INTRODUCTION TO TISSUE PROCESSING HISTOPATHOLOGY LECTURE

PRE-ANALYTIC FACTORS
• HISTOLOGY
- Microscopic study of NORMAL tissues
• HISTOPATHOLOGY
- Microscopic study of tissues affected by
disease
- Examined and diagnosed by a Pathologist

SURGICAL PROCEDURES PERFORMED TO OBTAIN

SPECIFIC-TYPES OF TISSUE
1. Fine needle aspiration
▪ Simplest, least invasive test Fresh tissue sample
▪ Uses smallest needle to remove cells - Fast and immediate need for evaluation
▪ Not always adequate to obtain a - Can examine tissues in living state
diagnosis
Fixed tissue sample
2. Core needle biopsy
- Majority of the specimens submitted in the
▪ Removes not only cells but also small
laboratory are fixed
amount of surrounding tissue
- Long process
▪ Provides additional information to assist
- Better and more effective means of studying
lesion examination
tissues to demonstrate specific structures
▪ Ultrasound or MRI guides process
3. Incisional biopsy Autolysis
▪ Takes out even more surrounding tissue - commonly known as self- digestion
▪ Slice into the lesion and remove only a - refers to the destruction of a cell through the
portion action of its own enzymes
4. Excisional biopsy - is retarded by cold and basically will accelerate
▪ Generally, removes the entire area in faster at room temperature
question - less severe in tissues that are rich in enzymes
5. Punch biopsy (e.g., liver, brain and the kidney)
▪ Considered the primary technique for - less rapid in elastic and collagen tissue
obtaining diagnostic full-thickness skin - it is important to minimize or prevent autolysis
specimens
▪ Requires basic general surgical and
suture-typing skills
▪ Involves the use of a circular blade
rotated down through the epidermis,
dermis and subcutaneous fat, yielding
3-4mm cylindrical core tissue sample
6. Shave biopsy
▪ Small fragments of tissue are “shaved”
from a surface
7. Curettings
▪ Tissue is scooped or spooned to remove
tissue or growths from body cavity

