Forte Diagnostics functions Becomes the Only Medical Laboratory who can cater all the latest and cutting edge Investigations and Technology in Sri Lanka.

Forte Diagnostics has a sophisticated and spacious laboratory that is spread over a total area of 5,000 sq.ft in a prime location. It has a team of dedicated, qualified and experience professionals who are competent to handle wide range of specialized tests Over 10,000 parameters can be processed per day, indicating the volume of operations. Forte Diagnostics is equipped with highly advanced and state-of-the-art technology & analyzers to process samples in various departments.

TEST PRINCIPLE

Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies (millions or billions!) of a particular region of DNA. This DNA region can be anything the experimenter is interested in. For example, it might be a gene whose function a researcher wants to understand, or a genetic marker used by forensic scientists to match crime scene DNA with suspects.

 

Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or clonedinto a plasmid for further experiments.

 

PCR is used in many areas of biology and medicine, including molecular biology research, medical diagnostics, and even some branches of ecology.

As the name suggests, real time PCR is a technique used to monitor the progress of a PCR reaction in real time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified. Real Time PCR is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds. This occurs due to the accumulation of the PCR product with each cycle of amplification. These fluorescent reporter molecules include dyes that bind to the double-stranded DNA (i.e. SYBR® Green ) or sequence specific probes (i.e. Molecular Beacons or TaqMan® Probes). Real time PCR facilitates the monitoring of the reaction as it progresses. One can start with minimal amounts of nucleic acid and quantify the end product accurately. Moreover, there is no need for the post PCR processing which saves the resources and the time. These advantages of the fluorescence based real time PCR technique have completely revolutionized the approach to PCR-based quantification of DNA and RNA. Real time PCR assays are now easy to perform, have high sensitivity, more specificity, and provide scope for automation. Real time PCR is also referred to as real time RT PCR which has the additional cycle of reverse transcription that leads to formation of a DNA molecule from a RNA molecule. This is done because RNA is less stable as compared to DNA

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies and hormones. In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measureable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.

 

ELISA’s are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISA’s so easy to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from non-bound material during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.

Fluorescent in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes to investigate the presence of small, submicroscopic chromosomal changes that are beyond the resolution of karyotype analysis.

Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies. These technologies allow for sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, and as such revolutionized the study of genomics and molecular biology. Such technologies include:

Illumina (Solexa) sequencing

Illumina sequencing works by simultaneously identifying DNA bases, as each base emits a unique fluorescent signal, and adding them to a nucleic acid chain.

Roche 454 sequencing

This method is based on pyrosequencing, a technique which detects pyrophosphate release, again using fluorescence, after nucleotides are incorporated by polymerase to a new strand of DNA.

Ion Torrent: Proton / PGM sequencing

Ion Torrent sequencing measures the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase and therefore differs from the previous two methods as it does not measure light.

MLPA (Multiplex Ligation-dependent Probe Amplification) is a multiplex PCR method detecting abnormal copy numbers of up to 50 different genomic DNA or RNA sequences, which is able to distinguish sequences differing in only one nucleotide (1). The MLPA technique is easy to use and can be performed in many laboratories, as it only requires a thermocycler and capillary electrophoresis equipment. Up to 96 samples can be handled simultaneously, with results being available within 24 hours. Although for most hereditary conditions, (partial) gene deletions or duplications account for less than 10 % of all disease-causing mutations, for many other disorders this is 10 to 30 % (2-8) or even higher still (9, 10). The inclusion of MLPA in clinical settings can therefore significantly increase the detection rate of many genetic disorders.

Chemiluminescence (CL) is defined as the emission of electromagnetic radiation caused by a chemical reaction to produce light. Chemiluminescence immunoassay (CLIA) is an assay that combine chemiluminescence technique with immunochemical reactions. Similar with other labeled immunoassays (RIA, FIA, ELISA), CLIA utilize chemical probes which could generate light emission through chemical reaction to label the antibody. In recent years, CLIA has gained increasing attention in different fields, including life science, clinical diagnosis, environmental monitoring, food safety and pharmaceutical analysis because of its high sensitivity, good specificity, wide range of applications, simple equipment and wide linear range.

Flow cytometry is a popular cell biology technique that utilizes laser-based technology to count, sort, and profile cells in a heterogeneous fluid mixture. Using a flow cytometer machine, cells or other particles suspended in a liquid stream are passed through a laser light beam in single file fashion, and interaction with the light is measured by an electronic detection apparatus as light scatter and fluorescence intensity. If a fluorescent label, or fluorochrome, is specifically and stoichiometrically bound to a cellular component, the fluorescence intensity will ideally represent the amount of that particular cell component.

