Running head: HISTOLOGICAL STAINS 1
Thehistory of Histology indicates that, there have been great changes inthe techniques used for histological staining through chemical,molecular biology assays and immunological techniques collectivelyreferred to as histochemistry. Early histologists used the readilyavailable chemicals to prepare tissues for microscopic studies theselaboratory chemicals were potassium dichromate, alcohol and themercuric chloride to hard cellular tissues.Staining techniques used were carmine, silver nitrate, Giemsa,Trichrome Stains,Gram Stainand Hematoxylinamong others.
Thepurpose of this research was to assess past and current literaturereviews as well as case studies in the aim of informing ways in whichhistological stains have been improved in the modern age.Results from literature review indicated that there has been animprovement in histopathology and histotechnology in stains used.There has been a rising need for efficient, accurate and less complexstaining procedures.Oldstain procedures are still in use today, and many others have beenreplaced with new immunostaining, molecular, non-culture and otheradvanced staining techniques. Some staining methods have beenabandoned because the chemicals required have been medically provento be toxic. The case studies indicated that, in the modern histologya combination of different stain techniques are used to enhance theeffectiveness of the staining process. In the modern histologic as away of improving histological stains, several stains have beenmodified and combined with other stains to improve theireffectiveness.
Keyterms:Fixatives, Histopathology, Histochemistry, Histological staining,Hematoxylin
Histologyis the microscopic study of animal and plant cell and tissues throughstaining and sectioning and examining them under a microscope(electron or light microscope). There are various methods used tostudy tissue characteristics and microscopic structures of the cells.Histological studies are used in forensic investigations, autopsy,diagnosis and in education. In addition, histology is usedextensively in medicine especially in the study of diseased tissuesto aid treatment(Black, 2012).
Histologicalstaining is a series of technique processes undertaken in thepreparation of sample tissues by staining using histological stainsto aid in the microscope study (Anderson, 2011). The process ofhistological staining takes five key stages which involve fixing,processing, embedding, sectioning and staining (Titford, 2009). Greatchanges have been done on techniques used for histological stainingthrough chemical, molecular biology assays and immunologicaltechniques collectively referred to as histochemistry and havefacilitated greatly in the study of organs and tissues(Shostak, 2013).
Ideally,histochemistry rely on frozen tissues that have not been fixed toenhance accessibility to proteins, antigens and the generic materialused in the study of cells and tissues (Titford & Bowman, 2012).In the conventional form, techniques used involve embedding andfixation using wax that improves the morphology of histochemistry(Young, O’Dowd & Stewart, 2010).
Specificaspects in histopathology
Stainingis used to highlight important features of the tissue as well as toenhance the tissue contrast. Hematoxylinis a basic dye that is commonly used in this process and stains thenuclei giving it a bluish color while eosin (another stain dye usedin histology) stains the cell’s nucleus giving it a pinkish stain.However, there are other several staining technics used forparticular cells and components (Black,2012).Staining is a commonly used medical process in the medical diagnosisof tumors in which a dye color is applied on the posterior andanterior border of the sample tissues to locate the diseased ortumorous cells or other pathological cells (Musumeci, 2014). Inbiological studies staining is used to mark cells and to flag nucleicacids, proteins or the gel electrophoresis to aid in the microscopicexamination (Jackson& Blythe, 2013).In some cases, various multiple staining methods are used such asdifferential staining, double staining or the multiple staining(Iyiola & Avwioro, 2011).
Inhistology Fixationrefersto the use of chemicals to preserve the natural tissue structure andmaintain the cell structure from degradation. Mostly, neutralbuffered formalin is used in this case when a light microscope is tobe used to conduct the study. However, when an electron microscope isto be used a glutaraldehyde fixative is used (Anderson, 2011).Fixatives enhance the preservation of tissues and cells through anirreversible process through cross linking proteins. However, whilethe process serves to preserve the structure of the cell for thepurpose of histological studies, it has been found to destroy anddenature proteins rendering them dysfunctional (Young, O’Dowd &Stewart, 2010). Formalin fixation denatures the DNA, miRNA and themRNA tissues and extraction of these components for the purpose ofhistology may lead to flawed results (Anderson, 2011).