LECTURE 1 (MIDTERMS) 1
 INTRODUCTION TO TISSUE PROCESSING
RECEIVING, LABELING, CUSTODY AND IDENTIFICATION
- the first and most important process in the
histopathology section
REQUEST FORM:
1. Histopath number assigned (patient &
specimen)
- S is for surgical pathology and A is for autopsies
2. Patient’s information
3. Clinical history
4. Description of the site of origin
5. Name of attending physician
Specimen Identification
- Identification number with year
ex: S-10-M4382
- Used to identify all specimen containers,
additional materials and paperwork
GROSS EXAMINATION AND DISSECTION
Identify all anatomic structure present
• Determining parts of the body or organ
• Will identify all the specimens being submitted
Orientation marker
• Anatomic or surgically designated orientation
must be identified
• Radiographic studies, operative notes or
additional information from the surgeon
• For surgical margins, when pertaining to
malignancies
Measurements
• Dimensions (metric units), weights must be
taken on intact specimens prior to dissection
and fixation
Inking margins
• Small simple specimens with known or potential
neoplasia are often inked in their entirety
before proceeding
• All margins of skin excisions are inked
• Care must be taken to avoid smearing of ink by
either blotting specimens dry or air dry before
sectioning
HISTOPATHOLOGY LECTURE
• Gives the pathologist a better view of the area
of the margins noted by the surgeons
• Not just an ordinary ink, but a specific ink
Dissection
• No specimen is adequately examined until it has
been completely dissected and serially
sectioned
• Open all hollow structures
• Tumors – determine the site, tumor size,
location and identify of structures invaded by
tumor, vascular invasion, distance invasion,
distance from resection margins and presence
of lymph nodes in specimen
Identification of pathologic process
• All pathologic lesions have characteristic gross
appearances
Histologic Sections
• Sections are taken that best demonstrate the
features seen on gross examination
• If you see an area with the most lightly
pathologic area then you get more sections of
this portion of this tissue
GENERAL PRINCIPLES OF GROSS DESCRIPTIONS
Diagnosis
- Gross descriptions provide important diagnostic
information that is used for staging and
prognosis
Correlation
- Good gross descriptions allow the pathologist to
correlate microscopic findings with the gross
findings
Documentation
- Each specimen and the condition in which it
arrived must be carefully documented for
medical and legal purposes
Training
- Accurate descriptions reveal the strengths and
limitations of the gross examination as
compared to microscopic examination
LECTURE 1 (MIDTERMS) 2
 INTRODUCTION TO TISSUE PROCESSING
GROSS DESCRIPTIONS
Succinct and to the point
- Important information can usually be captured
in few sentences
Good organization
- Information is easily overlooked if not readily
accessible and in the right anticipated location
Adequate dissection
- Specimen cannot be described accurately until
after it has been completely dissected and
examined
Standardization
- Minimizes the risk of omission of important
information
- Creative dictations should be reserved for the
very unusual or complicated specimens
Diagrams
- Diagrams of complicated specimens are helpful
to show the site of tissue blocks3
COMPONENTS TO A GROSS DESCRIPTION
1. Document patient’s name, specimen label, type
of specimen received, anatomic structures
present in the specimen
2. Description of the main pathologic findings that
caused the specimen to be resected
3. Describe any secondary pathology not
described in the second part
4. List frozen sections, photographs, radiographs
and any other special studies that were done
5. List of all the cassette and the type of tissue
sampled
FRESH TISSUE EXAMINATION
1. Teasing or Dissociation
2. Squash Preparation (Crushing)
3. Smear Preparation
a. Streaking
b. Spreading
c. Pull-Apart
4. Touch Preparation (Impression Smear)
HISTOPATHOLOGY LECTURE
Teasing or Dissociation
- 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
- ADVANTAGE: it allows the examination of cells
in the living state
- DISADVANTAGE: not permanent
Squash Preparation
- Process whereby small pieces of tissue not
more than 1 mm in diameter are placed in a
microscopic slide and forcible compressed with
another slide or with a cover glass
- Vital stain may be placed at the junction of the
slide and clover glass
Smear Preparation
- Process of examining sections or sediments,
