2. INDICATIONS FOR URINALYSIS
1. Suspected renal diseases like glomerulonephritis
nephrotic syndrome, pyelonephritis, and renal failure
2. Detection of urinary tract infection
3. Detection and management of metabolic disorders
like diabetes mellitus
4. Differential diagnosis of jaundice
5. Detection and management of plasma cell dyscrasias
6. Diagnosis of pregnancy.
Time of Collection
first morning voiding, a random specimen, or a post-prandial
specimen.
The first voided specimen in the morning is the
most concentrated and has acidic pH in which formed
elements (cells and casts) are well preserved. This
specimen is used for routine examination, fasting
glucose, proteins, nitrite, microscopic analysis for
cellular elements, pregnancy test, orthostatic
proteinuria, and bacteriological analysis.
3. Collection of urine sample
• First morning, midstream: Preferred for routine urine
examination.
• Random, midstream: Routine urine examination.
• First morning, midstream, clean catch: Bacteriological
examination.
• Postprandial: Estimation of glucose, urobilinogen
• 24-hour: Quantitative estimation of proteins or hormones.
• Catheterised: Bacteriological examination in infants,
bedridden patients, and in obstruction of urinary tract.
• Plastic bag (e.g. colostomy bag) tied around genitals:
Infants; incontinent adults.
4. Composition of normal urine (24 hour) in adults
Parameters Values
1. Volume 600-2000 ml
2. Specific gravity 1.003-1.030
3. Osmolality 300-900 mOsm/kg
4. pH 4.6-8.0
5. Glucose <0.5 gm
6. Proteins <150 mg
7. Urobilinogen 0.5-4.0 mg
8. Porphobilinogen 0-2 mg
9. Creatinine 14-26 mg/kg (men), 11-20 mg/kg (women)
10. Urea nitrogen 12-20 gm
11. Uric acid 250-750 mg
12. Sodium 40-220 mEq
13. Potassium 25-125 mEq
14. Chloride 110-250 mEq
15. Calcium (low calcium diet) 50-150 mg
16. Formiminoglutamic acid (FIGlu) < 3 mg
17. Red cells, epithelial cells, and white
blood cells <1-2/high power field <1-2/high power field
5. Nocturia, When excess urine is passed during night (>500ml). This is a sign
of early renal failure.
ii) Polyuria: When excess of urine is passed in 24 hours (>2500ml). Polyuria
can be physiological due to excess water intake, may be seasonal (e.g. in
winter) or can be pathological (c.g.In diabetes insipidus, diabetes mellitus).
Oliguria: When less than 500 ml of urine is passed in 24 hours. It can be due
to less intake of water, dehydration, renal ischemia,
iv) Anuria : When there is almost complete suppression of urine (< 150ml) in
24 hours. It can be due to renal stones, tumours, renal ischaemia.
6. Preservation of Urine Sample
• Hydrochloric acid: It is used for preservation of a 24- hour urine sample
for adrenaline, noradrenaline, vanillylmandelic acid, and steroids.
• Toluene: It forms a thin layer over the surface and
acts as a physical barrier for bacteria and air. It is used for measurement of
chemicals.
• Boric acid: A general preservative.
• Thymol: It inhibits bacteria and fungi.
• Formalin: It is an excellent chemical for preservation
of formed elements.
7. Colors Conditions
Colorless Dilute urine (diabetes mellitus,
diabetes insipidus, overhydration)
Red Hematuria, Hemoglobinuria,
Porphyria, Myoglobinuria
Dark brown or black (mousy or fishy order) Alkaptonuria, Melanoma
Brown Hemoglobinuria
Yellow Concentrated urine
Yellow-green or green Biliverdin
Deep yellow with
yellow foam
Bilirubin
Orange or orange-
brown
Urobilinogen
Porphobilinogen
Milky-white Chyluria
Red or orange fluorescence with
UV light
Porphyria
Note: Many drugs cause changes in urine color; drug history
should be obtained if there is abnormal coloration of urine
8. Odor
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). Some abnormal odors with associated
conditions are:
• Fruity: Ketoacidosis, starvation
• Mousy or musty: Phenylketonuria
• Fishy: Urinary tract infection with Proteus, tyrosinaemia.
