Sources/Clones
Both monoclonal and polyclonal antibodies to immunoglobulins of the various types are available from a wide variety of sources. Affinity-isolated F (ab')fragments to Igk and Igl are also available.
Ig kappa
Accurate (EA2-38), American Research Products (HK3), Becton Dickinson (TB28-2), Biodesign/Pharmingen (polyclonal), Biogenesis (HK3, polyclonal), Biosource (LOHK3), Caltag Laboratories (3B10, HP6062), Calbiochem (HP6062, polyclonal), Central Lab Netherlands Red Cross/Accurate (MH19), Cymbus Bioscience (24K6), Dako (R10-21-F3, A8B5, polyclonal), Eurodiagnostica/Accurate (MH19-1), Harlan Seralab/Accurate (SL.2, KAP3.B3), Immunotech (G6.42), Pharmingen (polyclonal, G20-193), Research Diagnostics (6KA4G7), Sanbio/Monosan/Accurate (2B7) and Zymed (HP6053).
Ig lambda
Accurate (AG7.47), American Research Products (HL2), Becton Dickinson (1-155-2), Biogenesis (polyclonal), Biosource (LOHL2), Caltag Laboratories/Sigma (HP6054), Central Lab Netherlands Red Cross/Accurate (MH292), Cymbus Bioscience (24L6), Harlan Seralab/Accurate (Lam2.G4), Pharmingen (JDC12, polyclonal), Research Diagnostics (4LA2G9) and Zymed (HP6054).
IgA
Accurate (GA1, SB14, A1-18), Accurate/Sigma (GA112), American Research Products (14A3E3, HA8, 14A1B12), American Research Products/Research Diagnostics (14A2B5), Becton Dickinson (1-155-1), Biodesign (polyclonal), Biogenesis (polyclonal, 15D6, 2E2), Biosource (LOHA8), Calbiochem (HP6130, HP6141), Caltag Laboratories (SB14), Central Lab Netherlands Red Cross/Accurate (MH14-1), Coulter (NIF2), Cymbus Bioscience (M24A), Dako 6E2C1, polyclonal), E-Y Labs (polyclonal), Immunotech/Immunotech SA (NIF2), Pharmingen (polyclonal), Research Diagnostics (polyclonal), Sanbio/Monsan/Accurate (MH14-1), Sigma (A1-18) and Zymed (WAN741).
IgD
American Research Products (HD11), Becton Dickinson (TA4.1), Biogenesis (polyclonal), Biogenex (NI158, polyclonal), Biosource (LOHD11), Dako (IgD26, polyclonal), EY Labs (polyclonal), Harlan SeraLab/Accurate (1AD86), Immunotech (JA11), SeraLab Ltd (12.1), and Sigma (HJ9).
IgE
Accurate (GE1, AMD-E), Accurate/Sigma (GE1), Biodesign (polyclonal), Biogenesis (0257), Dako A-E-7.12, E1, polyclonal), EY Labs (polyclonal), Fitzgerald (M94175, M94176, M94177, M94178, M94179) Research Diagnostics (polyclonal).
IgG
Accurate (polyclonal, 4.22D10, A57H, SL13), Accurate/Sigma (GG4), American Research Products (HE10, HE17, 1619-18, HG20, HG22), Becton Dickinson (C3-124), Becton Dickinson/Biodesign (polyclonal), Biodesign (polyclonal), Biogenesis (polyclonal, 2D7), Biosource (LOHE17), Calbiochem (HP6058, HP6019), Caltag Laboratories (SB15, SB16), Central Lab Netherlands Red Cross/Accurate (MH25-1, MH164), Coulter (679.1MC7, U7.27, LODNP16, MOPC195), Cymbus Bioscience (MR36G), Dako (A57H, polyclonal), EY Labs (polyclonal, NL16, GB7B), Harlan SeraLab/Accurate (ISE503, C3-8-80, C27-15), Pharmingen (G7-18, G18-145, G18-21, G18-3, polyclonal), Research Diagnostics (10G2C11, 22G5G12, 20G5C7), Sanbio/Monosan/Accurate (MH25-1, BL-G4-1) and Sigma (SH21, SK44).
IgG F(ab)
Accurate/Sigma (SG16) and EY Labs (HP6014).
IgM
Accurate (AMD-u, SB17), Accurate/Sigma (MB11), American Research Products (HM9), American Research Products/Biogenesis (HM7), Becton Dickinson (145-8), Biogenesis (polyclonal), Biosource (LOHM9), Coulter (GC323), Coulter/Immunotech (AF6), Cymbus Bioscience (RVS-M), Dako (R1/69, polyclonal), EY Labs (polyclonal), Pharmingen (G20-127, polyclonal) and Research Diagnostics (MH15-1-3).
