Neuron-specific enolase in mucosal endocrine cells and carcinoid tumours of the small intestine: A comparative study with neuron-specific enolase immunocytochemistry and silver stains

Summary Endocrine cells of human small intestinal mucosa, small intestinal carcinoids and carcinoid liver metastases were stained with an immunocytochemical technique using an antiserum against neuron-specific enolase (NSE), with the argyrophil technique of Grimelius and with the argentaffin technique of Masson. In the normal mucosa, scattered NSE-immunoreactive cells were seen mainly in the deeper parts of the crypts. These cells, as shown in the same sections, corresponded to the argentaffin and/or argyrophil cells indicating that they were of endocrine type.All intestinal carcinoids (16 cases) displayed NSE immunoreactivity. However, this reaction did not correlate on the cellular level with the silver techniques employed. Thus, many tumour cells were NSE immunoreactive but lacked an argentaffin or argyrophil reaction and vice versa. On the light microscopical level the silver techniques reveal the presence of neurohormonal granules in the tumour cells, while the NSE immunoreactivity appears to disclose neuroendocrine differentiation of the tumour cells irrespective of their hormone and granular content.Out of 13 carcinoid liver metastases, eight displayed strong NSE immunoreactivity, three were weakly stained and two were unreactive. Consecutive or the same tumour sections showed an argentaffin and argyrophil reaction in all carcinoid metastases. Since silver staining provides one type of information and NSE immunocytochemistry another, they provide in combination a good discriminator for neuroendocrine tumours.     

Silver stains for identification of neuroendocrine cells. A study of the chemical background

Summary The chemical background of silver stains used for visualization and characterization of peripheral neuroendocrine cells in the gastrointestinal tract and pancreas, and of their corresponding tumours, was studied in tissue sections and by a dot-blot technique. Sequential staining of pancreatic islets with an immunohistochemical procedure and silver staining of the same tissue section revealed that chromogranin A immunostained cells also displayed an argyrophil reaction with the Grimelius method, but no argentaffin reaction with the Masson technique. Accordingly, purified chromogranin A (15 g or less) treated in formalin and applied to nitrocellulose did not show any argentaffin reaction but displayed a dose-related argyrophil reaction. Equal quantities of other polypeptide components did not give rise to any silver reaction. Further dot-blot studies showed that the tryptophan and tyrosine metabolites, dopamine, norepinephrine, 5-hydroxytryptamine and 5-hydroxindole caused strongly argentaffin and argyrophil reactions while epinephrine, 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophan gave only the former reaction. Among other chemical components studied, only guanine displayed weak silver staining. The results indicate that the reaction products between aldehydes and the granular content of biogenic amines synthesized from tryptophan and tryosine display an argentaffin reaction and that the granular chromogranin A caused an argyrophil but no argentaffin reaction.     

Silver stains in the study of endocrine cells of the gut and pancreas

The usefulness of argentaffin (Masson) and argyrophil (Davenport, Sevier-Munger and Grimelius) staining methods for identification of endocrine cell types in the gastrointestinal tract and pancreas is discussed and comments are made on the techniques themselves. The applicability of silver impregnation methods in the histopathological investigations of endocrine tumours in the above-mentioned organs is outlined, and the chemical background of the silver reactions is briefly reviewed.     

Serotonin in cytologic samples of liver metastases of carcinoid tumors. An immunocytochemical study with a monoclonal antibody

Cytologic samples of malignant carcinoid tumors were examined with regard to the presence of serotonin by immunocytochemical staining with a monoclonal antibody. Serotonin immunoreactivity occurred in tumor cells derived from carcinoids of the small intestine while bronchial carcinoid tumor cells were nonreactive. Acetone-alcohol fixation of the cells was a prerequisite for an adequate staining. The serotonin-immunoreactive tumors were also argentaffin positive. The results indicate that cytologic specimens of neuroendocrine tumors, such as carcinoid, can be successfully assayed for the presence of serotonin by an immunocytochemical procedure.     

Immunocytochemical study of argyrophil (Sevier Munger) endocrine cells of adult human pancreas

Summary Bouin-fixed tissues from non-diabetic adult human pancreata display an argyrophil reaction mainly in the periphery of the islets with the silver technique of Sevier-Munger. The nature of these argyrophil cells was examined after restaining by an indirect immunocytochemical method using antibodies against insulin, glucagon, somatostatin and pancreatic polypeptide. After this procedure the argyrophil cells were identified as glucagon (A-) cells and pancreatic polypeptide (PP-) cells, although the latter exhibited a weaker reaction. The insulin (B-) cells and somatostatin (D-) cells were unreactive. The results show that the Sevier-Munger stain is of equal value to the Grimelius silver nitrate stain in adult human pancreatic islets after fixation in Bouin's fluid.Supported by grants from the Swedish Medical Research Council (Project No. 102)     

Silver stains demonstrating neuroendocrine cells

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.     

