Hematoxylin–eosin-stained slide preparation is one of the most durable techniques in medicine history, which has remained unchanged since implemented. It allows an accurate microscopic diagnosis of the vast majority of tissue samples. In many circumstances, this technique cannot answer all the questions posed at the initial diagnostic level. The pathologist has always been looking for additional ancillary techniques to answer pending questions. In our daily histopathology practice, we referred to those techniques as special stains, but nowadays, they are more than stains and are collectively called ancillary tests. They include a wide range of techniques starting from histochemical stains and ending in one or more advanced techniques, such as immunohistochemistry, immunofluorescence, molecular studies, cytogenetic studies, electron microscopy, flow cytometry, and polymerase chain reaction.
IntroductionThe daily histopathology practice mainly depends on simple and low-cost procedure of fixation and chemical processing of tissue samples to obtain the ultimate hematoxylin–eosin (H&E)-stained slides, which give rapid, comprehensive, and informative scope of the scene. In the H&E technique, hematoxylin stains the nuclei while eosin counterstains the cytoplasm and various extracellular materials. It is one of the most durable techniques in medicine history, which has remained unchanged since implemented. Moreover, it allows an accurate microscopic diagnosis of the vast majority of tissue samples sent for evaluation.
Meanwhile, in many cases, this technique cannot answer all the questions posed at the initial diagnostic level, and it is clearly insufficient when the pathologist is seeking for etiologic factors or a histogenetic and pathogenetic relationship. Therefore, the pathologist has always been looking for additional ancillary techniques to answer those questions. In our daily histopathology practice, we referred to those techniques as special stains, but nowadays, they are more than stains and are collectively called ancillary tests. In this review, the current techniques that are most popular in diagnostic pathology practice are discussed. Histochemical StainsAlmost all histochemical stains are suitable for formalin-fixed tissues, and they belong to different families of chemical stains designed for microscopic visualization of different types of human tissues, cells, body secretions, pigments, minerals, parasites, and microorganisms. They remain an important diagnostic tool for many pathologists providing powerful complementary information, which may be followed by one of other ancillary techniques such as immunohistochemistry (IHC), flow cytometry, in situ hybridization, and other diagnostic technologies. Most of the histopathology laboratories run the protocol of chemical stains manually, but nowadays, the protocols of chemical stains have become increasingly automated, thus resulting in higher levels of productivity and flexibility.
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In general, a histochemical stain consists of the main chemical reaction that demonstrates cellular or tissue element of interest followed by chemical reactions to stain the background. The histochemical stains were widely used prior to the advent of immunohistochemistry; nowadays, the pathologist will find a relatively minority of them to be of real diagnostic utility, while the majority are rendered obsolete. Lists the most widely used chemical stains in the daily clinical practice of histopathology and their clinical uses. CHEMICAL STAINAPPLICATIONCLINICAL USES1.(Ziehl-Neelsen Stain)Detects nocardioform-actinomycete, Mycobacterium SppTo settle the diagnosis of Tuberculosis2.Giemsa StainStains H. Pylori, Plasmodium vivax, Rickettsia, Trypanosoma cruzi, Giardia lambliaDemonstration of various hematolymphoid elements (including mast cells) and microorganisms3.Oil Red Ostains lipids in frozen sections and some lipoproteins in paraffin sectionsDetect neutral lipids5.sliver for reticulinidentification of reticulin fibers in tissue samplesTraditionally it used to differentiate epithelial tumors from different types of sarcoma.
ImmunohistochemistryIHC is a method for demonstrating the presence and location of proteins, such as antigen, in the tissue sections. In other words, it is the application of basic immunologic principles and techniques to demonstrate molecules in cells and tissues. However, it is quantitatively less sensitive than immunoassays, such as Western blotting or enzyme-linked immunosorbent assay, but it enables the observation of processes in the context of intact tissue, typically applicable for assessing the progression and treatment of diseases, such as breast cancer.
