Analysis of Normal Urine.pptx

INTRODUCTION
• Urine is the excretory waste product formed by the
kidneys
• It reflects the overall metabolic & kidney functions of the
body
• Its analysis, therefore is important in evaluating kidney
functions as well as in the diagnosis of many other
diseases

URINE ANALYSIS
• A complete analysis of urine includes the following:
• Bacteriological analysis
• Done by microbiologists to identify the infecting organism by
culture studies
• Microscopic analysis
• Done to detect presence of pus cells, RBCs, casts etc., which
may be pathological many a times

URINE ANALYSIS
• Biochemical analysis
A) Routine qualitative examination for detecting pathological
variations in physical properties & presence of abnormal
constituents
B) Quantitative estimation of many biochemical constituents
like proteins, creatinine, calcium, etc.
Routine qualitative examination is one of the most commonly
done examinations as a preliminary starting point in evaluating
kidney diseases.

1. Random specimen (at any time)
 Useful for routine screening but may give false results
due to dietary intake or physical activity just prior to the
collection of the specimen.
 It’s not useful for quantitative analysis.
2. First morning specimen
 Valuable as it’s concentrated to reveal abnormalities and
formed elements
 It’s free of dietary influences and changes due to
physical activities
 Prevents false negative pregnancy test
 Useful in evaluation of orthostatic proteinuria.
3. 24 hr’s collection
Used for quantitative determination and for evaluation the
kidney function.

URINE SAMPLE
• ‘Mid stream morning sample’
 To collect this sample, the patient is advised to discard the
initial part of the first voided urine in the morning after
getting up and then to collect 15-20 mL of mid stream urine
flow in a clean glass or plastic container.
 It should be immediately analysed or within a few hours of
collection.
 In the meantime it should be refrigerated

URINE SAMPLE
 This sample is commonly used in routine examination
 For bacteriological examination, a sample is collected in a
sterile container which is to be collected from the laboratory
• 24-hour Urinary Sample
 This sample is collected for 24 hours from the time after
discarding the first morning sample.
 A preservative is used while collecting the sample.
 Commonly, such a sample is used for quantitative
estimation of different constituents, e.g. creatinine, proteins,
calcium, etc.

URINE SAMPLE
• Random Urine Sample
 This sample is collected on the spot at any time of the day.
 It has limited utility and is generally used for detection of
glucose, ketone bodies and proteins.

URINARY PRESERVATIVES
Concentrated HCI
• 10 ml concentrated HCl is adequate for a 24-hour
specimen.
• It is suitable for urea, ammonia, protein. nitrogen and
calcium estimations.
• No preservative is needed for immediate analysis of a
spot urine sample.

URINARY PRESERVATIVES
• Thymol
• 5 mL of 100 g/L solution in isopropanol is suitable for
estimations of sodium, potassium chloride, bicarbonate,
calcium, phosphorus, urea, amino acids, creatinine,
protein, etc. but not for 17-ketosteroids.

Steps in basic urine analysis
Three steps analysis:
First: physical characteristics of urine are noted and
recorded.
Second: urine sediment is examined under microscope to
identify the components of sediments.
Third: series of chemical tests is run.

• direct visual observation.
• Normal fresh urine: Color: pale or dark yellow-amber, clear.
• Vol:800 - 2500 ml/24hr.
• Physical examination involves:
1. Color
2. Transparency
3. Odour
4. Volume
5. pH
6. Specific gravity

1. Appearance: (color and clarity)
Normal urine color has a wide range of variation
ranging from pale yellow, straw, light yellow,
yellow, dark yellow amber due to urochrome
pigment,
The color is affected By :
 Concentration of urine, pH, Metabolic activity,
Diet intake and Some Drugs

1- Color:
• Many things affect urine color, including fluid balance, diet,
medicines, and diseases.
• Color intensity of urine correlates to concentration.
• Darker color means more concentrated sample.
• Amber yellow Urochrome (derivative of urobilin, produce
from bilirubin degradation, is pigment found in normal urine).
• Colorless due to reduced concentration.
• Silver or milky appearance Pus, bacteria or epithelial cells
• Reddish brown Blood (Hemoglobin).
• Yellow foam Bile or medications.
• Orange, green, blue or red medications.
• Vitamin B supplements can turn urine bright yellow.

1. Colorless or pale yellow 2. Dark yellow, Amber, orange
 High fluid intake
 Reduction in perspiration.
 Using of diuretic.
 Nervousness
 Alcohol ingestion
 Diabetes Mellitus.
 Diabetes Insipidus
 (Low level of antidiuretic
hormone).
 Low fluid intake.
 Excessive sweating
 Carrots or vitamin (A)
 Dehydration (burns, fever).
 Pyridium and nitrofurantoin,
rifampicin (drugs).

