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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5539_Библиотеки_им_академика_М_И_Перельмана.pdf
Introduction
7
PCR-Amplified Immunoassay
This technique combines the versatility of ELISA and the high sensitivity
through amplification of nucleic acids in PCR. This method is useful
in early detection of infectious diseases by virus, bacteria and parasites
through their antigens (Mehta, Raj, Singh, & Khuller, 2014).
Histological Analysis
It is the study of patient tissues (thin film) under a microscope. The
histological analysis of tissues using different staining procedures
such as PAS, H&E, Azan or EvG can diagnose scars, pathological
vascular conditions, fibrosis, myocardial cell death, inflammatory cell
differentiation, granulomas, disarrays, and cardiomyocytes changes.
It can also detect storage disorders including iron deposits, glycogen,
amyloidosis and similar problems. Optional analysis by electron
microscope in combination with the histological analysis is also possible.
Immunohistochemical Examination on Intramyocardial
Inammation
This is based on using primary antibodies which are application-specific
on cryo-fixed tissues. This is followed by use of secondary antibody
conjugated with a particular enzyme complex to detect the coupled
primary antibody. The enzyme complex linked to the secondary antibody
gives a colored complex that precipitates out when a staining solution
is used. This can be measured using digital applications and image
analyzing software to have an accurate diagnosis (Noutsias et al., 2002).
Autoimmunity Testing by Indirect Immunouorescence
It is a chronic inflammation mediated by the immune system on the
myocardial tissue. Auto-antibodies are generated by inducing infection
of the heart muscle with the help of intramyocardial inflammation or by
viruses. This analytical method enables simultaneous screening of various
autoantibodies and often uses monkey tissues resembling close to human
tissues. The patient sera are reacted with the reference presented (autoantibodies exist against individual auto-antigens) (Willitzki et al., 2012).
Fluorescence microscopy is used to make the immunological analysis.
Advances in Molecular Diagnostics
8
Profiling Techniques for Gene Expression Systemic
Diagnosis
Myocardial infection or intramyocardial inflammation often goes
unnoticed in biopsies. To overcome this problem a stable and global
biomarker is needed to identify infections in the affected organ.
A god way to overcome this problem is to have quantitative PCR for
miRNAs or gene expression. This proling assay has high sensitivity
along with a broad dynamic detection range. Set-ups based on microplate
and low-density QPCR arrays are suitable diagnostic approaches to have
expression panels that can analyze 50 analytical parameters (Grigorenko,
Ortenberg, Hurley, Bond, & Munnelly, 2011).
Molecular Virology as Detection Tool
PCR-based methods are used successfully to determine, quantify and
sequence microbial genomes. Protocols consisting of nested-PCR with
two PCR assays are done sequentially using the amplicon from the first
assay as a template in the second. This is a highly sensitive process for
the detection of viral genomes with low copy numbers. The amplified
PCR products are separated by an agarose gel electrophoresis containing
ethidium bromide (intercalating dye) in the buffer which allows the
visualization of PCR product generated using UV fluorescence. The
positive PCR products are checked for quality control by sequencing and
analyzed for manual alignment with the existing NCBI database. This
confirms the subtypes and variants of the corresponding virus strains
present (Kühl et al., 2008).
Fluorescent In-situ Hybridization (FISH)
In FISH fluorescence-labeled oligonucleotides are attached specifically
to complementary DNA sequences which are the targets on the genome.
This way the target region acquires fluorescence color (such as acridine
orange, FITCI green or Texas red) enabling easy visualization using
fluorescence microscope. There are three types of probes currently
available:
• Painting probes which attach to the overlapping sequences
of the target and can recognize the whole chromosome.
Introduction
9
This is why they are called “painting” as it paints the whole
chromosome with the color of the chosen uorescence.
• The probes that can recognize only the centromeric region
of any particular chromosome is known as centromeric
probes. This probe helps in enumerating copy numbers of a
chromosome even when it is in the nondividing interphase
state.
• Probes those are allele-specic. These probes tend to adhere a
particular target in allele sequence (for example the HER2/neu
oncogene or the p53 tumor suppressor gene).
This technique has great advantages over the other conventional
methods involving the study of gene amplications and chromosomal
translocations and deletions (Min & Swansbury, 2003). Conventional
techniques of cytogenetics are elaborate needing time-consuming steps
of cell culture. They can only be made using fresh samples of tissues
whereas FISH can be done with dividing or resting cells (metaphase
and interphase), both freshly frozen or archive smears or tissue sections
embedded in parafn.
