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Immunofluorescence Studies on Pathologic Routine Material: Application to Malignant Lymphomas
Beitr. Path. Bd. 159,219-225 (197 6)
Practical Notes
Department of Pathology, University of Vienna, School of Medicine, Vienna, Austria (Director: Prof. Dr. J.-H. Holzner), and Department of Pathology, University of Munster, School of Medicine, Munster, Federal Republic of Germany (Director : Prof. Dr. E. Grundmann)
Immunofluorescence Studies on Pathologic Routine Material: Application to Malignant Lymphomas Immunfluoreszenzuntersuchungen an pathologischem Routinematerial: Anwendung bei malign en Lymphomen H. DENK, T. RADASZKIEWICZ, and CH. WITTING With
I
Figure· Received July 15, 1976
Key words: Immunofluorescence - Routine - Malignant lymphoma Diagnostic
Summary In an attempt to apply immunmorphologic techniques to pathologic routine material, malignant lymphomas of the Non-Hodgkin- and the Hodgkin-type were studied by indirect immunofluorescence. To minimize nonspecific background staining formal in fixed paraffin sections were treated with protease prior to incubation with the antisera. The usefullness of this procedure in providing additional diagnostic criteria was tested on specimens of malignant lymphomas (chronic lymphocytic leukemias, immunocytic lymphomas, plasmocytic lymphomas, centrocytic lymphomas, centroblastic-centrocyctic lymphomas, centroblastic lymphomas, immunoblastic lymphomas, lymphoblastic lymphomas of Burkitt, convoluted and unclassified type, Hodgkin's disease). In addition, cases of nonspecific lymphadenitis were studied. The disc;imination of single cells particularly of the B-cell line was facilitated using immunofluorescence and different patterns were easily recognized thus aiding the diagnosis based on light microscopic criteria. In addition, preliminary evidence was obtained that lymphoblastic lymphomas categorized as "others" may be further characteri7.ed.
The major obstacle to the general application of immunofluorescence in routine pathology is the need for frozen sections with often inferior
220'
H. Denk, T. Radaszkiewicz, and Ch. Witting
Fig. I a. Lymphoplasmocytoid lymphoma. Positive cytoplasmic fluorescence staInIng of plasmacells and their precursors which are sparse in this type of lymphoma. X 200. Fig. I b. Lymphoplasmocytic lymphoma composed of mostly positively stained cells. Note the intranuclear inclusion (arrow). X 100.
Immunofluorescence Studies of Malignant Lymphomas .
221
quality in most instances. Formalin fixation and paraffin embedding may alter antigenic properties (Pearse, 196 I; Deng and Beutner, 1974), and, in the case of resistant antigens, the high background staining in formalin fixed material usually interferes with specific immunofluorescence. The latter can be minimized by treatment of the tissue sections with protease (Huang, 1975). In the present communication it will be shown that protease treatment facilitates immunofluorescence studies on formalin fixed paraffin sections of lymphomas.
Material and Methods Material: Three chronic lymphocytic leukemias, 5 immunocytic lymphomas, 5 plasmocytic lymphomas, 5 centrocytic lymphomas, 5 centroblastic-centrocytic lymphomas, twO centroblastic lymphomas, 6 lymphoblastic lymphomas and 5 immunoblastic lymphomas were studied. One specimen with lymphocytic predominance (nodular paragranuloma), two specimens with nodular sclerosis and two of the mixed type represented the Hodgkinlymphoma category. In addition, 5 lymphnodes with the histologic picture of nonspecific lymphadenitis were studied. The histologic material was classified according to Lennert et al. (I975a, b). Methods: The paraffin sections were dewaxed in xylene, rehydrated, covered with 0.3% celloidine and treated with 0.1% pronase type VII (Sigma Chemical Comp., MO) in phosphate buffered saline (PBS), pH 7,4 for 15 min at 37° C. Pronase treatment for a longer time period was without further benefit. The slides were washed thereafter in icecold PBS for 60 min and then covered with a human globulin antiserum from the rabbit (ammoniumsulphate-precipitated globulin fraction) in a dilution of I : 40 (corresponding to 1/5 precipitation unit in double immunodiffusion). After 45 min incubation in a moist chamber at room temperature the slides were washed in several changes of PBS and then incubated with FITC-conjugated rabbit globulin antiserum (goat; Behring-Werke, Marburg/Lahn, Germany) in a dilution of I : 10 (corresponding to approx-
Fig. X
I.
c.
