MICROSCOPIC EXAMINATION OF URINE

MICROSCOPIC EXAMINATION OF URINE

to detect and to identify insoluble materials present in the urine.

- blood

- kidney

- lower genitourinary tract

- external contamination

formed elements contributor

Microscopic analysis

subject to several procedural variations, including the methods by which the sediment is prepared, the volume of sediment actually examined, the methods and equipment used to obtain visualization, and the manner in which the results are reported.

URINE SEDIMENT PREPARATION

undertaken to identify cells, casts, crystals, and/or microorganisms

decanted urine = concentrated

within 2 hours

Examination time

Dilute Alkaline

Formed elements disintegrate rapidly

Refrigeration

cause precipitation of amorphous urates and phosphates and other nonpathologic crystals

37C

Warming to _____ prior to centrifuging may dissolve crystals (heat)

Standard Volume: 10-15mL

provides an adequate volume from which to obtain a representative sample of the elements present in the specimen

Average Volume: 12mL

frequently used because multiparameter reagent strips are easily immersed in this volume, and capped centrifuge tubes are often calibrated to this volume.

Speed and Time

Consistency in ____________ is essential in centrifugation.

Standard Centrifugation: 400RCF (~1000rpm) for 5 minutes

Produces an optimum amount of sediment with the least chance of damaging the elements excessive speed/time may destroy elements.

0.5-1mL

decantation

Concentration Factor

Volume centrifuged ÷ Sediment volume

Resuspension

gentle agitation to evenly distribute sediment.

Coverslip

prevents evaporation and refractive artifact.

- ocular lens system

- objective lens system

Two-lens system:

Ocular lens system

typically 10× or 15× magnification.

▪ Binocular - reduces strain, better visualization.

▪ Field of view: diameter seen; decreases with higher magnification

Objective lens system

▪ 4× (scanning)

▪ 10× (low power)

▪ 40× (high power)

▪ 100× (oil immersion)

10× (low power)

cast

40× (high power)

cells and crystals

100× (oil immersion)

rarely used

Total magnification

ocular x objective

light source

usually in the base

rheostat

to adjust brightness

filters

modify illumination or wavelength.

field/iris diaphragm

controls diameter of light beam

condenser

beneath stage, focuses light on specimen.

Resolution ( resolving power)

The ability of the microscope to distinguish two closely spaced objects as separate.

o Wavelength of light (shorter wavelength = higher resolution).

o Numerical Aperture (NA) of the lens.

o Maximum resolution of a light microscope: about 0.2 μm.

o Important for studying fine structural details

Parfocal

Once focused under low power, only minor fine focusing is needed when switching to higher objectives. This prevents constant refocusing and minimizes the risk of damaging slides or the objective lens. (uses fine adjustment knob)

Coarse adjustment knob

for initial focusing

Fine adjustment knob

for sharpening details

lens paper or lint-free wipes

lens care

Camel-hair brush

Used to remove dust on the optical surfaces of the microscope

tissue, cotton, or cloth

can scratch lenses

lens paper

Remove oil immediately from the 100× oil immersion lens with _____

Commercial lens cleaner

used to clean any contaminated lenses

General handling

▪ Always carry with two hands (one on the arm, one on the base).

▪ Keep microscope covered when not in use.

▪ Store with the lowest power objective in place and stage lowered.

Kohler's illumination

ensures uniform illumination and resolution

Brightfield Microscopy

⚫ Standard method used in routine urinalysis for examining stained or unstained specimens. ⚫ works by transmitting light through the specimen, with denser areas appearing darker against a bright background.

⚫ simple, widely available, and suitable for general laboratory use

⚫ provides limited contrast for transparent structures unless staining is applied.

Phase Contrast Microscopy

- enhances the visibility of translucent and unstained specimens by converting phase differences in light into contrast.

- useful for observing living cells, urine sediments, and other lowcontrast elements without staining.

Phase Plate

requires phase objectives with a built-in _______ and a special condenser with an annular diaphragm.

Halo effect

effect around specimens in Phase Contrast

Polarizing Microscopy

- the ability of certain substances to refract light in two different directions at 90°.

- useful for identifying cholesterol in oval fat bodies, fatty casts, and various crystals in urine.

- enhances diagnostic accuracy by differentiating birefringent from non-birefringent structures.

Birefringence

Polarizing Microscopy is used to detect _______

polarizer and analyzer

requires two polarizing filters: one placed below the condenser (____________) and the other above the objective (_________).

Darkfield Microscopy

⚫ used to visualize very small or thin organisms, such as Treponema pallidum, that are not easily seen with brightfield microscopy.

⚫ employs a condenser with an opaque disc that blocks direct light, allowing only scattered light from the specimen to enter the objective lens.

⚫ produces a bright image of the organism against a dark background.

⚫ highly sensitive but requires careful alignment of the condenser for optimal results.

Fluorescence Microscopy

- used to detect substances or organisms that have been stained with fluorescent dyes such as acridine orange or fluorescein.

- valuable for identifying microorganisms, antigens, antibodies, and other cellular components with high sensitivity. - widely applied in microbiology, immunology, and clinical diagnostics.

