine sample should(urine sample should be submitted be submitted separately toseparately to cytology, cytology, void or 24 hrs.)void or 24 hrs.)
GlucoseGlucoseGlucoseGlucose
BilirubinBilirubinBilirubinBilirubin
KetonesKetonesKetonesKetones
Specific GravitySpecific GravitySpecific GravitySpecific Gravity
BloodBloodBloodBlood
pHpHpHpH
ProteinProteinProteinProtein
UrobilinogenUrobilinogenUrobilinogenUrobilinogen
NitriteNitriteNitriteNitrite
Leukocyte EsteraseLeukocyte EsteraseLeukocyte EsteraseLeukocyte Esterase
++
Bacterial Cystitis:
Urinalysis often shows increased protein and hemoglobin
Increased numbers of WBC, RBC, and/or bacteria are consistent with cystitis.
Urine Culture
Urine Culture
Purpose:To identify the specific infectious agent.Antibiotic sensitivity test.
Urine Culture
Media Descriptions:C.L.E.D. (Cystine Lactose Electrolyte
Deficient Agar) is a non-selective medium that supports the growth of Gram (+) and Gram (-) species, specifically for enumeration of bacteria in urine.
URINALYSIS REPORT: