ROUTINE

HISTOTECHNIQUES
PART I
 SPECIMEN HANDLING AND
IDENTIFICATION
• Each laboratory has its own way of specimen
identification.
> Giving the tissue a unique accession number
> May include the year and month the specimen was
received.
• If multiple specimens are received on the same patient
from the same operation/procedure --- the specimen
may be given the same number followed by a numerical
or alphabetical designation.
 SPECIMEN HANDLING AND
IDENTIFICATION

 Bar codes are frequently used by clinical

laboratories.
 The specimen container label and the
accompanying request form should
include:
a. Patient’s name
b. Age or birth date
c. A medical record number
 Label should be firmly attached to the
body of the container --- not to the lid of
the container.
 SPECIMEN HANDLING AND
IDENTIFICATION
• The request form should have a provisional diagnosis
and brief clinical details.
• Any discrepancies in specimen identification or labeling
should be resolved prior to processing.
• Incorrect identification of any specimen of any specimen
results in the wrong diagnoses and incorrect treatment.
 GROSSING
Major components in grossing a specimen:

1. Reliable and rapid transfer of the specimen from

surgery to pathology
2. Accurate identification of the specimen
3. Description of additional specimens received from
the same patient
4. Gross description of the specimen’s normal and
abnormal features
 GROSSING
Major components in grossing a specimen:

5. Recording the sites from which blocks of tissue

are taken
6. Recording markers that help with the correct
orientation
7. Identifying special studies requested and/or
needed.
 SPECIMEN FROM DERMATOLOGY
@CORE BIOPSIES
 Larger core biopsies (4mm) --- should be bisected eccentrically
and embedded with cut surfaces down.
 Small core biopsies (2mm) --- should be embedded totally
without cutting it.

@SHAVE BIOPSIES OF SKIN

 Depending upon the size of the biopsy, it may be bisected,
trisected, or cut into sections.
Excision biopsy
SPECIMEN FROM DERMATOLOGY
@SHAVE BIOPSIES OF SKIN
 Most specimens of skin or other epithelial surfaces should be
cut --- all aliquots are embedded on edge.
 Care should be taken with any pigmented lesions of the skin.

@EXCISIONAL BIOPSY
 Method of choice for surgical removal of melanomas but may be
sometimes removed by shaving
Shave skin biopsy
Excision biopsy
SPECIMEN FROM DERMATOLOGY
@ EXCISIONAL BIOPSY
 Biopsies of skin are examined to ensure that the lesion has
been completely removed and the original clinician’s diagnosis
was correct.
 Can be oriented using sutures or dyes.

@ RE-EXCISION SPECIMENS
 Original site of a lesion may need to be re-excised if:
> The margins are invaded by tumor
> Too close to the tumor --- melanoma or basal cell carcinoma.
 NON-SKIN SPECIMEN
• Excisional biopsies
• Operative specimens --- tumors, unidentifiable
inflammatory masses, tissues removed prior to
transplantation, traumatic, congenital
malformations, or cosmetic surgical specimens.
• All specimens must be examined carefully ---
may harbor unsuspected malignant tumors
 NON-SKIN SPECIMEN
• Important determinants of neoplastic specimens:
> Overall size of the tumor
> Depth of invasion into or through the tissue walls
> Involvement of margins and lymph nodes
 METHODS OF FRESH TISSUE
EXAMINATION
1. TEASING or DISSOCIATION
• A process whereby a selected tissue specimen is
immersed in a watch glass containing isotonic salt
solution, carefully dissected or separated, and examined
under the microscope.
2. SQUASH PREPARATION
• Crushing
• A process whereby small pieces of tissue not more than 1
mm in diameter are placed in a microscopic slide and
forcibly compressed with another slide or with a cover
glass.
 METHODS OF FRESH TISSUE
EXAMINATION
3. SMEAR PREPARATION
• The process of examining
sections or sediments, whereby
cellular materials are spread
lightly over a slide by a wire loop
or applicator, or by making an
apposition smear with another
slide.
• Useful in cytological examinations
--- particularly for cancer
diagnosis
 METHODS OF FRESH TISSUE
EXAMINATION

