Metastasis in Greek means displacement.

The term is used to denote the spread of a disease from one organ or part to another non-adjacent organ or part.

Malignant tumor cells and infections have the established capacity to metastasize. Until specified otherwise the term metastasis usually denotes malignant spread.

Why Does Metastasis occur?

Cancer cells can break away, leak, or spill from a primary tumor, enter lymphatic and blood vessels, circulate through the bloodstream, and be deposited within normal tissue elsewhere in the body.

Metastasis is one of three hallmarks of malignant tumors.

Different tumors have different capacity to metastasize.

When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor. This is done to differentiate it from original source or primary tumor.

However, the cells of  new secondary tumor are similar to the primary tumor

How Does Metastasis Occur?

The spread of metastases may occur via the blood or the lymphatics or through both routes.

Metastasis is a complex series of steps in which cancer cells leave the original tumor site and migrate to other parts of the body via the bloodstream or the lymphatic system.

To do so, malignant cells break away from the primary tumor and attach to and degrade proteins that make up the surrounding extracellular matrix, which separates the tumor from adjoining tissue. By degrading these proteins, cancer cells are able to breach the extracellular matrix

The body resists metastasis by a variety of mechanisms through the actions of a class of proteins known as metastasis suppressors, of which about a dozen are known.

What Are The Places Where Metastases Occur?

The most common places for the metastases to occur are the lungs, liver, brain, and the bones. Different tumors behave differently in their predilection to sites  for metastases

Common Sites Where Metastases Come From

• Lung
• Breast
• Skin: Melanoma*
• Colon
• Kidney
• Prostate
• Pancreas
• Cervix
• Bone

*Other skin tumors rarely metastasize.

Metastases With Unknown Primary

Almost 10% of patients presenting to oncology units will have metastases without a primary tumor found.

These are termed as unknown or occult primaries and the patient is said to have cancer of unknown primary origin (CUP) or Unknown Primary Tumors (UPT).

Approximately,  3% of all cancers are of unknown primary origin. In some of these cases a primary tumor may appear later.

Symptoms
Symptoms of cancer metastasis depend location of the tumor.

• In lymph nodes, a common symptom is lymphadenopathy.
• Lungs: cough, hemoptysis and dyspnea (shortness of breath)
• Liver: hepatomegaly (enlarged liver)and jaundice.
• Bones: bone pain, fracture of affected bones.
• Brain: neurological symptoms such as headaches,seizures, and vertigo.

Although advanced cancer may cause pain, it is often not the first symptom.

Treatment

Treatment and survival is determined by whether or not a cancer is local or has spread to other locations.

If the cancer spreads to other tissues and organs, it may decrease a patient’s likelihood of survival.

Radiosurgery, chemotherapy, radiation therapy, biological therapy, hormone therapy, surgery or a combination of these is used to.

The choice of treatment generally depends on the type of primary cancer, the size and location of the metastasis, the patient’s age and general health, and the types of treatments used previously.

The treatment options currently available are rarely able to cure metastatic cancer.

Text adapted from Wikipedia

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This is abrief article about various substances that are expressed in the body and are used as tumour markers too.

Epithelial Markers

Keratins are the markers most frequently used in the identification of epithelial phenotypes.

Epithelial membrane antigen (EMA)

EMA represents a membrane glucoprotein that is most likely similar or identifical to the casein fraction of human milk. It is expressed by virtually all epithelial cells and neoplasm of epithelial origin. It is also expressed on a wide range of tumors of mesenchymal origin and even on some lymphomas.

Muscle Markers

Desmin, actin and myoglobin are the most frequently used markers of muscle differentiation.

Actin

Actin is a collective term describing a group of contractile filaments. Actins are divided into three major subgroups: alfa, beta, and gama. The three forms of alfa actin are organ or tissue specific and are designated as alfa-skeltal, alfa-cardiac, and alfa-smooth muscle.

