ADVANCED MAXILLOFACIAL

PATHOLOGICAL
INVESTIGATIONS
 INTRODUCTION
The analysis of DNA,RNA & protiens obtained
from diagnostic specimen, is currently
revolutionizing the practice of surgical pathology.

The diagnosis of cancer & many other diseases

is fundamentally based on the microscopic study
of cells & tissues.
Histochemical methods are gradually being
replaced by advanced immunologic & molecular
tech’s by which complicated infectious,
inflammatory, metabolic & neoplastic diseases
are diagnosed & classified.
 DIAGNOSTIC METHODS

Molecular methods.

Immuno histochemical methods.

Direct immuno fluoresence methods

 MOLECULAR METHODS
Polymerase chain r
Hybridization methods – BLOTTING
Advanced Technologies
Flow cytometry
Fluoresence in situ hybridization [ FISH ]
DNA chips/ Microarrays
Laser capture microdissection
 Polymerized Chain Reaction
Polymerase chain reaction (PCR) is a molecular biology
technique for enzymatically replicating DNA .

The technique allows a small amount of the DNA molecule

to be amplified many times, in an exponential manner.

PCR is commonly used in medical and biological research

labs for a variety of tasks, such as the detection of
hereditary diseases, the identification of genetic fingerprints
, the diagnosis of infectious diseases, the cloning of genes,
paternity testing, and DNA computing.
 Discovered by Kary Mullis

Components of PCR are :

1. DNA template, or cDNA which contains the region
of the DNA fragment to be amplified
2. Two primers, which determine the beginning and
end of the region to be amplified (see following
section on primers)
3. Taq polymerase, which copies the region to be
amplified
4. Deoxynucleotides-triphosphate, from which the
DNA-Polymerase builds the new DNA
5. Buffer, which provides a suitable chemical
environment for the DNA-Polymerase
Primers
Primers are short, artificial DNA strands —
often not more than fifty and usually only 18
to 25 base pairs long nucleotides that are
complementary to the beginning and end of
the DNA fragment to be amplified.
They anneal by adhering to the DNA template
at these starting and ending points, where the
DNA-Polymerase binds and begins the
synthesis of the new DNA strand.
 PROCEDURE
1. DENATURATION
The double-stranded DNA has to be heated to
94-96°C in order to separate the strands. This
step is called denaturing; it breaks apart the
hydrogen bonds that connect the two DNA
strands.
The DNA is often denatured for an extended
time to ensure that both the template DNA and
the primers have completely separated and are
now single-strand only. Time: 1-2 minutes up to
5 minutes.
2. ANNEALING
After separating the DNA strands, the
temperature is lowered so the primers can
attach themselves to the single DNA
strands. This step is called annealing.
The temperature of this stage depends on the
primers and is usually 5°C below their
melting temperature (45-60°C).
 A wrong temperature during the annealing
step can result in primers not binding to the
template DNA at all, or binding at random.
 Time: 1-2 minutes.
3. EXTENSION
Finally, the DNA-Polymerase has to
copy the DNA strands. It starts at the
annealed primer and works its way
along the DNA strand. This step is
called extension.

Temperature required is 72oc for 45

sec.
 LIMATATIONS

Difficulties can be encountered when

studying small quantities of DNA, since the
ingredients necessary for PCR may be
exhausted b4 target is produced.
A major limitation is susceptibility of the
process to contamination particularly to
detect RARE DNA sequences
 USES OF PCR
Genetic Finger printing
Paternity testing
Diagnosis of disease- infectious and malignant
disease
Cloning of genes
Mutagenesis
Analysis of ancient DNA
Genotyping of specific mutations- used in amplifying
certain haplotypes or detection of recombinant
chromosomes and the study of meiotic
recombinations
 DNA SEQUENCING
DNA sequencing is the process of
determining the nucleotide order of a given
DNA fragment called the DNA sequence.

