Histology, Staining

Issues of Concern

Basic knowledge of tissue preparation, including staining, is important to know when interpreting pathology reports on either in-patient or out-patient biopsies.  It is not always the case that the interpreting pathologist has thoroughly analyzed the tissue sample by including appropriate histologic staining, and this deficiency can retard accurate diagnosis.

Structure

Four basic types of human tissue can be stained and viewed using various histological techniques. Epithelium, connective tissue, muscle tissue, and nervous tissue have commonalities but look very distinct structurally after staining. Each stain exists to highlight an important feature or component within a tissue type. For example, one of the most common stains, Hematoxylin, is a basic dye that stains proteins a blue color, while Eosin stains proteins a pink color. These two stains are commonly used together to define intracellular organelles and proteins. Because of the variety of the proteins that exist, some stains were created to highlight a particular protein, which this review will discuss in the following sections. The benefit of using a special stain is that it can highlight the specific protein very well. However, because of its specificity, the other structures will not be seen. For this reason, multiple slides will often be created from a given specimen so that multiple stains can be performed to gather the full range of needed information.

Function

Almost all tissue stains are performed on tissue that has been removed from the body.  However, in rare instances, very specialize stains called vital stains can work on tissue remaining in the body.  These stains are used for the identification of specific types of tissue and identification of abnormal tissue, so a subsequent biopsy can be more accurate in obtaining abnormal tissue.

Tissue Preparation

Before specific staining can occur, tissue samples must undergo preparation through the following stages: Fixation, processing, embedding, sectioning, and sometimes antigen retrieval. In modern histology laboratories, most of these steps are automated.

Fixation: Fixation uses chemicals to preserve the structure of the tissue in its natural form and protects it from degradation by irreversibly cross-linking proteins. Although several specialized fixatives are available, Neutral Buffered Formalin is a common choice for this step. The fixation step is vital to the rest of the histologic staining procedure because by retaining the chemical composition of the tissue, the sample is hardened and makes the sectioning phase easier. Paraffin-formalin is another effective fixative. Its benefit is that it is the fixative of choice for immunostaining; however, it requires preparation at the time of the fixation. Bouin is a fixative used for examining embryo and brain tissue because of its superior preservation of delicate nuclei and glycogen. Its downside is that it does not preserve kidney tissues well and also distorts mitochondrial structure.[1] 

Dehydration: The addition of ethanol accomplishes the dehydration of a sample. It removed water from the sample and further hardens the tissue for eventual light microscopy. After ethanol is applied, and following the completion of tissue dehydration, xylene is used to remove the ethanol.[1] 

Embedding: Embedding is the process of putting the sample into a paraffin wax or a plastic resin to enhance the process of extracting cellular structures. This step is to be performed with caution if the goal is to perform immunostaining because the paraffin wax will inhibit the penetration of antibodies, and lead to a false result.[1]

Sectioning: Sectioning involves mounting the specimen on a microtome and cutting it into sections. The preferred thickness is 4-5 micrometers so that it can be stained and put on a microscope slide for examination.[1]

Antigen Retrieval:  This step is to retrieve antigens that could have been covered in the fixation and embedding stages. If the cross-linking of proteins conceals the antigen sites, there may not be as robust of an immunohistochemical response. Antigen retrieval is achieved through heating and proteolytic methods to break down the cross-links and reveal the epitopes and antigens that were previously covered.[1] Although this step carries the risk of denaturing both the fixative and the antigens themselves, a successful antigen retrieval method can lead to a much more effective immunostaining intensity. 

Histochemistry and Cytochemistry

Hematoxylin and Eosin

As the name implies, it is two stains done in subsequent steps. The hematoxylin is a basic dye that stains acidic structures. The resulting color is a purple/blue hue, and structures that are targeted with this dye are named Basophilic. Basophilic structures include DNA in cell nuclei, RNA in ribosomes, and the rough endoplasmic reticulum.[1]

Eosin is a counterstain done after hematoxylin and is an acidic dye that targets basic structures. The resulting color is a pink/red hue, and structures that attract eosin are called eosinophilic.[1] The cytoplasm is an example of an eosinophilic structure.