The two forms of gama-actins are gama-smooth muscle and gama-cytoplasmic. There is only one form of beta-actin, and it is designated as beta-cytoplasmic. Beta and gama-cytoplasmic actins are ubiquitous and are expressed in virtually all cells. On the other hand, the expressions of alfa actins (skeletal, cardiac, and smooth muscle) as well as gama-smooth muscle actin are tissue specific.

As with desmin, actin is expressed on various cells that perform contractile functions such as myofibroblasts, myoepithelial cells, and pericytes. In bone tumors, antibodies against actin are frequently used in the differential diagnosis of primary and metastatic spindle and round-cell tumors.

Myoglobin

Myoglobin is the heme metalloprotein that binds oxygen and is expressed in skeletal muscle fibers. In contrast to other muscle markers, it is not expressed in smooth muscle cells.

It serves as a marker of skeletal muscle differentiation and is used in the diagnosis of rhabdomyosarcoma.

However, its expression is restricted to tumor cells with more differentiation, and most poorly differentiated tumors do not express sufficient amounts of this protein to be identified in paraffin-embedded, formalin-fixed tissue.

Vascular Markers

Factor VIII

Factor VIII-associated antigen, or von Willebrand factor can be demonstrated in all normal endothelial cells and is expressed by virtually all benign vascular lesions.

There is great variability of its expression in malignant vascular lesions.

High-grade angiosarcoma may show only focal positive staining. In skeletal pathology, it is most often used to disclose the endothelial nature of tumor cells in vascular lesions such as hemangioendothelioma.

Platelet-endothelial adhesion molecule (CD31)

CD31 is a member of the immunoglobulin family and represents a transmemebrane protein of endothelial cells. It is also expressed by megakaryocytes and platelets and can be detected in some plasma cells. It is expressed by virtually all benign vascular tumors and by a high percentage of malignant vascular lesions .

Human Progenitor cell antigen (CD34)

CD34 is a member of a large, superfamily of lymphoid markers. The expression of CD34 is not restricted to endothelial cells. Unfortunately, it is also present in a wide range of spindle cells and peripheral nerve sheath tumors. Therefore the applicability of CD34 in the differential diagnosis of vascular lesions is limited.

Neural Markers

S-100 protein

S-100 protein is an acidic nuclear protein that binds calcium and is composed of two (alfa and beta) subunits. S-100 protein is often used in the differential diagnosis of tumors of neural origin and melanoma.

In bone tumors, S-100 protein is more frequently used as a marker of cartilaginous differentiation. It can be identified in tumors exhibiting relatively primitive cartilaginous differentiation such as in chondromyxoid fibroma, chondroblastoma, and mesenchymal chondromyxoid fibroma, chondroblastoma, and mesenchymal chondrosarcoma.

S-100 protein is expressed by chordal tissue, and its expression is retained in chordomas. It is frequently used in the differential diagnosis of chordoma, chondrosarcoma, and the so-called chondroid chordoma.

Neuron-specific enolase

Neuron-specific enolase is an enzyme of the glycolytic pathway. Different isoenzymes of the group have tissue-specific expression to some extent.

Beta-beta dimeric form is expressed in skeletal muscles, the alfa-alfa form is present in glial cells, and the gama-gama isoform is present in neurons.

In bone tumors, it is most frequently used in the diagnosis of Ewing’s sarcoma and related conditions. It is also used in the diagnosis of neuroblastoma and other metastatic or primary bone tumors with potential neural differentiation.

Myelin basic protein

Myelin basic protein is a major polypeptide component of the myelin. Hence, it is expressed in tumors derived from the Schwann cells, both benign and malignant.

Leu-7

Leu-7 was identified as a marker of natural killer cells, but it is also present in the nerve sheath and neuroendocrine cells. It is typically used in the differential diagnosis of spindle-cell neoplasm with a presumed nerve sheath origin. Leu-7 is also expressed in tumors such as leiomyosarcoma, synovial sarcoma, and occasionally in rhabdomyosarcoma. It is also used in the differential diagnosis of small blue-cell neoplasms. Like neuron-specific enolase, it cannot be used as the sole marker of neural differentiation.

