EXAMINATION OF URINE

SPEAKER – DR. SHAMBHU SINGH

PG. PATHO.
MODERATOR – DR. MONILISHA JHA
ASSIST. PROF. PATHO.
COLLECTION OF URINE:

● TIME OF COLLECTION-
1. A SINGLE SPECIMEN – Can be -

THE FIRST VOIDED EARLY MORNING SAMPLE – Most

concentrated, has acidic pH, formed elements as casts and cells are
well preserved, so, it is the ideal specimen.
Used for routine examination, fasting glucose, proteins, nitrite, microscopic analysis
for cellular elements, pregnancy test ,orthostatic proteinuria and bacteriological
analysis.
 RANDOM SPECIMEN – Single specimen collected at any time. Used for routine urine
examination since bleeding, proteinuria or cast formation can occur at any time.
POST – PRANDIAL SPECIMEN – Collected 2 hours after a meal in the afternoon. For estimation
of glucose (to monitor insulin therapy in DM) or of urobilinogen.
Container - clean, dry, leakproof, with wide mouth and wide flat bottom, disposable, of clear
material for colour and transparency, of 50ml capacity.
2. 24 HOURS SPECIMEN – After getting up in the morning, the first urine is discarded. All the urine
voided subsequently during rest of the day and
the night is collected in a large clean bottle of 2 litres capacity. The first urine after getting up
next morning is also collected. Urine preserved at 4-6°C and transported to lab.
Used for quantitative estimation of proteins, hormones and their metabolites(catecholamines,
VMA, metanephrines, cortisol, estriol),creatinine and electrolytes. LIMITATION – Poor patient
compliance. Creatinine is also routinely measured to assess completeness of collection. In
adults excretion between 1.2 to 1.5 g/day. If creatinine less than 0.8g in 24 hour, some urine was
discarded .
COLLECTION METHODS-
● MIDSTREAM SPECIMEN- Used for all types of examination. Initial half of urine voided,discarded
and a part of urine is collected in the bottle. This flushes out contaminating cells and microbes
from urethra and perineum. Rest stream is from urinary bladder which is collected.
● CLEAN-CATCH SPECIMEN- Recommended for bacteriologic culture. Glans penis is sufficiently
exposed and thoroughly cleaned. Urethral opening in women is exposed, washed with soapy
cotton balls, and rinsed. The labia is held and initial urine is discarded and remaining is voided into
the bottle(amount-20-100ml). This method avoids contamination of urine with vaginal fluid.
● CATHETER SPECIMEN- For bacteriological study or culture and routine analysis in bed ridden, ill
patients and patients with urinary tract obstruction. Avoided in ambulatory patients –risk of
introduction of infection.
● PLASTIC BAG SPECIMEN- Plastic bag tied around genitals in infants and incontinent adults for
routine and quantitative examination.
● SUPRA PUBIC ASPIRATION- Used in infants for bacteriologic examination.
● “THREE GLASS” SPECIMEN- To determine prostate infection. Three consecutive midstream clean-
catch urine samples are collected. Prostate is massaged before collection in third container.
In UTI increased pus cells and bacteria in second and third containers.
In prostate infection pus cells and bacteria are more in third container.
 PRESERVATION OF URINE:

1. REFRIGERATION – Sample should be kept in the refrigerator for a max. of 24 hours. Refrigeration
is the best method of preservation upto 8 hours, though it raises specific gravity and precipitates
amorphous phosphates and urates. Used for routine urine analysis, if delay is expected.
2. CHEMICAL PRESERVATIVES – Chemical preservatives can be added to 24 hours urine sample
and to those sample which are to be transported over a long distance and refrigeration is
impossible. Avoided for routine urinalysis as interferes with reagent strip techniques and
chemical test for proteins.
● HYDROCHLORIC ACID- For 24 hour urine sample for adrenaline, noradrenaline, VMA and
steroids. Potential chemical hazard. Unacceptable for urinalysis.
● TOLUENE- Forms a thin layer and acts as a physical barrier for bacteria and air but not the
bacteria already present in urine. Preserves protein,reducing substances and ketones.
● BORIC ACID (0.8%)- Prevents bacterial growth and preserves protein and formed elements. For
24 hours sample 10g boric acid is used for estimation of aldosterone and cortisol. Interferes
with pH reading and drug and hormone analysis.
● THYMOL- Preserves glucose and cellular elements. One small crystal per 100 ml.
● FORMALIN- One drop per 30 ml. Excellent chemical for preservation of formed elements, but
interferes with copper reduction test and with chemical tests for glucose, blood and
leucocyte esterase.
● SODIUM CARBONATE- Quantitative analysis of porphyrins and porphobilinogen.
Unacceptable for urine analysis.
● COMMERCIAL PRESERVATIVE TABLETS- One tablet per 30 ml. Acts by releasing
formaldehyde.
 PHYSICAL EXAMINATION OF URINE:

