Sources/Clones
Dako (BM-1), Oncor (Apop Tag), Monosan (Annexin V - polyclonal) and Pharmingen (APO-BRDU, Annexin V-FITC).
Fixation/Preparation
Various methods of detection of apoptotic bodies are available. All methods can be used on formalin-fixed, paraffin-embedded tissue sections. Some require proteolytic digestion. Acetone-fixed cryostat sections and fixed-cell smears may also be used.
Background
Cell death may occur by necrosis or apoptosis. Necrosis results from direct physical or chemical damage to the plasma membrane or disturbances in the osmotic balance of a cell (Wyllie et al, 1980). With the entrance of extracellular fluid into the cell, resultant cell swelling and lysis precedes a subsequent inflammatory response. Furthermore, necrosis affects groups of cells, with consequent disruption of normal tissue architecture.
In contrast to necrosis, apoptotic cell death is a highly regulated physiologic process. The balance between apoptosis and cell proliferation results in the maintenance of cell homeostasis (Kerr et al, 1972). Apoptotic bodies are rapidly engulfed by neighboring cells or macrophages, without an inflammatory response being elicited. The nuclear structure alteration in apoptotic cells is induced by endonuclease DNA cleavage that results in the generation of large 50-300 kb fragments. This produces the characteristic DNA "ladders" of apoptosis as viewed on agarose gel electrophoresis (Oberhammer et al, 1993).
Recently, reliable methods have been developed that enable the rapid assessment of apoptosis on sections prepared from paraffin-embedded material, e.g. the TUNEL method for TdT-mediated dUTP-biotin nick end labeling (Sarkiss et al, 1996). The APO-BRDU kit utilizes the same principle. The enzyme TdT is used to catalyze a template-independent addition of bromolated deoxyribonucleotide triphosphates (Br-dUTP) to the 3' -hydroxyl ends of the numerous fragments of double- and single-stranded DNA present in apoptotic cells. This allows the labeling of the very high concentrations of 3'-OH ends that are localized in apoptotic bodies. Br-dUTP is claimed to be more readily incorporated into the genome of apoptotic cells than are deoxyribonucleotide triphosphates complexed to larger ligands like fluorescein, biotin or digoxigenin (Nagata & Golstein, 1995). Although rather specific for cells undergoing apoptosis, these techniques may also label cells undergoing necrosis. However, this is seldom a problem since the distinction between focal apoptotic events and necrosis is fairly clear. The histologic features of apoptosis include cell shrinkage and loss of junctional contact, resulting in a "halo" around the cell. The nucleus shows condensation and margination of the chromatin. This is followed by the fragmentation or "pinching off" of pieces of nuclear material, which are surrounded by cytoplasm with intact cytoplasmic organelles as shown at ultrastructural level, These apoptotic fragments of pyknotic nuclear material and cytoplasm are phagocytosed by adjacent cells or macrophages. Apoptotic cells have been called by various names in different tissues and include "Councilman bodies", "Civatte bodies", ''necrobiotic cells" and "nuclear dust". The BM-1 antibody is directed to the Lewisy antigen which has been identified phenotypically as a Lamant L, Meggetto F, Saati TA et al 1996. High incidence of the t(2;5) (p23;q35) translocation in anaplastic large cell lymphoma and its lack of detection in Hodgkin's disease. Comparison of cytogenetic analysis, reverse transcriptase-polymerase chain reaction, and Panaplastic large cell lymphoma and its lack of detection in Hodgkin's disease. Comparison of cytogenetic analysis, reverse transcriptase-polymerase chain reaction, and P marker of specific types of cells, and possibly specific stages of differentiation. Lewis is totally absent at the morula stage, but is highly expressed on the blastocyst surface and has been shown to play a role in the implantation process (Fenderson et al, 1991). Recently, Lewisy has been identified as a characteristic of cells undergoing apoptosis (Hiraishi et al, 1993). In Lewisy-positive areas of tissue sections, typical apoptotic morphological changes and DNA fragmentation were frequently observed in certain loci, although not all Lewisy positive cells showed such signs of apoptosis. Although the BM-1 antibody against the Lewisy antigen is reputed to detect apoptotic cells, further studies to test its efficacy, including a comparative analysis with the insitu end-labeling techniques, are awaited.