Synaptophysin

Synaptophysin is expressed in neural cells and is found in the presynaptic vesicles. It is also expressed in neuroendocrine cells. Synaptophysin is generally used as a sensitive marker to document neural or neuroendocrine differentiatial diagnosis of metastatic lesions such as neuroblastoma, paraganglioma and other neuroendocrine tumors.

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Intermediate filaments are ubiquitous cytoplasmic structures that are about 10 nm thick. They have a uniform appearance and represent a nonbranching, fine filamentous, cytoplasmic material.

On the basis of their chemical compositionthey can be separated into five major groups: vimentin, keratins, desmin, glial fibrillary acidic protein, and neurofilaments.

Vimentin

Vimentin is a filamentous protein universally expressed in mesenchymal cells and in some epithelial cells and their neoplasms. In epithelial cells, it is typically coexpressed with other epithelial markers, such as keratins. For these two reasons, the specific diagnostic applicability of vimentin in the differential diagnosis of tumors is minimal.

It is most often used to verify the antigenicity of cells in question when other markers are negative.

Keratins

Cytokeratin is a collective term for a group of 19 polypeptides. Keratins can be separated into two major categories, A and B (acidic and basic), on the basis of their electrophoretic behavior. Cytoplasmic keratin molecules consist of pairs of acidic and basic components.

The basic component is typically the larger molecule of the pair. Different epithelia have different keratin profiles. Keratins are markers of epithelial differentiation.

Tumors such as chordoma and, to some extent, chondrosarcoma express keratins. RNA that code for keratins are expressed in a wide range of mesenchymal cells and tumors of mesenchymal origin.

In skeletal pathology, keratins are used in the diagnosis of adamantinoma, chordoma, chondrosarcoma, epithelioid hemangioendothelioma, and sarcomatoid carcinoma and in the differential diagnosis of metastatic neoplasms.

Desmin

Desmin is a filamentous molecule expressed in smooth, skeletal, and cardiac muscle fibers. Desmin is a marker of muscle differentiation, but it is also present in other cells that have contractile properties such as myofibroblasts.

Desmin is also expressed in some fetal cells such as embryonal mesothelium, stromal cells of fetal kidney, and chorionic villi. In general pathology, desmin is used as a marker for the diagnosis of tumors that exhibit muscle-predominately skeletal differentiation.

It is typically used in the differential diagnosis of primary and metastatic spindle-cell neoplasms and in the documentation of rhabdomyoblastic differentiation in dedifferentiated chondrosarcoma. It is also frequently used in the differential diagnosis of small-cell tumors of bones.

Neurofilaments

Neurofilaments protein is expressed in most neuronal cells. It is subdivided into three distinct polypeptides that differ in their molecular weight. Typically, all three polypeptides are expressed, but some neuronal cells may lack all or some of the neurofilament proteins. In general pathology, neurofilaments are used as markers of neural differentiation.

With reference to bone tumors, neurofilament proteins are used in the differential diagnosis of primary and metastatic small-cell tumors (e.g., neuroblastoma). Poorly differentiated tumor cells may express undetectable levels of neurofilament protein. Moreover, fixation and paraffin embedding significantly reduce the stainability of cells for this marker.

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Immunohistochemistry has become a generally accepted and widely used auxiliary method of diagnostic pathology, including the pathology of bone tumors and tumor like lesions.

This method has emerged as a diagnostically useful technique because of the development of highly specific antibodies and the invention of sensitive immune and enzymatic detection systems. The fluorescence detection methods are more often used in investigative studies and are rarely sued in diagnostic pathology.

Basis

The Immunohistochemical method is based on binding of a specific cellular or extracellular substance by the antibody, with subsequent visualization of the bound antibody by a color-based detection system. Subsequent use of a counterstain such as hematoxyline or toluidine blue enables the precise microscopic localization of a positive reaction in various components of the tissue.

The avidin-biotin and peroxidased-based detection systems are most frequently used.

In formalin-fixed, paraffin-embedded tissue, the so-called antigen-retrieval techniques are important steps in increasing the sensitivity of the test. The goal of this step is to expose the epitopes of the studies antigen and to facilitate antigen antibody binding.

