Histochemistry as a tool in morphological analysis: a historical review

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Annals of Diagnostic Pathology 16 (2012) 71 – 78

History of Pathology

Histochemistry as a tool in morphological analysis: a historical review Mark R. Wick, MD⁎ Divisions of Surgical Pathology & Cytopathology and Autopsy Pathology, University of Virginia Medical Center, Charlottesville, VA, USA

Abstract

Keywords:

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. Histochemistry; Histology; Immunohistochemistry; Biochemistry

One can defensibly argue that biochemistry and histology originated from the same human interest, that is, a desire to know the basic structure and composition of living things. From the beginning of time, a series of observations—both scientific and fanciful—accrued in an effort to inform that topic. Such “data” emanated from several and diverse sources, such as hunter-gatherers, alchemists, mathematicians, abbatoir workers, physicians, anatomists, morticians, astrologers, sorcerers, philosophers, and theologians [1-4]. In ancient Greece, Hippocrates theorized that diseases were caused by imbalances in 4 basic body substances, called humors: phlegm, blood, black bile, and yellow bile [4,5]. Astoundingly, variations on that mechanistic scheme were accepted as dogma until the 19th century. Diets designed to “cleanse putrefied juices” were therapeutically joined with purging or venesection or both to reestablish a balance between the 4 humors [6]. Theophrastus Phillippus Aureolus Bombastus von Hohenheim (1493-1541; also known as Paracelsus) was among the first to challenge such views and practices [4]. He believed that illness was induced by factors originating without, rather than within, the body, and that it resulted—at least partly—from imbalances of indigenous chemicals and minerals [7]. As a corollary to that premise, Paracelsus encouraged investigations of the compounds and

⁎ University of Virginia Hospital, Charlottesville, VA 22908-0214, USA. Tel.: +1 434 242 2410. E-mail address: [email protected]. 1092-9134/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.anndiagpath.2011.10.010

elements that comprised plant and animal tissues. Moreover, he opened the door to the bona fide practice of pharmacy, in which prescribed external substances were taken into the body and targeted to the presumed sources of biochemical aberration or deficiency [4]. That approach clearly affected the primary focus of medicine in the middle ages, which was nonmorphological and primitively centered on biologic chemistry. Physiologic mechanisms and anatomical structure were regarded as relatively inconsequential during that period of history. Therefore, no disadvantage was attached to destructive (digestive) analysis of plants and animals, in efforts to discern their chemical constitution [8]. Beginning in the 16th century and through the efforts of Andreas Vesalius, William Harvey, Anton van Leeuwenhoek, and others, the study of anatomical structures grew steadily at gross and microscopic levels [1,3]. Botany was the principal scientific discipline in which such activities evolved; early textbooks on the subject of plant histochemistry included Essai de Chimie Microscopique Appliquee a la Physiologie and Nouveau Systeme de Chimie Organique, both by Francois-Vincent Raspail (Fig. 1) (1830 and 1833, respectively) [9,10]; Lehrbuch der physiologischen Chemie by Karl Gotthelf Lehmann (1842) [11]; and Handbuch der Experimental Physiologie der Pflanzen by Julius von Sachs (1865) [12]. Interestingly, botanists retained a basic interest in the cellular chemical processes that were illumined by histochemistry, whereas zoology-oriented histologists and histochemists used microscopy and staining techniques primarily

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a science can be traced to the introduction by Bencke, in 1852, of the aniline dyes which were in general use by 1880, followed closely by the development of paraffin-sectioning and photomicrography as routine techniques. Thus, by the end of the [19th] century, a fashion was set in histology which even today has not been completely supplanted. In retrospect, it may seem strange that the attention of histologists was concentrated for so long upon descriptive morphology without their making any serious attempt to study the chemistry of the structures they were staining… it is the use of a staining procedure with known chemical specificity that distinguishes a histochemical from a histological technique” [15]. The foregoing material sets the stage for a discussion of the 3 main categories into which “histochemists” can be assigned over the past 150 years. These are (1) investigators who were chemistry-oriented but not concerned with morphology; (2) those with contemporaneous interests in physiologic chemistry, histology, and technology; and (3) applied (diagnostic) histochemists (histopathologists). 1. “Histochemists” who were indifferent to morphology

