Microscopic Examination of Urine Lecture Notes (copy)

Microscopic Examination of Urine: Lighting and Microscopy Techniques

• Low light setting is essential to begin the examination. Use the rheostat to reduce light intensity.

• The goal is to identify a dark entity on a bright background.

• Use the condenser to adjust contrast levels.

• Polarizing Microscopy:

  • Utilizes polarizing filters through which light travels in waves.

  • Light is reduced to a single plane of vision ($360^{\circ}$).

  • This technique aids in the identification of specific structures by producing unique colors.

  • Primarily used for Crystals and Lipids.

  • Results in sharper images of shapes and unique color patterns.

• Darkfield Microscopy:

  • Produces a brighter entity on a dark background.

  • This is the primary method for identifying Treponema species (bacterial species).

• Immunofluorescence (Fluorescence Microscopy):

  • Uses antibodies with fluorescent labels attached to them.

  • A specific light hits the label, causing the label to fluoresce.

  • The antibodies attach to specific antigens, making this a highly specific identification technique.

Visual Enhancements: Stains and Chemical Additives

• Gram Stain:

  • Used when a random urine sample is received from the Emergency Department (ED).

  • Gram-positive ($+$): Stains purple.

  • Gram-negative ($-$): Stains pink.

  • QNS: Quantity Not Sufficient (used when the sample volume is too low for standard testing).

• Methylene Blue and Acetic Acid:

  • This combination stains nucleated cells.

  • 3 % Acetic Acid ($3\%\,AA$): This concentration lyses (removes) Red Blood Cells (RBCs).

  • This is useful for "narrowing the field" to see other entities.

  • Highly useful for manual counts on Cerebrospinal Fluid (CSF/Spinal Tap).

  • Used to distinguish RBCs from Yeast:

    • RBCs: Removed/lysed by acetic acid.

    • Yeast: Not removed/lysed by acetic acid.

• Lipid/Fat Stains:

  • Lipids are not proteins; they require specific stains like Sudan III or Sudan Black.

  • These identify Fats, Lipids, and casts containing fats.

• Protein Stains:

  • Examples include Bromocresol green and Bromocresol purple.

• Prussian Blue:

  • Used to stain iron-containing entities.

• Hansel Stain (Wright Stain as an option):

  • Used for the identification of Urinary Eosinophils.

  • Identifies granules and nuclei.

  • Eosinophils appear as "Raspberries" under this stain.

  • The presence of these cells is an important indicator of Acute Interstitial Nephritis.

Formed Elements: Red Blood Cells (RBCs)

• Indicates bleeding in the urinary tract.

• The quantity of RBCs determines the extent of the damage.

• Physical characteristics: Small, measuring approximately $6-10\,\mu m$.

• Differentiate between individual cells versus those incorporated into casts, as this indicates location and age of the bleeding.

• Differential Identification (Mistaken for RBCs):

  • Yeast: Look for budding structures; yeast is very similar in appearance to RBCs.

  • Oil Droplets: Distinguished by high refractility and variable size.

  • Air Bubbles: High refractility, variable size, and often located in a different plane of focus.

  • Starch: Highly refractile and polarizes (forming a Maltic Cross/specific pattern).

Formed Elements: White Blood Cells (WBCs)

• Normal Range: Typically <5 per high power field (HPF).

• Females: Often have a higher count than males naturally.

• Clinical Indicators: Infections, allergic reactions, or general damage.

• Glitter Cells:

  • Found in hypotonic urine.

  • Characterized by Brownian motion of the granules within the cell.

• Eosinophils:

  • Associated with allergic responses and drug-induced interstitial nephritis.

  • Identified specifically with Hansel or Wright stains.

• Monocytes and Lymphocytes:

  • Must be differentiated from Renal Tubular Epithelial (RTE) cells.

  • Lymphocytes are rare but may appear in early kidney transplant rejection.

  • Note: RTE cells have an eccentric (off-center) nucleus.

• Pyuria: The term for increased WBCs in the urine.

• Causes of Elevated WBCs (Bacterial vs. Non-bacterial):

  • Glomerulonephritis

  • Interstitial Nephritis

  • Lupus Erythematosus

  • Tumors

  • Cystitis (Bladder infection)

  • Pyelonephritis (Kidney infection)

  • Urethritis

  • Prostatitis

Microorganisms, Artifacts, and Other Structures

• Bacteria:

  • Urine is usually sterile; presence indicates true infection or contamination during exit.

  • Correlate with Chemistry: Positive Leukocyte Esterase (LE) and Nitrite ($NO_2^-$) tests suggest infection.

  • Nitrite test reduction: Nitrate ($NO_3^-$) is reduced to Nitrite ($NO_2^-$).

  • Report as few, moderate, or many per HPF.

• Yeast:

  • Single, oval structures (can be mistaken for RBCs).

  • Common in HIV/AIDS patients (immunocompromised) and Diabetics (moniliasis/Candida).

  • Diabetic Urinalysis Profile: High Glucose in urine ($+$, $++$, etc.) and Yeast presence.

