MICROSCOPIC EXAMINATION OF URINE

MACROSCOPIC SCREENING a.k.a Chemical Sieving

Microscopic examination of sediment is performed only on specimens
meeting specified criteria
ADDIS COUNT
1st procedure to standardize the quantitation of formed elements
(1926)
Uses a HEMOCYTOMETER to count the elements present in a 12 hr
specimen
Monitor the course of diagnosed cases of renal disease

Normal values of ADDIS COUNT

HEMOCYTOMETER RULES :
The two ruled areas on either side of the hemocytometer each have
an area of 9 mm
The depth of the chamber between coverslip and ruled area is 0.1 mm
All cells touching any one of the triple lines at the TOP or LEFT of the
square being evaluated are counted.
All cells touching any one of the triple lines at the BOTTOM or to the
RIGHT are excluded from the count.

Specimen Preparation
Examine specimen while fresh or adequately preserved
Formed elements disintegrate rapidly particularly in dilute alkaline
urine
REFRIGERATION causes precipitation of amorphous solutes and
other nonpathologic crystals that can obscure other elements in the
urine sediment
Warm specimen to 37C prior to centrifugation
SPECIMEN VOLUME
Volume recommended is 12 mL (10-15mL)
This volume must be from a well-mixed specimen must be mixed
prior to transferring a portion into a centrifuge tube
Contains a representative sampling of the formed elements
For Pediatric Patients: 6 mL
CENTRIFUGATION
Speed of centrifuge and length of time should be consistent
Recommended:
400 to 450 g for 5 minutes (speed is in RCF )
The speed in RPM required to obtain 400 to 450 g may vary in
different centrifuges.
Centrifugation speed of 450 g
Allows for OPTIMAL SEDIMENT CONCENTRATION without the
disruption of fragile formed elements such as cellular casts.
The centrifuge brake must not be used because it causes the
sediment to resuspend prematurely.
SEDIMENT CONCENTRATION
Following centrifugation, the covered urine specimens should be
carefully removed and all specimens concentrated equally.
Standardized commercial systems accomplish this through consistent
retention of a specific volume of urine.
Goal is to retain at least 1 mL of urine for sediment resuspension
Manual techniques strive toward a 12:1 concentration
---------- | Lorreine Denise Castaares, RMT, AMT

MICROSCOPIC EXAMINATION OF URINE

Uniform amount of urine and sediment should remain after decanting
0.5ml and 1 ml volume are frequently used
Specimen must be thoroughly resuspended by gentle agitation
Lightly tap bottom of tube with the forefinger
Urine should be aspirated off and not poured off.
DROP ON A SLIDE METHOD
Glass microscope slides and coverslips do not yield standardized,
reproducible results.
Recommended volume :
20 ul (.02 ml) covered by a 22x22 mm glass coverslip
Sediment should just fill the area beneath the coverslip without
excess.
Bubbles and uneven distribution of the sediment components can
result from application of the coverslip heavier components such as
casts are more concentrated near the coverslip edges
REPORTING OF RESULTS
Average number of formed elements present in 10 fields for both HPF
and LPF is reported
LPF
Detect casts and ascertain the general composition of the sediment
HPF
When elements such as casts that require identification are
encountered
REPORTING for MICROSCOPIC EXAMINATION
Casts
Average no./LPF in 10 fields
RBCs and WBCs
Average no./HPF in 10 fields
Epithelial cells, Normal crystals, Mucus threads, Amorphous solutes,
Bacteria
Rare, Few, Moderate or Many