Gastrointestinal carcinoid tumours. Histogenetic, histochemical, immunohistochemical, clinical and therapeutic aspects

The increased knowledge of the pathobiology of gastrointestinal carcinoid (neuroendocrine) tumours and the improved therapeutic possibilities have brought a demand for more precise diagnosis. Although the carcinoid tumours can often be tentatively recognized in routinely processed microscopic slides, their more accurate identification requires additional diagnostic procedures. General neuroendocrine markers such as the argyrophil reaction of Grimelius and immunohistochemistry with application of antibodies against chromogranin A and of neuron-specific enolase are discriminatory staining methods which are used to reveal the neuroendocrine origin of almost all highly differentiated carcinoid tumours of the gastrointestinal tract. Mid-gut carcinoids, which predominate among these tumours almost unexceptionally contain serotonin. This biogenic amine can be demonstrated by the argentaffin reaction of Masson, serotonin immunoreactively or by formalin-induced fluorescence. The characteristic staining pattern of mid-gut carcinoids is almost invariably preserved in the metastatic deposits and consequently the staining methods for identifying serotonin can also be used on metastases to reveal a primary mid-gut carcinoid. The enterochromaffin-like (ECL) cell carcinoids of the body and fundic area of the stomach often seen in association with pernicious anaemia are argyrophil with the Sevier-Munger silver stain. Other neuroendocrine tumours, viz. antral, duodenal and rectal carcinoids should be studied by a battery of relevant peptide hormone antisera for adequate diagnosis. During the last decade new peptide hormones have been found in circulation in patients with carcinoid tumours, but serotonin and urinary 5-HIAA are still the most important markers for carcinoids of the mid-gut origin. Other clinically useful tumour markers are chromogranin A + B, pancreatic polypeptide, human chorionic gonadotropin alpha and beta subunits. For localizing procedures, angiography is the most reliable investigative method for primary tumours in the gut, whereas CT-scan and ultrasound investigations are good for detection of liver metastases. During the last five years, the therapy for malignant carcinoid tumours has been considerably improved. Chemotherapy has only revealed objective response rates in about 10-30% of the patients giving median survivals from start of therapy of about 10 months. Recently treatment with alpha interferons and the new somatostatin analogue octreotide have given objective responses in 50-75% of patients with malignant mid-gut carcinoid tumours. These patients have now a median survival from start of therapy of 70 months when treated with alpha interferons. In the future new therapies will come into use such as monoclonal antibodies and perhaps also agents blocking different growth factors.     

Immunocytochemical study of argyrophil (Sevier Munger) endocrine cells of adult human pancreas

Bouin-fixed tissues from non-diabetic adult human pancreata display an argyrophil reaction mainly in the periphery of the islets with the silver technique of Sevier-Munger. The nature of these argyrophil cells was examined after restaining by an indirect immunocytochemical method using antibodies against insulin, glucagon, somatostatin and pancreatic polypeptide. After this procedure the argyrophil cells were identified as glucagon (A-) cells and pancreatic polypeptide (PP-) cells, although the latter exhibited a weaker reaction. The insulin (B-) cells and somatostatin (D-) cells were unreactive. The results show that the Seiver-Munger stain is of equal value to the Grimelius silver nitrate stain in adult human pancreatic islets after fixation in Bouin's fluid.     

Silver stains demonstrating neuroendocrine cells

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.     

Small intestinal chromaffin cells and carcinoid tumours: a study with silver stains, formalin-induced fluorescence and monoclonal antibodies to serotonin

Summary The enterochromaffin cells of the human small intestinal mucosa were stained immunocytochemically with monoclonal antibodies against serotonin. The staining results were compared with those obtained with other methods for identifying serotonin-containing endocrine cells such as the argentaffin reaction, formalin-induced fluorescence and the argyrophil reaction of Grimelius. The different techniques gave similar, but not identical, results. The serotonin-immunoreactive cells outnumbered the argentaffin cells by 7%. Almost all (99%) serotonin-immunoreactive cells showed formalin-induced fluorescence but only a small population (5%) were fluorescent. In a subsequent study, these techniques were applied to 14 small intestinal carcinoids. It was shown that formalin-induced fluorescence and the argentaffin reaction were positive in 14 and 13 tumours, respectively, while the monoclonal serotonin antibodies failed to stain seven of the tumours. It is concluded that formalin-induced fluorescence and the argentaffin reaction are more useful techniques than serotonin immunocytochemistry for defining these tumours in routine formalin-fixed surgical specimens.     

