Carcinoid Tumors of the Rectum Silver Reactions, Fluorescence

Brief Scientific Reports
Carcinoid Tumors of the Rectum
Silver Reactions, Fluorescence, and Serotonin Content
of the Cytoplasmic Granules
JEROME B. TAXY, M.D., GEOFFREY MENDELSOHN, M.B., B.CH., AND PRABODH K. GUPTA, M.D.
IN the currently accepted classification of carcinoid
tumors,'•'"•2I the hindgut or rectal neoplasms are
generally considered to be nonreactive with respect
to argyrophil and argentaffin staining reactions. Argentaffin-positive rectal carcinoids have been reported,
however,H-18-20 and the carcinoid syndrome associated
with a rectal tumor has been reported. 13 In recent
years, paralleling the detailed pathologic studies of
carcinoid tumors in which various peptide hormones
have been localized to the tumor cells, 1219 it has been
recognized that rectal carcinoids may also exhibit posiReceived January 14, 1980; received revised manuscript and
accepted for publication March 10, 1980.
Dr. Mendelsohn is the recipient of an American Cancer Society
Junior Faculty Clinical Fellowship.
Address reprint requests to Dr. Taxy: Department of Pathology,
The Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore,
Maryland 21205.
Department of Pathology, The Johns Hopkins University
School of Medicine, Baltimore, Maryland
tive immunohistochemical reactions for some peptide
hormones, 20 as well as a frequently positive argyrophil
reaction.,!18 -2" Rectal carcinoids are unusual tumors,
and the number of cases studied has been small. A
relatively large number of rectal carcinoids forms the
basis of this report, in which the silver-staining characteristics of the tumors are correlated with formalininduced fluorescence and immunoperoxidase staining
for serotonin.
Materials and Methods
Twenty-three examples of carcinoid tumors of the
rectum for the period 1959-1979 were retrieved from
the surgical pathology files of The Johns Hopkins
Hospital. Glass slides and paraffin blocks were available in all cases. Newly prepared slides were stained
with hematoxylin and eosin, two argyrophil methods
(Sevier-Munger and Grimelius) and the FontanaMasson argentaffin method. In the argyrophil methods,
the staining reaction was interpreted as positive when
a group of several tumor cells showed black or brown
cytoplasmic granules. A similar distribution of black
granules was regarded as positive with the argentaffin
stain. Unstained deparaffinized sections were examined for the presence of intracytoplasmic fluorescent granules according to the method of Grillo and
associates. 3 Briefly, an unstained slide was immersed
in Xylene® for approximately 30-60 sec, removed,
and immediately viewed with a Leitz® orthoplan microscope with 500-nm excitor and 530-nm barrier filters
and an epiilluminator with an HBO 100-watt light
0002-9173/80/1200/0791 $00.75 © American Society of Clinical Pathologists
791
Downloaded from http://ajcp.oxfordjournals.org/ by guest on October 12, 2016
Taxy, Jerome B., Mendelsohn, Geoffrey, and Gupta, Prabodh K.:
Carcinoid tumors of the rectum. Silver reactions, fluorescence,
and serotonin content of the cytoplasmic granules. Am J Clin
Pathol 74: 791-795, 1980. The argyrophil and argentaffin
staining reactions of the intracytoplasmic granules of 23 carcinoid tumors of the rectum were studied. The granules were
evaluated for the presence of amines by means of formalininduced fluorescence and an immunoperoxidase method for
serotonin. The results indicate that most rectal carcinoids
are argyrophil when the more sensitive Grimelius method is
employed. Only occasionally are rectal carcinoids argentaffin,
a feature that correlates with positive fluorescent reactions
and suggests amine content. Of three argentaffin tumors, one
stained positively for serotonin, and a second contained
clusters of pleomorphic electron-dense granules of the type
more typically seen in known amine-containing carcinoid
tumors. For practical purposes, the Grimelius argyrophil
stain is most useful in confirming a light-microscopic diagnosis of a rectal carcinoid. (Key words: Carcinoid; Rectum;
Argyrophil; Argentaffin; Grimelius; Fluorescence.)
TAXY, MENDELSOHN, AND GUPTA
792
Table I. Properties of Cytoplasmic Granules in
23 Rectal Carcinoids
Stains*
No.
Tumors
8
11
1
3
G
S-M
F-M
FF
_
_
-
—
-
_
-
+
+
+
+
+
+
+t
* G. Grimelius argyrophil slain: S-M. Sevier-Munger argyrophil stain; F-M, FontanaMasson argentaffin stain: FF. formalin-induced fluorescence.
t One tumor had pleomorphic granules by electron microscopy; a second stained
positively for serotonin.
