4.1 Historical Histological Staining Techniques in Medicine and Biological Studies
The history of staining indicates that the application of histological techniques is a relatively new area of diseases diagnosis (Rodrigues et al., 2009). Historical staining techniques by early pathologists and surgeons were borrowed from a seventeen scientist Leeuwenhoek, who was instrumental in histology using substances such as Madder, indigo and saffron to stain tissues and using rudimentary microscopes to study them (Titford, 2009). These categories of early researchers used the microanatomy to draw a relationship among differences in cells as well as delineating a normal plant cell structure from that of the animal (Bancroft & Layton, 2013).
Later, newer techniques were devised to enhance the study of cell structure in detail using various laboratory chemicals to preserve tissues in their natural form before staining (Titford & Bowman, 2012). Joseph Von Gerlach was viewed as the pioneer of microscopical staining in 1858 when he used ammoniacal carmine successfully to stain cerebellum cells (Costa, Brito, Gomes, & Caliari, 2010).
The early histologists used the readily available chemicals to prepare tissues for microscopic studies; these laboratory chemicals were potassium dichromate, alcohol and the mercuric chloride to hard cellular tissues (Iyiola & Avwioro, 2011). These fixatives and staining agents were ingenious and after a period colored staining agents were developed which are still applicable in current laboratory staining techniques (Black, 2012). Examples of these ingenious colored stains still in use include the trichrome that is used in the liver and renal biopsies as well as the silver nitrate that is used in other organisms (Musumeci, 2014).
Great development in histologic stains was shaped by the improved technologic development of microscopes and the establishment of the histologic stains (aniline dye) in 1856 in Germany which manufacture a variety of new histological stains (Shostak, 2013). At the same time, research and knowledge relating to anatomy and tissues of the human body increased, and this knowledge was used to further research into new-histological techniques for the study of diseased tissue (Titford, 2009).
In the wake of the nineteenth century, many medical centers hired physicians, pathologists and surgeons to handle surgical issues (Titford & Bowman, 2012). It is this crop of pathologists who devised intraoperative staining techniques for frozen tissues sections by adapting a special staining technique in histopathology. It is during this time that the paraffin infiltration staining technique was devised (Shostak, 2013). Owing to this achievement, the non-malignant and the malignant tumors were studied, and a bacterium was identified as the causal organism of the disease in the nineteenth century (Godwin, 2011).
The Gram staining method was named after a Danish inventor Hans Christian Gram, who invented it as an approach to differentiating bacteria species in 1875 (Anderson, 2011). It is while working at the city morgue with his colleagues that Gram devised the technique of staining for the purpose of distinguishing the type of bacterium infection and also as a way of making the bacteria visible on selected and stained lung tissues during examination (Black, 2012). Although this technique was found unsuitable for certain bacterium organisms, it is still used today and competes fairly with modern molecular techniques of histology (Shostak, 2013).
4.2 Important Histological Stains Used in the Past and Present
It is a commonly used stain in histology used by early botanists such as John Hill in their studies in 1770s (Jackson & Blythe, 2013). The stain was used to study microscopic tissue structures when in ammoniacal solution form and it is still used today in histologic studies. In particular, the stain was used widely by Rudolph Virchow (1821–1902) in microscopic studies; Virchow is considered as the ‘father of pathology’ (Musumeci, 2014).
Hematin and Hematoxylin
These are naturally occurring substances that have been in use in the history of histopathology (Titford, 2009). The stain was developed by Wilhelm von Waldeyer in 1863 and was obtained from a log tree found in Central America. Hematoxylin is a weak stain and is used with a combination of other solutions in oxidized form (Shostak, 2013).
In particular, the stain is combined with an oxidizer mordant to enhance its differentiating capacity of cell components; these solutions are called Hematoxylin. The versatility of the stain has enhanced the development of various Hematoxylin methods (Titford & Bowman, 2012). Historically, Hematoxylin was made into a nuclear stain that had shorter staining time and was resistant to acidic solutions; this made it suitable for histologic stain techniques requiring several steps (Anderson, 2011).
Silver Nitrate has had a long usage in historical staining techniques and is still used in modern pathology. Initially, early researchers used silver nitrate to enhance the visibility of the tissue structure while studying it; this was done by applying solid silver nitrate on a tissue and then studying it (Titford & Bowman, 2012). The stain substance has been developed for many compounds, and confirmatory tests are needed when silver nitrate is used (Shostak, 2013). Silver nitrate stain has also been found to be reduced by argentaffin cells found in the epithelial linings of lungs, intestines, melanin and others (Musumeci, 2014).
However, methods have been devised to ‘tailor’ these tissues to avoid argyrophilic reactions when silver nitrate is used during staining process (Titford, 2009). In particular, methods such as the Gomori reticulin methods and the Grocott-Gomori method were devised to assess missing tissues and diseases in the liver and the rectum (Nadworny, Wang, Tredget, & Robert, 2010).