Ultraviolet (UV)

When exposed to __________________ or specific wavelengths of light, these dyes emit visible fluorescence, making the target structures stand out brightly against a dark background.

Interference Contrast Microscopy

⚫ provides a 3 dimensional appearance of specimens

⚫ enhances contrast in layered or transparent structures without the need for staining

⚫ works by splitting and recombining polarized light to highlight differences in specimen thickness and refractive index. ⚫ makes it especially useful for studying live, unstained cells and fine structural details.

- Nomarski Differential

- Hoffman Modulation

types of Interference Contrast

Steinheimer Malbin

⚫ most commonly used supravital stain in urinalysis for enhancing visualization of urine sediment.

⚫ highlights the nuclei and cytoplasm of WBCs, epithelial cells, and casts, improving differentiation of cellular elements.

⚫ crystal violet + safranin O: crystal violet stains nuclei purple, while safranin O stains cytoplasm pink to orange.

⚫ dual staining provides better contrast and aids in accurate identification of urinary sediment components.

Toluidine blue

⚫ metachromatic supravital stain that enhances nuclear detail in urine sediment preparations. ⚫ useful in differentiating white blood cells (WBCs) from renal tubular epithelial (RTE) cells, which may appear similar under brightfield microscopy.

⚫ binds strongly to nucleic acids, highlighting nuclei with greater clarity.

⚫ confirms the presence of RTE cells in cases of renal tubular damage.

2% acetic acid

⚫ used in urine microscopy to lyse red blood cells (RBCs), which helps distinguish them from white blood cells (WBCs), yeast, oil droplets, and crystals that may appear similar in unstained sediments.

⚫ enhances the visibility of WBC nuclei, aiding in accurate identification.

⚫ helpful when RBCs are present in large numbers and obscure other formed elements.

Lipid Stains: Oil Red O and Sudan III

(milky urine)

⚫ used in urine microscopy to detect neutral fats and triglycerides, staining them an orange to red color

⚫ confirms the presence of lipids in oval fat bodies and fatty casts, which are associated with conditions like nephrotic syndrome.

Gram stain

⚫ differentiates gram-positive and gram-negative bacteria

⚫ identifies bacterial casts

Hansel stain

⚫ specialized stain used in urine microscopy for the detection of urinary eosinophils (parasites), which are important markers in allergic and drug-induced interstitial nephritis.

⚫ uses a combination of methylene blue and eosin Y, producing a distinctive appearance where eosinophil granules stain bright pink to red against a blue background.

⚫ Identifying eosinophils in urine helps support the diagnosis of hypersensitivity reactions, drug toxicity, and inflammatory renal conditions.

Prussian blue

⚫ used in urine microscopy to detect hemosiderin, an iron-storage pigment that appears in renal tubular epithelial (RTE) cells following chronic or recurrent hemoglobinuria.

⚫ reacts with ferric iron, producing a blue or blue-green coloration in hemosiderin granules

RBCs (0- 3/HPF)

✓ smooth, non-nucleated, biconcave disks

✓ about 6-8 µm in diameter.

✓ Dilute or hyposthenuria urine: ghost cells

✓ Concentrated or hypersthenuric urine: Crenated

✓ hematuria

Ghost cells

- lysed, colorless membranes

crenated

shrunken with spiky edges.

Hematuria

presence of increased RBCs in urine may indicate glomerular disease, urinary tract infection, trauma, or renal calculi.

WBCs (0- 8/HPF)

typically larger than RBCs, about 10-12 µm, with visible granular cytoplasm and sometimes lobed nuclei (especially neutrophils).

pyuria

An increased number of WBCs suggests infection or inflammation of the urinary tract.

neutrophil

most predominant

Brownian movement

In hypotonic urine, neutrophils may swell, and their cytoplasmic granules exhibit __________________,

glitter cells

producing a sparkling appearance known as "____________."

Eosinophil

Presence in urine is associated with interstitial nephritis

Mononuclear cells

Includes lymphocytes, monocytes, macrophages; Increased numbers suggest chronic inflammation, renal tubular injury, or viral infections.

Squamous Epithelial Cell

✓ largest cells found in urine sediment

✓ abundant, irregular cytoplasm and a centrally or eccentrically located prominent nucleus.

✓ from lining of the distal urethra and vagina

✓ usually considered contaminants in urine specimens, especially in females, due to their origin.

Clue cell and Gardnerella vaginalis

_______________ squamous epithelial cell covered with __________________- presence is significant in the diagnosis of bacterial vaginosis.

Renal Tubular Epithelial Cell

✓ most clinically significant epithelial cells

✓ from the nephrons, specifically the proximal and distal convoluted tubules, and collecting ducts.

✓ appear regular, polyhedral, cuboidal, or columnar, usually smaller than transitional cells, with an eccentric (off-center) nucleus.

✓ Increased numbers indicate renal tubular injury or necrosis

Oval fat bodies

When RTE cells absorb lipids, they become ____________, commonly seen in nephrotic syndrome.