Fig. 1. Etching of Francois-Vincent Raspail (1794-1878), one of the originators of histochemistry as a discipline. Raspail was also a naturalist and a politician.

to further the development of microanatomy, taxonomy, and nosology, more or less in vacuo. The latter situation led to an interesting, Darwinesque competition. Physiologic-cellular chemists began to disparage the efforts of histologists in the second half of the 19th century as unworthy of true scientific respect. Morphologists were regarded as little more than clerks and scribes who recorded their visual observations without correlating them to chemical findings [13]. That perception was furthered by the tendency of many histologists to embrace new stains and dyes as a means to an end (ie, morphological discrimination), rather than as ligands for cellular chemicals that had yet to be delineated. Pearse [8] framed this picture well, in saying, “although diagnostic significance was attached to many of the new color reactions, no attempt was made to put them on a physical or chemical basis.” Hence, in the era introduced by August Bencke in the 1860s and 1870s, with aniline dyes and similar reagents in hand, histology-based histochemists broke ranks, in philosophical and heuristic terms, with cellular biochemists [14]. A rancorous dichotomy persisted between the 2 groups well into the 20th century; indeed, as late as 1962, Lewis [15] stated that “the decay of histology as

As mentioned earlier, one, rather extreme, view of living organisms was that their structure is only important as a way of partitioning inorganic and organic substances, or chemical reactions. This was the credo of “pure” biochemists, who typically subscribed to “destructive” or “digestive” histochemistry. In such a context, the possible affinity of tissue for chemical laboratory reagents had “meaning” only if it illuminated the biochemical constitution of the substrate [8]. An example is represented in an early analysis, by FrancoisVincent Raspail, of starch in plant tissues, using the binding of iodine solution as an indicator [9]. The amylose in plant carbohydrates enters a colloidal suspension in water and comprises long polymeric chains of glucose units that are interconnected by alpha-acetal linkages, forming a 3dimensional coil. Iodine molecules can intercalate with the amylose coil, yielding a blue moiety [10,16]. The latter property obtains, regardless of whether the target is groundup plant material studied in a test tube, intact amylose-rich organs that are infused with potassium iodide (Fig. 2) or histologic sections of tissue that are “stained” with iodine and visualized with a microscope. To histochemists belonging to the pragmatic group under discussion, it would not matter—it would be sufficient to know that the target tissue did indeed contain starch, explaining its iodinophilia. Similar comments can be made regarding iron deposits (hemosiderin) in plants and animals. Perls [17] was among the first to show that acidified potassium ferrocyanide solution binds to iron in tissue, forming a relatively insoluble blue-purple precipitate with the chemical formula Fe7(CN)18(H2O)x, where 14 ≤ × ≤ 16. Again, in an egalitarian sense, it might be regarded as immaterial whether the iron was demonstrated in a glass beaker, an intact organism, or a

M.R. Wick / Annals of Diagnostic Pathology 16 (2012) 71–78

Fig. 2. Cut section of amyloidaceous myocardium, stained with Lugol's solution (elemental iodine and potassium iodide in solution). Because amyloid contains amylose-like carbohydrate sequences, the heart is stained blue.

microscope slide. Analogous models include the demonstrations of peroxidase in pus by Klebs [18] using tincture of guaiac in 1868, Ehrlich's [19] detection of cytochrome oxidase (originally called “Nadi oxidase”) in 1885 by intravenous injection of alpha-naphthol and p-phenylenediamine into animals, Rudolf Heidenhain's discovery that selected cells in the gastric mucosa would turn brown when exposed to chromic acid (“chromaffinity”) [20], and Miescher's [21] identification of DNA in cellular nuclei through its selective binding to methyl green. All of these assessments provided new information, but, from the perspective of current-day morphologists, they would be unsatisfying because the particular cellular locations of the chemical substances in question were not addressed. In that vein, microanatomy as a discipline was advancing in the 1800s as well, despite its being regarded as a “non-science” by biochemists of the period. The first attempt at a comprehensive textbook of histology was published in 1841 by Friedrich Gustav Henle [22], followed by a succession of additional works by Albert Donne, Arthur Hill Hassall, Rudolph Albert von Kolliker, Lionel Beale, Gottlieb Gluge, John Scott Burdon-Sanderson, Georg Eduard von Rindfleisch, Andre-Victor Cornil and LouisAntoine Ranvier, Edward Albert Schafer, Phillip Stohr, and other authors in the latter half of the 19th century [23]. The topical approach in several of those publications was to combine microanatomy with physiology, stressing both structure and function simultaneously. That orientation led to development of the next tier of histochemists, whose work occupied much of the 20th century.

