HTL Momentrix Staining PDF

HTL Momentrix Staining IONIC, COVALENT, AND HYDROGEN BONDS In ionic or electrostatic bonding, one atom donates or receives an electron from another. This kind of bonding creates acharged particle. An atom that donates an electron becomes positively charged and is called a ca...

HTL Momentrix Staining IONIC, COVALENT, AND HYDROGEN BONDS In ionic or electrostatic bonding, one atom donates or receives an electron from another. This kind of bonding creates acharged particle. An atom that donates an electron becomes positively charged and is called a cation; the atom that receives the electron becomes negatively charged, and is called an anion. This kind of bonding occurs between metals and non-metals. The charged particle attracts dyes with the opposite charge. This kind of binding is seen when salts bind. When two atoms share electrons they form a covalent bond. Covalent bonding is seen in organic compounds, where carbons share electrons with hydrogen, oxygen, nitrogen or other carbon atoms. Hydrogen bonding is a type of weak covalent bond where one of the atoms sharing electrons is hydrogen. Hydrogen bonding is seen in the bond between oxygen and hydrogen in water; it is also the bond between hydrogen and nitrogen in amino acids. HUMAN BODY TISSUE TYPES AND FUNCTIONS The four types of tissue found in the human body are epithelial, muscle, connective, and nerve: Epithelial tissue forms the skin and the interior lining of organs. Epithelial tissue absorbs and secretes materials, provides a protective layer over more delicate tissue, and is a bu ering layer between organs. Epithelial cells can be squamous, cuboidal, or columnar. Epithelial cells may be single layered or strati ed into multiple layers. Muscle tissue is specialized to contract and expand. The three types are cardiac, skeletal, and smooth. Connective tissue holds tissues together into an organ, and holds organs together into systems. Connective tissue includes tendons, ligaments, cartilage, bone and blood. Nerve tissue transmits impulses from the cells of the central nervous system (CNS), made up of the brain and spinal cord, to and from the peripheral nervous system. MUSCLE TYPES The following are the three types of muscle: Skeletal muscle is striated, meaning it appears striped under the microscope. The cells are quite long and usually the nucleus is located at the periphery. Skeletal muscle is a voluntary muscle. Skeletal muscles are attached to bones and are responsible for movement. Cardiac muscle is an involuntary, striated muscle. Each cell has one centrally located nucleus. Cardiac muscles branch on longitudinal sections. Cardiac muscle is only found in the heart, and is unique because of its automaticity. It keeps beating for a while after its removal from the chest, and two cardiac cells placed together beat in unison. Smooth muscle is not striated. It is involuntary and commonly found in layers. The cells are long and have a centrally located nucleus. Smooth muscle is found in many internal organs, including blood vessels and intestines. Muscle can be demonstrated under light microscopy using a basic H&E stain. However, special stains such as Mallory PTAH, which demonstrates striations or enzyme methods, provide further detail. TYPE I AND TYPE ll MUSCLE FIBERS Muscle bers are de ned by the speed at which they react, whether they are oxidative or not, and whether they are glycolytic or not. Type I muscle bers are "slow-twitch" bers that use an aerobic (oxidative) metabolism. They contain myoglobin, which is a protein that stores energy. They have a rich blood supply, are rich in oxidative enzymes, and low in glycolytic markers and ATPase activity. On average, one third of human muscle bers are Type I. Type Il muscle bers are "fast- twitch" bers that rely on anaerobic (no oxygen) metabolism. They do not have a good blood supply, or oxidative enzymes. However, they are rich in glycogen and the enzymes that help glycogen supply energy. They are also called fatigue resistant bers. Two thirds of muscle bers in humans are Type II. Type I bers are further divided into Type IA, 1IB, and IC based on their ATPase activity. fi fi fi fi fi fi fi fi fi fi ff fi  MYOFIBRILS, ACTIN, MYOSIN, SARCOLEMMA, SARCOPLASMA, PERIMYSIUM, ENDOMYSIUM, AND EPIMYSIUM Myo brils are cylindrical bundles of actomyosin laments found in muscle cells. Actin and myosin are the proteins responsible for cell contraction. The cell membrane of a muscle cell is called the sarcolemma. The cytoplasm of a muscle cell is called the sarcoplasma. The sarcoplasma is led with organelles, including mitochondria, endoplasmic reticula, microtubules, intermediate laments, ribosomes and Golgi apparatus. Muscle bers are gathered in bundles called fascicles, which are surrounded by a dense layer of collagen called the perimysium. The network of ne collagen bers that separate individual muscle bers is called the endomysium. The connective tissue sheath surrounding a whole muscle is called the epimysium. CONNECTIVE TISSUE Connective tissue is one of the four types of tissue found in the body. Its purpose is to support other tissues and organs, and as the name implies, connect them. Types of connective tissue include connective tissue proper, cartilage, bone, and blood. Connective tissue is made up of cels, bers, and an amorphous ground substance made of mucopolysaccharide. There are three types of bers: Collagen; elastic; and reticular. The cell types are: Fibroblasts; mesenchymal cells; adipose (fat) cells; mast cells; macrophages; plasma cells; and blood cells. The types and numbers of cells and bers found in each of the connective tissue types di er according to the function of the connective tissue. For example, blood is made up of red cells, white cells, platelets (cell fragments), and serum. Blood and lymph are in a liquid matrix, so unlike the other connective tissues, are not usually within the scope of the histology laboratory. FIBROBLASTS, MESENCHYMAL CELLS, ADIPOSE CELLS, MAST CELLS, MACROPHAGES, AND PLASMA CELLS Fibroblasts are the most common cells in connective tissue. They secrete bers and ground substance. Their cytoplasm is not usually visible without special staining. The nuclei are seen in sections as large, attened, oval shapes. Mesenchymal cells are similar to broblasts, but they are in a precursor stage. Mesenchymal cells will di erentiate into other types of connective tissue cells. Adipose cells store fat. Fat molecules occupy almost the entire cell, pushing the nucleus and cytoplasm to the edge, giving adipose cells a characteristic signet ring appearance. Mast cells contain granules that secret histamine and heparin. These granules often obscure the nucleus. They are often found along the lumens of blood vessels. Macrophages are phagocytic cells derived from monocytes. Digested material can often be seen inside of the macrophage. They appear as irregularly shaped cells with a small, darkly stained nucleus. Macrophages are found in connective tissue, lymphatic tissue, and in the liver. Plasma cells produce antibodies. They are oval cells with a basophilic cytoplasm and dense nucleus. CARBOHYDRATES Carbohydrates are organic compounds that contain carbon, hydrogen and oxygen. They include sugars, starches, and cellulose. They are commonly classi ed as either monosaccharides or polysaccharides, depending on the con guration of the compound. Monosaccharides are simple sugars made up of one base unit of sugar. Glucose is an example of a monosaccharide (simple sugar). Polysaccharides are more complex, made up of two or more simple sugars. Cellulose, starch, and glycogen are examples of polysaccharides. Glucose is soluble in water and can be chemically measured in blood. It cannot be demonstrated ni tissue. Long strings of glucose molecules are stored in the liver as the polysaccharide glycogen. When muscle cells require glucose, the liver releases glycogen. When glycogen reaches the muscle cells, it is broken down into glucose molecules and used for energy. Many carbohydrates are found in the body in combination with other molecules, such as proteins or lipids. NUCLEIC ACIDS FOUND IN CELLS Nucleic acids found in cells are either DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). DNA is found exclusively in the cell nucleus. There are several types of RNA. Small amounts of fi fi fi fi fl fi fi ff fi fi ff fi fi fi fi fi fi fi  RNA are found in the nucleolus inside of the nucleus. Most RNA is in the cytoplasm, speci cally in ribosomes and in association with granular endoplasmic reticula. The Feulgen method is used to demonstrate DNA. First, DNA is hydrolyzed with hydrochloric acid, and then the aldehyde groups generated by the hydrolysis are demonstrated using Schi reagent. RNA is not reactive in the Feulgen reaction. Do not use tissue xed with Bouin solution for the Feulgen reaction. Methyl Green- Pyronin Y reagent will stain both DNA and RNA, but they will stain di erent colors. The DNA will be stained with the methyl green. The RNA will be red from the pyronin. NERVOUS SYSTEM The nervous system is made up of neurons (nerve cells) and supportive glial cells. Each neuron has an axon ber up to 20 cm long that carries outgoing (e erent) messages to target cells. Each neuron has a short dendrite "tree" to receive incoming (a erent) chemical messages over synapses. The nervous system's cells and cel processes have special properties of irritability and conductivity, which generate and transmit impulses