The antigen-retrieval techniques are simple and typically include limited digestion with proteolytic enzymes, microwave treatment, or both.

At this level of advancement, Immunohistochemistry provides consistent results with most fixatives. Satisfactory results can be obtained in decalcified tissue or even on decolorized, previously stained microscopic sections. It can also be used in most cytologic preparations.

Constant daily comparison of the immunohistochemical results with external and internal positive and negative controls helps prevent misinterpretation of ambiguous results. The pitfalls of immunohistochemistry are in general caused by false-negative or false-positive results.

False-negative results:

1 The epitopes of antigens in the tissue are lost because of inadequate fixation. The amount of antigen is reduced by degradation and cannot be detected by immunohistochemistry.

2 The antigen are misplaced from the specific tissue or cellular location because of diffusion. This is most frequently observed in reference to endothelial antigens such as factor VIII-related antigen.

3 The antibody is inappropriately used (too low a concentration) or destroyed, or its affinity for the antigen is inadequate.

4 The components of the detection system are inadequate or are inappropriately used.

False-positive Results:

1 Cross-reactivity (lack of specificity) of the antibody with other antigens or its nonspecific binding to the tissue.

2 Nonspecific color reaction caused by the presence of unblocked endogenous peroxidase.

3 Nonspecific binding of detection system components, such as the avidin-biotin complex, to the tissue (This is typically caused by excessive use of detection system components).

5 Misplacement of the antigen from normal tissue that is subsequently absorbed or phagocytized by tumor cells False-positive results are in fact more misleading than false-negative results and probably occur more frequently.

immunohistochemistry is a powerful tool used to provide diagnostically valuable information on the histogenesis and differentiation of cells. Similar to other auxiliary techniques, the immunophenotypic profiles of cells are not used to distinguish among benign, malignant, and reactive conditions.

The number of antibodies with potential diagnostic applications is huge, and new antibodies are constantly being developed. The specific applications of immunohistochemical stains and the so-called immunophenotypic features of bone tumors are provided in the sections on special techniques that accompany the discussion of each specific bone tumors.

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Histomorphometry represents a microscopic planimetry or stereology. It is used to study homeostasis of the skeleton, mainly in metabolic disorders of bone. The technique is occasionally used to evaluate the skeletal status and treatment effect of rickets and osteomalacia associated with tumors.

For histomorphometry, undecalcified bone sections stained with techniques that enable the visualization of calcified and uncalcified osteoid such as von Kossa’s or Goldner’s (modified trichrome) stains are used. Another frequently used technique is tetracycline-pulse labeling for epifluorescence.

Two short cycles (3 days) of tetracycline approximately 2 weeks apart allow two separate mineralization fronts to be visualized as epifluorescence lines outlining the bone trabeculae of cancellous medullary and endosteal cortical bone. This is due to the ability of tetracyclines to incorporate at the border of osteoid, which is actively mineralized.

The width between the two lines of fluorescence (pulse labels) reflects the rate of mineralization.

A number of parameters can be assessed by histomorphometry of bone.

Total bone volumeThe content of mineralized and non-mineralized bone calculated as the percentage of the total area of tissue examined.

Cancellous bone volume

Mineralized and nonmineralized cancellous bone calculated as the percentage of the total area of tissue examined or as the percentage of the area of the medullary cavity.

Trabecular osteoid volume

Nonmineralized cancellous bone calculated as a percentage of total cancellous bone.

Trabecular osteoid surface: The percent of cancellous bone surface covered by osteoid.

Mineralizing surface: The proportion of trabecular osteoid surface that exhibits tetracycline labeling.

Mineral apposition value: The distance (in microns) between two tetracycline-pulse labels per day.

Bone formation value: The mineralized bone produced in 1 year.

Mineralization lag time: The time (delay) of mineralization of the matrix after its deposition by osteoblasts.

Trabecullar resorptive surface: The percent of bone surface that shows current or prior osteoclastic activity.

Cortical resorptive surface: The percent of the cortical surface that shows current or prior osteoclastic activity.

