Progress In The Development Of Diagnostic Histopathology Staining-By Benard Solomon

Dyes were the foundation for diagnostic histopathological staining. Most of the early methods for histopathological analysis of tissue sections have been overtaken by modern and safer methods like immunohistochemistry. Over time, some of the dyes used in the past are no longer available and the chemicals used now consider hazardous. Professionals who were familiar with these techniques are also rare to come by. New entrants to the field of histotechnology and pathology deserve to know the progressive development of histopathological diagnostic staining techniques and their wide range of applications.
This article attempt to bridge the information gap between the old and new generation of practitioners in the field of histopathology and pathology as regards staining techniques and their evolution.

Microscopical Staining Development In Histopathology Diagnosis

Naturally occurring substances such as madder, saffron and indigo were used to color tissues by early surgeons and pathologists. The staining techniques were developed by seventeenth century amateur scientists like Antoin Leeuwenhoek. The first microscopical staining was credited to Joseph Von Gerlach in 1858. He stained cerebellum with ammoniacal carmine. The mid 1800s brought about the development of improved microscopes with corrected spherical and chromatic aberration. The creation of the aniline dye industry in 1856 accelerated the development of histopathology staining.


Historical Facts Behind 3 Important Dyes In Histopathology Staining

1. Carmine: This is a commonly used dye in histology with wide applications. Carmine is most commonly used in an ammoniacal solution to study microscopic tissue structures. The colored compound is obtained by grinding the dried bodies of the female Coccus cacti insect found in Mexico. Cochineal from the insect contains carminic acid. Further treatment with alum and calcium salts results in carmine. Rudolph Virchow, the ‘Father of Pathology’ used carmine in his microscopy studies. Boiling carmine with acetic acid creates aceto-carmine used in staining plant chromosomes. In 1896, Mayer devised the mucicarmine stain by the addition of aluminium as mordant. The method was modified by Southgate in 1927. The mucicarmine stain is used to identify mucin production in adenocarcinomas, in signet ring carcinomas and some mucin-containing fungi such as Cryptococcus neoformans. Carmine was combined with potassium salt to stain glycogen as in Best’s carmine method published in 1906.
2. Haematoxylin and Haematin: Haematoxylin is a natural substance with useful properties and long history in histopathology. It was first used by Wilhelm von Waldeyer in 1863. It is obtained from the logwood tree , Haematoxylon campechianum, found mainly in Central America. It is a weak dye and the different staining solutions are based on its oxidized form, hematein. Hematein when combined with a mordant, an oxidizer and sometimes a differentiating agent, can be used to identify a wide variety of cellular components. Solutions prepared from hematein are usually called ‘hematoxylin’. Alum based hematoxylin solutions include Ehrlich-1886, Mayer-1903 and Harris-1900 just to mention a few. Weirgert’s iron hematoxlyin was introduced in 1904 and has a wide application as a nuclear stain in trichrome methods. This progressive nuclear stain is resistant to subsequently applied acidic solutions, making it suitable for procedures with multiple steps. Other hematoxylin solutions are Verhoeff elastic method –1908, Weil myelin method-1928 and PTAH-1897.
3. Silver Nitrate: Silver nitrate has extensive use in histopathology. The von Kossa reaction first published in 1901 was used in the demonstration of calcium carbonate and calcium phosphate. The Masson Fontana argentaffin reactions originated in 1914. Silver nitrate in ammoniacal solution is reduced by argentaffin cells found in the epithelial lining of intestine and lungs, melanin, chromaffin and some lipofuscins. Other substances stain with the ammociacal silver solution hence a need for confirmatory tests. Silver nitrate also react with argyrophils without being reduced as in Bielschowsky method published in 1904. External substance such as formalin is used to reduce the silver. Additional silver nitrate methods have been tailored for certain substances like Gomori reticulin method-1837, Grocott method for fungi-1955, Gomori method for basement membrane-1946, Steiner an dSteiner method for organisms-1944, Holmes method for nerves-1947. The Gordon and Sweets method-1936 is very popular in the United Kingdom.



Staining Methods And Other Substances Demonstrated In Histopathology.Hematoxylin And Eosin: This method was devised by Wissowzky in 1876. Numerous H&E methods exist but they follow the same general staining procedure of staining the nuclei in an alum or iron mordanted hematoxylin, differentiation in dilute acid alcohol and bluing in running tap water or slightly alkaline water. The cytoplasmic components are then stained in aqueous or alcoholic eosin. Standardization efforts has been limited in H&E staining as a result of variation in personal tastes.

