AUBF LEC MICRO

• First morning midstream clean-catch urine specimen is most recommended sample
• Refrigerated specimen must be warmed to room temperature before centrifugation
• Obtaining a urine specimen

  1. Obtain a 12-ml sample of urine specimen and put it in a conical tube

  2. Exemption rule- infants are not able to provide 12mL sample

  3. Its okay to accept <12mL sample if infant
• Centrifugation of urine specimen

  Centrifuge for 5 minutes at 400 relative centrifugal force
• Sediment preparation


  1. After centrifugation, decant the specimen leaving only 0.5-1.0 mL of sediments

  2. Shake gently and aspirate the sediments into a glass slide

  3. Recommended volume for microscopy is only 20uL to prevent overflowing
• Microscopic examination


  1. Examine using 10 low-power fields (First) and 10 high-power fields (Second) before concluding your analysis

  2. Subdued light is recommended to view low-refractive index sediments

  
• Casts


  Reported as the average number per 10 low-power fields
• Blood cells


  Reported as the average number per 10 high-power field
• Epithelial cells and crystals

  Frequently reported in semi-quantitative terms like few, occasional, moderate, and many following specific laboratory formats
• Sediments reported


  • RBCs, WBCs, Renal tubular epithelial cells, transitional epithelial cells, crystals, and parasites
  • Casts and squamous Epithelial cells
  • Bacteria, yeast, mucus, fat droplets, spermatozoa, and amorphous crystal
• Reporting of sediments


  • RBCs, WBCs, Renal tubular epithelial cells, transitional epithelial cells, crystals, and parasites are reported as none, seen or #/HPF
  • Cast and squamous Epithelial cells are reported as none seen or #/lpf
  • Bacteria, yeast, mucus, fat droplets, spermatozoa, and amorphous crystal are reported as: None seen, + (trace), 1+ (few), 2+ (moderate), 3+ ( many), 4+ (TNTC/ Too numerous to count)

  
• Artifacts, as a rule, are not reported
• Most common artifacts/contaminants


  • Hair (Can be mistaken as a parasite)
  • Pollen grain (Can be mistaken for Ova of a parasite)
  • Starch
  • Air bubbles (Can be confused with RBC)
  • Fat droplets
  • Cloth Fiber
• Importance of knowing these artifacts: they could be mistaken for some other sediments in urine
• Brownman movement


  Non- directional movement of cells
• Matix


  Outer layer of course cast
• Stains used for urine specimen

  • Sternheimer-Malbin stain (general stain for urine sediments composed of crystal violet and sarafin O)
  • 0.5% toluidine blue (Enhances nuclear details for differentiation of cells)
  • 2% acetic acid (Enhances nuclear detail of WBCs but lysed RBCs)
  • Oil red O and Sudan III (Lipid stains - Confirm the presence of triglycerides and nuetral fat stains, DOES NOT stain cholesterol)
  • Gram stain (Identifies presence of bacteria and distinguishes if Gram (+) or (-) bacteria)
  • Hansel stain (Detects presence of eosinophils)
  • Prussian blue stain (Stains presence of hemosiderin in cells and casts)

  
• Microscopy techniques


  • Bright-field microscopy (Used for routine body fluid analysis, the sedimenta appears dark against a light background)
  • Phase-contrast microscopy (Best for detection of sediments with low refractive index like hyaline casts, mixed cellular casts, mucus threads, and trichomonas)
  • Polarizing microscopy (Aids in identification of Triglycerides (TAGS), neutral fats and cholesterol that exhibit Maltese cross pattern)
  • Interference contrast microscopy (Three dimensional image and layer-by-layer imaging)
  • Dark-field microscopy (Used for those sediments easily viewed under bright like Treponema pallidum)
  • Fluorescence microscopy (Uses a fluorescent-dye to detect microorganisms, These dyes include labeled antigens and antibodies)

  
• Cholesterol lipids


  Exhibit maltese cross formation, Composed of four arrowheads or letter V's
• Neutral fats


  Do not produce the cross phenomenon, Lipid stains (Sudan III, Sudan IV, and oil red) are used to stain the neutral fats/ triglyceride lipids
• Erythrocytes (RBCs)