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enthusiastically. Nevertheless, as cited earlier, that practice was often unaccompanied by a clear understanding of exactly what was being labeled by tissue staining procedures. Moreover, the detailed chemical mechanisms for such techniques commonly went unstudied as well. Dyes that had entered into biologic use included “cochineal” agents such as mucicarmine [24], aniline dyes [25], hematoxylin and its congeners [26], precipitable silver solutions [27], Schiff-base derivatives [28], colloidal suspensions of metal ions [29], phthalocyanines [30], cotton dyes (eg, Congo red, Pagoda red) [31], methyl and ethyl green [32], and others. An international group of investigators increasingly focused on the biochemical processes and targets that were associated with the use of such reagents, in the 1890s and beyond. They included—but were not limited to—individuals such as Paul Ehrlich, Santiago Ramon y Cajal, Karl Weigert, Pio del RioHortega, Joseph von Gerlach, Paul Mayer, Friedrich Miescher, Alfred Fischer, Gustav Mann, Robert Feulgen, Julius von Kossa, Frank Burr Mallory, Lucien Lison, Clyde Mason, Maffo Vialli, Emile Chamot, David Glick, George Gomori, Anthony Guy Everson Pearse, and Ralph Lillie [8]. Some early explanations for the cellular affinities of dyes were scientifically infantile, for example, Fischer suggested in 1899 that all stains were merely absorbed passively by tissue [33]. Conversely, Ehrlich [34] and Miescher [21] correctly believed that specific chemical coupling was responsible, and in his text titled Physiological Histology—published in 1902—Mann

2. “In situ” biochemical histochemists Given the availability of dyes that evolved during the mid 1800s, histologists of the period began to use them

Fig. 3. Anthony Guy Everson Pearse, MA, MD, FRCPath, DCP, FRCP (1916-2003), Professor of Histochemistry at the Royal Postgraduate Medical School in London, England.

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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 microorganisms in granulomatous diseases Identification of acidic and neutral mucins in human cells and microorganisms Identification of neuronal axons Identification of neuronal axons Identification of bacteria and selected other microorganisms Labeling of acidic and neutral mucins in human cells and microorganisms Identification of amyloid Labeling of tissue copper deposits Labeling elastic tissue Identifying fibrin deposits Labeling nontuberculous mycobacteria and selected other microorganisms in tissue Identification of melanin

Identification of mast cell granules and lipofuscin

Alcian blue stain Bielschowsky silver technique Bodian silver technique Brown and Brenn (tissue Gram) stain Colloidal iron stain Congo red stain Copper stains (rhodanine and orcein) Elastic (Verhoeff-van Gieson) stain Fibrin (Fraser-Lendrum) stain Fite's acid-fast stain Fontana-Masson stain Giemsa stain Gridley silver method Grimelius silver stain Grocott methanamine-silver method Hall stain Iron (Perls; Prussian blue) stain Jones silver stain Leder stain Luxol fast blue Masson trichrome stain Methyl green–pyronin stain (MGP) Mucicarmine (Mayer) stain Nissl substance (cresyl violet) stain Oil-Red-O stain Periodic acid Schiff method

Phosphotungstic acid–hematoxylin stain Reticulin (Sweet) method Steiner technique Toluidine blue stain Trichrome (Masson) stain Urate (DeGalantha) stain Von Kossa stain Weigert stain

Identification of primary granules in myeloid and mast cells Identification of amoebae and fungi in tissue Identification of neurosecretory granules in neuroendocrine tumors Labeling of fungi (including Pneumocystis) Identification of bile pigment Identification of hemosiderin pigment Labeling of basement membranes Identification of myeloid-cell and mast-cell granules Labeling of myelin in central and peripheral nervous tissue Differentiation of collagen, elastic tissue, muscle, and epithelium Labeling of nucleic acids (DNA and RNA) Labeling of neutral (epithelial) mucins in tissues Identification of extranuclear ribonucleic acid in neurons and other cell types Identification of lipid deposits (requires use of frozen tissue) Identification of glycogen (undigested tissue) or neutral mucins and basement membrane material (after tissue digestion with diastase) Labeling of myofilaments, especially in striated muscle cells Identification of reticulin fibers (type III collagen) in connective tissue Silver-impregnation method for identification of spirochetes and Legionella bacteria in tissue Identification of myeloid and mastocytic granules in tissue sections or blood smears Differential staining of collagen (blue), muscle (red), and elastic tissue (purple) in tissue Labeling of urate deposits in tissue (best used with alcohol fixation) Identification of calcium salts in tissue Labeling of myelin in neural tissue