whereby cellular material is spread lightly over
a slide by means of a wire loop or applicator or
by making an apposition smear with another
slide
- Useful in cytological examinations
- Avoid too thin or too thick smears
- May be examined as fresh or fixed
a. Streaking
o 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
o Too thin or thick smears should be
avoided
b. Spreading
o 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
o Has the advantage of maintaining
cellular interrelationships of the
material to be examined
LECTURE 1 (MIDTERMS) 3
 INTRODUCTION TO TISSUE PROCESSING
c. Pull-Apart
o Done by placing a drop of secretion or
sediment upon one slide and facing it to
another clean slide
o Material disperses evenly over the
surface of the two slides
o 2 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
- 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
FROZEN SECTION
- This method is normally utilized when a rapid
diagnosis of the tissue in question is required
- It is especially recommended when lipids and
nervous tissue elements are to be
demonstrated
- Fresh tissue is frozen on a microtome with CO2
or on a cryostat, a chamber kept at an
atmospheric temperature of - 10° to -20°C
- Very thin slices are made around 10-15ų in
thickness
- Frozen sections are transferred to a slide
- Microscopic examination4
- ADVANTAGE:
▪ Rapid processing time with less
equipment
▪ Less need for ventilation
- DISADVANTAGE:
▪ Poor quality of the final slide
FROZEN SECTIONS APPLICATIONS IN
HISTOTECHNOLOGY
• Rapid pathologic diagnosis during surgery
• Diagnostic and research enzyme histochemistry
HISTOPATHOLOGY LECTURE
• Diagnostic and research demonstration of
soluble substances such as lipids and
carbohydrates
• Immunofluorescent and immunohistochemical
staining
• Some specialized silver stains
METHODS OF FREEZING
1. Liquid nitrogen
▪ Generally used in histochemistry &
during intra-operative procedures
▪ The most rapid of the commonly
available freezing agents
▪ Disadvantages:
o Soft tissue is liable to crack due
to rapid expansion of ice within
the tissue
o It causes a vapor phase to form
round the tissue – uneven
cooling
2. Isopentane
▪ Liquid at room temperature
▪ Pyrex glass beaker containing
isopentane is suspended in a flask of
liquid nitrogen until half-liquid & half-
solid
▪ Beaker is removed from liquid nitrogen
when small crystals start forming
▪ Tissue to be frozen is dropped
▪ An excellent method for freezing
muscle tissue
3. Carbon dioxide
▪ Convention freezing microtome gas
supply
4. Aerosol sprays
▪ Become increasingly popular method
▪ Adequate for freezing small pieces of
tissue except muscles
▪ Quick-freeze spray cans of fluorinated
hydrocarbons (e.g., Cryokwik)
▪ Rapidly freeze blocks of any type of
tissue
TWO METHODS OF PREPARING FROZEN SECTIONS
LECTURE 1 (MIDTERMS) 4
INTRODUCTION TO TISSUE PROCESSING
1. Cold knife procedure
• A piece of filter paper soaked in gum
syrup is placed on the microtome stage
• The success of this procedure depends
upon the ambient temperature and
humidity
• Short bursts of CO2 are applied
• Selected tissue block 3-5mm thick is
oriented on the stage
• Apply few drops of gum syrup and
frozen solid with several intermitted
bursts of CO2
• Tissue is lifted up to the knife manually
and trimmed until the surface is flat
• Surface is warmed with finger until hard
frozen tissue starts to thaw
o DewLine - sections may then be
cut at 10um thickness
• Sections do not form ribbons but stick
to knife blade
• Sections are transferred to a dish of
distilled water to separate and picked
up individually for mounting and
staining
• Optimum condition for sectioning:
o Knife -40˚ to -60˚C
o Tissue -5˚ to -10˚C
o Environment 0˚ to -10˚C
2. Cryostat procedure
• Makes use of Cryostat
• Cryostat consist of an insulated
microtome housed in an electrically
driven refrigerated chamber and
maintained at temperature near -20˚C
(microtome, knife, specimen &
atmosphere)
• Optimum working temperature -18˚ to -
20˚C
• Majority of sections can be cut in
isothermic condition
• Slow freezing can cause tissue
distortion due to ice crystal artifacts
HISTOPATHOLOGY LECTURE
MOUNTING OF TISSUE BLOCK
• Synthetic water-soluble glycols and resins are
generally used as mounting media for tissue
block sectioned on a cryostat
• -5˚to -15˚C – brain, lymph nodes, liver, spleen,
soft cellular tumors
• -15˚to -25˚C – non-fatty breast tissue, ovary,
prostate, tongue and GI tract
• -35˚C – fatty breast and omental tissue