• Ammoniacal: Urinary tract infection with Escherichia coli, old standing urine.
• Foul: Urinary tract infection
• Sulfurous: Cystinuria.
9. Causes of increase in SG of urine
•Diabetes mellitus (glycosuria),
•Nephrotic syndrome (proteinuria),
•Fever, and dehydration.
Causes of decrease in SG of urine are diabetes insipidus
(SG consistently between 1.002-1.003),
• chronic renal failure (low and fixed SG at 1.010 due to loss of concentrating
ability of tubules) and
compulsive water drinking.
10. Reaction and pH
• Normal pH range is 4.6 to 8.0 (average 6.0 or slightly acidic).
• Urine pH depends on diet, acid base balance, water balance, and renal
tubular function.
• Acidic urine is found in ketosis (diabetes mellitus, 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, old ammoniacal urine sample and chronic renal failure.
11. • Determining pH of urine helps in identifying various crystals in urine.
• Altering pH of urine may be useful in
•treatment of renal calculi (i.e. some stones form only in acid urine e.g. uric
acid calculi; in such cases urine is kept alkaline);
•urinary tract infection (urine should be kept acid); and treatment with
certain drugs (e.g. streptomycin is effective in urinary tract infection if urine
is kept alkaline).
• 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.
12. CHEMICAL EXAMINATION
Proteinuria refers to protein excretion in urine
greater than 150 mg/24 hours in adults
Glomerular proteinuria
Selective and nonselective proteinuria can be distinguished
by urine protein electrophoresis.
Causes of glomerular proteinuria are glomerular
diseases that cause increased permeability of glomerular
basement membrane. The degree of glomerular proteinuria
Nephrotic syndrome
• Massive proteinuria (>3.5 gm/24 hr)
• Hypoalbuminemia (<3.0 gm/dl)
• Generalised edema
• Hyperlipidemia (serum cholesterol >350 mg/dl)
• Lipiduria
13. Tubular proteinuria: Normally, glomerular membrane, although impermeable to
high molecular
weight proteins, allows ready passage to low molecular weight proteins like β2-
microglobulin,
retinol-binding protein, lysozyme, α1-microglobulin, and free immunoglobulin light
chains. These low
molecular weight proteins are actively reabsorbed by proximal renal tubules. In
diseases involving mainly
tubules, these proteins are excreted in urine while albumin excretion is minimal.
Overflow proteinuria: When concentration of a low
molecular weight protein rises in plasma, it “overflows” from plasma into the urine.
Such proteins are immunoglobulin light chains or Bence Jones proteins (plasma
cell dyscrasias), hemoglobin (intravascular hemolysis), myoglobin (skeletal muscle
trauma), and
lysozyme (acute myeloid leukemia type M4 or M5).
14. Hemodynamic proteinuria: Alteration of blood flow
through the glomeruli causes increased filtration of
proteins. Protein excretion, however, is transient. It
is seen in high fever, hypertension, heavy exercise,
congestive cardiac failure, seizures, and exposure to
Cold.
Post-renal proteinuria: This is caused by inflammatory
or neoplastic conditions in renal pelvis, ureter,
bladder, prostate, or urethra.
15. Heat and acetic acid test (Boiling test): This test is
based on the principle that proteins get precipitated when boiled in an acidic
solution.
•Method: Urine should be clear; if not, filter or use supernatant from a centrifuged
sample.
•Urine should be just acidic (check with litmus paper); if not, add 10% acetic acid
drop by drop until blue litmus paper turns red.
•A test tube is filled 2/3rds with urine. The tube is inclined at an angle and the
upper portion is boiled over the flame. (Only the upper portion is heated so that
convection currents generated by heat do not disturb the precipitate and the upper
portion can be compared with the lower clear portion).
•Compare the heated part with the lower part. Cloudiness or turbidity indicates
presence of either phosphates or proteins .
•Few drops of 10% acetic acid are added and the upper portion is boiled again.
Turbidity due to phosphates disappears while that due to proteins does not.
16. Sulphosalicylic acid test: Addition of sulphosalicylic acid to the urine
causes formation of a white precipitate if proteins are present (Proteins are
denatured by organic acids and precipitate out of solution).