Thefixation phase retains the chemical composition of the tissues,hardens the cells or tissues for sectioning and delays degradation(Titford, 2009). In addition, fixatives changes tissue penetrationand influence antigen exposures which may be productive ordetrimental (Iyiola & Avwioro, 2011). These fixatives areadministered in two ways through perfusion and immersion of theprepared tissue. These fixatives are infused in the animals’ bodythrough diffusion. Perfusion is a slower process, require more timeand only one fixative can be used at a time (Shostak,2013).Fixation- There are a number of fixatives in use, but theformaldehyde fixatives are the commonly used (Black,2012).The neutral buffered formalin (NBF) stabilizes amino acids inproteins and offer good tissues and cell structure preservation. Theparaffin-formalin (paraformaldehyde- PFA) is effective inimmunostaining but require to be freshly prepared to enhance itseffectiveness (Iyiola & Avwioro, 2011). The Bouin fixative hasbeen found to be effective in delicate and soft tissues such as smalltissues, embryo and brain tissues (Musumeci, 2014). Bouin fixativeoffers good preservation of nuclei and the glycogen but theirpenetrations is slow and distorts mitochondria and the kidney tissues(Weiss,Delcour, Meyer & Klopfleisch, 2010).
Dehydration:In this step, the aim is to remove water from the selected tissues tosolidify them and facilitate the cutting of thin sections of slidesthin used in light microscopes and thick for electron microscope.Water is removed from the tissues through the dehydration methodthrough ethanol (Shostak,2013).The process is repeated through a hydrophobic clearing substance suchas xylene to remove the alcohol and paraffin wax and the infiltratedagent. Resins are used to enhance cutting of thin sections of thetissues (Titford, 2009).
Embedding:In staining, the process of embedding is done using paraffin wax toenhance easier extraction of cellular structures. In complex cellulartissues plastic resin or wax is used, or combinations of fixativesare used to produce good morphology (Musumeci, 2014). However, thesefixatives may lead to degradation of the cell and tissue structuresdue to prolonged heating, and this may lead to problems whenconducting hybridization process arising from the unstable RNA. Inthe same line, the infiltration of paraffin wax leads to inhibitionof antibody, chemical and penetration of other fixatives. In order toalleviate this problem, freezing of tissues after the embedding,removing wax after staining and the use of PFA fixatives offers areliable solution to improved morphology (Titford, 2009).
Antigensretrieval:This is the next process after fixation and embedding and focuses onretrieving antigens that have been masked. When formalin fixativesare used as well as other aldehyde fixations the cross-linking ofproteins leads to masking of the antigen sites, and this leads toweaker immunohistochemical staining. Antigen retrieval process servesto break protein cross-links and unmask the epitopes and the antigensthat were fixed and embedded using formalin and paraffin (Titford,2009). The overall strategy is to improve on the staining intensityof the antibodies(Cai, Caswell, Prescott, 2014).
Thecommonly used antigen retrieval techniques are through heat-inducedand proteolytic retrieval methods. The proteolysis digestion processshould take the minimal dosage and time possible to avoid overdigestion that may denature the tissue structures and the epitopes(Musumeci, 2014). The heat method leads to protein denaturalizationand in some cases antigens are lost (Black,2012).Similarly, heating may lead to the reversal of the chemicalmodifications induced during the fixation period. Heating from suchdevices as microwaves leads to chemical reactions of the proteinstructure (Shostak,2013).However, a combination of enzymatic and heat retrieval methods leadto effective staining intensity (Godwin,2011).
Inhistology sectioning refers to the preparation of ‘ribbon’ likemicrotomes of a tissue for the purpose of mounting it on a microscopeslide for examination (Cai,Caswell, Prescott, 2014).In this case, a series of thin sections of tissues of requiredthickness are cut and prepared through the paraffin method.
Grossand microscopic examination
Grossexamination is a laboratory procedure in which pathological andmedical examination is done through visible aspects of the eye. Inmicroscopic examinations, pathological changes are done using amicroscope (light or electronic microscope)(Musumeci, 2014).In most aspects, gross examination precedes microscopic examinationlike in the identification of samples for microscopic examination.For instance, gross examination helps the pathologist identify thecells or tissues that have lumps (possibly cancer) but microscopicexamination is used to confirm.
Thisis a histopathological procedure in which antibody-based technique isused to assess the existence of a protein in a section. In this casethe Hematoxylin-Eosin(H&E) stains are used to stain and study cell and tissuestructures (Titford,2009).
