Para Lab

• Concentration techniques


  Procedures used to recover more parasites in cases of light infections, especially useful in confirming negative results from direct fecal smear
• Concentration techniques


  • Sedimentation techniques
  • Flotation techniques
• Sedimentation techniques


  Parasites have higher specific gravity than the reagent, so they sink to the bottom
• Formalin-Ether Concentration Technique (FECT)


  Uses 10% formalin as a fixative and ether to dissolve neutral fats in stool, can recover both helminth eggs and protozoan cysts
• FECT procedure


  1. Add stool to formalin

  2. Strain suspension

  3. Add more formalin

  4. Add ether and mix

  5. Centrifuge

  6. Pour off top layers

  7. Mix sediment

  8. Transfer to slide and examine
• Flotation techniques


  Parasites have lower specific gravity than the solution, so they float to the surface
• Brine flotation technique


  Uses saturated salt solution, stool is directly mixed with brine, no centrifugation required
• Brine flotation procedure


  1. Add brine solution to test tube

  2. Add stool and mix

  3. Add more brine to fill tube

  4. Lower coverslip

  5. Allow to stand

  6. Remove coverslip and examine
• Brine flotation


  • Cheap and simple, but Schistosome eggs become shrunken and operculated eggs don't float
• Quality assurance and quality control measures are important for concentration techniques
• QA/QC measures


  • Calibrate microscope
  • Perform multiple egg counts
  • Review organism appearance and size
  • Test specific gravity of solutions
  • Check reagents for contamination and expiration
• Cestodes (tapeworms) are hermaphroditic organisms with a scolex, neck, and proglottids
• Trematodes (flukes) are hermaphroditic organisms with an oral sucker, ventral sucker, and sometimes a gonotyl
• Protozoa are unicellular organisms with a nucleus, cytoplasm, membrane, and organelles
• Protozoan locomotory apparatus


  • Cilia
  • Flagella
  • Pseudopodia
• Protozoan life cycle stages

  • Cyst
  • Trophozoite
• Protozoans of medical importance include Amoebas, Ciliates, and Flagellates
• Artifacts are structures sometimes confused with parasites, also known as pseudoparasites, contaminants, or confusers
• Artifacts should be clearly distinguished from real parasites through careful examination of their differences
• Examples of artifacts


  • Oil droplets
  • Fat globules
  • Air bubbles
  • Muscle fibers
  • Yeast cells
  • Plant materials
  • Crystals
  • Cells
• Red blood cells


  • 7-8 um biconcave disc, round or oval, non-nucleated, colorless, may appear swollen or crenated
• Leukocytes/Pus cells/Polymorphonuclear Cells/PMNs


  • 10-21um, round or oval, irregular outline, cytoplasm is refractile, clear and granular with a lobed nucleus, large amount indicate inflammation, eosinophils indicate immune response to parasitic infection
• Oil/Fat droplets/Fat globules


  • Perfectly round, highly refractile, sizes vary, no internal structure, may come in several layers, stains orange to red with Sudan III, too much fats in stool is termed steatorrhea
• Yeast cells


  • 5-8um, oval bodies often with buds, usually appear in cluster, brownish red with iodine, little internal structure
• Starch granules


  • Rounded or angular, very refractile, no internal structure, stain pink to purple in iodine mounts
• Air bubbles


  • Trapped air in fecal smear, perfectly round (sometimes irregular), refractile, sizes are variable, no internal structure, confused with cyst or Taenia egg
• Pollen grains and Fungal spores

  • Size is variable, have peculiar and distinctive shapes, some have saw-like or rounded projections
• Vegetable/Plant cells


  • Colorless, hyaline rectangular or oval cells, with double contour and chlorophyll bodies, seldom possess the regular shape that characterizes helminth eggs
• Vegetable/Plant spirals


  • Spring-like bodies, component of plant xylem, easily recognized by the spiral appearance or rectangular markings
• Vegetable/Plant hairs or Fibers

  • 50-300um, colorless or pale yellow, curved, rather rigid, wide and clean cut at one end, narrow central canal between two transparent refractile layers
• Bacteria


  • Normal constituent of stool, small, no internal structure, may have terminal spores that resemble vacuoles in microsporidian spore
• Epithelial Cells


  • Shed from the anal mucosa, nucleus refractile and large, cytoplasm smooth, cell border distinct, mistaken with trophozoites
• Charcot-Leyden Crystals


  • Disintegrated products of eosinophil, diamond-shaped/Compass needle appearance, may indicate hypersensitivity or allergic reactions, may indicate parasitic infections
• Guaiac dye tests


  One type of Fecal Occult Blood (FOB) test commercially available
• Immunochemical Fecal Occult Blood Tests (iFOBT)


  Another type of Fecal Occult Blood (FOB) test commercially available
• Guaiac dye test principle

  Detection of the pseudoperoxidase activity of hemoglobin reacting with hydrogen peroxide to oxidize a colorless compound to a colored compound
• Several different indicator chromagens have been used to detect occult blood (e.g. benzidine, ortho-tolidine, and gum guaiac)
• Patients should avoid eating certain foods and taking certain medications prior to specimen collection to prevent false-positive and false-negative reactions with guaiac dye tests
• Immunochemical Fecal Occult Blood Tests (iFOBT)

  More sensitive and specific methods for the detection of occult blood based on lateral flow immunoassay specific for human hemoglobin
• iFOBT principle


  Specimen migrates chromatographically by capillary action across the cassette, human hemoglobin (if present at or higher than 25 ng/mL) binds to the anti-Hb conjugate which is then captured by the pre-coated reagent forming a colored Test (T) line