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DR.P.G.KONAPUR
PROFESSOR OF PATHOLOGY
V.M.K.V.MEDICAL COLLEGE
SALEM
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1.Formation of Urine
4.Preservation of urine
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• Urine is formed in the kidney.
• The functional unit of kidney is called nephrons,
• where in the ultrafilteration of plasma takes
place,
• followed by absorption of most of the water and
some of the solutes.
• The kidneys through the nephrons ---excrete
many of the waste products of the body
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3 NORMAL CONSTITUENTS AND COMPOSITION
OF URINE
Constituent’s grams/litre
A.Inorganic
• Chloride 9.0
• Phosphorous 2.0
• Sulfur 1.5
• Sodium 4.0
• Potassium 2.0
• Calcium 0.2
• Magnesium 0.2
• Iron 003
B.Organic
• Urea 25
• Uric acid 0.6
• Creatinine 1.5
• Ammonia 0.6
• Sugar not detected by benedicts test trace
• Ketone bodies trace
• Carbonates, bicarbonates & free carbonic acid trace
• Mucin & mucin like substances Diastase
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COLLECTION OF URINE SPECIMENS
• In. Improper collection---- may invalidate the results
• Containers for collection of urine should be wide mouthed, clean
and dry
• First morning sample –concentrated urine ---
chemical constituents
casts and crystals
• Random specimen
chemical screening
microscopic examinations
• 24 hours urine sample
quantitative estimation of proteins, sugars, electrolytes, and
hormones.
Urine specimen is collected in a clean 2 liters bottle with a
stopper. The first morning sample is not collected.
All the urine passed during the rest of the day and night and next
day I st sample is collected.
Volume is measured and immediately sent to the laboratory.
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Midstream urine specimen
• Urine should be collected in a clean, dry and,
preferably, sterilized container.
• urethral opening is cleaned with a moist cotton
swab.
• first 10 – 25 ml of urine is not collected
(discarded)
• since it contains urethral and prostatic
secretions which may be required if
investigating urethra and prostrate.
• First morning samples ---- these are the
concentrated samples and casts, crystals etc .if
present
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• Terminal urine sample patient voids the last portion of
urine in an open container.
• Urine specimen collection using a catheter. This
procedure is used for certain bacteriological tests
• Urine specimens from infants
• Urine can be collected into a plastic bag with an
adhesive mouth.
• The bag is fixed around the genitalia and left
in place for 1- 3 hours, depending on the
examination requested.
• Colostomy bags can be used
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PRESERVATION OF URINE
• Volume –
normal -- 1.2-2 L /day
the day is 3-4 times > night.
night is < 400 ml.
polyuria >2000ml / day.
Oliguria <500ml / day.
Anuria is total suppression of urine <100 ml per day.
• Appearance – color & turbidity
• 1. Color - normal ---- amber yellow ( to the presence
of urobilin,uroerythrin,and urochromes)
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1.Colorless---Very dilute urine
---Polyuria,diabetes
2.Yellow orange(high colored)--Concentrated urine
-----------Excess urobilin
------------Bile pigments
------------Intake of carrots
(Yellow foam ++)
3.Red/ smoky------Hemoglobin/
---- RBC
----MyoglobinBeetroot /
---------- anilinedyes
-------Menstrual contamination
-------porphyrins
4.Cloudy--------Phosphates & carbonates, Urates & uric acid
Pus cells
bacteria,yeast,spermatozoa
5.Milky-------Pyuria,Fat ,chyluria
6. Brown black----
Methemoglobin,,Homogenesticacid(alkaptonuria),Melanin
7. orange--------Bile pigments,Drugs – pyridium,rifampicin
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• 2. Turbidity – normal urine----clear.
d. pus
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• b. Odour: Normal-- aromatic odour
On standing--- ammonical
there is decomposition of urea
forming ammonia which gives a strong
ammonical smell..
• PROCEDURE
• Dip the litumus paper strips in the urine, remove and read the color
change immediately.
• Blue litmus turns red - acid
• Red litmus turns blue - alkaline
• Blue and red litmus turns purple - neutral
• nitrazine paper method :The Ph---- nitrazine paper which
are sensitive and specific in the pH range of 4.5 – 8.0 range.