Commercial systems
Purpose:
To surpass the outdated practice of using a drop of urine on a glass and
covering it with a coverslip.
Features a disposable plastic centrifuge tube with graduations for
consistent urine volume measurement.
The tubes are clear and conical facilitating sediment formation
during centrifugation.
Advantages:
Cost competitive
Easy to adapt to
Necessary to ensure reproducible and accurate results
Each system seeks consistently to :
1. Produce the same concentration of urine or sediment volume
2. Present the same volume of sediment for microscopic examination
3. Control microscopic variables such as focal planes and optical
properties of the slides
Enhancing urine sediment visualization
1. STAINING
changes refractive index of formed elements
2. TYPE OF MICROSCOPY
facilitate visualization of low-refractility components
STAINS
1. SUPRAVITAL STAINS
A. STERNHEIMER-MALBIN
Most commonly used
It consists of crystal-violet and safranin
Allows more detailed images of the internal structure, particularly of
white blood cells, epithelial cells and casts

---------- | Lorreine Denise Castaares, RMT, AMT

MICROSCOPIC EXAMINATION OF URINE

Disadvantage:
In strongly alkaline urines, this stain precipitates, thereby obstructing the
microscopic visualization of formed elements.
B. 0.5 % TOLUIDINE BLUE
metachromatic dye that stains various cell components differently.
differentiation between nucleus and cytoplasm becomes more
apparent
distinguishes between cells of similar size
2. ACETIC ACID
Although it is not a stain, it can be helpful in identifying white blood
cells.
Upon adding 1 to 2 drops to urine sediment, the nuclear pattern of
WBCs and Epithelial cells is accentuated, whereas red blood cells
lyse.
3. FAT or LIPID STAINS
SUDAN III and Oil Red O
Only Neutral fat (Triglycerides) will stain
Cholesterol and Cholesterol esters will NOT stain
4. GRAM STAIN
It provides means of positively identifying bacteria in the urine
Differentiates gram negative from gram positive
Use a dry preparation of Urine sediment
5. PRUSSIAN BLUE REACTION
Used to identify hemosiderin
Described by Rous in 1918 to identify Urinary siderosis.
It stains the iron of hemosiderin granules a characteristic blue
6. HANSEL STAIN
Methylene blue and Eosin- Y in methanol
Used to identify specifically Eosinophils in the urine
Determination of Acute Interstitial Nephritis

MICROSCOPY TECHNIQUES
1. PHASE CONTRAST MICROSCOPY
More detailed visualization of translucent or low refractile components
and living cells than is possible with brightfield microscopy.
2. POLARIZING MICROSCOPY
confirm the presence of fat, specifically Cholesterol.
3. INTERFERENCE CONTRAST MICROSCOPY
A. Differential interference contrast (Nomarski)
B. Modulation contrast microscopy (Hoffman)
FORMED ELEMENTS IN URINARYSEDIMENT

RED BLOOD CELLS

normal value = < 0-3 RBCs / HPF

8 m in diameter and 3 m in depth
smooth biconcave discs with no nucleus
in Hypertonic urine - crenate or develop spicules and crenations
in Hypotonic urine - swell and release their hemoglobin
GHOST CELLS
CORRELATION
MACROSCOPIC Red color of Sediment
(+) Chemical Test
( - ) Microscopic examination
o RBCs readily lyse and disintegrate in hypotonic and alkaline
urine
( - ) Chemical Test
(+ ) Microscopic examination
o Ascorbic acid interference
LOOK-ALIKES
Yeast
Monohydrate Calcium oxalate crystals
Oil droplets
---------- | Lorreine Denise Castaares, RMT, AMT

MICROSCOPIC EXAMINATION OF URINE

Air bubbles
WBCs
CLINICAL SIGNIFICANCE
Glomerulonephritis
Pyelonephritis
Cystitis
Calculi
Tumors
Trauma
Anticoagulant drugs
False positive : vaginal secretion and hemorrhoids

Eosinophils
Differentiation from neutrophils in routine exam is impossible
Use Hansel stain to distinguish
Eosinophiluria is seen in Acute Interstitial Nephritis
Lymphocytes
Single, round to slightly oval nucleus and scant cytoplasm
Seen in Renal Transplant rejections
Produce negative result on reagent strip
LOOK-ALIKES
Renal tubular epithelial cells
RBCs

