MLSBIO107: HISTOPATHOLOGIC TECHNIQUES
LABORATORY
TRANSCRIBED BY: CATALONIA, LARC JED, LLADONES, JOY
MIDTERM 001:
SPECIMEN HANDLING AND IDENTIFICATION
• Each laboratory has its own way of specimen identification.
Giving the tissue a unique accession number
o May include the year and month the specimen was
received.
o E.g., SDCA:2023:Skin:003
• 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.
• Bar codes are frequently used by clinical laboratories.
o Bar codes uses binary numbers and accession code is
no longer seen.
• 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 [accession number]
▪ These labels should be firmly attached to the body
of the container—not to the lid of the container.
▪ 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.
• The request form should have a provisional diagnosis and
brief clinical details.
o This is because the pathologist do not see the patients
and solely base on what we present to them.
GROSSING
Major components in grossing a specimen:
MAJOR COMPONENTS IN GROSSING A SPECIMEN
1. Reliable and rapid transfer of the specimen from
surgery to pathology
— This is done to avoid cell deterioration
2. Accurate identification of the specimen
3. Description of additional specimens received from
the same patient.
— For multiple surgeries, the alphanumeric
accession code is needed.
4. Gross description of the specimen’s normal and
abnormal features
— Color is usually checked in fluids [red usually
indicates the presence of blood]
— Shape
5. Recording the sites from which blocks of tissue are
taken
— Areas with lymph nodes because they are the
rapid responders to diseases.
6. Recording markers that help with the correct
orientation
7. Identifying special studies requested and/or
needed.
— Routine
— Special Test = for confirmation tests
SPECIMEN FROM DERMATOLOGY
CORE BIOPSIES
• Tumors are normally circular in nature unless malignant.
• Uses imaging results [CT scan—per slice or area and
Ultrasound—for the whole area]
• Alternatives include Fine needle aspiration biopsy.
o Fine needle aspiration is the simplest, least invasive
test.
o It uses the smallest needle to simply remove cells
from the area of abnormality.
o This is not always adequate to obtain a diagnosis,
depending on the area to be biopsied.
LARGER CORE BIOPSIES (4MM)
o Should be bisected eccentrically and embedded with
cut surfaces down.
o They are dissected first
SMALL CORE BIOPSIES (2MM)
o 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
• 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.
• Generally, removes the entire area in question.
• Method of choice for surgical removal of melanomas but
may be sometimes removed by shaving.
• Before closing the area, we should analyze it first.
INCISIONAL BIOPSY
• Takes out even more surrounding tissue.
• It takes out some of the abnormality, but not all.
• The doctor will slice into the lesion and remove only a
portion of it.
o If the lesion is found to be cancerous, further surgery
may be needed to remove or excise the entire lesion.
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SHAVE SKIN BIOPSY
• We should be careful of dark pigmentation!!!
• Where small fragments of tissue are “shaved” from a
surface (usually skin).
• 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.
PUNCH BIOPSY
• Considered the primary technique for obtaining diagnostic
full-thickness skin specimens.
• It requires basic general surgical and suture-tying skills and
is easy to learn.
o The technique involves the use of a circular blade
that is rotated down through the epidermis and
dermis, and into the subcutaneous fat, yielding a 3-
to 4- mm cylindrical core of tissue sample
NON-SKIN SPECIMEN
• Skin-deep samples
• Can also be done through 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 and may harbor
unsuspected malignant tumors. [Organs and fluid samples
are usually malignant]
• Important determinants of neoplastic specimens:
o Overall size of the tumor [Bigger=Malignant,
Smaller=Benign]
o Depth of invasion into or through the tissue walls [No
invasion=Benign, Has invasion/Metastasis=Malignant]
o Involvement of margins and lymph nodes
TISSUE EXAMINATION
• May vary according to the structural and chemical
components of the cells to be studied, the nature and
amount of the tissue to be evaluated, and the need for
immediate examination of a tissue structure.
• Examination may be done on fresh or preserved tissues,
depending upon necessity.
FRESH TISSUE EXAMINATION
• Time is essential—the faster the better.
• Done for rapid diagnosis
• Once tissues are removed from the body, their proteins and
cells are digested and broken down by their own enzymes,
independent of a bacterial action.
o This process is known as autolysis, which is retarded
by cold and accelerated at room temperature.