Results
Most of the tumors in this series were discovered
in patients who had rectal bleeding or nonspecific
bowel complaints. The patients' ages ranged from 18
to 67 years, and the tumors were small, ranging from
0.3 to 2.0 cm in diameter. Two patients when first seen
had hepatomegaly and metastases proven by liver
biopsy, however the sizes of the rectal lesions were
not noted.
The histopathologic features of all tumors were those
typical of rectal carcinoids. 812 The special studies are
summarized in Table 1. Eight tumors did not react with
any of the silver stains employed, did not exhibit
fluorescent granules, and were not reactive with the
serotonin-immunoperoxidase staining procedure. Fifteen tumors demonstrated positive Grimelius argyrophil reactions. In twelve tumors, the argyrophil cells
were focally distributed, and the granules, varying
in intensity, stained brown, in contrast to the darker
intensity of the normal enterochromaffin cells of the
overlying mucosa (Fig. 1A). In three tumors, the positive cells were more uniformly distributed, and the
argyrophil granules appeared more numerous and more
closely approximated the dark staining intensity of the
normal mucosal cells (Fig. IB). These three tumors
* Cappel Laboratories, Inc., Cochranville, Pennsylvania.
were also argentaffin and exhibited fluorescent granules
in the cytoplasm of many tumor cells (Fig. 2). Four
tumors, which included the three argentaffin tumors,
reacted positively with the Sevier-Munger argyrophil
stain. Only one tumor stained positively for serotonin
(Fig. 3). Electron microscopy of one argentaffin tumor
demonstrated focal collections of pleomorphic electron-dense granules (Fig. 4).
Discussion
The currently employed classification of carcinoid
tumors relates the anatomic sites of the tumors to a
presumed derivation from the appropriate embryonic
portion of the gut, i.e., forgut, midgut, hindgut. 11014,21
The presumed ability of the tumors to demonstrate
silver-positive cytoplasmic granules has supported
this classification, in which rectal or hindgut tumors
have been thought until recently to be nonreactive,
albeit ultrastructurally granulated. 1 A positive argentaffin reaction involves the reduction of ionic to metallic
silver by strong endogenous reducing substances. In
endocrine and neuronal cells, this reaction occurs
within secretory granules containing amines such as
serotonin or catecholamines. Argentaffinity also implies that the argyrophil reaction will be positive, although the exact basis of argyrophilia is unknown. After
the exposure of the tissue to the ionic silver solution,
an exogenous reducing substance must be added to
build up metallic silver on sites presumed to be already impregnated. 5,8 In the Grimelius method, the
reactive granules may contain either biogenic amines
or peptide hormones. 2 ' 4 ' 121719,20
In addition to a positive argentaffin stain, the
presence of amines in carcinoid tumors is often inferred by formalin-induced fluorescence or pleomorphic, electron-dense granules. 1,2,12 Among the infrequently documented argentaffin rectal carcinoids, 818,20
one has been found to contain pleomorphic electrondense granules and immunoreactive substance P, a
peptide. 20 In the present study, three argentaffin
rectal carcinoids were identified, and all three tumors
showed fluorescent cytoplasmic granules, although
only one demonstrated a positive immunoperoxidase
stain for serotonin. Another argentaffin tumor demonstrated foci of dense pleomorphic granules, as well as
more numerous round granules by electron microscopy, but failed to react with the serotonin stain.
These observations suggest that the argentaffin rectal
carcinoids reported here contain amines, although
the lack of uniformity of fluorescence, immunoperoxidase, and ultrastructure in these tumors may indicate
a heterogeneous amine content.
The argyrophil stains in common use are the SevierMunger and the Grimelius. The Grimelius method is
Downloaded from http://ajcp.oxfordjournals.org/ by guest on October 12, 2016
source. Two tumors could not be examined with this
technic, because the fixative initially employed was
other than formalin. The presence of serotonin was
evaluated by means of the standard unlabeled antibody immunoperoxidase method 915 with a specific
rabbit antiserum against serotonin* diluted 1:100. An
unstained section of an intestinal carcinoid from a
patient who was known to have carcinoid syndrome
was used as a control in both the fluorescence and
immunoperoxidase procedures. In one case, tissue for
electron microscopy was available, having been fixed
in the fresh state in glutaraldehyde and processed in
the usual manner.
A.J.C.P. • December 1980
Vol. 74 • No. 6
793
BRIEF SCIENTIFIC REPORTS
\
\
ri. i
v.
