Cellular gross examination which is examination of organs and

Cellular
pathology is the branch of pathology that involves the study of body organs and
tissues which are also known as
anatomical pathology. Its roles include determining the cause of certain
diseases and the effect that they are having on the body, assisting with the
choice of treatment that will be given, aiding in giving a prognosis and
determining what may have caused a person’s death. There are two main
subdivisions within cellular pathology which is histopathology and cytopathology Lab Tests Online UK,
2017.

 

In recent years, cellular pathology has become more closely involved in
the direct management of patients. This report shows how clinical practice has
been affected by these respective technologies and how further development will
give impact on the patient through targeted therapeutics and diagnostics Mary
Hannon, 2009.

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Advanced techniques in diagnostic cellular pathology that is explained
in this report are virtual microscopy, cytopathology, flow cytometry, immunohistochemistry
and tissue in situ hybridization.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DISCUSSION

 

Basic Techniques in Diagnostic Cellular Pathology

Before
the advanced techniques are widely used, some basic techniques are commonly
used. First is gross examination which is examination of organs and tissues macroscopically to select
relevant portions for microscopic examination. It can be accurately made in 90 % of specimens while the
remaining 10 % is depending on the pathologist’s skill. However, the
pathologist’s skills are rapidly declining thus lower the accuracy and precision
Geller,
SA., 2014.

 

Second
is histopathology technique which includes fixation,
embedding, and staining. Fixation is to preserve
tissues permanently. Tissue embedded in paraffin to be made into thin
microscopic sections and microtome will cut into sections. Staining uses a
variety of dyes to stain various cellular components of tissue Edward, C.,
2018.

Third is microscopy examination which are either light microscope or electron microscope can be used. Electron microscope utilizes beams of electrons
rather than visible light to magnify the cells in a tissue sample which allows
much greater magnification, enabling the visualization of organelles within the cells Lab Tests
Online UK, 2018.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Molecular Techniques

Molecular pathology focuses on the diagnosis
and study of disease through the examination of molecules within bodily fluids,
tissues or organs Harris TJ., 2010. First is Polymerase chain reaction (PCR)
which enables the amplification of specific sequences of nucleic acids from an
extremely small amount of genetic starting material which usually performed on
a variety of fresh specimens Bethesda, 2004. Second is Real-time PCR which detects pathogens in the research setting and diagnostic settings. It combines PCR chemistry
with either a fluorescent probe or DNA detection dyes such as Sybr green                                                     
                       Sumathi,
S., 2012. Third is Spectral
karyotyping which uses 23 sets of
chromosome-specific “painting” probes to identify chromosomal abnormalities George, J., 2003. Next is Microdissection of tissue sections and
cytological preparations which has been used increasingly for the isolation of
homogeneous since it allows precise examination Fend, F., 2000. Lastly is Forensic pathology focuses on the causes of death as opposed to
illnesses and their cures or treatments. A forensic pathologist must remove an
extremely thin section of the tissue for an autopsy to study the areas of
body tissue Mary Hannon, 2009.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Advanced Techniques in Diagnostic Cellular Pathology

For the past recent years, many
basic techniques for cellular pathology diagnosis have been replaced with
advanced techniques which have many benefits.

 

First technique is Virtual microscopy which is the new
technique which replaces telepathology system. It utilizes digitization of
whole-microscopic glass slides via computer-aided systems to produce virtual
microscopic slides for the interpretation of tissue sections Mary, H., 2009. The advantages are ease of navigation while maintaining orientation, produce better image quality, greater time efficiency and can view any
part of the tissue section at any magnification Ngozi, N., 2017.

 

Equipments of virtual microscopy are automated microscopes which is a High-specification
microscopes combine imaging and computational technology to produce the device Mary, H., 2009. 

 

 

 

 

 

 

 

 

Figure 1: Automated
Microscope Mary, H., 2009. 

 

 

 

 

 

 

 

 

 

 

Besides that is Scanners
such as Progressive scan CCD systems which has capability for slide observation and allows digital image
capture. It additionally has Internet communications capabilities which are the
major feature. Samples can be viewed on networked PCs at remote locations Mary, H., 2009. 

 

 

 

 

 

 

 

 

Figure 3: Scan CCD system LabWrench, 2018

 

Next is Aperio ScanScope system which offers a
slide capacity of 120 slides and is suitable for environments such as
hospitals, labs and research organizations. It
created an alternative way to tiling, termed ‘line scanning’ which accurately
moves the slide under a line-scan camera to acquire the image Mary, H., 2009. 

 

 

 

 

 

 

 

 

 

Figure 5: Aperio ScanScope scanning system Mary, H., 2009. 

