HUMAN HISTOLOGY LECTURE

TISSUE PROCESSING

Tissue Processing chamber kept at an atmospheric temperature of -10°C to -

SPECIMENS FOR TISSUE PROCESSING 20°C).
• INCISIONAL BIOPSY : the removal of the part of the lesion, • The tissue for freezing should be done as quickly as
it is perforemed whe I it is too big or too foxed to allow possible for slow freezing can cause distortion of tissue due
excision to ice crystal artifacts.
• CORE BIOPSY : is perforemed by using circular cutting
needle to retrieve a core of tissue and maybe guided by a Soilid structures and tissues must be preserved and carefuly
CT or ultrasound. processed in the following order:
• ENDOSCOPIC BIOPSY • Fixation
• Decalcification
FRESH TISSUE EXAMINATION • Dehydration
Examination in the living state where observation of • Clearing/Dealcoholization
protopasmic activities such as motion, mitosis, phagocytosis, • Impregnation/Infiltration
and pinocytosis can be done. • Embedding/Casting/Blocking
Methods: • Trimming
1. Teasing or Dissociation • Sectioning/Microtomy
• Selected tissue specimen is in isotonic salt solution, • Staining
dissected and examined under the microscope. • Mounting
2. Squash Preparation • Labeling (slides)
• Less than 1mm tissue pieces are compressed (stained when Mnemonics: FDCIETSSMoL
necessary) with another slide or coverslip, and examined
under the microscope. There are two important things that we must consider in
3. Smear Preparation tissue procesing : FIXATION and L ABELING
• Spread cellular material (secretions/sediments) is
examined. QUALITY MANAGEMENT SYSTEM
• Useful in cytological examinations. In the pre-analytical phase, what is probably the most critical
• Streaking step?
‒ With an applicator stick or a platinum loop, • FIXATION
the material is rapidly and gently applied in a • It is important to preserve the tissue at the exact
direct or zigzag line throughout the slide. moment when it is removed from the body to
• Spreading accurately represent the diseased state.
‒ A selected portion of the material is
transferred to a clean slide and gently spread STEPS IN TISSUE PROCESSING (from pre-ana to post-ana)
into a moderately thick film by teasing the 1. SPECIMEN ACCESSIONING
mucous strands apart with an applicator stick. • Tissue specimens received in the laboratory have a request
form that lists the patient information and history along
• Pull-apart with a description of the site of origin.
‒ Done by placing a drop of secretion or • The specimens are accessioned by giving them a number
sediment upon one slide and facing it to that will identify each specimen for each patient
another clean slide. A. SPECIMEN RECEPTION
‒ Useful in preparing serous fluids, • Received in the laboratory.
concentrated sputum, enzymatic lavage • Specimens: Biopsy (the tissue may either be cytological [in
samples from GIT, and blood smears. liquid form] or histological [in muscular form])
• Histopath | Cytopath
• Touch Preparation (Impression Smear) B. Requisition Form
‒ The surface of a freshly cut piece of tissue is • Patient's identity must be verified at the time of specimen
brought into contact and pressed on to the collection. Following points should be noted on the
surface of a clean glass slide, allowing the requisition form (Standard):
cells to be transferred directly to the slide for ‒ Date
examination. ‒ Patient's name, age, sex
5. Frozen Section ‒ Patient registration number (CPD/ID)
• Utilized when a rapid diagnosis of the tissue in question is ‒ Type of sample and room location
required. ‒ Number of samples received from one patient
• Especially recommended when lipids and nervous tissue ‒ Remarks/final diagnosis which may be
elements are to be demonstrated. entered later on
• 10-15 micrometers in thickness are cut from a fresh tissue ‒
frozen on a microtome with CO2 or on a cryostat (a cold • Sample form.
TRANSCRIBED BY: ALAMBRA, KIMBERLY KATE C.
 HUMAN HISTOLOGY LECTURE
TISSUE PROCESSING

• Note: It varies in different institutions. B. Clearing:

