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History of Pathology
Abstract Histochemistry has an interesting history, extending back to ancient times, in some ways. Man has
long had a desire to understand the workings of the human body and the roles that various humors
or chemicals have in those processes. This review traces the evolution of histochemistry as an
investigative and diagnostic discipline, beginning with the efforts of medicinal chemists and
extending through a period in which histology was increasingly paired with biochemistry. Those
developments served as the underpinnings for an eventual marriage of microscopy, chemistry,
immunology, and molecular biology, as realized in the current practice of anatomical pathology.
2012 Elsevier Inc. All rights reserved.
One can defensibly argue that biochemistry and histology elements that comprised plant and animal tissues. Moreover,
originated from the same human interest, that is, a desire to he opened the door to the bona fide practice of pharmacy, in
know the basic structure and composition of living things. which prescribed external substances were taken into the
From the beginning of time, a series of observationsboth body and targeted to the presumed sources of biochemical
scientific and fancifulaccrued in an effort to inform that aberration or deficiency [4].
topic. Such data emanated from several and diverse That approach clearly affected the primary focus of
sources, such as hunter-gatherers, alchemists, mathemati- medicine in the middle ages, which was nonmorphological
cians, abbatoir workers, physicians, anatomists, morticians, and primitively centered on biologic chemistry. Physiologic
astrologers, sorcerers, philosophers, and theologians [1-4]. mechanisms and anatomical structure were regarded as
In ancient Greece, Hippocrates theorized that diseases relatively inconsequential during that period of history.
were caused by imbalances in 4 basic body substances, called Therefore, no disadvantage was attached to destructive
humors: phlegm, blood, black bile, and yellow bile [4,5]. (digestive) analysis of plants and animals, in efforts to discern
Astoundingly, variations on that mechanistic scheme were their chemical constitution [8].
accepted as dogma until the 19th century. Diets designed to Beginning in the 16th century and through the efforts of
cleanse putrefied juices were therapeutically joined with Andreas Vesalius, William Harvey, Anton van Leeuwenhoek,
purging or venesection or both to reestablish a balance and others, the study of anatomical structures grew steadily at
between the 4 humors [6]. Theophrastus Phillippus Aureolus gross and microscopic levels [1,3]. Botany was the principal
Bombastus von Hohenheim (1493-1541; also known as scientific discipline in which such activities evolved; early
Paracelsus) was among the first to challenge such views and textbooks on the subject of plant histochemistry included
practices [4]. He believed that illness was induced by factors Essai de Chimie Microscopique Appliquee a la Physiologie
originating without, rather than within, the body, and that it and Nouveau Systeme de Chimie Organique, both by
resultedat least partlyfrom imbalances of indigenous Francois-Vincent Raspail (Fig. 1) (1830 and 1833, respec-
chemicals and minerals [7]. As a corollary to that premise, tively) [9,10]; Lehrbuch der physiologischen Chemie by Karl
Paracelsus encouraged investigations of the compounds and Gotthelf Lehmann (1842) [11]; and Handbuch der Experi-
mental Physiologie der Pflanzen by Julius von Sachs (1865)
[12]. Interestingly, botanists retained a basic interest in the
University of Virginia Hospital, Charlottesville, VA 22908-0214, cellular chemical processes that were illumined by histo-
USA. Tel.: +1 434 242 2410. chemistry, whereas zoology-oriented histologists and histo-
E-mail address: mrwick1@usa.net. chemists used microscopy and staining techniques primarily
1092-9134/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.anndiagpath.2011.10.010
72 M.R. Wick / Annals of Diagnostic Pathology 16 (2012) 7178
Table 1
Histochemical methods now in use in anatomical pathology
Histochemical stain Principal use Secondary use
Acid-fast bacilli (Ziehl-Neelsen) stain Identification of mycobacteria and selected other Identification of mast cell granules and lipofuscin