WHITE BLOOD CELLS

5 TYPES :
Neutrophils
Lymphocytes
Eosinophils
Basophils
Monocytes (Macrophages)
Neutrophils
Most common
Larger than RBCs (10-14m)
Spherical w/ lobed or segmented nuclei
NORMAL VALUE = 0-8/HPF
Brownian movement of refractile cytoplasmic granules in edemic
leukocytes in hypotonic urine Glitter cells
Aged or Degenerated Neutrophils
Fusion of lobed nuclei
Formation of blebs or vacuoles
Development of Myelin forms (finger or wormlike projections)

CORRELATION
MACROSCOPIC Cloudy and Sediment is gray-white
(+) Chemical examination
( - ) Microscopic examination
o Cellular lysis&disintegration
( - ) Chemical examination
( + ) Microscopic examination
o Not granulocytic leukocytes or amount of esterase not
sufficient
CLINICAL SIGNIFICANCE
BACTERIAL (Pyelonephritis, Cystitis, Urethritis, Prostatitis)
NONBACTERIAL (Nephritis, Glomerulonephritis, Chlamydia,
Mycoplasmosis, Tuberculosis, Trichomonads and Mycoses)
EPITHELIAL CELLS
FROM :
Normal cell turnover of aging cells
Epithelial damage caused by inflammation or Renal disease
---------- | Lorreine Denise Castaares, RMT, AMT

MICROSCOPIC EXAMINATION OF URINE

1. Squamous Epithelial Cells

Most common and the largest EC in urine
Has small, centrally nucleated nucleus, can be anucleated
Thin, flagstone shaped with large cytoplasm filled with fine granulation
(Keratohyalin granules)
The only EC assessed using Low power objective
Rarely have diagnostic significance
Usually indicate specimen contamination
2. Transitional (Urothelial) Epithelial Cells
Small, centrally located, oval to round nucleus
Variable shape :
Superficial layer - larger, round or pear shaped
Intermediate layer - smaller and rounder
Deep Basal layer - small, elongated or columnarlike
Increased in UTI
Clusters or sheets seen after catheterization or other types of
instrumentation procedures
But if w/o these procedures - Transitional Cell Carcinoma
3. Renal Tubular Epithelial Cells
Newborns have more RTEs than older children or adults
2 TYPES :
1. Convoluted Renal Tubular Cells
Proximal Convoluted Tubular Cells
Distal Convoluted Tubular Cells
2. Collecting Duct cells
Convoluted Renal Tubular Cells
1. Proximal Convoluted Tubular Cells
Large with granular cytoplasm, are oblong or cigar shaped
With usually eccentric nucleus, can be multinucleated

2. Distal Convoluted Tubular Cells

Smaller with granular cytoplasm and are round to oval shaped
Eccentric nucleus
3. Collecting Duct Cells
Are cuboidal, polygonal or columnar
Always look for a corner or straight edge on the cell
Single large nucleus that is 2/3 of its moderately smooth cytoplasm
Can be observed as fragments of undisrupted tubular epithelium
CLINICAL SIGNIFICANCE
Severe tubular injury and damage to epithelial basement membrane
Acute Ischemic or Toxic Renal Tubular Disease
Heavy metals or Drug Toxicity
Trauma
Shock
Sepsis
Renal Tubular Cells with Absorbed Fat
Called Oval Fat Bodies
Accompanied by increased amounts of Urinary Protein and Cast
formation
Identified using polarized microscopy and fat stains

CASTS
Formed in Distal and Collecting tubules
Made up of Tamm-Horsfall Protein or Uromodulin excreted by the
renal tubular epithelial cells in the distal convoluted tubule and upper
collecting duct
Cylindrical and thicker in the middle than along edges
Shape and size vary greatly depending on the diameter and shape of
the tubule in which they were formed

---------- | Lorreine Denise Castaares, RMT, AMT

MICROSCOPIC EXAMINATION OF URINE

CYLINDROIDS - casts that are well formed in one end and tapered or
have a tail at the other end
Formation of casts are enhanced by :
Increased pH
Increased solute concentration
Increased plasma proteins (particularly albumin)
Urinary stasis