● APPEARANCE:
URINE VOLUME- Average 24 hour urinary output in adults is 600-2000 ml. Volume varies according
to fluid intake, diet, and climate.
● POLYURIA- Urine volume more than 2000ml per 24 hours. Seen in DM(osmotic diuresis), Diabetes
insipidus(failure of secretion of ADH), chronic renal failure(loss of concentrating ability of kidneys)
or diuretic therapy.
● OLIGURIA- Urine volume less than 400ml per 24 hours. Causes febrile states, acute
glomerulonephritis(decreased GFR), congestive cardiac failure and dehydration(decreased renal
blood flow).
● ANURIA- Urine output is less than 100ml per 24 hours or complete cessation of urine
output. Occurs in acute tubular necrosis, acute glomerulonephritis, and complete
urinary tract obstruction.
● COLOUR – Normal urine colour in a fresh state is pale yellow or amber and due to presence
of pigment, urochrome. The intensity of urine colour is crude indicator of urine concentration
and hydration of body as urochrome is produced and excreted at a constant rate. So, urine is
colourless- in overhydration and dark yellow- in dehydration.
● Foamy urine- Large amount of stable white foam- develops in moderate to severe
proteinuria. Yellow to yellow- green foam develops with sufficient bilirubin in urine. Presence
and colour of foam- confirmatory test for proteins / bilirubin.
● Red- Haematuria, haemoglobinuria, porphyria, myoglobinuria.
● Colourless- Dilute urine(DM, diabetes insipidus, overhydration)
● Brown- Haemoglobinuria
● Cola coloured - Rhabdomyolysis and patients on L- dopa. Dark brown or black – Acid urine
containing haemoglobin, on standing turns dark brown – due to formation of [Link]
causes – Alkaptonuria and melanin.
● Yellow- Concentrated urine, B complex vitamins, nitrofurantoin, dehydration
● Yellow-green or green- Biliverdin
● Deep yellow with yellow foam- Bilirubin. In severe obstructive jaundice, urine – dark green.
● Milky-white- Chyluria, fat, pyuria, emulsified paraffin from vaginal creams for treatment of
Candida infection.
● Red or orange fluorescence with UV light (360 nm)- Porphyria (uroporphyrin, coproporphyrin).
● Orange or orange-brown- Urobilinogen, Porphobilinogen. Urobilinogen – colourless but
converted to urobilin in presence of light and low pH.
● DRUGS-
> Bright orange-red- Rifampin
> Bright yellow-Riboflavin(multivitamins)
> Darkening of urine, reddish brown- Metronidazole
● CLARITY(CHARACTER)- Normally freshly voided urine appears clear.
Causes of cloudy and turbid urine :
● Amorphous phosphates and carbonates – Precipitate in alkaline urine on standing,and
disappears on adding dilute acetic acid.
● Amorphous urates and uric acid - White, pink or orange cloud in acid urine. Redissolve on
warming to 60°C.
● Pus cells, red cells
● Bacteria- (Fresh urine) Uniformly cloudy urine
● ODOUR- Freshly voided urine has a typical aromatic odor due to volatile organic acids. After
standing, urine develops ammoniacal odor( formation of ammonia occurs when urea is
decomposed by bacteria).
● Fruity or sweet: Ketoacidosis, starvation
● Mousy/ musty: Phenylketonuria
● Fishy: UTI with Proteus, Tyrosinemia
● Ammoniacal: UTI with [Link], old standing urine
● Foul: UTI
● Rancid: Tyrosinemia
● Sweaty feet: Isovaleric acedemia
● Maple syrup or burnt sugar: Maple syrup urine disease
● SPECIFIC GRAVITY AND OSMOLALITY – Specific gravity is defined as density of a solution as
compared to density of distilled water at similar temperature. Specific gravity of distilled
water-1.000
Normal specific gravity of urine- 1.003-1.030 .
It depends on the state of hydration and is related to urea and sodium.
As solute concentration increases, specific gravity increases and it decreases when temperature
rises.(since volume expands with rise in temperature).
Specific gravity is the measure of concentrating ability of kidneys and gives info about tubular function.
● Causes of increase in specific gravity of urine- DM(glycosuria), nephrotic syndrome(proteinuria),
fever or dehydration- Hypersthenuria- SG above 1.010.
● Causes of decrease in specific gravity of urine- Diabetes insipidus, chronic renal failure and
compulsive water drinking- Hyposthenuria-SG below 1.010
Isosthenuria refers to urine of constant specific gravity of 1.010 which is same as that of plasma
which occurs in chronic renal failure- low and fixed SG- due to loss of concentrating ability of tubules.
Methods for measuring SG :
[Link]-Based on principle of buoyancy. SG affected by proteinuria and glucosuria, so, correction
needed.
2. REFRACTOMETER METHOD- Requires only 1-2 drops of urine. Higher the conc. of total dissolved solids
higher the refractive index.
3. REAGENT STRIP METHOD- Measures the concentration of ions in urine.
OSMOLALITY- Most specific measure of total solute conc. as colloids like proteins and lipids do not
affect osmolality. Measured by osmometer. After a period of dehydration , osmolality of urine should be
3-4 times that of the plasma.
 CHEMICAL EXAMINATION OF URINE:
REACTION AND pH –
On standing, the urine becomes alkaline because of loss of carbon dioxide and production of
ammonia from urea. So, for correct estimation of urine, fresh urine should be examined.
Various methods for determination of reaction of urine :
1. LITMUS PAPER TEST – A small strip of litmus paper is dipped in urine and any colour
change is noted. If blue litmus turns red, it indicates acid urine and if red litmus turns blue, it
indicates alkaline urine.
2. PH INDICATOR PAPER – Reagent area(impregnated with bromothymol blue and methyl
red)of indicator paper strip is dipped in the urine sample and the colour change is
compared with the colour guide provided.
3. PH METER – Used to know the exact pH. An electrode of pH meter is dipped in urine and
