Microscopic Analysis of Urine

• Addis Count - first procedure to standardize the quantitation of formed elements, used a hemocytometer

  • Specimen: 12 hours urine preserved in formalin
• Normal Value of Addis Count

  • RBCs = 0 to 500,000 cells / uL
  • WBCs and Epithelial Cells = 0 to 1,800,000 cells / uL
  • Hyaline casts = 0 to 5,000 cells / uL
• Specimen Preparation in Addis Count

  • Urine 10 to 15mL > centrifuge at 400 RCF for 5 minutes > Decant > Get the sediment (0.5 to 1.0mL) > Place the sediment on the microscopic slide (20uL or 0.02mL) > Covered by glass cover slip (22 x 22mm) > Observe under the microscope (bright field - reduced lighting)
• To correct for differences in the diameter of centrifuge heads, RCF rather than revolutions per minute (RPM) is used.

  • formula: RCF (g) = 1.118 x 10^-5 x radius (cm) x RPM^2
• Sternheimer malbin - supravital stain consisting of crystal violet and safranin

  • Action: delineates structure and contrasting colors of the nucleus and cytoplasm
  • function: identifies WBCs, Epithelial cells, and casts
• 0.5% Toluidine blue - metachromatic supravital stain

  • Action: Enhances nuclear detail
  • Function: differentiates WBCs and RTE cells
• 2% Acetic acid

  • Action: lyses RBCs and enhances nuclei of WBCs
  • Function: distinguishes RBCs from WBCs, yeast, oil droplets, and crystals
• Lipid stains: Oil red O and Sudan III

  • Action: stain triglycerides and neutral fats orange red
  • Function: identifies free fat droplets and lipid containing cells and casts
• Gram stain

  • Action: differentiates gram positive and gram negative bacteria
  • Function: Identifies bacterial casts
• Hansel stain

  • Action: Methylene Blue + EosinY (stains eosinophilic granules)
  • Function: Identifies urinary eosinophils
• Prussian Blue Stain - used mainly to confirm hemosiderinuria

  • Action: stains structures containing iron
  • Function: Identifies yellow-brown granules of hemosiderin in cells and casts
• Sedi and KOVA stain

  • Action:
  • Modified sternheimer malbin
  • dye is absorbed well by WBCs, ECs, and casts, providing clearer delineation of structure and contrasting colors of the nucleus and cytoplasm
  • Function:
  • Hyaline cast appears as pink
  • motile bacteria are unstained
  • non-motile bacteria stains purple
  • T. vaginalis stains light blue-green
• Disadvantage of sternheimer-malbin: in strongly alkaline urines, this stain can precipitate, which obstructs the visualization of sediment components
• In Oil Red O and Sudan III, cholesterol and cholesterol esters do not stain and must be confirmed by polarizing Microscopy
• Wright's stain or Giemsa stain also distinguishes urinary eosinophils, but Hansel stain is preferred.
• CYTODIAGNOSTIC URINALYSIS

  • Play an important role in the early detection of renal allograft rejection and in the differential diagnosis of renal disease.
  • Involves making a 10:1 concentration of a first morning urine specimen, followed by cytocentrifugation of the urine sediment and Papanicolaou's staining
• Commercial Systems for Urine Sediment Preparation

  1. UriSystem - The UriSystem tube is designed such that after centrifugation, it can be decanted with a quick smooth motion and consistently retains 0.4 mL of urine for sediment resuspension.
  2. KOVA System - The KOVA System uses a specially designed pipette that snuggly fits the diameter and shape of the tube to retain 1 mL of urine during decanting.
  3. Count-10 System - The Count-10 System offers several options to retain 0.8 mL for sediment resuspension
• Brightfield Microscopy

  • Used for routine urinalysis
  • Objects appear dark against a light background
  • Most frequently used in the clinical laboratory
  • The oldest and most common type of illumination system used on microscopes
  • All other types of microscopes are adapted to bright-field
• Phase Contrast Microscopy