▪ It is more severe in tissues that are rich in enzymes
(e.g., liver, brain, and kidney) and less rapid in elastic
and collagen tissues
• Fresh tissues are usually examined when there is an
immediate need for evaluation.
• Fresh tissues have the advantage of being examined in the
living state, thereby allowing protoplasmic activities such as
motion, mitosis, phagocytosis and pinocytosis to be
observed.
• Its use has been limited, however, because of the fact that
tissues examined in the fresh state are not permanent and
liable to develop changes that have usually been observed
after death.
What is a better and more effective means of studying
tissues, either normal or abnormal?
— Examination of adequately preserved sections
and smears that are stained to demonstrate
specific structure.
METHODS OF FRESH TISSUE EXAMINATION
• Teasing or dissociation
o A process whereby a selected tissue specimen is
immersed in isotonic salt solution such as normal
saline or Ringer’s solution in a petri dish or watch glass
▪ It is carefully dissected with a needle and
separated by direct or zigzag spread using an
applicator stick.
▪ It is examined under the microscope either
unstained by phase contrast or bright field
microscopy or stained with differential dyes.
• Phase Contrast greatly
enhances the structural detail of
the cells examined allowing
movement and mitotic division
to be observed.
o It has the advantage of permitting the cells to be
examined in the living state.
o This preparation is not permanent.
• Squash preparation (Crushing)
o Uses 2 slides placed perpendicularly to one another
o A process where small pieces of tissue not more than
one mm in diameter are placed in a microscopic slide
and forcibly compressed with another slide or with a
cover glass.
▪ A vital stain may be placed at the junction of the
slide and the cover glass and allowed to be
absorbed by the tissue through capillary attraction.
Lung tissue aspirates/samples with decalcification is usually
used in this method.
• Smear preparation
o The method of preparing the smear differs depending
on the nature of the material to be examined.
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o Smears are made either by spreading the selected
portion of the specimen over the surface of the slide
with a platinum loop.
o Examining sections or sediments whereby cellular
materials are spread lightly over a slide by means of a
wire loop or applicator, or by making an apposition
smear with another slide.
o Smear preparations can be made permanent by fixing
them while still wet, staining them to demonstrate
specific structures and inclusions, and mounting the
cleared specimen beneath a cover glass with a suitable
mounting medium.
o Useful in cytological examinations, particularly for
cancer diagnosis
o This is also useful for preparing smears of thick
secretions like serous fluid, concentrated sputum,
enzymatic lavage samples from the GI tract, blood
smears.
Apposition smear
- Can be made using a second slide to obtain a relatively
uniform distribution of secretion.
STREAKING
• Used in sputum samples [mucoid samples]
• 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.
• Too thin or too thick smears must be avoided, since they
make the tissues unsuitable for examination.
SPREADING
• Used in viscous samples [fluids, cytological samples]
• 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.
• More tedious than streaking but has the advantage of
maintaining cellular interrelationships of the material to be
examined.
• Recommended for smear preparations of fresh sputum and
bronchial aspirates, and for thick mucoid secretions.
PULL-APART
• Mostly used.
• Done by placing a drop of secretions or sediment upon one
slide and facing it to another clean slide. [parallel]
o The material disperses evenly over the surface of the
two slides.
• Slight movement of the two slides are necessary to initiate
the flow of materials.
• The two slides are then pulled apart with a single
uninterrupted motion, and the specimen placed under the
microscope for immediate examination or applied with vital
stain.
• Use for thick secretions such as serous fluids, concentrated
sputum, enzymatic lavage samples from the GI and blood
smears.
TOUCH IMPRINT (IMPRESSION SMEAR)
• A special method of smear preparation where the surface
of a freshly cut piece if 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 by phase contrast microscopy or stained for
light microscopic study.
• It has the edge of added advantage in that the cells may be
examined without destroying their actual intercellular
relationship and without separating them from their normal
surrounding.
FROZEN SECTION
• Used to know if the surgeon wants to know if the margins
of his resection are free from tumor before closing.
• Immediate diagnosis is accomplished through the use of a
frozen section, especially in intra-operative pathology.
o It is recommended when lipids and nervous tissue
elements are to be demonstrated.
o Usually done on muscle and nerve biopsies as well
as on surgically removed tumors.
