JOURNAL OF PATHOLOGY, VOL.
164: 37-40 (1991)
LOCALIZATION OF IMMUNOGLOBULIN LIGHT CHAIN mRNA EXPRESSION IN HODGKIN'S DISEASE BY IN SITU HYBRIDIZATION ANITA K. RUPRAI, J. H. PRINGLE, CAROLE A. ANGEL*, C. N. KIND? AND I. LAUDER
Depurtment oJ' Puthology, University of Leicester, Clinicul Sciences Buildiiig, P. 0.Box 65, Leicester Royal Injrnzary, Leicester LE2 7 LX,U.K.; * Depurtment of Histopathology, Shefield University Medical School, Beech Hill Road, Shefield SI0, U.K.: ?Department of Pathology, Fisons plc, Pharmaceuticals Division, Loughborough, Leicestershire LEI1 ORH, U.K. Received 16 August 1990 Accepted 23 November 1990
SUMMARY In situ hybridization techniques were used to detect immunoglobulin light chain messenger R N A (mRNA) in 28 formalin-fixed, paraffin-embedded samples of Hodgkin's disease. Cocktails of biotinylated oligonucleotide probes specific for the constant regions of kappa and lambda light chain mRNA were used. None of the Reed-Sternberg cells or their variants in any of the cases studied showed positive staining with either probe, in contrast to normal plasma cells which showed strong staining in the same sections. It was concluded, therefore, that the cytoplasmic immunoglobulin frequently detected within these cells by immunocytochemistry is present not as a result of synthesis, but as a result of some other mechanism. KEY
wom-Hodgkin's
disease, in situ hybridization, oligonucleotide, messenger RNA.
INTRODUCTION The neoplastic cell of Hodgkin's disease (HD) has been defined as the Reed-Sternberg (RS) cell and this, together with its mononuclear Hodgkin's (H) variant, has been well characterized in terms of its cellular morphology. Little is known, however, about the nature and cellular origin of the H/RS cell in spite of much research in this area. Immunohistochemical studies involving the use of large panels of monoclonal antibodies have been used to characterize the phenotype of the H/RS and as a result, many cells such as B- and Tlymphocytes,"' macro phage^,^ dendritic reticulum cells and interdigitating reticulum cells' have been provosed as giving rise to the H/RS cell. It has also been suggested that the origin of the H/RS cell may vary between the different subtypes of Hodgkin's disease, and that it may be possible for H/RS cells of Addressee for correspondence: Miss A. Ruprai, Department of Human Metabolism and Clinical Biochemistry, Shefield
University Medical School, Beech Hill Road, Sheffield S10, U.K.
0022-34 I7/9 I ,I0I003744 $05.00 0 1991 by John Wiley & Sons, Ltd.
more than one derivation to be present within the same case.'.' The presence of cytoplasmic immunoglobulin within H/RS cells led early investigators to propose a B-cell but the subsequent demonstration of both light chains within the same cell suggested that this immunoglobulin was present as a result of uptake by the cell rather than synthe~is.~," More recently, other B-cell markers have been detected in H/RS, providing some support for a B-cell origin, at least in some Minor immunoglobulin gene rearrangements have been detected in some s t ~ d i e s , ' ~but . ' ~others have failed to confirm these findings." There is, however, some support for a Bcell lineage for H/RS cells in the nodular lymphocyte predominant subtype of HD, which is thought by some to represent a lymphoma of follicle centre cells. "."However, immunoglobulin gene rearrangements have not been detected in this subtype of Hodgkin's disease. I' The aim of this study was to examine the possible B-cell origin of the H/RS cell by the use of biotinylated oligonucleotide probes specific for the