Enteroglucagon and substance P-like immunoreactivity in argentaffin and argyrophil rectal carcinoids.

A specific immunofluorescence for enteroglucagon or substance P or for both hormones was demonstrated in nine out of 12 examined rectal carcinoids. One tumor was argentaffin, contained ultrastructurally pleomorphic granules of the entero-chromaffin cell type, and showed immunofluorescence for substance P. The rest were non-argentaffin but were argyrophil with the Grimelius technique and contained round granules. The argyrophil carcinoids were immunoreactive to one or both hormones in eight cases and not fluorescent in three cases. In two of the non-argentaffin carcinoids a small number of argyrophil cells was found with the method of Sevier-Munger.     

Etude critique des distinctions tinctoriales effectuées parmi les cellules endocrines de la muqueuse fundique du Lapin

Résumé Si chez des animaux intacts ou ayant reçu de la L-Dopa on compare les images d'une même coupe traitée d'abord selon Falck, ensuite colorée ou imprégnée selon divers procédés, on peut constater les faits suivants: la formaldéhyde confère une fluorescence jaune terne (Jt) à des cellules GIC dépourvues d'argyrophilie selon Sevier et Munger mais imprégnées en technique de Grimélius, colorées par le picroponceau et l'hématoxyline au plomb. Il y a approximativement trois fois plus de cellules imprégnées selon Grimélius que de cellules fluorescentes; parmi elles 1/3 environ réagit fortement en technique de Sevier et Munger (cellules à sérotonine), 1.3 réagit faiblement (cellules SM), 1/3 ne réagit pas du tout (cellules D et Jt). Les cellules SM correspondent aux «ECL cells» bien que dépourvues d'amine et d'argentaffinité (Fontana-Masson); elles se divisent en GIC et non GIC, les premières sont ou non colorables par l'hématoxyline au plomb, les secondes ne le sont pas.

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Critical study of tinctorial distribution proposed among endocrine cells of the fundic mucosa in the rabbit

Summary Whether the same endocrine like cells are reactive towards different staining reactions has been hardly examined in normal and L-Dopa treated rabbits for distinction of cell types. Samples were first freeze-dried, exposed to formaldehyde vapours (standard Falck technic) and sections photographied. Thereafter they became further hydrated and were stained with silver according to Fontana-Masson or Grimelius or Sevier-Munger, or with ferric ferricyanide or lead hematoxylin or picroponceau.Formaldehyde condensation induced a bright yellow fluorescence in serotonin cells and revealed a dim yellow (jaune terne: Jt) unexpected fluorescence in others scarce cells. The laters were non argentaffin, unreactive with Sevier-Munger method but argyrophilic in Grimelius technic, stained blue black by lead hematoxylin and red by picroponceau; there are a part of the GIC or APUD cells group.Grimelius reaction stained three times as many cells as fluorescence did. With Sevier-Munger method, heavily darkened (serotonin cells), slightly reactive (Sevier-Munger or SM cells) and unreactive cells (D and Jt cells) roughly represented 1/3 each of all Grimelius positive cells. ECL cells according to Capella et al. (1969) apparently corresponded to SM cells; since all fluorescent cells are strongly argentaffin and reactive with Sevier-Munger method, since they are too much scanty to include EC and ECL cells it is conclude that ECL cells failed to exhibit fluorescence and to store serotonin: furthermore taking hypothetically ECL cells into fluorescent cells it would imply to blend D cells and SM cells which is at variance with earlier observations of unreactivity of D cells in Sevier-Munger technic at the ultrastructural level. SM cells population seemed heterogenous: only some of them were GIC and only some of the laters were stained by lead hematoxylin; however it is not clear how much the L-Dopa administration effect the second staining possibly causing erroneous reactions.

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Avec la collaboration technique de Mme A. de Gouyon.     

