MICROSCOPIC

EXAMINATION OF URINE
• Microscopic examination of urine is also called as
the “liquid biopsy of the urinary tract
• Urine consists of various microscopic, insoluble, solid
elements in suspension. These elements are classified as
organized or unorganized.

• Organized substances include red blood cells, white blood

cells, epithelial cells, casts, bacteria, and parasites.

• The unorganized substances are crystalline and

amorphous material. These elements are suspended in urine
and on standing they settle down and sediment at the bottom
of the container; therefore they are known as urinary
deposits or urinary sediments.
• The major aim of microscopic examination of urine is to
identify different types of cellular elements and casts.

• Most crystals have little clinical significance

Specimen:
• The cellular elements are best preserved in acid,
hypertonic urine; they deteriorate rapidly in alkaline,
hypotonic solution.

• A mid-stream, freshly voided, first morning specimen is

preferred since it is the most concentrated
• The specimen should be examined within 2 hours of
voiding because cells and casts degenerate upon
standing at room temperature.

• If preservative is required, then 1 crystal of thymol or 1

drop of formalin (40%) is added to about 10 ml of urine
Method:
• A well-mixed sample of urine (12 ml) is centrifuged in a
centrifuge tube for 5 minutes at 1500 rpm and
supernatant is poured off.

• The tube is tapped at the bottom to resuspend the

sediment (in 0.5 ml of urine).
• A drop of this is placed on a glass slide and covered
with a cover slip.

• The slide is examined immediately under the

microscope using first the low power and then the high
power objective.

• The condenser should be lowered to better visualize

the elements by reducing the illumination.
Preparation of urine sediment for
microscopic examination
Different types of urinary sediment
Cells
Red Blood Cells
• Normally there are no or an occasional red blood cell in
urine.

• In a fresh urine sample, red cells appear as small,

smooth, yellowish, anucleate biconcave disks about 7 μ
in diameter (called as isomorphic red cells).
• However, red cells may appear swollen (thin discs of
greater diameter, 9-10 μ) in dilute or hypotonic urine,
or may appear 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).
• They result from passage of red cells through the
damaged glomeruli. Presence of > 80% of dysmorphic
red cells is strongly suggestive of glomerular
pathology.
• The quantity of red cells can be reported as number of red
cells per high power field.
Causes of Hematuria

Diseases of urinary tract

• Glomerular diseases: Glomerulonephritis, Berger’s
disease, lupus nephritis, Henoch-Schonlein Purpura
• Nonglomerular diseases: Calculus, tumor, infection,
tuberculosis, pyelonephritis, hydronephrosis, polycystic
kidney disease, trauma, after strenuous physical
exercise, diseases of prostate (benign hyperplasia of
prostate, carcinoma of prostate)
Hematological conditions: Coagulation disorders, sickle
cell disease
Presence of red cell casts and proteinuria along with
hematuria suggests glomerular cause of hematuria
 White Blood Cells (Pus Cells)
• White blood cells are spherical, 10-15 μ in size, granular in
appearance in which nuclei may be visible.
• Degenerated white cells are distorted, smaller, and have
fewer granules.
• Clumps of numerous white cells are seen in infections.
• Presence of many white cells in urine is called as pyuria.
• In hypotonic urine white cells are swollen and the granules
are highly refractile and show Brownian movement; such
cells are called as glitter cells; large numbers are indicative
of injury to urinary tract
• Normally 0-2 white cells may be seen per high power
field.
• Pus cells greater than 10/HPF or presence of
clumps is suggestive of urinary tract infection.

• Increased numbers of white cells occur in fever,

pyelonephritis, lower urinary tract infection,
tubulointerstitial nephritis, and renal transplant
rejection
In urinary tract infection, following are usually seen in
combination:
• Clumps of pus cells or pus cells >10/HPF
• Bacteria
• Albuminuria
• Positive nitrite test
• Simultaneous presence of white cells and white cell casts
indicates presence of renal infection (pyelonephritis).
• Eosinophils (>1% of urinary leucocytes) are a
characteristic feature of acute interstitial nephritis due
to drug reaction (better appreciated with a Wright’s
stain).
 Renal Tubular Epithelial Cells
• Presence of 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
• These cells are small (about the same size or slightly larger
than white blood cell), polyhedral, columnar, or oval, and
have granular cytoplasm.