throughout the body. Structurally, the nervous system is divided into the Central Nervous System (CNS), which is made up of the brain and spinal cord, and the Peripheral Nervous System (PNS), which is the rest of the nervous system. Functionally, the nervous system is divided into the Somatic Nervous System, which is under voluntary control, and the Autonomic Nervous System, which is involuntary (visceral). Nerve tissue is referred to as gray matter, made up mostly of nerve cell bodies, or white matter, which are primarily myelinated bers. GLIAL CELLS Glial cells are the supportive cells of the nervous system, comparable to connective tissue, which have a variety of functions. They support neurons, produce myelin, act as phagocytes, and are responsible for transporting gases, uids, and nutrients in and out of cells and tissue. There are four types of cells glial cells: Oligodendroglia are small cells with a dense nucleus and cytoplasm. They produce and maintain myelin. Astrocytes or astroglia are star-shaped and larger than oligodendroglia. Their processes are often close to blood vessels, and they play a role in transporting gases, nutrients and uids within the central nervous system, as well as providing for nerves ber tracts. Microglia are phagocytes that remove debris from the central nervous system. Ependymal cells are epithelial cells that line that cavities of the brain and spinal cord. NEURONS Neurons are nerve cells found primarily in the gray matter of the Central Nervous System (CNS). Special staining techniques will di erentiate the various components of the neuron. Neurons are among the largest cells in the body, and they vary in shape depending on where they are located. There is usually only one nucleus, which frequently has many prominent nucleoli. Large aggregations of basophilic material are found in the cytoplasm. This is called Nissl substance, and is actually rough endoplasmic reticulum and RNA. The amount of Nissl substance varies with the activity of the cell. Neuronal processes are extensions of the cel body that function to conduct impulses to and away from the cel body. The two types of processes are axons and dendrite. Each cel has only one axon, which conducts impulses away from the cel body, often over very long distances. Dendrites are shorter, branched arms, and they bring impulses back to the cell. MYELIN Myelin is a lipid-protein axon insulator that increases impulse conduction speed along nerves. Multiple sclerosis patients' demyelinated nerves do not conduct impulses smoothly. Myelin lipid is lost during routine processing for para n embedding. Use either Weil stain or Luxor fast blue to demonstrate myelin. Cut sections for myelin staining 10-15 microns thick. The Weil method is a regressive stain that uses a mordant-hematoxylin solution to attach to the phospholipids of myelin. Two di erentiation methods are common:.1 Use ferric ammonium sulfate to remove the excess dye and monitor decolorization visually; 2. Use borax ferricyanide, an oxidizer, to remove excess hematoxylin lake and form a colorless oxidation product, and monitor decolorization microscopically. The myelin is blue and the background is tan. Luxor fi fi fi ff ff ff fl fi ffi ff ff fi ff fl  fast blue is a sulfonated copper phthalocyanine, similar to Alcian blue, but is alcohol-soluble. When myelin has an acid-base reaction with Luxor dye, it stains blue and the background is colorless. PROPERTIES OF ENZYMES TO CONSIDER NI HISTOCHEMICAL METHODS Enzymes are proteins. Each protein has an optimal pH, temperature and salt concentration at which it works best. Enzyme activity can be destroyed in tissue that does not provide the optimal environment for the desired enzyme. Most enzymes function best around Hp 70., with the exception of alkaline and acid phosphatase. Fixatives, especially strong acids, alkalis, and those that contain heavy metals, destroy enzyme functions. That is why cryostat sections are most often used for histology. Temperatures over 56 C° destroy proteins, and os does freezing. Many proteins are changed when frozen and thawed, os avoid freeze-thaw cycles. Enzymes only react with one speci c substrate, in a lock and key arrangement. fI a di erent substrate is added, or the substrate is less than optimal, on enzyme activity will be demonstrated. ENZYME PROCEDURES DONE NO MUSCLE NADH Diaphorase is a dehydrogenase used to identify Type I and Type I bers. Type I bers stain dark, while Type Il appear light. It can also be used to evaluate architectural changes from muscle disease. SDH is succinic dehydrogenase, an enzyme of the Krebs' citric acid cycle, which is a series of chemical reactions mediated by a series of enzymes that produces energy through aerobic respiration. Energy is produced in mitochondria so demonstrating SDH also identi es mitochondria, and is a way of show that respiration is taking place. ATPase is a phosphatase in muscle that is required for energy production. Evaluating the presence and amount of APase at di erent pH levels di erentiates types of muscle bers. Acdic phosphatase is present in necrotized or in amed tissue. It is a considered a lysosome marker. Alkaline phosphatase is present in regenerating muscle bers. FACTORS THAT INFLUENCE RATE OF ENZYME REACTIONS Heat in uences the rate of a chemical reaction. A reaction takes place fasters of higher temperature. Chemical reactions have an optimal temperature at which the reaction proceeds at a maximum rate. Chemical reactions in the human body are optimal at body temperature (98.6, 37). Catalysts can speed up or slow down the rate of a chemical reaction, without actually entering into it. The catalysts do not change during the reaction. Enzymes are proteins that act as biological catalysts. An enzyme temporarily combines with a substrate, producing a complex. The substrate changes in some way, and is then released from the complex as a new product. The enzyme is released unchanged, and is available to interact with a new supply of substrate. COENZYMES AND COFACTORS Coenzymes are organic compounds that work together with an enzyme to in uence the rate of a catalytic reaction. Some coenzymes are vitamins; some directly participate in the chemical reaction by acting as a cosubstrate. Cofactors are chemicals that speed up the action of an enzyme. They may be complex proteins or simple metallic ions. Trace amounts of metals, such as manganese, copper, zinc, iron, and cobalt are required for many enzyme reactions. Cofactors and coenzymes can change during the catalytic reaction; they are often the recipient of what is removed from the substrate, such as oxygen or hydrogen. OXIDOREDUCTASES Oxidoreductases are enzymes that catalyze oxidation and reduction reactions. Oxidation is rusting and ageing, the chemical process where oxygen is added to a compound or hydrogen, as electrons are removed. Reduction is where oxygen is removed and hydrogen, or electrons, are added. Oxidoreductases include oxidases, peroxidases, and dehydrogenases. NADH Diaphorase is a dehydrogenase used to identify Type I and Type Il bers. Type I bers will stain dark, while Type Il will appear light. It can also be used to evaluate architectural changes in muscle from disease. Un xed, air-dried, frozen sections cut at 10 microns are incubated in a solution of bu ered NADH (nicotinamide adenine dinucleotide) and a tetrazolium salt (NBT). Sites of enzyme activity will be dark blue because as the NBT receives hydrogen, ti forms a blue precipitate. Artifacts that form when the tissue is dehydrated in alcohol, cleared in xylene, and mounted in a synthetic medium can mimic the precipitate. fi fl ff ff fi fi fl ff fi fi fi fi ff fl fi fi  TRANSFERASES Transferases are enzymes that move a functional group from one compound to another. A transferase identi ed in muscle tissue is phosphorylase, used to diagnose McArdles' disease, a glycogen storage disease that results from de ciency of phosphorylase. When phosphorylase is incubated with glucose-1-phophate, an unbranched polysaccharide produced. The length of the chain is proportional to the amount of enzyme present. Alcohol is added to prevent the resulting chain from branching and glycogen is present as a primer. Gram's iodine solution is used as the stain to produce a brown, blue, or purple color that varies with the amount of enzyme present. The reaction is done on frozen tissue that has been xed in cold acetone for ve minutes immediately before incubation with substrate. HYDROLASES Hydrolases are enzymes that catalyze the addition or removal of water from a substrate. One group of hydrolases is esterase, which hydrolyze linkages in a classi cation of organic compounds called esters. Many esters are derived from carboxylic acid. Examples of an ester are Alpha Naphthyl acetate, which is often used as a substrate for demonstrating esterase activity. Adiazonium compound, such as hexazotized pararosaniline, is added to the substrate and forms an insoluble azo precipitate. Phosphatases are hydrolytic enzymes that act on substrates containing phosphate. ATPase is responsible for converting adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The reaction releases the energy for cellular metabolism. ATPase is found in the mitochondria of all cells, and bound to myosin in muscle. Alkaline and acid phosphatase are also examples of phosphatases present in muscle. Phosphatase activity is used to diagnosis muscle disease. ANTIGEN, ANTIBODY, AND IMMUNOGLOBULIN An antigen is a substance