stated that “it is not sufficient to content ourselves with using acid and basic dyes and speculating on the basic or acid nature of the tissues or to apply color radicals with oxidizing or reducing properties… we must endeavor to find staining reactions which will indicate not only the presence of certain

None Labeling of reticulin and mucosubstances Labeling of reticulin and mucosubstances Labeling of high-molecular-weight keratin None Labeling of foreign material containing cellulose Identification of hepatitis B virus in hepatocytes (orcein) Labeling collagen and enhancing nuclear detail Labeling collagen and high-molecular-weight keratin Labeling mastocytic granules and lipofuscin Labeling neurosecretory granules in argentaffin cells; labeling neuromelanin Labeling of protozoan microorganisms; identification of amyloid (with metachromasia) Labeling of reticulin fibers and mucins Labeling of mucins Labeling of mucins None None Labeling of mucins None Identification of neurolipofuscin Identification of selected protozoa None Identification of selected microbes Metachromatic labeling of amyloid Labeling of lipochrome Labeling of selected microorganisms, especially fungi

Labeling of fibrin deposits Labeling of mucins in tissue Labeling of mucins in tissue Labeling of protozoan organisms in tissue; metachromatic staining of amyloid Identification of selected microbes (eg, amoeba; helminths) None None None

elements such as iron or phosphorus, but the presence of organic complexes such as the carbohydrate groups, the nucleins, protamines, and others” [35]. Most scientists in the above-listed group took that directive to heart, as did others after them. Indeed, many

M.R. Wick / Annals of Diagnostic Pathology 16 (2012) 71–78 Table 2 Histochemical methods: undifferentiated large-cell neoplasms

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Table 4 Histochemical diagnosis of small round-cell tumors

Tumor

PAS

Mucin

MGP

FM

Retic

Tumor

PAS w/o

Pericellular reticulin

Carcinoma Germ-cell tumor Lymphoma Melanoma MESO

+/− + 0 +/− +/−

+/− 0 0 0 0

+/− +/− ++ +/− +/−

0 0 0 +/− 0

+−OP +−OP +−PCP +−OP +−OP

PNET RMS Lymphoma Neuroblastoma

+ to +++ + to +++ 0 0

0 + + to ++ 0

PAS indicates periodic acid–Schiff; MGP, methyl green–pyronin; FM, Fontana-Masson; Retic, reticulin; OP, organoid pattern; PCP, pericellular pattern; MESO, epithelioid mesothelioma.

microscopists became so engrossed by a characterization of in situ chemical reactions that the practical and diagnostic uses of histochemistry were given short shrift. The admonitions of Mann [35], and Lewis after him [15], became the marching orders of the day. Histochemical textbooks written by Lison [36] in 1936, Glick [37] in 1949, Gomori [38] in 1952, Pearse [39] in 1953, Lillie [40] in 1954, Bancroft [41] in 1967, Kiernan [42] in 1981, and Sumner [43] in 1988 were devoted largely to the chemistry of tissues as seen under the microscope. As a result, knowledge of cellular biochemistry grew exponentially during the 20th century. By 2000, Coleman [44] was able to say confidently that “histochemistry and cytochemistry… allow precise analysis of the chemistry of cells and tissues in relation to structural organization.” He also went on to state that histochemistry was still a useful and productive field of study and that “there are…few other disciplines in experimental biology or medicine that can make a similar claim.”