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SPECIFIC GRAVITY
• degree of concentration or dilution of the
specimen.
• specific gravity measures the
concentrating and diluting abilities of the
kidney.
• The normal --------------1.015 and 1.025
(in a 24 hours specimen).
ESTIMATION OF SPECIFIC GRAVITY BY
URINOMETER
• urinometer: This is a weighted bulb-
shaped
a scale calibrated----- from 1.000 to
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PROCEDURE
Urine is poured into a cylindrical or conical glass so that the vessel is
nearly full.
Froth-- removed with a filter
• The instrument is floated in the urine and care should be taken to
see that it does not touch the slides.
• read on the urinometer scale at the junction of the urine with the
air.
• The reading is taken at eye level, the lowest part of the meniscus
being taken.
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• Correction for temperature-
• The urinometer is calibrated to read 1.000 in distilled
water at a specific temperature, indicated on each
instrument e.g. 15 C or 20 C.
• There is a change in the specific gravity of 0.001 for
each 3 C above and below this temperature. Therefore
add 0.001 to the reading for each 3 C above the
temperature for which the urinometer is calibrated,
• substract 0.001 for each 3 C, the temperature below the
standard temperature.
• For example - urinometer calibrated for 20 C
• Specific gravity of urine at 32 C is 1.001
• Corrected specific gravity {(32 – 20)/ 3 X 0.001} +
1.001 = 1.005
• Correction is also recommended when glucose or protein
are present. It is recommended that .003 be subtracted
from the urinometer reading for each 1000 mg / dl of
glucose or protein.
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• 1. SUGARS IN URINE:
• This test is not specific for sugars and is affected by most of the
reducing substances.
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• Dissolve crystalline copper sulphate in 100 ml of
distilled water. Dissolve sodium carbonate and
sodium citrate in 700 cc. of distilled water.
Slowly add the latter to the former solution with
constant stirring. When complete, make up the
volume to 1000ml with distilled water.
Procedure
• Take 5ml of benedicts reagent
• Boil for 3 – 5 minutes
• add to it 0.5ml (8 drops)of protein free urine.
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• Rothera’s Test for Acetone and
Acetoacetic Acid:
• Procedure:
– Take 5ml of urine in a test tube and saturate
it with ammonium sulphate.
– Add 1 crystal of sodium nitroprusside.Run
liquor ammonia carefully at the side of the
tube so as to form a layer on top of the
saturated urine.
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Result: a permanganate calomel red (pink –
purple) ring forms at the junction of the
two layers------positive.
• Negative result shows no ring or a brown
ring.
• Ferric Chloride Test for Diacetic Acid –
Gerhardt’s Test
• Harts Test for Beta-Hydroxybutyric Acid
• Colorimetric Reagent Strip Test
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• 3. Proteins in urine:
Normal <30 mgms / 100 ml (30 – 50
mgms/24 hours)
Tests for Detection of Proteins
• Semiquantitative precipitation tests:
1.The heat and acetic acid method,
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Bence Jones Proteinuria:
• Reappear on cooling
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OCCULT BLOOD IN URINE:
red blood cells
or
haemoglobin
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• Tests for Detection of Blood:
• 1. Microscopic Examination:
sediment------------RBC’S/HPF
• 2. Benzidine Test
PRINCIPLE
Heme acts as a catalyst when hydrogen peroxide is
mixed with benzidine.
REAGENTS
A: Saturated solution of benzidine in glacial acetic acid
B: Hydrogen peroxide
PROCEDURE
Mix equal parts of A and B in a test tube and an
equal amount of the mixed reagent.
RESULTS
• A blue color indicates the presence of hemoglobin.
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Colorimetric reagent strip
method
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BILE IN THE URINE:
The constituents
1. bilirubin (bile pigments),
2. bile salts,
3. urobilin and urobilinogen.
breakdown of hemoglobin.
linked to albumin---------- liver. This albumin-bound form,
which is also known as unconjugated bilirubin( indirect bilirubin)
insoluble in water and does not appear in the urine.