Can be used to identify,diagnose and

potentially develop treatment fro genetic
diseases.
 How is DNA sequencing
done?
Chromosomes, which range in size from 50 million to
250 million bases, must first be broken into much
shorter pieces - subcloning step
Each short piece is used as a template to generate a
set of fragments that differ in length from each other
by a single base that will be identified in a later step -
template preparation and sequencing reaction steps
The fragments in a set are separated by gel
electrophoresis (separation step). New fluorescent
dyes allow separation of all four fragments in a
single lane on the gel.
The final base at the end of each fragment is
identified (base-calling step). This process
recreates the original sequence of As, Ts, Cs,
and Gs for each short piece generated in the
first step. Automated sequencers analyze the
resulting electropherograms, and the output
is a four-color chromatogram showing peaks
that represent each of the four DNA bases.
After the bases are "read," computers are
used to assemble the short sequences (in
blocks of about 500 bases each, called the
read length) into long continuous stretches
that are analyzed for errors, gene-coding
regions, and other characteristics
 HYBRIDIZATION METHODS
Hybridization refers to the pairing of
complementary RNA/DNA strands to produce a
double-stranded nucleic acid.
All Hybridization methods use radio/fluorescence
labeled DNA/RNA probe that binds to the target
DNA/RNA of interest, permitting visualization.
METHODS
Southern blotting
Northern & western blotting
 SOUTHERN BLOTTING
Described by Ed southern in 1975
This method is widely used for analyzing the
structure of DNA.
This involves the transfer or blotting of DNA
fragments onto a membrane.
DNA is 1st enzymatically cleaved into smaller
pieces by restriction endonucleases (enzymes
that are able to cut DNA at specific recognition
sites)
After fragment separated, the DNA is transferred
from the gel to a nylon or nitrocellulose
membrane.

Finally, specific DNA fragments can be identified

by hybridizing the membrane with labeled cDNA
or RNA probes, followed by detection of the
label on an x-ray film by autoradiography
 WESTERN BLOTTING

A powerful method for detecting a

particular protein in a complex
mixture,combines the superior resolving
power of gel electrophoresis,the specificity
of antibodies and the sensitivity of enzyme
assays.
Commonly used to separate proteins and the
identify a specific protein of interest

Formerly called as PROTEIN IMMUNOBLOT

and is used to determine the molecular wt. Of
protein and measure relative amount of
protein present in different samples

Technique for separating molecules in a

mixture under the influence of an electric field
 NORTHERN BLOTTING
Northern blotting is a simple extension of
Southern blotting - and derives its name from the
earlier technique.

It is used to detect cellular RNA rather than DNA.

It was shown that when RNA was denatured, that

it would also bind efficiently to nitrocellulose. This
means that the RNA has to be unfolded into a
linear strand before it will bind efficiently to
nitrocellulose.
.
Chemicals such as formaldehyde and
methylmercuric hydroxide can be used
to denature the RNA - breaking down
hydrogen bonding structure in the
molecule. Alkali is not used to denature
the RNA - since RNA is degraded under
alkaline conditions.
 IN SITU HYBRIDIZATION
FISH uses fluorescent molecules to vividly paint
genes or chromosomes.This technique is useful
for gene mapping and for identifying
chromosome abnormalities.

FISH (Fluorescent in situ hybridization) is a

cytogenetic technique which can be used to
detect and localize DNA sequences on
chromosomes.
The technical approaches to the identification of
DNA & RNA differ slightly but are conceptually
similar to blotting techniques

Here DNA probe, labeled with BIOTIN

FISH has a wide range of application in
pathology
It has been used to detect EBV particles in oral
hairy leukoplakia, a lesion often seen in HIV
It has other applications in microbiology,
embrology, cytogenetics & neurogenetics
 FLOW CTYOMETRY
Flow cytometry is an important method used to
analyze cell kinetics (the distribution of cells in
diff phases of cell cycle ) & protein expression in
normal & tumour cells

Large numbers of cells can be analyzed rapidly,

providing a distribution several thousands cells
at one time.
Flow cytometry permits analysis of tissue when it
is prepared as single-cell suspension stained
with a DNA-binding fluorescent dye.
The amount of fluorescence thereby
corresponds to the amount of DNA present in
the cell
The labeled cells are then directed, along a
charged column through a laser beam, which
excites the fluorescent emissions dye bound to
the cell.
The emissions are then collected by a
fluorescence detector & analyzed .
DNA CHIP TECHNOLOGY

A DNA chip (DNA microarray) is a

biosensor which analyzes gene
information from humans and bacteria.
This utilizes the complementation of the
four bases labeled A (adenine), T
(thymine), G (guanine) and C (cytosine)
in which A pairs with T and G pairs with
C through hydrogen bonding.
DNA fragments amplifies by PCR technique are
spotted on a microscopic glass slide coated with
polylysine prior to spotting process.
RNA are extracted from the cultures and mRNA
are the transformed in Cdna by reverse
transcription.
DNA from the first culture is labeled with a green
dye and DNA from the second culture is labeled
with a red dye.
Both the DNA from the culture are
mixed together (target) and put on the
matrix of spotted single strand DNA
(call the probe).The chip is then
incubated at 60oC for 1night.
A laser excites each spot and the
fluorescent emissions through a
photomultiplier coupled to a confocal
microscope gives two images – one
image composed of spots going from
green ones to red passing through the
yellow colour (where the DNA from the
2 conditions are fixed in equal amount).
 APPLICATIONS
Gene discovery
Disease diagnosis
Drug discovery
Toxicological research
LASER CAPTURE MICRODISSECTION
One major limitation of the application of
molecular biology to pathology has been the
heterogeneous nature of tissue samples.