•Take 2 ml of clear urine in a test tube. If reaction of urine is neutral or alkaline,
a drop of glacial acetic acid is added.
•Add 2-3 drops of sulphosalicylic acid (3 to 5%), and examine for turbidity
against a dark background
•This test is more sensitive and reliable than boiling test.
•The test can detect albumin, hemoglobin, myoglobin,
and Bence Jones proteins
17. GLUCOSE
Urine should be tested for glucose within 2 hours of collection (due to lowering of
glucose by glycolysis and by contaminating bacteria which degrade glucose
rapidly)
• Reagent strip test is a rapid, inexpensive, and semi-quantitative test
• Urine glucose cannot be used to monitor control of diabetes since renal
threshold is variable amongst individuals, no information about level of blood
glucose below renal threshold is obtained, and urinary glucose value is affected
by concentration of urine.
Benedict
1. Take 5 ml of Benedict’s qualitative reagent in a test
tube (composition of Benedict’s qualitative reagent: copper sulphate 17.3 gram,
sodium carbonate 100 gram, sodium citrate 173 gram, distilled water 1000 ml).
2. Add 0.5 ml (or 8 drops) of urine. Mix well.
3 . Boil over a flame for 2 minutes.
4. Allow to cool at room temperature.
5. Note the color change, if any.
•Sensitivity of the test is about 200 mg reducing substance per dl of urine.
Since Benedict’s test gives positive reaction with carbohydrates other than
glucose,
it is also used as a screening test (for detection of galactose, lactose, fructose,
maltose, and pentoses in urine) for inborn errors of carbohydrate metabolism in
infants and children. For testing urine only for glucose, reagent strips are
preferred .
18. The result is reported in grades as follows
Nil: no change from blue color
Trace: Green without precipitate
1+ (approx. 0.5 grams/dl): Green with precipitate
2+ (approx. 1.0 grams/dl): Brown precipitate
3+ (approx. 1.5 grams/dl: Yellow-orange precipitate
4+ (> 2.0 grams/dl): Brick- red precipitate.
19. KETONURIA Excretion of ketone bodies (acetoacetic acid, β-hydroxybutyric
acid, and acetone) in urine is called as ketonuria. Ketones are breakdown
products of fatty acids and their presence in urine is indicative of excessive fatty
acid metabolism to provide energy
1. Decreased utilization of carbohydrates
a. Uncontrolled diabetes mellitus with ketoacidosis
b. Glycogen storage disease (von Gierke’s disease)
2. Decreased availability of carbohydrates in the diet:
a. Starvation
b. Persistent vomiting in children
c. Weight reduction program (severe carbohydrate
restriction with normal fat intake)
3. Increased metabolic needs:
a. Fever in children
b. Severe thyrotoxicosis
c. Pregnancy
d. Protein calorie malnutrition
20. 1. Rothera’s’ test (Classic nitroprusside reaction )
Principle : Acetoacetic acid or acetone reacts with nitroprusside in
alkaline solution to form a purple-colored complex
Rothera’s test is sensitive to 1-5 mg/dl of acetoacetate and to 10-25
mg/dl of acetone.
Method
1. Take 5 ml of urine in a test tube and saturate it with ammonium sulphate.
2. Add a small crystal of sodium nitroprusside. Mix well.
3. Slowly run along the side of the test tube liquor ammonia to form a layer.
4. Immediate formation of a purple permanganate colored ring at the
junction of the two fluids indicates a positive test.
False-positive test can occur in the presence of L-dopa in urine and in
phenylketonuria.
Ferric chloride test (Gerhardt’s): Addition of 10% ferric chloride solution
drop by drop to urine causes solution to become reddish or purplish if
acetoacetic acid is present. Sensitivity of the test is 25-50 mg/dl.
4. Reagent strip test: Reagent strips tests are modifications of
nitroprusside test Their sensitivity is 5-10 mg/dl of acetoacetate. If exposed
to moisture, reagent strips often give false-negative result. Ketone pad on
the strip test is especially vulnerable to improper storage and easily gets
damaged.
