By Dr.

SUJIT DIVYA RANJAN BISWAL

URINE ANALYSIS

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WHAT IS URINE ANALYSIS?
 IT IS A SERIES OF URINE EXAMINATION
PERFORMED FOR MEDICAL DIAGNOSIS.
 ONE OF THE OLDEST LABORATORY
PROCEDURE PRACTISED IN THE FIELD
OF MEDICINE.
 ALSO KNOWN AS URINE R&M
(URINE ROUTINE AND MICROSCOPY)

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Unknown Author is
licensed under CC
BY-NC-ND
COMPOSITION OF URINE
INDICATION OF URINE EXAMINATION
 SUSPECTED RENAL DISEASE LIKE IN GLOMERULONEPHRITIS, KIDNEY FAILURE.
 DETECTION OF URINARY TRACT INFECTION.
 DETECTION OF METABOLIC SYNDROME LIKE IN DIABETES MELLITUS.
 DIAGNOSIS IN PREGNANCY.
 DIFFERENTIAL DIAGNOSIS FOR JAUNDICE LIKE DETECTION OF URINE
BILLINOGEN.
PRECAUTION FOR SAMPLE COLLECTIONS
 AVOID STRENOUS PHYSICAL EXERCISE WITHIN 72 HOURS OF URINE
COLLECTION.
 AVOID URINE ANALYSIS WHILE MENSTRUATION.
 COLLECT THE URINE AFTER DISCARDING FIRST PORTION OF MICTURITION
(MIDSTREAM TECHNIQUE) TO REDUCE CONTAMINATION OF FROM URETHRAL
AREA.
 CLOSE CONTAINER COMPLETELY AND WRITE PATIENT’S DETAILS CLEARLY ON
LABEL.
METHODS OF URINE COLLECTION
 Collection of freshly voided, clean catch, mid stream specimen of urine
via: -
a. Catheterization
b. Suprapubic Aspiration
 Should be analyzed within 2 hours of urine collection else requires
refrigeration.
COLLECTION VIA CATHETERIZATION

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COLLECTION VIA SUPRAPUBIC ASPIRATION
URINE STORAGE AND TRANSPORTAION KIT
ADEQUACY OF URINE SPECIMEN
 Capacity – 20 to 30 ml ( should have 50 ml capacity )
 Should be properly labelled and disposable container.
 Clean, leak proof, wide mouth, transparent.
 For routine examination – wide base container.
 For microbiological examination – sterile container.
FOR ROUTINE EXAMINATION

WIDE BASE CONTAINER

STERILE CONTAINER
URINE SHOULD BE PROCESSED WITHIN 2
HOURS TO PREVENT FOLLOWING CHANGES:-
 Increase in pH.
 Formation of crystals.
 Loss of ketone bodies.
 Decrease in glucose.
 Bacterial proliferation.
 Oxidation of some chemicals.
 Disintegration of cellular elements.
PRESERVATIVES FOR URINE SAMPLE
Why Preservatives are necessary?
 Ideally urine examination should be done within 1-2 hours of sample collection.
 Then, after sometime specimen are left in room temperature.
 As a result of which bacteria grows in urine sample leading to decomposition of urine sample.
 To prevent this decomposition, we need to add preservatives.
EXAMPLES OF PRESERVATIVES USED ARE: -
a) Toluene : 2ml/100 ml of urine (best preservative)
b) Thymol : one crystal
c) Boric acid : 1 gm/ 100 ml of urine
d) Formalin : 3 drop/100 ml of urine
e) Chloroform : 5ml/100ml of urine
f) Commercial preservative tablets which release formaldehyde : 3 tablet/100ml of urine
WHAT ALL INVESTIGATIONS ARE NEEDED
FOR URINE SAMPLE?
Routine examinations of urine (Urinanalysis)
 Physical examination of urine
 Chemical examination of urine
 Microscopic examination of urinary sediment

