Introduction: more closely involved in the direct management

Introduction:

In recent years, cellular pathology has
become more closely involved in the direct management of patients with the
introduction of molecular technologies and targeted therapies. Through this, we
have seen the introduction of specialist pathology. These concepts and the key
technologies that are influencing clinical practice today have been introduced.
It showed that how clinical practice has been affected by these respective
technologies and how further development will influence the practice and
delivery of cellular pathology, which will impact on the patient through
targeted therapeutics and diagnostics.

Discussion:

There are many types of advanced techniques
in cellular pathology.

Digital
(virtual) microscopy:

Automated microscopes represent a form
of hybrid technology in that they bring together various components within the
industry to form a device capable of creating virtual slides without being
dedicated to that purpose. High-specification microscopes are the starting
point for this technology and essentially combine imaging (CCD) and
computational technology to produce the device. Image acquisition as in the
systems described above is realized via a digital camera. The CCD chip acquires
the centre of the field given by the objective, thus reducing optical
aberration to a point considered negligible. The Olympus DotSlide system is
essentially an automated microscope (Olympus upright BX research microscope)
that is computer-driven over the slide to form an image from a Peltier-cooled
1379 × 1032 pixel camera. With
the virtual slides, the operator can defined accurate protocols and run
efficient algorithms on whole slides or specified area.

DNA
profiling:

That is 0.1% of the genomes in 3,000,000
base pairs of nucleotide are unique in all human being. This speciality in the
base sequences occur in repetitive DNA also known as satellite DNA as well as in
genes. Various small peaks are formed on the DNA which gives rise to
polymorphism due to density gradient configuration in the satellite DNA. Variable number tandem repeats (VNTR)
is one of the main satellite DNA having high degree of polymorphism. The number
VNTRs at a particular area of the DNA of the child will be different may be due
to insertion, deletion or mutation in the base pairs since a child receive 50%
of the DNA from its father and the other 50% from his mother.

Application:

DNA
profiling, as already indicated, has application in a broad cross section of
disciplines, including human forensic science, diagnostic medicine, family
relationship analysis, animal and plant sciences, and wildlife forensic
science. DNA profiling is applicable in a number of areas in medicine  including twin zygosity testing , bone marrow
transplantation marker analysis, detection of DNA changes in tumors, indication
of possible contamination of fetal by maternal tissue in chorionic villus
analysis, pathogen identification  and paternity
testing where family studies are being performed for antenatal diagnosis of
inherited diseases.
Polymerase chain reaction (PCR):

Principle:

The primer mediated enzymatic amplification of DNA is the principle of
PCR. The ability of DNA polymerase to synthesize new strand of DNA
complementary to the offered template strand are by using PCR. DNA polymerase
can add a nucleotide only onto a preexisting 3?-OH group to add the first
nucleotide, so the primer is needed. DNA polymerase then elongated its 3 end by
adding more nucleotides to generate an extended region of double stranded DNA.

Applications:

There are many applications of PCR
including to test the presence of genetic disease mutation such as cystic
fibrosis, hemoglobinopathies or other inborn errors of metabolism. It can used
to study the alteration to oncogene that causes cancer. It is also a tool used
in genetic fingerprinting. In a crime investigation, there may only be tiny DNA
sample to work with to identify anyone from the million.

Real-time PCR (RT-PCR):

Principle:

In a thermal cycler, the capacity to illuminate each sample with a
beam of light of at least one specified wavelength and detect the fluorescence
emitted by the excited fluorophore are carried out by real-time
PCR. Advantage of the physicochemical properties of the nucleic acids and DNA polymerase is also able to quick heat and cool samples.

Applications:

The
tool of choice for the rapid and sensitive determination and quantitation of
nucleic acid in various biological samples is real-time PCR assays. The detection
of genetically modified organisms in food, gene expression analysis and cancer
phenotyping are main applications of real-time PCR. RT-PCR assays are widely
used for the quantitative measurement of gene copy number (gene dosage) in
transformed cell lines or the presence of mutant genes in research laboratories.
It can be used to precisely quantitate changes in gene expression, for example,
an increase or decrease in expression in response to different environmental
conditions or drug treatment, by measuring changes in cellular mRNA levels.

Spectral
karyotyping:

Spectral
karyotyping (SKY) is a hybridization-based diagnostic technique originally
developed to diagnose chromosomal aberrations associated with cancer and
genetic disease. Specific inter and intra-chromosomal genomic rearrangements,
and unambiguously determine both the total number and individual identity of
all chromosomes in a metaphase nucleus can be detected by using SKY. Sky Paint (Applied
Spectral Imaging) is hybridized to metaphase chromosome spreads from the cells
of interest. Sky Paint is a mixture of probes specific to single chromosomes,
each of which contains a spectrally unique combination of fluorescent
nucleotides thus allowing the user to “paint” each chromosome a different
color. After acquiring a metaphase spread image using a microscope equipped
with an interferometer that reads emissions across the entire visible spectrum,
individual chromosomes are assigned using SkyView  software. SkyView analyzes the spectral image
in two dimensions and displays each chromosome with a distinct classification
color from which it creates a karyotype table.