3. Brownish yellow
 Bilirubin on shaking yellow foam will appear.
 Urobilin on shaking the foam has no color.
4. Yellow – green
 Bilirubin oxd. Biliverdin (greenish).
 Which give a yellow foam & (- ve) test for bilirubin
5. Blue – Green
 Pseudomonas Infection

5. Pink – Red
Due to the presence of
fresh blood or Hb, fresh
blood will give smoky
color while Hb gives clear
reddish urine.
Both may be due to
 Trauma
 Calculi
 Urinary tract infection
 Menstrual
contamination.
6. Dark brown
 Methemoglobin if
bloody sample long
standed, Hb will be
oxidized.
 Melanin
7. Black Urine
 Alkaptonurea, a
disease of tyrosine
metabolism.

2- Clarity (transparency)
Normal urine clear or transparent,
any turbidity will indicate.
 WBCs (pus).
 RBCs
 Epithelial cells
 Bacteria
 Casts
 Crystals
 Lymph
 Semen.

3. Volume
Adult urine volume = 800 – 2500 ml /24hr.
Children urine volume = 200 – 400ml /24hr.
(4ml / kg / hr).
Volume of urine depends on
1. Water intake
2. External temperature.
3. Mental and physical state.
4. Intake of fluid and diuretics
(Drugs, alcohol – tea).

Oligouria: marked decrease in urine flow < 300 ml.
Polyuria : Marked increase in urine flow > 2500 ml.
Anuria : complete stoppage of urine flow.
(No urine output in 24 hrs)
Nocturia: excessive urination during night.

4. Specific Gravity
 Specific gravity measures urine density, or the ability of the
kidney to concentrate or dilute the urine over that of plasma.
 Directly proportional to urine osmolality which measures
solute concentration
 Measure of number and size of molecules
 Hence, large molecules such as urea will contribute to
reading more than the small molecules, such as Na+ and K+
 Hence, osmolality may express this function with more
effectively because it’s the number of particles / kg of
substance.

Specific gravity between 1.010 and 1.025 on a
random sample should be considered normal if
kidney function is normal.
 Since the specific gravity of the glomerular filtrate
in Bowman's space ranges from 1.007 to 1.010,
any measurement below this range indicates
hydration and any measurement above it indicates
relative dehydration.

 Diabetes Insipidus
 Glomerulonephritis
 Sever renal damage
(diminish the concentration
ability of the kidney)
 Excessive water intake.
 Diabetes mellitus.
 Nephrosis
 Fever since urine is conc.
 Urine preservative
substance
Low specific gravity High specific gravity

PROPERTIES OF NORMAL URINE
• Urine of a normal healthy individual has definite physical
properties and chemical composition
• However, in many diseases, the properties and composition
of urine change.
• Several new metabolites indicating the presence of specific
diseases may also appear in the urine.
• Such metabolites which are not normally present in the
urine are called abnormal constituents.

• So, in order to detect abnormalities, an understanding of the
of the normal physical and chemical properties of urine is
necessary. A brief account of these is given below :
• Physical Properties
 Appearance: Normal urine is clear, transparent, without any
turbidity or sediment. However variations can occur and
may point to many diseases.
 Colour: Normal urine is light yellow or straw coloured.
Gross variations are seen in many diseases like jaundice
(deep yellow) and hematuria (red due to blood).

• Volume: Average urine output is 800 mL to 2.5 L per day,
depending upon the water intake.
• Wide variations in output volume are seen in many
diseases. Changes in volume of urine are called:
• Polyuria > 2.5 L per day
• Oliguria < 300 ml per day
• Anuria - No urine output in 24 hours

• Specific gravity: It is normally between 1.010 to 1.025.
However, it can vary widely depending on diet, fluid intake
and renal function. It is inversely related to urinary output
except in diabetes mellitus where it is increased (polyuria)
due to glucose excretion.

Determination of Specific Gravity
It is determined by an instrument called urinometer
• The bulb at the bottom contains some fixed amount of mercury.
• There are markings on the long neck at the upper end
• By virtue of the weight of the mercury, it dips into the urine to an
extent dependent on the specific gravity of the urine.
• The level of urine corresponding to the markings indicates the
specific gravity.
• It is calibrated at 20°C therefore temperature correction is applied
depending on the actual temperature.

