HEMATOPATHOLOGY Original Article
Enhanced Staining for Leu Ml (CD 15) in Hodgkin's Disease Using a Secondary Antibody Specific for Immunoglobulin M DAVID P. LEBRUN, M.D., ONSI W. KAMEL, M.D., RONALD F. DORFMAN, M.D., F.R.C.PATH, AND ROGER A. WARNKE, M.D.
suited in a greater number of cases with reactive Hodgkin's cells (7 versus 5), stained an average of more than three times as many neoplastic cells in each case (49% versus 14%), and usually produced staining that was distinctly more intense, often in a membrane and paranuclear distribution characteristic of Leu Ml in Hodgkin's cells. In the noLeu Ml in Hodgkin's cells. In the nodular L&H group, 1 case showed weak, focal staining with the newer method. None of the L&H cases stained using the traditional technique. It is concluded that use of a second-stage antibody that is directed specifically against mu heavy chains results in an improvement in immunohistochemical staining for Leu Ml in paraffin sections, which is of practical significance. (Key words: Hodgkin's disease; Leu Ml; CD15; Sensitivity; Mu heavy chains) Am J Clin Pathol 1992;97:135-138
Si nee the first report by Hsu and Jaffe' of the immunohistochemical reactivity of immunoglobulin M (IgM) monoclonal antibodies directed against Leu M1 (CD 15) with the Reed-Sternberg cells of Hodgkin's disease, the efficacy of anti-Leu M1 antibodies as a diagnostic aid in this disorder has been controversial. Initial enthusiasm has been tempered by reports of relative insensitivity and nonspecificity.2"4 Furthermore, interpretation can be made difficult by staining that is often weak and focal. Nonetheless, Leu Ml continues to be considered useful by many authors, especially when incorporated as part of a strategically constructed panel of immunohistochemical markers.5"7 Technical maneuvers that improve the sen-
sitivity and quality of Leu M1 staining might significantly enhance its utility in the pathologic diagnosis of Hodgkin's disease, a disorder for which few, if any, reliable immunohistochemical markers are available. In this report, we describe and evaluate an improved immunohistochemical method for the detection of Leu M1 on Hodgkin's cells in which the secondary, goat-derived antibody directed against mouse immunoglobulin is replaced by one that is specific for the Mu heavy chain of mouse IgM. MATERIALS AND METHODS Case Material
Representative, unstained slides from formalin-fixed, From the Department ofPathology, Stanford University Medical Cenparaffin-embedded biopsy material from 15 cases of ter, Stanford, California. Hodgkin's disease (9 nodular sclerosing [NSHD], 1 mixed Received February 1, 1991; received revised manuscript and accepted cellularity [MCHD], and 5 nodular lymphocytic and hisfor publication April 29, 1991. Supported in part by Grants No. 34233 and 33119 from the National tiocytic (L&H) type and 10 cases of non-Hodgkin's lymCancer Institute, National Institutes of Health. phoma (4 diffuse large cell, 2 follicular mixed large cell Address reprint requests to Dr. Warnke: Department of Pathology, and small cleaved cell, 3 follicular small cleaved cell, and Stanford University Medical Center, 300 Pasteur Drive, Stanford, Cal1 diffuse small lymphocytic type) were retrieved from the ifornia 94305. 135
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The utility of staining for Leu Ml (CD15) as a diagnostic aid in Hodgkin's disease has been questioned because of a relative lack of specificity and sensitivity. Furthermore, interpretation is often made difficult by staining that tends to be weak and focal. Because the murine monoclonal anti-Leu Ml antibody is of immunoglobulin M type, it is reasonable to wonder whether improved immunohistochemical staining might result from use of a secondary goat antibody specific for the mouse mu heavy chain instead of the traditional one against mouse immunoglobulin. The two methods were compared, using a biotin-avidin detection system, on paraffin sections from 15 cases of Hodgkin's disease: 9 nodular sclerosing, 1 mixed cellularity, and 5 of nodular lymphocytic and histiocytic (L&H) type. In the nodular sclerosing/ mixed cellularity group, the mu-specific detection method re-
136
HEMATOPATHOLOGY Article
files of the Laboratory of Surgical Pathology at Stanford University. One of the L&H cases originated at Stanford and the 14 remaining Hodgkin's cases represented consultation material referred from other institutions. Immunohistochemistry