pH is read off directly from the digital display.
4. REAGENT STRIP TEST – The test area contains polyionic polymer bound to H⁺, on
reaction with cations in urine, H⁺ is release causing change in the colour of the pH
sensitive dye.
● Normal pH range is 4.6 to 8.0(average 6.0 to slightly acidic).Urine pH depends on diet, acid
base balance, water balance, and renal tubular function. Lowest urinary pH to be produced
by the kidneys – is 4.5 as the tubular max. of H⁺ secretion is at this level. pH of fresh
normal or abnormal urine does not exceed 8.5.
● ACIDIC URINE – Found in ketosis(DM, starvation, fever),urinary tract infection by
Escherichia coli, and high protein diet.
● ALKALINE URINE – May result from urinary tract infection by bacteria that split urea to
ammonia (Proteus or Pseudomonas),severe vomiting, vegetarian diet, renal tubular
acidosis, old ammoniacal urine sample and chronic renal failure.
● CLINICAL USES OF MEASURING URINARY pH –
1. Determining pH – helps in identifying various crystals in urine.
2. Altering pH – may be useful in treatment of renal calculi(i.e. some stones form in acid urine only e.g.
uric acid calculi; in such cases – urine is kept alkaline);urinary tract infection caused by urea – splitting
organisms(urine – kept acid);and treatment with certain drugs(e.g. streptomycin is effective in UTI ,if
urine is kept alkaline).
3. In unexplained metabolic acidosis, measurement of urine pH is helpful in diagnosing renal tubular
acidosis; in renal tubular acidosis ,urine pH is consistently alkaline despite metabolic acidosis due to
defect in renal tubular reabsorption of bicarbonate. Urinary loss of sodium bicarbonate and potassium
bicarbonate results in alkaline urine.
PROTEIN – Normally, kidneys excrete scant amount of protein in urine(upto 150 mg/24hours).These
proteins include proteins from plasma(albumin) and proteins derived from urinary tract(Tamm –
Horsfall protein, secretory IgA, and proteins from tubular epithelial cells, leucocytes, and other
desquamated cells;this amount of proteinuria – not detected by routine tests.
● Proteinuria refers to protein excretion in urine greater than 150mg/24 hours in adults.
CAUSES OF PROTEINURIA –
● Pre-renal Proteinuria(Overflow proteinuria) – In pre-renal proteinuria, also known as over-flow
proteinuria, levels of low molecular weight proteins increase in plasma with their consequent
excretion in urine. Such proteins include haemoglobin, myoglobin, acute phase proteins, and Bence
Jones proteins.
● Proteins such as Ig light chains or Bence Jones proteins( plasma cell dyscrasias), hemoglobin
(intravascular hemolysis), myoglobin (skeletal muscle trauma),and lysozyme( AML type M4 or M5)-
overflow in urine from plasma.
● Renal proteinuria – It is of two main types : glomerular and tubular.
GLOMERULAR PROTEINURIA –Increased filtration of proteins by glomeruli due to changes in
glomeruli. Proteinuria due to increased permeability of glomerular capillary wall. Two types of
glomerular proteinuria : Selective and non selective.
● In early stages of glomerular disease, there is increased excretion of lower molecular weight proteins
like albumin and transferrin and larger molecular weight proteins are retained - called Selective
Proteinuria.
● With further glomerular damage, this selectivity is lost and larger molecular weight proteins(ƴ –
globulins) are also excreted along with albumin – called Non selective Proteinuria.
● Selective and nonselective proteinuria – distinguished by urine protein electrophoresis. In
selective, albumin and transferrin bands seen, while in non selective type, pattern resembles that
of serum(i.e. all protein are present).
● CAUSES OF GLOMERULAR PROTEINURIA – Glomerulonephritis, Minimal change disease,
Preeclampsia, Strenuous exercise, fever, exposure to extreme cold, orthostatic or postural
proteinuria. Most severe degree of proteinuria occurs in nephrotic syndrome.
● Alteration of blood flow through the glomeruli causes increased filtration of proteins and
transient excretion of proteins – seen in high fever, hypertension, heavy or strenuous exercise,
CCF, seizures and exposure to cold.
● Postural (orthostatic) proteinuria – occurs when subject is standing or ambulatory, but absent in
recumbent position. Common in adolescents (3-5%) probably due to lordotic posture that causes
inferior venacaval compression between liver and vertebral column. Disappears in adulthood.
Amount of proteinuria-<1000mg/day.
First - morning urine sample is negative for proteins ,while another urine sample collected after
patient performs normal activities is positive for proteins. So, periodic testing for proteins done
to rule out renal disease.
TUBULAR PROTEINURIA – Normally, glomerular membrane although impermeable to high
molecular weight proteins, allows ready passage to low molecular weight proteins like albumin,
transferrin,α₂- microglobulin, α₁ - microglobulin, β₂ - microglobulin,retinol –binding protein ,lysozyme.
These are actively reabsorbed by proximal renal tubules. In diseases involving mainly the tubules as
acute and chronic pyelonephritis, interstitial nephritis, cystinosis, Fanconi syndrome, TB of kidney, in
heavy metal poisoning, rejection of kidney transplant; proteinuria occurs due to decreased
reabsorption of filtered low molecular proteins by renal tubules but albumin excretion is minimal.
● Urine electrophoresis shows prominent α- and β- bands(where migration of low molecular weight
proteins occurs) and a faint albumin band.
Tubular proteinuria – not detected by reagent strip (as it is sensitive to albumin) ,but heat and
acetic acid test and sulfosalicylic acid test is positive.
● Post–renal Proteinuria – Caused by inflammatory or neoplastic conditions in renal pelvis, ureter,