In plasmocytic lymphoma all cells show cytoplasmic immunofluorescence.
200.
Fig. I d. Centroblastic lymphoma. Positive superficial and sometimes granular fluorescence staining outlines the tumor cells. Intensively stained cells were identified as plasmacells. >( 200. Fig. I e. In lymphoblastic lymphoma of the Burkitt type positive and also negative stained cells can be observed. X 200. Fig. I f. Immunoblastic lymphoma with variations of intensity of cytoplasmic immunofluorescence staining of the tumor cells. X 200. Fig. I h. Follicular hyperplasia. Granular outline and honeycomb-like pattern in germinal Fig. I h. Follicular hyperplasia. Granular outline and honeycomb-like pattern germinal center and some positive stained plasmacells and plasmacell-precursors in the interfollicular area. X 100. Fig. I i. Hodgkin's disease, mixed type - positive Reed-Sternberg cell and a Hodgkin cell (inset). X 200. 15 Beitr. Path. Bd. 159
lll'
H. Denk, T. Radaszkiewicz, and Ch. Witting
imately rlz precipitation unit; fluorescein/protein ratio 1.5 : I). After a second wash in PBS the sections were mounted in glycerol-PBS (r : 8) and read on a Leitz Orthoplan fluorescence microscope with incident light illumination (Ploem-Opak, filter combination 3/3). Fotomicrographs were taken with a Kodak Ektachrome High-Speed color film. Controls included normal rabbit serum and PBS, respectively, in the first layer.
Results In chronic lymphocytic leukemia a uniform immunofluorescence pattern was observed. The tumor cells were consistently negative. Positive specific fluorescence was only present in lymphnode areas not involved by the disease process, particularly in plasma cells and cellular elements in infiltrated sinuses. In immunocytic lymphomas a high percentage of the cells was negative. However, cells of small to intermediate size with specific cytoplasmic staining were haphazardly distributed throughout (Fig. I a) and were identified in Giemsa stained sections as lymphatic plasmacells, plasmoblasts and plasmacells (lymphoplasmocytoid lymphoma). In the lymphoplasmocytic type most of the cells gave a positive cytoplasmic immunofluorescence and occasionally showed positive intranuclear inclusions (Fig. I b). In plasmocytomas all cells exhibited cytoplasmic immunofluorescence of variable intensity (Fig. I c). In centrocytic lymphomM the cells were specifically outlined in a honeycomb-like fashion. It is unclear whether this is cytoplasmic, surface or intercellular staining. In centroblastic-centrocytic lymphomas the cells were specifically outlined in a honeycomb-like fashion and in the interfollicular areas in case of follicular pattern, brightly stained lymphatic plasmacells, plasmoblasts and plasmacells were seen. In one anaplastic specimen amorphous globular and irregular substances were found in the neoplastic follicles exhibiting fluorescence of medium intensity. Two of the very rare centroblastic lymphomas were included in this study. The majority of the cells was only outlined by specific fluorescence in a honeycomb-pattern. Occasionally larger cells with faint superficial granular fluorescence were seen (Fig. I d). The lymphoblastic lymphoma material consisted of two Brukitt type, two convoluted type, and two unclassifiable-("other")lymphomas The Burkitt type-lymphomas contained mostly small to medium size cells with a distinct cytoplasmic fluorescence (Fig. I e). Cells with superficial granular fluorescence and entirely negative cells were also encountered in smaller numbers. In the convoluted type-lymphomas, the tumor cells were
Immunofluorescence Studies of Malignant Lymphomas .
