Notes

1. When using the StrABC/HRP technique for each primary antibody, the pretreat-ment protocol and dilution required need to be established for each user, so that the best possible demonstration of antigen is achieved with minimal nonspecific binding, which is assessed using a negative control and by examination of internal cellular controls within the positive control and the test. Positive controls should be used for each antibody.

2. On paraffin sections, CD10 and Bcl-6 work optimally using the standard StrABC technique with the DAKO ChemMate Kit to detect the antigen; however, tyramide signal amplification can be used to intensify the staining in resin-embedded samples or if weak staining is observed by routine methods.

3. With the TSA technique, the Dako CD23 clone (MHM6) requires tyramide signal amplification to optimize staining; however, the clone available from Novocastra and Serotec (1B12) produce optimal staining using the standard StrABC technique.

4. The reliability of immunocytochemistry methods is critically dependent on the initial handing of the tissue specimen. It is important that lymph node biopsies are sent to the laboratory unfixed and as quickly as possible.

5. Placing a whole specimen in formalin results in major artifacts, with wide variation in fixation between the center and edge of the node. As a result, the morphology and intensity of tinctorial and immunocytochemical staining are highly inconsistent across the tissue section. This is particularly apparent with many of the antibodies used for demonstrating a germinal center phenotype, and in extreme cases the diagnosis may be compromised if the specimen is poorly fixed. To avoid this, tissue should be thinly sliced into pieces of no more than 2- to 3-mm thick.

6. There has been a great deal written on methods of fixation of lymphoid tissue for the optimization of immunocytochemical staining. When modern immunocy-tochemistry is used, 10% formalin or neutral buffered formalin is adequate for all routine applications, and "special" fixatives are no longer required.

7. The advantage of pressure-cooking is that multiple 25-slide racks can be pressure-cooked at the same time and, therefore, it is less time-consuming than microwaving. Pressure-cooking also can result in superior demonstration of certain antigens. Pressure-cooking can, however, lead to poor morphology and inaccurate staining in suboptimally fixed specimens, particularly in very small biopsies in which fixation times are reduced. Microwaving is recommended in these situations.

8. If the Dako ChemMate kit is to be used, prepare ChemMate DAB solution by adding 20 |L of DAB concentrate (reagent C) per 1 mL of substrate (reagent D), allowing 200 |L of DAB solution per slide. Alternatively, use DAB working solution as described in the immunocytochemistry materials section.

9. When performing flow cytometry lysis also may be performed at room temperature, but this may be suboptimal if samples are older than 24 h. Complete lysis may take longer than 5 min, but leucocyte degradation begins to occur after 20 min.

10. For Becton Dickinson flow cytometers, initial calibration is best performed using FACSComp with CALIBrite beads, and then further calibrated using relevant reagents from the panel on cells. In particular, for CD19 PE/Cy5 reagents, an aliquot of cells should be stained with this reagent alone, and compensation optimized accordingly.

11. This number of cells will be adequate for immunophenotyping B-lymphocyte populations that represent more than 1% of leucocytes. For smaller populations, more accurate results may be obtained if 50,000 events are acquired. However, more specific antibody combinations may be required to detect minimal disease.

12. T-cell contamination: these cells are rarely a problem, and frequent contamination of a B-cell region by T-cells is usually indicative of a poor CD19 reagent. However, the expression of CD19 may be weak in follicular lymphoma/DLBCL, and in some very rare cases it may be necessary to repeat the analysis using a different B-cell gating reagent such as CD20. T-cell contamination is identifiable as a population of cells that bind both CD3 FITC and CD3 PE in equal proportions. In tissue biopsies, T-cells are often present in the B-cell gate at low level because of cell:cell adhesion of B-cells to T-cells that occurs in follicles.: these cannot be excluded by this gating strategy, but it is extremely rare that these represent more than 1% of events in the B-cell gate.