DNA microarrays:

DNA
microarrays (sometimes called DNA chips) are in general characterized by a
structured immobilization of DNA targets in the free nucleic acid samples on
planar solid supports, on which different types of nucleic acids with known
sequences (known as “probes”) are fixed. A probe may be derived from
complementary DNA (cDNA), polymerase chain reaction (PCR) products, or
synthetic oligomers. Commonly, applications of DNA microarray technology
broadly include gene expression analysis (transcription analysis), which
analyzes the transcriptional activity of genes through hybridization between
DNA targets and probes; genotyping with oligonucleotide arrays, which is based
on the notion of combining the complete sequence of a DNA sample by presenting
all possible sequences as a complement on the chip; measurement of enzyme
activities on immobilized DNA, which is based on the finding that DNA-modifying
enzymes are capable of acting on immobilized DNA templates or oligonucleotides;
transcription on chip, which shows the transcription of a complete gene into
mRNA on the chip.

FISH
is a technique used to identify the presence of a single nucleic acid sequence
(often specific to a particular chromosome) through hybridization of fluorescently
labeled DNA probes to denatured chromosomal DNA in cytological material.
Interphase nuclei are hybridized with the FISH probe, though metaphase spreads
can be used as well. FISH probes can be purchased commercially. By using FISH, a
copy of aberrations numbers as well as specific cytogenetic abnormalities can
be sketched and enumerated because it can easily detected chromosomal micro-deletion,
amplification, and subsequently translocation. Compare with cytogenetic
metaphase karyotype analysis, FISH is less time consuming detect and monitor
the specific therapy with regards to the gene abnormalities.

Flow cytometer:

The flow cell is where the cellular
interrogation takes place, the objective being very simple: to allow the cell
of interest to pass through the laser (or lasers) interrogation point and then
for the product of that interrogation to be displayed as physical and
fluorescent properties of that cell.

Major application
where low
cytometry is used include the diagnosis and subclassification of acute
leukaemia and chronic lymphoproliferative disorders, including chronic
lymphocytic leukaemia and non-Hodgkin lymphoma, HIV monitoring and DNA
analysis.

Microarray-based
Comparative Genomic Hybridization

Array CGH is based on the same
principles as metaphase CGH, a technique that has been extensively used for the
genomic characterization of a number of solid tumours. Both techniques allow
the study of DNA copy-number alterations genome-wide, except that the targets
for hybridization are mapped clones in the aCGH technique instead of chromosomes
as in metaphase CGH. Propidium iodide, phycoerythrin and fluorescein are the
common dyes to used although many other dyes are available. Furthermore, the
frequency of the recurrent amplification and the specific tumour subgroup where
it is most prevalent can also be established in this manner. By interrogating
the genome to identify critical molecular drivers in cancer, aCGH offers the
means to identify the therapeutic target and hence the appropriate biomarker
assay as well in the process of drug development.

Tissue in
situ hybridization (ISH)

The basic requirements of a probe for
use in tissue in situ hybridization are that it is complementary to the target
nucleic acid sequence and can be labelled in such a way as to allow microscopic
visualization of the hybrid formed. The different types of probe include the
following. Double-stranded DNA probes are most commonly used to detect DNA
targets. They are generated by the cloning and amplification of specific
sequences of DNA or cDNA, derived by reverse transcription of mRNA employing
vectors (bacterial plasmids and cosmids). Single-stranded RNA probes, so-called
riboprobes, are most commonly used to detect RNA in tissue sections. Riboprobes
are usually generated by in vitro transcription from plasmids containing the
sequence of interest. The plasmids are designed with promoter sites for RNA
polymerases (e.g. T3, T7 and SP6) and can produce probes complementary to the
target RNA sequence (antisense) or identical to the target sequence (sense). Tissue ISH is used to detect nucleic
acid sequences in a wide variety of solid neoplastic and infectious conditions
and is becoming a crucial theranostic tool, helping to guide therapy by
identifying relevant targets for new drugs in the field of pharmacogenomics. The deciphering of the human genome,
in combination with recent developments in nucleic acid-based testing, has
positioned tissue ISH as a central tool in diagnosis and predictive therapy.
Molecular diagnostics including tissue ISH are widely used in the areas of
inherited genetic disorders and infectious diseases, as well as haematologic
and solid tumours. In addition, tissue ISH has a role to play in guiding
appropriate therapy.

Laser
capture microdissection (LMD):

A
technique for isolating specific and pure targets from microscopic
heterogeneous samples for downstream analysis (DNA, RNA & proteins) is
known as laser microdissection (LMD). It is based on
microscopic imaging and utilizing a laser. In contrast to other systems which
use a fixed laser focus for dissection, Leica Microsystems’ LMD
systems guide the laser focus for dissection. This unique feature allows highly
precise laser dissection independent of the stage accuracy. LCM is broadly used
in many medical research areas from neuroscience, forensic science research to
biomarker discovery, cancer and clinical diagnostics. Molecular biology such as
genetics and proteomics are the mainly applications. Many genetic materials
such as DNA, mRNA, and microRNA, can be extracted from tissues.  Neural stem cell therapy is another interesting
example of LCM application.