Virchows Arch. A Path. Anat. and Histol. 371, 177--181 (1976) 9 by Springer-Verlag 1976

Fluorescamine Fluorescence Detection of Growth Hormone-Producing Cells in Human Pituitary Adenomas Arie C. Nieuwenhuijzen K r u s e m a n and Fr6 T. Bosman Department of Pathology, University of Leiden, Leiden, The Netherlands Received April 24, 1976 Summary. Formalin-fixed and paraplast-embedded tissue specimens of human pituitary, thyroid, and pancreas were investigated using fluorescamine fluorescence and immunohistochemical methods. Growth hormone-producing cells present in normal and neoplastic pituitary tissue exhibited fluorescamine fluorescence. The other tissues examined showed no fluorescamine binding. Key words: Fluorescamine fluorescence - - Immunohistochemistry - - Growth hormone - Pituitary adenomas.

Introduction

Recently, fiuorescamine has been introduced as a reagent in the fluorometric detection of amines, amino acids, peptides, and proteins in solutions (Udenfriend et al., 1972 ; Weigele et al., 1972) .In tissue specimens fixed in formaldehyde vapor, fluorescamine was found to give strong and selective fluorescence of certain cell types, m a n y of which either proved to produce or store polypeptide hormones or have been assumed to belong to this category (Hs et al., 1974). Therefore,

it has been suggested that fluorescamine fluorescence may be helpful in the histopathologic diagnosis of certain endocrine tumors (Sundler et al., 1974). In human pituitaries, growth hormone-producing cells were found to bind fiuorescamine particularly (Larsson ct al., 1975). During the last decade, immunohistochemical methods, particularly the peroxidase-labeling technique, have proved to be valuable in the identification of peptide hormones in routinely formalin-fixed and paraffin-embedded tissue specimens. Growth hormone-producing cells in p i t u i t a r y adenomas can be specifically identified using this technique (Nieuwenhnijzen K r u s e m a n et al., 1976). The aim of the present investigation was to determine whether fluorescamine fluorescence could be used as a rapid screening m e t h o d to detect polypeptide hormone-producing or -containing cells in h u m a n tumors. The results of this technique were compared with the immunohistochemical reactions. Materials and Methods Tissues. SurgicMly removed pituitary adenomas from five patients with acromegMy, one patient with Cushing's syndrome and two patients without clinically apparent endocrine disorders were examined. As control tissues, specimens of two normal pituitaries, one medullary 1 VirchowsArch. A Path. Anat. and 1tistol.

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carcinoma of the thyroid, and one insuloma of the pancreas were added to this series. The control pituitaries were obtained at autopsy. One of these came from a woman who died of eelampsia and therefore could be expected to contain prolaetin-producing cells. _Fixation and Embedding. The tissue specimens were fixed in 10% phosphate-buffered neutral formalin for about 6 h at room temperature. Afterward, the tissues were routinely processed in paraplast at 56 ~ and cut at 4 ~zm. _Fluorescamine Staining. Deparaffinized and rehydrated sections were mounted in Tris HCl-buffered glycerin (1:9, pH 8.0) and were initially examined for autofluoreseenee. Subsequently, the eoverslips were removed and the slides briefly washed in a 0.2 M phosphate buffer, pH 8.0. Immediately after removal of the slides from the buffer, each section was drained and covered with a few drops of the fluoreseamine solution (2mg FluramI~, HoffmanLa Roche, Basel, Switzerland, in 10ml acetone) for about 15 s (Hs et al., 1974). The slides were then washed repeatedly in phosphate buffer, mounted in Tris HCl-buffered glycerin and examined with an Ortholux microscope (Leitz) equiped with a high-pressure mercury-arch ttBO 100 light source and a standard Ploemopak vertical illuminator in positionl (peak exitation at 365 nm) and a K 490 (Leitz) as barrier filter. Immunohistochemistry. The fluoreseamine-stained sections were subjected to an indirect immunofluoreseenee method with Tl%ITC-labeled antibodies (Nordic, Tilbury, The Netherlands). Adiaeent, unstained sections were subjected to an indirect immunoenzymemethod using peroxidase-labeled antibodies (Miles, Kankakee, Ill., USA). The first layer consisted either of antiserum against human growth hormone, porcine ACTH, human caleitonin, or bovine insulin. The growth hormone antibodies were purchased from Wellcome (Beekenham, Kent, England), the insulin antibodies from Miles. The ACTH and ealcitonin antibodies were prepared by immunization of adult New Zealand rabbits with a zinc preparation of porcine ACTH (Cortrophine-Z, a generous gift from Organon, Oss, The Netherlands) and human synthetic calcitonin (a generous gift from Ciba-Geigy, Basel, Switzerland), respectively. The TRITCtreated sections were mounted in Tris HCl-buffered glycerin and examined with an Ortholux microscope as outlined before using the standard filter setting in position 4 (peak excitation at 560 nm). The peroxidase-treated sections were stained according to Graham and Karnovsky with 0.075% 3.3'-diaminobenzidine-tetra-ttCl (Sigma Chemical Co., St. Louis, Mo., USA) and 0.01% hydrogen peroxide (Perhydrol, Merck, Darmstadt, Western Germany) in 0.05M Tris ttC1 buffer, pI-I 7.6. After passing through a graded alcohol series, the sections were mounted in malinol. Appropriate controls on the immunohistoehemieal preparations were performed (Nieuwenhuijzen Kruseman et al., 1976). Histologic Staining. The pituitary cells were characterized in adjacent sections using the triehrome methods of Cason (1950) and Brookes (1968). Other sections were stained with hematoxylin and eosin.