CLINICAL SIGNIFICANCE
NORMAL : a few Hyaline or finely granular casts
Increased numbers indicate Renal Disease
Both type of cast and their numbers provide valuable information to
the physician
2 EXCEPTIONS :
1. Athletic Pseudonephritis
After strenuous exercise, increased number of casts are found in
urine
Does not indicate renal disease
Due to exercise- induced albuminuria
2. Diuretic therapies
CLASSIFICATION OF CASTS
BASED ON :
Composition of their Matrix
Type of substances or cells enmeshed within
HOMOGENOUS MATRIX COMPOSITION
1. HYALINE CASTS
Composed of homogenous Uromodulin protein matrix
Most commonly observed casts
Has low refractive index similar to urine

Colorless with rounded ends in various shapes and sizes

NORMAL : <2/LPF
2. WAXY CASTS
Have high refractive index
Edges are well defined with sharp, blunt or uneven ends
Cracks or Fissures are often present
Indicate tubular obstruction with prolonged stasis
Renal Failure Casts
CELLULAR INCLUSION CASTS
1. Red Blood Cell Casts
May degenerate into blood casts
Indicate intrinsic renal disease
Often of glomerular origin but can also be from tubular damage
Accompanied by varying degrees of Proteinuria
2. White Blood Cell Casts
Indicates renal infection or inflammation
If origin is Glomerular, accompanied by greater number of RBC casts
Primary marker for distinguishing pyelonephritis (upper UTI) from lower
UTIs
3. Renal Tubular Cell Casts
Are nonspecific markers of Tubular injury
May appear as fragments of the tubular lining removed intact from the
tubule
Indicate severely damaged nephron
Accompanied by Proteinuria and Granular casts
4. Bacterial Casts
Visualizing is difficult, often reported as leukocyte casts (are actually
mixed casts)
Diagnostic of Pyelonephritis
Do gram staining

---------- | Lorreine Denise Castaares, RMT, AMT

MICROSCOPIC EXAMINATION OF URINE

CASTS WITH INCLUSIONS
1. Granular Casts
Range from small and fine to large and coarse granules byproducts of
protein metabolism
Cast granulation is not clinically significant
Accompanied by cellular casts in renal disease and may degenerate into
waxy casts during urinary stasis
2. Fatty Casts
Contain :
Free fat globules
Oval Fat Bodies
Or Both
Matrix :
Hyaline
Granular
Accompanied by significant Proteinuria
Indicates Renal Pathologic condition (particularly Nephrotic syndrome)
Other Inclusion Casts
Sulfonamide and Calcium oxalate are the most commonly encountered
Often accompanied by varying amounts of Hematuria
PIGMENTED CASTS
Usually of hyaline matrix with distinct coloration
Hemoglobin and Myoglobin casts = yellow to brown, differentiation
requires patient history
Bilirubin casts = yellow to brown, colors formed elements of sediment
SIZE
Wide or Broad casts indicate significant urine stasis because of
obstruction or disease

Indicates destruction (widening) of the tubular walls

a poor prognosis
all types of casts may occur in the broad form
CORRELATION
Presence of casts must be accompanied by proteinuria (in varying
degree)
RBC casts = chemical test for blood is (+)
WBC casts = chemical test for LE may be (+/-)
Often accompanied by bacteriuria (+) nitrite test)
Bilirubin casts = chemical test for bilirubin is (+)
Hemoglobin and Myoglobin casts = chemical test for blood is (+)
LOOK-ALIKES
Mucus threads
Cotton threads
Diaper fibers
Squamous ECs
Scratches on coverslip surface
Crystals (amorphous urates & phosphates)

CRYSTALS
From the precipitation of urinary solutes
Crystal formation in nephrons can cause significant tubular damage
Identified based on:
Microscopic appearance
pH at which they are present
FACTORS THAT INFLUENCE CRYSTAL FORMATION
1. Concentration of the solute in the urine
2. Urinary pH
3. Flow of urine through the tubules