Gram Stain

The gram stain is a sequential staining technique invented for differentiating bacterial species. Its major utility lies in determining the causative organism of bacterial infection by staining the cell wall.[2]  While not all bacteria have a cell wall and thus cannot be stained with this method, it is still a very useful and commonly performed stain. A bacterial sample can be heat-fixed and undergo gram stain with these four steps: Primary staining with crystal violet, secondary staining with grams iodine, decolorized with alcohol or acetone, and counterstained with safranin. Gram-positive bacteria are those that contain a thick layer of peptidoglycan, making them retain the violet stain and appear purple. Alternatively, the gram-negative bacteria have a thin layer of peptidoglycan and more lipids in the cell wall, so the decolorizing step washes out the violet more, and the sample appears pink.[2] 

Giemsa Stain

The Giemsa stain is commonly used in hematology for its superior ability to stain bone marrow, plasma cells, and mast cells. It is also very popular for identifying blood parasites.[3] The Giemsa stain can also help to visualize chromosome abnormalities through "Giemsa-Based Banding," or observing the alternating darker and lighter nucleotide portions on chromosomes during mitosis.[3]

Periodic Acid Schiff Reaction

The periodic acid Schiff Reaction Stain, often called the PAS stain, is a way to examine structures containing high amounts of carbohydrate molecules, such as the intestinal brush border, renal tubular cells, mucus, and reticular fibers of connective tissue. [4] The glycogen, glycoprotein, glycolipids, and mucins stain red or magenta color when the stain is complete. The periodic acid, a highly oxidized iodine, oxidizes the hydroxyl groups of adjacent sugar molecules to produce aldehydes. After this step, the Schiff reagent attaches to the aldehyde and forms a red magenta color for visualization.[5][6]

Masson's Trichrome

Masson's Trichrome Stain is a stain that can yield a multicolor result on the tissue. Even though it has red counterstains, it is popular for its ability to stain collagen fibers blue. Masson's Trichrome can identify cardiac fibrosis, pulmonary fibrosis, chronic kidney disease, and muscular dystrophy.[7]

Congo Red

Congo red is a water-soluble blue dye that produces a red solution at a pH of 3.0-5.0. Its many aromatic rings can stack together through hydrophobic interactions and collect in tissue. Most notably, Congo red can stain amyloid fibers red and orange color, making it a useful study in amyloidosis. When viewed under polarized light in a microscope, Congo-red-stained tissues high in amyloid will show with bright "apple" green birefringence. PMID:[8]

Prussian Blue

The Prussian blue stain is useful for identifying iron stores in the body.[7][9] The stain works by first staining the tissue with hydrochloric acid and then seeing the ferric ions react to form the insoluble bright blue pigment. It is useful in diagnosing iron accumulation states like hemochromatosis or hemosiderosis through staining liver tissue and seeing the build-up of iron near the peri-portal hepatocytes or along the sinusoidal lining.[10][11] An overabundance in iron stores within bone marrow could signal ineffective erythropoiesis, like in anemia of chronic disease. Alternatively, absent reaction to the Prussian blue stain could indicate low iron levels, like in iron deficiency anemia.

Mucicarmine

Mucicarmine stains mucin, a secretion produced in epithelial and connective tissue cells.[12] The aluminum and carmine combine to form a positively charged chelating complex. The newly positive charge binds the mucin, stains it red, and allows visualization. It is useful in identifying potential carcinomas and inflammatory conditions, where there is excessive mucin production.[12] In surgery, mucicarmine staining can also determine a primary tumor location by staining the mucus-secreting epithelium in a site not containing mucin-producing cells.[13] Mucicarmine also stains the gelatinous capsule of fungi Cryptococcus.[12]

Sudan Black

The Sudan Black dye stains lipid-containing structures like triglycerides and lipoproteins, a dark black or brown color.[7][6]The tissue preparation for Sudan Black and Oil Red O skips the alcohol dehydration step to avoid washing away the lipids to be stained. It may be used to diagnose atherosclerosis by staining atherosclerotic plaques and autosomal dominant leukodystrophy by staining macrophages in white matter after a post-mortem brain biopsy.[7]