microorganisms in granulomatous diseases
Alcian blue stain Identification of acidic and neutral mucins in None
human cells and microorganisms
Bielschowsky silver technique Identification of neuronal axons Labeling of reticulin and mucosubstances
Bodian silver technique Identification of neuronal axons Labeling of reticulin and mucosubstances
Brown and Brenn (tissue Gram) stain Identification of bacteria and selected other Labeling of high-molecular-weight keratin
microorganisms
Colloidal iron stain Labeling of acidic and neutral mucins in human None
cells and microorganisms
Congo red stain Identification of amyloid Labeling of foreign material containing cellulose
Copper stains (rhodanine and orcein) Labeling of tissue copper deposits Identification of hepatitis B virus in hepatocytes (orcein)
Elastic (Verhoeff-van Gieson) stain Labeling elastic tissue Labeling collagen and enhancing nuclear detail
Fibrin (Fraser-Lendrum) stain Identifying fibrin deposits Labeling collagen and high-molecular-weight keratin
Fite's acid-fast stain Labeling nontuberculous mycobacteria and Labeling mastocytic granules and lipofuscin
selected other microorganisms in tissue
Fontana-Masson stain Identification of melanin Labeling neurosecretory granules in argentaffin cells;
labeling neuromelanin
Giemsa stain Identification of primary granules in myeloid and Labeling of protozoan microorganisms; identification of
mast cells amyloid (with metachromasia)
Gridley silver method Identification of amoebae and fungi in tissue Labeling of reticulin fibers and mucins
Grimelius silver stain Identification of neurosecretory granules in Labeling of mucins
neuroendocrine tumors
Grocott methanamine-silver method Labeling of fungi (including Pneumocystis) Labeling of mucins
Hall stain Identification of bile pigment None
Iron (Perls; Prussian blue) stain Identification of hemosiderin pigment None
Jones silver stain Labeling of basement membranes Labeling of mucins
Leder stain Identification of myeloid-cell and mast-cell granules None
Luxol fast blue Labeling of myelin in central and peripheral Identification of neurolipofuscin
nervous tissue
Masson trichrome stain Differentiation of collagen, elastic tissue, muscle, Identification of selected protozoa
and epithelium
Methyl greenpyronin stain (MGP) Labeling of nucleic acids (DNA and RNA) None
Mucicarmine (Mayer) stain Labeling of neutral (epithelial) mucins in tissues Identification of selected microbes
Nissl substance (cresyl violet) stain Identification of extranuclear ribonucleic acid in Metachromatic labeling of amyloid
neurons and other cell types
Oil-Red-O stain Identification of lipid deposits (requires use of Labeling of lipochrome
frozen tissue)
Periodic acid Schiff method Identification of glycogen (undigested tissue) or Labeling of selected microorganisms, especially fungi
neutral mucins and basement membrane material
(after tissue digestion with diastase)
Phosphotungstic acidhematoxylin stain Labeling of myofilaments, especially in striated Labeling of fibrin deposits
muscle cells
Reticulin (Sweet) method Identification of reticulin fibers (type III collagen) Labeling of mucins in tissue
in connective tissue
Steiner technique Silver-impregnation method for identification of Labeling of mucins in tissue
spirochetes and Legionella bacteria in tissue
Toluidine blue stain Identification of myeloid and mastocytic granules Labeling of protozoan organisms in tissue; metachromatic
in tissue sections or blood smears staining of amyloid
Trichrome (Masson) stain Differential staining of collagen (blue), muscle Identification of selected microbes (eg, amoeba; helminths)
(red), and elastic tissue (purple) in tissue
Urate (DeGalantha) stain Labeling of urate deposits in tissue None
(best used with alcohol fixation)
Von Kossa stain Identification of calcium salts in tissue None
Weigert stain Labeling of myelin in neural tissue None
stated that it is not sufficient to content ourselves with using elements such as iron or phosphorus, but the presence of
acid and basic dyes and speculating on the basic or acid nature organic complexes such as the carbohydrate groups, the
of the tissues or to apply color radicals with oxidizing or nucleins, protamines, and others [35].