  • Enhances visualization of elements with low refractive indices, such as hyaline casts, mixed cellular casts, mucous threads, and Trichomonas
  • Type of microscopy in which variations in the specimen's refractive index are converted into variations in light intensity or contrast
• Phase Contrast Microscopy

  • Adaptation of a bright - field microscope with a phase-contrast objective lens and a matching condenser, Two phase rings that appear as "targets" are placed in the condenser and the
  • Light passes to the specimen through the clear circle in the phase ring in the condenser, forming a halo of light around the specimen
• Polarizing Microscopy

  • aids in identification of cholesterol in oval fat bodies, fatty casts, and crystals
  • uses halogen quartz lamp that produces light rays of many different waves
  • Positive birefringence - substance that rotates the plane of polarized light 90 degrees in a clockwise direction
  • negative birefringence - substance that rotates the plane in a counterclockwise direction
  • adapted from brightfield microscope by inserting two polarizing filter in a crossed configuration
• Dark field Microscopy

  • aids in identification of spirochetes such as Treponema pallidum
  • adapted by bright field by replacing the condenser containing opaque disk
  • specimen appears light against dark background
• Interference Contrast Microscopy

  • produces 3D microscopy image and layer by layer imaging of a specimen
  • difference in optical light paths through the specimen is converted into intensity differences in the specimen image
  • 2 types of Interference Contrast Microscopy:

  1. modulation contrast (hoffman)
  2. differential interference contrast (nomarski)

  • to convert from brightfield to interference:

  1. polarizer placed between light source and condenser
  2. special condenser with modified Wollaston prism in each objective and eyepiece
  3. polarizing filter behind prism and before eyepiece
• Fluorescence Microscopy

  • allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye
  • 2 filters: (1) to select wavelength of illumination light (excitation filter) that is absorbed by the specimen, and (2) barrier filter to transmit the different, longer-wavelength light emitted from the specimen to the eyepiece for viewing
• Aperture diaphragm


  microscope component that regulated the angle of light presented to the specimen
• birefringent / doubly refractile


  ability of a substance to refract light in 2 direction
• chromatic aberration


  unequal refraction of light rays by a lens that occurs because the different wavelengths of light refract or bend at different angles
• condenser


  microscope component that gathers and focuses the illumination light onto the specimen for viewing
• eyepiece


  microscope lens or system of lenses located closest to the viewer's eye. it produces the secondary image magnification of the specimen
• field diaphragm


  microscope component that controls / regulate the diameter of light beams that strike the specimen and hence reduces stray light
• field of view


  circular field observed through a microscope
• Kohler illumination


  microscopic illumination in which a lamp condenser (located above the light source) focuses the image of the light source (lamp filament) onto the front focal plane of the substage condenser (where aperture diaphragm is located)
• mechanical stage


  holds the microscope slide with the specimen for viewing
• objectives


  lens or system of lenses located closest to the specimen. produces the primary image magnification of the specimen
• parcenter


  objective lenses that retain the same field of view when the user switches from one objective to another of a differing magnification
• parfocal


  objective lenses that remain in focus when the user switches from one objective to another of a differing magnification
• resolution


  ability of a lens to distinguish two points or objects as separate
• cytocentrifugation


  technique used to produce permanent microscope slides of urine sediment and body fluids. the end result is a monolayer of the urine sediment components with their structural details greatly enhanced by staining
• magnification


  process of enlarging or magnifying an object's size without affecting its actual or physical size
• care of microscope

  1. carry microscope with two hands, supporting the base with one hand
  2. always hold the microscope in a vertical position
  3. clean optical surfaces only with a good quality lens tissue and commercial lens cleaner
  4. do not use the 10x and 40x objectives with oil
  5. clean the oil immersion lens after use
  6. always remove slides with the LPO raised
  7. store the microscope with the LPO in position and the stage centered