---------- | Lorreine Denise Castaares, RMT, AMT

MICROSCOPIC EXAMINATION OF URINE

ACIDIC URINE
1. Amorphous Urates
Ionized form of Uric acid ( at a pH of 5.7-7.0)
If added w/ conc. HAc, uric acid crystals form
Small, yellow brown granules like sand
Uroerythrin deposits - Brick dust
Alkali soluble
Dissolves when heated to 60C
Have no clinical significance
2. Acid Urates
Small, yellow brown balls or spheres
Alkaline counterpart - Ammonium biurate
Frequently not observed in fresh urine
3. Monosodium urates
Colorless to light yellow slender, pencil like prisms
4. Uric Acid
Orange to yellow brown ( color intensity varies w/ thickness of crystal)
Diamond shape (most common), cube shape, clustered to form
rosettes, may have four to six sides
Present only if pH is below 5.7
Originates from catabolism of purine nucleosides
Increased following administration of cytotoxic drugs and in Gout
5. Calcium oxalate
2 FORMS :
Monohydrate - small, ovoid and dumbbell-shaped
Dihydrate - octahedral or envelope shaped, 2 pyramids joined at their
bases
Are the most frequently observed crystals (can form in any pH)
Of the oxalate present in urine, 50% is from ascorbic acid
Increased numbers after ingestion of ethylene glycol (antifreeze) and
during severe chronic renal disease

6. Bilirubin crystals
Yellow brown small clusters of fine needles (but granules and plates
have been observed)
Correlated w/ chemical exam
Classified as an abnormal crystal
Form in urine after excretion and cooling, frequently not observed
7. & 8. Tyrosine and Leucine
Tyrosine - colorless or yellow fine delicate needles (form clusters or
sheaves but also appear singly)
Leucine - yellow to brown spheres with concentric circles and radial
striations
Require refrigeration to force them out of solution
Tyrosine is found more often because it is less soluble than leucine
Abnormal crystals that indicate overflow aminoaciduria, and rarely in
severe liver disease
9. Cystine
Colorless, hexagonal plates
Indicate congenital cystinosis or cystinuria
Deposit within tubules as calculi resulting in renal damage
Present in urine with pH less than 8.0
Thin hexagonal uric acid crystals can resemble cystine
Do Cyanide-Nitroprusside reaction.
Na cyanide - cystine cysteine - purple color
10. Cholesterol
Clear, flat rectangular plates with notched corners
Soluble in chloroform and ether
Accompanied by free floating fat globules, Fatty cast, Oval Fat bodies
and large amounts of protein
Seen in Nephrotic syndrome and in conditions resulting in chyluria
Intravenous radioopaque contrast media are morphologically similar,
correlate with chemical findings to differentiate

---------- | Lorreine Denise Castaares, RMT, AMT

MICROSCOPIC EXAMINATION OF URINE

11. Medications
Iatrogenic - induced in the patient as a result of a treatment
Proper identification and reporting is important
Treatment such as increased hydration or infusion of pH-adjusting
agents can be initiated
A. Ampicillin
Long, colorless thin prisms or needles
Indicate large doses of ampicillin
B. Sulfonamides
Yellow to brown bundles of needles or resemble sheaves of wheat
(constrictions result in fan formation)
C. Radiographic contrast media
Include Meglumine diatrizoate
May appear as long colorless pointed needles singly or in sheaves or
as flat, elongated rectangular plates
ALKALINE URINE
1. Amorphous phosphate
Microscopically indistinguishable from amorphous urates (differentiate
based on urine pH, solubility characteristics and macroscopic
appearance)
Acid soluble
Do not dissolve at 60C
2. Triple phosphate
Three to six sided prisms Coffin lids
With prolonged storage, crystals can dissolve and take on a feathery form
like a fern leaf
Have little clinical significance
3. Calcium phosphate