Cytological examination
Hormonal assay
Culture and sensitivity
Physical examination of urine : -
 Volume
 Color
 Appearance
 Sediment formation
 Odor
 Specific gravity
 pH
VOLUME OF URINE(FOR 24 HOURS) COLOR OF URINE
 Normal- 600 to 2000 ml  White- pus , phosphates
 < 500 ml- Oliguria  Pink to red- hemoglobinuria, myoglobinuria,
hematuria, excess beet root intake.
 < 100 ml- Anuria
 Brownish black- Alkaptonuria, intake of
 > 2500 ml- Polyuria chloroquine, iron, metronidazole
 Low volume can be seen in some renal and post  Blue to green- pseudomonas infection, biliverdin,
renal condition. intake of methylene blue and phenyl salicylate.
 High volume can be seen in some clinical  Yellow, dark yellow, brownish yellow to orange-
condition like diabetes. intake of food color(yellow) and vitamin b
complex, concentrated urine, presence of water
soluble bilirubin in hepatic and post hepatic
condition.
APPEARANCE OF URINE pH OF URINE

 Usually clear sometimes cloudy.  Usually acidic at the range of 4.8-7.5.

 Turbid- Presence of abnormal number of  pH < 4.8- more acidic urine seen in protein
leukocytes, epithelial cells, bacteria and rich diet, fevers, acidosis, ketosis.
precipitation of amorphous phosphates in
 pH > 7.5- intake of large quantities of
alkaline urine and amorphous urates in
acidic urine. citrus fruits, alkaline therapy, severe
vomiting, urinary retention
 Hazy- due to mucus
 Smoky- due to red blood cells.
SEDIMENT FORMATION AFTER
ODOR
COLLECTION
 Usually there is no or very little sediment  Usually it is characteristic aromatic.
formation.
 FRUITY- Acidosis, ketosis in severe
 Moderate to high sediment proportion occurs
in :- diabetes mellitus
a. Precipitation of amorphous phosphates in  AMMONIACAL- Cystitis, urinary retention
alkaline urine =>appears white color & decomposition of urea
b. Precipitation of amorphous urates in to ammonia by bacterial
acidic urine=>appears pinkish white color
c. Presence of leukocytes (pus cells) , action due to storage at
epithelial cells, red blood cells, casts. room temperature.
 FOUL SMELLING- UTI
LOW SPECIFIC GRAVITY HIGH SPECIFIC GRAVITY

 Diabetes Insipidus  Diabetes Mellitus

 Chronic Nephritis  Fever
 Acute Nephritis
PHYSICAL EXAMINATION OF URINE
 REQUIREMENTS
a) Pasteur pipettes
(b)
b) Ordinary filter papers
c) Measuring cylinder (a)
d) Litmus papers and pH papers
e) Urinometer