Fixation/Preparation
Immunostaining of cytoplasmic immunoglobulin can be performed in formalin-fixed paraffin-embedded sections, fresh frozen sections and cytologic preparations. Other fixatives and processing procedures such as AMEX (Sato et al, 1986) and freeze-drying (Stein et al, 1985) have been suggested to produce effective immunoglobulin staining.
Background
Surface membrane immunoglobulin (SIg) expression is the classic and specific marker of B lymphocytes and serves as the antigen recognition molecule for this lymphocyte population. Each of the heavy chain classes of Ig can be expressed on the B-cell membrane and more than one heavy chain class can be expressed on the same cell, the majority of peripheral B-cells expressing IgM with or without IgD, less than 10% expressing IgM or IgA.
IgM is the first heavy chain class to appear in B-cell ontogeny with the majority of immature B cells expressing IgM in high density. This decreases in density with maturation and increasing amounts of IgD appear on the cell membrane. The IgM and IgD molecules that coexist in the same membrane cap exist independently but share the same idiotype and have the same light chain. Following B-cell activation and differentiation, there is loss of IgM and IgD as the result of a productive isotype gene rearrangement switch. With the progression to antibody-forming plasma cells, different subpopulations of SIgM and/or SIgG-bearing memory B-cells may appear.
Clonality of a given B-cell population can be inferred from the uniformity of light chain class expression as individual B cells can express eitherk or l light chains but not both the ratio ofk to l bearing B cells being 2:1. A vast predominance ofk or l light chain-bearing B cells indicates monoclonality, generally implying a neoplastic proliferation, whereas a mixture of light chain-bearing cell types suggests polyclonality and a reactive or non-neoplastic proliferation of B cells. Direct immunofluorescent staining with heterologous antisera raised against whole or Fab fragments of human Ig molecules is the simplest method of identifying SIg. Class-specific antisera monospecific for individual heavy and light chain determinants (monovalent antisera) may be employed to determine the precise isotype of the SIg but these procedures require fresh cell preparations. Alternatively, immunoenzyme techniques can be used on cytocentrifuge preparations and imprints as well as frozen sections (Banks et al, 1983; Forbes & Leong, 1987). The latter procedures have suffered from the high level of background staining which can make interpretation difficult.
Ideally, the aim would be to be able to perform consistent staining of immunoglobulin in fixed, paraffin-embedded sections, allowing the advantage of retrospectivity as well as optimal cytomorphology. While many attempts have been made with special fixatives such as B5 and other mercury-based fixatives and the application of various enzymatic digestions, they have not met with much success. Coupled with the recent introduction of HIER, the use of 4 M urea as the retrieval solutions as produced consistent results, with the claim that the procedure allows the demonstration of not only cytoplasmic Ig but also surface Ig (Merz et al, 1993). mercury-based fixatives and the application of various enzymatic digestions, they have not met with much success. Coupled with the recent introduction of HIER, the use of 4 M urea as the retrieval solutions as produced consistent results, with the claim that the procedure allows the demonstration of not only cytoplasmic Ig but also surface Ig (Merz et al, 1993).
Applications
About 80% of non-Hodgkin's lymphoma cases in Western countries are of B cell lineage and the majority express monotypic SIg (Lukes et al, 1978; Tubbs et al, 1983). The examination of lymphoid proliferations for the presence and clonal nature of SIg expression is a common practice and forms the basis for traditional immunophenotypic analysis (Leong and Forbes, 1982). By convention, it is inferred that monoclonal B-cell proliferations are neoplastic. This analysis has traditionally been carried out by flow cytometry on cell suspensions, in cytospin preparations of disaggregated cells, or in frozen tissue sections. The SIg isotypes expressed by B-cell non-Hodgkin's lymphoma and lymphoid leukemias parallel those of normal B cells. The most common heavy chain class is IgM, with or without associated IgD, and IgG and IgA are expressed much less frequently. The ratio of Igk to Igk-bearing lymphomas is about 2:1.