Results After fluorescamine s t a i n i n g a n u m b e r of cells i n p i t u i t a r y adenomas of acromegalic p a t i e n t s as well as i n the n o r m a l control pituitaries revealed a distinct g r a n u l a r fluorescence. I m m n n o h i s t o c h e m i e a l e x a m i n a t i o n of these cells proved t h a t t h e y c o n t a i n e d growth hormone. I n the p i t u i t a r y of a w o m a n who died of eelampsia these cells could easily be differentiated from prolactin-producing cells which showed no fluoreseamine fluorescence b u t were earmoisinophilic i n a d j a c e n t Brookes' s t a i n e d sections. The i n t e n s i t y of fluorescamine fluorescence correlated well with the degree of immunohistochemicM g r a n u l a t i o n . No fluorescamine fluorescence was noticed i n the other p i t u i t a r y adenomas, the m e d u l l a r y thyroid carcinoma, a n d the pancreatic i n s u l o m a (Table 1). No differences i n i m m u n o s t a i n i n g i n t e n s i t y were observed b e t w e e n sections t h a t had been previously exposed to fluorescamine a n d unexposed controls. Similarly, i m m n n o s t a i n i n g did n o t appear to affect the i n t e n s i t y of s u b s e q u e n t flnoreseamine fluorescence. Therefore, both fluorescamine fluorescence a n d ira-

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Table l. Fluorescamine fluorescence and immunohistochemical staining of hormone-producing tumors

1. Acidophilic adenoma of the pituitary 2. Acidophilic adenoma of the pituitary 3. Acidophilic adenoma of the pituitary 4. Acidophilic adenoma of the pituitary 5. Acidophilic adenoma of the pituitary 6. Acidophilic adenoma of the pituitary 7. Chromophobe adenoma of the pituitary 8. Basophilic adenoma of the pituitary 9. Medullary carcinoma of the thyroid 10. Insul oma of the pancreas

Secretion product

Immunohistochemical granulation

Fluorescamine fluorescence

growth hormone growth hormone growth hormone growth hormone growth hormone

+++ ++ ++ + +

+++ ++ ++ + +

n o n e

- -

none ACTH calcitonin insulin

- -

++ +++ ++

munofluorescence can be carried out on one section, simultaneously. The preparations can be examined easily by switching the filter setting of the fluorescence microscope. In agreement with earlier observations (tIAkanson et al., 1974; Sundler et al., 1974), fluorescamine fluorescence was found to fade rapidly upon exposure to UV light. After storage of the sections for some hours protected from daylight fluorescence was restored. The fluorescamine-stained sections could be stored at --20~ without serious decrease of fluorescence intensity.

Discussion In solution, fluoreseamine reacts with primary amino groups (Udenfriend et al., 1972). The same reaction has been ascribed to formaldehyde. Fluorescamine fluorescence of certain cell types after fixation in formaldehyde vapor could therefore be explained by the presence in certain proteins of hidden amino groups which are not condensed after formaldehyde fixation and hence are still capable of binding fluorescamine (Its et al., 1974). In human tissues, these cell types include cells containing growth hormone, gastrin, insulin, and calcitonin (Sundler et al., 1974; Larsson et al., 1975). In our hands, pituitary cells containing growth hormone from normal as well as neoplastic tissues still exhibited fluorescamine fluorescence when routine formalin fixation and paraplast embedding was used instead of fixation in formaldehyde vapor. The other cell types examined were found to be devoid of fluorescamine affinity after routine histologic tissue processing. From these observations, it may be expected that fluorescamine binding is a simple and convenient way to determine rapidly whether or not acidophilic granulation of pituitary adenomas is correlated with the presence of growth hormone. I t remains to be determined whether this is so, and under what specific conditions fhioreseamine staining of other polypeptide hormoneproducing cells in routinely processed tissues can be achieved.