223
consistently negative. The two lymphoblastic lymphomas type "others" showed cytoplasmic staining of most of the cells suggesting their B-cell nature. Immunoblastic lymphomas featured an inhomogeneous immunofluorescence pattern consisting of cells of the plasmacell line with narrow or broad specifically stained cytoplasm. The immunoblasts showed variations in immunofluorescence from bright to negative staining (Fig. 1 f). Five cases of Hodgkin's disease (one lymphocytic predominance type, two nodular sclerosis and two of the mixed type) were included. Most of the tumor cells were negative and plasmacells abundantly present in these lymphnodes served as positive controls. Hodgkin cells and Sternberg-Reed cells were either positive (Fig. 1 i), weakly positive or negative, but even the positive cells never reached the intensity of fluorescence observed in plasmacells (Fig. 1 g). In lymphnodes with histologic criteria of nonspecific lymphadenitis and/or follicular hyperplasia larger cells with granular outline, presumably centroblasts, were found in the follicles together with smaller specifically outlined cells (honeycomb-like appearence) (Fig. 1 h). Cells belonging to the plasmacell line were sparse in the germinal centers but were observed more often in the interfollicular areas and in the medulla. The sinuses contained a large number of small- to medium-sized cells with bright cytoplasmic staining. The parafollicular areas were negative.
Discussion Immunofluorescence studies are usually performed on frozen material which, however, in most instances is difficult to obtain in a pathologic routine laboratory. Therefore, several authors have tried to adept immunofluorescence and other immunmorphologic techniques to formalin or alcohol fixed paraffin sections (Sainte-Marie, 1962; Brown et al., 1974; Burns et al., 1974; Garvin et al., 1974; Muller et al., 1974; Taylor and Burns, 1974; Watkins and Evans, 1974; Burns, 1975; Bussolati and Pich, 1975; Bussolati et al., 1975; Nayak and Sachdeva, 1975; Ray and Desmet, 1975; Taylor, 1976). The problem of high nonspecific background staining in formalin fixed material was overcome partly by Huang (1975) who found that pronase treatment greatly reduced background staining at least in liver biopsy material searched for hepatitis-B-antigens. In the present communication a modified Huang-technique was employed on routine material of malignant lymphomas and it was found that the immunoglobulin antigens are preserved under these conditions and readily demonstrable by indirect immunofluorescence. It was shown by Lennert et al.
224' H. Denk, T. Radaszkiewicz, and Ch. Witting
(1975 b) that determination of immunoglobulins in lymphoid tissue homogenates provides additional clues on the type and biologic behaviour of the tumor. Under these circumstances, however, no information is obtained on the different cells particularly in those lymphomas which consist of different types of cells like immunoblastic lymphomas. With immunofluorescence, not only retrospective studies of cases were possible, but also the discrimination of single cells, particularly of the B-cell line, proved to be greatly facilitated. Similar conclusions were reached by Taylor (1976) using the immunoperoxidase technique. In addition, different immunofluorescence patterns emerged which may favourably aid the diagnosis based on light microscopic criteria. It could also be shown that lymphoblastic lymphoma.s categorized as "others" (Lennert et a1., 1975 a) because of their nonspecific morphology may be further characterized by immunofluorescence. In agreement with the results obtained by Burns et al. (1974), Taylor (1974, 1976) and Taylor and Burns (1974) it was found that some of the Hodgkin cells and Sternberg-Reed cells contain immunoglobulins favouring a B-cell origin of these cells. Studies of a more comprehensive material and using antisera to different immunoglobulin types are presently in progress.
Zusammenfassung Formalin-fixiertes Paraffin material von Non-Hodgkin- und Hodgkin-Lymphomen wurde mittels der indirekten Immunfluoreszenz untersucht. Vor der Inkubation mit Humanglobulin-Antiserum wurden die Paraffinschnitte mit Protease behandelt, urn die unspezifische Fluoreszenz zu verringern. Folgende maligne Lymphome wurden untersrucht: chronische lymphatische Leukamie, immunozytisches Lymphom, plasmozytisches Lymphom, zentrozytisches Lymphom, zentroblastisch-zentrozytisches Lymphom, zentroblastisches Lymphom, lymphoblastisches Lymphom vom "Burkitt-type", "convoluted type" und "unclassified type", immunoblastisches I.ymphom und M. Hodgkin, zusatzlich Falle von unspezifischen Lymphadenitiden. Der immunmorphologische Nachweis von zytoplasmatischem Immunglobulin erleichtert die Klassifikation der Zelltypen in Lymphomen, wodurch zusatzliche diagnostische Kriterien gewonnen werden konnen.