 

 

 

Applications of
virtual microscopy in diagnosis of
disease is that it has very high interest, for
example, in cancer biomarker expression quantification (e.g. HER2/NEU) which
important for both patient health and financial considerations Mary, H., 2009. 

For education, traditionally it has been based on
printed micrographs and projection slides. Now, modern textbooks provide a
supplemental CD with digital images. This enables viewing of any part of a
specimen at any magnification Mary, H., 2009.

Lastly
are research studies.  All studies generally
show ‘proof of concept’ with small images captured from a traditional CCD
camera. In molecular pathology research, virtual
microscopy could provide the entire study material viewable and it would be
valuable for authors, journals, and readers. Lundin, M., 2004

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Second
technique is Liquid-Based Cytology. In an attempt to improve the traditional Pap smear,
Liquid-Based Cytology has been introduced. The aim is to improve both the
sensitivity and specificity of the cervical smear. One difference between LBC
and traditional technique is that the cells are rinsed into a vial of fixative,
which allows better preservation of cells so, clearer nuclear staining which is
the main principle for this technique
Mary, H., 2009. 

 

Figure 8: Cell
Processing Using LBC Mary, H., 2009. 

 

 

 

 

 

Advantages of
this technique is that the Cellular
material evenly distributed and spread on the slide, cellular material
well-preserved, reduces mucus, blood, and exudates on slide, and reduces
screening time Mary, H., 2009. 

 

Figure 9: Conventional Smear vs.
Liquid Based Michael, A., 2006

 

Applications for this technique in Cervical Screening is that it reduces the number of false-negative test results, invasive
cancer incidence, make diagnosis accurately and decrease in time taken to obtain
the smear Mahboobeh, S., 2007.

For oral brush, it improves cytodiagnostic accuracy and provides
an adequate sample of oral epithelium. LBC also removes most mucus, protein and
red cells from the microscope slides, distributes cells evenly, maintains
diagnostic clusters, reduces scanty preparations and eliminates air-drying
artifacts in oral samples Shwetha, N., 2016

 

 

 

 

 

 

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Next technique is Flow cytometry. It can be defined as a semi-automated
procedure for the interrogation of single cells in a continuous fluid stream,
enabling the derivation of simultaneous measurements of multiple extra- and
intracellular characteristics Mary, H.,
2009. 

 

 

 

 

 

 

 

 

 

Figure 10: Flow Cytometer Beckmen,
C., 2015

 

The principle
for this technique involves a passage of cells in single file in front of a
laser so they can be detected, counted and sorted. Cell components that are
fluorescently labeled are then excited by the laser to emit light at varying
wavelengths. The fluorescence measured is the amount and type of cells present
in a sample Ananya, M., 2014.

The advantages for
this technique in terms of heterogeneous
cell populations, it can analyze the subpopulations in a few minutes, data
produces are also detailed, highlighting any non-uniformity and
takes off any debris when providing the final data and can
measure large numbers of cells Martin,
C., 2017.

 

 

 

 

 

Applications of this technique in immunophenotyping of peripheral blood cells
includes the traffic of the B-cell subsets
between tissues through peripheral blood reflects the immune status of an
individual and potentially disorders like autoimmunity and lymph proliferative
diseases can be detected Artjoms, S., 2011.

For analysis of apoptosis,
quantification of apoptosis by microscopy is difficult. Flow cytometry
binds FITC-labeled annexin V to phosphatidylserine which exposed on the surface
of apoptotic cells so that easy to quantify apoptosis Crowley, LC., 2016.

Lastly for detection of cytokines,
flow cytometry detects intracellular cytokines
using specific fluorescence-labeled antibodies. It also allows for analyzing
the biological function of cytokines Qiu, JG., 2014

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fourth technique is Immunohistochemistry which uses
antibodies to test for certain antigens (markers) in a sample of tissue. The
antibodies are linked to an enzyme or a fluorescent dye. When the antibodies
bind to the antigen in the tissue sample, the enzyme or dye will be activated,
and the antigen can be seen under a microscope National Cancer Institute, 2018. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure
11: Immunohistochemistry Staining Equipment BioGenex, 2017

 

 

 

 

 

 

 

 

 

 

 

 

Principle for this technique is
that it is developed from the antigen-antibody binding reaction that visualizes
distribution and localization of specific antigen or cellular components in
separated tissues, or tissue sections. Fluorescent or chromogenic
signal for protein detection can be produce by direct or indirect detection
methods. Direct detection is when the primary antibody specific for the target
molecule is directly labeled while indirect detection uses an unconjugated
primary antibody Novus Biological, 2018.