Determination of Specific Gravity
 Procedure:
Take a clean 100 ml measuring cylinder
Fill it with urine to about 3/4 of its length.
Gently place the urinometer into the cylinder and allow it to dip.
Care should be taken that it does not touch the walls of
cylinder.
Note the reading corresponding to the urine level and calculate
the specific gravity.
A correction factor of one is added or subtracted for every 3°C
rise or fall of temperature over the standard temperature of
20°C respectively.
{corrected SG=Observed SG+(0.001x temp. difference/3)}

• pH and reaction: Normal urine is acidic though, pH can vary
between 4.0 to 8.0. Average pH is around 6.
• Nature of diet influences the urinary pH
• Frank variations of urinary pH occur due to acid-base disturbances
• Odour: Normal urine has a slight ammonical odour.
Commonly seen variations are:
• Fruity odour in ketoacidosis due to acetone
• Foul smell in infection by bacteria
• Mousy smell in phenylketonuria

Properties of a normal urine
A. Physical
Appearance: clear, no sediment
Volume: 800 mL to 2500 mL/day
pH: 4-8; usually acidic (6.0)
Colour: straw coloured
Sp. gravity: 1-010-1-025
Odour: Slightly ammonical

CHEMICAL COMPOSITION OF NORMAL
URINE
• Urine is an ultra filtrate of the plasma, containing organic and
inorganic substances.
• These include urea, uric acid, creatinine, calcium,
phosphates, sodium, potassium, chloride, etc

Properties of a normal urine
B. Chemical composition
Water: 90-95%
Solids: 5-10%
Urea: 25-30 g/day
Uric acid: 0-5–0-8 g/day
Creatinine: 1.0 to 1:5 g/day
Chlorides: 10–15 g/day
Sodium: 3–5 g/day
Potassium: 2-2.5 g/day
Calcium: 0.1-3.0 g/day
Phosphate: 0.8-1.3 g/day
Sulphate: 1-1.2 g/day
Ammonia: 0.7–0.8 g/day

Electrolytes
• Sodium, potassium and chloride are also excreted in the urine.
• Dietary salt intake influences the excretion of sodium and chloride,
• Daily excretion of chloride as sodium chloride is 10-15 gm/day
• Renal tubular disease lead to decrease in their capacity to
conserve or excrete sodium.
• Sodium excretion test in urine is used to assess the renal tubular
function under standard conditions of sodium intake or salt free
diet
• Abnormalities of aldosterone secretion lead to chat in urinary
excretion of electrolytes, as well as in their plasma levels,

Calcium and phosphate
 Calcium and phosphate are also excreted in
significant amounts
 Their excretion depends on many factors like
dietary intake, vitamin D stat and hormones like
calcitonin and PTH, Excessive calcium and
phosphate accumulation can lead to renal stone
formation of both calcium oxalate and calcium
phosphate.

TESTS DONE FOR NORMAL
CONSTITUENTS
 Silver nitrate test for chloride
 Test for sulfates
 Tests for calcium and phosphates
 Sodium hypobromite test for urea, specific urease test
 Jaffe's test for creatinine
 Phosphotungstic acid reduction test for uric acid
 Ehrlich’s test for urobilinogen

Test for chloride
PROCEDURE OBSERVATION INFERENCE
Take 2ml of urine
Add 0.5 ml of conc.
HNO3 (Nitric acid)
& add 1 ml of silver
nitrate (AgNO3)
White precipitate of
AgCl is formed
Presence of
chloride is
confirmed
Principle
A white precipitate of AgCl is formed
Nitric acid prevents precipitation of salts other than chloride
like silver phosphates & silver urates

Test for sulphates
Take 3 ml of urine
Add 1 ml of conc.
HCl
& 2 ml of barium
chloride (BaCl2)
BaSO4 is formed
Presence of
sulphate is
confirmed
Principle
A white precipitate of BaSO4 is formed
Hydrochloric acid prevents precipitation of phosphates

Test for inorganic phosphate
Take 3 ml of urine
Add few drops of
conc. HNO3 (Nitric
acid)& pinch of
ammonium
molybdate.
Warm it
Canary yellow
colour precipitate
of ammonium-
phospho-molybdate
is formed
Presence of
phosphate
Principle
Upon warming with ammonium molybdate in presence of
nitric acid, inorganic phosphate is precipitated as canary
yellow ammonium-phospho-molybdate

Test for calcium
Take 2 ml of urine
Add 5 drops of
acetic acid and 3 ml
of potassium
oxalate
calcium oxalate is
formed
Presence of calcium
Principle
With potassium oxalate in acidic medium, calcium is
precipitated as calcium oxalate

Tests for non protein nitrogenous
substances in urine
 Ammonia
 Urea
 Uric acid
 Creatinine
 Ammonia
Urinary ammonia is derived from glutamine & other amino
acids in the kidneys.
Acid forming foods increase ammonia excretion.