References Brown, P., Bourne, ]., and Steel, M.: Immunoperoxidase and Immunofluorescence Techniques in Pig Tissues. Histochemistry 40,343-348 (I974) Burns, ].: Background Staining and Sensitivity of the Unlabelled Antibody-Enzyme (PAP) Method. Comparison with the Peroxidase Labelled Antibody Sandwich Method Using Formalin Fixed Paraffin Embedded Material. Histochemistry 43, 29I-294 (I975) Burns, J, Hambridge, M., and Taylor, C. R.: Intracellular immunoglobulins. A comparative study on three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates. ]. din. Path. 27, 548-557 (I974)
Immunofluorescence Studies of Malignant Lymphomas . 225 Bussolati, G., and Pich, A.: Mammary and Extramammary Paget's Disease. An Immunocytochemical Study. Amer.]. Path. 80, 1I7-uS (1975) Bussolati, G., Pich, A., and Alfani, V.: Immunofluorescence Detection of Casein in Human Mammary Dysplastic and Neoplastic Tissues. Vir chows Arch. A Path. Anat. 3 65,15-21 (1975) Deng, ].-S., and Beutner, E. H.: Effect of Formaldehyde, Glutaraldehyde and Sucrose on the Tissue Antigenicity. Int. Arch. Allergy 47, 562-5 69 (1974) Garvin, A. ]., Spicer, S. S., Parmley, R. T., Hintz, D., and Munster, A. M.: Immunostaining of Immunoglobulin G (IgG) in Immunocytes and Hodgkin and Reed-Sternberg Cells in Hodgkin's Disease. J. Histochem. Cytochem. 22, 296 (1974) Huang, S.-N.: Immunohistochemical Demonstration of Hepatitis B Core and Surface Antigens in Paraffin Sections. Lab. Invest. 33, 88-95 (1975) Lennert, K., Mohri, N., Stein, H., and Kaiserling, E.: The Histopathology of Malignant Lymphoma. Brit. J. Haematol. 31 (Suppl.), 193-203 (197sa) Lennert, K., Stein, H., and Kaiserling, E.: Cytological and functional criteria for the classification of malignant lymphoma. Brit. J. Cancer 31 (Suppl. II), 29-43 (1975b) Miiller, W. H., Knop, B., und Felsch, G.: Zur Eignung paraffineingebetteten Gewebes als Substrat fUr den immunfluoreszenztechnischen Nachweis antinuklearer Faktoren. Acta histochem. 48, 291-300 (1974) Nayak, N. c., and Sachdeva, R.: Localization of Hepatitis B Surface Antigen in Conventional Paraffin Sections of the Liver: Comparison of Immunofluorescence, Immunoperoxidase, and Orcein Staining Methods With Regard to Their Specificity and Reliability as Antigen Marker. Amer. J. Path. 81,479-493 (1975) Pearse, A. G. E.: Histochemistry, Theoretical and Applied, p. 146. J. and A. Churchill, Ltd., London (1961) Ray, M. B., and Desmet, V. J.: Immunofluorescent detection of hepatitis B antigen in paraffin-embedded liver tissue. J. Immunol. Methods 6, 283-289 (1975) Sainte-Marie, G.: A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem. 10, 25°-256 (1962) Taylor, C. R.: The nature of Reed-Sternberg cells and other malignant "reticulum" cells. Lancet II, 802-807 (1974) Taylor, C. R.: Immunohistological study of follicular lymphoma, reticulum cell sarcoma and Hodgkin's disease. Europ. J. Cancer 12, 61-75 (1976) Taylor, C. R., and Burns, J.: The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidaselabelled antibody. J. clin. Path. 27, 14-20 (1974) Watkins, W. B., and Evans, J. J.: The use of formalin fixation in an improved method for immunofluorescence histochemical investigation of neurophysin in thy hypothalamus. J. Histochem. Cytochem. 22, 128-130 (1974) Doz. Dr. H. Denk, Dr. T. Radaszkiewicz, Pathologisches Institut der Universitat, Spitalgasse 4, A-109° Wien, Austria and Dr. C. Witting, Pathologisches Isntitut der Universitat, Westring 17, D-4400 Munster, W.-Germany
Practical Notes
Department of Pathology, University of Vienna, School of Medicine, Vienna, Austria (Director: Prof. Dr. J.-H. Holzner), and Department of Pathology, University of Munster, School of Medicine, Munster, Federal Republic of Germany (Director : Prof. Dr. E. Grundmann)
Immunofluorescence Studies on Pathologic Routine Material: Application to Malignant Lymphomas Immunfluoreszenzuntersuchungen an pathologischem Routinematerial: Anwendung bei malign en Lymphomen H. DENK, T. RADASZKIEWICZ, and CH. WITTING With