In general, the information gained from IHC combined together with the initial morphological information provided by H&E staining reveals that data cannot be obtained by using any other available ancillary methods. Immunohistochemical staining is carried out for treating the tissue or the cell, which contains the specific antigen with antibodies that recognize the target antigen. As antibodies are highly specific, the antibody will bind only to the protein of interest in the tissue section. The antibody–antigen interaction is then visualized using chromogenic detection, in which an enzyme conjugated to the secondary antibody cleaves a substrate, containing chromogen, to produce a colored precipitate at the location of the antigen and can be visualized using light microscopy.There is probably no other method that has so revolutionized the scene of clinical histopathology during the past 50 years as the immunohistochemical technique.
The obvious advantages are remarkable sensitivity and specificity; applicability to routinely processed tissue, which is the formalin-fixed, paraffin-embedded tissue section (FFPETS), even if stored for long periods; and ultimately the easiness and precise correlation with morphological features. The immunohistochemical technique is compatible with formalin, the most widely used fixative in routine clinical pathology and earlier in H&E-stained microscopic sections.
It is sometimes positive even in entirely necrotic material. It can also be used in cytology to detect antigen molecules in different types of cytologic preparations. It has replaced many of the conventional histochemical stains and rendered many of the ancillary diagnostic techniques, such as electron microscopy (EM), obsolete. In order to prevent this technique to be misleading rather than helpful, the pathologist should realize that it presents potential pitfalls, and hence all the stains must be interpreted in the context of morphological features. Many immunohistochemical detection techniques are available in the daily pathology practice, he most commonly used at present being the immunoperoxidase technique.By using the traditional light microscopy, tissue and cellular antigen molecules cannot be visualized unless they are labeled by chromogen to permit their visualization.
Detection systems attach certain labels to primary or secondary antibodies in order to visualize the target antibody–antigen localization in the tissue sections. There are two methods to visualize the antigen localization.
Direct methodThe direct method is a one-step staining method and involves a labeled antibody reacting directly with the antigen in the tissue sections. This technique utilizes only one antibody, and therefore, it is simple and rapid, the sensitivity is less than the indirect method due to no or little signal amplification, and it is less commonly used than the indirect methods. The direct conjugate procedure has the advantages of rapidity and ease of performance, while the practical disadvantage of the direct method is that it usually demands the primary antibody to be used at a relatively high concentration and it cannot be used to detect multiple antigens in the same section. Indirect methodThis is a two-step method; primary antibodies are raised against an antigen of interest and are typically unconjugated (unlabeled). Secondary antibodies are raised against immunoglobulins (Igs) of the primary antibody.
The secondary antibody is usually conjugated to a linker molecule, such as biotin, or enzyme–chromogen complex. It is the commonly used method in routine pathology practice. To amplify the outcome signal, various techniques have been developed to add more enzymes or fluorophores to the target of interest, such as avidin-biotin complex, labeled streptavidin biotin, HRP polymer, and Tyramide signal enhancing.
IHC Double StainingIHC double-staining technique is used to visualize two or more antigens in the same section and analyze the relationship of their localization, within the same cell or tissue section. Currently, this technique is widely used in clinical pathology practice especially in lymphoma and prostatic carcinoma. It is usually done by immunoperoxidase staining through sequential staining technique (see Sequential staining section); however, new polymer-based methods and polyvalent detection systems have made concurrent staining possible. There are two methods to carry out the double-staining technique.
Sequential stainingThis method is used when both the primary antibodies are produced in the same species of animals. It is an indirect technique using unlabeled primary antibody, the first sequence of staining for the first antigen is completed with the development of the peroxidase reaction using 3,3′-Di-amino-benzidine (DAB) as the substrate, and this procedure yields a brown color signal. Then, staining for the second antigen is carried out with a primary antibody of different specificity and a second different substrate and chromogen system for the peroxidase or phosphatase enzymes; this method yields a blue or red color signal. Clinical uses of IHCThe widespread application of IHC in clinical pathology has entirely transformed diagnostic surgical pathology “from something resembling an art into something more closely resembling a science.” It is a notably a cost-effective powerful tool to settle the final diagnosis and should be used judiciously. As a matter of fact, the daily clinical practice can no longer be afforded without IHC.