that produces a response from the body's immune system. Antigens are usually proteins or polysaccharides. They are usually foreign to the body, but they can be endogenous. Endogenous examples are the antigens present on red blood cell surfaces, which account for A, B, AB, and O blood types. Exogenous antigens include bacteria and viruses. The immune system responds to an antigen by producing antibodies. Antibodies are also called immunoglobulins because they are proteins (globulins) responsible for immunity. Antibodies are produced by B lymphocytes. Since antibodies are proteins, they can also be antigens when injected into a di erent species where they are foreign, and that species responds by producing anti-antibodies. For example, when a rabbit si injected with human IgG, the rabbit produces anti-human IgG for specimen testing. STRUCTURE AND CLASSES OF ANTIBODIES Antibodies are immunoglobulin proteins produced by B lymphocytes as protection for the body against an invader. An antibody is produced to react with a speci c antigen. Each antibody has a speci c structure and function, but all antibodies have a similar y-shape. The legs of the Y di er. The short legs of the antibody, called the light chains, can have one of two structures, either kappa or lambda. The light chains portion of the antibody react with the target antigen. The speci c site where the antigen and antibody react with each other is called the epitope. The longer chains of the Y, called the heavy chains, can be one of ve di erent classes: Gamma, alpha, mu, delta or epsilon. The heavy chain determines the classi cation of the antibody. Antibody classes are referred to as IgG, IgM, IgA, ID, and IgE. The classes have di erent functions in the immune system. MONOCLONAL AND POLYCLONAL ANTIBODIES A polyclonal antibody is a mixture of antibodies directed against portions of the same antigen. For example, the protein human cytokeratin is injected into a rabbit as an antigen. The rabbit produces a mixture of antibodies that respond to di erent parts of the cytokeratin molecule. Monoclonal antibodies are al produced by the same clone of cells, so they are all directed against one site on the antigen. They are produced in hybridomas grown in cell culture or mice. Monoclonal antibodies are preferred over polyclonals because the immunohistochemical stain will be more speci c. There is usually less background staining and less variability between fi ff fi fi fi fi ff fi fi fi ff ff ff fi fi fi  batches. Polyclonal antibodies are less expensive than monoclonals, but monoclonals are more speci c and give higher quality staining. CATEGORIES OF BACTERIA Round or spherical bacteria are called cocci. They may be found in pairs (diplococci), chains (streptococci) or clusters (staphylococci). Diseases caused by cocci include pneumonia (Streptococcus pneumoniae) and meningitis (Neisseria meningitides). Rod shaped bacteria are called bacilli. Perhaps the most well-known gram-negative bacillus is E. coli, which is part of the normal ora in the human intestine. Water contaminated with antibiotic-resistant E. coli 0 1 5 7 : 7 causes gastrointestinal disease, purpura, and kidney destruction, particularly in babies and the very old. Clostridium tetani, a gram-positive bacillus, causes tetanus. Spirochetes are very primitive, spiral-shaped bacteria that look like vibrating springs or phone cords. They move with axial sheaths. Treponema pallidum is a spirochete that causes syphilis. Borrelia burgdorferi spirochetes carried by ticks cause Lyme disease. MYCOLOGY, MYCOSIS, HYPHAE, SEPTA, MYCELIUM, AND PSEUDOHYPHAE Mycology is the science that studies fungi. There are several kinds of fungi, ranging from mushrooms to the yeast responsible for fermentation. Several kinds of fungi cause disease and therefore medically signi cant. Mycosis (plural mycoses) is a disease caused by a fungus (plural fungi). Pathogenic fungi species include Coccioides, Candida, and Histoplasma. Some fungi, such as Pneumocystis carinii, are classi ed as opportunistic because they only produce infection in an immune- compromised host, like an AIDS patient. Fungi are unicellular or multi- cellular microorganisms that have a true nucleus and a chitinous cell wall. Hyphae are lamentous outgrowths of the fungal cel wall that can further divide into partitions known as septa. When hyphae mesh together, the resulting network structure is called mycelium. Fungi can be further classi ed according to their reproductive mechanism. Yeasts are a kind of fungi that reproduce through budding. Cryptococci are yeast. Yeast-like fungi, such as Candida albicans, reproduce by forming pseudohyphae that produce long lamentous buds that do not detach from the parent cell. Chart on Page 72 DYES All dyes are aromatic compounds, indicating that they all possess a benzene ring at the core of their structure. Benzene is an organic compound with six carbon atoms arranged in a hexagonal shape. Benzene itself is colorless, or at least out of the range of human vision. When the hydrogen atoms attached to the six carbon atoms are replaced with other chemical groups, the properties of the ring change and produce visible color. The additional groups, called chromophores, are usually double bonded, and contain carbon, oxygen, sulfur, and nitrogen in various combinations. All chromophores have an a nity for hydrogen. Some will bind with substances to produces color without an additional chemical. Others require an auxochrome, which enhances the absorption of light in a speci c wavelength to improve color production. Amino and carboxyl groups are common auxochromes. ACID AND BASIC DYES The terms acid and base can be confusing when applied to dyes, because rather than referring to pH, they refer to the charge on the dye itself, and identify what kinds of tissue component the dyes will stain. Basic dyes are attracted to basophilic (base- loving) compounds, such as acids or acid proteins. A basic dye is one that has a positive charge, or is cationic. Three examples are hematoxylin, crystal violet and safranin. Basophilic cell components are DNA in the nucleus and ribosomes, which contain RNA, in the cytoplasm. Acid dyes are attracted to acidophilic compounds, such as the negatively charged proteins of the cytoplasm, muscle, and connective tissue. Acids dyes are negative or anionic. Eosin, picric acid, and Orange Gare acid dyes. Acidophilic cel components are the proteins of the cytoplasm, red blood cels, and connective tissue. AMPHOTERIC, METACHROMATIC, AND ORTHOCHROMATIC Amphoteric means that a substance can have a positive or negative charge depending on the pH of the solution it is in. Proteins are amphoteric because their overall charge depends on the pH of the solution they are in. A change in pH will result in a protein that attracts di erent dyes. fi fi fl fi fi fi fi ffi fi ff  Some dyes are amphoteric. The pH of the dye solution will determine whether it acts as an acid or basic dye. Examples of amphoteric dyes are hematein and lithium carminate. Metachromatic means that the color a dye imparts to the tissue or tissue component is not the same as the color of the dye itself. For example, toluidine blue stains mast cells a pink to rose color, even though the dye itself is blue. When the tissue stains the same color as the dye, it is said to be orthochromatic. STAINING MORDANT, LAKE, CHROMOPHORE, AND AUXOPHORE A mordant is a substance, usually a metal, that provides a link between the dye and the tissue. The mordant combines with the dye to form a dye lake. A chromophore, also called the chromogen, is the chemical group on a dye that gives the dye its color properties. Dyes are organic compounds that contain chromophores. These chromophores are combinations of the compounds in organic chemical such as carbon (C), oxygen (0), nitrogen (N), and sulfur (S). A chromophore needs an additional chemical group on the molecule that enables the chromophore to stain tissue components. This additional group is called an auxophore. PROGRESSIVE AND REGRESSIVE STAINING Most staining is progressive. A dye comes in contact with the tissue and remains in contact until the desired intensity of color is achieved. Then the reaction is stopped by removing the tissue from the staining solution. A progressive stain requires careful attention to both the concentration of dye and the mordant. Staining is highly selective, requires more careful control, and may require longer reactivity times. In a regressive stain, the tissue is rst overstained, and then stain is removed to the point where only the desired tissue component is stained. This is referred to as di erentiation, or decolorization. The amount of stain removed needs to be controlled, so that too much stain is not removed. This can be done by frequently observing the tissue under the microscope. The advantage of a regressive stain is that it can often be done very quickly, even with the decolorization step. POLYCHROMATIC STAINING A polychromatic stain contains stains of several colors, each of which stains cellular components selectively. The most well-known of the polychromatic stains combines the basic dye methylene blue with the acid dye eosin. As the stain ages, other dyes are formed from these, especially in an alkaline pH. These are called Romanowsky- type dyes, and there are several variations. Romanowsky stains give a wide variety of colors and are good for di erentiating blood cells in both bone marrow and peripheral blood smears. Giemsa stain