13. Apoptotic cell contamination: these are problematic because they show variable levels of nonspecific binding. Again, fluorescein isothiocyanate conjugates are most likely to bind nonspecifically. However, antibodies conjugated to other fluo-rochromes may also show very high levels of nonspecific binding to apoptotic cells, and there are also differences between identical antibodies conjugated by different companies. It is therefore necessary to exclude all apoptotic cells, including apoptotic B-cells, because their inclusion may lead to a false-positive result for certain antigens. Apoptotic cells frequently are present in bone marrow samples, particularly those in which normal B-cells also are present. In some tissue biopsies, virtually all of the B-cells may be apoptotic or necrotic, but this can be minimized by gentle disaggregation techniques and by analysis immediately after disaggregation. For definitive exclusion of apoptotic cells, a viability dye such as propidium iodide or preferably 7-AAD may be included, but this will limit the number of channels available to analyze antigen expression. In the panel used here, binding of CD3 FITC but not CD3 PE is indicative of contamination with apoptotic cells. In most case, they may be completely removed by adjusting the scatter region to gradually remove cells with the lowest forward scatter until there is no evidence of aberrant binding.

14. Monocyte contamination: these cells have high levels of nonspecific binding and as such may appear to have weak CD19 binding. The are readily separated from most B-LPDs by their high side scatter but often show overlapping characteristics with DLBCL cells. They also are present in bone marrow/peripheral blood samples or tissue samples with heavy blood contamination. Immunoglobulin is present on the surface of monocytes ligated to Fc receptors. Both normal and neoplastic B-cells express only one light chain or none at all, whereas mono-cytes will have both kappa and lambda bound to their cell surface. As such, assessment of kappa vs lambda binding will reveal a diagonal line if monocytes are present, with B-cells to the upper left, lower right, or lower left of the plot. In addition, monocytes tend to bind FITC antibodies nonspecifically to a greater extent than PE antibodies. Therefore, a population of cells with moderate side scatter, weak binding of several FITC reagents, and equivalent binding of both kappa and lambda antibodies should be excluded from analysis. This can be achieved by adjustment of gating regions allowing exclusion of monocytes without removal of any B-cells in the vast majority of cases.

References

1. Faili, A., Aoufouchi, S., Gueranger, Q., Zober, C., Leon, A., Bertocci, B.. et al. (2002) AID-dependent somatic hypermutation occurs as a DNA single-strand event in the BL2 cell line. Nat. Immunol. 3, 815-821

2. Harris, R. S., Sale, J. E., Petersen-Mahrt, S. K., and Neuberger, M. S. (2002) AID is essential for immunoglobulin V gene conversion in a cultured B-cell line. Curr. Biol. 12, 435-438.

3. Dunn-Walters, D., Thiede, C., Alpen, B., and Spencer, J. (2001) Somatic hypermutation and B-cell lymphoma. Phil. Trans. R. Soc. Lond. B. Biol. Sci. 356, 73-82.

4. Klein, U., Goossens, T., Fischer, M., Kanzler, H., Braeuninger, A., Rajewsky, K., et al. (1998) Somatic hypermutation in normal and transformed human B-cells. Immunol. Rev. 162, 261-280.

5. Nadel, B., Marculescu, R., Le, T., Rudnicki, M., Bocskor, S., and Jager, U. (2001) Novel insights into the mechanism of t(14;18)(q32;q21) translocation in follicular lymphoma. Leuk. Lymphoma 42, 1181-1194.

6. Iida, S., Rao, P. H., Butler, M., Corradini, P., Boccadoro, M., Klein, B., et al. (1997) Deregulation of MUM1/IRF4 by chromosomal translocation in multiple myeloma. Nat. Genet. 17, 226-230.

7. Bishop, P. C., Rao, V. K., and Wilson, W. H. (2000)Burkitt's lymphoma: molecular pathogenesis and treatment. Cancer Invest. 18, 574-83.

8. Hecht, J. L., and Aster, J. C. (2000) Molecular biology of Burkitt's lymphoma. J. Clin. Oncol. 18, 3707-3721.

9. Fenton, J. A., Pratt, G., Rawstron, A. C., and Morgan, G. J. (2002) Isotype class switching and the pathogenesis of multiple myeloma. Hematol. Oncol. 20, 75-85.