bladder, prostate or urethra.
TESTS FOR DETECTION OF PROTEINURIA –
1. Heat and acetic acid test /Modified boiling test – Based on principle that proteins get
precipitated when boiled in an acidic solution. Cloudiness or turbidity indicates presence of
either phosphates or proteins. Turbidity due to phosphates disappears while that due to proteins
does not.
False positive – tolbutamide and large doses of penicillin.
2. Reagent strip test – Reagent strip impregnated with an indicator dye – bromothymol blue and
buffered to an acid pH of 3.0 with citrate which changes color in presence of proteins due to
change in ionization(and hence pH).The test is semi quantitative as well as intensity of color is
proportional to conc. of protein.
● Overload(Bence Jones) proteinuria and tubular proteinuria may be missed by reagent strip;as it is
sensitive mainly to albumin.
● So, this test is followed by sulfosalicylic acid test which is confirmatory test.
● False positive – highly alkaline urine,quaternary ammonium compounds(detergents),gross
hematuria,and examination with vaginal secretions.
3. Sulfosalicylic acid test – Addition of sulfosalicylic acid to urine forms a white precipitate if
proteins present.(Proteins are denatured by organic acids and precipitated out).Turbidity checked
against a dark background.
False positive- salicylates, penicillins, radiographic contrast media,tolbutamides, sulphonamides,
excess uric acid.
● Not affected by highly alkaline urine.
QUANTITATIVE ESTIMATION OF PROTEINS –
● For detection of nephrotic syndrome
● Detection of microalbuminuria or early diabetic nephropathy.
● To follow response to therapy in renal disease.
Proteinuria >1500mg/24hours – indicates Glomerular disease- Moderate proteinuria
>3500mg/24hours –Nephrotic proteinuria range – Marked/severe proteinuria
<1500mg/24hours-Tubular,hemodynamic,postrenal proteinuria – Mild proteinuria
● Quantitative estimation methods – [Link] of proteins in a 24 hours sample.
2. Estimation of protein/creatinine ratio in a random urine sample.
MICROALBUMINURIA –Defined as urinary excretion of 30 to 300mg/24 hours of albumin in
[Link] sign of renal damage in DM(Diabetic nephropathy)-Due to increased capillary
permeability to [Link] blood sugar and HTN are tightly controlled at this stage by aggressive
treatment,progression to irreversible renal disease and renal failure can be prevented.
Also risk factor for cardiovascular disaease in DM.
Detection- Measurement of albumin-creatinine ratio in a random sample.
Measurement of albumin in early morning sample.
Measurement of albumin in a 24 hour [Link] assay done and strips also available.
● BENCE JONES PROTEINURIA-Bence Jones are monoclonal immunoglobulin light chains (either
kappa or lambda) that are synthesized by neoplastic plasma cells – in plasma cell dyscrasias as
multiple myeloma and primary [Link]flow proteinuria occurs – because of low
molecular weight and high conc.
● Characteristic thermal behavior ,basis for screening test-When heated,they precipitate at
temperatures between 40̊-60̊ C(other proteins between 60-70̊ C),and precipitate disappears
around 100 C. When cooled to around 60̊ C,there is reappearance of Bence Jones [Link]
positive and negative can occur. Replaced by protein electrophoresis of conc. urine sample.
GLUCOSE – The main indication for testing glucose in urine is detection of unsuspected DM or follow-
up of known diabetic [Link] all of the glucose filtered by glomeruli are reabsorbed by
proximal renal tubules and returned to [Link] normal renal threshold for glucose is
165-180mg/dl.
When the carrier transporting glucose from tubular lumen to vasa recta gets saturated,the tubular
maximum is reached and glucose is excreted in [Link],small quantity (<500mg/24hours or <
15mg/dl) is excreted,that cannot be detected by routine [Link] – presence of detectable
amounts of glucose in urine,which results if filtered glucose load exceeds the capacity of renal tubular
[Link] common cause for hyperglycemia – DM.
Causes of hyperglycemia –
1. Glycosuria with hyperglycemia:
-Endocrine disease – DM,acromegaly,Cushing’s syndrome,hyperthyroidism,pancreatic disease
-Non endocrine diseases- Central nervous system diseases ,liver disorders.
-Drugs- Adrenocorticotropic hormone,corticosteroids,thiazides.
-Alimentary glycosuria (Lag –storage glycosuria)-Glucose tolerance test reveals a peak at 1
hour above renal threshold due to rapid intestinal absorption following a [Link] in persons
with gastrectomy or gastrojejunostomy and in hyperthyroidism.
2. Glycosuria without hyperglycemia:
-Renal glycosuria – 5% of [Link] to the renal threshold level,glucose filtered by glomeruli is
efficiently reabsorbed by the [Link] glycosuria is a benign condition in which renal threshold
is set below 165 – 180mg/dl but glucose tolerance is normal. Transmitted as autosomal dominant
disorder.
- Other conditions – Fanconi syndrome , toxic tubular damage
- During pregnancy, threshold decreased due to increased GFR and impaired tubular glucose
reabsorption.
TESTS FOR DETECTION OF GLUCOSE IN URINE –
1. Copper reduction methods – a) Benedict’s qualitative test –
-Alkaline copper sulfate is reduced to red – brown cuprous oxide if reducing agent is
present,when boiled in Benedict’s qualitative [Link] extent of reduction depends on
conc. of reducing substance in urine.
-Other carbohydrates([Link],fructose,galactose,pentoses),ceratin
metabolites(glucuronic acid,homogentisic acid,uric acid,creatinine) and drugs (ascorbic
acid ,salicylate,cephalosporin,penicillin, streptomycin,isoniazid,nalidixic acid) also reduce
alkaline copper sulfate [Link], not specific for glucose.
Sensitivity of test – about 200mg reducing per dl of urine.
● For only glucose estimation,reagent strips used.