21. Bilirubin is converted to non-reactive biliverdin on exposure to light (daylight or
fluorescent light) and on standing at room temperature. Biliverdin cannot be
detected by tests that detect bilirubin. Therefore fresh sample that is kept
protected from light is required. Findings associated with bilirubinuria are shown
in
1. Foam test: About 5 ml of urine in a test tube is shaken
and observed for development of yellowish foam.
Similar result is also obtained with proteins and highly concentrated urine. In
normal urine, foam is white.
2. Gmelin’s test: Take 3 ml of concentrated nitric acid in a test tube and
slowly place equal quantity of urine over it. The tube is shaken gently; play of
colors (yellow, red, violet, blue, and green) indicates positive test.
3. Lugol iodine test: Take 4 ml of Lugol iodine solution
(Iodine 1 gm, potassium iodide 2 gm, and distilled water to make 100 ml) in a test
tube and add 4 drops of urine. Mix by shaking. Development of green color
indicates positive test.
Hemolytic
Jaundice
Hepatocellular
Jaundice
Obstructive
Jaundice
1. Bilirubin Absent Present Present
2. Urobilinogen Increased Increased Absent
BILIRUBIN
22. Fouchet’s test: This is a simple and sensitive test.
i. Take 5 ml of fresh urine in a test tube, add 2.5 ml of 10% of barium chloride,
and mix well. A precipitate of sulphates appears to which bilirubin is bound
(barium sulphate-bilirubin complex).
ii. Filter to obtain the precipitate on a filter paper.
iii. To the precipitate on the filter paper, add 1drop of Fouchet’s reagent.
(Fouchet’s reagent consists of 25 grams of trichloroacetic acid, 10 ml of 10%
ferric chloride, and distilled water 100 ml).
iv. Immediate development of blue-green color around the drop indicates
presence of bilirubin
5. Reagent strips or tablets impregnated with diazo reagent: These tests are
based on reaction of bilirubin with diazo reagent; color change is proportional
to the concentration of bilirubin. reagent strip tests are less sensitive than icotest
tab (0.5 mg/dl).
Positive Gmelin’s showing play of
colors
Positive Fouchet’s test for bilirubin in urine
23. Bile Salts Take some fresh urine in a conical glass tube. Urine should be at the
room temperature. Sprinkle on the surface particles of sulphur. If bile salts are
present, sulphur particles sink to the bottom because of lowering of surface
tension by bile salts. If sulphur particles remain on the surface of urine, bile salts
are absent.
Thymol (used as a preservative) gives false positive
test.
Urobilinogen
Ehrlich’s aldehyde test: Ehrlich’s reagent
(pdimethylaminobenzaldehyde,PDMAB)
reacts with urobilinogen in urine to produce a pink color. Intensity of color
developed depends on the amount of urobilinogen present. Presence of bilirubin
interferes with the reaction, and therefore if present, should be removed. For this,
equal volumes of urine and 10% barium chloride are mixed and then filtered. Test
for urobilinogen is carried out on the filtrate.
Method: Take 5 ml of fresh urine in a test tube. Add 0.5
ml of Ehrlich’s aldehyde reagent (which consists of hydrochloric acid 20 ml,
distilled water 80 ml, and paradimethylaminobenzaldehyde 2 gm).
Allow to stand at room temperature for 5 minutes.
Development of pink color indicates normal amount of urobilinogen. Dark
red color means increased amount of urobilinogen
24. Since both urobilinogen and porphobilinogen produce similar reaction, further
testing is required to distinguish between the two.
For this, Watson-Schwartz test is used. Add 1-2 ml of chloroform, shake for
2 minutes and allow to stand. Pink color in the chloroform layer indicates
presence of urobilinogen, while pink coloration of aqueous portion indicates
presence of porphobilinogen. Pink layer is then decanted and shaken with
butanol. A pink color in the aqueous layer indicates porphobilinogen.
False-negative reaction can occur in the presence of
(i) urinary tract infection (nitrites oxidize urobilinogen to urobilin), and
(ii) antibiotic therapy (gut bacteria which produce urobilinogen are destroyed).
2. Reagent strip method: This method is specific for
urobilinogen. Test area is impregnated with either
p-dimethylaminobenzaldehyde or 4-methoxybenzene
diazonium tetrafluoroborate.
25.