Molecularbiology histology Stain
Inthis case antibodies are used as staining components. The methodtargets the enzymes in the cells by staining them so that they areeasily detectable (Musumeci, 2014). In this case, various stains suchas the fluorescent dyes and the alkaline phosphates are used toenhance the visibility of the cells morphology (Shostak,2013).
Inthe modern age of histologic there have been great improvements inhistological stains and techniques. Advanced histological techniquesare immunohistochemistry, antibody binding and electron microscopy(Titford, 2009). In the same line advanced stains includeimmunohistochemical (IHC), routine hematoxylin eosin (H&E) andthe insituhybridization (Musumeci, 2014). Modern stains used are
Masson`s Stain used in Connective Tissues,
Golgi Stain used in Neuronal Fibers
Immunological Labeling that have Fluorescent or Enzymatic stains
Kluver-Barrera Stain used in Lipofuscin
Mallory`s CT Stain
Periodic Acid-Schiff (PAS) Stain used in Carbohydrates
Regulationsof histologic in different countries
Mostcountries have standards and organizations that collaborate withnational and international groups involved in the control andstandardization of biological staining methods. Standardization isimportant in setting uniform criteria, methods and technicalspecifications of the stains used. The objective is to enhanceestablishment of procedures that produce stain substances thatproduce microscopic results capable of been reproducible in differentcountries in areas of cytology, bacteriology, histopathology andhematology(Lyon & Horobin, 2010).
Formalregulatory bodies that standardize stains and are independent ofmanufacturers are International Organization for Standardization(ISO), European Committee for Standardization (CEN) and the AmericanNational Standards Institute (ANSI). Other bodies involved in thestandardization of staining substances are the USA ClinicalLaboratory Standards Institute (CLSI), the World Health Organization(WHO) and the European Diagnostic Manufacturers Association (EDMA)among others. These regulatory bodies accredit, evaluate and approvemanufactured and the use of staining dyes, antibodies, fluorochromesand the nucleic acid probes (Lyon& Horobin, 2010).
Objectivesof the study
Backgroundstudy on commonly used histological staining techniques and stainsindicate that while some fixatives and techniques used in thehistological processes are effective. Some stains and processes areineffective and this leads to denaturalization of tissues and cellswhich inhibit effective histological studies. The objective of thisresearch was to assess past and current literature reviews and casesin the aim of informing ways in which histological stains have beenimproved in the modern histopathology. As a result, this studyfocuses on conducting an extensive and qualitative case study on pastand present histological processes in the aim of understanding howhistological strains could be improved.
Theresearch used an extensive exploration and review of historical,recent and current medical research studies and case studies in orderto collect quantitative and qualitative data in regard tohistological stains used in the past and recent cases (Silverman,2011). In this case, a database of clinical pathology journalsinvolving past and recent usage of histological stains was made. Theidentified pathological journals, articles and case studies werereviewed, analyzed, and important trends in the use of histologicalstains was made. As such, through an integrative and intensiveliterature and case study reviews rich data was collected in regardto stains used in the past and present to make a distinct on howhistological stains should be improved. This triangulation helps togather and assess in-depth data on past, present and future stain andstaining techniques (Silverman, 2011).
Historicalhistological staining techniques in medicine and biological studies
Thehistory of staining indicates that the application of histologicaltechniques is a relatively new area of diseases diagnosis (Rodrigues,Costa, Penha, Melo, Bottós, Furlani, Lima, Maia, Meyer, Höfling-Lima& Farah, 2009).Historical staining techniques by early pathologists and surgeonswere borrowed from a seventeen scientist Leeuwenhoek, who wasinstrumental in histologic using substances such as Madder, indigo,saffron to stain tissues and using rudimentary microscopes to studythem (Titford, 2009). These categories of early researchers used themicroanatomy to draw relationship of differential in cells as well asdelineating a normal plant cell structure from that of the animal(Bancroft& Layton, 2013).
Later,newer techniques were devised to enhance the study of cell structurein details using various laboratory chemicals to preserve tissues intheir natural for while waiting staining (Titford & Bowman,2012). Joseph Von Gerlach was viewed as the pioneer of microscopicalstaining in 1858 when he used ammoniacal carmine successfullyto stain cerebellum (Costa, Brito, Gomes & Caliari, 2010).