10. Pasqualucci, L., Migliazza, A., Fracchiolla, N., William, C., Neri, A., Baldini, L., et al. (1998) BCL-6 mutations in normal germinal center B-cells: evidence of somatic hypermutation acting outside Ig loci. Proc. Natl. Acad. Sci. USA 95, 11816-11821.

11. Pasqualucci, L., Neumeister, P., Goossens, T., Nanjangud, G., Chaganti, R. S., Kuppers, R., et al. (2001) Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature 412, 341-346.

12. Kuppers, R., Hansmann, M. L., and Rajewsky, K. Clonality and germinal center B-cell derivation of Hodgkin/Reed- Sternberg cells in Hodgkin's disease. Ann. Oncol. 9(Suppl 5), S17-S20.

13. Kuppers, R., Schwering, I., Brauninger, A., Rajewsky, K., AND Hansmann, M. L. (2002) Biology of Hodgkin's lymphoma. Ann. Oncol. 13(Suppl 1), 11-18.

14. Dunphy, C. H., Polski, J. M., Lance, Evans H., and Gardner, L. J. (2001) Paraffin immunoreactivity of CD10, CDw75, and Bcl-6 in follicle center cell lymphoma. Leuk. Lymphoma 41, 585-592.

15. Abou-Elella, A., Shafer, M. T., Wan, X. Y., Velanker, M., Weisenburger, D. D., Nathwani, B. N., et al. (2000) Lymphomas with follicular and monocytoid B-cell components. Evidence for a common clonal origin from follicle center cells. Am. J. Clin. Pathol. 114, 516-522.

16. Ott, G., Katzenberger, T., Lohr, A., Kindelberger, S., Rudiger, T., Wilhelm, M., et al. (2002) Cytomorphologic, immunohistochemical, and cytogenetic profiles of follicular lymphoma: 2 types of follicular lymphoma grade 3. Blood 99, 3806-3812.

17. Alizadeh, A. A., Eisen, M. B., Davis, R. E., Ma, C., Lossos, I. S., Rosenwald, A., et al. (2000) Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503-511.

18. Barrans, S. L., Carter, I., Owen, R. G., Davies, F. E., Patmore, R. D., Haynes, A. P., et al. (2002) Germinal center phenotype and bcl-2 expression combined with the International Prognostic Index improves patient risk stratification in diffuse large B-cell lymphoma. Blood 99, 1136-1143.

19. King, B. E., Chen, C., Locker, J., Kant, J., Okuyama, K., Falini, B., et al. (2000) Immunophenotypic and genotypic markers of follicular center cell neoplasia in diffuse large B-cell lymphomas. Mod. Pathol. 13, 1219-1231.

20. Kramer, M. H., Hermans, J., Wijburg, E., Philippo, K., Geelen, E., van Krieken, J. H., et al. (1998) Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma. Blood 92, 3152-3162.

21. Barrans, S. L., O'Connor, S. J., Evans, P. A., Davies, F. E., Owen, R. G., Haynes, A. P., et al. (2002) Rearrangement of the BCL6 locus at 3q27 is an independent poor prognostic factor in nodal diffuse large B-cell lymphoma. Br. J. Haematol. 117, 322-332.

22. Chang, C. C., Liu, Y. C., Cleveland, R. P., and Perkins, S. L. (2000) Expression of c-Myc and p53 correlates with clinical outcome in diffuse large B-cell lymphomas. Am. J. Clin. Pathol. 113, 512-518.

23. Leroy, K., Haioun, C., Lepage, E., Le Metayer, N., Berger, F., Labouyrie, E., et al. (2002) p53 gene mutations are associated with poor survival in low and low- intermediate risk diffuse large B-cell lymphomas. Ann. Oncol. 13, 1108-1115.