Other Staining Procedures That Were Developed Recently
The Hematoxylin and Eosin Procedures
Although historically used, there have been great laboratory changes in Hematoxylin stains; nearly all tissue specimens are treated with Hematoxylin and Eosin today (Bancroft & Layton, 2013). In addition, various Hematoxylin methods have been developed but all follow the same approach of staining tissue specimens in a hematoxylin, alcohol and tap or alkaline water to clear argentaffin agents. It has been found that most histopathological processes could be studied using the Hematoxylin and Eosin procedures (Titford & Bowman, 2012). In the same line, the method is quick to execute, cheap and can be altered. However, the Hematoxylin and Eosin are inefficient in that not all features of a substance can be received and special stains must be used (Musumeci, 2014).
Romanowsky Stains–Giemsa Stains
They were developed in the 1891 by Dimitri Romanowsky and popular for its multicolor in identifying blood parasites. The Giemsa Stains procedure is still used today. There has been great improvement in the stains, and its various methods make it applicable in paraffin-embedded, formalin-fixed and bone marrow biopsies (Musumeci, 2014).
The Gram staining method was named after a Danish inventor Hans Christian Gram, who invented it as an approach to differentiating bacteria species in 1875 (Musumeci, 2014). Gram devised the technique of staining for the purpose of distinguishing the type of bacterium infection and also as a way of making the bacteria visible on selected and stained lung tissues during examination (Shostak, 2013). Although this technique was found unsuitable for certain bacterium organisms, it is still used today and competes fairly with modern molecular techniques of histology (Rudijanto, 2007). However, Gram technique is infallibly limited in the application on matters of environmental microbiology (Titford, 2009). That aside, Gram techniques had had success when performed on biopsy of infected parts and produced results quickly especially when there is a significant difference in prognosis and treatment. The method is often used in modern histology especially in paraffin fixatives for tissue sectioning (Titford & Bowman, 2012). In a recent case in Kuwait, the Gram staining technique was particularly effective in the diagnosis of Gonorrhea giving 99.4% effective results (Iyiola & Avwioro, 2011). The method is still used today especially with paraffin sections and has been modified to suit different substances.
Historical assessment on the use of various stains in histology indicates that most pathologists were attracted by stains that gave multicolored results on the tissue specimens. As such, trichrome stains were developed from this need (Shostak, 2013). There have been various multiple stains such as blue–eosin, “triacid stain” by Ehrlich's (1888) and Masson's trichrome stain that has been popular in the modern histology. Trichrome stains show how complex the staining methods have become in the search of an efficient and consistent stain that would show fine, differentiated tissues (Musumeci, 2014).
4.3 Case Study Reviews
Case Study 1
This study was done in order to compare different staining methods and assess their effectiveness. The specific aim was to assess if the newly developed staining methods, the Helicobacter pylori silver stain HpSS methods and the modified McMullen's methods in the identification of H pylori organism. The method involved selecting tissue sections of gastric biopsies of 63 patients diagnosed with dyspepsia. The section tissues were stained using the four staining methods. In all the 63 cases, 30 sections tested positive for Helicobacter pylori while 30 tested negative for all cases of pylori infection while the remaining were tested using a combination of five histological tests (Anderson, 2011). The results indicated that, the interobserver stain method was the best for antibodies at 98%, followed by Giemsa at 87%, then the HpSS at 85%. At gold standard level, it was found that the Giemsa stain method was the best followed by McMullen's method (Rotimi, Cairns, Gray, Moayyedi, & Dixon, 2000). The study conclusions were that in all cases of staining, the H pylori infection was revealed; however, the modified Giemsa stain was the most effective for its sensitivity, ease of use, reproducibility and cost-effectiveness.
Case Study 2
The aim was to investigate the difference in capacity among different stains: Hematoxylin and Eosin, toluidine blue Stain, neuron-specific enolase (NSE) immunostaining and the S 100 protein. These stains were applied to assess the presence of neurons and mast cells in acute appendices Specimens were collected from clinically acute appendices categorized as histologically positive and negative. In the study all the 50 appendix specimens sections were subjected to Hematoxylin and Eosin, toluidine blue Stain, neuron-specific enolase (NSE) immunostaining and the S 100 protein. Hematoxylin and Eosin were applied as a routine stain for general study of the tissues while Toluidine blue stain was applied to enhance the easier study of mast cells. In addition, neuron-specific enolase (NSE) immunostaining was used as a marker and as well as the S 100 protein.