• The tissue block should be 2 – 4mm thick in
order to maximize the size of the knife
• Frozen tissue is mounted on the microtome
• Both microtome knife and tissue block are left
in cryostat for 15 mins at -20˚C
• Sections between 5-10um are slowly cut and
steadily removed from the knife, attached
directly to slides of cover-glasses, air dried and
fixed
• Cryostat sections provide the simplest, quickest
and least labor-intensive method and routinely
used for intraoperative and rapid diagnosis
• Cryostat cut only individual sections
• Paraffin blocks are able to perform ribbons
during cutting or sectioning
FREEZING PREVIOUSLY FIXED TISSUE
• Cryostat sections of fresh unfixed tissue usually
attach easily to the slide even without
adhesives and will preserve enzymes and other
substances that may be studied by
histochemical techniques
• Cryostat is also recommended for any
technique requiring cold sectioning of fixed
material
o Fats, lipids and some special methods
for the nervous system
• Formalin-fixed sections may not adhere to
slides
• Clean slides should be coated with albumin or
chrome-glycerin jelly
• Immersed tissue block in boiling 10% buffered
formalin for 1 to 2mins before freezing and
sectioning
LECTURE 1 (MIDTERMS) 5
 INTRODUCTION TO TISSUE PROCESSING
• Special fixatives – 10% formol calcium at 4˚C
may be used in histochemistry for lipid
demonstration
• Fixed tissue or stored in alcohol should be
washed in water for 12-24hrs before sectioning
SPECIAL PROCESSING TECHNIQUES
- Histochemical evaluation involving enzyme
studies
o Tissues: chemically active chemical
constituents are present, unaltered nor
displaced
- METHODS USED IF CHEMICAL FIXATION MUST
BE AVOIDED:
o Freeze-drying
o Freeze substitution
Freeze-drying
• A special way of preserving tissues by rapid
freezing (quenching) of tissue at -160˚C
• Subsequent removing of ice water (dessication)
by transferring to vacuum chamber at higher
temp ex. -40˚C without use of chemical fixative
• Generally, time consuming and expensive
o Drying – most time-consuming part
• Advantages:
o Produces minimum tissue shrinkage
o Allows tissue to be processed in fresh
state
o Causes minimal change on the cells and
less displacement of tissue and cellular
constituents
o Avoids chemical alteration of cellular
components and loss of tissue
constitutions
• Disadvantages:
o Freeze-dried materials are generally
more difficult to section than ordinary
paraffin blocks
o Tissue is brittle and inadequately
supported
• Immunocytochemistry
HISTOPATHOLOGY LECTURE
• Fluorescent antibody studies of polypeptide and
polypeptide hormones
• Autoradiography
• Microspectrofluorimetry of auto fluorescent
substances
• Formaldehyde-induced fluorescence of biogenic
amines
• Scanning electron microscopy
Freeze-Substitution
• Process of dehydration, performed at low
temperatures to avoid ice crystal formation and
circumvent damaging effects observed after
ambient temperature dehydration
o Fixation – Rossman’s formula or
1%Acetone
o Dehydration – absolute alcohol
• Based on rapid freezing of tissue followed by
solution (substitution) of ice at temp below 0˚C
• 1mm to 3 mm specimen is thrown into 3:1
propane-isopentane (super cooled by liquid
nitrogen)
• Cryostat sections are cut 8-10um and
transferred to water-free acetone and cooled to
-70˚C for 12 hrs. to 1 week
• Advantage:
o More economical and less time-
consuming
• For best morphological and histochemical
preservation:
o Substituting fluids must contain fixing
and solvent for ice
o 1% solutions of Osmium tetroxide in
acetone
o Mercuric chloride in ethanol
o Picric acid in ethanol
CONVENTIONAL TISSUE PROCESSING
• Histotechnologists needs to produce a tissue
section of good quality that will allow adequate
interpretation of cellular changes in the
microscopic view
• Solid tissue needs to be fixed and processed to:
LECTURE 1 (MIDTERMS) 6
 INTRODUCTION TO TISSUE PROCESSING HISTOPATHOLOGY LECTURE

o Preserve tissue structure

o Impregnate with appropriate hardening
substance for:
➢ tissue sectioning
➢ microscopic evaluation
• Tissue processing
o Describes the various steps required to
take the tissue from fixation to the state
where it is completely infiltrated with
suitable histological wax and ready for
section cutting

STEPS OF PROCESSING SOLID STRUCTURES

1. Fixation
2. Decalcification (Optional)
3. Dehydration Clearing
4. Impregnation (Infiltration)
5. Embedding
6. Trimming
7. Section-Cutting (Microtomy)
8. Staining
9. Mounting
10. Labeling

LECTURE 1 (MIDTERMS) 7