In the liver cells,--conjugated with glucuronic and sulfuric acids
to form water soluble conjugated bilirubin (direct bilirubin).
It is secreted into the bile and then excreted into the intestinal
tract through the bile duct.
This conjugated bilirubin -------------to urobilinogen.
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• certain liver diseases --unable to
conjugate all the bilirubin-----an increase
in both conjugated and unconjugated
bilirubin --bilirubinuria.
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Tests for detection of bile salts:
Hay Test
Procedure:
flowers of sulphur --sprinkled on the surface
of the urine,
Results:if bile salts are present they sink to
the bottom.
Otherwise they float on the surface.
This is due to the property of bile salts to
lower surface tension
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Tests for detection of bile pigments
1. Foam Test:
Shake urine in a test tube. If the foam on
top is yellow, bile pigments are present.
2. Gmelins test:
PROCEDURE:
1. Place ½ inch column of yellow nitric acid
in a test tube.
2. Overlay with equal amounts of urine
RESULT:
A play of colored rings, the most distinct being
green indicates the presence of bile pigments.
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3. Fouchets Test:
FOUCHETS REAGENT
Trichloroacetic acid – 25 gms
Distilled water - 100 ml
10% Ferric chloride solution – 10 ml.
PROCEDURE
1. Place 10 ml of acidified urine in a test tube.
2. Add 2.5ml of 10 % barium chloride.
3. Mix and filter.
4. Unfold the filter paper and spread it on a dry
filter.
5. Add 1 drop of Fouchets reagent to the residual
precipitate.
RESULT: A green or blue color indicates the presence of
bilirubin.
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Colorimetric Strip Reagent Test
• Principle: this test is base on the coupling of
bilirubin with diazotized 2, 4-
• dichloroaniline in a strong acid
medium to form a brown purple azobilirubin
• Compound. The color ranges
through various shades of tan.
• Procedure: the reagent strip is dipped into
fresh, uncentrifuged urine tapped to
• remove excess urine and after 20
seconds, compared to the color chart
• on the reagent strip bottle
• Result: the results are interpreted as negative,
small (+), moderate (++), and large (+++)
amounts of bilirubin.
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Tests for detection of
urobilinogen:
1. Qualitative Ehrlich’s Test
EHRLICH’S REAGENT
Paradimethylaminobenzaldehyde – 2 gms
20%5 HCL - 100 ml
PROCEDURE
a. Place 10 ml of urine in a test tube.
b. Add 2.5 ml of barium chloride (to remove
bilirubin).
c. Mix well and filter.
d. Add 0.5 ml of Ehrlich’s reagent
e. Allow it stand for 3 – 5 minutes.
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Results:
pink color
observable when viewed from the top of
the test tube. Against a white background
placed beneath the bottom of the test
tube.
cherry red color
Abnormally high amounts of This test
must be done with fresh urine or else
urobilinogen is oxidized on exposure to air
urobilin. Excessively cold water should
not be used in diluting
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MICROSCOPIC EXAMINATION
Qualitative technique:
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10-15 ml of urine ----from freshly mixed urine
specimen and centrifuged at a standard speed,
usually 1500 to 2000 rpm for 5 minutes.
• unorganized sediment
• Organized sediment
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A. Unorganized sediment - these are the crystals of various
substances present in the urine and they vary with the
pH of the urine .crystals of normal urine is formed as
the specimen cools.
1. Crystals in acidic urine:
a. Uric acid and Urates; –
crystals are seen when the urine is allowed to stand for
sometime and
are not seen in freshly passed urine.
Amorphous urates appear as red granules and are
dissolved by heat and sodium hydroxide but not acetic
acid.
Uric acid crystals vary in shape and are yellow brown
in colour and are not dissolved by heat, acetic acid or
HCL but are soluble when heated with sodium
hydroxide.
disturbances of uric acid metabolism
fevers where the urine is concentrated.