is a common polychromatic stain used in histology. May-Grunwald Giemsa can also be used for some microorganisms. Wright's stain is the most common for peripheral blood smears in routine hematology. It stains red blood cells pink; platelets violet to purple; lilac acidophilic and dark basophilic granules in the cytoplasm of white blood cells; and produces various colors of blue in lymphocytes. EXOGENOUS AND ENDOGENOUS PIGMENTS Exogenous pigments are colored substances that come from outside the body to form deposits in tissues. Exogenous pigments are not normal inclusions. Examples are: Carbon (air pollution); asbestos bers (miner's lung); tattoo ink; silica and talc used to cut street drugs. Endogenous pigments are produced by the body and are categorized by their origin. Endogenous pigments are either hematogenous or non- hematogenous. Non-hematogenous pigments are divided into two categories: Those that derive from lipids, and those that do not. Lipofuscin is a yellowish-brown lipid pigment found in aged cells when the patient lacks Vitamin E. Use Oil 0 red and Sudan Black stains to see lipofuscin in old cells. Melanin is a non- lipid pigment made by melanocytes that protects us from sunburn by UV radiation. ANTHRACOTIC, HEMATOGENOUS, HEMOSIDERIN, BILIRUBIN, AND JAUNDICE Anthracotic pigments derive from carbon, such as coal dust, tobacco smoke, and industrial pollution. Anthracotic pigments are usually found ni the lungs and lymph nodes. Carbon ff fi ff fi particles are not soluble in concentrated acid, which helps to di erentiate them from other pigments. A hematogenous pigment derives from blood. The main pigment of blood is hemoglobin, which is made up of heme and globulin. The heme portion contains iron. When iron is removed from heme, it binds with proteins to form the yellowish-brown pigment hemosiderin. Hemosiderin is found primarily in bone marrow. Heme is broken down in the liver, where old red blood cells go for recycling, and produces various bile pigments, including biliverdin and bilirubin. The yellow color of bilirubin is responsible for jaundice (icterus) in liver patients. Normal bilirubin is 0.5 mg/dl. When it builds up to 1.5 mg/dl in hepatitis, cirrhosis, malaria, pancreatic cancer, and hemolytic uremic syndrome, the skin and eye sclera turn yellow. ENZYMES AND SUBSTRATES An enzyme is a biological catalyst that changes the rate of chemical reactions in a living organism. The chemical reaction might still occur without an enzyme, but the speed of the reaction would be di erent. A substrate is the compound on which an enzymes acts. Each enzyme recognizes and catalyzes a speci c substrate. Enzymes are proteins. Naming conventions add the su x -ase to the name of the speci c substrate the enzyme acts on. For example, hydrogenase catalyzes the addition or removal of hydrogen, and protease acts on protein. Histological enzyme techniques identify and measure the activity of some diagnostically signi cant enzymes, especially in muscle tissue. Immunohistochemical (IHC) methods use antibodies as reagents to identify speci c antigens, and enzyme-labeled molecules detect the antigen-antibody complex. Enzymes are also sometimes used in IHC to open up antigenic sites that may be masked during xation. ENZYME HISTOCHEMICAL METHODS Enzyme histochemical techniques use chemical reactions to demonstrate enzyme physiological activity, primarily in muscle tissue. They can be used to show the presence, activity, or amount of an enzyme, as well as to study changes in pH, ion concentration, and other physiological determinants. Some pathological changes in muscle can be seen with an H&E, but others, such as the identi cation of ber types, require histochemical methods. The enzyme reaction takes place directly on the tissue to be evaluated. Enzyme remain active in frozen tissue (and sometimes in para n, although not as much). Enzyme methods require a substrate, energy source, and away to visualize the reaction. WAYS HYDROLYTIC ENZYMES CAN BE DEMONSTRATED NI TISSUE When an enzyme acts on a substrate, simultaneous capture or coupling releases a reaction product. The product is then captured by a diazonium salt or a metalic ion. The resulting product may be visible as a colored precipitate, or require further development for visualization. If the reaction products are both insoluble and visible, they require no further agents to be added. This is called a self-colored substrate. The incubation of an enzyme and substrate may produce an insoluble and invisible reaction product. Another step following incubation is required to visualize the reaction product. This is called post incubation coupling. An intramolecular rearrangement may result when a soluble substrate is hydrolyzed to an insoluble, colored end product. TEP OF PROTEINS The isoelectric point (IEP) i s the pH a t which a protein is neutral. When a neutral protein is placed in an electric eld, it is not attracted to either the positive or negative pole. Changing the pH will make the protein more positive or more negative and the protein will migrate. A protein with an overall positive charge will migrate to the negative pole; a negatively charged protein will migrate to the positive pole. The IEP for most proteins is approximately 6. In staining, this means that the pH of the solution will a ect the charge on the proteins and staining results. fI the tissue is in a solution is below pH 6, the proteins will be acidophilic and attract an acid dye, such as eosin. If an eosin solution is not below pH 6, the eosin will not attach to the protein, and the cytoplasm will not stain. Optimal pH for staining with eosin is 4.6-5.0. fi ff fi ffi fi fi ff fi fi fi ff fi ffi  EFECTS FOPH, TEMPERATURE, DYE CONCENTRATION, AND SALT CONCENTRATION ON STAINING Most staining takes place because of the attraction of dye to tissue components, based on the charge of the component. Acid dyes are attracted to basic components and vice versa. The charge on proteins in tissue depends on the pH of its surroundings. There is an optimal pH at which the tissue will not overstain or understain. Chemical reactions speed up with an increase in temperature. Therefore, staining will take place at a faster rate in higher temperatures because tissue swells when it is higher than body temperature (37 °C), and it becomes more penetrable. The greater the concentration of dye, the faster the reaction will occur. Salts can have both positive and negative e ects on staining. Salt ions other than the dye can bind with tissue components, making them unavailable for reaction with the dye. In other cases, salts may act as catalysts to increase the speed of a staining reaction. BASEMENT MEMBRANE The basement membrane, also called basal lamina, separates epithelial and connective tissue. The basement membrane supports epithelial tissue and attaches tissues to one another. It also lters molecules. For example, the basement membrane of the capillary endothelium of the kidney allows some molecules across and provides a barrier to others. Muscle cells contain a structure similar to the basement membrane. Although the basement membrane is made up of both collagen and glycoproteins, PAS, which demonstrates carbohydrates, is often used to stain it. To visualize the glomerular basement membrane of the kidney, cut sections of 1-2 microns in thickness. Use 10% neutral bu ed formalin or Bouin solution as the xative. Kidney can also be used as a control when staining for the basement membrane in other tissue. Periodic acid- methenamine silver reagent can also be used to stain the carbohydrate components of basement membranes. Silver ions bind to the carbohydrates, and then the aldehyde groups reduce the silver, producing a visible silver precipitate. DYES FOR LIPIDS Commonly used fat stains are Oil Red 0 and a variety of dyes known as Sudan dyes, such as Sudan Black B or Sudan I. These highly-colored dyes are more soluble in fat than in the other solvents generally used to prepare dye solutions, such as ethanol or water. Fats stains are physical stains, meaning that the dye is soaked up and dissolved in the fat. No chemical reaction takes place. Frozen sections are used to demonstrate fat in tissue because the fat is soluble in the reagents needed for para n processing, and it would dissolve. Similarly, the sections cannot be mounted in resinous materials. An aqueous mounting media must be used. Osmium tetroxide is the only fat stain that can be used in para n tissue. The osmium tetroxide combines chemically with the fat in a reaction that is not totally understood. However, the fat is not always well preserved, and staining is sometimes less than optimal. MELANIN Melanin is brownish-black pigment that imparts color to skin, eyes, hair, and parts of the CNS. Melanocytes produce melanin. Skin cancer (melanoma) that spreads to the liver sheds melanin in the patient's urine. Melanin obscures cell structures if it is present in large amounts. Bleach the melanin with oxidizing agents, such as 10% hydrogen peroxide or 0.25% potassium permanganate, to decolorize it. Immunohistochemical stains are commonly used today to identify melanin and melanomas. Melanin dissolves ni strong alkali, but not in weak alkali, acid, or organic solvents. Melanin is strongly argenta n when Fontana-Masson Stain is used; however, Fontana-Masson is not speci c for melanin. Demonstrate melanocytes in frozen tissue using the reagent DOPA-oxidase. Schmorl Technique takes advantage of the reducing properties