1. The result is reported in grades as follows:

2. Nil – no change from blue colour
3. Trace – green without precipitate
4. 1+(approx. 0.5g/dl) – green with precipitate
5. 2+ (approx. 1.0g/dl) – brown precipitate
6. 3+ (approx. 1.5g/dl) – yellow-orange precipitate
7. 4+ (>/=2g/dl)-Brick red precipitate

b)Clinitest tablet method – Copper reduction tablet test

2. Reagent strip method – Preferred over Benedict’s and Clinitest [Link] is based on
glucose oxidase –peroxidase [Link] area is impregnated with 2 enzymes (glucose
oxidase and peroxidase) and a [Link]fic for [Link] reducing agents give
negative [Link] positive in presence of oxidizing agents and negative in large
amounts of ketones, salicylates, ascorbic acid and severe [Link] infection (catalase
produced by organism inactivates H₂O₂).
A positive reagent strip test and negative for reducing sugars occur in true glycosuria.
KETONES – Excretion of ketone bodies(acetoacetic acid, β – hydroxybutyric acid, and
acetone).Ketones are breakdown products of fatty acids and their presence in urine is indicative of
excessive fatty acid metabolism to provide energy.
Causes of ketonuria –Normally, ketone bodies are not found in urine of healthy persons. If energy
requirements cannot be met by metabolism of glucose (due to defective carbohydrate metabolism,
low carbohydrate intake, or increased metabolic needs),then energy is derived from breakdown of
fats – this leads to formation of ketone bodies.
1. Decreased utilization of carbohydrates – a) Uncontrolled diabetes mellitus with ketoacidosis –
Poor glucose utilization leads to compensatory increased lipolysis, which leads to increased free
fatty acids in plasma. Their degradation