Romanowsky Stains-Giemsa StainsThe Romanowsky stain-1891 replaced the Ehrlich’s stain as a popular multicolored stain in the field of hematopathology. Ehrlich created the field of hematopathology when he demonstrated that certain white blood cell structures had different affinities for the same dyes based on their acid or alkaline composition.
The Romanowsky method uses eosin, methylene blue, and oxidation products of methylene blue to stain blood smears, resulting in a multicolor of pink, through orange, to red-purple and an almost black color. The boiling or aging of the methylene blue(polychromin) generates products of demethylation(azures) that provide a purple rather than blue coloration of leukocyte nuclei when present in a solution that also contains eosin.
Modifications were made to the Romanowsky method and include, Unna-1891, Jenner-1899, Lieshman-1901, Wright-1902, and Giemsa-1902. Giemsa is the most popular. Some of these stains are adaptable to formalin-fixed , paraffin-embedded sections and used for cell maturation studies in bone marrow biopsies, organism detection etc.

Acid Fast Bacilli
Robert Koch in 1882 first developed a working method for the demonstration of Mycobacterium tuberculosis. His method laid the foundation for all other methods. Paul Erhlich stained AFB with a solution of either aniline basic fuchsine or aniline methylene violet and decolorizing with a mineral acid. Franz Ziehl recommended the use of carbolic acid(phenol) to aid in dye penetration and Rindfleisch(1882) recommended heating the slide. Neelsen in 1883 joined Erhlich’s basic fuchsine stain and Ziehl’s phenol. This method is still widely used today and applicable to paraffin sections. Faraco in 1938 recommended olive oil to retain acid-fastness of the organism lost in routine paraffin processing. Fite etal in 1947 recommended 30% vegetable oil such as cotton seed or peanut oil for dewaxing slides during the staining of Mycobacterium leprae. Later in 1952, Wade recommended rectified turpentine mixed with liquid petroleum. In the modern histopathology laboratory, a fluorescent method using auramine-rhodamine is favored for its ability to expose sparse, acid fast bacilli. It is seen as a reddish yellow organism on a green-black background.

Mallory’s Phosphotungstic Acid Hematoxylin(PTAH)This method was devised by Mallory in 1897. This method is used to demonstrate muscle striations, intercalated discs, nervous tissue and fibrin using ripened hematoxylin which stains polychromatically with phosphotungstic acid as mordant.

Gram Stain
Christian Gram devised this method in 1884. This method is widely used and applicable to paraffin sections. Modifications of the Gram method used in histopathology laboratories include the Gram-Twort method-1924, Brown and Brenn method-1931 and the Brown and Hopps method-1973.

Other Organism StainsThe Warthin-Starry method for Syphilis was introduced in 1920. Fungi could be demonstrated by the Gridley stain-1953 and Grocott-Gomori method-1955. Inclusion bodies such as cytomegalovirus(CMV) and rabies is demonstrated by Macchiavello’s stain developed for Rickettsia in 1937. The Leung method –1996 is recommended for Helicobacter pylori.

The PMC-LEARN-MN Structures And Substances Demonstrated In Histopathology
These are
P-Pigments
M-Mucins
C-Carbohydrates
L-Lipids
E-Elastic Fibres
A-Amyloid
E-Endocrine Glands
R-RNA&DNA
N-Nerves
M-Myelin
N-Neuroglia

Pigments: Pigments can be found in the human body normally. Others are introduced pathologically. The use of nonbuffered formalin creates formalin pigment in tissues especially in blood containing organs. Mercuric and dichromate pigments are introduced when tissues are fixed in those chemicals. Exogenous pigments such as carbon is found in lungs of smokers and Asbestos fibers found in the lungs of shipyard workers. Endogenous pigments are of most interest to pathologists. These include hemosiderin, melanin, chromaffin substance, lipochrome, lipofuschin, hemoglobin, and Dublin-Johnson pigment.
The Perls’ Prussian blue method for hemosiderin was published in 1867. Melanin demonstration method of Fontana was published in 1912 and Modified by Masson in 1914. Melanin is a naturally occurring pigment found in the skin and substancia nigra of the midbrain.
The argentaffin cells of the intestinal and respiratory tract epithelium can form carcinoid tumours. Diazo method was introduced for the demonstration of argentaffin cells in 1931. Generally, the argyrophil cells are demonstrated by silver methods. These include the Hellman method-1960, the Grimelius method-1968, the Churukian and Schenk method 1979.
Bile pigment is demonstrated by Fouchet method introduced in 1917. The rubeanic method for cupper was introduced in 1958 while the rhodamine method was developed in 1969. The leuco patent blue method for hemoglobin was developed in 1938 while the benzidine method was introduced in 1941.