of melanin and other reducing substances in tissue to stain light green with a ferric chloride-potassium ferricyanide solution. MINERALS NORMALlY FOUND IN CELLS Minerals are non-organic materials, usually metals, that have a speci c chemical content and a characteristic crystalline structure. Three minerals found in tissue are iron in its ferrous and ferric forms, calcium, and copper. Do not use xatives that contain metals for mineral detection, as they produce a false-positive result. Use these preferred xatives: Formalin- alcohol or 10% neutral bu ered formalin. Microincinerate a tissue section at 650C° to obtain an fi ff ff fi ffi ff fi ffi ffi fi fi fi  inorganic residue. Detect calcium with Alizarin Red S or with von Kossa Calcium Stain. The Alizarin Red S chelates the calcium at an acid pH and forms an orange-red complex, which is birefringent. Von Kossa is an indirect reaction for calcium, because it does not detect calcium itself, but causes calcium salts in the tissue to react with silver nitrate, which is then reduced by bright light to form a metallic silver deposit. Detect copper in tissue with the Rhodanine method. The copper stains red. CHROMAFFIN AND ARGENTAFFIN GRANULES Chroma n cells contain granules that stain well with chromium. An example of chroma n cells is those that release adrenalin from the adrenal gland. Argenta n cells contain granules that stain well with silver. An example of argenta n cells is epithelium from the small intestine and appendix. Fixation is an important concern for preserving these cytoplasmic granules. Chroma n granules must be xed in dichromate solution, so the stained granules turn brown. Chroma n can be seen using a modi ed Giemsa, Schmorl's, or Wiesel's stain. Argenta n granules in the GI tract are destroyed by alcoholic xatives. Use Fontana-Masson, Diazo, or Schmorl's stains, or auto uorescence on argenta n cells, and the granules will turn very dark brown or black. FERRIC AND FERROUS IRON Iron is found in two forms, ferrous (Fe?+) and ferric (Fe3+). Ferrous iron is toxic and not normally stored in cells. It usually appears in children poisoned with iron supplement tablets. Ferrous i r o n stains with Turnbull blue reaction. The reagent is acidic potassium ferricyanide, and the ferrous iron forms a bright blue precipitate called Turnbull blue. Ferric iron is normally present in tissue, usually bound to protein to form hemosiderin. Ferric iron is concentrated in bone marrow, the spleen, and in decaying, hemorrhagic material. Excess ferric iron interferes with organ function, and is seen in the disease hemochromatosis. The Prussian blue reaction uses potassium ferrocyanide in acid, also known as Perl's iron stain, and ferric iron precipitates out as a blue deposit. (Note that the reagents in these two methods are very similar, but not identical.) Hemochromatosis patients require monthly venipunctures to draw a pint of blood and reduce the iron build-up. STAIN PROBLEMS NUCLEI TO LIGHTLY OR DARKLY STAINED, HAZY, RO REDDISH-BROWN Nuclei may not stain dark enough if the slide is not left in hematoxylin long enough, or if di erentiated for too long, or if the hematoxylin is over-oxidized. If the nuclei are too dark, it is just the opposite: The time in hematoxylin is too long, and the di erentiation time is too short. Dark nuclei may also be seen in sections that are cut too thick. Hazy nuclei can be the result of too much heat in an automated tissue processor, or if the tissue is kept in hot para n too long. Hazy nuclei could also be caused by incomplete xation, where the xative has not had time to penetrate into the nucleus. Old hematoxylin that has become oxidized will stain nuclei reddish- brown. PROBLEMS SEEN FOLLOWING COVERSLIPPING THE SECTION The following problems may be seen after coverslipping the section: If stain leaches from the slide following coverslipping, you used the wrong mounting media. For example, precipitates that are soluble in organic solvents form as the end products of many immunohistochemical reactions, so an aqueous mounting media must be used. If water bubbles appear under the coverslip, the sections are not completely dehydrated. Water is left on the slide and trapped in the mounting media. Remove the coverslip, dehydrate, clear, and remount the slide again. If the slide will not focus, it may simply be because there is mounting media on top of the coverslip. Remove the dirty coverslip and apply a new, clean one. If mounting media retracts from the edge of the coverslip, then the coverslip may have warped, or the resinous mounting media was thinned with too much xylene. Remove the coverslip and remount with an appropriate prepared media. PALE CYTOPLASMIC STAINING AND BRIGHT (RIGHT) CYTOPLASMIC STAINING fi ff ffi ffi ffi fi fl fi ffi fi ffi ffi fi ffi ff ffi ffi  If the cytoplasm on an H&E-stained slide is very pale pink, and red blood cells also stain very lightly, the most likely reason is that the eosin solution is not at the correct pH. Eosin should be between pH 4.6-5. When the pH is above 5, the staining is pale. Incorrect pH results if the solution was incorrectly prepared, or because excess ammonia from blueing is carried over into the eosin. Correct the pH by adding acetic acid. Pale staining can also result from sections that are too thinly cut or left in eosin for too short a time. Overstained cytoplasm results when the eosin solution is too concentrated or the time in eosin is too long. It may also be seen when slides are incompletely dehydrated or from sections that are too thick. Bright (right) cytoplasmic staining indicates Paget's disease. SMOOTH MUSCLE STAINS FAINTLY WITH MASSON TRICHROME If epithelial cells in a section stained with Mason trichrome stain red, but smooth muscle stains very faintly, there is either a problem with stale reagents, or the tissue was left in the nal acetic acid solution too long. Try another slice with fresh reagents and decrease the vinegar bath time. The smooth muscle should stain red. SLIDES STAINED FOR RETICULIN SHOW IRREGULAR GRANULAR STAINING There are variations of silver stains used for staining reticulin. With al variations, the reticulin should stand out sharply, in a well-de ned, linear pattern. Irregular precipitate that appears granular instead of linear results from dirty glassware or -------------------------------------------------------- old reagents. Silver stains are notoriously nicky. Clean glassware used for silver stains with bleach or a commercial cleaning agent. Incorporate treatment with sodium thiosulfate (hypo solution) to remove silver that is non-speci cally bound to reticulin. PRUSSIAN BLUE STAIN FOR IRON SHOWS NO IRON NI BONE MARROW Iron may dissolve in acid xatives or decalci cation solutions. The removal of iron is an artifact. Bone marrow contains iron and can be used as a positive control. Alcohol or 10% neutral bu ered formalin are the best xatives, although Zenker solution with 3% acetic acid can also be used. SKIN SHOWS NO STAINING WITH FONTANA-MASSON STAIN Fontana-Masson is a stain to demonstrate melanin and argenta n pigments. The preferred xative is 10% neutral bu ered formalin, as alcohols will dissolve the pigments. Also consider over-processing or bad reagents. NO CALCIUM IDENTIFIED NI SECTION STAINED WITH VON KOSSA TECHNIQUE Either no calcium is present in the tissue or there is a problem with the method. Use a control known to have calcium to verify that the stain is working properly. fI the control shows calcium, it may be that the tissue was not xed in alcohol or 10% neutral bu ered formalin. GOBLET CELLS NI THE SMALL INTESTINE DO NOT STAIN WITH MAYER MUCICARMINE Goblet cells are epithelial cells present in the intestine and respiratory tract that secrete mucin, and therefore should stain red with Mayer's mucicarmine. If no staining is seen, consider the quality of the sections used. Autolyzed tissue will not stain. In addition, if the counterstain is too strong, or the tissue has been left in it too long, the mucin will be masked. Metanil Yellow solution is commonly used as a counterstain and produces an intense yellow color. Hematoxylin lakes are often used. GLOMERULAR BASEMENT MEMBRANES STAIN WEAKLY WITH PAS Kidney is used as a positive control for PAS because its glomerular basement membrane contains carbohydrates. fI the membrane does not stain red, showing a positive PAS, carbohydrate residues may have been incompletely oxidized to aldehyde groups by the periodic acid. Also, the Schi reagent, which produces the color, may have expired. The xative used may have contained chromate, which will overoxidize the reactive groups during xation and reduce the number of reactive groups available for staining. ALKALINE AND ACID PHOSPHATASE Phosphatases are hydrolytic enzymes that break the bond between a phosphate group and an alcohol. Phosphatases are found throughout the body, and serum measurements often are used to assist in diagnoses. Acid and alkaline phosphatase (ALP) are both found in lysosomes. As their names indicate, they di er in their optimal pH levels. Acid phosphatase is present in fi fi fi ff fi ff fi ff fi ff fi fi fi fi fi ffi ff fi necrotized or in amed muscle tissue. It is a considered a lysosome marker. Alkaline phosphatase is present in ------------------------------------------------------- regenerating muscle bers. To demonstrate both enzymes, cut un xed frozen sections to10 microns. Incubate the sections in substrate with a solution containing Naphthol-AS-BI Phosphate. Adjust the