I
Figure· Received July 15, 1976
Key words: Immunofluorescence - Routine - Malignant lymphoma Diagnostic
Summary In an attempt to apply immunmorphologic techniques to pathologic routine material, malignant lymphomas of the Non-Hodgkin- and the Hodgkin-type were studied by indirect immunofluorescence. To minimize nonspecific background staining formal in fixed paraffin sections were treated with protease prior to incubation with the antisera. The usefullness of this procedure in providing additional diagnostic criteria was tested on specimens of malignant lymphomas (chronic lymphocytic leukemias, immunocytic lymphomas, plasmocytic lymphomas, centrocytic lymphomas, centroblastic-centrocyctic lymphomas, centroblastic lymphomas, immunoblastic lymphomas, lymphoblastic lymphomas of Burkitt, convoluted and unclassified type, Hodgkin's disease). In addition, cases of nonspecific lymphadenitis were studied. The disc;imination of single cells particularly of the B-cell line was facilitated using immunofluorescence and different patterns were easily recognized thus aiding the diagnosis based on light microscopic criteria. In addition, preliminary evidence was obtained that lymphoblastic lymphomas categorized as "others" may be further characteri7.ed.
The major obstacle to the general application of immunofluorescence in routine pathology is the need for frozen sections with often inferior
220'
H. Denk, T. Radaszkiewicz, and Ch. Witting
Fig. I a. Lymphoplasmocytoid lymphoma. Positive cytoplasmic fluorescence staInIng of plasmacells and their precursors which are sparse in this type of lymphoma. X 200. Fig. I b. Lymphoplasmocytic lymphoma composed of mostly positively stained cells. Note the intranuclear inclusion (arrow). X 100.
Immunofluorescence Studies of Malignant Lymphomas .
221
quality in most instances. Formalin fixation and paraffin embedding may alter antigenic properties (Pearse, 196 I; Deng and Beutner, 1974), and, in the case of resistant antigens, the high background staining in formalin fixed material usually interferes with specific immunofluorescence. The latter can be minimized by treatment of the tissue sections with protease (Huang, 1975). In the present communication it will be shown that protease treatment facilitates immunofluorescence studies on formalin fixed paraffin sections of lymphomas.
Material and Methods Material: Three chronic lymphocytic leukemias, 5 immunocytic lymphomas, 5 plasmocytic lymphomas, 5 centrocytic lymphomas, 5 centroblastic-centrocytic lymphomas, twO centroblastic lymphomas, 6 lymphoblastic lymphomas and 5 immunoblastic lymphomas were studied. One specimen with lymphocytic predominance (nodular paragranuloma), two specimens with nodular sclerosis and two of the mixed type represented the Hodgkinlymphoma category. In addition, 5 lymphnodes with the histologic picture of nonspecific lymphadenitis were studied. The histologic material was classified according to Lennert et al. (I975a, b). Methods: The paraffin sections were dewaxed in xylene, rehydrated, covered with 0.3% celloidine and treated with 0.1% pronase type VII (Sigma Chemical Comp., MO) in phosphate buffered saline (PBS), pH 7,4 for 15 min at 37° C. Pronase treatment for a longer time period was without further benefit. The slides were washed thereafter in icecold PBS for 60 min and then covered with a human globulin antiserum from the rabbit (ammoniumsulphate-precipitated globulin fraction) in a dilution of I : 40 (corresponding to 1/5 precipitation unit in double immunodiffusion). After 45 min incubation in a moist chamber at room temperature the slides were washed in several changes of PBS and then incubated with FITC-conjugated rabbit globulin antiserum (goat; Behring-Werke, Marburg/Lahn, Germany) in a dilution of I : 10 (corresponding to approx-
Fig. X
I.
c.