Urea
 Urea is the major catabolic waste product of both
exogenous (dietary) and endogenous proteins
 It constitutes more than 75% of total non-protein
nitrogen excreted by the body.
 It is mainly excreted (> 90%) by the kidneys, the
remainder being excreted through the GIT and skin.
 Normal excretion of urea in urine is 25-30 gms/day.
 Its excretion is dependent on protein intake.
 In renal failure, excretion of urea decreases in urine,
and the plasma level increases.

Test for urea (urease test)
Take 5 ml of urine
Add 1-2 drops of
phenol red indicator
& add a pinch of
urease powder.
Shake the contents &
allow it to stand for
10 min
Pink colour obtained Presence of urea
This is the specific test for urea. Urease is an enzyme that
occurs in horse gram, jack beans & water melon seeds

Principle
Clinical significance
When urea is treated with enzyme urease, it liberates ammonia
and carbonic acid. Under the pH conditions of the reaction,
ammonium carbonate is formed which raises the pH to 8.5 since
it is alkaline. At this pH, phenolphthalein gives pink colour.
Increased – The urinary content of urea is elevated whenever
protein intake and catabolism are increased, for eg. fevers,
diabetes mellitus & hyper function of the adrenal cortex
Decreased – cirrhosis of liver, nephritis & severe acidosis
Urea Ammonia + carbonic acid
Ammonia + carbonic acid ammonium carbonate
urease
At pH 8.5

Uric acid
 Uric acid is the end product of catabolism of purine bases
present in the nucleoproteins.
 Therefore, formation of uric acid is principally endogenous,
mainly of tissue nucleoprotein breakdown, but some amount
is also formed from purine containing compounds present in
the food.
 However, serum uric acid levels are only marginally affected
by diet.
 Chemically, uric acid is 2,6,8 trihydroxy purine.
 It acts like a dibasic acid and can form mono and disodium
salts depending on the pH.
 These salts are deposited in the joints causing arthritis
(gout).
 Normal excretion is 0.5-0.8 g/day

Test for uric acid (Benedict’s uric acid test)
Take 3 ml of urine
Add 1-2 drops of
Benedict’s uric acid
reagent & add a
pinch of Na2CO3
powder & mix
Emergence of blue
colour.
Presence of uric acid
Also called phosphotungstic acid reductase test

Principle
Uric acid, being a reducing agent, reduces phosphotungstate to
tungsten blue in an alkaline medium giving rise to blue colour
solution.
Hyperuricemia (elevated uric acid level) –
is mostly associated with gout. It is also seen in certain leukemias &
Wilson’s disease (defect in copper metabolism).
Accumulation of uric acid crystals in the synovial fluid causes
inflammatory changes, resulting in gouty arthritis.
Sodium urate crystals are deposited in the form of tophi in peripheral
joints, making them extremely painful.
Uric acid crystals are also deposited in the collecting tubules and
lower urinary tract, resulting in calculi (stones) & causing renal
damage.

Creatinine
 Creatinine is the catabolic waste product of tissue proteins that
is excreted by the kidneys.
 Creatinine is produced in muscles from creatine by non-
enzymatic irreversible dehydration.
 Creatine, synthesized in the liver and kidneys, passes into the
circulation and is taken up almost entirely by skeletal muscles
for conversion to creatine phosphate
 About 2% of the total creatine is converted daily into creatinine.
 Serum creatinine level is a sensitive index of renal function
because it is dependent on the muscle mass and renal
excretion.

Creatinine
 It does not vary with the dietary intake of proteins
 Normal excretion is 1-1.5 gm/day.
 Urinary creatinine excretion is constant on a day-to-day
basis because the muscle mass does not change.

Test for creatinine (Jaffe’s test)
Take 3 ml of urine
Add 1 ml of
saturated picric acid
solution & 1 ml of
10% NaOH
Orange-red colour is
seen
Presence of
creatinine
This is the specific test for creatinine. It is used for both
detection & estimation of creatinine level

Principle
Creatine react with picric acid in alkaline medium to form
creatine picrate complex which is orange-red colour
The amount of creatinine produced is related to the total
muscle mass and remains approximately the same in the
plasma and urine on a day-to-day basis unless muscle mass
changes.
Increased creatinine excretion is seen in muscular dystrophy,
hyperthyroidism, diabetes mellitus & starvation
Creatinine + picric acid creatinine picrate complex
(orange-red)

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