Evaluation of Leu Ml Staining The stained sections were examined at high magnification (400X) using a standard light microscope. For each case, an area of the section that contained relatively abundant Reed-Sternberg cells and variants was selected. To allow comparison of the techniques, the same area was chosen on each of the two sections from a given case. One hundred of the neoplastic cells were counted within this area and the number of staining cells was recorded. RESULTS Compared with the traditional method, use of the mu chain-specific secondary antibody generally resulted in more intense Leu M1 staining of Reed-Sternberg cells and their variants and detection of a greater number of cells (Fig. 1). Among the 10 NSHD/MCHD cases, stained cells were present among the cells counted in 7 cases using
A.J.C.P. - J ;
DISCUSSION Although infrequently recognized by diagnostic immunopathologists, the isotype of monoclonal primary antibodies appears to have significant implications for their immunohistochemical detection. The usual practice of using a secondary antibody directed against mouse immunoglobulin G heavy and light chains is generally appropriate because most of the monoclonal antibodies used in diagnostic immunopathologic procedures are of the immunoglobulin G isotype. All of the readily obtainable, commercially produced monoclonal antibodies against Leu M1, as well as several other monoclonal antibodies, are of IgM type. Our observation that Leu Ml is detected more effectively by a specific anti-mu secondary antibody therefore is not surprising. Recently, similar results have been obtained with an IgM monoclonal antibody directed against proliferating cell nuclear antigen/cyclin (PCNA/ cyclin), a cell-cycle-associated nuclear protein for which reliable detection in formalin-fixed, paraffin-embedded tissue appears to require the use of a mu chain-specific secondary antibody.8 The absence of Leu Ml staining in 5 of 10 cases in the NSHD/MCHD group using the traditional detection method represents a higher rate of negativity than generally has been reported.2 Granulocyte staining in these nonreactive cases was weak to absent, indicating poor antigen preservation. Variations in tissue handling may have
1992
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Two sections from each case of Hodgkin's disease were deparaffinized and rehydrated. Endogenous peroxidase was blocked by exposure to 3% H 2 0 2 for 5 minutes, followed by a brief wash in phosphate-buffered saline. Sections were incubated with the anti-Leu M1 monoclonal antibody (Becton-Dickinson, Mountain View, CA) at a dilution of 1:20 in a humidified box for 30 minutes at room temperature. One section from each case was incubated with a biotinylated goat anti-mouse, mu chainspecific antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) used at a dilution of 1:360 in phosphate-buffered saline. For comparison, the other section was incubated with biotinylated goat anti-mouse immunoglobulin (Jackson Immunoresearch Laboratories) at a dilution of 1:360. Incubations with both secondary antibodies lasted 45 minutes. This was followed by incubation with peroxidase-conjugated streptavidin (Jackson Immunoresearch Laboratories), used at a dilution of 1:360 in phosphate-buffered saline, for 45 minutes at 4 °C. Diaminobenzidine was used as a chromogen followed by copper sulfate to darken the reaction product and absolute methanol to fix the precipitate. Each step was followed by a brief wash in phosphate-buffered saline. The sections were lightly counterstained with hematoxylin. The 10 cases of non-Hodgkin's lymphoma were stained using the anti-IgM detection method only.
the newer method compared with only 5 cases using the traditional technique (Table 1). Three cases remained unreactive with both methods. The newer method stained from 21 to 81% (mean, 49%) of recognizable neoplastic cells in the NSHD/MCHD group compared to from 1 to 36% (mean, 14%) with the older method. Among the five nodular L&H cases, no L&H cells were stained using the older method, whereas rare cells became weakly positive in one case using the mu chain-specific secondary antibody. None of the cases of non-Hodgkin's lymphoma showed Leu M1 reactivity with the newer method. In addition to an increased number of Leu Ml-reactive cells with the newer approach, these cells tended to stain with distinctly greater intensity, often rendering them clearly visible at low magnification. Staining often was localized to the cell membrane or to the paranuclear region in a pattern that is characteristic of staining for Leu M1. As an internal control, granulocyte staining was evaluated in each case. Using the traditional method, staining of granulocytes was present in the five cases of NSHD that contained staining Hodgkin's cells and was either absent or minimal in the five Leu Ml-negative cases. Granulocytes in all of the latter cases became weakly to moderately positive when mu-specific detection was used.
LEBRUN ET
AL.