In plasmocytic lymphoma all cells show cytoplasmic immunofluorescence.
200.
Fig. I d. Centroblastic lymphoma. Positive superficial and sometimes granular fluorescence staining outlines the tumor cells. Intensively stained cells were identified as plasmacells. >( 200. Fig. I e. In lymphoblastic lymphoma of the Burkitt type positive and also negative stained cells can be observed. X 200. Fig. I f. Immunoblastic lymphoma with variations of intensity of cytoplasmic immunofluorescence staining of the tumor cells. X 200. Fig. I h. Follicular hyperplasia. Granular outline and honeycomb-like pattern in germinal Fig. I h. Follicular hyperplasia. Granular outline and honeycomb-like pattern germinal center and some positive stained plasmacells and plasmacell-precursors in the interfollicular area. X 100. Fig. I i. Hodgkin's disease, mixed type - positive Reed-Sternberg cell and a Hodgkin cell (inset). X 200. 15 Beitr. Path. Bd. 159
lll'
H. Denk, T. Radaszkiewicz, and Ch. Witting
imately rlz precipitation unit; fluorescein/protein ratio 1.5 : I). After a second wash in PBS the sections were mounted in glycerol-PBS (r : 8) and read on a Leitz Orthoplan fluorescence microscope with incident light illumination (Ploem-Opak, filter combination 3/3). Fotomicrographs were taken with a Kodak Ektachrome High-Speed color film. Controls included normal rabbit serum and PBS, respectively, in the first layer.
Results In chronic lymphocytic leukemia a uniform immunofluorescence pattern was observed. The tumor cells were consistently negative. Positive specific fluorescence was only present in lymphnode areas not involved by the disease process, particularly in plasma cells and cellular elements in infiltrated sinuses. In immunocytic lymphomas a high percentage of the cells was negative. However, cells of small to intermediate size with specific cytoplasmic staining were haphazardly distributed throughout (Fig. I a) and were identified in Giemsa stained sections as lymphatic plasmacells, plasmoblasts and plasmacells (lymphoplasmocytoid lymphoma). In the lymphoplasmocytic type most of the cells gave a positive cytoplasmic immunofluorescence and occasionally showed positive intranuclear inclusions (Fig. I b). In plasmocytomas all cells exhibited cytoplasmic immunofluorescence of variable intensity (Fig. I c). In centrocytic lymphomM the cells were specifically outlined in a honeycomb-like fashion. It is unclear whether this is cytoplasmic, surface or intercellular staining. In centroblastic-centrocytic lymphomas the cells were specifically outlined in a honeycomb-like fashion and in the interfollicular areas in case of follicular pattern, brightly stained lymphatic plasmacells, plasmoblasts and plasmacells were seen. In one anaplastic specimen amorphous globular and irregular substances were found in the neoplastic follicles exhibiting fluorescence of medium intensity. Two of the very rare centroblastic lymphomas were included in this study. The majority of the cells was only outlined by specific fluorescence in a honeycomb-pattern. Occasionally larger cells with faint superficial granular fluorescence were seen (Fig. I d). The lymphoblastic lymphoma material consisted of two Brukitt type, two convoluted type, and two unclassifiable-("other")lymphomas The Burkitt type-lymphomas contained mostly small to medium size cells with a distinct cytoplasmic fluorescence (Fig. I e). Cells with superficial granular fluorescence and entirely negative cells were also encountered in smaller numbers. In the convoluted type-lymphomas, the tumor cells were
Immunofluorescence Studies of Malignant Lymphomas .