38
A. K . RUPRAI ETAL.
constant regions of kappa ( K ) and lambda (A) light chain messenger RNA (mRNA) in in situ hybridization experiments. This technique has been documented as a useful method for the detection of mRNA species in fixed tissue,”-*’ and a suitable protocol for the detection of light chain immunoglobulin mRNA in formalin-fixed tissue has been developed in our department.22 MATERIALS AND METHODS Case selection Of the 28 cases of Hodgkin’s disease studied, the disease was classified as nodular sclerosis in 18, mixed cellularity in 4, lymphocyte predominant nodular in 5, and lymphocyte predominant diffuse in 1. Criteria for selection included availability of sufficient tissue for analysis, and good mRNA preservation as demonstrated by probing the tissue sections with an oligo d(T) p r ~ b e . ’A~ previous ,~~ study had shown that cytoplasmic immunoglobulin could be demonstrated by routine immunocytochemistry within the H/RS cells in 18 of these cases.’ Of these 18 cases, 13 showed both light chains in the same H/RS cell in serial sections. The remaining 10 cases had not been subjected to immunocytochernical analysis.
Methods Tissue had been fixed in 10 per cent formol saline for 2 4 4 8 h, routinely processed, and paraffinembedded. Four ,um sections were then cut onto 3aminopropyl triethoxysilane treated slides and dried overnight at 37°C. In situ hybridization was performed by the method previously described by Pringle et a1.*2 Briefly, cocktails of biotinylated oligonucleotide probes specific for K and A mRNA were prepared. The specificity of these probes had been confirmed by Northern blotting and by dual labelling experiments in which in situ hybridization and light chain immunocytochemistry were compared. Further evidence for the specificity of the probes was shown by the complete elimination of the signal in control sections treated with RNase A. Pretreatments involved a 20 min incubation in 0.2 N HCI at room temperature, followed by a 2 x SSC wash at 70°C for 10 min. The sections were then incubated for 1 h at 37°C with 10pg/ml of proteinase K. Hybridization took place overnight with the probe used at a concentration of 2 ,ug/ml, and was followed by stringent post-hybridization
washes of 50 per cent formamide/2 x SSC at 37°C for 10 min, repeated three times. Detection of the probe was performed by a four-stage streptavidin/ biotin/alkaline phosphatase technique as previously described.” The alkaline phosphatase was visualized using Fast Red TR; sections were counterstained with Mayer’s haematoxylin and mounted in Apathy’s. Positive controls were provided by examining sections from a case of plasmacytoma, and of reactive tonsil. Polytypic plasma cells were also present in most of the cases of H D studied, providing an internal positive control. Negative controls were provided by incubating sections in hybridization solution to which no probe had been added, RESULTS None of the H/RS cells in any of the cases of H D studied showed positive staining with either probe. In all the cases, background polytypic plasma cells were strongly stained, the signal being comparable to that seen in the control tonsil tissue and in the plasmacytoma (Fig. I). Non-specific staining was,
Fig. I-Photomicrograph showing a typical case of Hodgkin’s disease stained for light chain immunoglobulin mRNA with strongly positive plasma cells surrounding negative H/RS cells
LIGHT CHAIN mRNA I N HODGKIN’S DISEASE
however, present within H/RS cells in both the test and the negative control sections. This staining was generally weak and granular, in contrast to the stronger, more homogeneous staining seen in plasma cells. In the control tonsil tissue, reactive polyclonal plasma cells were strongly stained. A positive signal could also be detected in some follicle centre cells, predominantly small centrocytes, although occasional centroblasts were also positive. Non-specific staining was present in some tingible body macrophages, this staining also being present in the negative control sections. The case of plasmacytoma studied showed clear demonstration of monoclonality, the strength of signal being comparable to that seen in reactive plasma cells.