• Utilized when rapid diagnosis of the tissue in question is
required.
• A fresh tissue is frozen on a microtome with C02, or on a
cryostat, a cold chamber kept at an atmospheric
temperature of -10° to -20° C.
o The thin frozen sections are mounted on a glass
slide, fixed immediately and briefly in liquid fixative,
and stained using similar staining techniques as in
traditional wax embedded sections.
• The frozen sections are then transferred to a slide and
processed for light microscopic study.
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• For histochemistry, cryostat sections give much faster • Most commonly used
results than paraffin sections. • Freezes sample
o However, the morphological detail and resolution of • Tissue blocks can also be frozen by adapting a
frozen sections are usually inferior compared to the conventional freezing microtome gas supply of carbon
quality of tissue that has been embedded in paraffin. dioxide gas from a CO2 cylinder, or by using a specially
• The advantage of the frozen section method is rapid made piece of equipment.
processing time with less equipment requirement, and less
need for ventilation in the laboratory. AEROSOL SPRAYS
o The disadvantage is the relatively poor quality of the • Widely used in cytological samples
final slide. • The use of aerosol sprays has become increasingly popular
in recent years and is adequate for freezing small pieces of
USES OF FROZEN SECTION tissue except muscle.
• Frozen sections, both fixed and unfixed, have many • Quick- freezing spray cans of fluorinated hydrocarbons
applications in histotechnology, and are commonly used (e.g., Cryokwik) have a distinct advantage of rapidly
for: freezing blocks of any type of tissue.
1. Rapid diagnosis during surgery
2. Diagnostic and research enzyme histochemistry Fresh, completely unfixed tissues, or tissues that have been
3. Diagnostic and research demonstration of soluble briefly treated with formalin may not require embedding
substances such as lipids and carbohydrates. anymore; instead they may be frozen and cut in a freezing
4. Immunoflourescent and immunohistochemical staining microtome or cryostat.
5. Some specialized silver stains, particularly in
neuropathy. • Two methods of preparing frozen sections may be resorted
to:
COMMONLY USED METHOD OF FREEZING 1. Cold Knife procedure
The tissue for freezing should be fresh, and freezing should 2. Cryostat procedure (Cold Microtome)
be done as quickly as possible.
Slow freezing can cause distortion of tissue due to ice
crystal artifacts. The more commonly used methods of freezing PROCESSING OF TISSUES
include: • Fresh tissues are usually examined when there is an
immediate need for evaluation.
• Liquid nitrogen • A better and more effective means, however, of studying
• Isopentane cooled by liquid nitrogen tissues, whether normal or abnormal, is by examination of
• Carbon dioxide gas—most commonly used their sections and smears which have been permanently
• Aerosol sprays preserved, stained for demonstration of specific structures,
and mounted on glass slides with coverslips for permanent
LIQUID NITROGEN keeping.
• Generally used in histochemistry and during operative 1. Fixation
procedures and is the most rapid of commonly available 2. Dehydration
freezing agents. 3. Clearing
• Its main disadvantage is that soft tissue is liable to crack 4. Infiltration (impregnation)
due to rapid expansion of the ice within the tissue, 5. Embedding
producing ice crystals or freeze artifacts—causes spaces in 6. Trimming
the tissue 7. Section-cutting
• It also overcools urgent biopsy blocks, causing damage to 8. Staining
both block and blade if sectioning is done at -70 C or below. 9. Mounting
o Majority of non-fatty unfixed tissues are sectioned well 10. Labeling
at temperatures between -10C and -25C.
• One problem with the use of liquid nitrogen is that it causes
a vapor phase to form around the tissue, acting as an
insulator that causes uneven cooling of tissue, particularly
of muscle biopsies, and making diagnostic interpretation
difficult.
o This problem can be overcome by freezing the tissue
in Isopentane, OCT, Freon 2.2 that has a high thermal
conductivity.

ISOPENTANE
• Can hold extreme temperature/coldness
• It is liquid at room temperature.
• A pyrex glass beaker is usually suspended in a flask of
liquid nitrogen until half-liquid and half solid stage is
reached.
o The beaker is removed from the liquid nitrogen when
crystals start forming on the side of the beaker
(approximately -170C), and the tissue to be frozen is
dropped into the cooled liquid isopentane.

CARBON DIOXIDE GAS

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