Theearly histologists used the readily available chemicals to preparetissues for microscopic studies these laboratory chemicals werepotassium dichromate, alcohol and the mercuric chloride to hardcellular tissues (Iyiola & Avwioro, 2011). These fixatives andstaining agents were ingenious and after a period colored stainingagents were developed which are still applicable in the laboratorystaining techniques (Black,2012).Examples of these ingenious colored stains still in use include thetrichrome that is used in the liver and renal biopsies as well as thesilver nitrate which is used in other organisms (Musumeci, 2014).
Greatdevelopment in histologic stains was shaped by the improvedtechnologic development of microscopes and the establishment of thehistologic stains (aniline dye) in 1856 in Germany which manufacturea variety of new histological stains (Shostak,2013).In the same line, as developmental research and knowledge in theanatomical and tissues knowledge of the human body gained momentum,this knowledge was used in further research of new-histologicaltechniques in the study of diseased tissue (Titford, 2009).
Inthe wake of the nineteenth century many medical centers hiredphysicians, pathologists and surgeon to handle surgical issues(Titford & Bowman, 2012). It is this crop of pathologist whodevised intraoperative staining techniques for frozen tissuessections by adapting a special staining technique in histopathology.It is during this time that the paraffin infiltration stainingtechnique was devised (Shostak,2013).Owing to this achievement, the non-malignant and the malignant tumorswere studied, and a bacterium was identified as the causal organismof the disease in the nineteenth century (Godwin,2011).
TheGram staining method was named after a Danish inventor Hans ChristianGram, who invented it as an approach of differentiating bacteriaspecies in 1875 (Anderson, 2011). It is while working at the citymorgue with his colleagues that Gram devised the technique ofstaining for the purpose of distinguishing the type of bacteriuminfection and also as a way of making the bacteria visible onselected and stained lung tissues during examination (Black,2012).Although this technique was found unsuitable for certain bacteriumorganisms it is still used today and competes fairly with modernmolecular techniques of histology (Shostak,2013).
Importanthistological stains used in the past and present
Itis a commonly used stain in histology used by early Botanists such asJohn Hill in their studies in 1770s (Jackson& Blythe, 2013).The stain was used to study microscopic tissue structures when inammoniacal solution form and it is still used today in histologicstudies. In particular, the stain was used widely by Rudolph Virchow(1821–1902) in microscopic studies Rudolph is considered as the‘father of pathology’ (Musumeci, 2014).
Theseare naturally occurring substances that have been in use in thehistory of histopathology (Titford, 2009). The stain was developed byWilhelm von Waldeyer in 1863 and was obtained from a log tree foundin Central America. Hematoxylinis a weak stain, and is used with a combination of other solutions inoxidized form (Shostak,2013).
Inparticular, the stain is combined with an oxidizer mordant to enhanceits differentiating capacity of cell components these solutions arecalled Hematoxylin. The versatility of the stain has enhanced thedevelopment of various Hematoxylin methods (Titford& Bowman, 2012).Historically, Hematoxylin was made into a nuclear stain that hadshorter staining time and resistant to acidic solutions this made itsuitable for histologic stain techniques requiring several steps(Anderson,2011).
SilverNitrate has had a long usage in historical staining techniques and isstill used in the modern pathology. Initially, early researchers usedsilver nitrate to enhance the visibility of the tissue structurewhile studying it this was done by applying solid silver nitrate ona tissue and the studying it (Titford & Bowman, 2012). The stainsubstance has been developed into many compounds, and confirmatorytests are needed when silver nitrate is used (Shostak,2013).Silver Nitrate stain has also been found to be reduced by argentaffincells found in the epithelial linings of lungs, intestines, melaninand others (Musumeci, 2014).
However,methods have been devised to ‘tailor’ these tissues to avoidargyrophilic reactions when Silver nitrate is used during stainingprocess (Titford, 2009). In particular, methods such as the Gomorireticulin methods and the Grocott-Gomori method were devised toassess missing tissues and diseases in the liver and the rectum(Nadworny, Wang, Tredget, Robert, 2010).