Dibasic calcium phosphate - stellar phosphates, colorless, thin wedgelike

prisms - one tapered or pointed end, the other end squared off, or thin
long needles in bundles or sheaves
Monobasic calcium phosphate - irregular granular sheets or flat plates
4. Ammonium biurate
Yellow brown spheres with striations or spicules and thornlike projections
- thorny apple
Dissolve in acetic acid or heating to 60C
Presence often indicates inadequate hydration of patient
When encountered, determine whether :
Integrity of specimen has been compromised
In vivo formation is taking place (cause renal tubular damage)
5. Calcium carbonate
Small, colorless granular crystals
Sometimes misidentified as bacteria (size and occasional rod shape)
No clinical significance
MISCELLANEOUS FORMED ELEMENTS
1. Mucus threads
Delicate, ribbonlike strands with low refractive index
Some are derived partially from tubules, others from genitourinary tract
(vaginal epithelium)
2. Bacteria
Most commonly encountered are rod shaped but coccoid forms are also
present
Motility distinguishes them from amorphous substances
Presence is only clinically significant if urine is properly collected and
stored
3. Yeast
Ovoid, colorless that closely resemble RBCs (do not dissolve in acid and
do not stain with supravital stains)
Often have budding and pseudohyphae
---------- | Lorreine Denise Castaares, RMT, AMT

MICROSCOPIC EXAMINATION OF URINE

Often indicates vaginal infections (pregnancy, oral contraceptives and
Diabetes mellitus)
Most common is Candida albicans (characteristic budding and devt of
pseudohyphae)
Only presence of yeast is determined, to Identify species fungal culture is
done.
KOH preparation used to detect yeast, hyphae and fungal cells in
vaginal secretions
4. Fat
Lipiduria is always clinically significant
May occur following severe crush injuries
Characteristic feature of Nephrotic syndrome (severe proteinemia,
hypoprotenemia, hyperlipidemia and edema)
In preeclampsia patients
LOOK-ALIKES
Starch
RBCs
Oils and fats from lubricants, ointments, creams, lotions etc.
To differentiate:
Present only as free floating fat globules
Homogeneity
Lack of structure
Size
5. Hemosiderin
Form of Iron from Ferritin denaturation
Found in urine 2-3 days after a severe hemolytic episode
The Prussian blue reaction or Rous test urine + potassium ferricyanideHCl, after 10mins at RT coarse blue granules
6. Spermatozoa
Indicates recent intercourse or ejaculation
7. Vaginal Contaminants
Trichomonas vaginalis

Characteristic flitting or jerky movement in wet preparations

Most common cause of parasitic gynecological infections in female
patients
In males, usually asymptomatic
8. Clue cells and Gardnerella vaginalis
Clue Cells - squamous ECs from vaginal mucosa with large numbers of
bacteria adhering to them
Indicative of BV - synergistic infection by Gardnerella vaginalis and an
anaerobe Mobiluncus spp.

9. Fecal Contaminants
Through improper collection technique
Abnormal connection or fistula between urinary tract and the bowel
10. Starch
From body powder or in gloves
Vary in size and have a centrally located dimple
Exhibit maltese cross appearance under polarizing microscopy
11. Parasites
Entamoeba histolytica
Enterobius vermicularis
Giardia lamblia
Schistosoma haematobium

10

---------- | Lorreine Denise Castaares, RMT, AMT

MICROSCOPIC EXAMINATION OF URINE

HOMOGENOUS
MATRIX

Hyaline
Waxy
Red Blood Cells

CELLULAR
INCLUSIONS

White Blood Cells

Renal Tubular Epithelial Cells

Mixed Cells
Bacteria
Granular

OTHER
INCLUSIONS

Fat Globules (Cholesterol, Triglycerides)

Hemosiderin Granules
Crystals
Bilirubin

PIGMENTED

Hemoglobin
Myoglobin

SIZE

Broad

11

---------- | Lorreine Denise Castaares, RMT, AMT