Sources/Clones
Accurate (polyclonal), Biodesign (monoclonal), Biogenex (6A6.09), Chemicon, Dako (HT1, HT3, HT4, polyclonal), Gentrak, Immunotech (HTdT, polyclonal), Seralab (HTdT-1, polyclonal), Sigma (8-1 E4) and Supertechs (polyclonal).
Fixation/Preparation
While both immunofluorescent and immunoenzyme techniques were initially applied to cryostat sections and cell suspensions, immunohistochemical staining of formalin-fixed, paraffin-embedded sections is now possible. Paraffin section immunostaining is greatly enhanced by heat-induced antigen retrieval so that terminal deoxynucleotidyl transferase (TdT) can be demonstrated on routine and archival specimens without the need for DNAse digestion and prolonged incubation previously necessary. Both 4 M urea and citrate buffer pH 6.0 are suitable retrieval solutions (Orazi et al, 1994). Polyclonal antibodies are preferable to monoclonal antibodies for formalin-fixed, paraffin-embedded sections.
Background
Terminal deoxynucleotidyl transferase (TdT) is a 58 kD protein encoded by a 35 kb gene on chromosome 10q23-25. It is a nuclear enzyme that catalyzes the random addition of deoxynucleotidyl residues on the 3'OH termini of single-stranded DNA and of oligo-deoxynucleotide primers and differs from other DNA polymerases by not requiring template instruction for polymerization.
TdT is recognized to exert its DNA polymerase function during the early variation of genes coding for T and B cells, perhaps by resulting in the addition of non-germline-encoded nucleotides (N-regions) although its function is still debated.
TdT is normally present only in hematopoietic tissues such as thymus and bone marrow, where it is restricted to a proportion of multipotent cell precursors and immature T and B lymphocytes. TdT positivity is never observed in normal peripheral blood cells.
Approximately 1-2% (more in young individuals) of bone marrow cells show TdT positivity and these mostly express B-cell precursor phenotype in cell suspension studies. In trephine biopsies, TdT-+ cells do not display preferential localization and are sparsely dispersed in interstitial spaces.
In the thymus, T lymphocytes can be classified into three maturation stages corresponding to their microenvironment. Stage I thymocytes, accounting for 0.5-5% of thymocytes, reside in the subcapsular zone of the thymus and comprise large TdT blast cells which express CD 7, CD 2, CD 5, and cCD 3 (cytoplasmic). Stage II thymocytes, accounting for 60-80% of thymocytes, are TdT and express CD 7,
CD 5, cCD 3, CD 2, CD 1, CD 4 and CD 8. Stage II thymocytes, accounting for 15-20% of thymocytes, reside in the medulla, do not express TdT nor CD 1 and show differentiation into either CD 4+ or CD 8+ cells.
Applications
TdT as a marker is mostly used in the diagnosis of lymphomas and leukemias. TdT activity is seen in acute lymphoblastic leukemias (ALL) of both B- and T-cell lineages so that TdT is a useful diagnostic marker for lymphoblastic leukemias (Chilosi & Pizzolo, 1995). In addition, as many as 30% of patients with chronic granulocytic leukemia develop a lymphoid blast crisis which is characterized by a lymphoblastic phenotype including nuclear TdT expression.
These TdT lymphoblastic crisis have a better prognosis than TdT-nonlymphoid blast crisis and respond to ALL-like therapy.
About 20% of cases of acute non-lymphoid leukemias also express TdT in which the proportion of TdT blasts coexpressing various myeloid markers is variable. It has been suggested that the expression of TdT in such cases is a marker of poor prognosis but this is controversial. Such cases often show the phenomenon of phenotypic and genotypic ''lineage infidelity" in which there is expression of lymphoid antigens such as CD 7 and rearrangement of Ig and T-cell receptor genes.
The L3 ALL in the FAB classification which represents Burkitt-type leukemia is an exception as the blast cells of this type of leukemia represent a "mature" B-cell phenotype with surface immunoglobulin expression.