Another method of detection of apoptotic bodies is the use of Annexin V, which is a 35-36 kD Ca2+ -dependent phospholipid-binding protein that has a high affinity for the membrane phospholipid phosphatidylserine (PS). In apoptotic cells, PS is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment and allowing its binding to Annexin V. Binding to a signal system such as fluorescein isothiocyanate allows the easy identification of apoptotic cells (in frozen sections and cell preparations). Annexin V is thought to identify cells at an earlier stage of apoptosis than assays based on DNA fragmentation because externalization of PS occurs earlier than the nuclear changes associated with apoptosis (Raynal & Pollard, 1994).
Applications
BM-1 antibody may be applied to neoplasms in general to assess the apoptotic index, e.g., endometrial adenocarcinoma (Kuwashima et al, 1995). Recently, apoptosis has been considered to be a key event in oncogenesis, e.g. apoptosis has been reported to be promoted by tumorsuppressor gene p53 and inhibited by oncogene bcl-2 (Arends & Wyllie, 1991). Although apparent cell loss by apoptosis occurs in carcinomatous tissue (Hiraishi et al, 1993), the physiological significance is unclear (Umansky, 1982). BM-1 positivity has been found to be as high as 25-35% in T cells of lymph nodes of patients with AIDS-related complex (ARC), in contrast to healthy controls who had less than 5% (Adachi et al, 1988).
Comments
Strong BM-1 immunoreactivity is observed in the apical surface of tubular urothelium, basal cells (glandular foveoli) of gastric and esophageal mucosa and these tissues may be employed as controls.
The optimal method for the identification of apoptotic cells depends on the experimental system and the mode of induction of apoptosis. The degree of DNA degradation can vary according to the cell type, the nature of the inducing agent and the stage of apoptosis. The application of multiple methods, each based on a different feature of the apoptotic process, may provide more information about the cell population than any one method would give alone.
References
•Adachi M, Hayami M, Kashlwagi N et al 1988. Expression of ley antigen in human immunodeficiency virus-infected human T cell lines and in peripheral lymphocytes of patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC). Journal of Experimental Medicine 167:233-331.
•Arends MJ, Wyllie AH 1991. Apoptosis: mechanism and roles in pathology. International Reviews in Experimental Pathology 32:223-254.
•Fenderson BA, Killma N, Stroud MR et al 1991. Specific interaction between Ley and H as a possible basis for trophectoderm-endometrium recognition during implantation. Glycoconjugate Journal, 8:179 (abstract 8.5).
•Hiraishi K, Suzuki K, Hakomori S, Adachi M 1993. Ley antigen expression is correlated with apoptosis (programmed cell death). Glycobiology 3:381-390.
•Kerr JFR, Wyllie AH, Currie AR 1972. Apoptosis: a basic biological phenomenon with wider ranging implications in tissue kinetics. British Journal of Cancer 26: 239-257. implications in tissue kinetics. British Journal of Cancer 26: 239-257.
•Kuwashima Y, Uehara T, Kishi K, et al 1995. Proliferative and apoptotic status in endometrial adenocarcinoma. International Journal of Gynecological Pathology 14:45-49.
•Nagata S, Golstein P 1995. The Fas death factor. Science 267: 1445-1449.
•Oberhammer F, Wilson JW, Dive C et al 1993. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO Journal 12:679-684.
•Raynal P, Pollard HB 1994. Annexins. The problem of assessing the biological role for a gene family of multifunctional calcium and phospholipid-binding proteins.
•Journal of Biological Chemistry 265:4923-4928.
•Sarkiss M, Hsu B, El-Naggar AK, McDonnell TJ 1996. The clinical relevance and assessment of apoptotic cell death. Advances in Anatomical Pathology 3:205-211.
•Umansky SR 1982. The genetic program of cell death: hypothesis and some applications: transformation, carcinogenesis, and aging. Journal of Theoretical Biology 97:591-602.