223
consistently negative. The two lymphoblastic lymphomas type "others" showed cytoplasmic staining of most of the cells suggesting their B-cell nature. Immunoblastic lymphomas featured an inhomogeneous immunofluorescence pattern consisting of cells of the plasmacell line with narrow or broad specifically stained cytoplasm. The immunoblasts showed variations in immunofluorescence from bright to negative staining (Fig. 1 f). Five cases of Hodgkin's disease (one lymphocytic predominance type, two nodular sclerosis and two of the mixed type) were included. Most of the tumor cells were negative and plasmacells abundantly present in these lymphnodes served as positive controls. Hodgkin cells and Sternberg-Reed cells were either positive (Fig. 1 i), weakly positive or negative, but even the positive cells never reached the intensity of fluorescence observed in plasmacells (Fig. 1 g). In lymphnodes with histologic criteria of nonspecific lymphadenitis and/or follicular hyperplasia larger cells with granular outline, presumably centroblasts, were found in the follicles together with smaller specifically outlined cells (honeycomb-like appearence) (Fig. 1 h). Cells belonging to the plasmacell line were sparse in the germinal centers but were observed more often in the interfollicular areas and in the medulla. The sinuses contained a large number of small- to medium-sized cells with bright cytoplasmic staining. The parafollicular areas were negative.
Discussion Immunofluorescence studies are usually performed on frozen material which, however, in most instances is difficult to obtain in a pathologic routine laboratory. Therefore, several authors have tried to adept immunofluorescence and other immunmorphologic techniques to formalin or alcohol fixed paraffin sections (Sainte-Marie, 1962; Brown et al., 1974; Burns et al., 1974; Garvin et al., 1974; Muller et al., 1974; Taylor and Burns, 1974; Watkins and Evans, 1974; Burns, 1975; Bussolati and Pich, 1975; Bussolati et al., 1975; Nayak and Sachdeva, 1975; Ray and Desmet, 1975; Taylor, 1976). The problem of high nonspecific background staining in formalin fixed material was overcome partly by Huang (1975) who found that pronase treatment greatly reduced background staining at least in liver biopsy material searched for hepatitis-B-antigens. In the present communication a modified Huang-technique was employed on routine material of malignant lymphomas and it was found that the immunoglobulin antigens are preserved under these conditions and readily demonstrable by indirect immunofluorescence. It was shown by Lennert et al.
224' H. Denk, T. Radaszkiewicz, and Ch. Witting
(1975 b) that determination of immunoglobulins in lymphoid tissue homogenates provides additional clues on the type and biologic behaviour of the tumor. Under these circumstances, however, no information is obtained on the different cells particularly in those lymphomas which consist of different types of cells like immunoblastic lymphomas. With immunofluorescence, not only retrospective studies of cases were possible, but also the discrimination of single cells, particularly of the B-cell line, proved to be greatly facilitated. Similar conclusions were reached by Taylor (1976) using the immunoperoxidase technique. In addition, different immunofluorescence patterns emerged which may favourably aid the diagnosis based on light microscopic criteria. It could also be shown that lymphoblastic lymphoma.s categorized as "others" (Lennert et a1., 1975 a) because of their nonspecific morphology may be further characterized by immunofluorescence. In agreement with the results obtained by Burns et al. (1974), Taylor (1974, 1976) and Taylor and Burns (1974) it was found that some of the Hodgkin cells and Sternberg-Reed cells contain immunoglobulins favouring a B-cell origin of these cells. Studies of a more comprehensive material and using antisera to different immunoglobulin types are presently in progress.
Zusammenfassung Formalin-fixiertes Paraffin material von Non-Hodgkin- und Hodgkin-Lymphomen wurde mittels der indirekten Immunfluoreszenz untersucht. Vor der Inkubation mit Humanglobulin-Antiserum wurden die Paraffinschnitte mit Protease behandelt, urn die unspezifische Fluoreszenz zu verringern. Folgende maligne Lymphome wurden untersrucht: chronische lymphatische Leukamie, immunozytisches Lymphom, plasmozytisches Lymphom, zentrozytisches Lymphom, zentroblastisch-zentrozytisches Lymphom, zentroblastisches Lymphom, lymphoblastisches Lymphom vom "Burkitt-type", "convoluted type" und "unclassified type", immunoblastisches I.ymphom und M. Hodgkin, zusatzlich Falle von unspezifischen Lymphadenitiden. Der immunmorphologische Nachweis von zytoplasmatischem Immunglobulin erleichtert die Klassifikation der Zelltypen in Lymphomen, wodurch zusatzliche diagnostische Kriterien gewonnen werden konnen.