a. STREAKING – with an applicator stick or 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.
b. SPREADING – a selected portion of the material is
transferred to a clean slide and gently spread into a
moderately thick film by teasing the mucous strands
apart with an applicator stick
 METHODS OF FRESH TISSUE
EXAMINATION

b. SPREADING
• Little more tedious but with an advantage of
maintaining cellular interrelationships of the materials
to be examined.
• Recommended for smears preparations of fresh
sputum and bronchial aspirates and for thick mucoid
secretions.
 METHODS OF FRESH TISSUE
EXAMINATION

c. PULL-APART – done by placing a drop of secretion or

sediment upon one slide and facing it to another clean
slide.
• The material disperses evenly over the surface of two
slides.
• Slight movement of the two slides in opposite
directions may be necessary to initiate the flow of
materials.
 METHODS OF FRESH TISSUE
EXAMINATION

c. PULL-APART
• The two slides are then pulled apart with a single
uninterrupted motion, and the specimen placed under
microscope for immediate examination, or applied with
vital stains.
• Useful for preparing smears of thick secretions such as
serous fluid, concentrated sputum, enzymatic lavage
samples from GIT, and blood smear
 METHODS OF FRESH TISSUE
EXAMINATION

d. TOUCH PREPARATION – Impression smear

• A special method of smear preparation whereby the
surface of a freshly cut piece of tissue is brought into
contact and pressed on to the surface of a clean glass
slide, allowing the cells to be transferred directly to the
slide for examination.
• Advantage: Cells may be examined without destroying
their actual intercellular relationship
 METHODS OF FRESH TISSUE
EXAMINATION
4. FROZEN SECTION – is normally utilized when a rapid
diagnosis of the tissue in question is required.
• Especially recommended when lipids and nervous tissue
elements are to be demonstrated
 METHODS OF FRESH TISSUE
EXAMINATION
4. FROZEN SECTION
• Applications:
a. Rapid pathologic diagnosis during surgery
b. Diagnostic and research enzyme histochemistry
c. Diagnostic and research demonstration of soluble
substances such as lipids and carbohydrates
d. Immunofluorescent and immunohistochemical staining
e. Some specialized silver stains --- particularly in
neuropathology
 METHODS OF FRESH TISSUE
EXAMINATION
4. FROZEN SECTION
• LIQUID NITROGEN – generally used in
histochemistry and during operative procedures
> Most rapid of the commonly available freezing
agents
> DISADVANTAGE:
* Soft tissue is liable to crack due to rapid
expansion of the ice within the tissue --- producing
ice crystals or freeze artifacts
 METHODS OF FRESH TISSUE
EXAMINATION
4. FROZEN SECTION
• PROBLEM: Use of liquid nitrogen can cause a vapor
phase to form around the tissue --- acting as an
insulator that causes uneven cooling of tissue
(particularly in muscle biopsies) and making diagnostic
interpretation difficult.
> Can be overcome by freezing the tissue in
Isopentane, OCT, or Freon 2.2 --- has a high thermal
conductivity.
 HISTOTECHNOLOGY
• Is the art and science performed by the
histotechnologist to produce a tissue section of good
quality that will enable the pathologist to diagnose the
presence or absence of disease.
• FIXATION – first and most critical step in
histotechnology
> Fixing or preserving fresh tissue for examination.
> The quality of the section on the slide is only as good
as the quality of the fixed tissue specimen.
 FIXATION
• FIXATION – the process by which
the constituents of the cells and
tissues are fixed in a physical,
and partly in a chemical state, so
that they will withstand
subsequent treatment with
various reagents with a minimum
of loss, significant distortion, or
decomposition.
 FIXATION
• Inadequate or poor fixation will result in a poorly
processed tissue and will make it difficult for the
pathologist to render a proper diagnosis.
• With fixation, the shape, structure, intercellular
relationship and chemical constituents of tissues
are preserved.
• Fixation prevents degeneration, decomposition,
putrefaction, and distortion of tissues after
removal from the body.
 FIXATION
• To Preserve the tissue --- by stopping all cellular
activities so that cells can be viewed under the
microscope as if they are still in their original
living state.
> Do not leave the tissue specimen in air for
prolonged period of time --- dry out and will result
in distortion of its morphologic appearance
> Leaving the tissue in water ---cause the cell to
swell while a strong salt --- cause the cell to
shrink.
 FIXATION

