7 Fresh Tissue Examination & Biopsy + Fixation

• Histology is the microscopic study of the normal tissues of the body
• Histopathology is the microscopic study of tissues affected by disease.
• Label should be firmly attached to the body of the container—not to the lid of the container
• Bar codes are frequently used by clinical laboratories.
• Any discrepancies in specimen identification or labeling should be resolved prior to processing.
• Biopsy is the ante-mortem examination of tissues or the examination of tissue sample from the living.
• Fresh tissue does not undergo tissue processing; direct observation under the microscope.
• Fixed tissue undergoes whole tissue processing before viewing under the microscope
• Fine needle aspiration is the simplest, least invasive test that uses smallest needle to remove cells from the area of abnormality.

  • Not always adequate to obtain a diagnosis.
  • Will depend mainly on the area to be biopsied.
• Fine needle aspiration

  
• Core needle biopsy removes not only cells but also small amount of surrounding tissue.

  • Provides additional info to assist lesion examination
• Core needle biopsy

  
• Incisional biopsy takes out even more surrounding tissue; slices into the lesion and remove only a portion.

  • Takes out some of the abnormality, but not all.
  • If lesion is found to be cancerous, then further surgery may be needed to remove the entire lesion
• Excisional biopsy generally removes the entire area in question.
• Punch biopsy is considered the primary technique for obtaining diagnostic full-thickness skin specimens.

  • Requires basic general surgical and suture-typing skills.
  • Involves the use of a circular blade rotated down through epidermis, dermis, and subcutaneous fat, yielding 3-4 mm cylindrical core tissue sample.
• Punch biopsy

  
• Shave biopsy is where small fragments of tissue are “shaved” from a surface, usually the skin.
• Shave biopsy

  
• Curettings or Curettage is where tissue is scooped or spooned to remove tissue or growths from body cavity such as endometrium or cervical canal.
• Gross examination consists of describing the specimen and placing all or parts of it into a plastic cassette, in preparation for tissue processing.

  • One of the basis of pathologists' diagnosis.
  • Involves selection of elements that appear to be of clinical significance for histologic examination.
• Gross examination is also known as the macro examination or gross pathology that refers to the visual inspection and examination of tissues or specimen without a need of a microscope.
• The purpose of gross examination is to identify any macroscopic abnormalities such as tumor, inflammation, hemorrhage, necrosis, or other structural changes.
• Findings in gross examination provides important information that guides further diagnostic test, and helps determine cause of disease or death, for comprehensive diagnosis.
• Two major gross examination processes:

  • Fresh Tissue Examination
  • Preserved Tissue Examination

  
• Fresh tissues are usually examined when there is an immediate need for evaluation.

  • They have the advantage of being examined in the living state, thereby allowing protoplasmic activities such as motion, mitosis, and phagocytosis to be observed.
• Teasing or Dissociation is a fresh tissue exam method where:

  • Tissue is immersed in isotonic salt solution, carefully dissected with a needle, and separated by direct or zigzag spread using an applicator stick.
  • selected pieces of the tissue are transferred carefully to a microscope slide
  • either stained with a supravital dye or examined unstained by phase-contrast microscopy.
• Teasing or Dissociation

  • Isotonic salt solution used: Normal saline solution or Ringer’s solution
  • Mounted as a wet preparation in microscope, care being taken to avoid forming bubbles
• Squash Preparation or Crushing is a fresh tissue exam method where tissues less than or equal to 1mm in diameter are pressed between two slides or between a slide and a cover glass.

  • Vital stain is applied between the slides.
  • Viewed using a Phase-contrast or Bright Field microscope.
• Smear preparation is a process of examining sections or sediments where cellular materials are spread lightly over a slide by means of a wire loop, applicator stick or another slide.

  • Includes streaking, spreading, and pull-apart.
  • This technique is especially useful in cytological examinations especially for cancer diagnosis.
• Streaking is a smear preparation done with an applicator stick or a platinum loop, the material is rapidly and gently applied in a direct or zigzag line throughout the slide, attempting to obtain a relatively uniform distribution of secretion.

  • Too thin or too thick smears should be avoided.
• Spreading is a smear preparation where selected portion of the material is transferred into a clean slide and gently spread into moderately thick film by teasing mucous strands w/ applicator stick.

  • A little more tedious than streaking, but has the advantage of maintaining cellular interrelationships of material to be examined.
  • Specially recommended for smear preparation of fresh sputum, bronchial aspirates, and thick mucoid secretions
• Pull-apart is a smear preparation done by placing a drop of secretion or sediment upon one slide and facing to another clean slide.

  • Material dispenses evenly over the surface of the two slides
  • Two slides are then pulled apart with a single uninterrupted motion, and specimen placed under the microscope for immediate examination or applied with vital stains
• Touch Preparation or Impression Smear is a special method of smear preparation whereby the surface of a freshly cut piece of tissue is brought into contact and pressed on the surface of a clan glass slide allowing the cells to be transferred directly to the slide for examination by phase-contrast microscopy or staining for light microscopic study.

  • It is also done in frozen section biopsy.
• Tissue processing describes the various steps required to take the tissue from fixation to the state it is completely infiltrated with suitable histological wax and ready for section cutting
• Common agents/equipment used in each tissue process:

  • Fixation – formalin
  • Dehydration – alcohol
  • Clearing – xylene/toluene
  • Impregnation & Embedding – paraffin
  • Section-cutting & Trimming – rotary microtome
  • Staining – Hematoxylin-Eosin stain
• Fixation is the first and most critical step in histotechnology; it is a process that preserves tissues from decay, thereby preventing autolysis or putrefaction.
• In fixation,

  • The quality of the section on the slide is only as good as the quality of the fixed tissue specimen.
  • A poorly processed tissue will make it difficult for the pathologist to render a proper diagnosis
• The primary goal of fixation is to preserve the morphologic and chemical integrity of the cell.

  • With fixation, shape structure, intercellular relationship, and chemical constituent of tissues are preserved.
  • It is important to prevent degeneration, decomposition, putrefaction, and distortion of tissues after removal in the body.
• Formalin preserves the structure of cells & extracellular components by reacting with amino groups of proteins, cross-linking them
• Fixation preserves the tissues by stopping all cellular activities to view it under the microscope, as if in their original state.

  • They must be placed in a fixative as soon as it leaves the body, because degeneration, decay, autolysis, and putrefaction will occur once the tissue leaves the body