OtherStainingProcedures that were developed recently
TheHematoxylin and Eosin procedures
Althoughhistorically used, there have been great laboratory changes inHematoxylin stains nearly all tissue specimens are treated withHematoxylin and Eosin today(Bancroft & Layton, 2013).In addition, various Hematoxylin methods have been developed but allfollow the same approach of staining tissue specimens in ahematoxylin,alcohol and tap or alkaline water to clear argentaffin agents. Ithas been found that most histopathological processes could be studiedusing the Hematoxylinand Eosin procedures (Titford& Bowman, 2012).In the same line, the method is quick to execute, cheap and can bealtered. However, the Hematoxylin and Eosinisinefficient in that, not all features of a substance can be receivedand special stains must be used (Musumeci,2014).In other studies thehematoxylin and eosin procedures were found effective when assessingthe nucleic processes in RNAand DNA(Shostak,2013).
Theywere developed in the 1891 by Dimitri Romanowsky and popular for itsmulticolor in identifying blood parasites. The GiemsaStains procedure is still used today. There has been greatimprovement on the stains, and its various methods make it applicablein paraffin-embedded, formalin-fixed and bone marrow biopsies(Musumeci,2014).
TheGram staining method was named after a Danish inventor Hans ChristianGram, who invented it as an approach of differentiating bacteriaspecies in 1875 (Musumeci, 2014). Gram devised the technique ofstaining for the purpose of distinguishing the type of bacteriuminfection and also as a way of making the bacteria visible onselected and stained lung tissues during examination (Shostak,2013).Although this technique was found unsuitable for certain bacteriumorganisms it is still used today and competes fairly with modernmolecular techniques of histology(Rudijanto, 2007).
However,Gram technique is infallibly limited in application on matters ofenvironmental microbiology (Titford, 2009). Far from this, Gramtechniques have had success when performed on biopsy of infectedparts and produced results quickly especially when there is asignificant difference on prognosis and treatment. The method isoften used in the modern histologic especially in paraffin fixativesfor tissue sectioning (Titford & Bowman, 2012). In a recent casein Kuwait, the Gram staining technique was particularly effective inthe diagnosis of Gonorrhea giving 99.4% effective results (Iyiola &Avwioro, 2011). The method is still used today especially withparaffin sections and has been modified to suit different substances
Historicalassessment on the use of various stains in histologic indicates thatmost pathologists were appealed by stains that gave multicoloredresults on the tissue specimens. As such, trichrome stains weredeveloped from this need (Shostak, 2013). There have been variousmultiple stains such as blue–eosin,“triacid stain” by Ehrlich’s (1888) and Masson’s trichromestain which has been popular in the modern histology.Trichrome Stains show how complex the staining methods has become inthe search of efficient, and consistent stain that would show fine,differentiated tissues (Musumeci,2014).
Thisstudy was done in order to compare different staining methods andassess their effectiveness. The specific aim was to assess if thenewly developed staining methodsthe Helicobacterpylorisilver stain HpSS methods and the modified McMullen`s methods in theidentification of H pylori organism. The method involved selectingtissue sections of gastric biopsies of 63 patients diagnosed withdyspepsia. The section tissues were stained using the four stainingmethods. In all the 63 cases, 30 sections tested positive forHelicobacterpylori while 30 tested negative for all cases of pylori infectionwhile the remaining were tested using a combination of fivehistological tests(Anderson, 2011).The results indicated that, the interobserver stain method was thebest for antibody at 98% followed by Giemsaat 87%, then the HpSS at 85%. At gold standard level, it was foundthat the Giemsa stain method was the best followed by McMullen`smethod (Rotimi,Cairns, Gray, MoayyediP.,DixonMF.(2000).The study conclusions were that, in all cases of stain, the Hpylori infectionwas revealed however, the modified Giemsastain was the most effective for its sensitivity, ease of using,reproducible and cheap.
Theaim was to investigate the difference in capacity among differentstains Hematoxylinand Eosin, toluidineblue Stain, neuron-specificenolase (NSE) immunostainingand the S100 protein. These stains were applied toassess the presence of neurons and mast cells in acute appendices.Specimens were collected from clinically acute appendices categorizedas histologically positive and negative. In the study all the 50appendix specimens sections were subjected to Hematoxylinand Eosin, toluidineblue Stain, neuron-specificenolase (NSE) immunostainingand the S100 protein. Hematoxylinand Eosinwas applied as a routine stain for general study of the tissues,while Toluidine blue stain was applied to enhance easier study ofmast cells. In addition, neuron-specific enolase (NSE) immunostainingwas used as a marker and as well as the S 100 protein.