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B.Calcium Oxalate:
They are commonly found in diets rich in
tomatoes, spinach etc. they are typically
envelope shaped crystals but occasionally
appear dumb- bell shaped .they are insoluble in
strong Hcl.
c. Cystine Crystals:
highly refractile, hexagonal plates and are
soluble in Hcl but insoluble in acetic acid. They
are seen cystinosis which is an inborn error of
metabolism in which cystine crystals are found in
the urine, reticuloendothelial system and eyes.
d.Leucine:
slightly yellow, oily looking spheres with radial
and concentric striations .they are not soluble in
Hcl or ether .they are found in liver disorders.
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• e.Tyrosine:–
fine needles arranged in concentric sheaves, constriction at the
middle.
liver disorders.
• f.Sulpha crystals :-
patients taking sulphonamides.
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Alkaline Urine
Calcium Carbonate-Colorless
--Needles,Spheres,Dumbbells
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A- represents the residue of normal human urine, as seen under the
microscope.
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epithelial cells
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• Organized Sediment: the components of organized
sediment include casts, red blood cells, white blood cells,
epithelial cells, bacteria, yeast, parasites, spermatozoa
and artifacts.
• a. Casts:
• Casts are formed in the tubules and is composed of
proteinaceous material. They are washed out by the
glomerular secretion into the collecting tubules and the
bladder. They are cylindrical in shape with round or
broken ends with uniform diameter but varying in
length. They require acidic conditions, high salt
concentration, reduced urine flow and protein to be
formed. Practically all casts have a hyaline matrix, which
may or may not contain inclusions such as desquamated
cells.
• The casts are named according to the matrix of the
inclusions contained in them e.g. red blood cell casts.
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1. Hyaline casts:
are colorless,
semi-transparent and
occasionally refractile cylinders and are soluble in acetic
acid. They are seen when there is damage to the
glomerular capillary membrane, permitting leakage of
proteins through the glomerular filtrate. They are seen
in fever, orthostatic proteinuria, and emotional stress or
strenuous exercise.
2. Granular casts: are casts containing large or fine
granules embedded in coagulated protein. They are not
found in normal urine and their presences indicate
pyelonephritis. They are also seen in chronic lead
poisoning.
3. Epithelial casts: are formed of fused desquamated
tubular cells. They are coagulated protein in which are
embedded desquamated epithelial cells from the renal
tubules .they are seen in diseases where there is
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and heavy metal poisoning. 69
4. Red Blood Cell Casts:
are casts with red blood cells embedded
in the coagulated protein in the tubule.
Their presences indicate acute
inflammation or vascular disorder in the
glomerulus causing hematuria. They are
seen in pathological conditions such as
acute glomerulonephritis, renal infarction
and collagen vascular disorder.
5. White Blood Cell Casts (Pus cell)
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• CELLS:
• a. Red blood cells: Normally 1-2 red blood cell
are found per high power field
• .they appear pale , light refractive, biconcave
discs when viewed under high power
magnification .they have no nuclei. Red blood
cells in fresh, unstained sediment appear pale in
color; in urine that is not fresh, they are pale or
colorless shadow cells .in concentrated urine,
they may be small and crenated; and in dilute
urine, they are large and swollen and sometimes
rupture to produce ghost cells.
• b. White cells
• c. Epithelial Cells: Normally a few epithelial
cells occur in the urine .A marked increase
• is these cells in the urine is seen destruction
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urinary tract. 71
• Quantitative Evaluation of the urine
sediment – Addis count
• The Addis count is a quantitative
measurement of the excretion of red cells,
leucocytes and casts in the urine during a
12 hour period.
• e. Bacteria:
Bacteruria is considered significant when
there is the presence of 100,000 or more
bacteria per ml of urine specimen.
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DETECTION OF BACTERIA
• Microscopic Examination:
sediment - >20 or more bacteria per high
power field may indicate a urinary tract infection
Reagent strips:
PRINCIPLE:
This test depends upon the conversion of nitrate
to nitrite by the action of gram negative bacteria
in urine. At the acid pH of the reagent area,
nitrite in the urine reacts with p-arsanilic acid to
form diazonium compound. This compound in
turn couples with 1, 2, 3, 4-tetra
hydrobenzoquinolin-3-ol to produce pink color.
• Procedure: The strip is dipped in the urine
specimen for 5 seconds.
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Results:
uniform pink color --positive result
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