in the liver leads to formation of acetyl CoA which then forms ketone bodies.
● Ketone bodies are strong acids and produce H+ ions, which are neutralized
by bicarbonate ions; fall in the bicarbonate ([Link]) level produces
[Link] also increase plasma osmolality and cause cellular
[Link] and young adults with type 1 diabetes are especially
prone to ketoacidosis during acute illness and stress.
b) Glycogen storage disease.(von Gierke’s disease).
2. Decreased availability of carbohydrates in diet –
a) Starvation
b)Persistent vomiting in children
c) Weight reduction program (severe carbohydrate restriction with normal fat intake)
3. Increased metabolic needs –
d)Fever in children
e) Severe thyrotoxicosis
f) Pregnancy
g) Protein calorie intake
TESTS FOR DETECTION OF KETONURIA -
● The proportion of ketone bodies in urine in ketosis is variable – β –hydroxybutyric acid
-78%,acetoacetic acid-20%,and acetone -2%.
● No method reacts with all the ketone bodies .
● Rothera’s nitroprusside method and methods based on it detects acetoacetic acid and acetone
(10-20 times more sensitive to acetoacetic acid than acetone).
● Ferric chloride test detects only acetoacetic acid.
● Β- hydroxybutyric acid is not detected by any of the screening [Link] detection, if desired, it has
to be oxidized by adding H₂O₂ to acetoacetic acid.
● Tests for ketone bodies may underestimate presence of ketosis as acidosis is
associated with increased synthesis of β – hydroxybutyric acid,which is not detected by these tests.
● Methods of detection of ketone bodies in urine are –
1. Rothera’s test(Classical nitroprusside test) – Acetoacetic acid or acetone reacts with
nitroprusside in alkaline solution to form purple coloured complex. False positive in presence of L-
dopa in urine and in [Link] to 1-5 mg/dl of acetoacetic acid and to 10-25 mg/dl
of acetone.
2. Acetest tablet test – This is Rothera’s test in the form of a tablet. Consists of sodium
nitroprusside,glycine and an alkaline [Link] sensitive than reagent strip for ketones.
3. Ferric chloride test (Gerhardt’s test) -10% ferric chloride solution added to urine, which turns
reddish or purplish, if acetoacetic acid is [Link] positive by salicylates and [Link]
– 25-50 mg/dl.
4. Reagent strip test – Modifications of nitroprusside test. Sensitivity – 5-10 mg/dl of
[Link] negative, if exposed to moisture.
 DETECTION OF β – HYDROXYBUTYRIC ACID -
● Add 2-3 drops of acetic acid to 5-6 ml of 1:10 diluted urine [Link] for 3-4 minutes to drive away
acetone and and acetoacetic acid in [Link] add 1ml of hydrogen peroxide,warm gently ,cool and
perform Rothera’s test.
● However,enzymatic assay for β – hydroxybutyric acid recommended for diagnosis of ketoacidosis in
plasma.
● BILE PIGMENT (BILIRUBIN) – Bilirubin (a breakdown product of hemoglobin) is undetectable in normal
[Link] of bilirubin urine is called as bilirubinuria. Two forms of bilirubin – Conjugated and
Unconjugated.
● Bilirubin after formation from hemoglobin in reticuloendothelial cells,circulates in blood, bound to
albumin, is unconjugated and is not water soluble ;cannot pass through the glomeruli as bound to
albumin, therefore, does not appear in appear.
● The liver takes up unconjugated bilirubin and combines it with glucuronic acid to form bilirubin
diglucuronide(conjugated bilirubin),which is water – soluble, filtered by glomeruli and appears in urine.
● In acute viral hepatitis, bilirubin appears in urine even before jaundice is clinically [Link] a fever or
pyrexia of unknown origin,bilirubinuria suggests hepatitis.
● Presence of bilirubin in urine – indicates conjugated hyperbilirubinemia (obstructive or
hepatocellular jaundice)as only conjugated bilirubin is water soluble.
● Bilirubin in urine is absent in hemolytic jaundice as unconjugated bilirubin is water -
insoluble.
 TESTS FOR DETECTION OF BILIRUBINURIA -

● Bilirubin is converted to nonreactive biliverdin on exposure to light and on standing at room