39
conceivable that such a signal may mask a weak true positive reaction in H/RS cells. To overcome this problem, alternative labelling protocols where other haptens are used to label the oligonucleotide probe are currently being investigated. In conclusion, this study has shown that light chain mRNA is not present in sufficient amounts in H/RS cells to account for the often intense staining seen when immunocytochemical methods of detecting immunoglobulin light chain are employed. It must, however, be noted that given the sensitivity of this technique, the possibility that Reed-Sternberg cells and their variants are derived from B-cells cannot be excluded. REFERENCES
DISCUSSION This study has shown that the cytoplasmic immunoglobulin detected in H/RS cells is not present as a result of synthesis. In particular, no evidence could be found to suggest that the H/RS cells in the nodular lymphocyte predominant subtype of H D synthesize immunoglobulin light chain. The technique used in this study has been previously shown to be sufficiently sensitive to detect light chain mRNA in a range of normal B-cells, including some follicle centre cells. In immunocytochemically stained sections, the intensity of immunoglobulin staining of H/RS cells, whilst generally less than that in plasma cells, is usually greater than that observed in follicle centre cells. If the cytoplasmic immunoglobulin in H/RS cells were present as a result of synthesis, it would be expected that sufficient light chain mRNA would be present to be detected by this technique. The results of this study therefore suggest that immunoglobulin is present within H/RS cells as a result of uptake. At present, the level of sensitivity of this technique is such that only secretory immunoglobulin can be reliably detected within B-cells, and that Bcells synthesizing surface or perinuclear space immunoglobulin cannot be detected. Therefore, the possibility that H/RS cells are derived from early Bcells, in which the copy number of light chain mRNA is below the limit of sensitivity of this test, cannot be excluded. Work is in progress to increase the sensitivity of this technique whilst retaining the morphological detail necessary to reliably identify H/RS cells in stained sections. The weak non-specific signal seen in H/RS cells in both the negative control and test sections may represent endogenous biotin within these cells, and it is
1. Angel CA, Warford A, Campbell AC, Pringle JH, Lauder I. The
immunohistology of Hodgkin’s disease-Reed-Sternberg cells and their variants. J Patholl987;153:21-30. 2. Chittal SM, Caveriviere P, Schwarting R, el a/. Monoclonal antibodies in the diagnosis of Hodgkin’s disease: the search for a rational panel. Am J Surg Pathol 1988;1 2 9-21. 3. Hall PA, DArdenne AJ, Stansfield AG. Paraffin section immunohistochemistry II. Hodgkin’s disease and large cell anaplastic (Ki-I) lymphoma. Hi.stopathology 1988; 13: 161-169. 4. Kadin ME, Muramoto L, Said J. Expression of T-cell antigens on Reed-sternberg cells in subset of patients with nodular sclerosing and mixed cellularity Hodgkin’s disease. Am J Pathol1988;130 345-353. 5. Payne SV, Wright DH, Jones KJM, Judd MA. Macrophage origin of Reed-Sternberg cells: an immunohistochemical study. J Clin Pathol 1982;35: 159-166. 6. Kennedy ICS, Hart DNJ, Colls BM, Nimmo JC, Willis DA, Angus HB. Nodular sclerosing, mixed cellularity and lymphocyte-depleted variants of Hodgkin’s disease are probable dendriticcell malignancies. Clin Exp Immunol1989;76 324331. 7. Kadin ME. Possible origin of the Reed-Sternberg cells from an interdigitating reticulum cell. Cancer Treat Rep 1982;6 6 601-608. 8. Anastasi J, Variakojis D. Heterogeneity in Hodgkin’s disease: no simple answer for a complex disorder. Hum Pathol 1988; 1 9 125 1-1254. 9. Garvin AJ, Spicer S S , McKeever PE. The cytochemical demonstration of intracellular immunoglobulin in neoplasms of lymphoreticular tissue. A m J Parhol1976;82 457478. 10. Gladkowska-Dura MJ, Dura TW, Johnson WW. Light and immunoelectron microscopic study of Hodgkin’s disease: evidence of immunoglobulin synthesis by tumor cells. Virchows Arch 5 [Cell Parhol] 1981;37: 109-124. I I . Curran RC, Jones EL. Immunoglobulin in Reed-Sternberg and Hodgkin’scells. J Pathol1978;126: 35-37. 12. Norton AJ, Isaacson PG. Monoclonal antibody L26:an antibody that is reactive with normal and neoplastic B lymphocytes in routinely fixed and paraffin wax embedded tissues. J Clin Pathol 1987; 4 0 140s-I41 2. 13. Timens W, Visser L, Poppema S . Nodular lymphocyte predominance type Hodgkin’s disease is a germinal centre lymphoma. Lab Invesr 1986;54: 457461. 14. Weiss LM, Strickler JG, Hu E, Warnke RA, Sklar J. Immunoglobulin gene rearrangements in Hodgkin’s disease. Hum Puthol 1986; 17: 1009-1014. 15. Brinker MGL, Poppema S , Buys CHCM, Timens W, Osinga J, Visser L. Clonal immunoglobulin gene rearrangements in tissues involved by Hodgkin’s disease. Blood 1987;70 186191. 16. Villa A, Cairo G, Pozzi MR, et a/. Lack of TdT and immunoglobulin T-cell receptor gene rearrangements in Hodgkin’s disease. Int J 5iol Markers 1987;2 65-70.