References Brown, P., Bourne, ]., and Steel, M.: Immunoperoxidase and Immunofluorescence Techniques in Pig Tissues. Histochemistry 40,343-348 (I974) Burns, ].: Background Staining and Sensitivity of the Unlabelled Antibody-Enzyme (PAP) Method. Comparison with the Peroxidase Labelled Antibody Sandwich Method Using Formalin Fixed Paraffin Embedded Material. Histochemistry 43, 29I-294 (I975) Burns, J, Hambridge, M., and Taylor, C. R.: Intracellular immunoglobulins. A comparative study on three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates. ]. din. Path. 27, 548-557 (I974)
Immunofluorescence Studies of Malignant Lymphomas . 225 Bussolati, G., and Pich, A.: Mammary and Extramammary Paget's Disease. An Immunocytochemical Study. Amer.]. Path. 80, 1I7-uS (1975) Bussolati, G., Pich, A., and Alfani, V.: Immunofluorescence Detection of Casein in Human Mammary Dysplastic and Neoplastic Tissues. Vir chows Arch. A Path. Anat. 3 65,15-21 (1975) Deng, ].-S., and Beutner, E. H.: Effect of Formaldehyde, Glutaraldehyde and Sucrose on the Tissue Antigenicity. Int. Arch. Allergy 47, 562-5 69 (1974) Garvin, A. ]., Spicer, S. S., Parmley, R. T., Hintz, D., and Munster, A. M.: Immunostaining of Immunoglobulin G (IgG) in Immunocytes and Hodgkin and Reed-Sternberg Cells in Hodgkin's Disease. J. Histochem. Cytochem. 22, 296 (1974) Huang, S.-N.: Immunohistochemical Demonstration of Hepatitis B Core and Surface Antigens in Paraffin Sections. Lab. Invest. 33, 88-95 (1975) Lennert, K., Mohri, N., Stein, H., and Kaiserling, E.: The Histopathology of Malignant Lymphoma. Brit. J. Haematol. 31 (Suppl.), 193-203 (197sa) Lennert, K., Stein, H., and Kaiserling, E.: Cytological and functional criteria for the classification of malignant lymphoma. Brit. J. Cancer 31 (Suppl. II), 29-43 (1975b) Miiller, W. H., Knop, B., und Felsch, G.: Zur Eignung paraffineingebetteten Gewebes als Substrat fUr den immunfluoreszenztechnischen Nachweis antinuklearer Faktoren. Acta histochem. 48, 291-300 (1974) Nayak, N. c., and Sachdeva, R.: Localization of Hepatitis B Surface Antigen in Conventional Paraffin Sections of the Liver: Comparison of Immunofluorescence, Immunoperoxidase, and Orcein Staining Methods With Regard to Their Specificity and Reliability as Antigen Marker. Amer. J. Path. 81,479-493 (1975) Pearse, A. G. E.: Histochemistry, Theoretical and Applied, p. 146. J. and A. Churchill, Ltd., London (1961) Ray, M. B., and Desmet, V. J.: Immunofluorescent detection of hepatitis B antigen in paraffin-embedded liver tissue. J. Immunol. Methods 6, 283-289 (1975) Sainte-Marie, G.: A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem. 10, 25°-256 (1962) Taylor, C. R.: The nature of Reed-Sternberg cells and other malignant "reticulum" cells. Lancet II, 802-807 (1974) Taylor, C. R.: Immunohistological study of follicular lymphoma, reticulum cell sarcoma and Hodgkin's disease. Europ. J. Cancer 12, 61-75 (1976) Taylor, C. R., and Burns, J.: The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidaselabelled antibody. J. clin. Path. 27, 14-20 (1974) Watkins, W. B., and Evans, J. J.: The use of formalin fixation in an improved method for immunofluorescence histochemical investigation of neurophysin in thy hypothalamus. J. Histochem. Cytochem. 22, 128-130 (1974) Doz. Dr. H. Denk, Dr. T. Radaszkiewicz, Pathologisches Institut der Universitat, Spitalgasse 4, A-109° Wien, Austria and Dr. C. Witting, Pathologisches Isntitut der Universitat, Westring 17, D-4400 Munster, W.-Germany