(e)
 PROCEDURES
a) Observe some of the physical aspects of the urine specimen and note down in the note book
like color, appearance, odor, sediment (if present).
b) Measure the volume of urine and note down in the note book.
c) For reaction and pH : Place a drop of urine by using a Pasteur pipette ( or a glass rod ) on a
blue litmus paper and note down the reaction.
 SPECIFIC GRAVITY DETERMINATION
a) Mix urine well and fill the container three fourth full of urine.
b) Remove all foam by using a filter paper.
c) Float the urinometer in the urine. Rotate it carefully so that it can be prevented from
touching the bottom or sides of the container
d) Note the specific gravity reading from the scale
CHEMICAL EXAMINATION OF URINE
ROUTINE URINALYSIS INCLUDES CHEMICAL EXAMINATION OF FOLLOWING:-
a. Determination of protein
b. Determination of glucose
c. Determination of ketone bodies
d. Determination of occult blood
e. Determination of bile pigment
f. Determination of bile salts
g. Determination of urobillinogen
REQUIREMENTS FOR CHEMICAL EXAMINATION OF URINE ARE : -
a) Centrifuge tubes, Pasteur pipettes, test tubes( 10 x 75, 15 x 125, 20 x 150 mm), beakers of
250 or 500 ml, graduated pipettes ( 5 ml ) and test tube racks
b) Benedict’s qualitative reagent, Fouchet’s reagent, Ehrlich reagent
c) Sulfosalicylic acid
d) Sulfur powder, Benzidine powder, Hydrogen peroxide, Glacial acetic acid
 Sulfur powder is used for bile salts detection
 Benzidine powder, hydrogen peroxide and glacial acetic acid are used for occult blood
detection
DETERMINATION OF PROTEIN
HEAT COAGULATION METHOD AND ACID METHOD
PRINCIPLE- Heat coagulates the proteins present in soluble form. Denatured protein is less soluble
and is precipitated. In case of acid method, protein coagulates by adding sulfosalicylic acid.
NOTES: -
a) Urine should be clear. If it is turbid, it is necessary, to centrifuge it. In that case supernatant is
tested for protein and sediment for the microscopic examination. Even after centrifugation if the
urine is turbid then filter it.
b) If urine is alkaline, then add few drops of glacial acetic acid and make it slightly acidc.
REAGENTS REQUIRED: -
a) 3 g/dl of sulfosalicylic acid
b) Glacial acetic acid
PROCEDURES: -
 ACID METHOD
a) Transfer 3-4 ml of centrifuged ( or filtered ) urine to a small test tube ( 10 x 75 mm ).
b) Add 2-3 drops of sulfosalicylic acid on the top of the specimen
c) Observe for turbidity after 5 minutes.
 HEAT COAGULATION METHOD
a) Place 5-10 ml of clear urine in a test tube ( 20 x 150 mm ).
b) Boil the upper portion over a flame.
c) If turbidity develops, add 1-2 drops of glacial acetic acid => If the turbidity is due to phosphate
precipitation it will be cleared.
d) Reboil the specimen.
 OBSERVATIONS: -
i. No formation of turbidity=> Protein is absent
ii. Formation of turbidity=> Protein is present.
iii. If proteins are present, grade the result according to degree of turbidity as trace, +, + +,
+ + + and + + + +.
Conditions leading to proteinuria are: -
a) Excessive muscular exercise, excessive protein ingestion, prolonged cold bath
b) Dehydration, heart disease, severe diarrhea
c) Any kidney diseases
d) Lesions of renal pelvis, bladder and prostate
DETERMINATION OF GLUCOSE
BENEDICT’S QUALITATIVE TEST
PRINCIPLE- When Benedict’s reagent is heated with urine, glucose present in the urine
reduces cupric ions ( present in the reagent ) to the cuprous ions. Alkaline medium is provided to
the reaction by sodium carbonate present in the reagent. Original color of reagent is blue.
NOTE: -
This test is non specific as this test can show positive result by any reducing substances other
than glucose. Hence if Benedict’s reagent is positive, the it is necessary to perform glucose
oxidase ( uristix ) test to confirm wether it is due to only glucose
 PROCEDURE: -
1. Pipette 5 ml of Benedict’s reagent in a test tube.