The results indicated that when comparing Hematoxylin and Eosin stain with S 100 they? showed 100% accuracy in identifying the denatured mucosal cells. However, the combination of these different staining methods resulted in a supplementary technique effective than the conventional staining method in observing changes and the pattern of diseased cells as well as the morphological shape of nerve fibers in the inflamed appendices (Russell & Gordon, 2009). In addition, the use of the several staining methods aided in confirming results of earlier stain diagnosis.
Fixation - types of fixatives
The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology) or soon after death (with autopsy) to prevent autolysis. There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated.
There are five major groups of fixatives, classified according to mechanism of action:
- Oxidizing agents
Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost. Therefore, formaldehyde is good for immunohistochemical techniques. Formalin penetrates tissue well, but is relatively slow. The standard solution is 10% neutral buffered formalin. A buffer prevents acidity that would promote autolysis and cause precipitation of formol-heme pigment in the tissues.
Glutaraldehyde causes deformation of alpha-helix structure in proteins so is not good for immunohistochemical staining. However, it fixes very quickly so is good for electron microscopy. It penetrates very poorly, but gives best overallcytoplasmic and nuclear detail. The standard solution is a 2% buffered glutaraldehyde
Mercurials fix tissue by an unknown mechanism. They contain mercuricchloride and include such well-known fixatives as B-5 and Zenker's. These fixatives penetrate relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear detail. Their best application is for fixation of hematopoietic and reticuloendothelial tissues. Since they contain mercury, they must be disposed of carefully.
Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness. However, they are very good for cytologic smears because they act quickly and give good nuclear detail. Spray cans of alcohol fixatives are marketed to physicians doing PAP smears, but cheap hairsprays do just as well.
Oxidizing agents include permanganate fixatives (potassium permanganate), dichromate fixatives (potassium dichromate), and osmium tetroxide. They cross-link proteins, but cause extensive denaturation. Some of them have specialized applications, but are used very infrequently.
Picrates include fixatives with picric acid. Foremost among these is Bouin's solution. It has an unknown mechanism of action. It does almost as well as mercurials with nuclear detail but does not cause as much hardness. Picric acid is an explosion hazard in dry form. As a solution, it stains everything it touches yellow, including skin.
Fixation - factors affecting fixation
There are a number of factors that will affect the fixation process:
- Time interval
Fixation is best carried out close to neutral pH, in the range of 6-8. Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity. Acidity favors formation offormalin-heme pigment that appears as black, polarizable deposits in tissue. Common buffers include phosphate, bicarbonate, cacodylate, and veronal. Commercial formalin is buffered with phosphate at a pH of 7.
Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant. Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between. Oneway to get around this problem is sectioning the tissues thinly (2 to 3 mm). Penetration into a thin section will occur more rapidly than for a thick section.
The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. Obviously, we often get away with less than this, but may not get ideal fixation. One way to partially solve the problem is to change thefixative at intervals to avoid exhaustion of the fixative. Agitation of the specimen in the fixative will also enhance fixation.
Increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as you don't cook the tissue. Hot formalin will fix tissues faster, and this is often the first step on an automated tissue processor.
Concentration of fixative should be adjusted down to the lowest level possible, because you will expend less money for the fixative. Formalin is best at 10%; glutaraldehyde is generally made up at 0.25% to 4%. Too high aconcentration may adversely affect the tissues and produce artefact similar to excessive heat.
Also very important is time interval from of removal of tissues to fixation. The faster you can get the tissue and fix it, the better. Artefact will be introduced by drying, so if tissue is left out, please keep it moist with saline. The longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage and artefactual clumping will occur.
Fixatives - general usage
There are common usages for fixatives in the pathology laboratory based upon the nature of the fixatives, the type of tissue, and the histologic details to be demonstrated.
Formalin is used for all routine surgical pathology and autopsy tissues when an H and E slide is to be produced. Formalin is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly. Most clinicians and nurses can understand what formalin is and does and it smells bad enough that they are careful handling it.
Zenker's fixatives are recommended for reticuloendothelial tissues including lymph nodes, spleen, thymus, and bone marrow. Zenker's fixes nuclei very well and gives good detail. However, the mercury deposits must be removed(dezenkerized) before staining or black deposits will result in the sections.
Bouin's solution is sometimes recommended for fixation of testis, GI tract, and endocrine tissue. It does not do a bad job on hematopoietic tissues either, and doesn't require dezenkerizing before staining.
Glutaraldehyde is recommended for fixation of tissues for electron microscopy. The glutaraldehyde must be cold and buffered and not more than 3 months old. The tissue must be as fresh as possible and preferably sectionedwithin the glutaraldehyde at a thickness no more than 1 mm to enhance fixation.
Alcohols, specifically ethanol, are used primarily for cytologic smears. Ethanol (95%) is fast and cheap. Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are not sectioned,there is no problem from induced brittleness.
For fixing frozen sections, you can use just about anything--though methanol and ethanol are the best.