M AUBF: MICROSCOPIC EXAMINATION

• Microscopic examination of urine

  Detect and identify insoluble material present in the urine sediment or sample

  
• Macroscopic screening and microscopic correlation

  Different parameters: physical examination→ chemical examination
• Screening Test


  • Color
  • Clarity (Clear, Hazy or Turbid)
  • Blood
  • Protein
  • Nitrite
  • Leukoctre esterase
  • Glucose
• Significance


  Blood: Hematuria versus hemoglobinuria

  Clarity: Confirm pathologic or non-pathologic cause of turbidity

  Blood: RBCs/RBC casts

  Protein: Casts/cells

  Nitrite: Bacteria/WBCs

  Leukoctre esterase: WBCs/WBC casts/ bacteria

  Glucose: Yeast
• Preparation of the Urine Sediment

  1. Freshly voided urine

  2. Midstream clean-catch specimen

  3. 10-15 mL (12 mL)

  4. Centrifuged for 5 mins at 400 RCF

  5. 0.5-1.0 mL → left after decantation

  6. Agitate

  7. Place a drop (20 µL or 0.02 mL) of centrifuged urine in a slide + glass cover slip (22 X 22 mm)

  8. Read microscopically
• Examination of the Urine Sediment

  Observe minimum of 10 fields (both low & high PF)

  LPF (10x): detect casts and to ascertain the general composition of the sediment

  HPF (40x): Identification

  Casts have a tendency to locate near the edges of the cover slip (LPF scanning around the cover slip perimeter)

  Bright field microscopy (unstained sediments)

  Artifacts are larger than urinary sediments

  
• Reporting of Microscopic Sediments

  Casts: LPF 10 fields

  RBCs, WBCs: HPF

  Epithelial cells, crystals, and other elements: Semi-quantitative terms (rare, few, moderate, and many, or as 1+, 2+, 3+, and 4+, following laboratory format as to LPF or HPF use)

  
• Manner of Reporting


  • Occasional: 0-1, 0-3/HPF at 10 fields

  Few: 0-4, 2-5/HPF at 10 fields

  Some: 3-5, 8-10/HPF at 10 fields

  Moderate: More than 10/HPF at 10 fields

  Many: Abnudant/HPF

  TNTC: Too numerous to count (loaded)
• Routine Urinalysis Correlations


  Microscopic Elements: RBCs, WBCs, Epithelial cells, Casts, Bacteria, Crystals

  Physical: Turbidity, Color

  Chemical: Blood, Protein, Nitrite, Leukocyte esterase, pH, Bilirubin

  Exceptions: Hemolysis, Lysis, Number and type
• Sediment Examination Techniques (Stains)

  • Sternheimer-Malbin (Crystal violet & Safranin O)

  Toluidine blue (metachromatic stain)

  2% acetic acid

  Lipid Stains: Oil Red O and Sudan III

  Gram stain

  Hansel stain

  Prussian blue stain

  
• Expected Staining Reactions of Sediment Constituents

  RBCs, Hyaline casts, Coarse granular inclusion casts, Finely granular inclusion casts, WBCs, Glitter cells, Renal tubular epithelial cells, Bladder tubular epithelial cells, Squamous epithelial cells, Waxy casts, Fat inclusion casts, Red cell inclusion, Blood (hemoglobin) casts, Bacteria, Trichomonas vaginalis, Mucus, Background

  
• Cytodiagnostic Urine Testing


  Preparation of permanent slides using cytocentrifugation

  Staining with Papanicolaou stain

  Detection of malignancies of the lower urinary tract

  First morning specimen

  Provides more definitive information about renal tubular changes associated with transplants rejection; viral, fungal, and parasitic infections; cellular inclusions; pathologic casts; and inflammatory conditions.
• Urinalysis Microscopic Techniques


  • Bright-field microscopy

  Phase-contrast microscopy

  Polarizing microscopy

  Dark-field microscopy

  Fluorescence microscopy

  Interference-contrast microscopy
• Red Blood Cell (RBC)


  Non-nucleated biconcave disks

  Crenated in hypertonic urine

  Ghost cells in hypotonic/hyposthenuric urine

  Dysmorphic with glomerular membrane damage

  Sources of Identification error: Oil droplets, Yeast cells, Air bubbles

  Reporting: Average number per 10 hpfs

  Clinical Significance: Damage to the glomerular membrane or vascular injury within the genitourinary tract
• White Blood Cell (WBC)