Periosteal resorptive surface: The percent of periosteal surface with osteoclastic activity.

Trabecular osteoclast count: The number of osteoclasts per area (millimeters squared) of medullary cavity or the number of osteoclasts per length (in centimeters) of the trabecular bone.

Osteoclastic index: The number of osteoclasts per length of the trabecular surface with evidence of present or prior resorptive activity.

Cortical porosity: The percentage of the cortex that contains pores without osteocytic cells.

Accurate measurements of bone resorption usually require specimens taken from two consecutive biopsies.

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Cytophotometry, or image analysis, is conceptually similar to flow cytometery but requires a different preparations of cells for the measurements. The principle of this technique is the measurement of the optical density of cells in histological section or, even better, of whole cells spread in histological section or, even better, of whole cells spread on the glass.

Compared with flow cytometry, the technique is much slower, and fewer objects can be measured. The main advantage of this approach is the ability to identify a measured object microscopically and to perform the measurements separately on different cell populations and tissue components.

The technique is uniquely suited for the measurements of distinct tumor cell populations. Recent developments of computerized tissue reconstruction programs have made it possible to perform the quantitations on histologic sections.

The information provided is somewhat similar to data gained by flow cytometry. In pathology including that of bone tumors, this technique is most frequently used to generate DNA ploidy distribution patterns and to perform cell-cycle analysis. For this type of analysis, the cells are typically stained with Feulgen reaction.

As with flow cytometry, any cellular component that can be visualized directly by an appropriate color reaction or with the use of antibodies can be quantitated for investigative purposes. In addition, several other parameters, such as nuclear size, shape, volume, and chromatin texture, can be measured. In summary, the main advantage of this technique is its ability to verify the measured objects microscopically in standard cytologic and histologic preparations.

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Flow cytometers are machines constructed to measure and record flurescence on particles or cells stained with fluorochrome and flowing in suspension past an excitation source, typically a laser beam.

The fluorescence levels of the individual cells are captured by a photomultiplier tube, converted into an electric pulse, and stored and analyzed by a computer.

The cells can also be stained with multiple fluorochromes and can be excited by two different laser beams. This technique known as multiparameter flow cytometry, helps analyze several cellular components and their relationships.

Some other cellular features, such as light scattering at small angles, pulse width, or electrical conductivity, related to some extent to cell seize, can also be measured.

Flow cytometry does not permit morphologic corroboration of the identity of the measured objects. Cells that normally lack intercellular junctions like peripheral blood cells, bone marrow cells are ideal for this technique.

The measurement of cytoplasmic components of solid tumors is more difficult and less accurate because the preparation of The techniques of nuclear isolation from formalin-fixed, paraffin-embedded tissue are widely used for retrospective studies.

Analysis of the cell cycle and of DNA ploidy distribution patterns are the most frequent applications of this technique.

Propidium iodide, ethidium bromide, and diamidine phynylindole (DAPI) are normally used fluorochromes.

Acridine orange, which bonds differentially to DNA and ribonucleic acid (RNA), can be used for the simultaneous analysis of both components.

The results of single-parameter measurements are displayed as frequency histograms. With simultaneous measurement of several parameters, the results are presented as scattergraphs or contour maps that show the relationship between the measured parameters.

In immunofluorescence studies, the properties of cells binding a given fluorescent antibody are displayed as percentage of cell population screened.

In contrast to normal tissues, neoplastic lesions often undergo chromosomal aberrations that result in the appearance of aneuploid clones.; From a practical point of view, human tumors can be divided into two major groups: diploid and aneuploid.

Tumors with such a normal or near-normal DNA histogram are classified as diploid or, better, near-diploid. Many tumors exhibit an abnormal position of one or several cell populations that correspond to the abnormal chromosome complement of their cell populations. Such tumors are classified as aneuploid.

Cell proliferation can also be analyzed. Cell-cycle analysis based on DNA histograms is not always easy or possible, especially in aneuploid tumors in which several overlapping abnormal cell populations may be present. The DNA ploidy and cell-cycle analyses have been repeatedly proved to provide reliable prognostic information in many tumors of different organs, including some bone tumors.