TdT is a reliable marker to distinguish lymphoblastic lymphoma (LL) from other lymphomas that are always TdT -. LL are related to T-ALL and their distinction from the latter can be difficult, but, clinical and phenotypic differences have been observed with the latter tending to show a more immature immunophenotype. While LL is frequent in children, forming about one third of all non-Hodgkin's lymphoma cases, it also makes up about 5% of cases in adults and cases of non-T, non-B or pre-B-cell LL have been reported in extranodal sites in both children and adults.
TdT is thus a useful marker for diagnosis as well as for staging as it helps identify tumor cells from reactive lymphocytes. TdT staining can be used for the detection of early involvement and in staging, especially in extranodal sites such as the testes, CNS (through cerebrospinal fluid examination), skin, liver, kidney and other sites of extramedullary involvement and for monitoring minimal residual disease following chemotherapy.
Comments
TdT represents a powerful tool in leukemia and lymphoma diagnosis but it should be used in the context of a complete panel of markers and relevant histochemical enzyme stains. The ability to stain for this DNA polymerase in paraffin-embedded tissues, especially with polyclonal antibodies, following heat-induced antigen retrieval has greatly enhanced its diagnostic utility (Orazi et al, 1994). When immunostaining cryostat sections, it is necessary to employ brief fixation in buffered formalin or Zamboni's fixative and to reduce diffusion of the enzyme the sections must be immersed in fixative immediately after cryosectioning (Chilosi et al, 1983). We employ the rabbit anti-TdT from Dako.
References
•Chilosi M, Pizzolo G 1995. Review of terminal deoxynucleotidyl transferase. Biological aspects, methods of detection, and selected diagnostic applications. Applied Immunohistochemistry 3: 209-221.
•Chilosi M, Pizzolo G, Fiore-Donati L et al 1983. Routine immunofluorescent and histochemical analysis of bone marrow involvement of lymphoma/leukemia: the use of cryostat sections. British Journal of Cancer 48: 763-775.
•Orazi A, Cattoretti G, Joh K, Neiman RS 1994. Terminal deoxynucleotidyl transferase staining of malignant lymphomas in paraffin sections. Modern Pathology 7: 582-586.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Accurate (polyclonal), Biodesign (monoclonal), Biogenex (6A6.09), Chemicon, Dako (HT1, HT3, HT4, polyclonal), Gentrak, Immunotech (HTdT, polyclonal), Seralab (HTdT-1, polyclonal), Sigma (8-1 E4) and Supertechs (polyclonal).
Fixation/Preparation
While both immunofluorescent and immunoenzyme techniques were initially applied to cryostat sections and cell suspensions, immunohistochemical staining of formalin-fixed, paraffin-embedded sections is now possible. Paraffin section immunostaining is greatly enhanced by heat-induced antigen retrieval so that terminal deoxynucleotidyl transferase (TdT) can be demonstrated on routine and archival specimens without the need for DNAse digestion and prolonged incubation previously necessary. Both 4 M urea and citrate buffer pH 6.0 are suitable retrieval solutions (Orazi et al, 1994). Polyclonal antibodies are preferable to monoclonal antibodies for formalin-fixed, paraffin-embedded sections.
Background
Terminal deoxynucleotidyl transferase (TdT) is a 58 kD protein encoded by a 35 kb gene on chromosome 10q23-25. It is a nuclear enzyme that catalyzes the random addition of deoxynucleotidyl residues on the 3'OH termini of single-stranded DNA and of oligo-deoxynucleotide primers and differs from other DNA polymerases by not requiring template instruction for polymerization.
TdT is recognized to exert its DNA polymerase function during the early variation of genes coding for T and B cells, perhaps by resulting in the addition of non-germline-encoded nucleotides (N-regions) although its function is still debated.
TdT is normally present only in hematopoietic tissues such as thymus and bone marrow, where it is restricted to a proportion of multipotent cell precursors and immature T and B lymphocytes. TdT positivity is never observed in normal peripheral blood cells.
Approximately 1-2% (more in young individuals) of bone marrow cells show TdT positivity and these mostly express B-cell precursor phenotype in cell suspension studies. In trephine biopsies, TdT-+ cells do not display preferential localization and are sparsely dispersed in interstitial spaces.