Thus this technique offers great versatility as well as a topographic
advantage because of the examination by uorescent microscopy. This
distinctly allows differentiation between tumorous and nontumorous cell
signals (Truong et al., 2003).
Spectral Karyotype Imaging
This method was introduced by Schrock et al (Schrock, et al, 1996),
where they used painting probes for all the 23 sets of chromosomes.
Every probe is labeled with 3 fluorescent dyes with varying proportions.
This provides a unique spectral emission of light for each labeled
chromosome. Using the “interferometer” like the ones employed by the
astronomers to distinguish light spectral emission from different stars
is a critical component of this method. It can detect even the slightest
variations in color that remains undetected by the naked human eye and
the computer programming redesigns it to easily distinguishable visual
colour for each chromosome pair.
Currently, this technique is used to complement conventional
methods of cytogenetics. It is used to study cells at the metaphase
10
Advances in Molecular Diagnostics
stage (dividing cells). The karyotype of cells aligned as per numeric
order and chromosome pair color is portrayed on the digital display
allowing pathologists to identify readily the numerical abnormalities
in chromosomes (aneusomy) or any irregularities/ shifting in colored
portions of chromosomes (translocations).
Earlier indecipherable complex tumor cell translocations (for
example, cells of breast cancer) are difcult to resolve, but with SKI they
can be determined easily enabling “marker chromosome” analysis which
is composed of different chromosome amalgam fragments.
DNA Microarrays
This method has great promise in profiling gene expressions in molecular
diagnosis by allowing simultaneous evaluation of thousands of genes’
expression rate in one assay for a specific sample. There are two types of
DNA microarrays, which are in use widely: the cDNA microarrays and
the oligonucleotide or DNA chips.
Here complementary DNA sequences from an mRNA library
consisting thousands of genes are placed mechanically on one glass
slide. These immobilized cDNA sequences work as the anchoring probes
where the extracted mRNAs from the test samples will bind specically
at the time of hybridization.
Figure: Outline of DNA Microarray.
Image adapted from http://www.acfs2000.com/pgd-23-chromosome-microarray-why-its-done.html.
Introduction
A uorescent dye is tagged with the tested mRNA and uorescence
intensity, location at every anchoring probe will proportionally represent
the total mRNA (degree of expression) of that gene present at that
location. The uorescence intensity is displayed in a grid in a microarray
reader as a colored dot for every gene location (Pusztai, et al., 2003).
DNA microarrays is used in diagnosis of genes relevant to diseases
by genotyping and in detection of disease causing agents, analysis
of mutation, screening of SNPs, chromosomal abnormalities and
posttranslational modication (Hamels et al., 2001; Naqvi, Goldfarb,
Hanmer, & Bryant, 2016).
11
Assessment of Antimicrobial Resistance by Genetic
Techniques
Rather than the conventional method of susceptibility the genetic
methods to determine antibiotic resistance offer advantages. They
include rapid and more reliable assessment; can be done directly using
clinical specimens (no need to isolate from the organism after culturing);
here the organism’s genotype is assayed instead assessing the phenotype
or genotype expression in the laboratory conditions in conventional
techniques. Additional advantages are that it can evade the problem of
slow growth of organisms in conventional process (as genotypes may
discern earlier than phenotypes); many organisms may not be cultured in
laboratory conditions and genetic assessment is vital for these organisms;
genetic method is also less hazardous (no biohazard production) compared
to conventional test processes (Su, 2002).
CONCLUSIONS
In recent years many molecular technologies have made their projected
transitions into the diagnostic arena for clinical purposes. In fact,
molecular diagnostics is now a fundamental part of every clinical
practice. The advents of advanced molecular diagnostic techniques,
detection of diseases (particularly those related to mutations and immune
responses) have become more accurate and faster at a lower cost. PCRbased techniques are highly popular and the detection choice depends on
various factors such as the spectrum of the disease, sample volume, and
12
equipment available in the diagnostic center. These diagnostic approaches
have improved the process of diagnosis and monitoring various diseases
and further helped in timely initiation of proper therapeutic procedures.
These also minimize the sampling errors and improved early detection.
Advances in Molecular Diagnostics
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