137
Enhanced Staining for Leu Ml in Hodgkin's Disease
been at least partially responsible because all of these cases had been received in consultation from other institutions. Evidently, the enhanced sensitivity of mu-specinc detection can compensate for such reduced antigen accessibility in some cases. Despite the assertion by some authors that, because of its relative insensitivity and nonspecificity, Leu Ml is of little use in the diagnosis of Hodgkin's disease,2"4 others
TABLE 1. COMPARISON OF SECOND-STAGE ANTIBODIES IN NON-L & H HODGKIN'S DISEASE % Positive (Intensity*) Case
Diagnosis
Anti-Gamma
Anti-Mu
1 2 3 4 5 6 7 8 9 10
NSHD NSHD MCHD NSHD NSHD NSHD NSHD NSHD NSHD NSHD
19(1)
53 (III) 33 (II) 64 (III) 31 (III) 63(1) 21(1) 81 (III) 0 0 0
KD
36(11) 10(11) 4(1) 0 0 0 0 0
* I = barely perceptable; II = moderately intense; III = easily visible at low magnification. NSHD = nodular sclerosing Hodgkin's disease: MCND = mixed cellularily Hodgkin's disease.
advocate its use as part of a panel of monoclonal antibodies that includes antibodies intended to exclude other diagnostic possibilities, especially non-Hodgkin's lymphoma.5"7 Thus the usual phenotype of Reed-Sternberg cells and variants in non-L&H Hodgkin's disease includes reactivity for BerH2 (CD30) and absence of reactivity for leukocyte common and B-cell- and T-cell-associated antigens, with the exception of occasional reactivity for Bcell-associated antigen CD20. The technical improvement that we describe enhances the sensitivity and ease of interpretation of Leu M1 staining, with a greater proportion of Hodgkin's cases containing positive cells as well as more intense staining of a greater proportion of cells per case. The possibility that increased sensitivity may have been achieved at the expense of specificity is of concern. The neoplastic cells in the nodular L&H subtype of Hodgkin's disease are typically Leu M1 negative. Although one of the five cases of nodular L&H Hodgkin's disease in this study became Leu M1 positive using the newer detection method, the staining intensity was weak and involved only rare cells. Of the 10 cases of non-Hodgkin's lymphoma that were stained using the newer method, none showed reactivity for Leu M1. It appears, therefore, that the use of an anti-IgM secondary antibody does not significantly reduce the specificity of Leu Ml detection.
Vol. 97 • No. I
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FIGS. 1A and B. Hodgkin's cells (arrow) and granulocytes stain more intensely and in greater number for Leu M1 using a mu chain-specific secondary antibody (B) compared with one directed against mouse light chains and gamma heavy chains (A) (X400).
138
HEMATOPATHOLOGY Article
In conclusion, the use of a secondary antibody that is specifically directed against the mu heavy chain of antiLeu Ml results in improved immunohistochemical detection of this widely used marker to a degree that should be helpful in the routine pathologic diagnosis of Hodgkin's disease. REFERENCES 1. Hsu S-M, Jaffe ES. Leu M1 and peanut agglutinin stain the neoplastic cells of Hodgkin's diesease. Am J Clin Pathol 1984;82:29-32. 2. Hall PA, D'Ardenne AJ. Value of CD 15 immunostaining in diagnosing Hodgkin's disease: A review of published literature. J Clin Pathol 1987;40:1298-1304. 3. Sheibani K, Battifora H, Burke JS, Rappaport H. Leu Ml antigen
4. 5. 6.
7.
8.
I in human neoplasms. An immunohistologic study of 400 cases. Am J Surg Pathol 1986;10(4):227-236. Wieczorek R, Burke JS, Knowles DM. Leu M1 antigen expression in T-cell neoplasia. Am J Pathol 1985;121:374-380. Chittal SM, Caveriviere P, Schwarting R, et al. Monoclonal antibodies in the diagnosis of Hodgkin's disease. The search for a rational panel. Am J Surg Pathol 1988; 12(1):9-21. Dorfman RF, Gatter KC, Pulford KAF, Mason DY. An evaluation of the utility of anti-granulocyte and anti-leukocyte monoclonal antibodies in the diagnosis of Hodgkin's disease. Am J Pathol 1986;123(3):508-519. Medeiros LJ, Weiss LM, Warnke RA, Dorfman RF. Utility of combining antigranulocyte with antileukocyte antibodies in differentiating Hodgkin's disease from non-Hodgkin's lymphoma. Cancer 1988;62:2475-2481. Kamel OW, LeBrun DP, Davis RE, Berry GJ, Warnke RA. Grow fraction estimation of malignant lymphomas in formalin-fixed paraffin-embedded tissue using anti-PCNA/Cyclin 19A2. Am J Pathol 1991; 138(6): 1471-1477.
«* Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Enhanced Staining for Leu Ml (CD 15) in Hodgkin's Disease Using a Secondary Antibody Specific for Immunoglobulin M DAVID P. LEBRUN, M.D., ONSI W. KAMEL, M.D., RONALD F. DORFMAN, M.D., F.R.C.PATH, AND ROGER A. WARNKE, M.D.
suited in a greater number of cases with reactive Hodgkin's cells (7 versus 5), stained an average of more than three times as many neoplastic cells in each case (49% versus 14%), and usually produced staining that was distinctly more intense, often in a membrane and paranuclear distribution characteristic of Leu Ml in Hodgkin's cells. In the noLeu Ml in Hodgkin's cells. In the nodular L&H group, 1 case showed weak, focal staining with the newer method. None of the L&H cases stained using the traditional technique. It is concluded that use of a second-stage antibody that is directed specifically against mu heavy chains results in an improvement in immunohistochemical staining for Leu Ml in paraffin sections, which is of practical significance. (Key words: Hodgkin's disease; Leu Ml; CD15; Sensitivity; Mu heavy chains) Am J Clin Pathol 1992;97:135-138
Si nee the first report by Hsu and Jaffe' of the immunohistochemical reactivity of immunoglobulin M (IgM) monoclonal antibodies directed against Leu M1 (CD 15) with the Reed-Sternberg cells of Hodgkin's disease, the efficacy of anti-Leu M1 antibodies as a diagnostic aid in this disorder has been controversial. Initial enthusiasm has been tempered by reports of relative insensitivity and nonspecificity.2"4 Furthermore, interpretation can be made difficult by staining that is often weak and focal. Nonetheless, Leu Ml continues to be considered useful by many authors, especially when incorporated as part of a strategically constructed panel of immunohistochemical markers.5"7 Technical maneuvers that improve the sen-
sitivity and quality of Leu M1 staining might significantly enhance its utility in the pathologic diagnosis of Hodgkin's disease, a disorder for which few, if any, reliable immunohistochemical markers are available. In this report, we describe and evaluate an improved immunohistochemical method for the detection of Leu M1 on Hodgkin's cells in which the secondary, goat-derived antibody directed against mouse immunoglobulin is replaced by one that is specific for the Mu heavy chain of mouse IgM. MATERIALS AND METHODS Case Material
Representative, unstained slides from formalin-fixed, From the Department ofPathology, Stanford University Medical Cenparaffin-embedded biopsy material from 15 cases of ter, Stanford, California. Hodgkin's disease (9 nodular sclerosing [NSHD], 1 mixed Received February 1, 1991; received revised manuscript and accepted cellularity [MCHD], and 5 nodular lymphocytic and hisfor publication April 29, 1991. Supported in part by Grants No. 34233 and 33119 from the National tiocytic (L&H) type and 10 cases of non-Hodgkin's lymCancer Institute, National Institutes of Health. phoma (4 diffuse large cell, 2 follicular mixed large cell Address reprint requests to Dr. Warnke: Department of Pathology, and small cleaved cell, 3 follicular small cleaved cell, and Stanford University Medical Center, 300 Pasteur Drive, Stanford, Cal1 diffuse small lymphocytic type) were retrieved from the ifornia 94305. 135
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
The utility of staining for Leu Ml (CD15) as a diagnostic aid in Hodgkin's disease has been questioned because of a relative lack of specificity and sensitivity. Furthermore, interpretation is often made difficult by staining that tends to be weak and focal. Because the murine monoclonal anti-Leu Ml antibody is of immunoglobulin M type, it is reasonable to wonder whether improved immunohistochemical staining might result from use of a secondary goat antibody specific for the mouse mu heavy chain instead of the traditional one against mouse immunoglobulin. The two methods were compared, using a biotin-avidin detection system, on paraffin sections from 15 cases of Hodgkin's disease: 9 nodular sclerosing, 1 mixed cellularity, and 5 of nodular lymphocytic and histiocytic (L&H) type. In the nodular sclerosing/ mixed cellularity group, the mu-specific detection method re-
136
HEMATOPATHOLOGY Article
files of the Laboratory of Surgical Pathology at Stanford University. One of the L&H cases originated at Stanford and the 14 remaining Hodgkin's cases represented consultation material referred from other institutions. Immunohistochemistry