• A single, large, refractile, eccentric nucleus is often seen.

• Renal tubular epithelial cells are difficult to distinguish from

pus cells in unstained preparations
Squamous Epithelial Cells
• Squamous epithelial cells line the lower urethra and
vagina.
• They are best seen under low power objective (×10).
Presence of large numbers of squamous cells in urine
indicates contamination of urine with vaginal fluid.
• These are large cells, rectangular in shape, flat with
abundant cytoplasm and a small, central nucleus.
Transitional Epithelial Cells
• Transitional cells line renal pelvis, ureters, urinary
bladder, and upper urethra.
• These cells are large, and diamond- or pear-shaped
(caudate cells).

• Large numbers or sheets of these cells in urine occur

after catheterization and in transitional cell carcinoma
Oval Fat Bodies
• These are degenerated renal tubular epithelial cells
filled with highly refractile lipid (cholesterol) droplets.
Under polarized light, they show a characteristic
“Maltese cross” pattern.

• They can be stained with a fat stain such as Sudan III

or Oil Red O. They are seen in nephrotic syndrome in
which there is lipiduria
Spermatozoa
• They may sometimes be seen in urine of men
• Telescoped urinary sediment: This refers to urinary
sediment consisting of red blood cells, white blood
cells, oval fat bodies, and all types of casts in roughly
equal proportion. It occurs in lupus nephritis,
malignant hypertension, rapidly proliferative
glomerulonephritis, and diabetic glomerulosclerosis
Organisms

Organisms in urine:
(A) Bacteria,
(B) Yeasts,
(C) Trichomonas,
and
(D) Egg of
Schistosoma
haematobium
Bacteria
• Bacteria in urine can be detected by microscopic
examination, reagent strip tests for significant bacteriuria
(nitrite test, leucocyte esterase test), and culture

• Significant bacteriuria exists when there are >10 5

bacterial colony forming units/ml of urine in a clean

catch midstream sample, >10 colony forming
4

units/ml of urine in catheterized sample, and >10 3

colony forming units/ml of urine in a suprapubic

aspiration sample.
Collection for bacterial culture
• Use sterile container
• Collect midstream, clean catch sample
• Must be plated within 2 hours of collection
• If refrigerated, must be plated within 24 hours of collection
• No preservative should be added
 Microscopic examination

• In a wet preparation, presence of bacteria should be

reported only when urine is fresh.
• Bacteria occur in combination with pus cells.
• Gram’s-stained smear of uncentrifuged urine showing 1 or
more bacteria per oil-immersion field suggests presence of >
10 bacterial colony forming units/ml of urine.
5

• If many squamous cells are present, then urine is probably

contaminated with vaginal flora. Also, presence of only
bacteria without pus cells indicates contamination with
vaginal or skin flora
Chemical or reagent strip tests for significant
bacteriuria
• In addition to direct microscopic examination of urine
sample, chemical tests are commercially available in a
reagent strip format that can detect significant
bacteriuria: nitrite test and leucocyte esterase test.

• These tests are helpful at places where urine

microscopy is not available. If these tests are positive,
urine culture is indicated.
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 through the action of bacterial
enzyme nitrate reductase.

• 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
 The test detects about 70% cases of
urinary tract infections
• 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.
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

Culture:
• On culture, a colony count of >10 /ml is strongly
5

suggestive of urinary tract infection, even in

asymptomatic females.
• Positive culture is followed by sensitivity test. Most
infections are due to Gram negative enteric bacteria,
particularly Escherichia coli.
• If three or more species of bacteria are identified on
culture, it almost always indicates contamination by
vaginal flora
• Negative culture in the presence of pyuria (‘sterile’
pyuria) occurs with prior antibiotic therapy, renal
tuberculosis, prostatitis, renal calculi, catheterization,
fever in children (irrespective of cause), female genital
tract infection, and non-specific urethritis in males
Yeast Cells (Candida)
• These are round or oval structures of approximately the
same size as red blood cells. In contrast to red cells, they
show budding, are oval and more refractile, and are not
soluble in 2% acetic acid
• Presence of Candida in urine may suggest
immunocompromised state, vaginal candidiasis, or
diabetes mellitus. Usually, pyuria is present if there is
infection by Candida.