• Consists of removal of the dehydrant with a substance that
will be miscible with the embedding medium (paraffin). The
C. Accessioning commonest clearing agent is xylene.
• After matching the given information from the request C. Impregnation:
form, accession number will be given by the laboratory. • The tissue is infiltrated with the embedding agent, almost
• Specimens are accessioned by giving them a specific always paraffin.
number that will identify each specimen for each patient. 5. TISSUE EMBEDDING
• For example: “H124-2001” write in the requisition form as • The process by which the impregnated tissue is placed into
well as on the specimen container. precisely arranged position.
• Write A, B, C or 1, 2, 3 if more than one container for 6. TISSUE SECTIONING
specimens. • Once the tissues have been embedded, they must be cut
D. ( di ko na pic) into sections that can be placed on a slide. This is done with
E. Specimen Consideration a microtome.
• Check if the financial matters have been taken care of. Do
not process if the financial matters have not yet settled. 7. SLIDE STAINING
• Make the entries in the biopsy register and give the • Staining is the process of applying dyes on the sections for
specimen a pathology number called the accession the pathologist to see and show the architectural patterns
number. The unique accession number must be consistent of the tissue and physical characteristics on the cell.
across all applications: specimen container, requisition 8. MOUNTING
form, cassettes, paraffin blocks, and slides. • The stained section on the slide must be covered with a
thin glass plastic or glass to protect the tissue from being
2. GROSS EXAMINATION contaminated, to provide better optical quality for viewing
• Gross examination consists of describing the specimen and under the microscope, and to preserve the tissue section
placing all or parts of it into a small plastic cassette which for years to come.
holds the tissue while it is being processed to a paraffin
block. Initially, the cassettes are placed into a fixative. 9. SLIDE READING
10. REPORTING OF THE RESULTS
11. STORAGE
12. SAFE DISPOSAL

FIXATION
3. TISSUE FIXATION • Most critical step
• The purpose of fixation is to preserve tissues permanently • pH:Ideally between 6.8 - 7.4 (near physiological pH) for
in as life-like a state as possible. Fixation should be carried most fixatives
out as soon as possible after removal of the tissues (in the • Aldehyde, mercuric, picric acid, alcoholic, heat fixatives
case of surgical pathology) or soon after death (with • Best general fixative: Formaldehyde
autopsy) to prevent autolysis. There is no perfect fixative, • Factors affecting fixation of tissue:
though formaldehyde comes the closest. Therefore, a • Size and thickness
variety of fixatives are available for use, depending on the • Temperature
type of tissue present and features to be demonstrated. • Mucus
• Fat tissues
• Blood
• Agitation

DEHYDRATION
• Best dehydrating agent: Ethanol ( Ethyl Alcohol)

4. TISSUE PROCESSING CLEARING/DEALCOHOLIZATION

A. Dehydration: • Most commonly used: Xylene
• Wet fixed tissues (in aqueous solutions) cannot be directly
infiltrated with paraffin. First, the water from the tissues IMPREGNATION/INFILTRATION
must be removed by dehydration. This is usually done with • Ratio: Paraffin to tissue (at least 25:1) to ensure proper
a series of alcohols. infiltration
Ascending order- 25%-75% absolute • Most commonly used: Paraffin wax
TRANSCRIBED BY: ALAMBRA, KIMBERLY KATE C.
 HUMAN HISTOLOGY LECTURE
TISSUE PROCESSING

EMBEDDING/CASTING/BLOCKING
• Blocking out molds:
- Metal molds (stainless steel)
- Plastic molds
- Paper boats (temporary molds)

SECTIONING/MICROTOMY
• Honing vs. Stropping
- Honing: Sharpens the knife by removing nicks (done on
coarse stone).
- Stropping: Polishes and aligns the blade edge (done on
leather strop).

STAINING
• Most used stain : Hematoxylin and Eosin Staining( HNE)
• Principle:
• Natural vs. Synthetic
- Natural Dyes: Extracted from plants/animals
- Synthetic Dyes: Chemically manufactured (
• According to:
• presence of mordant
• presence of differentiator
• resultant color
• Vital staining
• Hematoxylin-eosin stain:
• Basic dye
• Acidic dye
• Other dyes:
• Benzidine
• Acridine orange
• Gentian violet
• Congo red
• Iodine
• Malachite green
• Janus green
• Crystal violet
• Saffranin
• Carbol fuchsin
• Methylene blue
• Night blue
• Victoria blue
• New methylene blue
• Sudan black
• Periodic acid schiff

TRANSCRIBED BY: ALAMBRA, KIMBERLY KATE C.