40
A. K. R U P R A I E T A L .
17. Pinkus GS, Said J. Hodgkin’s disease, lymphocyte predominance type, nodular-further evidence for a B-cell derivation. Am J Parhol 1988; 133211-217. 18. Linden MD, Fishleder AJ, Katzin WE, Tubbs RR. Absence of B-cell o r T-cell clonal expansion in nodular. lymphocyte predominant Hodgkin’s disease. Hum Parhol 1988; 1 9 591-594. 19. Pringle JH, Primrose L, Kind C N , Talbot IC, Lauder 1. In siru hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed sections using a biotinylated oligonucleotide poly(dT) probe. J Pnrhol 1989; 158: 279-286. 20. Close PM, Pringle JH, Ruprai AK, West KP, Lauder I. Zonal distribution of immunoglobulin-synthesising cells within the germinal
centres: an in situ hybridisation and immunohistochemical study. J . Porhol1990; 1 6 2 209-216. 21. Akhtar N, Ruprai AK, Pringle JH, Lauder I, Durrant S. In situ hybridization detection of light chain mRNA in routine bone marrow trephines from patients with suspected myeloma. Er J Hrrrmatol1989; 73: 296301 22. Pringle JH, Ruprai AK, Keyte J, rr al. In siru hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten labelled oligonucleotide probes. J Parhol 1990; 162: 197-207. 23. West KP, Pringle J H , Angel CA, Naylor JP, Lauder I Detection of RNA in paraffin sections of lymphoid tissue. J Pnrhol 1988; 154: 57A.
164: 37-40 (1991)
LOCALIZATION OF IMMUNOGLOBULIN LIGHT CHAIN mRNA EXPRESSION IN HODGKIN'S DISEASE BY IN SITU HYBRIDIZATION ANITA K. RUPRAI, J. H. PRINGLE, CAROLE A. ANGEL*, C. N. KIND? AND I. LAUDER
Depurtment oJ' Puthology, University of Leicester, Clinicul Sciences Buildiiig, P. 0.Box 65, Leicester Royal Injrnzary, Leicester LE2 7 LX,U.K.; * Depurtment of Histopathology, Shefield University Medical School, Beech Hill Road, Shefield SI0, U.K.: ?Department of Pathology, Fisons plc, Pharmaceuticals Division, Loughborough, Leicestershire LEI1 ORH, U.K. Received 16 August 1990 Accepted 23 November 1990
SUMMARY In situ hybridization techniques were used to detect immunoglobulin light chain messenger R N A (mRNA) in 28 formalin-fixed, paraffin-embedded samples of Hodgkin's disease. Cocktails of biotinylated oligonucleotide probes specific for the constant regions of kappa and lambda light chain mRNA were used. None of the Reed-Sternberg cells or their variants in any of the cases studied showed positive staining with either probe, in contrast to normal plasma cells which showed strong staining in the same sections. It was concluded, therefore, that the cytoplasmic immunoglobulin frequently detected within these cells by immunocytochemistry is present not as a result of synthesis, but as a result of some other mechanism. KEY
wom-Hodgkin's
disease, in situ hybridization, oligonucleotide, messenger RNA.