• Candida may also be a contaminant in the sample and

therefore urine sample must be examined in a fresh state
Trichomonas vaginalis
• These are motile organisms with pear shape,
undulating membrane on one side, and four flagellae.
• They cause vaginitis in females and are thus
contaminants in urine.
• They are easily detected in fresh urine due to their
motility
Eggs of Schistosoma haematobium
• Infection by this organism is prevalent in Egypt
Microfilariae
• They may be seen in urine in chyluria due to rupture of
a urogenital lymphatic vessel
 Casts
• Urinary casts are cylindrical, cigar-shaped microscopic
structures that form in distal renal tubules and collecting
ducts. They take the shape and diameter of the lumina (molds
or ‘casts’) of the renal tubules.
• They have parallel sides and rounded ends. Their length and
width may be variable. Casts are basically composed of a
precipitate of a protein that is secreted by tubules (Tamm-
Horsfall protein).
• Since casts form only in renal tubules their presence is
indicative of disease of the renal parenchyma.
• Although there are several types of casts, all urine casts
are basically hyaline; various types of casts are formed
when different elements get deposited on the hyaline
material.
• Casts are best seen under low power objective (×10) with
condenser lowered down to reduce the illumination
• Casts are the only elements in the urinary
sediment that are specifically of renal origin.
Casts are of two main types:
• Noncellular: Hyaline, granular, waxy, fatty
• Cellular: Red blood cell, white blood cell, renal
tubular epithelial cell.
• Hyaline and granular casts may appear in normal or
diseased states. All other casts are found in kidney
diseases.
Non-cellular Casts
 Hyaline casts
• These are the most common type of casts in urine and are
homogenous, colorless, transparent, and refractile.
• They are cylindrical with parallel sides and blunt, rounded
ends and low refractive index.
• Presence of occasional hyaline cast is considered as normal.
Their presence in increased numbers (“cylinduria”) is
abnormal. They are composed primarily of Tamm-Horsfall
protein. They occur transiently after strenuous muscle
exercise in healthy persons and during fever.
• Increased numbers are found in conditions causing
glomerular proteinuria
Granular casts:
• Presence of degenerated cellular debris in a cast
makes it granular in appearance.
• These are cylindrical structures with coarse or fine
granules (which represent degenerated renal tubular
epithelial cells) embedded in Tamm-Horsfall protein
matrix.
• They are seen after strenuous muscle exercise and in
fever, acute glomerulonephritis, and pyelonephritis.
Waxy cast:
• These are the most easily recognized of all casts. They
form when hyaline casts remain in renal tubules for
long time (prolonged stasis).

• They have homogenous, smooth glassy appearance,

cracked or serrated margins and irregular broken-off
ends. The ends are straight and sharp and not rounded
as in other casts. They are light yellow in color. They
are most commonly seen in end-stage renal failure
Fatty casts:
• These are cylindrical structures filled with highly
refractile fat globules (triglycerides and cholesterol
esters) in Tamm-Horsfall protein matrix.

• They are seen in nephrotic syndrome

Broad casts:
• Broad casts form in dilated distal tubules and are seen
in chronic renal failure and severe renal tubular
obstruction. Both waxy and broad casts are associated
with poor prognosis
Cellular Casts
• To be called as cellular, casts should contain at least
three cells in the matrix.

• Cellular casts are named according to the type of cells

entrapped in the matrix
Red cell casts
• These are cylindrical structures with red cells in Tamm-
Horsfall protein matrix. They may appear brown in
color due to hemoglobin pigmentation.
• These have greater diagnostic importance than any
other cast.
• If present, they help to differentiate hematuria due to
glomerular disease from hematuria due to other
causes.
• RBC casts usually denote glomerular pathology e.g.
acute glomerulonephritis
White cell casts:
• These are cylindrical structures with white blood cells
embedded in Tamm-Horsfall protein matrix.
• Leucocytes usually enter into tubules from the
interstitium and therefore, presence of leucocyte casts
indicates tubulointerstitial disease like pyelonephritis
Renal tubular epithelial cell casts:
• These are composed of renal tubular epithelial cells
that have been sloughed off.
• They are seen in acute tubular necrosis, viral renal
disease, heavy metal poisoning, and acute allograft
rejection. Even an occasional renal tubular cast is a
significant finding
Crystals
• Crystals are refractile structures with a definite
geometric shape due to orderly 3-dimensional
arrangement of its atoms and molecules.