3. Histochemists with a diagnostic orientation As mentioned earlier in this discussion, philosophical tension has existed between “basic” and “applied” histochemists for well over 100 years. This is not a novel situation, and in fact, it has applied to every one of the “translational” scientific techniques used in morphology-oriented areas of laboratory medicine. Electron microscopy, immunohistology, in situ hybridization, polymerase chain reaction–based procedures, and other “blotting” technologies have served as comparable battlegrounds for purists and practitioners [45]. In 1955, Jonas Friedenwald—a “basic” researcher in ophthalmology at Johns Hopkins University—published a review of applied histochemistry, including in it several Table 3 Histochemical methods in selected dermatological conditions • Lupus erythematosus (stromal mucin stains; PAS-D to show thick EBM) • Granuloma annulare (stromal mucin stains show increased interstitial mucin) • Porphyria cutanea tarda (PAS-D stain shows EBM abnormalities) • Perforating dermatoses (trichrome and VVG stains demonstrate extrusion of dermal connective tissue through epidermis) Abbreviations: PAS-D, perioric acid Schiff stain with diastase digestion; EBM, epidermal basement membrane; VVG, Verhoeff-van Gieson stain.

PAS w/o means periodic acid–Schiff stain without diastase. PNET indicates primitive neuroectodermal tumor; RMS, rhabdomyosarcoma.

maxims that are still true [46]. In regard to criticisms that focused on the “nonspecificity” or crudeness of some histochemical reactions, he said “criteria of specificity [in histochemistry] are similar to those in qualitative chemistry in general… [they] can be very much enhanced if two or more different reactions can be applied and compared.” Presciently, he went to opine that “analysis in-situ is a non-quantitative procedure. Sensitivity, therefore, merely concerns the limits at which the reaction is discernible.” The latter comments apply equally well today, in reference to modern attempts at “quantitative” immunohistochemistry [47]. Three years earlier, Robert Stowell—chair of pathology at the University of California-Davis—had suggested that “fundamental, critical research on new cytochemical techniques will do more to advance our eventual understanding of normal tissues and neoplasia than the application of the relatively few and often none-too-satisfactory histochemical and cytochemical techniques now available” [48]. In counterpoint, Pearse—arguably the most well-versed histochemist 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 investigators in the pure sciences. It is applied forthwith to…patients by the practitioners of medicine, and it is often by their observations and research that real advancement in the use of the remedy, and in knowledge of its mechanism and meaning, is brought about. I believe very strongly, therefore, that the methods of modern histochemistry, despite their imperfections, should be applied by all practitioners in the biological, cytological, and pathological sciences” [49]. After the passage of another 20 years, Pearse could further state that “histopathology can be transformed by the application of any technology which confers upon its observations an increase in objectivity. Foremost in the field comes histochemistry, for a variety of reasons. These include sheer breadth of scope and overwhelming numerical superiority in respect of techniques” [50]. Pearse [39], Bancroft and Stevens [51], and Filipe and Lake [52] took such tenets and built textbooks around them in the latter part of the twentieth century. With such perspectives by experienced hospital pathologists, the place of histochemistry as a valuable clinical method was solidified. Despite refinement and flux in the nosologic categorization of some human diseases, histochemical analysis continues to offer important information in regard to histopathologic diagnosis and differential diagnosis. A

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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 pathology is presented in Table 1. Tables 2 to 4 and Fig. 4 show selected practical applications of selected stains in Table 5 Selected infectious organisms requiring special histochemical techniques for identification • Spirochetes, Legionella, Bartonella, and Yersinia—require use of the Dieterle or Warthin-Starry silver stains and will not label with BrownHopps method • “Atypical” mycobacteria—best recognized with the Fite procedure • Rhodococcus, Legionella micdadeii, and Nocardia are also acid fast with the Ziehl-Neelsen procedure • Dematiaceous fungi (as in chromoblastomycosis and phaeohyphomycosis) are Fontana-Masson positive because of melanin content • Viruses can be labeled with the methyl green–pyronin e stain, as well as Macchiavello method and Lendrum technique

well-defined histologic contexts. Table 5 shows selected infectious organisms requiring special histochemical techniques for identification.

4. The nexus of histochemistry with immunology and molecular biology Dr Albert Coons was still a house-officer at Massachusetts General Hospital when he conceived a simple but revolutionary idea. His thought was to label antibodies with a chemical tag, so that their binding to predefined antigens in tissue could be visualized microscopically. Despite the fact that antibody structure was only primitively understood at that time and the lack of a proven technique for artificially

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However, in place of antibody probes to which chemical indicators can be joined, the basic investigative tools in in situ hybridization are specific sequences of nucleic acid that are complementary to DNA or RNA targets of interest in tissue sections [57,58] (Fig. 6).

