ADVANCED TECHNIQUES IN HISTOLOGY AND CYTOLOGY LABORATORIES
PREPARED BY MR. ABHIJIT DAS
IMMUNOHISTOCHEMISTRY/IMMUNOCYTOCHEMISTRY
Immunohistochemistry is a technique used to detect
specific antigens (proteins)
in cells within tissue sections by using antibodies. It plays a crucial role in
diagnosing diseases, especially cancers, by
identifying specific proteins that can act as biomarkers.
Key Steps in IHC:
1.
Fixation:
Tissue samples are preserved using fixatives like formalin to maintain their
structure.
2.
Embedding:
Fixed tissues are embedded in paraffin wax and sectioned into thin slices.
3.
Antigen Retrieval:
A process to unmask
antigens that might be hidden due to fixation.
4.
Antibody Application:
A primary antibody
binds to the target
antigen. A secondary
antibody, often linked
to an enzyme or dye, binds to the primary antibody.
5.
Visualization:
Enzyme-linked antibodies cause a colour change when a substrate is added, indicating the presence
of the antigen.
Applications:
- Cancer
Diagnosis: Used to detect specific markers
(e.g., HER2 in breast cancer).
- Infectious
Diseases: Identifies pathogens in tissues.
POLYMERASE CHAIN REACTION
Introduction:
Polymerase Chain Reaction (PCR) is a technique used to
copy specific DNA sequences, making millions of identical copies. It is
essential in research, medical diagnostics, and forensic science.
Principle:
PCR works by mimicking natural DNA replication. It uses a special enzyme (DNA polymerase) to amplify a targeted DNA segment
through repeated cycles of heating and cooling.
Procedure:
1.
Denaturation:
Heat the DNA to
separate its two strands (about 94-98°C).
2.
Annealing:
Cool the mixture to
allow short DNA primers to attach to the target sequences (about
50-65°C).
3.
Extension:
Heat again to let DNA
polymerase add nucleotides, creating
new DNA strands (about 72°C).
4.
Cycling:
Repeat the steps 20-40
times to exponentially amplify the target DNA.
Conclusion:
PCR is a simple and effective method for producing large amounts of specific
DNA, crucial for various applications in biology and medicine.
FLOW CYTOMETRY
Flow Cytometry is
a lab technique used to analyze cells in a liquid. It helps study cell size, shape, and specific markers on the cell
surface.
Basic Principle
Cells pass one by one through a laser beam. As they go
through, the light scatters,
and fluorescent dyes
attached to cell markers emit light.
The way light scatters tells us about the cell’s size
and internal complexity (like granules or structures within the cell).
And the emitted light helps identify specific markers
the dyes attach to.
Procedure
1.
Prepare Cells:
Cells are put in a liquid
(usually
a saline or buffer solution) and stained with fluorescent dyes.
2.
Run through Machine:
The flow cytometer sends cells through a laser, one at a time.
3.
Analyze Data:
The light signals are
detected, converted into data, and shown as graphs or plots to interpret
cell types and properties.
Applications
- Identifying
different cell types (like immune cells).
- Measuring
DNA content.
- Studying
cell health and behavior.
NOTE:
· Fluorescent
dyes
are special chemical compounds that absorb light at a specific wavelength and
then emit it at a different, usually longer, wavelength.
· When
cells pass through a laser beam in flow cytometry, two main things happen:
1.
Light Scatters:
The laser light hits the cell and scatters in different directions. The way
light scatters tells us about the cell’s size and internal complexity (like
granules or structures within the cell).
2.
Fluorescent Dyes Emit Light:
Cells are often tagged with fluorescent dyes that stick to specific
markers on the cell’s surface or inside it. When the laser hits these dyes,
they absorb the laser light and then emit it as a different color of light.
This emitted light helps identify particular cell features or types based on the
specific markers the dyes attach to.
FLUORESCENT IN SITU
HYBRIDIZATION
Introduction to FISH (Fluorescent In Situ
Hybridization)
FISH is a lab technique used to find specific genes or parts of DNA in
cells. It helps scientists and doctors see where certain DNA sequences are
located in the cell by making them glow under a special microscope.
Basic Principle
FISH uses fluorescent probes (tiny, glowing pieces of DNA) that attach to specific genes or DNA sections
in the cell. When the probe finds and binds to its matching DNA sequence, it
glows, making that part of the DNA easy to see.
Procedure
1.
Prepare Sample:
Cells are placed on a slide.
2.
Add Probes:
Fluorescent probes are added to the sample.
3.
Allow Binding:
Probes attach to their matching DNA.
4.
View Under Microscope:
The specific DNA parts glow, showing exactly where they are in the cell.