Basics in Pathology

By Dr. Ankush Nayyar

INTRODUCTION
Cells are the building blocks of life
Cells unite and form tissues which perform specific function
Microscopic study of individual cells in a smear is called CYTOLOGY
Microscopic study of cells in a tissue is called HISTOLOGY
Microscopic study of diseased tissues is called HISTOPATHOLOGY
Patient care is increasingly based on information provided
by examination of surgical specimens and biopsies.
Histopathological report must contain all the relevant
information and if possible, a diagnosis necessary for
patient care
The ultimate goal is to obtain all information relevant to
clinical management of patient.
Tissues from the body taken for diagnosis of disease
processes are processed in the histology laboratory to
produce microscopic slides that are viewed under the
microscope by pathologists.
The tissues processed may be biopsies, larger specimens
removed at surgery, surgical margins or tissues from
autopsy.
The basic steps in specimen processing include-
•Fixation- Preserving the tissue in its natural state as nearly as
possible
•Embedding- Provides necessary hardness to cut sections
•Microtomy- Thin sections of the tissue arecut using an
equipment- (Microtome)
•Staining- Parrafin is removed from section by drying and
dewaxing followed by rehydration. The rehydrated tissues are
stained.
•Mounting- After staining the sections on slides are placed on
mounting medium and then finally a cover slip is placed over it.
 FIXATION
Fixation is a chemical process by which biological tissues are preserved from
decay (through autolysis or putrefaction).
Fixation terminates any ongoing biochemical reactions, and may also increases
the mechanical strength or stability of the treated tissues.

The aim of fixation:

First, a fixative usually acts to disable intrinsic biomolecules (proteolytic enzymes ) which would
otherwise digest or damage the sample.
Second, a fixative will typically protect a sample from extrinsic damage. Fixatives are toxic to most
common microorganisms (bacteria) which might exist in a tissue sample or which might colonise the
fixed tissue.
Third, it renders cell constituents insoluble and stabilizes them.

Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical
strength or stability.
 CHOICE OF FIXATIVE
No single fixative is the best, so choice depends on
nature of tissue and tests to be performed.
Fixative Color Use
10% formalin Clear Routine
Bouin’s Yellow GI Biopsy, testis
Normal saline Clear Frozen, Squash, Imprint,
Crystals
Alcohol Clear Slides

NBF Clear Prevents formalin pigment

Rapid spray fix Clear PAP

Glutaraldehyde Clear EM study

 MOLECULAR DIAGNOSTIC
TECHNIQUES
Common methods used in molecular biology,
biochemistry, genetics and biophysics which
generally involve manipulation and analysis of
DNA, RNA and Proteins.
• These techniques have utility in virtually every
area of diagnostic pathology – neoplastic
disorders, infectious diseases, inherited
conditions and identity determination.
 TYPES
• Amplification techniques –
PCR and its modifications

• Blotting techniques –
Southern blot, Dot blot, RFLP
Northern blot
Western blot

• Hybridization techniques –
In situ hybridization
FISH and its modifications
Microarray
 Polymerase chain reaction
• Polymerase chain reaction, or PCR, is a technique to make
many copies of a specific DNA region in vitro.
• PCR relies on a thermostable DNA
polymerase, Taq polymerase, and requires
DNA primers designed specifically for the DNA region of
interest (template).
• In PCR, the reaction is repeatedly cycled through a series of
temperature changes, which allow many copies of the target
region to be produced.
 BLOTTING TECHNIQUES
The analytical techniques used for the identification of specific
DNA, RNA or a Protein
Blot type Material analysed Electrophoresis Probe used
required

Southern DNA Yes 32P-DNA

Northern RNA Yes 32P-DNA

Western Protein Yes 131I or enzyme

linked antibody

Dot blot Any of the three No Any of the above

IN SITU HYBRIDISATION
• Metaphase chromosomes
• Radioactive probe
FLUORESCENT IN SITU HYBRIDISATION
(FISH)
• Metaphase chromosomes
• Fluorescent probe
• Very sensitive
 FISH

• Description - Use of a fluorescently labeled

DNA probe to detect specific DNA
sequences in chromosome
• Detection –
– Gene number alterations,
– DNA rearrangements
• Example assays - HER2 FISH pharmDX Kit
• Sample requirement - Tissue sections
 Gene Microarray technique
• Description - Detection of specific DNA
sequences or cDNAs (for RNA analysis)
by hybridization to an array of DNA
probes
• Detection - Gene number alterations, DNA
rearrangements, gene expression levels, RNA
editing
• Sample requirement - As low as 75 ng DNA
from FFPE sample can be used
Gene Microarray technique