2. By using Pasteur pipette, add 8 drops of urine
3. Heat carefully on the flame of a gas burner or spirit lamp for 5-10 minutes.
4. Cool under tap water or by placing in a beaker containing tap water.
 OBSERVATIONS: -
Conditions leading to glucosuria are: -
a) Diabetes mellitus
b) Renal glycosuria
c) Brain injury
d) Hyperactivity of some of the endocrine glands
DETERMINATION OF LACTOSE
OSAZONE TEST
PRINCIPLE- Monosaccharides and the two disaccharides i.e. lactose and maltose react with
phenylhydrazine hydrochloride in acidic medium and after placing in boiling water bath, to form
characteristic phenylhydrazone crystals. Lactose gives lactosazone crystals which are specific
and can be identified by microscopic examination. These crystal look different from osazone
crystals formed by monosaccharide and maltose.
PROCEDURE: -
 Transfer about 5 ml of urine to a test tube.
 Make it just acidic to litmus paper by adding few drops of glacial acetic acid
 Add about one gram of the powder mixture of sodium acetate and phenylhydrazine
hydrochloride.
 Place in a boiling waterbath for 30 minutes.
 Cool by placing in a beaker containing tap water.
 Observe collected deposit under microscope by making coverslip preparations.
DETERMINATION OF GALACTOSE
ORTHOTOLUIDINE TEST
PRINCIPLE- Orthotoluidine reagent reacts with galactose in hot acidic medium to form a green
colored compound
PROCEDURE: -
 Pipette 5 ml of orthotoluidine reagent in a test tube.
 Add 0.5 ml of urine.
 Place in a boiling waterbath for 10 minutes.
 Observe the color.
OBSERVATIONS: -
 No green color ( original color of reagent persists i.e. pale yellow color ) : No Galactose
 Green color : Galactose is present
DETERMINATION OF KETONES
ROTHERA TEST: -
PRINCIPLE- Nitroprusside used in this test reacts with both acetone and acetoacetic acid in the
presence of alkali to produce a purple colored compound.
REQUIREMENTS
 Test tubes
 Rothera’s powder mixture ( 0.75 g of sodium nitroprusside + 20 g of ammonium sulfate ).
Stored in a wide mouth glass container at room temperature.
 Liquor ammonia solution
PROCEDURE: -
1. Transfer about 5 ml of urine to a test tube using a Pasteur pipette
2. Add about 1 g of Rothera’s powder mixture and mix well
3. Layer over the urine 1- 2 ml of concentrated ammonium hydroxide ( allow it to flow gently
down the side of the inclined test tube )
4. Observe for pink purple ring at the interface
OBSERVATIONS
1. No appearance of purple ring – Ketone bodies absent
2. Appearance of purple ring – Ketone bodies present
 Conditions leading to ketonuria are: -
a) Severe diabetes
b) Starvation
c) Fevers
d) After prolonged diarrhea and vomiting
DETERMINATION OF OCCULT BLOOD
OCCULT BLOOD TEST: -
PRINCIPLE- The peroxidase activity of hemoglobin ( present in urine ) decomposes hydrogen
peroxide and the liberated oxygen oxidises benzidine to form a green-blue colored complex.
REQUIREMENTS: -
a) Test tubes
b) Graduated pipettes of 5 ml
c) Pasteur pipettes
d) Benzidine powder, Glacial acetic acid, Hydrogen peroxide ( 30 % , v/v )
PROCEDURES: -
1. Place pinch of benzidine powder in a test tube
2. Add 2 – 3 drops of glacial acetic acid and mix well.
3. Add about 2 ml of hydrogen peroxide solution and mix well. Transfer 1 ml of supernatant to
a test tube labeled as T.
4. Add 0.5 ml of urine and mix well.
5. Observe the color of the mixture after 5 minutes.
NEGATIVE
Conditions seen in hematuria: -
 Acute nephritis
 Calculi
 Renal cancer
 TB of Kidney and other chronic infections
Conditions seen in hemoglobinuria: -
 Hemolytic poisons
 Severe burns
 Blood transfusion reaction
DETERMINATION OF BILE SALTS
( HAYS TEST )
PRINCIPLE- Bile salts ( when present ) lower the surface tension of urine. When sulfur powder
is added on the surface of urine, sulfur particles sink to the bottom of the test tube. In the case of
a normal urine sample, sulfur particles float on the surface of urine.