Oil Red O

Similar to the Sudan Black dye, Oil Red O is the most common dye used on hydrophobic fat or lipids, substances that are traditionally difficult to stain. Oil Red O has high utility in visualizing atherosclerotic plaques and hepatic and muscular lipid accumulation.[7][6][14]

Silver Stain

Silver stains are a larger category of stains used for the histopathological study of accumulation-based diseases in neurology. There are several methods to silver staining, including Bielschowsky, Gallyas, Bodian, and Campbell-Switzer. The staining method chosen is dependent on the neurological lesion in question, as each method speaks to a differing sensitivity and specificity. In general, the methods attach silver ions or salt complexes to the target tissue. Then, they must be reduced in situ, and the subsequent silver particles accumulate and can be analyzed.[15] Recently, there has been the use of fluorolabeling, where a fluorogenic semiconductor releases small 6nm nanoparticles at the silver depositions and produces colors. The diameter range of the silver particles that form correlates to different colors. For example, ranges in the 10 to 20 nm yield range of yellow colors whereas diameters exceeding 100nm yield a black color.[16]

The silver stains are very known for detecting amyloid beta-protein (Aß) in Alzheimer's disease and Pick bodies in Picks Disease. When dyed, the amyloid plaques become darker. They can range from yellow to black, depending on the size or amount of amyloid plaques.[15]

Nissl Stain

The Nissl Stain, also called the Cresyl Violet Stain, uses basic aniline dye to study neuronal structure in the brain and spinal cord. Neuropil stains blueish purple and granular. The Nissl substance has a high amount of ribosomal RNA, thus attracting the dye, appearing dark blue, and making the cytoplasm appear mottled. The advantage of using a Nissl stain for evaluating neuronal pathology is that it will recognizably stain the neuronal cytoplasm without staining the perikarya of other cell structures, like astrocytes.[17]

Papanicolaou Stain

The Papanicolaou stain colloquially referred to as the Pap smear is a cytological staining technique best known for detecting cervical cancer in female patients. The cells to be stained are collected from gynecological smears, sputum samples, brushings, fine needle aspiration materials, and washings. The multichromatic stain involves five dyes: Hematoxylin for the nucleus, Orange G for keratin, eosin for superficial structures, Light Green SF for cytoplasm, and Bismarck Brown.[18] In the setting of a cervical cancer screening Pap smear, the resulting stain of the epithelial cells from the transitional zone of the cervix undergo analysis for precancerous and cancerous processes. Often a second slide will be prepared for the immunostaining with the biomarker p16INK4a for identifying dysplasia.[19]

Microscopy, Light

The light microscope, also called optical microscope, can be used to view living or dead specimens. The magnification is lower than electron microscopes at 1500 times. The lighting method is a light on the microscope rather than a beam of electrons.[20] The light microscope is used for the majority of the stains described in this article, like the Gram Stain, H & E, and Giemsa. 

Microscopy, Electron

The electron microscope is useful for viewing intracellular components not visible via light microscopy, which aids in clarifying the biology of abnormal tissues and cells. Electron microscopy commonly magnifies 100 to 300 times more than the highest magnification of light microscopy.[20] Typically, the sections must be ultra-thin cut to allow adequate penetration of the electrons. The histological stains most useful for electron microscopy are those with heavy metal salts, as they create a phase-contrast needed to visualize structures.[21] Transmission electron microscopes are better for investigating intracellular structures, while scanning electron microscopes are typically the choice for surface structures. 

Pathophysiology

Use of special stains for tissues not only aids in distinguishing structural alterations of tissues but also alerts the physician to alterations in tissue function highly relevant to making a diagnosis, such as abnormal deposition or iron, abnormal deposition of protein [amyloidosis, paraproteinemia, etc., abnormal accumulation of glycogen or other carbohydrates and abnormal accumulation of fat. Highly specialized staining can detect many other alterations in cellular physiology.

Clinical Significance

Histological staining and examination hold a very high clinical significance in medical diagnosis and treatment in almost every field of medicine. Histological examination is a gold standard for the diagnosis of many pathological diseases, for which staining is an essential component. The histochemical analysis of a tissue specimen allows the pathologist to not only diagnose but to determine the severity of disease and issue a prognosis.