1. ADDITIVE FIXATION
• The chemical constituent of the fixative is taken in
and becomes part of the tissue by forming cross-
links or molecular complexes and giving stability
to protein.
E.g. Formalin Osmium tetroxide
Mercury
 FIXATION

2. NON-ADDITIVE FIXATION
• The fixing agent is not incorporated into the
tissue, but alters the tissue composition and
stabilizes the tissue by removing the bound water
attached to H-bonds.
E.g. Alcoholic fixatives
 FIXATION

1. Hydrogen Ion Concentration

2. 2. Temperature
 FIXATION

3. Thickness of section
4. Osmolality
 FIXATION

5. Concentration
• Low concentrations of glutaraldehyde have
been found to be an ideal concentration for
immuno-electron microscopy.
6. Duration of fixation
• Buffered formalin – 2 to 6 hours during the day
the specimen is obtained
 FIXATION

6. Duration of fixation
• Prolonged fixation:
* May cause shrinkage and hardening of tissue
* May severely inhibit enzyme activity and
immunological reactions
• For electron microscopy, tissues should be
fixed for 3 hours and then placed in holding
buffer.
 FIXATION

@ SPEED: Specimen should be placed in fixative as

soon as it is removed from the body.
* Done to prevent autolysis and putrefaction
@ PENETRATION: Time of fixation varies with
different types of tissue
* Formalin diffuses into the tissue at the rate of
1mm per hour.
 FIXATION

@ VOLUME: The amount of fixative used has been 10

– 25 times the volume of tissue to be fixed
* Maximum effectiveness of fixation: 20x the
tissue volume
@DURATION OF FIXATION: Some tissues take longer
to fix than others, depending on their structure.
* Fixation time can be cut down by using heat,
vacuum, agitation or microwave
 FIXATION

• Tissue selected for sectioning should be thin

enough to allow penetration by fixative within a
reasonable amount of time.
• To maintain an adequate fixation time of 4 – 6
hours, the recommended size of the tissue is 2
square cm, and no more than 4 mm thick.
• Most tissue can be cut and trimmed without prior
fixation, EXCEPT for the brain.
 FIXATIVES
• Effects of Fixatives in General:
a. Harden soft and friable tissues and make the handling
and cutting of sections easier.
* Usually accelerated by the action of alcohol during
dehydration process.
b. Make cells resistant to damage and distortion caused
by hypotonic and hypertonic solutions used during
tissue processing
c. Inhibit bacterial decomposition
 FIXATIVES
• Effects of Fixatives in General:
d. Increase the optical differentiation of cells and
tissue components thereby rendering them
more readily visible during examination.
e. Act as mordants or accentuators to promote
and hasten staining or inhibit certain dyes in
favor of another.
f. Reduce the risk of infections during handling
and actual processing.
 FIXATIVES
• CHARACTERISTICS OF A GOOD FIXATIVE:
1. Must be cheap, stable and safe to handle.
2. Must kill the cell quickly thereby producing
minimum distortion of cell constituents
3. Must inhibit bacterial decomposition and
autolysis
4. Must produce minimum shrinkage of tissue.
5. Must permit rapid and even penetration of
tissues.
 FIXATIVES
• CHARACTERISTICS OF A GOOD FIXATIVE:
6. Must harden tissues thereby making the cutting of sections
easier.
7. Must be isotonic --- causing minimal physical and chemical
alteration of the cells and their constituents.
8. Must make cellular components insoluble to hypotonic
solutions and render them insensitive to subsequent
processing.
9. Must permit the subsequent application of many staining
procedures to facilitate easier and more profitable
examinations.
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