  • Appear as smooth, biconcave, non-nucleated disks measuring 7 mm in diameter
  • In concentrated urine they appear created or irregularly shaped due to loss of water
  • In diluted urine, there is an influx of water and RBCs tend to swell and lyse rapidly (ghost cells)
  • Dysmorphic cells appear fragmented or full with cellular protrusion

  
• Differentiating erythrocytes against yeast cells and artifacts

  • Yeast cells usually exhibiting budding
  • Air bubbles are highly refractile
  • WBCs are nucleated and much larger
  • RBCs lyse upon application of 2% acetic acid while yeast, bubbles and leukocytes are resistant
• Dysmorphic RBCs


  Irregular RBCs with distorted appearance, Contain variable amounts of hemoglobin, Associated with glomerulonephritis
• Glomerular bleeding


  Predominant dysmorphic RBCs
• Damage to the vascular integrity of the urinary tract

  Caused by infection, trauma or inflammation
• Renal calculi


  Presence of hyaline casts, granular casts and RBC casts
• Strenuous exercises


  Presence of hyaline casts, granular casts and RBC casts
• Correlation of microscopic RBC

  • Appearance: Hazy to cloudy
  • Color: Look for pink to red to smokey coloration
  • Sediment button: Presence of a red button (Centrifuged)
  • Reagent strip test: Blood pad will be positive, Protein pad may be positive

  
• Leukocytes (WBCs)


  • Appear larger than RBCs measuring 12 mm in diameter
  • In alkaline urine, there is an influx of water and WBCs begin to lose their nuclear detail
  • In hypotonic urine, WBCs absorb water causing them to swell
  • Granules inside these cells produce a sparkling appearance and exhibit brownian movement (Glitter cells)
  • Neutrophils, eosinophils and mononuclear cells are commonly found with the former as the predominant
• Differentiating leukocytes against epithelial cells

  • Addition of supravital stain or 2% acetic acid
  • RTE cells are large with eccentrically-located round nuclei
  • WBCs exhibit ameboid motion
• Types of leukocytes


  • Neutrophils
  • Lymphocytes
  • Monocytes
  • Eosinophils
• Drug-induced interstitial nephritis


  Increased eosinophils (>1%)
• Renal transplant rejection


  Presence of lymphocytes, monocyte, macrophages and histiocytes
• Infection or inflammation in the genitourinary system

  Bacteria infections (pyelonephritis, cystitis, prostatitis, urethritis) and non- bacterial disorder (Glomerulonephritis, lupus erythematosus, interstitial nephritis, and tumors)
• Correlation of WBCs to other parameters

  • Appearance will be hazy to cloudy if >400 WBC/mm3
  • Odor may be strong, pungent, or foul
  • A grey button will appear in the bottom of the centrifuged tube
  • Leukocyte esterase test: positive (WBC > 100,00/mm3)
  • Nitrate (nitrite) test: positive ( if infective bacteria is a nitrate reducer) (enterics bacterias)
  • Blood test: positive (If lesions are present due to infection)
• Squamous epithelial cells


  • Largest cell found in urine sediment, flat, irregularly shaped cells with small central nuclei and abundant cytoplasm
  • Majority derived from the linings of vagina and urethra, small portion from the vulva
  • Characterized by an abundance of cytoplasm and a small eccentric nucleus
  • Diameter ranger from 40-60 uM
  • Normal amounts of sloughing will be characterized by < cells/LPF
  • More abundant in female
  • Cytoplasm and nuclear ratios will help to identify the squamous, being > 12 to 1
• Clue cells


  Variant of squamous epithelial cells which indicates vaginal infection by the bacterium Gardnerella vaginalis
• Gardnerella vaginalis


  Squamous cells completely covered with the said coccobacillus
• Transitional epithelial cells (urothelial cells)

  • Appearing quite smaller than squamous cells and larger than leukocytes, spherical, polyhedral and caudate in shape
  • Cells in direct contact with urine absorb water, altering their shape
  • Occasionally, these cells may contain two nuclei
  • Originates from the lining of the renal pelvis, calyces, ureter, urinary bladder and upper portion of male urethra
• Malignancy or viral infections


  Transitional cells with abnormal morphology such as vacuoles and irregular nuclei