Introduction: become more closely involved in the
Introduction:In recent years, cellular pathology hasbecome more closely involved in the direct management of patients with theintroduction of molecular technologies and targeted therapies. Through this, wehave seen the introduction of specialist pathology. These concepts and the keytechnologies that are influencing clinical practice today have been introduced.
It showed that how clinical practice has been affected by these respectivetechnologies and how further development will influence the practice anddelivery of cellular pathology, which will impact on the patient throughtargeted therapeutics and diagnostics. Discussion:There are many types of advanced techniquesin cellular pathology.Digital(virtual) microscopy:Automated microscopes represent a formof hybrid technology in that they bring together various components within theindustry to form a device capable of creating virtual slides without beingdedicated to that purpose. High-specification microscopes are the startingpoint for this technology and essentially combine imaging (CCD) andcomputational technology to produce the device. Image acquisition as in thesystems described above is realized via a digital camera. The CCD chip acquiresthe centre of the field given by the objective, thus reducing opticalaberration to a point considered negligible. The Olympus DotSlide system isessentially an automated microscope (Olympus upright BX research microscope)that is computer-driven over the slide to form an image from a Peltier-cooled1379 × 1032 pixel camera.
Withthe virtual slides, the operator can defined accurate protocols and runefficient algorithms on whole slides or specified area. DNAprofiling:That is 0.1% of the genomes in 3,000,000base pairs of nucleotide are unique in all human being. This speciality in thebase sequences occur in repetitive DNA also known as satellite DNA as well as ingenes. Various small peaks are formed on the DNA which gives rise topolymorphism 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 numberVNTRs at a particular area of the DNA of the child will be different may be dueto 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:DNAprofiling, as already indicated, has application in a broad cross section ofdisciplines, including human forensic science, diagnostic medicine, familyrelationship analysis, animal and plant sciences, and wildlife forensicscience. DNA profiling is applicable in a number of areas in medicine including twin zygosity testing , bone marrowtransplantation marker analysis, detection of DNA changes in tumors, indicationof possible contamination of fetal by maternal tissue in chorionic villusanalysis, pathogen identification and paternitytesting where family studies are being performed for antenatal diagnosis ofinherited diseases.Polymerase chain reaction (PCR):Principle:The primer mediated enzymatic amplification of DNA is the principle ofPCR.
The ability of DNA polymerase to synthesize new strand of DNAcomplementary to the offered template strand are by using PCR. DNA polymerasecan add a nucleotide only onto a preexisting 3?-OH group to add the firstnucleotide, so the primer is needed. DNA polymerase then elongated its 3 end byadding more nucleotides to generate an extended region of double stranded DNA.Applications:There are many applications of PCRincluding to test the presence of genetic disease mutation such as cysticfibrosis, hemoglobinopathies or other inborn errors of metabolism. It can usedto study the alteration to oncogene that causes cancer. It is also a tool usedin genetic fingerprinting.
In a crime investigation, there may only be tiny DNAsample 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 abeam of light of at least one specified wavelength and detect the fluorescenceemitted by the excited fluorophore are carried out by real-timePCR. Advantage of the physicochemical properties of the nucleic acids and DNA polymerase is also able to quick heat and cool samples.Applications:Thetool of choice for the rapid and sensitive determination and quantitation ofnucleic acid in various biological samples is real-time PCR assays. The detectionof genetically modified organisms in food, gene expression analysis and cancerphenotyping are main applications of real-time PCR.
RT-PCR assays are widelyused for the quantitative measurement of gene copy number (gene dosage) intransformed 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 environmentalconditions or drug treatment, by measuring changes in cellular mRNA levels.Spectralkaryotyping:Spectralkaryotyping (SKY) is a hybridization-based diagnostic technique originallydeveloped to diagnose chromosomal aberrations associated with cancer andgenetic disease.
Specific inter and intra-chromosomal genomic rearrangements,and unambiguously determine both the total number and individual identity ofall chromosomes in a metaphase nucleus can be detected by using SKY. Sky Paint (AppliedSpectral Imaging) is hybridized to metaphase chromosome spreads from the cellsof interest. Sky Paint is a mixture of probes specific to single chromosomes,each of which contains a spectrally unique combination of fluorescentnucleotides thus allowing the user to “paint” each chromosome a differentcolor. After acquiring a metaphase spread image using a microscope equippedwith an interferometer that reads emissions across the entire visible spectrum,individual chromosomes are assigned using SkyView software. SkyView analyzes the spectral imagein two dimensions and displays each chromosome with a distinct classificationcolor from which it creates a karyotype table.DNA microarrays:DNAmicroarrays (sometimes called DNA chips) are in general characterized by astructured immobilization of DNA targets in the free nucleic acid samples onplanar solid supports, on which different types of nucleic acids with knownsequences (known as “probes”) are fixed.