•Wyllie AH, Kerr JFR, Currie AR 1980. Cell death: the significance of apoptosis. International Reviews in Cytology 68:251-306.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Dako (BM-1), Oncor (Apop Tag), Monosan (Annexin V - polyclonal) and Pharmingen (APO-BRDU, Annexin V-FITC).
Fixation/Preparation
Various methods of detection of apoptotic bodies are available. All methods can be used on formalin-fixed, paraffin-embedded tissue sections. Some require proteolytic digestion. Acetone-fixed cryostat sections and fixed-cell smears may also be used.
Background
Cell death may occur by necrosis or apoptosis. Necrosis results from direct physical or chemical damage to the plasma membrane or disturbances in the osmotic balance of a cell (Wyllie et al, 1980). With the entrance of extracellular fluid into the cell, resultant cell swelling and lysis precedes a subsequent inflammatory response. Furthermore, necrosis affects groups of cells, with consequent disruption of normal tissue architecture.
In contrast to necrosis, apoptotic cell death is a highly regulated physiologic process. The balance between apoptosis and cell proliferation results in the maintenance of cell homeostasis (Kerr et al, 1972). Apoptotic bodies are rapidly engulfed by neighboring cells or macrophages, without an inflammatory response being elicited. The nuclear structure alteration in apoptotic cells is induced by endonuclease DNA cleavage that results in the generation of large 50-300 kb fragments. This produces the characteristic DNA "ladders" of apoptosis as viewed on agarose gel electrophoresis (Oberhammer et al, 1993).
Recently, reliable methods have been developed that enable the rapid assessment of apoptosis on sections prepared from paraffin-embedded material, e.g. the TUNEL method for TdT-mediated dUTP-biotin nick end labeling (Sarkiss et al, 1996). The APO-BRDU kit utilizes the same principle. The enzyme TdT is used to catalyze a template-independent addition of bromolated deoxyribonucleotide triphosphates (Br-dUTP) to the 3' -hydroxyl ends of the numerous fragments of double- and single-stranded DNA present in apoptotic cells. This allows the labeling of the very high concentrations of 3'-OH ends that are localized in apoptotic bodies. Br-dUTP is claimed to be more readily incorporated into the genome of apoptotic cells than are deoxyribonucleotide triphosphates complexed to larger ligands like fluorescein, biotin or digoxigenin (Nagata & Golstein, 1995). Although rather specific for cells undergoing apoptosis, these techniques may also label cells undergoing necrosis. However, this is seldom a problem since the distinction between focal apoptotic events and necrosis is fairly clear. The histologic features of apoptosis include cell shrinkage and loss of junctional contact, resulting in a "halo" around the cell. The nucleus shows condensation and margination of the chromatin. This is followed by the fragmentation or "pinching off" of pieces of nuclear material, which are surrounded by cytoplasm with intact cytoplasmic organelles as shown at ultrastructural level, These apoptotic fragments of pyknotic nuclear material and cytoplasm are phagocytosed by adjacent cells or macrophages. Apoptotic cells have been called by various names in different tissues and include "Councilman bodies", "Civatte bodies", ''necrobiotic cells" and "nuclear dust". The BM-1 antibody is directed to the Lewisy antigen which has been identified phenotypically as a Lamant L, Meggetto F, Saati TA et al 1996. High incidence of the t(2;5) (p23;q35) translocation in anaplastic large cell lymphoma and its lack of detection in Hodgkin's disease. Comparison of cytogenetic analysis, reverse transcriptase-polymerase chain reaction, and Panaplastic large cell lymphoma and its lack of detection in Hodgkin's disease. Comparison of cytogenetic analysis, reverse transcriptase-polymerase chain reaction, and P marker of specific types of cells, and possibly specific stages of differentiation. Lewis is totally absent at the morula stage, but is highly expressed on the blastocyst surface and has been shown to play a role in the implantation process (Fenderson et al, 1991). Recently, Lewisy has been identified as a characteristic of cells undergoing apoptosis (Hiraishi et al, 1993). In Lewisy-positive areas of tissue sections, typical apoptotic morphological changes and DNA fragmentation were frequently observed in certain loci, although not all Lewisy positive cells showed such signs of apoptosis. Although the BM-1 antibody against the Lewisy antigen is reputed to detect apoptotic cells, further studies to test its efficacy, including a comparative analysis with the insitu end-labeling techniques, are awaited.