26. Microscopic examination of urinary sediment:
• Definition of microscopic hematuria is presence of 3 or more number of red blood cells
per high power field on microscopic examination of urinary sediment in two out of three
properly collected samples
Chemical tests are positive in hematuria, hemoglobinuria, and myoglobinuria.
• Benzidine test: Make saturated solution of benzidine in glacial acetic acid. Mix 1 ml of
this solution with 1 ml of hydrogen peroxide in a test tube. Add 2 ml of urine. If green or
blue color develops within 5 minutes, the test is positive.
• Orthotoluidine test: In this test, instead of benzidine, orthotoluidine is used. It is more
sensitive than benzidine test.
• Reagent strip test: Various reagent strips are commercially available which use different
chromogens (o-toluidine, tetramethylbenzidine).
Tests for Detection of Hemoglobinuria are benzidine test, orthotoluidine test, and
reagent strip test.
27. Hemosiderin appears as blue granules when urine sediment is stained with
Prussian blue stain.
Ammonium sulfate solubility test is used as a screening test for myoglobinuria
(Myoglobin is soluble in 80% saturated solution of ammonium sulfate, while
hemoglobin is insoluble and is precipitated. A positive chemical test for blood done
on supernatant indicates myoglobinuria).
Tests for Significant Bacteriuria
Nitrite test: Nitrites are not present in normal urine;
ingested nitrites are converted to nitrate and excreted in urine. If gram-negative
bacteria (e.g. E.coli, Salmonella, Proteus, Klebsiella, etc.) are present in urine,
they will reduce the nitrates to nitrites Nitrites are then detected in urine by
reagent strip tests. As E. coli is the commonest organism causing urinary tract
infection, this test is helpful as a screening test for urinary tract infection. Some
organisms like Staphylococci or Pseudomonas do
not reduce nitrate to nitrite and therefore in such infections nitrite test is negative.
Also, urine must be retained in the bladder for minimum of 4 hours for conversion
of nitrate to nitrite to occur; therefore, fresh early morning specimen is preferred.
Sufficient dietary
intake of nitrate is necessary. Therefore a negative nitrite test does not
necessarily indicate absence of urinary tract infection. The test detects about
70% cases of urinary tract infections.
2. Leucocyte esterase test: It detects esterase enzyme
released in urine from granules of leucocytes. Thus the test is positive in pyuria. If
this test is positive, urine culture should be done. The test is not sensitive to
leucocytes < 5/HPF
28. MICROSCOPIC EXAMINATION
Cells in urine (1) Isomorphic red blood cells, (2) Crenated red cells, (3) Swollen red
cells, (4) Dysmorphic red cells, (5) White blood cells (pus cells), (6) Squamous
epithelial cell, (7) Transitional epithelial cells, (8) Renal tubular epithelial cells, (9)
Oval fat bodies, (10) Maltese cross pattern of oval fat bodies, and (11) spermatozoa
29. SWOLLEN RBCS(thin discs of greater diameter, 9-10 μ) in dilute or hypotonic
urine, or
CRENATED (smaller diameter with spikey surface) in hypertonic urine. In
glomerulonephritis, red cells are typically described as being DYSMORPHIC (i.e.
Markedly variable in size and shape).
Presence of > 80% of dysmorphic red cells is strongly suggestive of
glomerular pathology.
RENAL TUBULAR EPITHELIAL CELLS is a significant
finding. Increased numbers are found in conditions causing tubular damage like
acute tubular necrosis, pyelonephritis, viral infection of kidney, allograft
rejection, and salicylate or heavy metal poisoning.
OVAL FAT BODIES are seen in nephrotic syndrome in which there is lipiduria.
Significant bacteriuria exists when there are >105
bacterial colony forming units/ml of urine in a cleancatch
midstream sample, >104 colony forming units/ml
of urine in catheterized sample, and >103 colonyforming
units/ml of urine in a suprapubic aspiration
sample.
30. Organisms in urine: (A) Bacteria, (B) Yeasts,
(C) Trichomonas, and (D) Egg of Schistosoma
haematobium
31. Urinary casts: (A) Hyaline cast, (B) Granular cast, (C) Waxy cast, (D) Fatty cast,
(E) Red cell cast,
(F) White cell cast, and (G) Epithelial cast
