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Urine Examination & Analysis

Assigned by: Dr.Javeria Khan Presented by: Dr.Noor-ul-Ain Sarwar

Contents
1. 2. 3. 4. 5. 6. Collection of sample and preservation. Gross Examination. Determination of specific gravity. Biochemical Analysis. Microscopic Examination. 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: Abnormal specific gravity Proteinurea. Cast Leukocyte Erythrocytes

A. Kidney diseases:

B. Bladder infection:
Proteinuria Leukocytes Bacteria

C.

Neoplasia:
Exfoliated neoplastic cells Hematuria

C. 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. Acidosis I. Alkalosis

Collection of Urine

1. Collection of urine sample


Collection 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 hours It slightly increases specific gravity. It also interferes with tests using enzymes for reaction.

Chemical preservation
Certain 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.
a) Volume: species

Gross Examination

Normal values in liters 4.7 14.2 0.9

Horse Cattle Sheep, Goat and Dog

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 Acute renal disease Urolithiases fever Shock Severe nephritis Edema

Pathological:

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.

Interpretation
Various 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 Biliverdin Phenol poisoning

vi. Green:

c. Odor Interpretation
Ammonia-like : Urea-splitting bacteria Foul, offensive : Old specimen, pus or inflammation Sweet : Glucose Fruity : Ketones Maple syrup-like: Maple Syrup Urine Disease

d. Color:
Colorless Deep Yellow Yellow-Green Red Brownish-red Brownish-black Diluted urine Conc. Urine, Riboflavin. Bilirubin / Biliverdin Blood / Hemoglobin Acidified Blood (Actute GN) Homogentisic acid (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 20-25C. 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
Horse Cattle Sheep & Goat Dog Birds

Specific Gravity
1.020 1.050 1.025 1.045 1.015 1.024 1.015 1.045 1.005 1.020

Interpretation
i. Increased specific gravity:
Acute interstitial nephritis Cystitis Liver failure Diabetes mellitus Glomerulonephritis Chronic interstitial nephritis Diabetes insipidus Pylonephritis uremia

ii. Decreased specific gravity:

Chemical 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 Horses Cattle Sheep & Goat Dog & Cat Birds pH 8 7.4-8.4 7-8.2 5.5-7 6-8

b.

Protein Determination:

For Protein determination: a) Reagent strips(dip sticks) b) Acid prepitation Tests: i. Nitric Acid Precipitation Test OR Roberts Tests: Principle: Precipitation of protein occur by concentrated acid

Procedure:
Take 2ml of Roberts 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 Urolithiases Inflammation, Hemorrhage, Glomerular disease.

c. Glycosuria determination
Now a days strips and glucometers are available. Chemical method is Benedicts test. Benedicts 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 Benedicts Reagent


Copper sulphate Sodium citrate Sodium carbonate Distilled water (To make volume) 17.3 g 173 g 100 g 1000 mL

Procedure:
Take 5mL of Benedicts 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 Negative Blue color Orange to brick red or brown

Interpretation
Hyperglycemia After general anesthesia Chronic liver diseases Enterotoxaemia in sheep.

d. Ketonurea Determination
Ross test:
Principle: It is based on that the sodium nitroprusside is decomposed to: Sodium ferrocyanide Sodium nitrate Ferric hydroxide Results: 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 mellitus High fat diet Starvation Impaired liver functions After ether chloroform anesthesia Milk 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 urine Add 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. Neoplasia Obstruction 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. Hyperthyroidism Hypervitaminosis 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:
Urolithiases Ethylene glycol toxicosis

Cytological Examination
Staining:
Papanicolau Wrights Immunoperoxidase Immunofluorescence

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 - Histology

RBCs Cast

WBCs Cast

Tubular Epith. Cast

Tubular Epith. Cast

Granular Cast

Hyaline Cast

Waxy Cast

Fatty Cast

Crystals

Calcium Oxalate Crystals

Calcium Oxalate Crystals

Triple Phosphate Crystals

Urate Crystals

Leucine Crystals

Cystine Crystals

Bilirubin

Ammonium Biurate Crystals

Cholesterol Crystals

Cytology

carcinoma

Cytology: Polyoma (Decoy Cell)

Cytology: Squamous Cell Ca.

Cytology: Renal Cell Ca.

Cytology: Prostatic Carcinoma

Cytology

WBCs

Cytology: Normal

Cytology: Normal

Cytology: Reactive

Cytology: Reactive

Tubular Epithelial Cells

WBCs

RBCs

Cocci

Hematuria

Transitional Cells

Oval Fat Body

Transitional Cells

LE Cell

Squamous cell

Cytomegalovirus

Yeasts

Yeasts

Bacteria

Amorphous Substance

Bacilli

Mucous

Interpretations of Urine Analysis


Proteinuria Casts & cells Hematuria Hemoglobinuria Myoglobinuria Pyuria Bacteriuria Crystalluria Glycosuria Ketonuria Parasites

6.Interpretation Of
Diseases of

Urinary System

Common Findings in:

Acute Tubular Necrosis


Microscopic: Renal tubular epithelial cells Pathological casts.
Glucose Bilirubin Ketones Decreased +/S.G. Blood pH +/Protein Urobilinogen

Common Findings in:

Acute Glomerulonephritis
Microscopic: Erythrocytes (dysmorphic) Erythrocyte casts Mixed cellular casts
Glucose Glucose Bilirubin Ketones Specific Gravity Specific Gravity Blood Increased Blood pH Protein Increased Protein Urobilinogen Nitrite Leukocyte Esterase Leukocyte Esterase

Common Findings in:

Chronic Glomerulonephritis
Microscopic: Pathological casts (broad waxy casts, RBCs)
Glucose Glucose Bilirubin Bilirubin Ketones Ketones Specific GravityDecreased Blood pH pH Protein Protein
Increased Increased

Urobilinogen Urobilinogen Nitrite Nitrite Leukocyte Esterase Leukocyte

Common Findings in:

Acute Pyelonephritis
Microscopic: Bacteria Leukocytes Leukocyte, granular, and waxy casts Renal tubular epithelial cell casts
Glucose Glucose Bilirubin Bilirubin Ketones Ketones Specific Gravity Blood pH pH Protein Protein
Trace

Urobilinogen Urobilinogen Nitrite Nitrite


Positive

Leukocyte Esterase Leukocyte

Common Findings in:

Nephrotic Syndrome
Microscopic: Oval fat bodies Fatty casts Waxy casts
Glucose Glucose Bilirubin Bilirubin Ketones Ketones Specific Gravity Blood pH pH Protein Protein
++++

Urobilinogen Urobilinogen Nitrite Nitrite Leukocyte Esterase Leukocyte

Common Findings in:

Eosinophilic Cystitis
Microscopic: Numerous eosinophils (Hansels stain) NO significant casts.
Glucose Glucose Bilirubin Bilirubin Ketones Ketones Specific Gravity Blood pH pH Protein Protein Urobilinogen Urobilinogen Nitrite Nitrite Leukocyte Esterase Leukocyte
+

Common Findings in:

Urothelial Carcinoma
Microscopic: Malignant cells on urine cytology (urine sample should be submitted separately to cytology, void or 24 hrs.)
Glucose Glucose Bilirubin Bilirubin Ketones Ketones Specific Gravity Blood pH pH Protein Protein Urobilinogen Urobilinogen Nitrite Nitrite Leukocyte Esterase Leukocyte
+

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:

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