[AUBF] Microscopic Analysis

• Specimens should be examined while fresh or adequately preserved
• If amorphous materials have precipitated, warm the specimen to 37oC to dissolve these crystals
• Midstream clean-catch is preferred; dilute random specimens may cause false negative readings
• Specimen Volume


  12 ml ( 10-15 mL ) centrifuged in a conical tube
• Centrifugation


  Relative centrifugal force (RCF) of 400 for 5 minutes
• Sediment Preparation


  • Uniform amount of urine and sediment should remain in the tube after decantation (0.5-1.0ml)
  • Concentration factor= volume of urine centrifuged divided by the sediment volume; used when quantitating the number of elements present per milliliter
• Volume of sediment examined


  20 µl covered by a 22x22 mm glass cover slip
• Examination of the sediment


  • Should include observation of a minimum of 10 fields under both low and high power
  • Amount of light should be reduced when using bright-field microscopy; certain sediment constituents have a refractive index similar to urine
• Reporting


  • Casts are reported as the average number per lpf following examination of 10 fields, and RBCs and WBCs, as average number per 10 hpf
  • Epithelial cells, crystals, and other elements may be reported in semiquantitative terms (rare, few, moderate, and many) or as 1+,2+,3+ and 4+ following laboratory format as to lpf or hpf use
• Sternheimer-Malbin stain


  Delineates structure and contrasting colors of the nucleus and cytoplasm; identifies WBCs, epithelial cells, and casts
• Toluidine blue stain


  Enhances nuclear details, differentiates WBCs and RTE cells
• 2% Acetic acid


  Lyses RBCs and enhances nuclei of WBCs, distinguishes RBCs from WBCs, yeast, oil droplets, and crystals
• Lipid stains: Oil Red O and Sudan III


  Stains triglycerides and neutral fats orange red, identifies free fat droplets and lipid-containing cells and casts
• Gram Stain


  Differentiates gram-positive and gram-negative bacteria; identifies bacterial casts
• Hansel Stain


  Composed of methylene blue and Eosin Y; stains granules of urinary eosinophils
• Prussian blue stain


  Stains structures containing iron, identifies hemosiderin granules in cells and casts
• Cytodiagnostic Urine testing


  • Involves cytocentrifugation followed by staining with Papanicolou stain for the detection of malignancies of the lower urinary tract
  • Provides more definitive information about renal tubular changes associated with transplant rejection; viral, fungal, and parasitic infections; cellular inclusions, pathologic casts and inflammatory conditions
  • First morning urine specimen is recommended for testing
• Microscopy Types


  • Bright-Field
  • Phase-contrast
  • Polarizing
  • Dark-field
  • Fluorescence
  • Interference-contrast
• Bright-Field microscopy


  Used for routine urinalysis
• Phase-contrast microscopy


  Enhances visualization of elements with low refractive indices, such as hyaline casts, mixed cellular casts, mucous threads, and Trichomonas vaginalis
• Polarizing microscopy


  Aids in identification of cholesterol in oval fat bodies, fatty casts and crystals
• Dark-field microscopy


  Enhances visualization of specimens that cannot be easily viewed with a bright-field microscope; often used for unstained specimens; and aids in identification of T.pallidum
• Fluorescence microscopy


  Used to visualize naturally fluorescent microorganisms or those stained by a fluorescent dye
• Interference-contrast microscopy


  Produces a three dimensional microscopy image and layer-by-layer imaging of a specimen
• Red Blood Cells (RBCs)


  Smooth, non-nucleated, biconcave disks measuring approximately 7 µm in diameter; frequently confused with yeast cells, oil droplets, and air bubbles
• Variations in RBC appearance


  • Crenated cells
  • Ghost cells
  • Dysmorphic cells
• Hematuria


  Associated with damage to the glomerular membrane or vascular injury within the genitourinary tract
• Macroscopic hematuria


  Cloudy, red-brown urine; associated with advanced glomerular damage and damage to the vascular integrity of the urinary tract caused by trauma, acute infection or inflammation, and coagulation disorders
• Microscopic hematuria


  Associated with glomerular disorders and malignancy of the urinary tract and the presence of renal calculi
• Nonpathologic causes of hematuria