Most reliable data are available in reference to several common epithelial malignancies such as carcinomas of the breast, urinary bladder, prostate, and colon.

In osteosarcoma and chondrosarcoma – DNA ploidy provides prognostically valuable information.

Generally, tumors with modal patterns close to diploid have a more favorable prognosis compared with tumors that have an aneuploid DNA distribution. Normal or near-normal total DNA content correlates with a high degree of histologic differentiation. In bone tumors, most high grade conventional osteosarcomas are aneuploid.

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Examination of ultrastructural features of cellular and extracellular structures is a powerful diagnostic tool. The introduction of the transmission electron microscope in the early decades of the twentieth century dramatically expanded the investigative and diagnostic capabilities to study the submicroscopic details of diseased tissue including bone tumors and tumor like condition.

Use of the transmission through a thin section of tissue impregnated with electrondense material and embedded in plastic material and embedded in plastic medium is the most applicable for diagnostic purposes.

The scanning electron microscope provides an opportunity to examine the cell surface and intracytoplasmic membranous structures, as well as the three-dimensional composition of extracellular components. The so-called scanning and analytical instruments have little use in routine diagnosis.

Even the most enthusiastic electron microscopist must admit that as far as diagnostic pathology is concerned, the immunohistochemistry era is now upon us. The emergence of immunohistochemistry has significantly changed the approach to the diagnosis of bone tumors, and many diagnostic questions that in the past required ultrastructural studies are now addressed with the use of antibodies.

However, electron microscopy continues to provide unique, diagnostically useful information and should be used in addition to, not instead of, immunohistochemistry.

Diagnostically useful information can be obtained from cytologic preparations such as fine-needle aspirates. Limited ultrastructural studies can also be performed on tissue retrieved from paraffin-embedded material.

In bone tumors, as in general tumor pathology, ultrastructural studies provide valuable information about tumor histogenesis and differentiation pathways. Unfortunately, like other special techniques, ultrastructural studies are not useful in the differential diagnosis of benign versus malignant tumors and reactive conditions.

The most frequent applications of this technique in bone tumor pathology are in the differential diagnosis of:

1. Spindle-cell neoplasms
2. Small blue-cell neoplasms
3. Vascular versus nonvascular neoplasm and
4. A wide range of metastatic tumors of bone.

These four main areas of electron microscopy have numerous specific applications. Ultrastructural features of potential diagnostic significance are described with the individual conditions in their respective chapters.

Generally, tumors that cannot be diagnosed by light microscopy and that also have an inconclusive immunohistochemical profile very likely are not diagnosable by electron microsopcy. Ultrastructural studies conducted without a definite list of differential diagnosis and specific questions are likely to provide disappointing results.

Such studies have the best chance of being useful if conducted along with light microscopy and other applicable special techniques, with the formulation of specific questions that need to be answered.

The major limitations of ultrastructural studies in the diagnosis of bone tumors are similarly to those listed in general pathology textbooks:

1. Only a small proportion of tissue can be sampled and examined.

2. Relatively few ultrastructural features are diagnostically specific; as with light microscopy, the entire picture is more informative.

3. It is occasionally difficult to distinguish neoplastic from nonneoplastic cells in ultrastructural studies. Therefore the entrapment of normal elements with specific ultrastructural features of cellular differentiation can be misleading. This pitfall can be largely eliminated by careful examination of the so-called semi-thin section.

Electron microscopy is informative if it is used by an individual experienced in surgical pathology who also examines the light microscopic sections and knows the specific differential diagnostic problems related to the case under investigation.

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Amyloid stain

Staining for amyloid is one of the most useful special techniques. The Congo red stain and examination with nonpolarized and polarized light are the most frequently used techniques. The dye binds the beta-pleated arrangement of amyloid and has no chemical specificity.

This is seen as a reddish deposit under nonpolarized light. Green birefringence is present when the sections are examined under polarized light. Excessive or prolonged exposure to Congo red can cause binding to the tissue that is unrelated to the presence of amyloid.