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Fig. 1. Densely granulated growth hormone-secreting pituitary adenoma showing fluorescamine fluorescence. • 1,250 Fig. 2. Same adenoma as depicted in Figure 1, after immunostaining for growth hormone activity with peroxidase-labeled antibodies. X 1,250

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Fig. 3. Normal pituitary showing fluorescamine fluorescence of growth hormone cells. • 1,250 Fig. 4. Same section as depicted in Figure 3, after immunostaining for ACTH activity with Tt~ITC-labeled antibodies. Note that immunoreactive cells lack fluorescamine fluorescence. )< 1,250

A c c o r d i n g to our i m m u n o h i s t o c h e m i c M studies, t h e i n t e n s i t y of fluorescamine fluorescence a p p e a r e d to c o r r e s p o n d to t h e degree of g r a n u l a t i o n of t h e g r o w t h h o r m o n e cells. This f i n d i n g is in a g r e e m e n t with o b s e r v a t i o n s of L a r s s o n et al. (1975) who suggested t h ~ t h e fluorophore b i n d s to s e c r e t o r y granules. These a u t h o r s also n o t i c e d t h a t t r e a t m e n t with fluoreseamine g r e a t l y decreased i m m u n o r e a c t i v i t y . I n contrast, we f o u n d t h a t i m m u n o s t M n i n g a n d fluoreseamine fluorescence were n o t m u t u a l l y antagonistic. This o b s e r v a t i o n suggests t h a t a f t e r formalin f i x a t i o n fluorescamine b i n d i n g does no~ ~ake place a t t h e s a m e m o l e c u l a r site as ~hat a t which a n t i b o d i e s are b o u n d . Consequently, this i m p l i e s t h a t fluoreseamine is n o t n e c e s s a r i l y b o u n d b y t h e h o r m o n e itself. As i m m u n o h i s t o c h e m i e a l demons t r a t i o n of h o r m o n e s is r e l a t e d to t h e i m m u n o l o g i c a c t i v i t y of t h e i d e n t i f i e d hormone, t h e l a t t e r m e t h o d is s~ill p r e f e r a b l e for definitive confirmation of diagnosis of g r o w t h h o r m o n e - p r o d u c i n g p i t u i t a r y a d e n o m a s . The authors are indebted to Mr. Klaas van der Ham for preparing the microphotographs.

Reierenees Brookcs, L. D. : A stain for differentiating two types of acidophil cells in the rat pituitary. Stain Technol. 43, 42-43 (1968) Cason, J. E. A. : I~apid one-step MMlory-Iteidenhain stain for connective tissue. Stain Teehnol. 25, 225~226 (1950) H~kanson, 1%., Larsson, L.-L, Sundler, F. : Fluorescamine: A novel reagent for the histochemical detection of amino groups, ttistochemistry 39, 15-23 (1974) Larsson, L.-I., Sundler, F., I-I~kenson, g. : Fluorescamine as a histoehemical reagent: Demonstration of polypeptide hormone-secreting cells. Histochemistry 44, 245-251 (1975) Nieuwenhuijzen Kruseman, A. C., Bots, G. Th. A.M., Lindeman, J., Schaberg, A.: Use of immunohistochemieal and morphological methods for the identification of human growth hormone-producing pituitary adenom~s. Cancer (Philad.) In press (1976) Sundler, F., Larsson, L.-I., I-Igkanson, R., Ljungberg, O. : Fluoreseamine-induced fluorescence in C cells tumours of the thyroid. Virehows Arch. Abt. A 363, 17-20 (1974) Udenfriend, S., Stein, S., B6hlen, P., /)airman, W., Leimgruber, W., Weigele, M. : Fluorescamine: A reagent for assay of amino acids, peptides, proteins and prima.ry amines in tile picomole range. Science 178, 871-872 (1972) Weigele, M., Benardo, S. L. De, Tengi, J. P., Leimgruber, W. : A novel reagent for the fluoromettle assay of primary amines. J. Amer. chem. Soc. 9~, 5972 (1972) Arie C. Nieuwenhuijzen Kruseman Pathologisch Laboratorium der i~ijksuniversiteit te Leiden Wassenaarseweg 62 Leiden, The Netherlands

Fluorescamine fluorescence detection of growth hormone-producing cells in human pituitary adenomas.

Virchows Arch. A Path. Anat. and Histol. 371, 177--181 (1976) 9 by Springer-Verlag 1976 Fluorescamine Fluorescence Detection of Growth Hormone-Produc...
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