Nowadays, it is rare to issue an informative and conclusive histopathological report without the usage of IHC, especially for tumor cases.An ongoing major drawback of IHC is the lack of objective quantitative measurement for target antigens under investigation, especially for proper use of the different prognostic markers of many cancers. Immunofluorescence (IF)This ancillary study is used to support fixed tissue diagnosis and to provide additional diagnostic and prognostic information, particularly in autoimmune diseases, vesiculobullous skin diseases, tissue and organ transplantation, and renal glomerular diseases. IF is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections usually fresh tissue, but FFPETS also can be used by binding a specific antibody chemically conjugated with a fluorescent dye (fluorochrome), such as fluorescein isothiocyanate, and the antigen–antibody complex is visualized using fluorescent microscopy.
Fluorochromes are dyes that absorb ultraviolet rays and emit visible light. This process is called fluorescence. Commonly used fluorochromes are acridine orange, rhodamine, lissamine, and calcofluor-white. There are two major types of IF staining methods. Direct methodIt is a one-step histochemical staining in which the primary antibody is labeled with fluorescence dye.
This technique is used to detect antigen in clinical specimens using a specific fluorochrome-labeled antibody. It can be used to detect viral, parasitic, and tumor antigens from specimens or monolayer of cells of patients.
Another application is the identification of anatomic distribution of an antigen within a tissue or within compartments of a cell, particularly in renal diseases and immunobullous disorders of the skin. Indirect methodThis is a two-step histological staining in which the secondary antibody is labeled with fluorochrome. This technique is mainly used to detect circulating antibodies in the body fluid; meanwhile, it is a highly sensitive technique currently used to recognize a primary antibody in FFPETS.
Indirect IF uses two antibodies: the unlabeled first (primary) antibody specifically binds the target molecule and the secondary antibody, which carries the fluorophores, recognizes the primary antibody and binds to it. Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing the number of fluorophore molecules per antigen.
This protocol is more complex and time-consuming than the abovementioned primary (or direct) protocol, but it allows more flexibility because various different secondary antibodies and detection techniques can be used for a given primary antibody.Different cytological preparation and tissue sections (FFPETS) can be stained with IF staining. Then, the antibody–antigen interaction is visualized using fluorescein detection, in which a fluorescein, which is conjugated to the secondary antibody to produce a colored precipitate at the location of the antigen–antibody reaction, can be visualized using fluorescence microscopy.IF applications can label antigens in cell suspensions, cultured cells, tissues, beads, and microarrays for the detection of specific proteins.
IF techniques can label both fresh and fixed samples. Molecular StudiesThere were tremendous advancements during the past three decades made in molecular biology, reflecting a major impact on the clinical practice of pathology. In fact, the latest molecular techniques have the potential to alter the diagnostic and therapeutic profiles of cancers in a way that no other techniques ever had before. Molecular techniques can be performed in tissues handled as part of the routine pathology practice, such as FFPETS and frozen sections. Pathologists have been practicing molecular morphology (the microscopic localization of protein, DNA, and RNA) since the late 1970s. Most of the molecular studies depend on hybridization techniques based on the application of recombinant DNA technology, by using a synthetic probes of synthetic oligo(deoxyribo) nucleotides, single-stranded cDNA, and single-stranded antisense RNA.