24. Braziel, R. M., Arber, D. A., Slovak, M. L., Gulley, M. L., Spier, C., Kjeldsberg, C., et al. (2001) The Burkitt-like lymphomas: a Southwest Oncology Group study delineating phenotypic, genotypic, and clinical features. Blood 97, 3713-3720.

25. Nakamura, N., Nakamine, H., Tamaru, J., Nakamura, S., Yoshino, T., Ohshima, K., et al. (2002) The distinction between Burkitt lymphoma and diffuse large B-cell lymphoma with c-myc rearrangement. Mod. Pathol. 15, 771-776.

26. Macpherson, N., Lesack, D., Klasa, R., Horsman, D., Connors, J. M., Barnett, M. et al. (1999) Small noncleaved, non-Burkitt's (Burkit-Like) lymphoma: cytogenetics predict outcome and reflect clinical presentation. J. Clin. Oncol. 17, 1558-1567.

27. Nagai, J., Kigasawa, H., Koga, N., Katoh, A., Nishihira, H., and Nagao, T. (1998) Clinical significance of detecting p53 protein in Burkitt lymphoma and B-cell acute lymphoblastic leukemia using immunocytochemistry. Leuk. Lymphoma 28, 591-597.

28. Lindstrom, M., and Wiman, K. (2002) Role of genetic and epigenetic changes in Burkitt lymphoma. Semin. Cancer Biol. 12, 381.

29. Sullivan, M. P., and Ramirez, I. (1985) Curability of Burkitt's lymphoma with high-dose cyclophosphamide-high- dose methotrexate therapy and intrathecal chemoprophylaxis. J. Clin. Oncol. 3, 627-636.

30. Thomas, D. A., Cortes, J., O'Brien, S., Pierce, S., Faderl, S., Albitar, M., et al. (1999) Hyper-CVAD program in Burkitt's-type adult acute lymphoblastic leukemia. J. Clin. Oncol. 17, 2461-2470.

31. Attygalle, A., Al Jehani, R., Diss, T. C., Munson, P., Liu, H., Du, M. Q., et al. (2002) Neoplastic T-cells in angioimmunoblastic T-cell lymphoma express CD10. Blood 99, 627-633.

32. Ye, B. H., Cattoretti, G., Shen, Q., Zhang, J., Hawe, N., de Waard, R., et al. (1997) The BCL-6 proto-oncogene controls germinal-center formation and Th2-type inflammation. Nat. Genet. 16, 161-170.

33. Ree, H. J., Kadin, M. E., Kikuchi, M., Ko, Y. H., Suzumiya, J., and Go, J. H. (1999) Bcl-6 expression in reactive follicular hyperplasia, follicular lymphoma, and angioimmunoblastic T-cell lymphoma with hyperplastic germinal centers: heterogeneity of intrafollicular T-cells and their altered distribution in the patho-genesis of angioimmunoblastic T-cell lymphoma. Hum. Pathol. 30, 403-411.

34. Chaganti, S. R., Rao, P. H., Chen, W., Dyomin, V., Jhanwar, S. C., Parsa, N. Z., et al. (1998) Deregulation of BCL6 in non-Hodgkin lymphoma by insertion of IGH sequences in complex translocations involving band 3q27. Genes Chromosomes Cancer 23, 328-336.

35. Akasaka, T., Ueda, C., Kurata, M., Akasaka, H., Yamabe, H., Uchiyama, T., and Ohno, H. (2000) Nonimmunoglobulin (non-Ig)/BCL6 gene fusion in diffuse large B-cell lymphoma results in worse prognosis than Ig/BCL6. Blood 96, 2907-2909.

36. Artiga, M. J., Saez, A. I., Romero, C., Sanchez-Beato, M., Mateo, M. S., Navas, C., et al. (2002) A short mutational hot spot in the first intron of BCL-6 is associated with increased BCL-6 expression and with longer overall survival in large B-cell lymphomas. Am. J. Pathol. 160, 1371-1380.

37. Vitolo, U., Botto, B., Capello, D., Vivenza, D., Zagonel, V., Gloghini, A., et al. (2002) Point mutations of the BCL-6 gene: clinical and prognostic correlation in B-diffuse large cell lymphoma. Leukemia 16, 268-275.

