Subject: GENERAL

PATHOLOGY AND
HISTOPATHOLOGIC AND
CYTOLOGIC TECHNIQUE
 HISTOPATHOLOGIC
TECHNIQUE
• Histopathology – basic component of
tertiary hospital laboratory where
human tissues and body fluids are
processed into slides for microscopic
examination by the anatomic
pathologist. (Lo, et. al. Basic
Histopathologic Techniques)
 Cytopathologic Techniques
• Preparation and examination of
cells collected by fine needle
aspiration, bronchial washings,
and other techniques that will aid
in the diagnosis of diseases.
 ROUTINE
HISTOTECHNIQUES
PART I
 HISTOLOGICAL TECHNIQUE
• Deals with the preparation of
tissues for microscopic
examination.
• Accomplished by submitting the
total or a selected part of the
tissue presented for examination
to a series of processes:
a. Fixation d. Embedding
b. Dehydration e. Cutting
c. Clearing f. Staining
 HISTOLOGICAL TECHNIQUE
• As soon as a tissue is removed from the body or
cut off from its blood supply, tissue degradation
starts or decompose.
• Reason:
– Lack of oxygen hence, series of biochemical changes
eventually lead to poor tissue quality.
– Initial anoxic insult suffered by tissues – Warm
ischemia
 HISTOLOGICAL TECHNIQUE
• Cold ischemia – lack of oxygen once tissue
sample is removed from the patient’s body until
all metabolic functions are stopped by fixation.
• Delay in the fixation ( specimen left on the OR
tray until the surgery is done) extends the cold
ischemia time which results in poor tissue
fixation.
• Further aggravated if the tissue is large and
not sectioned at regular intervals before
immersing in the fixative.
 HISTOLOGICAL TECHNIQUE
• Decomposition results from:
a. Deprivation of oxygen and essential
metabolites
b. Accumulation of carbon dioxide and other
products of cell metabolism
c. Action of various enzymes --- autolysis
 HISTOLOGICAL TECHNIQUE
• The speed of decomposition appears to be
proportional to the natural metabolic activity of the
tissue.
> Rapid decomposition occurs in the following
organs:
a. Kidney b. Liver c. Pancreas
• To preserve the natural state of tissue cells --- it is
essential to check these processes of
decomposition with a minimum of delay.
 SPECIMEN HANDLING AND
IDENTIFICATION
• Identification of the specimen and all of
its components – first step in
specimen processing.
• Each laboratory has its own way of
specimen identification.
> Giving the tissue a unique accession
number
> Date and time the specimen is
received
 SPECIMEN HANDLING AND
IDENTIFICATION
• The specimen container must bear the same
name and accession number as those in the
acquisition form.
• 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
• The specimen container
label and the
accompanying request
form should include:
a. Patient’s name
(Includes maiden or
middle name)
b. Age or birth date
c. A medical record or
hospital number
d. Bar codes are also
frequently used by
clinical laboratories.
• 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 results in
wrong diagnosis and incorrect treatment.
 SPECIMEN HANDLING AND
IDENTIFICATION
• Criteria for rejecting of gross
specimens:
• 1. discrepancies between the requisition
and specimen labels
• 2. No labels or mislabeled specimens
• 3. Leaking specimen containers
• 4. Absent clinical data or history
• 5. Inappropriately identified specimens
• GROSS ROOM –
Specimen Reception
Laboratory
> Is where tissue
specimens from the
operating theaters
and clinics are
received.
 GROSSING
The following can gross specimens:
a. Pathologist d. Histotechnologist
b. Resident e. Biomedical scientist
c. Physician assistant
Gross descriptions are important
The type of biopsy and the number of fragments
received should be documented.
Most laboratories have developed standardized
formats that describe the gross examination and
processing of specimen.
 GROSSING
• Material used for
gross examination
• Cutting tools –
standard fits/sets
– scissors
– forceps
– blades
– blade holders)
• Measuring tool
• Casette
 GROSSING
• Describing specimens
• 1. Markers for orientation:
– Some surgeons use orientation markers such
as inks, nicking and suturing which are also
specified in the requisition form.
– One or 2 ink colors maybe used to identify
and orient the specimen’s components.
– Nicking is done for indicating laterality.
– Sutures attached may be represented as:
LL – long lateral
SS – short superior
 GROSSING
• 2. Weight of intact specimen is rounded to the
nearest 0.1g
– In some specimens, the weight maybe more
important than the histopathologic characteristics in
arriving at a diagnosis.
– Example: Hyperplastic thymic tissue
• 3. Dimensions are rounded to the nearest 1 cm.
• 4. Color
• 5. Consistency
• Minute specimens are inked to ensure that the
entire face of the specimen is present in the
slide.
 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

• Often small and can be excisional, shave,

core, or re-excisional biopsies.
• Each type of biopsy is handled differently
and is often processed separately from
other tissues.

@ SMALL SPECIMENS
• Should NOT be cut, bisected, or inked
while fresh and infixed.
• Are processed in cassettes either with a
fine mesh, in lens paper, or in a “tea bag”.
 SPECIMEN FROM DERMATOLOGY
@ SMALL SPECIMENS
• Use of sponges is an alternative ---- but may dry
out, harden and may stick to sponge.

@CORE BIOPSIES
• Usually taken with a larger lesion or of a
generalized inflammatory or other disease
process.
• Should be taken with the lesion at its center
 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.
 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
 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.
 SPECIMEN FROM DERMATOLOGY
• For skin specimens,
vertical orientation of the
specimen should always
be maintained at all times.
• Objective of the
pathologist is to visualize
the epidermis in a
perpendicular section.
• Punch biopsies:
– 3mm or less are submitted
as a whole.
– 4 mm or more are bisected
or trisected depending on
the size.
 SPECIMEN FROM DERMATOLOGY
• For skin ellipses, the
entire specimen is
serially cut along the
short axis at 2 to 3
mm intervals.
• The two tips are
submitted in separate
cassettes. And the
remainder are
submitted in one or
more cassesttes.
 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
• Gross examination and processing of pediatric
biopsies requires special care due to diagnostic
difficulties of pediatric lesions/diseases.
• Pediatric tumors are rare --- often necessary to
use immunohistochemistry, electron
microscopy, flow cytometry, cytogenetics, and
molecular genetics to confirm diagnosis.
• These studies may require fresh, frozen or other
specifically processes tissues.
 FRESH TISSUE EXAMINATION
• *
• Methods of tissue examination may vary according to:
a. Structural and chemical components of the cells to
be studied
b. Nature and amount of the tissue to be evaluated
c. Need for an immediate examination of a tissue
structure
• Examination may be done on fresh or preserved
tissues**
 FRESH TISSUE EXAMINATION
• Advantage
• For the study of protoplasmic activities
• such as: Motion
Mitosis
Phagocytosis
Pinocytosis
Disadvantage: Not permanent and tissues develop
changes that are observed after death
(decomposition).
 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)
• - 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, wherein 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.
 METHODS OF FRESH TISSUE
EXAMINATION
• 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
• 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