  Strenuous exercise (characterized by the presence of RBCs, hyaline, granular, and RBC casts), menstrual contamination
• White Blood Cells (WBCs)


  Larger than RBCs, about 12 mm in diameter, must be differentiated from RTE cells
• Normal WBC count


  < 5/hpf
• Pyuria


  Increase in urinary WBCs indicating the presence of an infection or inflammation in the genitourinary system; seen in bacterial infections, indicating pyelonephritis, cystitis, prostatitis, and urethritis and in nonbacterial disorders, such as glomerulonephritis, lupus erythematosus, interstitial nephritis and tumors
• Neutrophils


  • Predominant type found in the urine sediment; granular cytoplasm and with multilobed nuclei
  • Glitter cells - swollen neutrophils in dilute alkaline (hypotonic) urine exhibiting Brownian movement of the granules and producing a sparkling appearance; light blue when stained with Sternheimer-Malbin; no pathologic significance
• Eosinophils


  • Primarily associated with drug-induced interstitial nephritis
  • May be seen with urinary tract infection and renal transplant rejection
  • Demonstrated using cytocentrifugation and Hansel staining; percentage in 100 to 500 cells is determined: > 1% is considered significant
• Mononuclear cells


  • Lymphocytes, monocytes, macrophages, and histiocytes; may be present in small numbers
  • Lymphocytes - increased in the early stages of renal transplant rejection
  • Monocytes, macrophages, and histiocytes - large cells which may appear vacuolated or contain inclusions
• Squamous Epithelial cells


  • Largest cells found in the urine sediment; contain abundant, irregular cytoplasm and a prominent nucleus about the size of an RBC
  • Originate from the linings of the vagina and female urethra and the lower portion of the male urethra; no pathologic significance
  • Clue cells - squamous epithelial cells covered with the Gardnerella coccobacillus indicative of vaginal infection by the bacterium G. vaginalis
• Transitional Epithelial (Urothelial) cells


  • Smaller than squamous cells with distinct, centrally located nuclei, and appear in several forms including spherical, polyhedral, and caudate due to their ability to absorb water
  • Originate from the lining of the renal pelvis, calyces, ureters, and bladder, and from the upper portion of the male urethra
  • Increased numbers occurring singly, in pairs, or in clumps (syncytia) seen following invasive urologic procedures
  • May be indicative of malignancy or viral infection when they exhibit abnormal morphology
• Renal Tubular Epithelial (RTE) cells


  • Most clinically significant of the epithelial cells
  • Vary in size and shape depending on the area of the renal tubules from which they originate
  • PCT: RTE cells are large and have a rectangular shape (columnar or convoluted cells); cytoplasm is coarsely granular and often resembles casts
  • DCT: smaller than those from the PCT and are round or oval; can be mistaken for WBCs and spherical transitional epithelial cells
  • Collecting Duct: cuboidal with at least one straight edge; appear in groups of three or more (renal fragments) or as sheets of cells
• Clinical significance of RTE cells


  • 2/hpf indicates tubular injury
  • Increased amounts indicate necrosis of the renal tubules caused by exposure to heavy metals; drug-induced toxicity; hemoglobin and myoglobin toxicity, viral infections, pyelonephritis, allergic reactions, malignant infiltrations, salicylate poisoning, and acute allogenic transplant rejection or as secondary effects of glomerular disorders
  • Renal fragments - indicate severe tubular injury with basement membrane disruption
  • Bilirubin-laden RTE cells - deep yellow color, seen in liver disease e.g. viral hepatitis
  • Hemosiderin-laden RTE cells - yellow brown granules; seen following hemolytic episodes (transfusion reactions, PNH, malaria); stain blue with Prussian blue dye
  • Oval fat bodies - highly refractile lipid containing RTE cells; confirmed by Sudan III or Oil Red O fat staining and polarized microscopy; droplets are composed of triglycerides, neutral fats, and cholesterol; seen in lipiduria (nephrotic syndrome), severe tubular necrosis, diabetes mellitus, and in trauma cases that cause release of bone marrow fat from the long bones
  • Bubble cells - RTE cells containing large, non-lipid vacuoles; represent injured cells in which the endoplasmic reticulum has dilated prior to cell death; seen in cases of acute tubular necrosis