A probe may be derived fromcomplementary DNA (cDNA), polymerase chain reaction (PCR) products, orsynthetic oligomers. Commonly, applications of DNA microarray technologybroadly include gene expression analysis (transcription analysis), whichanalyzes the transcriptional activity of genes through hybridization betweenDNA targets and probes; genotyping with oligonucleotide arrays, which is basedon the notion of combining the complete sequence of a DNA sample by presentingall possible sequences as a complement on the chip; measurement of enzymeactivities on immobilized DNA, which is based on the finding that DNA-modifyingenzymes are capable of acting on immobilized DNA templates or oligonucleotides;transcription on chip, which shows the transcription of a complete gene intomRNA on the chip.FISHis a technique used to identify the presence of a single nucleic acid sequence(often specific to a particular chromosome) through hybridization of fluorescentlylabeled DNA probes to denatured chromosomal DNA in cytological material.Interphase nuclei are hybridized with the FISH probe, though metaphase spreadscan be used as well. FISH probes can be purchased commercially. By using FISH, acopy of aberrations numbers as well as specific cytogenetic abnormalities canbe sketched and enumerated because it can easily detected chromosomal micro-deletion,amplification, and subsequently translocation. Compare with cytogeneticmetaphase karyotype analysis, FISH is less time consuming detect and monitorthe specific therapy with regards to the gene abnormalities.
Flow cytometer:The flow cell is where the cellularinterrogation takes place, the objective being very simple: to allow the cellof interest to pass through the laser (or lasers) interrogation point and thenfor the product of that interrogation to be displayed as physical andfluorescent properties of that cell.Major applicationwhere lowcytometry is used include the diagnosis and subclassification of acuteleukaemia and chronic lymphoproliferative disorders, including chroniclymphocytic leukaemia and non-Hodgkin lymphoma, HIV monitoring and DNAanalysis. Microarray-basedComparative Genomic HybridizationArray CGH is based on the sameprinciples as metaphase CGH, a technique that has been extensively used for thegenomic characterization of a number of solid tumours. Both techniques allowthe study of DNA copy-number alterations genome-wide, except that the targetsfor hybridization are mapped clones in the aCGH technique instead of chromosomesas in metaphase CGH.
Propidium iodide, phycoerythrin and fluorescein are thecommon dyes to used although many other dyes are available. Furthermore, thefrequency of the recurrent amplification and the specific tumour subgroup whereit is most prevalent can also be established in this manner. By interrogatingthe genome to identify critical molecular drivers in cancer, aCGH offers themeans to identify the therapeutic target and hence the appropriate biomarkerassay as well in the process of drug development.
Tissue insitu hybridization (ISH) The basic requirements of a probe foruse in tissue in situ hybridization are that it is complementary to the targetnucleic acid sequence and can be labelled in such a way as to allow microscopicvisualization of the hybrid formed. The different types of probe include thefollowing. Double-stranded DNA probes are most commonly used to detect DNAtargets. They are generated by the cloning and amplification of specificsequences of DNA or cDNA, derived by reverse transcription of mRNA employingvectors (bacterial plasmids and cosmids). Single-stranded RNA probes, so-calledriboprobes, are most commonly used to detect RNA in tissue sections. Riboprobesare usually generated by in vitro transcription from plasmids containing thesequence of interest. The plasmids are designed with promoter sites for RNApolymerases (e.g.
T3, T7 and SP6) and can produce probes complementary to thetarget RNA sequence (antisense) or identical to the target sequence (sense). Tissue ISH is used to detect nucleicacid sequences in a wide variety of solid neoplastic and infectious conditionsand is becoming a crucial theranostic tool, helping to guide therapy byidentifying 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, haspositioned tissue ISH as a central tool in diagnosis and predictive therapy.Molecular diagnostics including tissue ISH are widely used in the areas ofinherited genetic disorders and infectious diseases, as well as haematologicand solid tumours.
In addition, tissue ISH has a role to play in guidingappropriate therapy.Lasercapture microdissection (LMD):Atechnique for isolating specific and pure targets from microscopicheterogeneous samples for downstream analysis (DNA, RNA & proteins) isknown as laser microdissection (LMD). It is based onmicroscopic imaging and utilizing a laser. In contrast to other systems whichuse a fixed laser focus for dissection, Leica Microsystems’ LMDsystems guide the laser focus for dissection. This unique feature allows highlyprecise laser dissection independent of the stage accuracy.
LCM is broadly usedin many medical research areas from neuroscience, forensic science research tobiomarker discovery, cancer and clinical diagnostics. Molecular biology such asgenetics and proteomics are the mainly applications. Many genetic materialssuch as DNA, mRNA, and microRNA, can be extracted from tissues.
Neural stem cell therapy is another interestingexample of LCM application.