• Amorphous material (or deposit) has no definite shape

and is commonly seen in the form of granular
aggregates or clumps
• Crystals in urine can be divided into two main types:
(1) Normal (seen in normal urinary sediment), and (2)
Abnormal (seen in diseased states).
• However, crystals found in normal urine can also be
seen in some diseases in increased numbers
• Most crystals have no clinical importance (particularly
phosphates, urates, and oxalates).

• Crystals can be identified in urine by their morphology.

• However, before reporting presence of any abnormal

crystals, it is necessary to confirm them by chemical
tests.
Normal Crystals
Crystals present in acid urine
Uric acid crystals:
• These are variable in shape (diamond, rosette, plates),
and yellow or red-brown in color (due to urinary
pigment).
• They are soluble in alkali, and insoluble in acid.

• Increased numbers are found in gout and leukemia.

Flat hexagonal uric acid crystals may be mistaken for
cysteine crystals that also form in acid urine
 Calcium oxalate crystals:
• These are colorless, refractile, and envelope-shaped.
Sometimes dumbbell-shaped or peanut-like forms are seen.
• They are soluble in dilute hydrochloric acid. Ingestion of
certain foods like tomatoes, spinach, cabbage, asparagus,
and rhubarb causes increase in their numbers.
• Their increased number in fresh urine (oxaluria) may also
suggest oxalate stones. A large number are seen in ethylene
glycol poisoning
Amorphous urates:
• These are urate salts of potassium, magnesium, or
calcium in acid urine. They are usually yellow, fine
granules in compact masses.

• They are soluble in alkali or saline at 60°C.

Crystals present in alkaline urine
Calcium carbonate crystals:
• These are small, colorless, and grouped in pairs. They
are soluble in acetic acid and give off bubbles of gas
when they dissolve
Phosphates:
• Phosphates may occur as crystals (triple phosphates,
calcium hydrogen phosphate), or as amorphous deposits.
• • Phosphate crystals
•
Triple phosphates (ammonium magnesium phosphate):


They are colorless, shiny, 3-6 sided prisms with oblique

surfaces at the ends (“coffinlids”), or may have a
feathery fern-like appearance.
•
Calcium hydrogen phosphate (stellar phosphate):


• These are colorless, and of variable shape (starshaped,

plates or prisms).
• Amorphous phosphates: These occur as colorless small
granules, often dispersed. All phosphates are soluble in
dilute acetic acid
Ammonium urate crystals
• These occur as cactus-like (covered with spines) and
called as ‘thornapple’ crystals.

• They are yellow-brown and soluble in acetic acid at

60°C.
Abnormal Crystals
• They are rare, but result from a pathological process.
• These occur in acid pH, often in large amounts.
Abnormal crystals should not be reported on
microscopy alone; additional chemical tests are done
for confirmation
• Cysteine crystals: These are colorless, clear, hexagonal
(having 6 sides), very refractile plates in acid urine.
• They often occur in layers. They are soluble in 30%
hydrochloric acid. They are seen in cysteinuria, an
inborn error of metabolism. Cysteine crystals are often
associated with formation of cysteine stones
• Cholesterol crystals: These are colorless, refractile, flat
rectangular plates with notched (missing) corners and
appear stacked in a stair-step arrangement. They are
soluble in ether, chloroform, or alcohol. They are seen
in lipiduria e.g. nephrotic syndrome and
hypercholesterolemia.
• They can be positively identified by polarizing
microscope
• Bilirubin crystals: These are small (5 μ), brown crystals
of variable shape (square, bead-like, or fine needles).
• Their presence can be confirmed by doing reagent strip
or chemical test for bilirubin. These crystals are soluble
in strong acid or alkali. They are seen in severe
obstructive liver disease.
• Leucine crystals: These are refractile, yellow or brown,
spheres with radial or concentric striations.

• They are soluble in alkali. They are usually found in

urine along with tyrosine in severe liver disease
(cirrhosis).
• Tyrosine crystals: They appear as clusters of fine,
delicate, colorless or yellow needles and are seen in
liver disease and tyrosinemia (an inborn error of
metabolism). They dissolve in alkali
• Sulfonamide crystals: They are variably shaped
crystals, but usually appear as sheaves of needles.

• They occur following sulfonamide therapy. They are

soluble in acetone.