*iT
r*
,, ^ V
^ * " >,
-V•<'
f «v- •***
I'
S
^
FIG. IA (upper, left). An area of an argyrophil tumor in which the brown cytoplasmic granules vary in intensity but are not as dark as the
normal colonic enterochromaffin cells (inset). The argentaffin stain was negative. Grimelius argyrophil stain. x600.
FIG. IB (lower, left). The argyrophil granules of an argentaffin tumor in which the granules were more numerous, uniformly distributed, and
of uniformly darker intensity. Grimelius argyrophil stain. x600.
FIG. 2 (upper, right). An unstained section of an argentaffin rectal carcinoid exhibiting spontaneous fluorescence of cytoplasmic granules.
Nuclear areas are pale or dark; cytoplasm is granular and bright. x600.
FIG. 3 (lower, right). Positive immunoperoxidase-serotonin reaction. The dark cytoplasmic granules are focally distributed and are
contrasted against the negative staining nuclei. x460.
now becoming widely used and appears to be the more
sensitive stain in that the rectal carcinoids examined
have all given positive reactions."1820 This sensitivity
is supported by the present study, in which 15 of 23
tumors demonstrated argyrophil granules when the
Grimelius method was used. Four tumors were positive
with the Sevier-Munger method, but in no tumor was
the Sevier-Munger positive and the Grimelius negative.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on October 12, 2016
•v»
TAXY, MENDELSOHN, AND GUPTA
A.J.C.P. • December I9K0
The present findings emphasize that rectal carcinoids
are frequently argyrophil tumors, and that for the surgical pathologist confronted with the differential diagnosis of carcinoid tumor or adenocarcinoma, the
Grimelius method affords the best light-microscopic
confirmation.
The criteria for the interpretation of a positive
Grimelius argyrophil stain may vary from one observer to another. For example, the presence of isolated argyrophil cells has been liberally interpreted
to confer such a property to the entire tumor. 20 However, in that study, the silver stains were correlated
with immunohistochemical data, which are not available in most routine laboratories. It has been noted
that argyrophil granules may appear in exocrine cells4
and that isolated positive cells may occur in conventional types of adenocarcinomas. 716 Given prevalent
diagnostic circumstances, in which uncertainties of
surgical sampling, small biopsy fragments, and artifactual distortion are often encountered, it would seem
prudent to require a greater number of argyrophil cells,
such as a group of several cells, to justify a positive
interpretation.
The theoretical implications of these argyrophil
properties are unclear. The Sevier-Munger stain, in
the present series, was not very sensitive, but correlated best with argentaffin positivity and fluorescence.
The Grimelius stain was more sensitive, and the intensity of the Grimelius argyrophil granules was darker
in those tumors that were also argentaffin. Eight
tumors, however, were interpreted as nonargyrophil
when the Grimelius method was used. These differences in staining intensity, or lack of staining, from
the one argyrophil method to the other may reflect
the presence of different substances in the cytoplasmic
granules. An understanding of the mechanism of the
argyrophil reaction and the availability of specific antibodies to the growing list of gastrointestinal peptide
hormones" may help to clarify these issues.
Acknowledgment. Paul F. Guerin, M.D., Franklin Square Hospital, Baltimore, Maryland, contributed cases to this study.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on October 12, 2016
FIG. 4. Electron micrograph of a group of cells from
an argentaffin rectal carcinoid showing a focus of electron-dense pleomorphic granules (arrow), highly suggestive of amine content, x 8,600.
Vol. 74 • No. 6
BRIEF SCIENTIFIC REPORTS
References
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Immunohistochemistry
of Warthin's Tumor
RAYMOND R. TUBBS, D. O., KHALIL SHEIBANI, M.D., ROBERT A. WEISS, M.D.,
VICTOR LEE, M.D., BRUCE A. SEBEK, M.D., AND RAFAEL VALENZUELA, M.D.
Tubbs, Raymond R., Sheibani, Khalil, Weiss, Robert A., Lee,
Victor, Sebek, Bruce A., and Valenzuela, Rafael: Immunohistochemistry of Warthin's tumor. Am J Clin Pathol 74:
7 9 5 - 7 9 7 , 1980. This study describes results of fresh-frozen
immunohistochemistry applied to two typical Warthin's
tumors. Polyclonal follicular center cell staining and an apparent reduction in the number of T-lymphocytes were found.
These observations tend to support the concept that the
lymphoid infiltrate in Warthin's tumor represents a reactive
cellular proliferation. (Key words: Immunohistochemistry;
Immunoperoxidase; Immunofluorescence; Warthin's tumor;
Lymphoproliferative disorders.)
