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Cellularpathology is the branch of pathology that involves the study of body organs andtissues which are also known asanatomical pathology. Its roles include determining the cause of certaindiseases and the effect that they are having on the body, assisting with thechoice of treatment that will be given, aiding in giving a prognosis anddetermining what may have caused a person’s death. There are two mainsubdivisions within cellular pathology which is histopathology and cytopathology Lab Tests Online UK,2017.

 In recent years, cellular pathology has become more closely involved inthe direct management of patients. This report shows how clinical practice hasbeen affected by these respective technologies and how further development willgive impact on the patient through targeted therapeutics and diagnostics MaryHannon, 2009. Advanced techniques in diagnostic cellular pathology that is explainedin this report are virtual microscopy, cytopathology, flow cytometry, immunohistochemistryand tissue in situ hybridization.                DISCUSSION Basic Techniques in Diagnostic Cellular PathologyBeforethe advanced techniques are widely used, some basic techniques are commonlyused. First is gross examination which is examination of organs and tissues macroscopically to selectrelevant portions for microscopic examination. It can be accurately made in 90 % of specimens while theremaining 10 % is depending on the pathologist’s skill. However, thepathologist’s skills are rapidly declining thus lower the accuracy and precisionGeller,SA., 2014.

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 Secondis histopathology technique which includes fixation,embedding, and staining. Fixation is to preservetissues permanently. Tissue embedded in paraffin to be made into thinmicroscopic sections and microtome will cut into sections.

Staining uses avariety 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 electronsrather than visible light to magnify the cells in a tissue sample which allowsmuch greater magnification, enabling the visualization of organelles within the cells Lab TestsOnline UK, 2018.

              Molecular TechniquesMolecular pathology focuses on the diagnosisand 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 anextremely small amount of genetic starting material which usually performed ona variety of fresh specimens Bethesda, 2004. Second is Real-time PCR which detects pathogens in the research setting and diagnostic settings. It combines PCR chemistrywith either a fluorescent probe or DNA detection dyes such as Sybr green                                                                            Sumathi,S., 2012.

 Third is Spectralkaryotyping which uses 23 sets ofchromosome-specific “painting” probes to identify chromosomal abnormalities George, J., 2003. Next is Microdissection of tissue sections andcytological preparations which has been used increasingly for the isolation ofhomogeneous since it allows precise examination Fend, F., 2000. Lastly is Forensic pathology focuses on the causes of death as opposed toillnesses and their cures or treatments. A forensic pathologist must remove anextremely thin section of the tissue for an autopsy to study the areas ofbody tissue Mary Hannon, 2009.

                 Advanced Techniques in Diagnostic Cellular PathologyFor the past recent years, manybasic techniques for cellular pathology diagnosis have been replaced withadvanced techniques which have many benefits.  First technique is Virtual microscopy which is the newtechnique which replaces telepathology system. It utilizes digitization ofwhole-microscopic glass slides via computer-aided systems to produce virtualmicroscopic 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 anypart of the tissue section at any magnification Ngozi, N., 2017. Equipments of virtual microscopy are automated microscopes which is a High-specificationmicroscopes combine imaging and computational technology to produce the device Mary, H.

, 2009.         Figure 1: AutomatedMicroscope Mary, H., 2009.           Besides that is Scannerssuch as Progressive scan CCD systems which has capability for slide observation and allows digital imagecapture. It additionally has Internet communications capabilities which are themajor 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 aslide capacity of 120 slides and is suitable for environments such ashospitals, labs and research organizations. Itcreated an alternative way to tiling, termed ‘line scanning’ which accuratelymoves the slide under a line-scan camera to acquire the image Mary, H., 2009.          Figure 5: Aperio ScanScope scanning system Mary, H.

, 2009.    Applications ofvirtual microscopy in diagnosis ofdisease is that it has very high interest, forexample, in cancer biomarker expression quantification (e.g. HER2/NEU) whichimportant for both patient health and financial considerations Mary, H.

, 2009. For education, traditionally it has been based onprinted micrographs and projection slides. Now, modern textbooks provide asupplemental CD with digital images. This enables viewing of any part of aspecimen at any magnification Mary, H., 2009. Lastlyare research studies.  All studies generallyshow ‘proof of concept’ with small images captured from a traditional CCDcamera. In molecular pathology research, virtualmicroscopy could provide the entire study material viewable and it would bevaluable for authors, journals, and readers.

Lundin, M., 2004                        Secondtechnique 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 thesensitivity and specificity of the cervical smear. One difference between LBCand traditional technique is that the cells are rinsed into a vial of fixative,which allows better preservation of cells so, clearer nuclear staining which isthe main principle for this techniqueMary, H., 2009.  Figure 8: CellProcessing Using LBC Mary, H., 2009.      Advantages ofthis technique is that the Cellularmaterial evenly distributed and spread on the slide, cellular materialwell-preserved, reduces mucus, blood, and exudates on slide, and reducesscreening 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, invasivecancer incidence, make diagnosis accurately and decrease in time taken to obtainthe smear Mahboobeh, S., 2007.

For oral brush, it improves cytodiagnostic accuracy and providesan adequate sample of oral epithelium. LBC also removes most mucus, protein andred cells from the microscope slides, distributes cells evenly, maintainsdiagnostic clusters, reduces scanty preparations and eliminates air-dryingartifacts in oral samples Shwetha, N., 2016      .     Next technique is Flow cytometry. It can be defined as a semi-automatedprocedure for the interrogation of single cells in a continuous fluid stream,enabling the derivation of simultaneous measurements of multiple extra- andintracellular characteristics Mary, H.,2009.

