Histopathology

• Histopathologic Techniques: involves different procedures that have been adopted for the preparation of materials and tissue for microscopic examination
• 12 steps in Histopathologic Techniques:

  1. Numbering
  2. Fixation
  3. Dehydration
  4. Clearing
  5. Wax impregnation
  6. Embedding
  7. Blocking
  8. Trimming
  9. Sectioning
  10. Staining
  11. Mounting
  12. Labelling

  • There is a 13th step which is decalcification (done or placed in between fixation and dehydration; for bones including teeth)
  • All in all, it will take 24-48 hours to do
• RECEPTIONIST

  • Validates if the specimen is adequate or good for tissue processing

  • 1st person that will receive the specimen and put it into the container
• Container: contain fixatives

  • Proportional to the size of the specimen [larger than the organ; the organ or specimen should be fully submerged]
  • If the specimen is the lungs, wrap it in gauze pads to ensure that the air inside of it wouldn’t let the organ float, hindering it to submerged fully to the fixative agent
  • Clear [to see the organ]
  • Unbreakable [no leaking]
  • Wide mouthed bottle [easy access]
  • The receptionist must not accept specimens that are only placed in plastic
  • Medical Technologists assist pathologists in autopsies
• Specimen Source: Bilateral organs - Examples: Extremities, kidneys, lungs and ovaries)
• Specimen Source:

  Miscellaneous - Examples: age, sex, ward
• NUMBERING

  • Basic information needed:

  1. Date and time
  2. Name of the patient
  3. Specimen Number

  • C- Cytology specimen
  • A - Anatomical specimen
  • S- Surgical specimen
  • Example: S-09-2111
• FIXATION

  • Most critical step in histopathological techniques
  • If you mess this step, you will mess up everything
• Fixation Primary aim: preserve the morphology and chemical constituents of the tissue [tissue should be alive as possible]
• Fixation Secondary aim: protect and harden the specimen for further handling
• Effects Of Fixatives

  • Inhibit bacterial growth and reduce the risk of infections (Make sure that there are no bacterial growth from decomposing flesh or tissue)
  • Act as mordant or accentuator- accelerating the staining process (Enhances stain/allows C.W to chemically bond with the stain)
• Microanatomic Fixative - Involves small tissue organ

  • 10% Formol Saline
  • 10% Neutral Buffered Formalin
• Cytological Fixative

  • Involves body fluid or secretion
  • Nuclear fixatives: Flemming’s Fluid, Bouin’s Fluid, Heidehain’s Susa
  • Cytoplasmic Fixatives: Kelly’s Fluid, Orth’s Fluid
• Histochemical Fixative

  • Involves tissue containing labile substances
  • 10% Fomol Saline
  • Absolute Ethyl Alcohol
  • Acetone
• Simple Fixative

  • Uses only on chemical for fixation
  • Aldehydes: Formaldehyde, Glutaraldehyde
  • Metallic Fixative: Mercuric Chloride, Chromate Fixatives, Lead Fixatives
• Compound Fixative

  • The use of two or more chemicals for fixation
• CLEARING

  • Removing of dehydrating agents
  • Xylene – most commonly used [organic solvents]
• DEHYDRATION

  • Utilizing chemical known as dehydrating agents
  • Removing of intracellular and extracellular water and fixatives in the tissue
  • Water can be a source of bacterial growth and may interfere with the staining process afterwards
• IMPREGNATION

  • Also known as INFILTRATION OR WAX IMPREGNATION
  • The process whereby the clearing agent is completely removed from the tissue and replace by a medium that will completely fill all the tissue cavities.
• PARAFFIN WAX IMPREGNATION

  • PARAFFIN: the simplest, most common and best embedding medium used for routine tissue processing
  • Surface area of the tissue is larger; clearing agent is removed; specimen is further hardened
• EMBEDDING

  • Also known as CASTING OR BLOCKING
  • The process by which the impregnated tissue is placed into a precisely arrange position in a mold containing medium which is then allowed to solidify
  • Larger surface area of wax or tissue block; larger piece to hold
• FOUR TYPES OF TISSUE IMPREGNATION:

  1. Paraffin wax
  2. Celloidin
  3. Gelatin
  4. Plastic
• BLOCKING

  • Allows the medium to solidify to produce tissue

  block
• TRIMMING

  • Process of removing excess wax after embedding

  • Can use knife/blade or heated spatula
• SECTIONING

  • Also known as CUTTING OR MICROTOMY
  • The process by which processed tissue is cut into uniformly thin slices to facilitate studies under microscope
  • Making tissue ribbons [very thin segments of wax and tissue]
• MICROTOME: machine or instrument used for cutting sections of tissue

  Blade and holder

  • Thin slices; cannot do manually
  • In this step, you will see the flaws and mistakes you’ve made throughout the process
• Rocking Microtome - Simplest and oldest type of microtome
• Rotary Microtome

  • for cutting serial sections of tissue specimen [common]
• Sliding Microtome

  • Most dangerous type of microtome
• Freezing Microtome

  • For urgent surgical biopsies specimen
• Ultrathin Microtome

  • Specimen for electron microscope
• STAINING

  • Tissue constituent are demonstrated in sections by direct interaction with dye or staining solution producing coloration of the active tissue
  • HEMATOXYLIN AND EOSIN STAINING - Utilizes micro anatomical studies of tissue. It is a regressive staining.
  • Hematoxylin is used to stain nuclear components
  • Eosin is used for cytoplasmic components
  • Can be used as a pair
• MOUNTING

  • MOUNTING MEDIUM: the solution in which the specimen is embedded, generally under a cover glass.
  • Adhesive agent to protect the specimen
  • It may be liquid, gum or resinous, soluble in water, alcohol or other solvents and be sealed from the external atmosphere by non- soluble ringing media
  • Sealed to ensure that no dust or insect can pass through the slideIndefinite shelf life
• LABELLING

  • Date and time

  • Name of the patient

  • Specimen Number

  1. C- Cytology specimen
  2. A - Anatomical specimen
  3. S - Surgical specimen

  o Example: S-09-2111
• GYNECOLOGICAL SPECIMEN

  • Performed regularly even in pregnant women without undue risk
  • EXAMPLE: Vaginal smear
• NON-GYNECOLOGICAL SPECIMEN

  • EXAMPLE: Respiratory Tract specimens: Sputum
  • Bronchoalveolar lavage (BAL)
• URINE

  • Determine the presence of urethral cancer
  • Pattern called “ferning”
• flemming's fluid - osmium tetroxide
• bouin's fluid - nitric acid
• heidehain's susa - mercuric chloride