Received December 27, 1979; received revised manuscript and
accepted for publication May 22, 1980.
Address reprint requests to Dr. Tubbs: Department of Pathology,
Cleveland Clinic Foundation, Cleveland, Ohio 44106.
Departments of Pathology and Immunology,
Cleveland Clinic Foundation, Cleveland, Ohio
SEVERAL TUMORS are characterized by a prominent lymphocytic component. Among the more interesting of such neoplasms is the Warthin's tumor. The
lymphocytic infiltrate in this tumor has been variously
classified as a cellular response to an epithelial neoplasm, residual normal lymph node involved by a
purely epithelial proliferation, or a lymphoid-epithelial
hamartoma.
Previous studies using immunoadherence technics
have demonstrated complement receptors on lymphoid
0002-9173/80/1200/0795 $00.65 © American Society of Clinical Pathologists
Downloaded from http://ajcp.oxfordjournals.org/ by guest on October 12, 2016
Black WC: Enterochromaffin cell types and corresponding
carcinoid tumors. Lab Invest 19:473-486, 1968
Buffa R, Capella E, Fontana P, et al: Types of endocrine cells
in the human colon and rectum. Cell Tissue Res 192:227-240,
1978
Grillo MA, Jacobs L, Comroe JH Jr: A combined fluorescence
histochemical and electron microscopic method for studying
special monoamine-containing cells (SIF cells). J Comp
Neurol 153:1-14, 1974
Grimelius L, Strand A: Ultrastructural studies of the argyrophil
reaction in a, cells in human pancreatic islets. Virchows
Arch (Pathol Anat| 364:129-135, 1974
Hakanson R, Owman C, Sporrong B, et al: Electron microscopic classification of amine-producing endocrine cells by
selective staining of ultra-thin sections. Histochemie 27:
226-242, 1971
Hosoda S, Kito H, Nogaki M, et al: Is rectal carcinoid argyrophilic? Application of Grimelius silver nitrate stain in four
cases. Dis Colon Rectum 18:386-390, 1975
Kubo T, Watanabe H: Neoplastic argentaffin cells in gastric
and intestinal carcinomas. Cancer 27:447-454, 1971
Lillie RD, Glenner GG: Histochemical reactions in carcinoid
tumors of the human gastrointestinal tract. Am J Pathol 36:
623-649, 1960
Mason TE, Phifer RF, Spicer SS, et al: An immunoglobulinenzyme bridge method for localizing tissue antigens. J Histo10. chem Cytochem 17:563-569, 1969
Orloff MJ: Carcinoid tumors of the rectum. Cancer 28:175-180,
1971
Pearse AGE. Polak JM, Bloom SR: The newer gut hormones:
795
cellular sources, physiology, pathology and clinical aspects.
Gastroenterology 72:746-761. 1977
Pearse AGE, Polak JM. Heath CM: Polypeptide hormone
production by "carcinoid" apudomas and their relevant
cytochemistry. Virchows Arch (Cell Pathol] 16:95-109. 1974
Saegesser F, Gross M: Carcinoid syndrome and carcinoid
tumours of the rectum. Am J Proctol 20:27-32, 1969
Soga J, Tazawa K: Pathologic analysis of carcinoids: histologic
re-evaluation of 62 cases. Cancer 28:990-998. 1971
Sternberger LA. Hardy PH Jr. Cuculis JJ, et al: The unlabeled
antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex
(horseradish peroxidase-anti horseradish peroxidase) and its
use in identification of spirochetes. J Histochem Cytochem
18:315-333. 1970
Taxy JB, Tischler AS. Insalaco SJ. et al: Carcinoid tumor of the
breast: a possible variant of lobular carcinoma (Abstract). Lab
Invest 40:287-288. 1979
Vassallo G, Capella C, Solcia E: Grimelius" silver stain for
endocrine cell granules as shown by electron microscopy.
Stain Technol 46:7-13, 1971
Wilander E, Portela-Gomes G. Grimelius L. et al: Argentaffin
and argyrophil reactions of human gastrointestinal carcinoids.
Gastroenterology 73:733-736. 1977
Wilander E. Grimelius L, Lundquist G. et al: Polypeptide
hormones in argentaffin and argyrophil gastroduodenal endocrine tumors. Am J Pathol 96:519-530. 1979
Wilander E, Portela-Gomes G. Grimelius L. et al: Enteroglucagon and substance P-like immunoreactivity in argentaffin
and argyrophil rectal carcinoids. Virchows Arch [Cell Pathol|
25: 117-124. 1977
Williams ED, Sandler M: The classification of carcinoid tumors.
Lancet 1:238-239. 1963