temperature and biliverdin can’t be detected by tests that detect bilirubin. So,fresh sample
should be protected from light.
● Methods for detection –
1. Foam test – Yellowish foam on [Link] result – proteins and highly conc. urine.
2. Gmelin’s test – Conc. nitric acid placed over equal quantity of urine and shaken [Link]
of colours (yellow,red,violet,blue and green)-positive test.
3. Lugol’s iodine test – 4 drops of urine added to 4ml of Lugol’s iodine solution and mixed by
[Link] of green colour – positive test.
4. Fouchet’s test – Simple and sensitive test.5 ml of fresh urine taken in a test tube,and 2.5ml of 10%
barium chloride added,and mixed well. A precipitate of sulfate (barium- sulfate –bilirubin complex)
[Link] and obtain the precipitate on a filter [Link] the precipitate, add 1 drop of Fouchet’s
reagent. Immediate development of blue – green colour around the drop – indicates presence of
bilirubin.
5. Reagent strips or tablets(Ictotest) impregnated with diazo reagent – Ictotest more sensitive than
reagent strip test –detects 0.05 -0.1 mg of bilirubin/dl of urine.
BILE SALTS –Bile salts are salts of 4 different types of bile acids –
cholic,deoxycholic,chenodeoxycholic,and [Link] combine with glycine or taurine to
form complex salts or acids. Bile salts enter the small intestine through the bile and act as
detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes, lipases
can breakdown the [Link] are taken up the liver and re- excreted in bile , after their absorption
by the terminal ileum(enterohepatic circulation).
Bile salts along with bilirubin can be detected in urine in obstructive [Link] regurgitate
into blood from biliary canaliculi and are excreted in [Link] property of bile salts to reduce
surface tension is utilized in HAY’S SURFACE TENSION [Link] sink to the bottom,if bile salts
are present.
UROBILINOGEN – Conjugated bilirubin excreted into duodenum through bile is converted to
urobilinogen by bacterial action in the [Link] part -in faeces. A portion undergoes
recycling enterohepatic circulation and a small part, not taken up by liver is excreted in
[Link] to urobilin,which is orange- yellow in colour.(urobilinogen is colourless).0.5-4mg is
excreted in urine in 24 hours.
Diurnal variation with highest peak in afternoon.A 2 hour post meal sample is preferred.
Causes of increased urobilinogen in urine – Hemolysis and hemorrhage in tissues.
Causes of decreased urobilinogen in urine – Obstructive jaundice and reduction of intestinal flora.
● TESTS FOR DETECTION –
1. Ehrlich’s aldehyde test – Pink colour due to reaction of the ehrlich’s aldehyde with
urobilinogen. Dark red colour if urobilinogen is increased.
Watson – Schwartz test to distinguish between the urobilinogen and porphobilinogen
2. Reagent strip test – Specific for urobilinogen.
MICROSCOPIC EXAMINATION:

● Also known as “liquid biopsy of the urinary tract”.

● The microscopic, insoluble and solid elements suspended in urine - classified as organized or
unorganized.
● Organized substances – Red blood cells, white blood cells, casts, epithelial cells, bacteria, and
parasites. Unorganized substances – crystalline and amorphous material.
● Remain suspended in urine and on standing, settle down and sediment at the bottom of the
container. So, known as urinary deposits or sediments.
● Urinary deposit examination – Diagnosis of urinary tract diseases.
SPECIMEN: The cellular elements are best preserved in acid, hypertonic urine. Mid-stream, freshly
voided, early morning specimen preferred- as most concentrated.
Examined within 2 hours of voiding.
● Preservative if required- 1 crystal of thymol or 1 drop of formalin(40%) to 10ml of urine.
METHOD:A well- mixed sample of urine(12ml) is centrifuged in a centrifuge tube for 5 mins. at 1500
rpm and supernatant is poured off. Tube is then tapped at the bottom to resuspend the sediment( in
0.5ml of urine).A drop of this placed on a glass slide and covered with a cover slip.
● Immediately examine under low power then under high power objective.
● Biochemical results can be reported even if sample volume < 12ml.
CELLS –
1. RED BLOOD CELLS – Normally no or occasional RBCs appear in urine. In fresh sample, appear as –
small, smooth, biconcave discs-7µm(called as isomorphic red cells),or may be swollen in dilute or
hypotonic urine and appear crenated (smaller with spikey surface) in hypertonic urine.
● In glomerulonephritis - dysmorphic - passage through damaged glomeruli.
● PRESENCE OF >80% OF DYSMORPHIC CELLS – STRONGLY SUGGESTIVE OF GLOMERULAR
PATHOLOGY.
● Quantity of RBCs – reported as their no. per high power field.
2. WHITE BLOOD CELLS (PUS CELLS) – spherical, 10-15µm size, granular in appearance and nuclei
visible. Degenerated ones - distorted, smaller and with fewer granules. Clumps of WBCs are seen in
infections.
● Presence of many white cells in urine – PYURIA .
● Glitter cells - In hypotonic urine, WBCs are swollen and granules – are highly refractile, and show
Brownian movement.
 LARGE NO. of glitter cells – INDICATIVE OF URINARY TRACT INJURY.
● Normally, 0-2 white cells – seen per high power field. PUS CELLS - >10/HPF OR PRESENCE OF
CLUMPS – SUGGESTIVE OF UTI.
● Increased no. of white cells – occur in fever, pyelonephritis, lower urinary tract infection, tubulo –
interstitial nephritis, and renal transplant rejection.
● In UTI, combination of these are seen – Clumps of pus cells or pus cells > 10/HPF, -
Bacteria, - Albuminuria and - Positive nitrite test.
● Simultaneous presence of white cells and white cell casts – Presence of renal
infection(pyelonephritis).
● Eosinophils(>1%of urinary leucocytes) – characteristic of Acute interstitial nephritis due to drug
reaction.(Wright’s stain better).
● Presence of bacteria in urine without white cell - contamination of sample with vaginal or skin flora.
3. RENAL TUBULAR EPITHELIAL CELLS – Increased numbers - in tubular damage conditions like acute
tubular necrosis, pyelonephritis, viral infection of kidney, allograft rejection, and salicylate or heavy
metal poisoning.
● Large no. of cells are shed in urine in viral infection of kidney (eg. In CMV) Renal neoplasms – do
not shed cells in urine.
● Renal tubular epithelial cells are difficult to distinguish from pus cells in unstained preparations. Renal
epithelial cells - small, polyhedral, columnar, or oval, and have granular [Link], large, refractile,
eccentric nucleus often seen.
4. SQUAMOUS EPITHELIAL CELLS – Squamous epithelial cells line the lower urethra and vagina. Best seen
under low power. Large no. squamous cells in urine indicates contamination with vaginal fl[Link]
cells ,rectangular with abundant cytoplasm and a small, central nucleus.
5. TRANSITIONAL EPITHELIAL CELLS – Transitional cells line renal pelvis, ureters, urinary bladder, and
upper urethra. Large, spherical, polyhedral ,or caudate. Small no. – a normal finding. Large no. or sheets
– occur after catheterization and in transitional cell carcinoma.
6. OVAL FAT BODIES – Degenerated renal tubular epithelial cells filled with highly refractile
lipid(cholesterol) droplets. Under polarized light, show a characteristic “MALTESE CROSS” pattern. Can
be stained with a fat stain such as Sudan III or Oil Red O. Seen in nephrotic syndrome – due to lipiduria.
7. SPERMATOZOA – May sometimes be seen in urine of men.