INTRODUCTION The neoplastic cell of Hodgkin's disease (HD) has been defined as the Reed-Sternberg (RS) cell and this, together with its mononuclear Hodgkin's (H) variant, has been well characterized in terms of its cellular morphology. Little is known, however, about the nature and cellular origin of the H/RS cell in spite of much research in this area. Immunohistochemical studies involving the use of large panels of monoclonal antibodies have been used to characterize the phenotype of the H/RS and as a result, many cells such as B- and Tlymphocytes,"' macro phage^,^ dendritic reticulum cells and interdigitating reticulum cells' have been provosed as giving rise to the H/RS cell. It has also been suggested that the origin of the H/RS cell may vary between the different subtypes of Hodgkin's disease, and that it may be possible for H/RS cells of Addressee for correspondence: Miss A. Ruprai, Department of Human Metabolism and Clinical Biochemistry, Shefield
University Medical School, Beech Hill Road, Sheffield S10, U.K.
0022-34 I7/9 I ,I0I003744 $05.00 0 1991 by John Wiley & Sons, Ltd.
more than one derivation to be present within the same case.'.' The presence of cytoplasmic immunoglobulin within H/RS cells led early investigators to propose a B-cell but the subsequent demonstration of both light chains within the same cell suggested that this immunoglobulin was present as a result of uptake by the cell rather than synthe~is.~," More recently, other B-cell markers have been detected in H/RS, providing some support for a B-cell origin, at least in some Minor immunoglobulin gene rearrangements have been detected in some s t ~ d i e s , ' ~but . ' ~others have failed to confirm these findings." There is, however, some support for a Bcell lineage for H/RS cells in the nodular lymphocyte predominant subtype of HD, which is thought by some to represent a lymphoma of follicle centre cells. "."However, immunoglobulin gene rearrangements have not been detected in this subtype of Hodgkin's disease. I' The aim of this study was to examine the possible B-cell origin of the H/RS cell by the use of biotinylated oligonucleotide probes specific for the
38
A. K . RUPRAI ETAL.
constant regions of kappa ( K ) and lambda (A) light chain messenger RNA (mRNA) in in situ hybridization experiments. This technique has been documented as a useful method for the detection of mRNA species in fixed tissue,”-*’ and a suitable protocol for the detection of light chain immunoglobulin mRNA in formalin-fixed tissue has been developed in our department.22 MATERIALS AND METHODS Case selection Of the 28 cases of Hodgkin’s disease studied, the disease was classified as nodular sclerosis in 18, mixed cellularity in 4, lymphocyte predominant nodular in 5, and lymphocyte predominant diffuse in 1. Criteria for selection included availability of sufficient tissue for analysis, and good mRNA preservation as demonstrated by probing the tissue sections with an oligo d(T) p r ~ b e . ’A~ previous ,~~ study had shown that cytoplasmic immunoglobulin could be demonstrated by routine immunocytochemistry within the H/RS cells in 18 of these cases.’ Of these 18 cases, 13 showed both light chains in the same H/RS cell in serial sections. The remaining 10 cases had not been subjected to immunocytochernical analysis.
Methods Tissue had been fixed in 10 per cent formol saline for 2 4 4 8 h, routinely processed, and paraffinembedded. Four ,um sections were then cut onto 3aminopropyl triethoxysilane treated slides and dried overnight at 37°C. In situ hybridization was performed by the method previously described by Pringle et a1.*2 Briefly, cocktails of biotinylated oligonucleotide probes specific for K and A mRNA were prepared. The specificity of these probes had been confirmed by Northern blotting and by dual labelling experiments in which in situ hybridization and light chain immunocytochemistry were compared. Further evidence for the specificity of the probes was shown by the complete elimination of the signal in control sections treated with RNase A. Pretreatments involved a 20 min incubation in 0.2 N HCI at room temperature, followed by a 2 x SSC wash at 70°C for 10 min. The sections were then incubated for 1 h at 37°C with 10pg/ml of proteinase K. Hybridization took place overnight with the probe used at a concentration of 2 ,ug/ml, and was followed by stringent post-hybridization