• Trichomonas:

  • A parasite with a pear-shaped flagellate body.

  • Note: The flagella and the undulating membrane (the "eye") help in identification.

• Sperm:

  • Characterized by oval, tapered heads and long tails.

  • Urine is toxic to sperm; they are often found dead/immobile.

  • Presence may cause a false positive protein on the reagent strip.

• Mucus:

  • Protein material originating from glands and squamous cells.

  • More common in female specimens.

• Fibers:

  • Synthetic fibers appear as "perfect" lines.

  • May resemble mucus, but are usually much coarser.

• Other Artifacts: Fecal matter, Parasite eggs, or cysts.

Epithelial Cells: Pathological and Non-Pathological

• Squamous Epithelial Cells:

  • Non-pathological; origin is the normal sloughing of skin from the Vagina or Urethra (the "last stop").

  • Nucleated; useful for initial focusing at $10\times$.

  • Common in females; usually not seen in clean-catch male specimens.

  • Clue Cells: Squamous cells covered in Coccobacillus sp. (Gardnerella vaginalis) making the cell look "fuzzy." Found in vaginal infections.

• Transitional Epithelial (Treps):

  • Origin: Urethra and Bladder.

  • Identification: Central nucleus.

  • Shapes: Spherical, Polyhedral, or Caudate (having a tail).

  • Spherical Treps: Generally non-pathological; common in catheterized samples.

  • Syncytia: Clumps of transitional cells.

    • Normal if following catheterization.

    • If no catheterization occurred: May indicate malignancy (refer to a pathologist).

• Renal Tubular Epithelial (RTE) Cells:

  • Origin: The Kidney tubules (Pathological).

  • Shapes indicate location of origin:

    • Round: Distal Convoluted Tubule (DCT).

    • Columnar/Cylindrical: Proximal Convoluted Tubule (PCT).

  • Characteristic: The nucleus is eccentric (off-center).

  • Associated Conditions:

    • Pyelonephritis: WBC clumps/casts.

    • Tubular Necrosis: Presence of RTE cells.

    • Glomerular Nephritis: RBC casts.

    • Nephrotic Syndrome: Fatty casts and Oval Fat Bodies (OFBs).

Urinary Casts: Formation and General Characteristics

• Formation Site: Distal Convoluted Tubule (DCT) and the collecting duct.

• Identification: Detect under low power ($10\times$) but identify specific type under high power ($40\times$).

• Structure: Parallel sides with rounded ends.

• Location: Typically found on the edges of the glass cover slip.

• Reporting: Document the number seen per low power field (LPF).

• Uromodulin (Tamm-Horsfall Protein): The major constituent of the cast matrix.

• Ideal Conditions for Cast Formation:

  • Urine Stasis (the matrix needs time to form).

  • Acidity (Low $pH$ urine).

  • Elevated protein levels.

  • Elevated concentrations of Sodium ($Na^+$) and Calcium ($Ca^{2+}$).

Categorization and Specific Types of Urinary Casts

• Cast Maturity Progression: Hyaline (youngest) $→$ Granular $→$ Waxy (oldest).

• Hyaline Casts:

  • Composed of only uromodulin protein.

  • Non-pathological (generally); low refractive index, colorless.

  • May be present due to dehydration or heavy exertion.

• Red Blood Cell (RBC) Casts:

  • Orange-Red color; indicates glomerular damage (e.g., Glomerulonephritis).

  • Look for the cast matrix to avoid mistaking a clump of cells for a cast.

  • Reagent strip will be positive for blood.

• White Blood Cell (WBC) Casts:

  • Indicates infection or inflammation (e.g., Bacterial Pyelonephritis or Acute Interstitial Nephritis).

  • Distinguishes Upper UTI (Casts present) from Lower UTI (Free WBCs, no casts).

  • May be accompanied by RBC casts in Glomerulonephritis.

• Bacterial Casts:

  • May be pure bacteria or mixed with WBCs.

  • Difficult to identify; confirm with Gram stain.

  • Differentiate from Granular casts by looking for bacteria specifically.

• Renal Tubular Epithelial (RTE) Casts:

  • Contain small, round cells with eccentric nuclei.

  • Associated with heavy metal poisoning, viral infections, drug toxicity, or graft rejection.

• Fatty Casts:

  • Highly refractile; associated with Nephrotic Syndrome, diabetes, and crush injuries.

  • Contain fat droplets and Oval Fat Bodies (OFBs).

  • Under polarized microscopy, triglycerides and neutral lipids stain orange.

• Granular Casts:

  • Result from the breakdown of cellular casts due to urinary stasis or long transit times.

• Waxy Casts:

  • The oldest stage of cast development; indicates extreme urinary stasis.

  • Brittle, highly refractile, often fragmented with jagged ends and notches.

  • Stains a homogenous dark pink.

• Broad Casts:

  • Also called "Renal Failure Casts."

  • Formed in widened/distended DCTs.

  • Can be granular or waxy.

  • May appear bile-stained in cases of viral hepatitis.