The probes are labeled by nonradioactive labeling – particularly with biotin and fluorescein. Fluorescence in Situ HybridizationFluorescence in situ hybridization (FISH) uses tagged probes that bind to chromosome-specific DNA sequences of interest, thereby allowing for the identification of both structural and numeric aberrations that specify certain hematopoietic and nonhematopoietic malignancies. FISH is usually performed on dividing (metaphase) cells as well as on nondividing (interphase) cells. It is superior to the cytogenetic study in that it can be used to diagnose many congenital anomalies, as listed in. It can be performed on air-dried cytological preparation, fresh tissue, frozen sections, and FFPETS, it can detect molecular abnormalities of many tumors, and it can facilitate the detection of genetic and chromosomal numeric abnormalities.
The principal use of FISH in daily practice of pathology is to detect somatic changes associated with cancers, which have a known diagnostic, prognostic, or therapeutic implication. Cytogenetic StudyCytogenetic technique is a conventional karyotypic analysis; it is the identification and study of each chromosome to detect specific chromosomal abnormalities associated with hereditary diseases and tumors. It has been only 50 years since the correct number of human chromosomes was established.Cytogenetic study is usually performed to settle the diagnosis of congenital disorders at pre- and postnatal time. Meanwhile, it is a powerful technique to study the tumors and provides clues to the molecular mechanisms involved in their pathogenesis. Flow CytometryFlow cytometry is a complex field that draws people from diverse scientific backgrounds. It is a biophysical technology based on laser light beam, widely and routinely used nowadays in clinical hematology and research purposes. It allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second; one or more laser beams interrogate each particle and, at a minimum, the system measures the degree and direction of the scattered light – indicators of the particles’ size, shape, and structure.
If a particle is stained with one or more fluorescent dyes, known as fluorochromes, the light source excites these dyes to provide additional biological information about each particle.The unique power of flow cytometers is that they can rapidly and quantitatively measure multiple simultaneous parameters on individual live cells and then isolate the cells of interest. General uses of flow cytometry include measurement of cell size, cytoplasmic granularity, cell viability, cell cycle time (S-phase fraction), DNA content (DNA ploidy), detection of surface marker phenotype, enzyme content, cell counting, and cell sorting.The test sample should be a single-cell suspension with a maximum cell dimension of 50 microns, which is regarded as the major limitation of this device; in other words, only body fluid and blood can be used as samples. A number of protocols are available for disaggregating tissue samples from solid tumors into suitable single-cell suspensions. These protocols typically involve enzymatic digestion and mechanical chopping and filtering. Hedley et al. Prescribed the preparation of single-cell suspension from FFPES for flow cytometric analysis, and the original method has been subjected to a wide range of technical modifications, either to simplify the procedure or to improve single-cell harvesting. A modified method to prepare single-cell suspension from FFPES was described by Makki et al.
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Electron MicroscopyIt has been more than 50 years since the admission of EM to the anatomical pathology field for the diagnosis of body’s different organ diseases and tumor pathologies. EM can visualize ultra cellular structures and any other cellular structural abnormalities that cannot be appreciated by light microscopy. The main diagnostic uses of EM in clinical pathology are in the fields of renal biopsy, peripheral nerve biopsies, and muscle biopsies.
EM is useful in the evaluation of metabolic and inherited diseases as well as demonstration of infectious agents or any evidence of drug toxicity, in addition to its major role in tumor pathology, where the ultrastructural features have proved very useful in the determination of tumor differentiation. However, reactive conditions of benign tumors share malignant tumors to those ultrastructural changes of the same cell type.Nowadays, the role of EM in clinical pathology has reduced remarkably since the advent of IHC and molecular techniques. EM has provided a great utility and remains a powerful tool to settle the diagnosis of controversial nature of some tumors, including granular cell tumor, schwannoma, Langerhans cell histiocytosis, spindle cell (sarcomatoid) carcinoma, mesothelioma, melanomas, spindle cell thymoma, neuroendocrine tumors, spermatocytic seminoma through the detection of tumor-specific cytoplasmic filaments, granules, melanosomes, mesaxons, membrane-bounded crystals, or other ultrastructural changes.