washes of 50 per cent formamide/2 x SSC at 37°C for 10 min, repeated three times. Detection of the probe was performed by a four-stage streptavidin/ biotin/alkaline phosphatase technique as previously described.” The alkaline phosphatase was visualized using Fast Red TR; sections were counterstained with Mayer’s haematoxylin and mounted in Apathy’s. Positive controls were provided by examining sections from a case of plasmacytoma, and of reactive tonsil. Polytypic plasma cells were also present in most of the cases of H D studied, providing an internal positive control. Negative controls were provided by incubating sections in hybridization solution to which no probe had been added, RESULTS None of the H/RS cells in any of the cases of H D studied showed positive staining with either probe. In all the cases, background polytypic plasma cells were strongly stained, the signal being comparable to that seen in the control tonsil tissue and in the plasmacytoma (Fig. I). Non-specific staining was,
Fig. I-Photomicrograph showing a typical case of Hodgkin’s disease stained for light chain immunoglobulin mRNA with strongly positive plasma cells surrounding negative H/RS cells
LIGHT CHAIN mRNA I N HODGKIN’S DISEASE
however, present within H/RS cells in both the test and the negative control sections. This staining was generally weak and granular, in contrast to the stronger, more homogeneous staining seen in plasma cells. In the control tonsil tissue, reactive polyclonal plasma cells were strongly stained. A positive signal could also be detected in some follicle centre cells, predominantly small centrocytes, although occasional centroblasts were also positive. Non-specific staining was present in some tingible body macrophages, this staining also being present in the negative control sections. The case of plasmacytoma studied showed clear demonstration of monoclonality, the strength of signal being comparable to that seen in reactive plasma cells.
39
conceivable that such a signal may mask a weak true positive reaction in H/RS cells. To overcome this problem, alternative labelling protocols where other haptens are used to label the oligonucleotide probe are currently being investigated. In conclusion, this study has shown that light chain mRNA is not present in sufficient amounts in H/RS cells to account for the often intense staining seen when immunocytochemical methods of detecting immunoglobulin light chain are employed. It must, however, be noted that given the sensitivity of this technique, the possibility that Reed-Sternberg cells and their variants are derived from B-cells cannot be excluded. REFERENCES
DISCUSSION This study has shown that the cytoplasmic immunoglobulin detected in H/RS cells is not present as a result of synthesis. In particular, no evidence could be found to suggest that the H/RS cells in the nodular lymphocyte predominant subtype of H D synthesize immunoglobulin light chain. The technique used in this study has been previously shown to be sufficiently sensitive to detect light chain mRNA in a range of normal B-cells, including some follicle centre cells. In immunocytochemically stained sections, the intensity of immunoglobulin staining of H/RS cells, whilst generally less than that in plasma cells, is usually greater than that observed in follicle centre cells. If the cytoplasmic immunoglobulin in H/RS cells were present as a result of synthesis, it would be expected that sufficient light chain mRNA would be present to be detected by this technique. The results of this study therefore suggest that immunoglobulin is present within H/RS cells as a result of uptake. At present, the level of sensitivity of this technique is such that only secretory immunoglobulin can be reliably detected within B-cells, and that Bcells synthesizing surface or perinuclear space immunoglobulin cannot be detected. Therefore, the possibility that H/RS cells are derived from early Bcells, in which the copy number of light chain mRNA is below the limit of sensitivity of this test, cannot be excluded. Work is in progress to increase the sensitivity of this technique whilst retaining the morphological detail necessary to reliably identify H/RS cells in stained sections. The weak non-specific signal seen in H/RS cells in both the negative control and test sections may represent endogenous biotin within these cells, and it is