PROCEDURE: -
a) Place about 10 ml urine in a test tube.
b) Sprinkle a little dry sulfur powder on to the surface of urine.
c) Observe the sulfur particles.
OBSERVATIONS: -
a) Sulfur particles sink to bottom : Bile salts are present
b) Sulfur particles remain floating : Bile salts are absent
NEGATIVE TEST POSITIVE TEST
DETERMINATION OF BILE PIGMENTS AND
UROBILLINOGEN
PRINCIPLES OF THE TESTS
1) BILE PIGMENT DETERMINATION ( HARRISON SPOT TEST ) – When barium
chloride reagent is added to urine , it combines with sulfate radicals in urine and precipitate
of barium sulfate is formed. If bile pigments are present in urine, they will adhere to these
large molecules. Ferric chloride present in Fouchet’s reagent then oxidizes yellow bilirubin
( in the presence of trichloroacetic acid ) to green biliverdin.
2) UROBILINOGEN DETERMINATION (EHRLICH’S TEST ) – Urobilinogen reacts
with p-dimethylaminobenzaldehyde in acidic medium to form pink colored compound.
REQUIREMENTS: -
1) Centrifuge tubes or test tubes
2) Pasteur pipette
3) 10 g/dl barium chloride
4) Fouchet’s reagent
5) Ehrlich reagent
6) Whatman No. 1 filter paper
PROCEDURE:-
1) Place 3 – 4 ml of urine in a centrifuge tube ( about 1/3rd full ) by using a Pasteur pipette.
2) Add equal amount of 10 g/dl of barium chloride, mix well.
3) Centrifuge at 1500 RPM for 10 minutes ( or filter by using Whatman No.1 filter paper )
4) Place supernatant in another test tube for urobilinogen test.
5) Add 1 – 2 drops of Fouchet’s reagent to the sediment ( or to the precipitate on a filter paper )
6) Add about 0.5 ml of Ehrlich reagent to the supernatant.
CAUSES OF INCREASED UROBILINOGEN ARE: -
 Cirrhosis
 Hemolytic jaundice
 Hepatic congestion
 Paralytic enterocolitis
CHEMICAL EXAMINATION OF URINE BY
USING MULTISTIX REAGENT STRIP
REQUIREMENTS-
 Uncentrifuged urine
 Multistix reagent strips
PROCEDURE-
 Dip the test areas of the strip in the urine specimen ( fresh, well mixed and uncentrifuged )
 Remove excess of the urine by tapping the edge of the strip against the container.
 Compare the test areas closely with corresponding color charts.
 Note down the findings.
CHEMICAL EXAMINATION OF URINE BY A
REFLECTANCE PHOTOMETER
Chemical examination of urine by urine strip methods may be affected by the following
variables: -
 Differences in lightning conditions
 Discoloration of the urine by bilirubin, blood or other constituents.
 Differences in individual skill at color matching and sometimes lack of concentration and
failure to keep to a specified time.
Hence to avoid above variables, reflectance photometer is used such as UROTRON RL 9
SYSTEM.
UROTRON RL 9 SYSTEM
 11 channel reflectance photometer.
 Involves evaluation of urine test strips in a reflectance photometer.
 On every strip there are 11 various reaction areas and according to the urinary component determined they are for: -
a. Erythrocytes
b. Bilirubin
c. Urobilinogen
d. Ketones
e. Glucose
f. Proteins
g. pH
h. Nitrite
i. Leukocytes
j. C & M component
MICROSCOPIC EXAMINATION OF URINE
PRINCIPLE- Microscopic elements present in urine ( in suspension ) are collected in the form
of deposit by centrifugation. A small drop of sediment is examined by making a coverslip
preparation under microscope.
REQUIREMENTS: -
 Centrifuge tubes or test tubes
 Glass slides
 Coverslips
 Pasteur pipettes
INSTRUMENTS: -
 Centrifuge
 Microscope
SPECIMEN- Freshly voided midstream morning urine sample.
PROCEDURES:-
 Mix the urine and pour into a centrifuge tube ( or small test tube ) until it is ¾ th full
 Centrifuge with another balanced test tube for 5 minutes at 2500 RPM
 Pour off the supernatant quickly and completely into another test tube
 Resuspend the deposit by shaking the tube.
 Place one drop of deposit on a glass slide.
 Cover it with a coverslip and mark it with the identification number.
 Observe under low power microcope.