acid incubating medium to a pH of 4.7-5.0. Adjust the alkaline medium to a pH of 8.6-8.8. The product of the alkaline reaction will react with fast red violet to produce an insoluble blue azo dye. The acid reacts with hexazotized pararosaniline to produce a red azo dye. ATPASE STAIN INCUBATED AT DIFFERENT PH LEVELS ATPase is used to di erentiate types of muscle bers. Muscle ATPase is pH- dependent. Incubate sections of tissue in barbital acetate bu er solutions, one with a pH of 4.3 and the other with a pH of 4.6. Incubate sections in sodium barbital solutions with a pH of 9.4. Use calcium in the incubating solutions because only ATPase in muscle will stain. Mitochondrial ATPase is inhibited by calcium. At Ph 4.6, Type I bers will stain dark, Type IA are very light, and Type IB are intermediate in intensity of stain. Normal tissue will have near equal amounts of each type, and will have a characteristic checkerboard pattern. At pH 4.3, all the Type Il bers stain light or are unstained, while Type I remain dark. At pH 9.4, only Type Il bers are stained. METHODS OF DIFFERENTIATION FOR REGRESSIVE STAINING PROTOCOL In a regressive stain, the tissue is rst overstained and then color is removed by di erentiation (decolorization) to the desired intensity. Use this method to stain by regression: 1. Rinse the stained tissue in a solution with a di erent pH (e.g., rinse basic dyed tissue in a weak acid solution, which alters the pH and releases some of the dye). Similarly, use a weak alkaline solution to decolor a basic dye. Prepare a weak acid in 70% alcohol to improve control of the decolorization process. 2. Add excess mordant to dissociate the dye from the tissue (e.g., di erentiation after staining with Harris hematoxylin uses ammonium aluminum sulfate as a mordant, to place the section in solution of excess of aluminum ions). 3. Add an oxidizing agent to stop the decolorization that occurs from the oxidization when the color desired has been reached. MUCOPOLYSACCHARIDES The categories of mucopolysaccharides are neutral, acid, and glycoproteins. Another term for mucopolysaccharides is glycosaminoglycan. Neutral polysaccharides contain glucose. Glycogen, starch and cellulose are examples of neutral polysaccharides. Neutral polysaccharides stain with PAS, but not with other carbohydrate stains, such as Alcian blue. Some acid polysaccharides are carboxylated (contain aCO group), such as hyaluronic acid, which is found in connective tissue. Some contain sulfate groups (OSOH) and others contain both sulfate and carboxyl groups. The latter category is more complex and includes chondroitins, keratins, and substances found in tissue stroma, cartilage and bone. The acid mucopolysaccharides do not stain with PAS, but do stain with Alcian blue. -------------------------------------------------------- Alcian blue is a basic dye that is water-soluble, and so is attracted to the acid groups of mucopolysaccharides. The blue color is due to the presence of copper. Glycolipids bind a fatty residue with glycogen. Examples are cerebrosides and phosphatides. This category of mucopolysaccharides is PAS positive. DIASTASE Diastase is an enzyme that breaks down glycogen. Incubate two tissue slides, one with the diastase, and one without. Any glycogen present on the diastase-treated slide will be digested into simple sugars. Wash these simple sugars out of the section and it will not stain with PAS, demonstrating that the color from the undigested section was due to the presence of glycogen. Cervix and liver sections both make good controls for this reaction. Cut two sections for the controls. Like the tested specimen, one will undergo the digestion; therefore, it should not stain with PAS. The other is the undigested slide and remains PAS positive. The xative used on tissue for diastase digestion is important, fl ff fi fi fi fi ff ff fi fi ff fi ff fi  as tissue xed in Boun will resist diastase digestion. Use 10% neutral bu ered formalin, or a formalin-alcohol solution, or absolute alcohol as the xative. IMPORTANCE OF PH NI STAINING WITH ALCIAN BLUE AND EFFECTS OF HYALURONIDASE ON STAINING Alcian blue is a basic dye containing copper, which gives ti the blue color. The dye will stain mucopolysaccharides di erently at various pH levels. At pH 2.5, Alcian blue dye demonstrates acid mucopolysaccharides that are both sulfated and carboxylated, as well as sulfated and carboxylated glycoproteins. At pH 1.0, Alcian blue will stain only sulfated substances. Hyaluronidase is an enzyme that digests the mucopolysaccharides found in connective tissue. Treating the tissue with hyaluronidase can help distinguish between epithelial tissue, which will still stain after treatment, and connective tissue, which will show diminished or no staining. STAINS FOR DIFFERENT CARBOHYDRATES (chart on page 81) ------------------------------------------------------- SCHIFF REAGENT Schi reagent is made from fuchsin, which is pararosaniline and sulfurous acid. Sulfurous acid is made by dissolving sulfur dioxide in water. The resulting reagent is colorless and is sometimes called leucofuchsin. In the Schi reaction, the reagent combines with aldehydes in the tissue to produce a bright red color. The most common use of Schi reagent is in the PAS (Periodic Acid Schi ) reaction t o demonstrate carbohydrates. The periodic acid oxidizes some of the carbohydrates to produce aldehyde groups. Not all carbohydrates will react with periodic acid. Polysaccharides, mucopolysaccharides, glycoproteins and glycolipids will give a positive PAS. These are substances found in connective tissue, membranes and mucus. Hematoxylin is generally used as the counterstain to demonstrate other tissue components. AMYLOID Amyloid is a complex group of proteins that contains a small amount of carbohydrate, mostly acid mucopolysaccharides. Its name is somewhat misleading, as it sounds like it should be a starch, but it is mostly brous protein. Amyloid deposits are found in disease conditions, known as amyloidosis, where amyloid replaces other cellular elements. Deposits are also associated with certain types of tumors. Alkaline Congo red can be used to demonstrate amyloid. The amyloid is seen under polarized light as a green birefringence. This is the most speci c method for detecting amyloid. Crystal violet can also be used. The amyloid will stain purplish blue. However, the method is not as speci c as Congo Red. A third method uses Thio avine T, a uorescent dye that attaches to the amyloid. This method is not as speci c as Congo red and naturally occurring uorescence must be quenched as part of the protocol. STAINING FIBERS IN CONNECTING TISSUE Collagen bers are al long, brous proteins, usually found in bundles that provide support and strength to surrounding tissue. Collagen is the major protein in the body, and the major component of tendons, organ capsules, ligaments, bone, and many other structures. It is also found within cells. Collagen is very eosinophilic and easily identi ed on an H&E stained Slide. It can also be stained with trichrome stains, such as Masson or Gomori. Elastic bers impart exibility to tissue. They are found in connective tissue proper, arteries, veins, lungs and other organs. Special stains are required to demonstrate them, such as Verhoe or Gomori stains. Reticular bers are a delicate type of collagen that form a ne, supportive mesh for tissues. These bers cannot be seen on an H&E Slide. Special silver staining techniques are needed, such as Gomori or Gordon and Sweets stain. TRICHROME STAINING METHODS BASIC MECHANISM Trichrome methods use two or more dyes of contrasting color to selectively stain tissue components. Most trichrome methods are used are used to di erentiate collagenous connective tissue from muscle. Al the methods take place at an acid pH, usually in dilute acetic acid. The acid pH accentuates the attachment of the dye to ------------------------------------------------------- fl fl ff fi fi fi fi fi fl ff fi ff fi fl ff fi fi fi ff fi ff ff ff fi fi  The proteins. Most techniques stain the cytoplasm with a red dye, often called the plasma dye. The collagen is stained with a blue or green dye, referred to as the ber dye. Some techniques incorporate all the dyes in one solution; other rely on a multi-step procedure. A nuclear counterstain imparts a third color. Because aluminum hematoxylins exhibit atypical colors at acid pH, they must be removed, or Weigert iron hematoxylin must be used as the nuclear counterstain. Mason Trichrome and Gomori are both trichrome stains. Although most xatives can be used for trichrome staining, Bouin solution enhances color by acting as a mordant. MASONS' TRCIHROME AND GOMORI TRCIHROME STAINING METHODS Masons' trichrome is amulti-step method. In the rst step, tissue is stained with an acid dye, such as Biebrich scarlet that binds to cytoplasm, muscle, and collagen. In the second step, the sections are immersed in phosphotungstic or phosphomolybdic acid, which removes the stain from collagen. The cytoplasm and muscle bers remain red. The sections are then stained in aniline blue, which imparts a blue color to the collagen and mucus. Nuclei are counterstained with an iron hematoxylin, such as Weigert. In some modi cations of the protocol, light green dye may replace the aniline blue and impart a green color to collagen and mucus. Gomori trichrome stain is a one-step procedure. It combines a cytoplasmic stain and a connective ber stain in one solution, which is in a phosphotungstic