To develop useful, reproducible data , it is

desirable to study pure population of cells
obtained from actual biopsy samples of tissues
both normal & diseased.
Laser Capture Microdissection (LCM) allows
researchers to obtain homogeneous cell types
and multi-cellular structures isolated from whole
tissue or cytology samples.

LCM is a new technology for rapid pprn of

relatively pure cell samples from tissue sections
 PRINCIPLE
 LCM is based on the preferential adherence of
identified cells to a plastic membrane activated
by a low energy infra-red laser pulse.

 APPARATUS
 Inverted microscope
 Infra red laser diode
 Laser controls
 Microscope stage controlled by joysticks
 Slide immobilizer by vacuum,charge couple
device camera
 Color monitor
.
 METHOD
 LCM utilizes an infrared laser integrated into a
standard microscope. A transparent cap is
attached to a thermoplastic transparent
membrane which lies directly on the surface of a
routinely prepared tissue section on a glass
slide.
 The investigator examines the tissue section
microscopically and activates the laser when the
desired cells underlie the target.
 This in turn activates the membrane with
subsequent binding and procurement of the cells
of interest.
 The laser pulse is very brief (approximately 5
msec) and the membrane is activated at
90°C, thus the tissue experiences only a very
brief thermal transient as the heat generated
from the membrane is rapidly dissipated.
 LCM allows for visualization and image
capturing of tissue as it is microdissected,
including images of the tissue before and
after microdissection. This is critical in
maintaining an accurate record of each
dissection and correlating histopathology with
subsequent molecular results.
 IMMUNOHISTOCHEMICAL METHODS

Immunohistochemistry has been shown to be an

effective adjunct to H&E diagnosis in majority of
tumour cases.

IHC is applied typically to cases when the

definitive diagnosis cannot be established solely
on the basis of findings in H&E sections
 MOLECULAR MARKERS
Epithelial marker:
IHC staining for cytokeratins, known as
keratins, is frequently done to confirm the
epithelial lineage of a poorly differentiated
adenocarcinoma, or spindle cell carcinoma

Keratin antibodies are typically used as part of a

general screening panel foe undifferentiated
tumors
Cytokeratins reoresent a group of structurally
related intermediate filament proteins. They are
divided into 19 subtypes depending on their
molecular wt
Abnormal expression has been seen in
eplithelial dysplasia & carcinoma insitu lesions of
oral mucosa, suggesting possibility of
premalignancy
 MUSCLE MARKERS

Muscle diff in a neoplasm can be established by

demonstrating the expression of desmin, actin or
myoglobin proteins.

Desmin & myoglobin immunoreactivity are helpful in the

diagnosis of tumors of muscle origin, especially
rhabdomyosarcoma
Anti-muscle-specific actin is effective in staining
myoepithelium of salivary glands. Because most salivary
glands neoplasms contain myoepithelium cells
 ENDOTHELIAL MARKERS
Vascular diff of a tumour can be established with
the antibodies to CD31, CD34

In diagnostic problems in which kaposi’s

sarcoma or other neoplasms of vascular origin,
any of these antibodies may be used

Anti CD34 most consistent marker of

endothelial cells in kaposi’s sarcoma.
 MINOR SALIVARY GLAND TUMOR MARKERS

S-100 PROTEIN & ACTINS

S-100- expressed to greater degree in

adenocarcimona

Muscle-specific actins: expressed in adenocystic

carcinoma
 DIRECT IMMUNOFLUORESENCE

Like IHC, DIF uses known prepared

antibodies to identify antigens in tissue
sections.
The antigens stained with DIF are
autoantibodies & inflammatory proteins.
This method is used predominantly in the
confirmation of microscopic diagnosis of a
small number of vesiculobullous ulcerative
disease
 APPLICATIONS
HELPFUL IN DIAGNOSIS OF
Pemphigus vulgaris

Mucous membrane phemphigoid

Lupus erythematosus

Lichen planus