reducing properties we must endeavor to find staining Most scientists in the above-listed group took that
reactions which will indicate not only the presence of certain directive to heart, as did others after them. Indeed, many
M.R. Wick / Annals of Diagnostic Pathology 16 (2012) 7178 75
Table 2 Table 4
Histochemical methods: undifferentiated large-cell neoplasms Histochemical diagnosis of small round-cell tumors
Tumor PAS Mucin MGP FM Retic Tumor PAS w/o Pericellular reticulin
Carcinoma +/ +/ +/ 0 +OP PNET + to +++ 0
Germ-cell tumor + 0 +/ 0 +OP RMS + to +++ +
Lymphoma 0 0 ++ 0 +PCP Lymphoma 0 + to ++
Melanoma +/ 0 +/ +/ +OP Neuroblastoma 0 0
MESO +/ 0 +/ 0 +OP
PAS w/o means periodic acidSchiff stain without diastase.
PAS indicates periodic acidSchiff; MGP, methyl greenpyronin; FM, PNET indicates primitive neuroectodermal tumor; RMS, rhabdomyosarcoma.
Fontana-Masson; Retic, reticulin; OP, organoid pattern; PCP, pericellular
pattern; MESO, epithelioid mesothelioma.
maxims that are still true [46]. In regard to criticisms that
focused on the nonspecificity or crudeness of some
microscopists became so engrossed by a characterization of histochemical reactions, he said criteria of specificity [in
in situ chemical reactions that the practical and diagnostic histochemistry] are similar to those in qualitative chemistry in
uses of histochemistry were given short shrift. The general [they] can be very much enhanced if two or more
admonitions of Mann [35], and Lewis after him [15], different reactions can be applied and compared. Prescient-
became the marching orders of the day. Histochemical ly, he went to opine that analysis in-situ is a non-quantitative
textbooks written by Lison [36] in 1936, Glick [37] in 1949, procedure. Sensitivity, therefore, merely concerns the limits
Gomori [38] in 1952, Pearse [39] in 1953, Lillie [40] in at which the reaction is discernible. The latter comments
1954, Bancroft [41] in 1967, Kiernan [42] in 1981, and apply equally well today, in reference to modern attempts at
Sumner [43] in 1988 were devoted largely to the chemistry quantitative immunohistochemistry [47].
of tissues as seen under the microscope. As a result, Three years earlier, Robert Stowellchair of pathology
knowledge of cellular biochemistry grew exponentially at the University of California-Davishad suggested that
during the 20th century. By 2000, Coleman [44] was able fundamental, critical research on new cytochemical tech-
to say confidently that histochemistry and cytochemistry niques will do more to advance our eventual understanding
allow precise analysis of the chemistry of cells and tissues in of normal tissues and neoplasia than the application of the
relation to structural organization. He also went on to state relatively few and often none-too-satisfactory histochemical
that histochemistry was still a useful and productive field of and cytochemical techniques now available [48]. In
study and that there arefew other disciplines in experi- counterpoint, Pearsearguably the most well-versed histo-
mental biology or medicine that can make a similar claim. chemist of all (Fig. 3)responded thus to Dr Stowell: in
medicine the new and imperfect remedy does not await
perfection by the research of groups of collaborating
3. Histochemists with a diagnostic orientation investigators in the pure sciences. It is applied forthwith
topatients by the practitioners of medicine, and it is often
As mentioned earlier in this discussion, philosophical by their observations and research that real advancement in
tension has existed between basic and applied histoche- the use of the remedy, and in knowledge of its mechanism
mists for well over 100 years. This is not a novel situation, and and meaning, is brought about. I believe very strongly,
in fact, it has applied to every one of the translational therefore, that the methods of modern histochemistry,
scientific techniques used in morphology-oriented areas of despite their imperfections, should be applied by all
laboratory medicine. Electron microscopy, immunohistol- practitioners in the biological, cytological, and pathological
ogy, in situ hybridization, polymerase chain reactionbased sciences [49]. After the passage of another 20 years, Pearse
procedures, and other blotting technologies have served as could further state that histopathology can be transformed
comparable battlegrounds for purists and practitioners [45]. by the application of any technology which confers upon its
In 1955, Jonas Friedenwalda basic researcher in observations an increase in objectivity. Foremost in the field
ophthalmology at Johns Hopkins Universitypublished a comes histochemistry, for a variety of reasons. These include
review of applied histochemistry, including in it several sheer breadth of scope and overwhelming numerical
superiority in respect of techniques [50].