38. Huang, J. Z., Sanger, W. G., Greiner, T. C., Staudt, L. M., Weisenburger, D. D., Pickering, D. L., et al. (2002) The t(14;18) defines a unique subset of diffuse large B-cell lymphoma with a germinal center B-cell gene expression profile. Blood 99, 2285-2290.

39. Pallesen, G. (1987) The Distribution of CD23 in normal human tissues and in malignant lymphomas, in Leucocyte Typing III. White Cell Differentiation Antigens (McMichael A. J., ed), Oxford University Press, Oxford, pp. 383-386.

40. Aoyagi, K., Kohfuji, K., Yano, S., Murakami, N., Miyagi, M., Takeda, J., et al. (2002) The expression of proliferating cell nuclear antigen, p53, p21, and apoptosis in primary gastric lymphoma. Surgery 132, 20-26.

41. Eischen, C. M., Weber, J. D., Roussel, M. F., Sherr, C. J., and Cleveland, J. L. (1999) Disruption of the ARF-Mdm2-p53 tumor suppressor pathway in Myc-in-duced lymphomagenesis. Genes Dev. 13, 2658-2669.

42. Adams, J. (1992) Biotin amplification of biotin horseradish peroxidase signals in histochemical stains. J. Histochem. Cytochem. 40, 1457-1463.

43. Rawstron, A. C., Kennedy, B., Evans, P. A., Davies, F. E., Richards, S. J., Haynes, A. P., et al. (2001) Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy. Blood 98, 29-35.

44. Rawstron, A. C., Owen, R. G., Davies, F. E., Johnson, R. J., Jones, R. A., Richards, S. J., et al. (1997) Circulating plasma cells in multiple myeloma: characterization and correlation with disease stage. Br. J. Haematol. 97, 46-55.

45. Pasqualucci, L., Neumeister, P., Goossens, T., Nanjangud, G., Chaganti, R. S., Kuppers, R., et al. (2001) Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature 412, 341-346.

46. Angelin-Duclos, C., Cattoretti, G., Lin, K. I., and Calame, K. (2000) Commitment of B lymphocytes to a plasma cell fate is associated with Blimp-1 expression in vivo. J. Immunol. 165, 5462-5471.

47. Shaffer, A. L., Lin, K. I., Kuo, T. C., Yu, X., Hurt, E. M., Rosenwald, A., et al. (2002) Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B-cell gene expression program. Immunity 17, 51-62.

48. Tsuboi, K., Iida, S., Inagaki, H., Kato, M., Hayami, Y., Hanamura, I., et al. (2000) MUM1/IRF4 expression as a frequent event in mature lymphoid malignancies. Leukemia 14, 449-456.

49. Cerutti, A., Schaffer, A., Shah, S., Zan, H., Liou, H. C., Goodwin, R. G., et al. (1998) CD30 is a CD40-inducible molecule that negatively regulates CD40-mediated immunoglobulin class switching in non-antigen-selected human B-cells. Immunity 9, 247-256.

50. Cerutti, A., Schaffer, A., Goodwin, R. G., Shah, S., Zan, H., Ely, S., et al. (2000) Engagement of CD153 (CD30 ligand) by CD30+ T-cells inhibits class switch DNA recombination and antibody production in human IgD+ IgM+ B-cells. J. Immunol. 165, 786-794.

51. Aizawa, S., Nakano, H., Ishida, T., Horie, R., Nagai, M., Ito, K., et al. (1997) Tumor necrosis factor receptor-associated factor (TRAF) 5 and TRAF2 are involved in CD30-mediated NFkappaB activation. J. Biol. Chem. 272, 2042-2045.

52. Clodi, K., Asgary, Z., Zhao, S., Kliche, K. O., Cabanillas, F., Andreeff, M., et al. Coexpression of CD40 and CD40 ligand in B-cell lymphoma cells. Br. J. Haematol. 103, 270-275.

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