 

Major
components in immunohistochemistry includes primary antibody binds to specific
antigen, the antibody-antigen complex is formed by incubation with a secondary,
enzyme-conjugated, antibody and lastly, with the presence of substrate and
chromogen, the enzyme catalyzes to generate colored deposits at the sites of
antibody-antigen binding Novus Biological, 2018.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 12: Immunohistochemistry Process ImmunohistochemistryUS, 2018.

 

 

 

 

 

The Advantages for
fluorescent detection include easy multiplexing, better target co-localization,
higher dynamic range and fewer steps. For chromogenic detection, it has greater
sensitivity and longer lasting signal Novus
Biological, 2018

 

For applications, this technique involves in Prognostic markers in cancer. Physicians diagnose cancer as
benign or malignant by using specific tumor markers. This technique can also
determine the grade and stage of a tumor, and identify the cell type and origin
of a metastasis to find the site of the primary tumor Jeyapradha, D., 2012. 

For genetics, it determines the function of specific gene products in fundamental biological
processes such as apoptosis and development. A custom made monoclonal antibody
was used against p53 homologue of the pro-apoptotic pathways of p53 was
identified Jeyapradha, D., 2012. 

Lastly is Tumors of
uncertain histogenesis which diagnose tumors of uncertain
origin, primary as well as metastatic from unknown primary tumor. A panel of
antibodies is chosen to resolve such diagnostic problem cases. Jeyapradha, D., 2012.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Last technique is Tissue in Situ Hybridization. Before this, researches are focus on the identification of
specific DNA targets using RNA or DNA labeled with radioisotopes.  Now, the use of the in situ hybridization
(ISH) technique is popular. The use of interphase FISH in routinely formalin
fixed paraffin-embedded (FFPE) tissue to study cytogenetic abnormalities has
become common Mary, H., 2009. 

 

Types of Probes used are DNA Probes which detect DNA targets. They are generated by the cloning and
amplification of specific sequences of DNA or cDNA Mary, H., 2009. Second is RNA Probes which detect RNA in tissue sections. It is known as
Riboprobes which generated by in vitro transcription from plasmids containing
the sequence of interest Mary, H.,
2009.
Next is Oligodeoxynucleotide
probes which are short sequences of DNA generated
on an automated DNA synthesizer Mary,
H., 2009. Lastly
is PNA (polypeptide nucleic acid) Probes which is a DNA analogue that
forms very stable duplexes with complementary DNA or RNA sequences Mary, H., 2009. 

 

Figure 13: FISH Digital Scanner Leica Biosystem, 2018

 

 

 

The principle for this technique is that it identifies where mRNAs are present in a fixed tissue samples and detect nucleic acid sequences in solid neoplastic and infectious
conditions. Furthermore, it also detects microRNA in cytological preparations
and tissue sections recently Mary, H.,
2009. 

 

 

 

 

 

 

 

 

 

 

 

 

Figure
14: FISH Techniques Steps VeterianKey, 2017.

 

 

 

 

 

 

 

 

 

 

 

 

Figure
15: FISH using oligonucleotide probes VeterianKey, 2017.

 

The advantages is detect protein and  mRNA of interest or cells phenotype, detect
more than one nucleic acid sequences using different labeling methods, enables
maximum use of tissue that is difficult to obtain (embryos and biopsies) and
perform hundreds of different hybridizations on the same tissue Ellen, J.,
2014.

 

Applications for this technique are in Genomic abnormalities in cancer which provides information on the
locations of important cancer genes and can have clinical use in diagnosis and
cancer classification. It also
applied for monitoring the progression of tumors Bassem,
A., 2006.

It also involves in Submicroscopic aberrations. Prader–Willi syndrome (PWS) is a paternal structural
abnormality involving 15q11-13, while a maternal aberration in the same region
causes Angelman syndrome. These small aberrations can detected using array CGH Hassan, M., 2016. 

Lastly
is in prenatal genetic diagnosis which uses array CGH in
preimplantation genetic screening is becoming an increasingly popular. It has
the potential to detect CNVs and aneuploidy in eggs, sperm or embryos which may
contribute to failure successfully implant or miscarriage Evangelidou, P., 2013.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Conclusion

 

As a conclusion, advanced techniques
that are available right now are very advantageous and can replace the old
technique. However, there are also drawbacks in the advanced techniques.
Manufacturers and researches can make a team to improve the advanced techniques
so that it can make improve the diagnosing quality in future to benefits the
patients.

 

Furthermore, scientist can make a
new technique that combines the old technique and advanced technique to produce
a new technique that benefits in diagnosis and research because the old
techniques has many advantages also.

 

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