Comments
When staining terminally differentiated B cells such as plasma cells, it is important to remember that, unlike SIg, which is detectable in viable cells in suspension or in minimally fixed frozen sections, the staining of CIg requires permeabilization of the cell membrane by the fixative to allow penetration of the anti-Ig reagents. Therefore, sections fixed by a gentle fixative such as acetone will not allow the demonstration of CIg and plasma cells may show false-negative staining. Alcohol and formalin are suitable fixatives for the demonstration of CIg in cell preparations and tissue sections respectively. We have found that fixation in 10% buffered formalin of freshly prepared or air-dried smears and cell preparations followed by HIER in 4 M urea produces excellent staining of CIg in lymphoid cells through a wide range of differentiation. Formalin-fixed, paraffin-embedded sections also show consistent staining for both CIg as well as SIg following HIER in 4 M urea and trypsin digestion.
References
•Banks PM, Caron BL, Morgan TW 1983. Use of imprints for monoclonal antibody studies: Suitability of air-dried preparations from lymphoid tissues with an immunohistochemical method. American Journal of Clinical Pathology 79:438-442.
•Forbes IJ, Leong AS-Y 1987. Essential oncology of the lymphocyte. London: Springer-Verlag, pp 184-188.
•Leong AS-Y, Forbes IJ 1982. Immunological and histochemical techniques in the study of the malignant lymphomas: A review. Pathology 14: 247-254.
•Lukes RJ, Parker JW, Taylor CR et al 1978. Immunologic approach to non-Hodgkin's lymphomas and related leukemias. Analysis of the results of multiparameter studies of 425 cases. Seminars in Hematology 15: 322-335.
•Merz H, Richers O, Schrimel S, et al 1993. Constant detection of surface and cytoplasmic immunoglobulin heavy and light chain expression in formalin-fixed and paraffin-embedded material. Journal of Pathology 170: 257-264.
•Sato Y, Mukai K, Watanabe S et al 1986. The AMEX method. A simplified technique of tissue processing and paraffin-embedding with improved preservation of antigens for immunostaining. American Journal of Pathology 125: 431-435.
•Stein H, Gatter K, Asbahr H, Mason DY 1985. Use of freeze-dried paraffin-embedded sections for immunohistologic staining with monoclonal antibodies. Laboratory Investigation 52: 676-683.
•Tubbs RR, Fishleder A, Weiss RA et al 1983. Immunohistologic cellular phenotypes of lymphoproliferative disorders. Comprehensive evaluation of 564 cases including 257 non-Hodgkin's lymphomas classified by the International Formulation. American Journal of Pathology 113: 207-221.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Both monoclonal and polyclonal antibodies to immunoglobulins of the various types are available from a wide variety of sources. Affinity-isolated F (ab')fragments to Igk and Igl are also available.
Ig kappa
Accurate (EA2-38), American Research Products (HK3), Becton Dickinson (TB28-2), Biodesign/Pharmingen (polyclonal), Biogenesis (HK3, polyclonal), Biosource (LOHK3), Caltag Laboratories (3B10, HP6062), Calbiochem (HP6062, polyclonal), Central Lab Netherlands Red Cross/Accurate (MH19), Cymbus Bioscience (24K6), Dako (R10-21-F3, A8B5, polyclonal), Eurodiagnostica/Accurate (MH19-1), Harlan Seralab/Accurate (SL.2, KAP3.B3), Immunotech (G6.42), Pharmingen (polyclonal, G20-193), Research Diagnostics (6KA4G7), Sanbio/Monosan/Accurate (2B7) and Zymed (HP6053).
Ig lambda
Accurate (AG7.47), American Research Products (HL2), Becton Dickinson (1-155-2), Biogenesis (polyclonal), Biosource (LOHL2), Caltag Laboratories/Sigma (HP6054), Central Lab Netherlands Red Cross/Accurate (MH292), Cymbus Bioscience (24L6), Harlan Seralab/Accurate (Lam2.G4), Pharmingen (JDC12, polyclonal), Research Diagnostics (4LA2G9) and Zymed (HP6054).
IgA
Accurate (GA1, SB14, A1-18), Accurate/Sigma (GA112), American Research Products (14A3E3, HA8, 14A1B12), American Research Products/Research Diagnostics (14A2B5), Becton Dickinson (1-155-1), Biodesign (polyclonal), Biogenesis (polyclonal, 15D6, 2E2), Biosource (LOHA8), Calbiochem (HP6130, HP6141), Caltag Laboratories (SB14), Central Lab Netherlands Red Cross/Accurate (MH14-1), Coulter (NIF2), Cymbus Bioscience (M24A), Dako 6E2C1, polyclonal), E-Y Labs (polyclonal), Immunotech/Immunotech SA (NIF2), Pharmingen (polyclonal), Research Diagnostics (polyclonal), Sanbio/Monsan/Accurate (MH14-1), Sigma (A1-18) and Zymed (WAN741).