1. Angel CA, Warford A, Campbell AC, Pringle JH, Lauder I. The
immunohistology of Hodgkin’s disease-Reed-Sternberg cells and their variants. J Patholl987;153:21-30. 2. Chittal SM, Caveriviere P, Schwarting R, el a/. Monoclonal antibodies in the diagnosis of Hodgkin’s disease: the search for a rational panel. Am J Surg Pathol 1988;1 2 9-21. 3. Hall PA, DArdenne AJ, Stansfield AG. Paraffin section immunohistochemistry II. Hodgkin’s disease and large cell anaplastic (Ki-I) lymphoma. Hi.stopathology 1988; 13: 161-169. 4. Kadin ME, Muramoto L, Said J. Expression of T-cell antigens on Reed-sternberg cells in subset of patients with nodular sclerosing and mixed cellularity Hodgkin’s disease. Am J Pathol1988;130 345-353. 5. Payne SV, Wright DH, Jones KJM, Judd MA. Macrophage origin of Reed-Sternberg cells: an immunohistochemical study. J Clin Pathol 1982;35: 159-166. 6. Kennedy ICS, Hart DNJ, Colls BM, Nimmo JC, Willis DA, Angus HB. Nodular sclerosing, mixed cellularity and lymphocyte-depleted variants of Hodgkin’s disease are probable dendriticcell malignancies. Clin Exp Immunol1989;76 324331. 7. Kadin ME. Possible origin of the Reed-Sternberg cells from an interdigitating reticulum cell. Cancer Treat Rep 1982;6 6 601-608. 8. Anastasi J, Variakojis D. Heterogeneity in Hodgkin’s disease: no simple answer for a complex disorder. Hum Pathol 1988; 1 9 125 1-1254. 9. Garvin AJ, Spicer S S , McKeever PE. The cytochemical demonstration of intracellular immunoglobulin in neoplasms of lymphoreticular tissue. A m J Parhol1976;82 457478. 10. Gladkowska-Dura MJ, Dura TW, Johnson WW. Light and immunoelectron microscopic study of Hodgkin’s disease: evidence of immunoglobulin synthesis by tumor cells. Virchows Arch 5 [Cell Parhol] 1981;37: 109-124. I I . Curran RC, Jones EL. Immunoglobulin in Reed-Sternberg and Hodgkin’scells. J Pathol1978;126: 35-37. 12. Norton AJ, Isaacson PG. Monoclonal antibody L26:an antibody that is reactive with normal and neoplastic B lymphocytes in routinely fixed and paraffin wax embedded tissues. J Clin Pathol 1987; 4 0 140s-I41 2. 13. Timens W, Visser L, Poppema S . Nodular lymphocyte predominance type Hodgkin’s disease is a germinal centre lymphoma. Lab Invesr 1986;54: 457461. 14. Weiss LM, Strickler JG, Hu E, Warnke RA, Sklar J. Immunoglobulin gene rearrangements in Hodgkin’s disease. Hum Puthol 1986; 17: 1009-1014. 15. Brinker MGL, Poppema S , Buys CHCM, Timens W, Osinga J, Visser L. Clonal immunoglobulin gene rearrangements in tissues involved by Hodgkin’s disease. Blood 1987;70 186191. 16. Villa A, Cairo G, Pozzi MR, et a/. Lack of TdT and immunoglobulin T-cell receptor gene rearrangements in Hodgkin’s disease. Int J 5iol Markers 1987;2 65-70.
40
A. K. R U P R A I E T A L .
17. Pinkus GS, Said J. Hodgkin’s disease, lymphocyte predominance type, nodular-further evidence for a B-cell derivation. Am J Parhol 1988; 133211-217. 18. Linden MD, Fishleder AJ, Katzin WE, Tubbs RR. Absence of B-cell o r T-cell clonal expansion in nodular. lymphocyte predominant Hodgkin’s disease. Hum Parhol 1988; 1 9 591-594. 19. Pringle JH, Primrose L, Kind C N , Talbot IC, Lauder 1. In siru hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed sections using a biotinylated oligonucleotide poly(dT) probe. J Pnrhol 1989; 158: 279-286. 20. Close PM, Pringle JH, Ruprai AK, West KP, Lauder I. Zonal distribution of immunoglobulin-synthesising cells within the germinal
centres: an in situ hybridisation and immunohistochemical study. J . Porhol1990; 1 6 2 209-216. 21. Akhtar N, Ruprai AK, Pringle JH, Lauder I, Durrant S. In situ hybridization detection of light chain mRNA in routine bone marrow trephines from patients with suspected myeloma. Er J Hrrrmatol1989; 73: 296301 22. Pringle JH, Ruprai AK, Keyte J, rr al. In siru hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten labelled oligonucleotide probes. J Parhol 1990; 162: 197-207. 23. West KP, Pringle J H , Angel CA, Naylor JP, Lauder I Detection of RNA in paraffin sections of lymphoid tissue. J Pnrhol 1988; 154: 57A.