In the thymus, T lymphocytes can be classified into three maturation stages corresponding to their microenvironment. Stage I thymocytes, accounting for 0.5-5% of thymocytes, reside in the subcapsular zone of the thymus and comprise large TdT blast cells which express CD 7, CD 2, CD 5, and cCD 3 (cytoplasmic). Stage II thymocytes, accounting for 60-80% of thymocytes, are TdT and express CD 7,
CD 5, cCD 3, CD 2, CD 1, CD 4 and CD 8. Stage II thymocytes, accounting for 15-20% of thymocytes, reside in the medulla, do not express TdT nor CD 1 and show differentiation into either CD 4+ or CD 8+ cells.
Applications
TdT as a marker is mostly used in the diagnosis of lymphomas and leukemias. TdT activity is seen in acute lymphoblastic leukemias (ALL) of both B- and T-cell lineages so that TdT is a useful diagnostic marker for lymphoblastic leukemias (Chilosi & Pizzolo, 1995). In addition, as many as 30% of patients with chronic granulocytic leukemia develop a lymphoid blast crisis which is characterized by a lymphoblastic phenotype including nuclear TdT expression.
These TdT lymphoblastic crisis have a better prognosis than TdT-nonlymphoid blast crisis and respond to ALL-like therapy.
About 20% of cases of acute non-lymphoid leukemias also express TdT in which the proportion of TdT blasts coexpressing various myeloid markers is variable. It has been suggested that the expression of TdT in such cases is a marker of poor prognosis but this is controversial. Such cases often show the phenomenon of phenotypic and genotypic ''lineage infidelity" in which there is expression of lymphoid antigens such as CD 7 and rearrangement of Ig and T-cell receptor genes.
The L3 ALL in the FAB classification which represents Burkitt-type leukemia is an exception as the blast cells of this type of leukemia represent a "mature" B-cell phenotype with surface immunoglobulin expression.
TdT is a reliable marker to distinguish lymphoblastic lymphoma (LL) from other lymphomas that are always TdT -. LL are related to T-ALL and their distinction from the latter can be difficult, but, clinical and phenotypic differences have been observed with the latter tending to show a more immature immunophenotype. While LL is frequent in children, forming about one third of all non-Hodgkin's lymphoma cases, it also makes up about 5% of cases in adults and cases of non-T, non-B or pre-B-cell LL have been reported in extranodal sites in both children and adults.
TdT is thus a useful marker for diagnosis as well as for staging as it helps identify tumor cells from reactive lymphocytes. TdT staining can be used for the detection of early involvement and in staging, especially in extranodal sites such as the testes, CNS (through cerebrospinal fluid examination), skin, liver, kidney and other sites of extramedullary involvement and for monitoring minimal residual disease following chemotherapy.
Comments
TdT represents a powerful tool in leukemia and lymphoma diagnosis but it should be used in the context of a complete panel of markers and relevant histochemical enzyme stains. The ability to stain for this DNA polymerase in paraffin-embedded tissues, especially with polyclonal antibodies, following heat-induced antigen retrieval has greatly enhanced its diagnostic utility (Orazi et al, 1994). When immunostaining cryostat sections, it is necessary to employ brief fixation in buffered formalin or Zamboni's fixative and to reduce diffusion of the enzyme the sections must be immersed in fixative immediately after cryosectioning (Chilosi et al, 1983). We employ the rabbit anti-TdT from Dako.
References
•Chilosi M, Pizzolo G 1995. Review of terminal deoxynucleotidyl transferase. Biological aspects, methods of detection, and selected diagnostic applications. Applied Immunohistochemistry 3: 209-221.
•Chilosi M, Pizzolo G, Fiore-Donati L et al 1983. Routine immunofluorescent and histochemical analysis of bone marrow involvement of lymphoma/leukemia: the use of cryostat sections. British Journal of Cancer 48: 763-775.
•Orazi A, Cattoretti G, Joh K, Neiman RS 1994. Terminal deoxynucleotidyl transferase staining of malignant lymphomas in paraffin sections. Modern Pathology 7: 582-586.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
