HISTOPATH COACHING

• Histology - Normal tissue
• Histopathology - tissues affected by the disease
• Core Biopsy - surgical pathology
• Shave biopsy - removal of mole
• Histopath reports:

  1. provided with 3 reports;

  • Doctor
  • Patient
  • Lab/Patho

  2. Signatories for request form: REQUESTING PHYSICIAN

  3. Result form: PATHOLOGIST
• Certification for Safety:

  • ASCP through MSDS & OSHA
• Current file for Laboratory Equipment:

  1. Name of instrument, manufacturer, model no. and serial no., certification of product registration & cert. of calibration.
  2. Record of preventive maintenance
  3. Record of Service & Repair
  4. Copy of Operating manual
• Labelling Basics:

  1. Name of chemical/ all the ingredients
  2. Manufacturer
  3. Date of purchase
  4. Expiration Date
  5. Hazard & Safety Warnings
• Instrumentation in Histotech (operated with electricity):

  • Cryostat
  • Autotechnicon
  • Microtome
  • Automated H&E Staining
  • Microscope
  • Automated cover slipper
• Standard Manual Processing Technique: FDCIE

  • Fixation
  • Dehydration
  • Clearing
  • Impregnation
  • Embedding
• Volume of Reagents:

  • Fixative - 20x
  • Decalcifying agent - 20x
  • Dehydrating agent - 10x
  • Clearing agent - 10x
  • Impregnating agent - 25x
• Practical Considerations of Fixation:

  1. Speed
  2. Penetration
  3. Volume
  4. Duration
• Volume of fixative for prolonged fixation: 50-100x
• Fixative for minute specimens/fragmentary: Picric acid
• Formol Calcium

  • a specialized fixative used in frozen section which serves to localize antigens and hydrolyze enzymes and used for preservation of lipids.
• Formalin usage by-products:

  1. Paraformaldehyde
  2. Formic acid
• Fixative not for H&E: Osmium Tetroxide
• Decalcifying agents:

  • ACID (most common: Nitric acid);(most rapid: Phloroglucin)
  • EDTA
  • ELECTROPHORESIS
  • ION EXCHANGE RESIN
• EDTA-decalcified tissue rinsing

  • rinse in running tap water
• Methods of determining the extent of Decalcification

  1. Physical/ Mechanical
  2. Radiologic/X-ray
  3. Chemical/Ca Oxalate
• Double Embedding Method Requirement:

  1. 2 MEDIA

  • Celloidin
  • Paraffin
• Methods of Freezing:

  1. Liquid Nitrogen
  2. Isopentane
  3. CO2
  4. Aerosol sprays
• It is promoted by slow-freezing:

  • Ice Crystal Artifacts
• Temperature for Rapid Substitution:

  • -160 to -180 or -170 degrees C
• Floating-Out Bath:

  • sections are floated on a water bath set at 45-50 degrees C or approx. 6-10 degrees C LOWER than the melting point of the wax.
• Paraffin changes in manual processing:

  • 4 changes with 15 mins. interval = 1 hr.
• Most commonly used Microtome:

  • Rotary Microtome
• Knife for Celloidin: Plane-concave
• Best stain for Glomerular Basement Membrane

  • Jones Methenamine Silver Stain (EM)
• Common Supravital Dyes: 3T2NJ

  • Toluidine Blue
  • Trypan Blue
  • Thionine
  • Neutral red
  • Nile Blue
  • Janus Green B
• H&E Staining End-Color Results:

  1. Pale Pink

  • proteins in edema fluid
  • cytoplasm

  2. Pink

  • decalcified bone
  • osteoid
  • collagen

  3. Deep Pink

  • muscle fiber

  4. Purplish pink

  • plasma cells
  • osteoblast
  • basophilic cytoplasm
• H&E Staining End-color results:

  5. Bright Orange-Red

  • RBC
  • Eosinophil Granules
  • Keratin

  6. Purplish Blue

  • Calcium
  • Calcified Bone

  7. Dark Blue

  • Karyosome

  8. Blue to Blue-black

  • Nucleus

  9. Light-Dark Blue

  • Cartilage
• Adhesives used in Tissue Processing:

  • Starch paste
  • Plasma
  • Gelatin, Gelatin formaldehyde
  • Mayer's Egg Albumin
  • Dried Albumin
  • 3-APES (Aminoproprylthriethoxysaline) - 2nd best
  • Poly-L-Lysine - Best
• Signs of Deat:

  Primary

  1. CNS Failure
  2. Cardiovascular
  3. Respiratory Failure

  Secondary

  1. Algor Mortis -
  2. Rigor Mortis
  3. Livor Mortis
  4. Autolysis
  5. Putrefaction
  6. Dessication
  7. Post-mortem clotting
• ALGOR MORTIS

  • Cooling of the body
• RIGOR MORTIS

  • stiffening of the skeletal muscles
• LIVOR MORTIS

  • purplish discoloration
• Autolysis

  • self-digestion of cells(lysozyme)
• PUTREFACTION

  • invasion of intestinal microorganisms
• DESSICATION

  • Drying and wrinkling of the anterior chamber of the eye