Bubble cells

"____________": RTE cells with large, nonlipid vacuoles associated with acute tubular necrosis.

Transitional Epithelial Cell

✓appear spherical, polyhedral, or caudate (taillike projection), typically with a centrally located nucleus. lining of the renal pelvis, calyces, ureters, and bladder, upper portion of the male urethra.

✓A few cells may normally be present in urine

✓Increased numbers may indicate infection, inflammation, or catheterization.

Urothelial carcinoma

_______________: large clusters of transitional cells

Bacteria

✓ Bacteria without WBCs: Often indicates contamination or an old specimen.

✓ Tiny rods (bacilli) or cocci that may form chains or clusters, often moving due to Brownian motion (not true motility).

✓ Clinical significance: Can lead to cystitis, pyelonephritis, prostatitis, or sepsis if untreated

Bacteria and WBC

True urinary tract infection (UTI)

Eschericia coli

most common cause; major pathogen in uncomplicated UTIs

Yeasts

✓Associated conditions: Diabetes mellitus, prolonged antibiotic therapy, immunosuppression, pregnancy, and vaginal moniliasis.

✓small, oval, budding cells; may form pseudohyphae, helping differentiate them from RBCs.

Candida albicans

✓ Most common speciesof yeast: _______________; opportunistic pathogen.

Yeast and WBC

✓ True Yeast infection:

Trichomonas vaginalis

✓ Most frequently encountered parasite in urine.

✓ Motile, flagellated protozoan with a jerky, tumbling movement.

✓ Associated with sexually transmitted infection (Trichomoniasis, also called "Ping-Pong disease" due to reinfection between partners).

Enterobius vermicularis

✓ Most common fecal contaminant in urine sediment.

✓ Eggs or occasionally adult worms may appear.

✓ Indicates poor hygiene or contamination during specimen collection.

Schistosoma haematobium

✓ Eggs with terminal spines may be seen in urine.

✓ Endemic in parts of Africa and the Middle East.

✓ Associated with urinary schistosomiasis, which can lead to hematuria, chronic cystitis, hydronephrosis, and bladder carcinoma.

Spermatozoa

✓ May be present after sexual intercourse, masturbation, or retrograde ejaculation.

✓ Oval heads with long, thin tails; may clump together.

✓ Generally not pathologic, but can be significant in fertility studies, forensic cases, and pediatric patients (possible sexual abuse concerns)

✓ In post-vasectomy, detection may indicate incomplete procedure or recanalization.

Mucus threads

✓ Appearance: Long, thin, thread-like, wavy, or ribbon-like strands with low refractive index, often overlooked unless stained.

✓ Primarily from the genitourinary tract epithelium

✓ Increased amounts may be seen in UTIs, inflammation, or irritation of the urinary tract.

Tamm-Horsfall protein (Uromodulin)

✓ ________________________: glycoprotein secreted by the thick ascending loop of Henle and distal convoluted tubules.

Casts

- These are cylindrical structures formed in the renal tubules and detected in urine sediment.

- Presence and type of these help localize renal pathology

Cylinduria

The excretion of casts in urine.

Renal diseases

Significant indicators since casts form only within the renal tubules

- distal convoluted tubule (DCT)

- collecting ducts (CD)

site of formation of casts

Tamm-Horsfall protein (uromodulin)

casts are composed mainly of what?

HYALINE (0-2/LPF)

◆ Most commonly encountered & most difficult cast to discover

◆ Colorless, transparent, homogeneous cylinders with smooth edges; often difficult to detect due to low refractive index.

Non-pathologic hyaline cast

May appear after strenuous exercise, dehydration, fever, or stress.

Pathologic hyaline cast

Increased numbers seen in glomerulonephritis, pyelonephritis, chronic renal disease, and congestive heart failure.

RBC cast

◆ Cylindrical casts containing intact or fragmented RBCs, giving a yellow to brown appearance; often granular as cells degenerate.

◆ most fragile type of cast; may break down easily, so careful examination is needed.

◆ Strongly indicates bleeding within the nephron

◆ Seen in glomerulonephritis, vasculitis, malignant hypertension, and strenuous exercise.

BLOOD CAST (MUDDY BROWN CAST)

◆ appear as brown to muddy brown, pigmented casts without intact RBCs; color results from the incorporation of hemoglobin or myoglobin into the cast matrix.

◆ formed when RBCs lyse within the tubules, releasing hemoglobin, or when myoglobin is filtered due to muscle injury.

◆ may also appear in acute tubular necrosis due to pigment-induced tubular damage.

◆ sometimes described as pigmented casts and distinguished from granular casts by their uniform brown coloration.

hemoglobinuria and myoglobinuria

◆ seen in severe intravascular hemolysis (____________) or rhabdomyolysis (____________).

WBC CAST (PUS CAST)

◆ Cylindrical casts containing intact or degenerated WBCs; may appear granular as cells break down.

◆ Indicates inflammation or infection within the nephron, differentiating upper urinary tract infections from lower tract infections.

◆ Commonly seen in pyelonephritis and acute interstitial nephritis (often drug induced)