It is of great benefit that an expert anatomical pathologist should handle EM examination who has carried out the light microscopy examination of the controversial case and should conclude that EM is indicated for that specific reason. This is because conclusive and informative EM report can be achieved only with close correlation with light microscopy findings. EM studies require fresh tissue to be fixed in a special fixative; however, formalin-fixed wet tissue can also be used, and even FFPES is useful. Moreover, from H&E-stained section, tissue for EM examination can be obtained when the diagnostic feature is present only focally. Different cytological preparations are suitable for EM studies.The clinical diagnostic uses in daily practice of pathology are summarized as follows (modified ):.Poorly differentiated malignancy to differentiate between carcinoma, melanoma, and sarcoma, –.Differential diagnosis between adenocarcinoma and mesothelioma,.Origin of anterior mediastinal tumors, either thymic tumors, malignant lymphoma, or germ cell tumor,.Differential diagnosis of small round cell tumors, including neuroendocrine tumors, and.Differentiation between spindle cell tumors of soft tissues. Polymerase Chain Reaction (PCR)PCR is an advanced molecular diagnostic technique that was developed during the mid 80 s of the last century.
It is quick, reliable, highly sensitive, and a specific central technology for much of clinical molecular genetic testing. It generates millions of copies of any specific DNA sequence within a few hours.
PCR relies on the ability of DNA polymerases to copy a DNA strand using a short complementary DNA fragment as an initiating template., The PCR technique can also be used to amplify RNA, so that gene expression can be analyzed. Real-time (RT) PCR represents a new generation of PCR for quantitative measure, in which the incorporation of fluorescent markers in the reaction mixture permits RT monitoring of the amplification process. The major advantages of RT PCR are shorter procedural time, higher sensitivity and specificity, and low risk of laboratory contamination. Specimens for PCR could be fresh blood, bone marrow, fresh tissue biopsies, frozen sections, and FFPETS, as all are sources of nucleic acids for molecular analysis.
Specimens should be collected and transported to the molecular pathology laboratory using aseptic techniques, if possible. Transport on ice reduces cell lysis, minimizes nuclease activity, and reduces nucleic acid degradation. Specimen contamination is a major drawback of this technique. Fresh tissue sampling is superior to fixed samples because all fixatives, including formalin, induce chemical degradation of nucleic acid.
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Nevertheless, fixed specimens are more easily stored and transported. Conclusion and RecommendationClinical ancillary diagnostic methods have been integrated not only into histopathology but also into many laboratory disciplines. Guidelines and recommendations from both professional institutions and regulatory agencies have been developed to assist in the development and performance of ancillary testing.
Diagnostic Immunohistochemistry Dabbs Pdf To Word Free
Most of the ancillary techniques are performed in surgical pathology for detecting markers specific for a particular disease or a tumor and for focusing on somatic or acquired DNA variations in the cells, providing essential information about the diagnosis, prognosis, prediction of effective treatment, with monitoring the treatment response and the patients at high risk of disease. Rapidshare library search engine download. ACADEMIC EDITOR: Dama Laxminarayana, Editor in ChiefPEER REVIEW: Six peer reviewers contributed to the peer review report. Reviewers’ reports totaled 1002 words, excluding any confidential comments to the academic editor.FUNDING: Author discloses no external funding sources.COMPETING INTERESTS: Author discloses no potential conflicts of interest.Paper subject to independent expert blind peer review.
All editorial decisions made by independent academic editor. Upon submission manuscript was subject to anti-plagiarism scanning. Prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties.
Diagnostic Immunohistochemistry Dabbs Pdf To Word Document
This journal is a member of the Committee on Publication Ethics (COPE). Provenance: the author was invited to submit this paper.Author ContributionsConceived the concepts: JSM. Analyzed the data: JSM. Wrote the first draft of the manuscript: JSM.
Developed the structure and arguments for the paper: JSM. Made critical revisions: JSM. The author reviewed and approved of the final manuscript.