  Larger than RBCs (12μ diameter)

  Granulated, multilobed neutrophils

  Glitter cells in hypotonic urine

  Mononuclear cells with abundant cytoplasm

  Sources of Identification error: Renal tubular epithelial cells

  Reporting: Average number per 10 hpfs

  Complete urinalysis correlation: Leukocyte esterase, Nitrite, Specific gravity, pH
• Epithelial Cells (Squamous)


  Largest cells in the sediment with abundant, irregular cytoplasm and prominent nuclei

  Sources of Identification error: Rarely encountered, folded cells may resemble casts

  Reporting: Rare, few, moderate, or many per lpf

  Complete urinalysis correlation: Clarity

  
• Epithelial Cells (Transitional/Urothelial)


  Spherical, polyheral, or caudate with centrally located nucleus

  Sources of Identification error: Spherical forms resemble RTE cells

  Reporting: Rare, few, moderate, or many per hpf

  Complete urinalysis correlation: Clarity, Blood (if malignancy-associated)
• Epithelial Cells (Renal Tubular)


  Rectangular, columnar, round, oval or, cuboidal with an eccentric nucleus possibly bilirubin-stained or hemosiderin-laden

  Sources of Identification error: Spherical transitional cells, Granular casts

  Reporting: Average number per 10 hpfs

  Complete urinalysis correlation: Leukocyte esterase and nitrite (pyelonephritis), Color, Clarity, Protein, Bilirubin (hepatitis), Blood
• Oval Fat Bodies


  Highly refractile RTE cells

  Sources of Identification error: Confirm with fat stains and polarized microscopy

  Reporting: Average number per hpfs

  Complete urinalysis correlation: Clarity, Blood, Protein, Free fat droplets/fatty casts
• Bacteria


  Small spherical and rod-shaped structures

  Sources of Identification error: Amorphous phosphates and urates

  Reporting: Few, moderate, or many per hpf, the presence of WBCs may be required

  Complete urinalysis correlation: pH, Nitrite, Leukocyte esterase, WBCs

  
• Yeast


  Small, oval, refractile structures with buds and/or mycelia

  Sources of Identification error: RBCs

  Reporting: Rare, few, moderate, or many per hpf, the presence of WBCs may be required

  Complete urinalysis correlation: Glucose, Leukocyte esterase, WBCs
• Parasite


  Pear-shaped, motile, flagellated

  Sources of Identification error: WBCs, renal tubular epithelial cells

  Reporting: Rare, few, moderate, or many per hpf

  Complete urinalysis correlation: Leukocyte esterase, WBCs
• Spermatozoa


  Tapered oval head with long, thin tail

  Sources of Identification error: None

  Reporting: Present, based on laboratory protocol

  Complete urinalysis correlation: Protein
• Mucus


  Single or clumped threads
• Identification error


  RBCs
• Reporting


  Rare, few, moderate, or many per hpf
• Presence of WBCs


  May be required
• Complete urinalysis correlation


  • Glucose
  • LE
  • WBCs
• Parasite


  T. vaginalis
• T. vaginalis


  • STP associated with vaginal inflammation
  • Asymptomatic - male urethra and prostate
• Schistosoa haematobiumm ova


  Often contained in the last few drops of urine expelled from the bladder
• Enterobius vernicularis ova


  Fecal contamination
• Spermatozoa


  Tapered oval head with long, thin tail
• Spermatozoa identification error


  None
• Spermatozoa reporting


  Present, based on laboratory protocol
• Spermatozoa complete urinalysis correlation

  Protein
• Urine casts


  • Unique to kidney
  • Formed within the lumen of DCT & CD
  • Provides microscopic view of condition within nephron
  • LPF → detection/scanning around cover slip edge
  • Cast matrix – low refractive index → observe in subdued light
  • Reporting: Ave. #/10 LPF
• Casts composition and formation

  • Tamm-Horsfall protein (glycoprotein excreted by the RTE cells of the CDT and upper collecting ducts) - major constituent of casts
  • Albumin, immunoglobulin
  • Protein gel → urine-flow stasis, acidity, Na+, Ca++
  • Tapered end → formed at the junction at ALH & DCT - cylindroids
  • Cylindria → presence of urinary cast
• Hyaline casts


  Colorless homogenous matrix
• Hyaline casts identification error

  Mucus, fibers, hair, increased lighting