IgD
American Research Products (HD11), Becton Dickinson (TA4.1), Biogenesis (polyclonal), Biogenex (NI158, polyclonal), Biosource (LOHD11), Dako (IgD26, polyclonal), EY Labs (polyclonal), Harlan SeraLab/Accurate (1AD86), Immunotech (JA11), SeraLab Ltd (12.1), and Sigma (HJ9).
IgE
Accurate (GE1, AMD-E), Accurate/Sigma (GE1), Biodesign (polyclonal), Biogenesis (0257), Dako A-E-7.12, E1, polyclonal), EY Labs (polyclonal), Fitzgerald (M94175, M94176, M94177, M94178, M94179) Research Diagnostics (polyclonal).
IgG
Accurate (polyclonal, 4.22D10, A57H, SL13), Accurate/Sigma (GG4), American Research Products (HE10, HE17, 1619-18, HG20, HG22), Becton Dickinson (C3-124), Becton Dickinson/Biodesign (polyclonal), Biodesign (polyclonal), Biogenesis (polyclonal, 2D7), Biosource (LOHE17), Calbiochem (HP6058, HP6019), Caltag Laboratories (SB15, SB16), Central Lab Netherlands Red Cross/Accurate (MH25-1, MH164), Coulter (679.1MC7, U7.27, LODNP16, MOPC195), Cymbus Bioscience (MR36G), Dako (A57H, polyclonal), EY Labs (polyclonal, NL16, GB7B), Harlan SeraLab/Accurate (ISE503, C3-8-80, C27-15), Pharmingen (G7-18, G18-145, G18-21, G18-3, polyclonal), Research Diagnostics (10G2C11, 22G5G12, 20G5C7), Sanbio/Monosan/Accurate (MH25-1, BL-G4-1) and Sigma (SH21, SK44).
IgG F(ab)
Accurate/Sigma (SG16) and EY Labs (HP6014).
IgM
Accurate (AMD-u, SB17), Accurate/Sigma (MB11), American Research Products (HM9), American Research Products/Biogenesis (HM7), Becton Dickinson (145-8), Biogenesis (polyclonal), Biosource (LOHM9), Coulter (GC323), Coulter/Immunotech (AF6), Cymbus Bioscience (RVS-M), Dako (R1/69, polyclonal), EY Labs (polyclonal), Pharmingen (G20-127, polyclonal) and Research Diagnostics (MH15-1-3).
Fixation/Preparation
Immunostaining of cytoplasmic immunoglobulin can be performed in formalin-fixed paraffin-embedded sections, fresh frozen sections and cytologic preparations. Other fixatives and processing procedures such as AMEX (Sato et al, 1986) and freeze-drying (Stein et al, 1985) have been suggested to produce effective immunoglobulin staining.
Background
Surface membrane immunoglobulin (SIg) expression is the classic and specific marker of B lymphocytes and serves as the antigen recognition molecule for this lymphocyte population. Each of the heavy chain classes of Ig can be expressed on the B-cell membrane and more than one heavy chain class can be expressed on the same cell, the majority of peripheral B-cells expressing IgM with or without IgD, less than 10% expressing IgM or IgA.
IgM is the first heavy chain class to appear in B-cell ontogeny with the majority of immature B cells expressing IgM in high density. This decreases in density with maturation and increasing amounts of IgD appear on the cell membrane. The IgM and IgD molecules that coexist in the same membrane cap exist independently but share the same idiotype and have the same light chain. Following B-cell activation and differentiation, there is loss of IgM and IgD as the result of a productive isotype gene rearrangement switch. With the progression to antibody-forming plasma cells, different subpopulations of SIgM and/or SIgG-bearing memory B-cells may appear.
Clonality of a given B-cell population can be inferred from the uniformity of light chain class expression as individual B cells can express eitherk or l light chains but not both the ratio ofk to l bearing B cells being 2:1. A vast predominance ofk or l light chain-bearing B cells indicates monoclonality, generally implying a neoplastic proliferation, whereas a mixture of light chain-bearing cell types suggests polyclonality and a reactive or non-neoplastic proliferation of B cells. Direct immunofluorescent staining with heterologous antisera raised against whole or Fab fragments of human Ig molecules is the simplest method of identifying SIg. Class-specific antisera monospecific for individual heavy and light chain determinants (monovalent antisera) may be employed to determine the precise isotype of the SIg but these procedures require fresh cell preparations. Alternatively, immunoenzyme techniques can be used on cytocentrifuge preparations and imprints as well as frozen sections (Banks et al, 1983; Forbes & Leong, 1987). The latter procedures have suffered from the high level of background staining which can make interpretation difficult.