Diagnostic Immunohistochemistry presents the latest information and most reliable guidance on immunohistological diagnoses in surgical pathology. Dabbs, MD and other leading experts bring you state-of-the-art coverage on genomic and theranostic applications, molecular anatomic pathology, immunocytology, Non-Hodgkin’s lymphoma, and more. Additional features such as tables discussing antibody specifications, differential diagnosis boxes, ancillary anatomic molecular diagnostics, and full-color histological images ensure user-friendly coverage that makes key information easy to find and apply.
The fully searchable text is also available online at.com, along with a downloadable image bank and access to Path Consult. This concise and complete resource is today’s indispensable guide to the effective use of immunohistochemical diagnosis.Detail + Download. Diagnostic Immunohistochemistry4th Edition Theranostic and Genomic Applications, Expert Consult: Online and PrintBy David J Dabbs, MD, Professor and Director of Pathology Professor and Chief of Pathology, Department of Pathology, Magee-Women?s Hospital, Pittsburg, PADabbs: Diagnostic Immunohistochemistry, 4/e1.
Techniques of Immunohistochemistry: Principles, Pitfalls and Standardization2. Molecular Anatomic Pathology: Principles, Technique and Application to Immunohistologic Diagnosis3. Immunohistology of Infectious Diseases4. Immunohistology of Neoplasms of Soft Tissue and Bone5. Immunohistology of Hodgkin Lymphoma6. Immunohistology of Non-Hodgkin Lymphoma7. Immunohistology of Melanocytic Neoplasms8.
Immunohistology of Metastatic Carcinoma of Unknown Primary9. Immunohistology of Head and Neck Lesions10. Immunohistology of Endocrine Tumors11. Immunohistology of the Mediastinum12. Immunohistology of Lung and Pleural Neoplasms13. Immunohistology of Skin Tumors14.
Immunohistology of the Gastrointestinal Tract15. Immunohistology of the Pancreas and Hepatobiliary Tract16. Immunohistology of the Prostate17. Immunohistology of the Urinary Bladder, Kidney, and Testis18. Immunohistology of the Female Genital Tract19. Immunohistology of the Breast20. Immunohistology of the Nervous System21.
Immunohistology of Pediatric Neoplasms23. Imaging and Quantitative Immunohistochemistry.
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Contents.Naming The term 'cytokeratin' began to be used in the late 1970s, when the of keratin inside were first being identified and characterized. In 2006 a new systematic nomenclature for mammalian keratins was created, and the proteins previously called 'cytokeratins' are simply called keratins (human epithelial category). For example, cytokeratin-4 (CK-4) has been renamed keratin-4 (K4).
However, they are still commonly referred to as cytokeratins in clinical practice.Types. Showing low molecular weight cytokeratin (LMWCK) staining of and endometrial glands.There are two categories of cytokeratins: the and the or neutral.
Within each category, cytokeratins are numbered in order of decreasing size, from low molecular weight (LMWCKs) to high molecular weight (HMWCKs). Cytokeratins are usually found in heterodimeric pairs of acidic and basic subunits of similar size.Basic CK(Type B / Class II)Acidic CK(Type A / Class I)HMWCK' squamous keratins'CK-11LMWCK' simple keratins'Expression of these cytokeratins is largely organ or tissue specific. The subsets of cytokeratins which an epithelial cell expresses depends mainly on the type of epithelium, the moment in the course of terminal differentiation and the stage of development. Thus a specific cytokeratin expression profile allows the identification of epithelial cells. Furthermore, this applies also to the malignant counterparts of the epithelia, , as the cytokeratin profile is generally retained. Thus the study of cytokeratin expression by techniques is a tool of immense value widely used for tumor diagnosis and characterization in surgical.
Cytokeratin filaments in the human epithelial cell Cell biology In the, the keratin filaments conform a complex network which extends from the surface of the nucleus to the cell membrane. Numerous accessory proteins are involved in the genesis and maintenance of such structure.This association between the and the nuclear surface provides important implications for the organization of the cytoplasm and cellular communication mechanisms.
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