Solid-Phase enzyme immunoassay for terminal deoxynucleotidyl transferase (abbott tdt-eia) in extracts of whole blood and mononuclear cells isolated from bone marrow and blood

Abstract We evaluated a newly developed solid-phase immunoassay (EIA) of terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.3) and compared it with the enzymatic assay of TdT involving DNA polymerase. We assessed the precision, performance characteristics, and clinical efficacy of the EIA procedure, using 249 specimens of peripheral blood and bone marrow and 118 specimens of whole blood. On linear regression analysis of results for these 249 samples as measured by the two procedures, the correlation coefficient was 0.87. Distribution of TdT in mononuclear cells isolated from whole blood and bone marrow of subjects in several disease categories indicated good concordance between the two assay procedures. The EIA procedure is precise, can be performed on whole blood without first isolating mononuclear cells, is nonisotopic, and shows potential as a quantitative indicator for the differential diagnosis and monitoring of human leukemia.

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Prognostic significance of terminal transferase activity in childhood acute lymphoblastic leukemia: a prospective analysis of 164 patients

Blood ◽

10.1182/blood.v60.6.1267.1267 ◽

1982 ◽

Vol 60 (6) ◽

pp. 1267-1276 ◽

Cited By ~ 15

Author(s):

JJ Hutton ◽

MS Coleman ◽

S Moffitt ◽

MF Greenwood ◽

P Holland ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Complete Remission ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Lymphoblastic Leukemia ◽

Prognostic Significance ◽

Terminal Deoxynucleotidyl Transferase ◽

Central Nervous System Relapse ◽

Transferase Activity

Abstract Whether the level of terminal deoxynucleotidyl transferase (TdT) activity in mononuclear cells from bone marrow and peripheral blood has prognostic significance has been analyzed prospectively in 164 children with T and non-T, non-B marked acute lymphoblastic leukemia (ALL). TdT was measured at diagnosis to assess its value as a predictor of duration of remission and length of survival. It was measured repeatedly during remission to assess whether it could predict relapse. Ninety-seven percent of the children achieved a complete remission of their disease, and 40% relapsed during the study. The level of TdT activity in blasts at diagnosis varied 1000-fold from patient to patient. There was no statistically significant relationship between TdT activity in cells at diagnosis and the achievement of complete remission, the duration of remission, or length of survival. TdT activity was significantly increased in the bone marrow of 65% of patients at the time of marrow morphological relapse, but was rarely increased in marrow from patients with isolated testicular or central nervous system relapse. Wide fluctuations in TdT activity were characteristically seen in mononuclear cells from the marrow and peripheral blood of patients with ALL at all stages of their disease. An isolated high value of TdT activity in the bone marrow or peripheral blood cannot be taken as evidence of impending relapse. Quantitative measurements of TdT activity alone on mononuclear cells from bone marrow and peripheral blood are helpful in differential diagnosis, but cannot guide therapy of children with ALL.

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Normal human bone marrow precursors that express terminal deoxynucleotidyl transferase include T-cell precursors and possible lymphoid stem cells

Blood ◽

10.1182/blood.v77.8.1681.bloodjournal7781681 ◽

1991 ◽

Vol 77 (8) ◽

pp. 1681-1690

Author(s):

SD Gore ◽

MB Kastan ◽

CI Civin

Keyword(s):

Bone Marrow ◽

T Cell ◽

Mononuclear Cells ◽

Cell Receptor ◽

Small Population ◽

Terminal Deoxynucleotidyl Transferase ◽

Immune Adherence ◽

Hla Dr ◽

Major Subset ◽

B Lymphoid

To compare the differentiation of early B- and T-lymphoid precursors, we have used immune adherence combined with analytical flow cytometric techniques to enrich and characterize subsets of the small population of bone marrow mononuclear cells that express the enzyme terminal deoxynucleotidyl transferase (TdT) but lack the CD19 B-lymphoid marker. Two percent to five percent of bone marrow TdT + mononuclear cells belong to the T-lymphoid lineage by virtue of expression of CD7 or CD5. Three-color immunofluorescence studies showed that, like early B- lymphoid precursors, most bone marrow TdT + T cells express HLA-DR and the progenitor cell antigen CD34, and about half express CD10. All CD5 + TdT + cells express surface CD3 and T-cell receptor alpha, beta, while a subset of CD7 + TdT + cells lack these “mature” T cell features. CD2 is low or absent on CD5 + TdT + cells. Examination of isolated CD34 + cells showed that approximately 70% of CD34 + TdT + cells expressed neither CD19, CD22, CD7, nor CD5, and 15% to 50% also lacked CD10. Thus, a major subset of CD34 + TdT + cells lack lineage- specific surface antigens. TdT expression may be the earliest available marker of lymphoid differentiation, and CD34 + TdT + cells are likely to include progenitor cells for both the B and T lineages.