Another method of detection of apoptotic bodies is the use of Annexin V, which is a 35-36 kD Ca2+ -dependent phospholipid-binding protein that has a high affinity for the membrane phospholipid phosphatidylserine (PS). In apoptotic cells, PS is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment and allowing its binding to Annexin V. Binding to a signal system such as fluorescein isothiocyanate allows the easy identification of apoptotic cells (in frozen sections and cell preparations). Annexin V is thought to identify cells at an earlier stage of apoptosis than assays based on DNA fragmentation because externalization of PS occurs earlier than the nuclear changes associated with apoptosis (Raynal & Pollard, 1994).
Applications
BM-1 antibody may be applied to neoplasms in general to assess the apoptotic index, e.g., endometrial adenocarcinoma (Kuwashima et al, 1995). Recently, apoptosis has been considered to be a key event in oncogenesis, e.g. apoptosis has been reported to be promoted by tumorsuppressor gene p53 and inhibited by oncogene bcl-2 (Arends & Wyllie, 1991). Although apparent cell loss by apoptosis occurs in carcinomatous tissue (Hiraishi et al, 1993), the physiological significance is unclear (Umansky, 1982). BM-1 positivity has been found to be as high as 25-35% in T cells of lymph nodes of patients with AIDS-related complex (ARC), in contrast to healthy controls who had less than 5% (Adachi et al, 1988).
Comments
Strong BM-1 immunoreactivity is observed in the apical surface of tubular urothelium, basal cells (glandular foveoli) of gastric and esophageal mucosa and these tissues may be employed as controls.
The optimal method for the identification of apoptotic cells depends on the experimental system and the mode of induction of apoptosis. The degree of DNA degradation can vary according to the cell type, the nature of the inducing agent and the stage of apoptosis. The application of multiple methods, each based on a different feature of the apoptotic process, may provide more information about the cell population than any one method would give alone.
References
•Adachi M, Hayami M, Kashlwagi N et al 1988. Expression of ley antigen in human immunodeficiency virus-infected human T cell lines and in peripheral lymphocytes of patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC). Journal of Experimental Medicine 167:233-331.
•Arends MJ, Wyllie AH 1991. Apoptosis: mechanism and roles in pathology. International Reviews in Experimental Pathology 32:223-254.
•Fenderson BA, Killma N, Stroud MR et al 1991. Specific interaction between Ley and H as a possible basis for trophectoderm-endometrium recognition during implantation. Glycoconjugate Journal, 8:179 (abstract 8.5).
•Hiraishi K, Suzuki K, Hakomori S, Adachi M 1993. Ley antigen expression is correlated with apoptosis (programmed cell death). Glycobiology 3:381-390.
•Kerr JFR, Wyllie AH, Currie AR 1972. Apoptosis: a basic biological phenomenon with wider ranging implications in tissue kinetics. British Journal of Cancer 26: 239-257. implications in tissue kinetics. British Journal of Cancer 26: 239-257.
•Kuwashima Y, Uehara T, Kishi K, et al 1995. Proliferative and apoptotic status in endometrial adenocarcinoma. International Journal of Gynecological Pathology 14:45-49.
•Nagata S, Golstein P 1995. The Fas death factor. Science 267: 1445-1449.
•Oberhammer F, Wilson JW, Dive C et al 1993. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO Journal 12:679-684.
•Raynal P, Pollard HB 1994. Annexins. The problem of assessing the biological role for a gene family of multifunctional calcium and phospholipid-binding proteins.
•Journal of Biological Chemistry 265:4923-4928.
•Sarkiss M, Hsu B, El-Naggar AK, McDonnell TJ 1996. The clinical relevance and assessment of apoptotic cell death. Advances in Anatomical Pathology 3:205-211.
•Umansky SR 1982. The genetic program of cell death: hypothesis and some applications: transformation, carcinogenesis, and aging. Journal of Theoretical Biology 97:591-602.
•Wyllie AH, Kerr JFR, Currie AR 1980. Cell death: the significance of apoptosis. International Reviews in Cytology 68:251-306.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