or phosphomolybdic acid solution. Almost any tissue can be used for controls on trichrome stains. If the staining does not show clear di erentiation and distinct color separation, it may be due to old reagents. VERHOEFF ELASTIC STAIN Verhoe elastic stain will demonstrate normal and pathological elastic bers. Tissues with elastic bers include blood vessels, so often a section of artery is used as a control. Verhoe is a regressive stain. The sections are overstained with a hematoxylin- ferric chloride-iodine solution. Ferric chloride and iodine are mordants and also act as oxidizing agents to convert hematoxylin to hematein. Excess mordant is added t o remove and di erentiate the stain. The elastic bers remain deeply colored, while other tissue elements are decolorized. Van Gieson picric acid -acid fuchsin stain is often used as a counterstain and colors the other tissue elements yellow, providing good contrast with the blue-black elastic bers. Collagen bers stain red in this procedure. Proper di erentiation in 2% ferric chloride is a critical step in this method. Results vary depending on the length of time the slides are di erentiated, and variations in the preparation of the van Gieson reagent. The xative is not critical, although Zenker or neutral bu ered formalin is preferred STAINS FOR RETICULAR FIBERS Two staining methods for reticular bers are Gomori and Gordon and Sweets. They are similar in many ways. They both use potassium permanganate as the oxidizing agent, ferrous ammonium sulfate as the sensitizer, and tone with gold chloride. Gomori removes excess oxidizing agent with potassium metabisul te; Gordon and Sweets uses oxalic acid. Silver stains for reticulin are often di cult, and good ------------------------------------------------------------ staining relies on careful attention to timing and detail. Fresh reagents, at the correct concentration, are essential in silver stains. The presence of excess metallic ions, which may come from incompletely cleaned glassware or the use of metallic forceps, will a ect the result. The washing steps are also important. To little washing may leave undesired precipitation and excess background stain; too much will diminish speci c staining. When a counterstain is used, it might obscure the delicate pattern often critical to a diagnosis. Some pathologists prefer not to use a counterstain. SILVER STAINS FOR RETICULIN FIBERS The basic steps in silver stains for reticulin bers are as follows: 1. Oxidation: An oxidizing agent changes the glycol groups of the sugars in reticular bers to aldehydes. Phosphomolybdic acid, periodic acid, and potassium permanganate are commonly used reagents for this step. Sensitization and impregnation: The sensitization step prepares the tissue for impregnation with the silver by depositing metallic salts on the tissue. Iron salts are ff fi fi ff ff fi ffi fi ff fi fi fi fi fi fi fi fi fi fi ff ff fi ff fi fi ff  frequently used for sensitization. A silver complex, usually coupled with ammonia or diamine, is then added. This deposits silver at the site of the aldehyde groups. 3. Reduction: The tissue is transferred ot formaldehyde, which reduces the residual diamine to metallic silver and deposits additional silver. The reduction step is also called developing. 4. Toning: The color of the bound silver changes from brown to black when it is treated with gold chloride. The gold compound is crisper and more stable than the original silver. 5. Washing: Unreduced silver is removed by washing with sodium thiosulfate, which is called hypo solution. MALLORY PTAH STAIN Mallory PTAH (phosphotungstic acid-hematoxylin) is a polychromatic stain used to demonstrate cross-striations in cardiac and skeletal muscle. Less commonly, it is used for staining glial bers. The ratio of the phosphotungstic acid to hematein is 20:1. Chemical ripening using potassium permanganate speeds up the preparation of Malory PTAH reagent, although many think abetter reagent is prepared when it is allowed to ripen naturally. However, complete ripening may require four to six months. Mallory PTAH forms a blue lake, which stains the striated tissue, glial bers, and brin blue, while other tissue components stain reddish- brown. This stain is not as commonly used today as it once was, as it has been replaced by immunohistochemical methods. ---------------------------------------------------------- STANINIG METHODS OFR DEMONSTRATNIG NERVE FBIERS Three methods used to demonstrate nerve bers are: Bodian; Holmes; and Bielschowsky. Bodian method impregnates tissue with a commercial silver protein then adds copper to destain connective tissue. Hydroquinone reduce ate, and silver salt, and gold chloride is used to tone the reaction, providings the intense staining. Sodium thiosul te removes unreduced silver. Holmes method modi es Bodian by using silver nitrate in the impregnating solution, and bu ering that solution with pyridine to increase alkalinity, which according to Holmes, provides more consistent staining results. Bielschowsky method is used to demonstrate neuro brillary tangles and senile plaques. The method uses ammonicial silver that is then reduced in formalin. The remaining steps are similar to other silver stains. Variations of this method use PAS to stain basement membranes and amyloid in the plaques. A microwave modi cation requires less silver nitrate and enhances staining. TISSUE COMPONENTS THAT UPTAKE STAINS (chart on page 85) GRAM STAIN Gram stain is the most commonly used stain in microbiology. It is used to di erentiate between gram-negative and gram-positive bacteria. The di erence in staining is due to di erent components in the cell wall. Gram-positive bacteria have a thick cell wall containing telchoic acid, and stain blue with Gram stain. Gram-negative have a thinner cell wall, which contains lipopolysaccharides, which does not retain a blue color. Gram stain reagent is made up of crystal violet and iodine. The latter acts as a mordant. Fuchsin is used as a counterstain to color the gram-negative bacteria red. DIFF-QUIK® STAIN D f- Quik is a rapid commercial staining kit that is a modi cation of hematology stains like Wright, Giemsa, and Romanowsky. A Dif-Quik® kit contains three solutions a 10-second xative, 20-second stain, and 20-second counterstain. It is often used for staining blood and cervical smears. In tissue sections, Di -Quick is ------------------------------------------------------------------------- used to identify Helicobacter pylori, which causes some stomach and duodenal ulcers. Formalin- xed, para n-embedded sections can be stained for demonstration of H. Pylori which is a risk factor for developing gastric carcinoma. H pylori and related bacteria will stain blue. DEMONSTRATION OF URATES IN TISSUE fi fi ff fi fi ffi fi fi fi fi fi ff fi ff fi fi ff ff  Uric acid crystals are normally excreted in the urine. Sodium urate crystals can precipitate out of the blood, build up around the great toe joint, and cause very painful gouty arthritis. Gout is treated with colchicine, an extremely toxic drug. You will need to verify the presence of urate crystals, so the patient can avoid these side e ects of colchicine: Baldness; muscle and nerve pain; purpura (bruising); complete lack of sperm production; nausea; vomiting; hematuria; bloody diarrhea; delirium; paralysis; seizures, and respiratory arrest. Use absolute alcohol as the xative for urate stains, because urates are soluble in aqueous solutions. Use the Gomori's silver method with methenamine-silver nitrate to reduce the urate and stain it black. Apply a light green counterstain, so the urates appear black against a green background. Urate crystals are birefringent, so you can see them on an H&E-stained slide by using a polarizing microscope. MOVAT's PENTACHROME STAIN PROCEDURE Movat's pentachrome stain procedure uses multiple dyes and selective di erentiation to identify mucin, brin, elastic bers, muscle and collagen, all on the same section. The rst step uses Alcian blue to stain acidic monosaccharides. Alcian blue is converted to Monastral Fast Blue by alkaline alcohol, creating a blue precipitate. Iron hematoxylin then stains elastic bers, using ferric chloride as a di erentiation agent. Sodium thiosulfate removes residual iodine. Crocein scarlet- acid fuchsin reagent then stains muscle, cytoplasm, collagen, and background connective tissue. Further di erentiation with phosphotungstic acid removes the red from collagen and ground substance, which is nally counterstained with safran. The resulting tissue is stained with up to ve colors. Nuclei and elastic bers stain black Collagen is yellow. Ground substance and mucin are blue. Fibrin is deep red and muscle a lighter red. This stain can be used to identify Cryptococcus neoformans, which because of the presence of mucopolysaccharides, stains a bright blue color. PAPANICOLAOU STAINING Papanicolaou stains are used to di erentiate types of cells found in the sample. Most often associated with gynecological PAP smears, it is a staining technique that utilizes multiple dyes to highlight speci c cell types. Three solutions are applied to achieve the nal polychromatic stain; hematoxylin, orange green 6, eosin azure. The hematoxylin stains the nuclei blue, orange green 6 stains the cytoplasm orange in mature cells, and eosin azure stains the cytoplasm of squamous cells pink and of metabolically active cells green. ---------------------------------------------------------------------- SMEAR WTIH ACID-FAST STAIN TO DETECT TUBERCULOSIS The steps in making a smear with acid-fast stain to detect tuberculosis are listed below: 1. Label glass slide with pencil 2. Place one drop of water on slide 3. Pick up a bacteria colony from an LJ egg plate with a innoculating loop 4. Mix colony in water drop until it becomes a milky suspension 5. Air dry smear.6 Heat x smear by quickly passing it over a Bunsen burner several times.7 Do not lyze the cells by cooking them-if the slide feels too hot when placed on the back of your hand, then heat killed the TB 8. Submerge slide in Kinyoun carbol fuchsin stain for ve minutes 9. Remove slide from Kinyoun stain 10. Hold slide at an angle. Rinse slide with deionized water decanted in a squirt bottle. 