Pearse [39], Bancroft and Stevens [51], and Filipe and
Table 3
Lake [52] took such tenets and built textbooks around them
Histochemical methods in selected dermatological conditions
in the latter part of the twentieth century. With such
Lupus erythematosus (stromal mucin stains; PAS-D to show thick EBM)
perspectives by experienced hospital pathologists, the place
Granuloma annulare (stromal mucin stains show increased interstitial mucin)
Porphyria cutanea tarda (PAS-D stain shows EBM abnormalities) of histochemistry as a valuable clinical method was
Perforating dermatoses (trichrome and VVG stains demonstrate extrusion solidified. Despite refinement and flux in the nosologic
of dermal connective tissue through epidermis) categorization of some human diseases, histochemical
Abbreviations: PAS-D, perioric acid Schiff stain with diastase digestion; analysis continues to offer important information in regard
EBM, epidermal basement membrane; VVG, Verhoeff-van Gieson stain. to histopathologic diagnosis and differential diagnosis. A
76 M.R. Wick / Annals of Diagnostic Pathology 16 (2012) 7178
Fig. 4. A, This tumor of the lung is labeled with the Best mucicarmine method, demonstrating the presence of abundant intracellular epithelial mucin and
supporting a diagnosis of adenocarcinoma (mucicarmine, 300). B, The liver in sickle-cell disease shows easily seen hemosiderin deposits, using Perls' method
(Perls stain, 200). C, This epithelioid neoplasm of the dermis exhibits diffuse reactivity with the chloroacetate estrase (Leder) stain, consonant with its identity
as a granulocytic sarcoma (Leder stain, 200). D, chromaffinity is observed in this ileal carcinoid tumor, using the Fontana-Masson technique (Fontana-Masson
stain, 200).
sampling of histochemical methods now in use in anatomical well-defined histologic contexts. Table 5 shows selected
pathology is presented in Table 1. Tables 2 to 4 and Fig. 4 infectious organisms requiring special histochemical tech-
show selected practical applications of selected stains in niques for identification.
Table 5
Selected infectious organisms requiring special histochemical techniques for
identification 4. The nexus of histochemistry with immunology and
Spirochetes, Legionella, Bartonella, and Yersiniarequire use of the molecular biology
Dieterle or Warthin-Starry silver stains and will not label with Brown-
Hopps method Dr Albert Coons was still a house-officer at Massachu-
Atypical mycobacteriabest recognized with the Fite procedure
setts General Hospital when he conceived a simple but
Rhodococcus, Legionella micdadeii, and Nocardia are also acid fast with
the Ziehl-Neelsen procedure revolutionary idea. His thought was to label antibodies with a
Dematiaceous fungi (as in chromoblastomycosis and phaeohyphomycosis) chemical tag, so that their binding to predefined antigens in
are Fontana-Masson positive because of melanin content tissue could be visualized microscopically. Despite the fact
Viruses can be labeled with the methyl greenpyronin e stain, as well as that antibody structure was only primitively understood at
Macchiavello method and Lendrum technique
that time and the lack of a proven technique for artificially
M.R. Wick / Annals of Diagnostic Pathology 16 (2012) 7178 77
5. Conclusions
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