Theresults indicated thatwhen comparing Hematoxylin and Eosinstainwith S 100 showed 100% accuracy in identifying the denatured mucosalcells. However, the combination of these different staining methodsresulted in a supplementarytechnique effective than the conventional staining method inobserving changes and the pattern of diseased cells as well as themorphological shape of nerve fibers in the inflamed and inflamedappendices(Russell & Gordon, 2009).In addition, the use of the several staining method aided inconfirming results of earlier stain diagnosis.
Theliterature review on staining techniques indicates that there hasbeen great improvement in the histopathology and histotechnology.Historically, staining techniques used were carmine, silver nitrate,Giemsa,TrichromeStains,Gram Stainand Hematoxylinamong others (Titford& Bowman, 2012).These staining techniques are still in use although severalmodifications have been made to improve their efficiency. In othercases, some stain methods used earlier have been abandoned as theywere toxic.Several staining techniques have been established to improve thestaining methods.
Therehas been a rising need for efficient, accurate and less complexstaining procedures(Harris & McCormick, 2010).The histopathology lab today is laden with much work load anddifferent types of histological assignments (Musumeci, 2014). Assuch, most histologists are more trained on special stains forparticular works to give efficient results(Morelli, Porazzi, Ruspini, Restelli & Banfi, 2013).Inthe history of histologic, greatshift and development in histologic stains was shaped by improvedtechnologic development of microscopes and the establishment of thehistologic stains factory (aniline dye) in 1856 in Germany whichmanufactured variety of new-histological stains (Godwin,2011).
Asdevelopmental research and knowledge in anatomical and tissuesknowledge of human body gained momentum, further research on newhistological techniques in the study of tissues (Victor, 2013). Inthe wake of the nineteenth century many medical centers hiredphysicians, pathologists and surgeon to handle surgical issues(Titford & Bowman, 2012). These pathologists devisedintraoperative staining techniques for frozen tissues sections byadapting a special staining technique in histopathology(Loreto, Leonardi, Musumeci, Pannone & Castorina, 2013).It is during this time that the paraffin infiltration stainingtechnique was devised (Titford, 2009). Whilethese changes have taken place, there are old stain procedures thatare still in use today and many others have been replaced with newimmunal or staining techniques.
Somestaining methods have been abandoned because the chemicals requiredhave been medically proven to be toxic(Titford & Bowman, 2012).Similarly, there have been great changes in workload requiring moreadvanced technics of staining(Serrano, Hegge, Sato, Richmond & Stahnke, 2010).The case studies indicate that, in the modern histology a combinationof different stain techniques are used to enhance the effectivenessof the staining process. Furthermore, unlike in the past there havebeen great efforts in improving stains by modification to enhancetheir effectiveness (Victor,2013).Additionally, the complexity of stains has been enhanced for thepurpose of efficient and consistent staining processes that show fineand differentiated tissues(Ntziachristos, 2010).
Histologicalstaining is a commonly used medical process in the pathologicaldiagnosis and forensic studies. The process of histological stainingtakes five key stages and they include fixing, processing,embedding, sectioning and staining. Early histologists used thereadily available chemicals to prepare tissues for microscopicstudies these laboratory chemicals were potassium dichromate,alcohol and the mercuric chloride to hard cellular tissues. Thesefixatives and staining agents were ingenious and after a periodcolored staining agents were developed which are still applicable inthe laboratory staining techniques today.
Stainingtechniques used were carmine, silver nitrate, Giemsa,TrichromeStains,Gram Stainand Hematoxylinamong others.There has been great changes in the techniques used for histologicalstaining through chemical, molecular biology assays and immunologicaltechniques collectively referred to us histochemistry and havefacilitated greatly in the study of organs and tissues. Hematoxylinis a basic dye that is commonly used in this process and stains thenuclei giving it a bluish color while eosin (another stain dye usedin histology) stains the cell’s nucleus giving it a pinkish stain(Victor, 2013). Whilethese changes have taken place, there are old stain procedures thatare still in use today and many others have been replaced with newimmunalstaining or staining techniques(Sine, 2014).
Somestaining methods have been abandoned because the chemicals requiredhave been medically proven to be toxic. Similarly, there have beengreat changes in workload requiring more advanced technics ofstaining. The case studies indicate that, in the modern histology acombination of different stain techniques are used to enhance theeffectiveness of the staining process. In the modern histologic as away of improving histological stains, several stains have beenmodified and combined with other stains to improve theireffectiveness.
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