TELESCOPED URINARY SEDIMENT – Urinary sediment consisting of RBCs, WBCs, oval fat bodies, and all
types of casts in roughly equal proportion. Occurs in lupus nephritis, malignant hypertension, rapidly
proliferative glomerulonephritis, and diabetic glomerulosclerosis.
ORGANISMS –
1. BACTERIA – Detected by microscopic examination, reagent strip tests for significant bacteriuria
(nitrite test, leucocyte esterase test),and culture.
SIGNIFICANT BACTERIURIA – When there are > 10⁵ bacterial colony forming units/ml of urine in a
clean - catch midstream sample, and > colony – forming units/ml of urine in a suprapubic aspiration
sample.
a) CHEMICAL OR REAGENT STRIP TESTS – 1. NITRITE TEST –Nitrites – not present in normal urine.
Ingested nitrites are converted to nitrate and excreted in [Link] Gram-negative bacteria ([Link],
Salmonella, Proteus, Klebsiella, etc).present in urine ,they will reduce the nitrates to nitrites through
the action of bacterial enzyme, nitrate reductase. As [Link] – commonest organism causing UTI, so ,
screening done through this test.
● Nitrite test – NEGATIVE for Staphylococcus or Pseudomonas – don’t reduce.
● Urine must be retained for minimum of 4 hours for conversion of nitrate to nitrite. So, fresh early
sample preferred. Dietary intake – should be sufficient. False negative with ascorbate and highly acid
urine.
● Detects about 70% cases of UTIs.
2. LEUKOCYTE ESTERASE TEST – Detects esterase enzyme released in urine from granules of
leucocytes. Test positive in – PYURIA. If positive, urine culture should be done. Not sensitive to
leucocytes < 5/HPF.
b)MICROSCOPIC EXAMINATION – In a wet preparation,presence of bacteria is reported only when urine
is fresh. Bacteria occur in combo with pus cells. Gram- stained smear of uncentrifuged urine showing
1 or more bacteria per oil- immersion field suggests presence of ≥ 10⁵ bacterial colony forming
units / ml of urine.
c) CULTURE – On culture, a colony count of >10⁵/ml - suggestive of urinary tract infection, even in
asymptomatic females. Positive culture is followed by sensitivity test. Most infections are due to
particularly [Link]. or more species of bacteria are identified in culture by culture, it almost always
indicates contamination by vaginal flora.
 CASTS :
● Cylindrical,cigar shaped microscopic structures – formed in distal tubules and collecting ducts.
● Take shape and diameter of lumina of renal tubules – so called – casts or molds .
● Are precipitates of a mucoprotein i.e Tam Horsfall protein, secreted by tubules.
● So casts – present only in diseases of renal parenchyma, are hyaline.
● Different elements get deposited on the hyaline material to form other types of casts.
● Seen under low power,with illumination reduced.
● Cylindriuria
● Cellular and noncellular casts
.
CASTS -
CRYSTALS :

● Crystals are refractile substances,of 3 D orderly arrangement.

● Cyrstalluria – crystals in urine
● Normal and abnormal crystals
● THANK YOU