 

Flow cytometry is a powerful tool because it allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. This makes it a rapid and quantitative method for analysis and purification of cells in suspension. Using flow, we can determine the phenotype and function and even sort live cells.

 

FACS is an abbreviation for fluorescence-activated cell sorting, which is a flow cytometry technique that further adds a degree of functionality. By utilizing highly specific antibodies labeled with fluorescent conjugates, FACS analysis allows us to simultaneously collect data on, and sort a biological sample by a nearly limitless number of different parameters. Just like in conventional flow cytometry, forward-scatter, side-scatter, and fluorescent signal data are collected. The user defines the parameters on how cells should be sorted and then the machine imposes an electrical charge on each cell so that cells will be sorted by charge (using electromagnets) into separate vessels upon exiting the flow chamber. The technology to physically sort a heterogeneous mixture of cells into different populations is useful for a wide range of scientific fields from research to clinical. Nowadays the terms “flow cytometry” and “FACS” are often used interchangeably to describe this laser-based biophysical technique. The diagram below illustrates the experimental setup and general procedure of a typical FACS experiment. A population of mixed cells is sorted into a negative sample and a positive sample containing cells of interest by the flow cytometer.

In 1977, Frederick Sanger developed a new method for DNA sequencing based on the chain termination method, where nucleotides in a single-stranded DNA molecules are determined by complementary synthesis of polynucleotide chains, based on the selective incorporation of chain-terminating dideoxynucleotides driven by the DNA polymerase enzyme . For this method, Sanger was awarded in 1980 with a second Nobel Prize in Chemistry, and nowadays this method is still known as the Sanger method of DNA sequencing, becoming a standard method in clinical genetics. The present opinion article wants to remark that, targeted SSM is still effective in specific clinical scenarios at a lower cost as a diagnostic method compared to new technologies for sequencing, one example is the detection of Andersen-Tawil syndrome (ATS).

Applications are those in which fluorescent fragments of DNA (produced by PCR using primers designed for a specific experiment) are separated using capillary electrophoresis and sized by comparison to a size standard.

Most fragment analysis applications have simplified workflows—straightforward sample preparation (without the purification required for sequencing samples), ability to multiplex, and applications can all be separated using the same capillary electrophoresis polymer.

Karyotyping is the process of pairing and ordering all the chromosomes of an organism, which gives a genome wide idea of any individual’s chromosomes.

Chromosome analysis is a labor-intensive endeavor that requires the culture of cells, the arrest of the mitotic cell cycle in the metaphase stage, and special staining, which bands each chromosome so that chromosome pairs can be distinguished from one another.

Immunohistochemistry (IHC) is a method for detecting antigens or haptens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. The antibody-antigen binding can be visualized in different manners. Enzymes, such as Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP), are commonly used to catalyze a color-producing reaction.

 

IHC is widely used in many research and clinical laboratories because this technique makes it possible to visualize the distribution and localization of specific cellular components within cells and in proper tissue context. There are numerous IHC methods that can be used to localize antigens. The method selected should include consideration of parameters such as the specimen types and assay sensitivity.

Immunofluorescence (IF) microscopy is a particularly robust and broadly applicable method generally used by researchers to assess both the localization and endogenous expression levels of proteins of interest. Immunofluorescence microscopy is a widely used example of immunostaining and is a form of immunohistochemistry based on the use of fluorophores to visualize the location of bound antibodies. The effective application of this methodcomprises several considerations, including the nature of the antigen, specificity and sensitivity of the primary antibody, properties of the fluorescent label, permeabilization and fixation technique of the sample, and fluorescence imaging of the cell. Although each protocol will require fine‐tuning depending on the cell type, the antibody, and the antigen, there are steps common to nearly all applications. For more information see our technical tips for successful IF microscopy.

 

Immunofluorescence can be used on tissue sections, cultured cells or individual cells that are fixed by a variety of methods. Antibodies can be used in this method to analyze the distribution of proteins, glycoproteins and other antigen targets including small biological and non-biological molecules.

The electron microscope is a type of microscope that uses electrons to create an image of the target.

 

It has much higher magnification or resolving power than a normal light microscope.

 

Although modern electron microscopes can magnify objects up to two million times, they are still based upon Ruska’s prototype and his correlation between wavelength and resolution.

 

The electron microscope is an integral part of many laboratories.

Researchers use it to examine biological materials (such as microorganisms and cells), a variety of large molecules, medical biopsy samples, metals and crystalline structures, and the characteristics of various surfaces.