Hemosiderin

The most frequently used stain for hemosiderin is an iron stain. In this technique the acid hydrolysis separates the iron from the protein. Potassium ferrocyanide is subsequently used and forms an insoluble ferric ferrocyanide also known as Prussian blue.

Melanin stain

The most frequently used Fontana-Masson technique is a silver stain without an external reducing substance (argentaffin reaction). In this technique, metallic silver is directly reduced by melanin..

Calcium stain

Von Kossa’s technique for calcium is another variant of a silver stain. Calcified tissue such as mineralized osteoid is identified by the substitution of calcium with potassium and is reduced to an insoluble black silver precipitate with the photographic developer. Von Kossa’s technique is frequently used on nondeclacified bone sections for histomorphometric assessment of metabolic bone disorders.

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Trichrome Stain

The trichrome stain is frequently used to demonstrate the presence of extracellular substances such as collagen. It is sometimes used in research investigation of soft and skeletal tissue, but is has minimal diagnostic applicabilities. As the name implies, the technique uses three dyes that stain nuclei, cytoplasm, and extracellular matrix, primarily the collagen.

This technique is rarely used in the diagnosis of tumors, but is frequently used for histomorphometric evaluation of mineralized versus nonmineralized osteoid in metabolic bone disease.

Stains for microorganisms

Of a long list of stains used to visualize infectious organisms, only a few are routinely used in diagnostic pathology.

Grams’s stain is used to classify bacteria into two major categories: those that retain crystal violet-iodine dye after alcohol and acetone treatment (gram positive) and those that are decolorized by this technique (gram negative).

The acid-fast stain (Ziehl-Neelsen) is used to document the presence of mycobacteria. The degree of resistance of acid hydrolysis depends on the lipid content in the wall of the bacteria. Bacteria with a high content of complex lipids retain carbolfuchsin after decolorization with acid alcohol.

Other stains occasionally used to document the presence of infectious organisms are

• Grocott’s silver Fungi
• Periodic acid-Schiff-Fungi
• Dieterle’s-spirochetes
• Warthin-Starry -Spirochetes and Rickettsiae.

Argentaffin and argyrophilic stains

Before the advent of immunohistochemistry, argentaffin and argyrophilic reactions were frequently used to disclose the neuroendocrine and neural differentiation of cells. In skeletal pathology, these reactions are used mainly in the diagnosis of metastatic tumors. The Fontana-Masson modification of the argentaffin reaction is used as a stain for melanin.

In the argentaffin reaction, the presence of the phenolic groups, mainly in catecholamines and indolamines, reduces the silver salt concentration and generates an insoluble black precipitate. The argyrophilic reaction requires an external reducing agent, usually hydroquinone or formalin.

The argyrophilic reaction produces the best results when tissue is fixed in formalin-free fixative such as Bouin’s solution. The most frequently used argyrophilic reaction in Grimelius stain.

Reticulin Stains

Reticulin fibers represent fine fibrillary material deposited in the intercellular matrix throughout the body. The major component of these fibres is type III collagen. The histochemical stains used to identify reticulin fibers are silver based, such as Gomori’s, Wilder’s, or Gordon and Sweet’s stains. These stains are nonspecific, and positive reaction is related to the presence of interstitial proteoglycans.

Therefore all elements rich in proteoglycans stain positive with these stains. Before the discovery of cell markers and immunohistochemistry, reticulin stains were used to disclose the patterns of reticulin fibers, to distinguish epithelial and nonepithelial neoplasia, and to subclassify some of the mesenchymal tumors. Epithelial neoplasms are characterized by the presence of reticulin fibers surrounding nests of tumor cells.

In mesenchymal tumors, reticulin fibers are sparsely distributed among individual tumor cells. In addition, some of the mesenchymal tumors have a distinct pattern of reticulin fibers (e.g., longitudinal parallel arrangement in tumors of Schwann cells). In fibrosarcoma, reticulin fibers encircle individual tumors cells. In the era of immuno-histochemistry, reticulin stains are seldom used for diagnostic purposes.

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