Effect of Temperature on Argyrophil Impregnation: Development of A High Temperature Rapid Argyrophil Procedure

Out studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

Effect of temperature on argyrophil impregnation: development of a high temperature rapid argyrophil procedure

Our studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

褐马鸡幽门区内分泌细胞的免疫组织化学研究

在褐马鸡的幽门区显示出了大量嗜银细胞和亲银细胞。免疫组织化学染色表明,褐马鸡的幽门区内存在有大量G细胞和D细胞,少量EC细胞和极少量PP细胞,其中EC细胞是迄今为止首次在鸟类幽门区得到证实的一个内分泌细胞类型。规察到G细胞、D细胞和EC细胞伸出长的胞质突起与邻近细胞相接触。GRP细胞、Mo细胞、A细胞和B细胞均未在幽门区检出。     

Electron microscopic classification of amine-producing endocrine cells by selective staining of ultra-thin sections

Summary The argyrophil, argentaffin and chromaffin reactions were performed directly on ultra-thin sections for examination in the electron microscope. Glutaraldehyde fixation was appropriate for the argentaffin and chromaffin reactions; additional fixation with osmium tetroxide, however, caused impairment of these reactions. Fixation with formaldehyde, but not with glutaraldehyde, was adequate for the argyrophil reaction; post-fixation with osmium tetroxide did not affect this staining. At the light microscopic level the staining reactions were correlated with fluorescence histochemistry according to the method of Falck and Hillarp. The techniques described were used to study certain amine-producing endocrine cell systems: adrenal medullary cells and thyroid parafollicular cells of the mouse, gastric endocrine cells from the oxyntic gland area of the mouse, rat and rabbit. All these cells stained argyrophil. The adrenal medullary cells and one cell type in the oxyntic gland area of the rabbit were strongly argentaffin and chromaffin. The remainder of the cells were non-argentaffin and non-chromaffin but could be induced to give an argentaffin (and chromaffin) reaction after injection of the animals with l-3,4-dihydroxyphenylalanine or l-5-hydroxytryptophan, a treatment which is known to result in the accumulation of the highly reducing dopamine and 5-hydroxytryptamine, respectively, in these endocrine cells. Without exception the precipitates formed in all the staining reactions accumulated selectively over the secretory granules of the cells.The techniques described permit differential staining of consecutive ultra-thin sections for electron microscopic characterization of one and the same cell. They will provide information necessary for correlative studies of the stainable cells at the light and electron microscopic levels.     

Age and changes in the number of endocrine cells in the stomach and their role in senile atrophy of the gastric glands

In the gastric mucous membrane of 40 rats representing 4 age groups (pubertal, mature, old and very old age) per 10 animals in each, thickness of the mucous membrane has been measured by the argyrophil method of Grimelius and the argentaffin method of Masson - Hamperl and the number of the endocrine cells has been counted. The thickness of the mucous membrane and the length of its glands, as well as the number of argyrophil cells increase from the pubertal towards the mature age. From the mature up to the old age the number of argyrophil cells rises in the antral part, and in the acid-producing zone it significantly decreases. The number of argentaffin cells in the stomach does not change from the pubertal towards the old age. In the very old age, against the background of atrophy of gastric glands, the number of both argyrophil and argentaffin cells decreases in both parts of the stomach studied. The data obtained are discussed taking into consideration functional role of various types of endocrine cells, producing biologically active peptides and monoamines.     

Pulmonary neuroendocrine cells: Decreased serotonin fluorescence and stable argyrophil-cell numbers in acute hypoxia

Summary We recorded serotonin-emission intensity using formalin induced fluorescence and counted the number of neuroendocrine cells (NEC's) and neuroepithelial bodies (NEB's) containing argyrophil granules. Comparison of serotonin-emission intensity in NEB's of acutely hypoxic neonates (520 mmHg for 2–21/2 h) and normoxic controls showed significantly lower fluorescence in hypoxic animals. Neuroendocrine cell numbers as shown with Grimelius silver-nitrate stain did not change. This suggests either that the Grimelius stain reacts with compounds other than serotonin, or that the decrease in serotonin in acutely hypoxic rabbits is not sufficient to alter argyrophil stainability.Supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and a grant from The Council for Tobacco Research, Inc. USA Number 1036We are grateful to Ms. Sharon DeFoliart and Ms. Maureen Decorah for technical assistance. We also thank Dr. Walter Flaut who kindly let us use the fluorescence microscope, and contributed helpful advice. The use of the hypobaric chamber at the Biotron, University of Wisconsin-Madison, was essential for the study     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

Cytologic diagnosis of a medullary carcinoma of the thyroid by Sevier-Munger silver staining and calcitonin immunocytochemistry

A medullary carcinoma of the thyroid was preoperatively diagnosed on ultrasonically guided fine needle aspiration biopsies. After cytocentrifugation, the tumor cells displayed a dense cytoplasmic silver granulation with the Sevier-Munger technique when applied to air-dried or acetone-ethanol-fixed samples and an obvious calcitonin immunoreactivity after fixation in Bouin's fluid. These methods may prove useful in the identification of nonpalpable metastases and recurrences of medullary carcinomas of the thyroid, especially since the cytologic typing of medullary thyroid carcinoma cells may be difficult with routine stainings.