          Figure 10: Flow Cytometer Beckmen,C., 2015 The principlefor this technique involves a passage of cells in single file in front of alaser so they can be detected, counted and sorted. Cell components that arefluorescently labeled are then excited by the laser to emit light at varyingwavelengths. The fluorescence measured is the amount and type of cells presentin a sample Ananya, M., 2014. The advantages forthis technique in terms of heterogeneouscell populations, it can analyze the subpopulations in a few minutes, dataproduces are also detailed, highlighting any non-uniformity andtakes off any debris when providing the final data and canmeasure large numbers of cells Martin,C., 2017.     Applications of this technique in immunophenotyping of peripheral blood cellsincludes the traffic of the B-cell subsetsbetween tissues through peripheral blood reflects the immune status of anindividual and potentially disorders like autoimmunity and lymph proliferativediseases can be detected Artjoms, S.

, 2011. For analysis of apoptosis,quantification of apoptosis by microscopy is difficult. Flow cytometrybinds FITC-labeled annexin V to phosphatidylserine which exposed on the surfaceof apoptotic cells so that easy to quantify apoptosis Crowley, LC., 2016.

Lastly for detection of cytokines,flow cytometry detects intracellular cytokinesusing specific fluorescence-labeled antibodies. It also allows for analyzingthe biological function of cytokines Qiu, JG., 2014                         Fourth technique is Immunohistochemistry which usesantibodies to test for certain antigens (markers) in a sample of tissue. Theantibodies are linked to an enzyme or a fluorescent dye. When the antibodiesbind 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.                  Figure11: Immunohistochemistry Staining Equipment BioGenex, 2017            Principle for this technique isthat it is developed from the antigen-antibody binding reaction that visualizesdistribution and localization of specific antigen or cellular components inseparated tissues, or tissue sections. Fluorescent or chromogenicsignal for protein detection can be produce by direct or indirect detectionmethods.

Direct detection is when the primary antibody specific for the targetmolecule is directly labeled while indirect detection uses an unconjugatedprimary antibody Novus Biological, 2018.  Majorcomponents in immunohistochemistry includes primary antibody binds to specificantigen, the antibody-antigen complex is formed by incubation with a secondary,enzyme-conjugated, antibody and lastly, with the presence of substrate andchromogen, the enzyme catalyzes to generate colored deposits at the sites ofantibody-antigen binding Novus Biological, 2018.                Figure 12: Immunohistochemistry Process ImmunohistochemistryUS, 2018.     The Advantages forfluorescent detection include easy multiplexing, better target co-localization,higher dynamic range and fewer steps. For chromogenic detection, it has greatersensitivity and longer lasting signal NovusBiological, 2018 For applications, this technique involves in Prognostic markers in cancer. Physicians diagnose cancer asbenign or malignant by using specific tumor markers.

This technique can alsodetermine the grade and stage of a tumor, and identify the cell type and originof 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 biologicalprocesses such as apoptosis and development. A custom made monoclonal antibodywas used against p53 homologue of the pro-apoptotic pathways of p53 wasidentified Jeyapradha, D., 2012. Lastly is Tumors ofuncertain histogenesis which diagnose tumors of uncertainorigin, primary as well as metastatic from unknown primary tumor.

A panel ofantibodies 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 ofspecific 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 formalinfixed paraffin-embedded (FFPE) tissue to study cytogenetic abnormalities hasbecome common Mary, H., 2009.

  Types of Probes used are DNA Probes which detect DNA targets. They are generated by the cloning andamplification of specific sequences of DNA or cDNA Mary, H., 2009. Second is RNA Probes which detect RNA in tissue sections. It is known asRiboprobes which generated by in vitro transcription from plasmids containingthe sequence of interest Mary, H.,2009.Next is Oligodeoxynucleotideprobes which are short sequences of DNA generatedon an automated DNA synthesizer Mary,H.

, 2009. Lastlyis PNA (polypeptide nucleic acid) Probes which is a DNA analogue thatforms 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 infectiousconditions. Furthermore, it also detects microRNA in cytological preparationsand tissue sections recently Mary, H.,2009.             Figure14: FISH Techniques Steps VeterianKey, 2017.            Figure15: FISH using oligonucleotide probes VeterianKey, 2017.

 The advantages is detect protein and  mRNA of interest or cells phenotype, detectmore than one nucleic acid sequences using different labeling methods, enablesmaximum use of tissue that is difficult to obtain (embryos and biopsies) andperform 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 thelocations of important cancer genes and can have clinical use in diagnosis andcancer classification.

 It alsoapplied for monitoring the progression of tumors Bassem,A., 2006. It also involves in Submicroscopic aberrations. Prader–Willi syndrome (PWS) is a paternal structuralabnormality involving 15q11-13, while a maternal aberration in the same regioncauses Angelman syndrome. These small aberrations can detected using array CGH Hassan, M., 2016. Lastlyis in prenatal genetic diagnosis which uses array CGH inpreimplantation genetic screening is becoming an increasingly popular. It hasthe potential to detect CNVs and aneuploidy in eggs, sperm or embryos which maycontribute to failure successfully implant or miscarriage Evangelidou, P.

, 2013.                        Conclusion As a conclusion, advanced techniquesthat are available right now are very advantageous and can replace the oldtechnique. However, there are also drawbacks in the advanced techniques.

Manufacturers and researches can make a team to improve the advanced techniquesso that it can make improve the diagnosing quality in future to benefits thepatients. Furthermore, scientist can make anew technique that combines the old technique and advanced technique to producea new technique that benefits in diagnosis and research because the oldtechniques has many advantages also. 

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