Evaluation of Leu Ml Staining The stained sections were examined at high magnification (400X) using a standard light microscope. For each case, an area of the section that contained relatively abundant Reed-Sternberg cells and variants was selected. To allow comparison of the techniques, the same area was chosen on each of the two sections from a given case. One hundred of the neoplastic cells were counted within this area and the number of staining cells was recorded. RESULTS Compared with the traditional method, use of the mu chain-specific secondary antibody generally resulted in more intense Leu M1 staining of Reed-Sternberg cells and their variants and detection of a greater number of cells (Fig. 1). Among the 10 NSHD/MCHD cases, stained cells were present among the cells counted in 7 cases using
A.J.C.P. - J ;
DISCUSSION Although infrequently recognized by diagnostic immunopathologists, the isotype of monoclonal primary antibodies appears to have significant implications for their immunohistochemical detection. The usual practice of using a secondary antibody directed against mouse immunoglobulin G heavy and light chains is generally appropriate because most of the monoclonal antibodies used in diagnostic immunopathologic procedures are of the immunoglobulin G isotype. All of the readily obtainable, commercially produced monoclonal antibodies against Leu M1, as well as several other monoclonal antibodies, are of IgM type. Our observation that Leu Ml is detected more effectively by a specific anti-mu secondary antibody therefore is not surprising. Recently, similar results have been obtained with an IgM monoclonal antibody directed against proliferating cell nuclear antigen/cyclin (PCNA/ cyclin), a cell-cycle-associated nuclear protein for which reliable detection in formalin-fixed, paraffin-embedded tissue appears to require the use of a mu chain-specific secondary antibody.8 The absence of Leu Ml staining in 5 of 10 cases in the NSHD/MCHD group using the traditional detection method represents a higher rate of negativity than generally has been reported.2 Granulocyte staining in these nonreactive cases was weak to absent, indicating poor antigen preservation. Variations in tissue handling may have
1992
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Two sections from each case of Hodgkin's disease were deparaffinized and rehydrated. Endogenous peroxidase was blocked by exposure to 3% H 2 0 2 for 5 minutes, followed by a brief wash in phosphate-buffered saline. Sections were incubated with the anti-Leu M1 monoclonal antibody (Becton-Dickinson, Mountain View, CA) at a dilution of 1:20 in a humidified box for 30 minutes at room temperature. One section from each case was incubated with a biotinylated goat anti-mouse, mu chainspecific antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) used at a dilution of 1:360 in phosphate-buffered saline. For comparison, the other section was incubated with biotinylated goat anti-mouse immunoglobulin (Jackson Immunoresearch Laboratories) at a dilution of 1:360. Incubations with both secondary antibodies lasted 45 minutes. This was followed by incubation with peroxidase-conjugated streptavidin (Jackson Immunoresearch Laboratories), used at a dilution of 1:360 in phosphate-buffered saline, for 45 minutes at 4 °C. Diaminobenzidine was used as a chromogen followed by copper sulfate to darken the reaction product and absolute methanol to fix the precipitate. Each step was followed by a brief wash in phosphate-buffered saline. The sections were lightly counterstained with hematoxylin. The 10 cases of non-Hodgkin's lymphoma were stained using the anti-IgM detection method only.
the newer method compared with only 5 cases using the traditional technique (Table 1). Three cases remained unreactive with both methods. The newer method stained from 21 to 81% (mean, 49%) of recognizable neoplastic cells in the NSHD/MCHD group compared to from 1 to 36% (mean, 14%) with the older method. Among the five nodular L&H cases, no L&H cells were stained using the older method, whereas rare cells became weakly positive in one case using the mu chain-specific secondary antibody. None of the cases of non-Hodgkin's lymphoma showed Leu M1 reactivity with the newer method. In addition to an increased number of Leu Ml-reactive cells with the newer approach, these cells tended to stain with distinctly greater intensity, often rendering them clearly visible at low magnification. Staining often was localized to the cell membrane or to the paranuclear region in a pattern that is characteristic of staining for Leu M1. As an internal control, granulocyte staining was evaluated in each case. Using the traditional method, staining of granulocytes was present in the five cases of NSHD that contained staining Hodgkin's cells and was either absent or minimal in the five Leu Ml-negative cases. Granulocytes in all of the latter cases became weakly to moderately positive when mu-specific detection was used.
LEBRUN ET
AL.