Mucins: Hale’s colloidal iron method introduced in 1946was the first solution for the demonstration of mucins. Other modifications of this method exist. In 1965, the high iron diamine method was introduced to identify sulfated mucins with a brown black color.
Steedman in 1950 developed the alcian blue method for acid mucins. Alcian blue method combine well with other techniques such as H&E, PAS, Trichrome methods etc.
Carbohydrates: Histopathologists look for the absence or increase in carbohydrates, its staining reations and distribution. Recently, John Kiernan classified them into polysaccharides, proteoglycans and glycoproteins. In 1946, the P.A.S. was developed by McManus for carbohydrates which became one of the most useful stains in histology.

Lipids: In the histology community, lipids stains are commonly called ‘fat stains’.Normally, fats are found surrounding certain organs of the body and in the skin as packing. When found inside any organ, it becomes a pathological process. Sudan III was the first fat stain to be introduced in 1896 by Daddi. In 1901, Sudan IV was introduced; Oil red O in 1926 and Sudan black in 1935.
Most fat stains work on preferential solubility. The stain is more soluble in the fat than in the organic solvent. 70% was initially used as solvent for fat stains but it has been found to dissolve out most of the fat. Alternative methods were devised using other solvents. Such methods include Lillie and Ashburn’s method published in 1943 using a supersaturated solution of isopropyl alcohol and the Chiffelle and Putt’s method of 1951 which uses propylene glycol as solvent.
Having a good understanding of lipids classification is important in the choice of staining methods. Lipids are often classified as simple lipids, compound lipids and derived lipids. Baker’s acid hematein method was introduced in 1946 for phospholipids. The Luxol fast blue method was also published in 1953 for phospholipids and neuronal storage disorders. The Nile blue sulphate method was published in 1947 for the staining of neutral lipids and free fatty acids. More complicated lipids methods exist. They include the OTAN(osmium tetroxide alpha naphthylamine) for cholesterol esters was published in 1959 and the gold hydroxamate method for phosphoglycerides was published in 1963.

Elastic Fibers: The Orcein elastic stain was developed in 1890. It was very popular in early times and highly recommended for delicate elastic fibers in skin and was favored by dermatopathologists. Verhoeff’s elastic method was introduced in 1908 and is the most popular stain used today in histopathology laboratories. It is often combined with Van Gieson.

Amyloid Fibrils: Amyloid fibrils are demonstrated in amyloidosis-a group of diseases resulting in the deposition of insoluble protein in the interstitial spaces of blood vessel and various organs. Congo red is the most popular routine stain for amyloid. It was developed in 1922. The thioflavine T fluorescence method for amyloid was introduced in 1959.

Endocrine Glands:Chromaffin reaction is the method of choice for adrenal glands. For the pancreas, the Gomori’s chrome alum hematoxylin phloxine stain introduced in 1952 stains beta cells blue and alpha cells red. The aldehyde fuchsin method introduced in 1950 stains beta cells deep blue. The orange fuchsine green method of 1961 is suitable for pituitary staining. The Tripas method of Pearse introduced in 1949 stains beta cells magenta, acidophil cells orange and nuclei blue-black.

RNA and DNA:The feulgen reaction was the earliest method for nucleic acids. It was introduced in 1924. In 1932, Einarson introduced the chrome alum gallocyanine method to stain nucleic acid. The methyl green-pyronine method devised in 1899 and improved by Unna in 1902 stains DNA and RNA differentially and invaluable in study of cell population of tumours.

Myelin: At one time in the past, hematoxylin was used to demonstrate myelin in tissue sections. Later, the Weigert pal method developed in 1886, Kultschitsky method of 1890 and Loyez method of 1910 were commonly used. PTAH method of 1897 also demonstrates myelin. In modern times, common methods include Luxol fast blue –1953, Solochrome cyanine-1965, Swank and Davneport-1935 and Luxol fast blue/oil red O method of 1956.

Nerve Cells, Axons, and Neurofibrils: The classical stain for neurofibrils in the Bielschowsky silver method published in 1904. Other methods are Marsland, Glees and Erikson-1954. The Palmgren method was published in 1948. More methods include
Davenport-1936 and Holmes-1947. The Gally’s silver stain published in 1971 is said to demonstrate more pathological lesions than any other method.

Neuroglia: These are the supporting cells of the CNS. Early methods for glia demonstration are Holtzer’s stain introduced in 1921, Victoria blue method introduced in 1929. Also introduced was the Cajal’s gold chloride method of 1913 and the Hortega’s silver carbonate method of 1918.

Trichrome Stains: The most popular trichrome stain in recent time is Masson Trichrome Stain published in 1929. The Mallory stain was introduced in 1949 while the MSB method was published in 1962.

Conclusion: Different staining methods in histopathology and how they evolved has been highlighted. Future changes are expected but practitioners now have a deeper knowledge of the history behind the techniques they employ in their day-to-day practice. Such knowledge could serve as a foundation for future discoveries and improvements.