5. Conclusions

Fig. 5. Direct immunofluorescence microscopy of a skin biopsy in bullous pemphigoid, which was labeled with fluorescein-tagged antibody to immunoglobulin G (EgG). Linear reactivity is seen at the epidermal basement membrand (Anti-IgG immunofluorescence, ×200).

binding other substances to them, Coons pursued the concept doggedly over several years [53]. Eventually, specific antibodies were produced in vivo in animal hosts that were specific for particular proteins. They were coupled successfully with fluorescein isocyanate and proved to be effective in localizing polypeptide targets in histologic sections that were illuminated by ultraviolet light with a special microscope [54,55] (Fig. 5). Hence, the field of immunofluorescence-based histochemistry was thereby established by Coons, who won the prestigious Albert Lasker award in 1959 for that contribution [56]. Today, many different chemical tags can be linked with a plethora of antibody reagents that are clinically relevant in pathology. In addition, the facet of molecular biology known as in situ hybridization is predicated on a similar construct.

Fig. 6. Chromagenic in situ hybridization (CISH) of a uterine cervical squamous carcinoma for human papillomavirus type 16 DNA. Several gene copies are seen in each tumor cell nucleus (CISH, ×250).

Histochemistry has had a long history as well as a broad interface with many of the other life sciences. Because the in situ chemical reactions it concerns have been thoroughly studied over many years, histochemistry is now one of the most objective methods in biology and medicine. That fact should not be forgotten in the current fervor over “new” techniques in pathology, nor should one slight the practical use of histochemistry in a variety of clinical differential diagnostic settings. The rapidity, reproducibility, and relatively low expense attached to this form of biomedical analysis continue to recommend it as a valuable enterprise, after nearly 200 years of existence. References [1] Persaud TVN. The early history of human anatomy: from antiquity to the beginning of the modern era, CC Thomas Books. IL: Springfield; 1984. [2] Dyer GS, Thorndike ME. Quidne mortui vivos docent? The evolving purpose of human dissection in medical education. Acad Med 2000;75:969-79. [3] McLachlan JC, Patten D. Anatomy teaching: ghosts of the past, present, and future. Med Educ 2006;40:243-53. [4] Nuland S. The mysteries within. New York: Simon & Schuster; 2000. p. 50-130. [5] Gill NS. Hippocratic method and the four humors in medicine. http:// ancienthistory.about.com/cs/hippocrates/a/hippocraticmeds.htm. Accessed 1-14-2010. [6] Shryock RH. Medicine & society in America: 1660-1860. Ithaca: Cornell University Press; 1972. p. 69-72. [7] Anonymous. Paracelsus. http://en.wikipedia.org/wiki/Paracelsus. Accessed 1-4-2010. [8] Pearse AGE. The history of histochemistry. In: & Pearse AGE, editor. Histochemistry. London: Churchill; 1960. p. 1-12. [9] Raspail F-V. Essai de Chimie Microscopique Appliquee a la Physiologie. Paris: Meihac Publishers; 1830. [10] Raspail F-V. Nouveau Systeme de Chimie Organique. Paris: JB Bailliere Publishers; 1833. [11] Lehmann KG. Lehrbuch der physiologischen Chemie. Leipzig: Engelmann Publishers; 1842. [12] von Sachs J. Handbuch der Experimental Physiologie der Pflanzen. Leipzig: Engelmann Publishers; 1865. [13] von Bunge GB. Lehrbuch der physiologischen und pathologischen Chemie. Leipzig: Vogel Publishers; 1887. [14] Kornhauser SI. The history of staining: the development of cytological staining. Stain Technol 1930;5:117-25. [15] Lewis PR. Histochemistry in biology. In: & Garthy JD, editor. Viewpoints in Biology. London: Butterworths; 1962. p. 49-88. [16] Yu X, Houtman C, Atalla RH. The complex of amylose and iodine. Carbohydrate Res 1996;22:129-41. [17] Perls M. Nachweis von Eisenoxyd in gewissen pigmenten. Virchows Arch 1867;39:42-8. [18] Klebs E. Die pyrogene substanz. Z Med Wiss 1868;6:417-37.

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