137
Enhanced Staining for Leu Ml in Hodgkin's Disease
been at least partially responsible because all of these cases had been received in consultation from other institutions. Evidently, the enhanced sensitivity of mu-specinc detection can compensate for such reduced antigen accessibility in some cases. Despite the assertion by some authors that, because of its relative insensitivity and nonspecificity, Leu Ml is of little use in the diagnosis of Hodgkin's disease,2"4 others
TABLE 1. COMPARISON OF SECOND-STAGE ANTIBODIES IN NON-L & H HODGKIN'S DISEASE % Positive (Intensity*) Case
Diagnosis
Anti-Gamma
Anti-Mu
1 2 3 4 5 6 7 8 9 10
NSHD NSHD MCHD NSHD NSHD NSHD NSHD NSHD NSHD NSHD
19(1)
53 (III) 33 (II) 64 (III) 31 (III) 63(1) 21(1) 81 (III) 0 0 0
KD
36(11) 10(11) 4(1) 0 0 0 0 0
* I = barely perceptable; II = moderately intense; III = easily visible at low magnification. NSHD = nodular sclerosing Hodgkin's disease: MCND = mixed cellularily Hodgkin's disease.
advocate its use as part of a panel of monoclonal antibodies that includes antibodies intended to exclude other diagnostic possibilities, especially non-Hodgkin's lymphoma.5"7 Thus the usual phenotype of Reed-Sternberg cells and variants in non-L&H Hodgkin's disease includes reactivity for BerH2 (CD30) and absence of reactivity for leukocyte common and B-cell- and T-cell-associated antigens, with the exception of occasional reactivity for Bcell-associated antigen CD20. The technical improvement that we describe enhances the sensitivity and ease of interpretation of Leu M1 staining, with a greater proportion of Hodgkin's cases containing positive cells as well as more intense staining of a greater proportion of cells per case. The possibility that increased sensitivity may have been achieved at the expense of specificity is of concern. The neoplastic cells in the nodular L&H subtype of Hodgkin's disease are typically Leu M1 negative. Although one of the five cases of nodular L&H Hodgkin's disease in this study became Leu M1 positive using the newer detection method, the staining intensity was weak and involved only rare cells. Of the 10 cases of non-Hodgkin's lymphoma that were stained using the newer method, none showed reactivity for Leu M1. It appears, therefore, that the use of an anti-IgM secondary antibody does not significantly reduce the specificity of Leu Ml detection.
Vol. 97 • No. I
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
FIGS. 1A and B. Hodgkin's cells (arrow) and granulocytes stain more intensely and in greater number for Leu M1 using a mu chain-specific secondary antibody (B) compared with one directed against mouse light chains and gamma heavy chains (A) (X400).
138
HEMATOPATHOLOGY Article
In conclusion, the use of a secondary antibody that is specifically directed against the mu heavy chain of antiLeu Ml results in improved immunohistochemical detection of this widely used marker to a degree that should be helpful in the routine pathologic diagnosis of Hodgkin's disease. REFERENCES 1. Hsu S-M, Jaffe ES. Leu M1 and peanut agglutinin stain the neoplastic cells of Hodgkin's diesease. Am J Clin Pathol 1984;82:29-32. 2. Hall PA, D'Ardenne AJ. Value of CD 15 immunostaining in diagnosing Hodgkin's disease: A review of published literature. J Clin Pathol 1987;40:1298-1304. 3. Sheibani K, Battifora H, Burke JS, Rappaport H. Leu Ml antigen
4. 5. 6.
7.
8.
I in human neoplasms. An immunohistologic study of 400 cases. Am J Surg Pathol 1986;10(4):227-236. Wieczorek R, Burke JS, Knowles DM. Leu M1 antigen expression in T-cell neoplasia. Am J Pathol 1985;121:374-380. Chittal SM, Caveriviere P, Schwarting R, et al. Monoclonal antibodies in the diagnosis of Hodgkin's disease. The search for a rational panel. Am J Surg Pathol 1988; 12(1):9-21. Dorfman RF, Gatter KC, Pulford KAF, Mason DY. An evaluation of the utility of anti-granulocyte and anti-leukocyte monoclonal antibodies in the diagnosis of Hodgkin's disease. Am J Pathol 1986;123(3):508-519. Medeiros LJ, Weiss LM, Warnke RA, Dorfman RF. Utility of combining antigranulocyte with antileukocyte antibodies in differentiating Hodgkin's disease from non-Hodgkin's lymphoma. Cancer 1988;62:2475-2481. Kamel OW, LeBrun DP, Davis RE, Berry GJ, Warnke RA. Grow fraction estimation of malignant lymphomas in formalin-fixed paraffin-embedded tissue using anti-PCNA/Cyclin 19A2. Am J Pathol 1991; 138(6): 1471-1477.
«* Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