11. Decolorize with a 70% ethanol/0.5% hydrochloric acid solution until it runs clear 12. Rinse slide with deionized water 13. Submerge slide in 1% methylene blue stain for 1 minute 14. Rinse with deionized water 15. Air dry or tissue blot 16. Look for red bacteria with oil immersion lens SDH SDH is succinic dehydrogenase. SDH is an enzyme of the Kreb's citric acid cycle, which is a series of chemical reactions mediated by a series of enzymes that produces energy through fi fi fi fi fi ff ff fi ff fi ff fi fi fi ff fi fi  aerobic (using oxygen) respiration. Energy is produced in mitochondria. Demonstrating SDH also identi es mitochondria, and is away of show that respiration is taking place. Frozen sections of un xed tissue are incubated with substrate. A bu er that contains nitroblue tetrazolium (NBT) is added to produce a color reaction. Unlike NADH diaphorase, SDH is only present in mitochondrion and this can be used to di erentiate stained cellular components. Type Il bers stain only lightly with SDH; Type I wil appear dark because they have more mitochondria. ISH In situ hybridization (ISH) is a process used to indicate the expression of speci ed genes via labeled complementary DNA/RNA. ISH can be used to localize endogenous or exogenous genetic material and is therefore excellent for investigating either expression patterns or the structure of chromosomes. Analysis can be performed with light or electron microscopy. Prepare the probe solution for the desired nucleotide target. Fixate the sample as required per tissue and hybridization type. Chemically expose target nucleotides and heat to temperature for hybridization. Add probe solution to sample no slide. ------------------------------------------------------------------- Hybridize following prescribed temperature and time requirements. Cool slide to room temperature and wash of the excess probe solution. Use antibody detection to locate the probe labels. SPIROCHETES, HELICOBACTER PYLORI, ARGYROPHIL, ARGENTAFFIN, WARTHIN- STARRY TECHNIQUE, AND STEINER AND STEINER PROCEDURE Spirochetes are primitive, spiral-shaped bacteria. They are the cause of systemic diseases like leptospirosis, Lyme disease, syphilis, yaws, and relapsing fever. Helicobacter pylori is a spirochete that causes peptic ulcers of the stomach and duodenum by eating through the protective mucous coating. Spirochetes are argyrophilic, which means that they will bind to silver if a reducing agent is added to the solution. Argenta n is asubstance that binds to silver and reduces it to a visible metallic deposit. The Warthin-Starry technique is a method for demonstrating spirochetes in tissue that uses hydroquinone as a reducer. Warthin- Starry also stains other bacteria in the tissue, and can be used to stain small bacteria that stain weakly gram-negative, such as Legionella. Spirochetes can be identi ed in the tissue as they are larger and have a characteristic corkscrew shape. Steiner and Steiner procedure is a similar protocol to Warthin-Starry that uses a microwave protocol. STAINS THAT CAN DEMONSTRATE FUNGI IN TISSUE Fungi cannot be seen with an H&E, although the H&E will show the tissue's in ammatory response to the fungus, and can be used in combination with fungal stains. PAS is not speci c for fungi, but will demonstrate the polysaccharides present in their cell walls. Gridley fungus stain is similar to PAS, except it uses a stronger oxidizing agent, chromic acid. Most of the aldehyde groups in the connective tissue are destroyed by the strong acid, but the aldehydes formed from the high concentration of polysaccharide in the fungal cell walls remain. Fungi are stained deep red to purple with Schi reagent. Light Green is a good counterstain for viewing fungi. Methenamine silver methods, including Gomori and Grocott, also use chromic acid to produce aldehyde groups. Methenamine-silver at an alkaline pH is then reduced to visible silver, followed by toning with gold chloride, similar to other silver staining methods. Sodium thiosulfate removes unreduced silver. Microwave modi cations of methenamine silver stains have been developed that provide faster results. STAINING METHOD TO DEMONSTRATE MYCOBACTERIUM LEPRAE The causative agent of leprosy is Mycobacterium leprae, which is an acid-fast bacterium. An acid-fast technique is used to demonstrate the lipid in the cell wall. But the variations of acid- fast stains such as Kinyoun Acid-fast stain and Ziehl- Neelson method are not suitable. The Fite Acid Fast Stain is a better choice. This method uses a peanut oil-xylene mixture to depara nize the section, as xylene used alone adversely a ects the stain. The peanut oil protects the lipid capsule. For al acid-fast stains, the counterstain is critical, as overstaining can mask the presence of the leprosy bacteria, which are di cult to see even under high magni cation. fi fi ffi fi fi ff ff fi ffi ff ffi ff fi fl fi fi  ---------------------------------------------------------------------- Acid fast stain is used to identify Mycobacteria. Mycobacteria are rod shaped bacteria that resemble fungi because they grow extensions and protuberances like fungi. Acid fast bacteria contain lipids in the cell wall that will take up carbol- fuchsin, a stain made from basic fuchsin and phenol. The bright red color will not be washed out with dilute acids. Mycobacterium tuberculosis, the organism that causes tuberculosis and Mycobacterium leprae, which causes leprosy, are both acid fast bacteria. They do not stain with Gram stain. Two variations of the acid fast stain are the Kinyoun method and the Ziehl-Neelson method. Tissue stained for acid fast bacili are usually counterstained with methylene blue, although the methylene blue must not be too intense, or i t will mask the bright red bacilli. When staining for M. leprae, the routine organic solvents used may dissolve the lipid capsule of the bacteria. The Fite modi cation of the basic acid-fast stain uses a xylene-peanut oil mixture to protect the capsule. HEMATOXYLIN Hematoxylin is a naturally-occurring substance derived from logwood. It does not bind to tissue, but the oxidation product of hematoxylin, hematein, does. Hematein, which binds to nuclear components, is a weak basic dye. Although hematein is the active ingredient, we still refer to the dye as hematoxylin and not hematin. Do not confuse hematein with hematin, which is an artifact sometimes seen in formalin- xed tissue. The two, although similar in spelling, are di erent. Hematoxylin is oxidized to hematein in a process called ripening. Hematoxylin can be ripened by exposing it to air, but more commonly an oxidizing agent, such as sodium iodate, mercuric chloride, or potassium permanganate, is added. The ripened hematoxylin will not bind to tissue unless a metallic mordant is added. Iron can be used as both an oxidizer and a mordant. However, the resulting solution is not as stable as those where a non-oxidizing mordant, such as aluminum, is used in the form of ammonium or potassium aluminum sulfate. Several types of hematoxylins are used for nuclear staining. Common formulations are named for their originator. These include Mayer, Harris, Gil, Weigert, Ehrlich and Dela eld. Di erences in the hematoxylins are the mordants used with them,the methods and compounds that ripen them, and the nal pH adjustment step before reading. Mayer, Harris,Dela eld and Ehrlich solutions use aluminumas a mordant; Weigert uses iron mordant; Malory uses tungsten mordant. Dela eld and Ehrlich ripen naturally by exposure to air. As a result, these take longer to prepare. Mayer and Ehrlich formulations use sodium iodate. Harris uses mercuric oxide with boiling, although because mercury is a neurotoxin, sodium iodate is recommended as a replacement. Hematein forms more rapidly in alkaline solutions. Alcohol can be added both as a preservative and to retard oxidation. Some formulations ad acid for more selective nuclear staining. Harris hematoxylin adds glacial acetic acid in the nal step and Mayer adds citric acid. Gill prevents the formation of surface precipitates by using ethylene glycol as the solvent. The only hematoxylin that will stain goblet cells is Gill. --------------------------------------------------------------------- H&E-STAINED CELLS INCLUSIONS THAT MIGHT BE FOUND IN CYTOPLASM Lysosomes are small, round, darkly stained bodies within the cytoplasm. There may be only a few or many, depending on the type of cell, its function, and longevity. Lysosomes contain hydrolytic enzymes, and their purpose is to digest food, worn out organelles, and debris such as bacteria. Incompletely digested particles may also be seen in the cytoplasm in vacuoles called residual bodies. Secretory granules, which are proteins produced by ribosomes and tra