Ideally, the aim would be to be able to perform consistent staining of immunoglobulin in fixed, paraffin-embedded sections, allowing the advantage of retrospectivity as well as optimal cytomorphology. While many attempts have been made with special fixatives such as B5 and other mercury-based fixatives and the application of various enzymatic digestions, they have not met with much success. Coupled with the recent introduction of HIER, the use of 4 M urea as the retrieval solutions as produced consistent results, with the claim that the procedure allows the demonstration of not only cytoplasmic Ig but also surface Ig (Merz et al, 1993). mercury-based fixatives and the application of various enzymatic digestions, they have not met with much success. Coupled with the recent introduction of HIER, the use of 4 M urea as the retrieval solutions as produced consistent results, with the claim that the procedure allows the demonstration of not only cytoplasmic Ig but also surface Ig (Merz et al, 1993).
Applications
About 80% of non-Hodgkin's lymphoma cases in Western countries are of B cell lineage and the majority express monotypic SIg (Lukes et al, 1978; Tubbs et al, 1983). The examination of lymphoid proliferations for the presence and clonal nature of SIg expression is a common practice and forms the basis for traditional immunophenotypic analysis (Leong and Forbes, 1982). By convention, it is inferred that monoclonal B-cell proliferations are neoplastic. This analysis has traditionally been carried out by flow cytometry on cell suspensions, in cytospin preparations of disaggregated cells, or in frozen tissue sections. The SIg isotypes expressed by B-cell non-Hodgkin's lymphoma and lymphoid leukemias parallel those of normal B cells. The most common heavy chain class is IgM, with or without associated IgD, and IgG and IgA are expressed much less frequently. The ratio of Igk to Igk-bearing lymphomas is about 2:1.
Comments
When staining terminally differentiated B cells such as plasma cells, it is important to remember that, unlike SIg, which is detectable in viable cells in suspension or in minimally fixed frozen sections, the staining of CIg requires permeabilization of the cell membrane by the fixative to allow penetration of the anti-Ig reagents. Therefore, sections fixed by a gentle fixative such as acetone will not allow the demonstration of CIg and plasma cells may show false-negative staining. Alcohol and formalin are suitable fixatives for the demonstration of CIg in cell preparations and tissue sections respectively. We have found that fixation in 10% buffered formalin of freshly prepared or air-dried smears and cell preparations followed by HIER in 4 M urea produces excellent staining of CIg in lymphoid cells through a wide range of differentiation. Formalin-fixed, paraffin-embedded sections also show consistent staining for both CIg as well as SIg following HIER in 4 M urea and trypsin digestion.
References
•Banks PM, Caron BL, Morgan TW 1983. Use of imprints for monoclonal antibody studies: Suitability of air-dried preparations from lymphoid tissues with an immunohistochemical method. American Journal of Clinical Pathology 79:438-442.
•Forbes IJ, Leong AS-Y 1987. Essential oncology of the lymphocyte. London: Springer-Verlag, pp 184-188.
•Leong AS-Y, Forbes IJ 1982. Immunological and histochemical techniques in the study of the malignant lymphomas: A review. Pathology 14: 247-254.
•Lukes RJ, Parker JW, Taylor CR et al 1978. Immunologic approach to non-Hodgkin's lymphomas and related leukemias. Analysis of the results of multiparameter studies of 425 cases. Seminars in Hematology 15: 322-335.
•Merz H, Richers O, Schrimel S, et al 1993. Constant detection of surface and cytoplasmic immunoglobulin heavy and light chain expression in formalin-fixed and paraffin-embedded material. Journal of Pathology 170: 257-264.
•Sato Y, Mukai K, Watanabe S et al 1986. The AMEX method. A simplified technique of tissue processing and paraffin-embedding with improved preservation of antigens for immunostaining. American Journal of Pathology 125: 431-435.
•Stein H, Gatter K, Asbahr H, Mason DY 1985. Use of freeze-dried paraffin-embedded sections for immunohistologic staining with monoclonal antibodies. Laboratory Investigation 52: 676-683.
•Tubbs RR, Fishleder A, Weiss RA et al 1983. Immunohistologic cellular phenotypes of lymphoproliferative disorders. Comprehensive evaluation of 564 cases including 257 non-Hodgkin's lymphomas classified by the International Formulation. American Journal of Pathology 113: 207-221.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