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Prognostic significance of terminal transferase activity in childhood acute lymphoblastic leukemia: a prospective analysis of 164 patients

Blood ◽

10.1182/blood.v60.6.1267.bloodjournal6061267 ◽

1982 ◽

Vol 60 (6) ◽

pp. 1267-1276

Author(s):

JJ Hutton ◽

MS Coleman ◽

S Moffitt ◽

MF Greenwood ◽

P Holland ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Complete Remission ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Lymphoblastic Leukemia ◽

Prognostic Significance ◽

Terminal Deoxynucleotidyl Transferase ◽

Central Nervous System Relapse ◽

Transferase Activity

Whether the level of terminal deoxynucleotidyl transferase (TdT) activity in mononuclear cells from bone marrow and peripheral blood has prognostic significance has been analyzed prospectively in 164 children with T and non-T, non-B marked acute lymphoblastic leukemia (ALL). TdT was measured at diagnosis to assess its value as a predictor of duration of remission and length of survival. It was measured repeatedly during remission to assess whether it could predict relapse. Ninety-seven percent of the children achieved a complete remission of their disease, and 40% relapsed during the study. The level of TdT activity in blasts at diagnosis varied 1000-fold from patient to patient. There was no statistically significant relationship between TdT activity in cells at diagnosis and the achievement of complete remission, the duration of remission, or length of survival. TdT activity was significantly increased in the bone marrow of 65% of patients at the time of marrow morphological relapse, but was rarely increased in marrow from patients with isolated testicular or central nervous system relapse. Wide fluctuations in TdT activity were characteristically seen in mononuclear cells from the marrow and peripheral blood of patients with ALL at all stages of their disease. An isolated high value of TdT activity in the bone marrow or peripheral blood cannot be taken as evidence of impending relapse. Quantitative measurements of TdT activity alone on mononuclear cells from bone marrow and peripheral blood are helpful in differential diagnosis, but cannot guide therapy of children with ALL.

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Highly sensitive solid-phase enzyme immunoassay for terminal deoxynucleotidyl transferase

Analytical Biochemistry ◽

10.1016/0003-2697(82)90523-1 ◽

1982 ◽

Vol 126 (2) ◽

pp. 327-334 ◽

Cited By ~ 3

Author(s):

Tsuguhiro Kaneda ◽

Saiko Kuroda ◽

Yutaka Hirota ◽

Kanefusa Kato

Keyword(s):

Enzyme Immunoassay ◽

Solid Phase ◽

Terminal Deoxynucleotidyl Transferase ◽

Highly Sensitive

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Normal human bone marrow precursors that express terminal deoxynucleotidyl transferase include T-cell precursors and possible lymphoid stem cells

Blood ◽

10.1182/blood.v77.8.1681.1681 ◽

1991 ◽

Vol 77 (8) ◽

pp. 1681-1690 ◽

Cited By ~ 54

Author(s):

SD Gore ◽

MB Kastan ◽

CI Civin

Keyword(s):

Bone Marrow ◽

T Cell ◽

Mononuclear Cells ◽

Cell Receptor ◽

Small Population ◽

Terminal Deoxynucleotidyl Transferase ◽

Immune Adherence ◽

Hla Dr ◽

Major Subset ◽

B Lymphoid

Abstract To compare the differentiation of early B- and T-lymphoid precursors, we have used immune adherence combined with analytical flow cytometric techniques to enrich and characterize subsets of the small population of bone marrow mononuclear cells that express the enzyme terminal deoxynucleotidyl transferase (TdT) but lack the CD19 B-lymphoid marker. Two percent to five percent of bone marrow TdT + mononuclear cells belong to the T-lymphoid lineage by virtue of expression of CD7 or CD5. Three-color immunofluorescence studies showed that, like early B- lymphoid precursors, most bone marrow TdT + T cells express HLA-DR and the progenitor cell antigen CD34, and about half express CD10. All CD5 + TdT + cells express surface CD3 and T-cell receptor alpha, beta, while a subset of CD7 + TdT + cells lack these “mature” T cell features. CD2 is low or absent on CD5 + TdT + cells. Examination of isolated CD34 + cells showed that approximately 70% of CD34 + TdT + cells expressed neither CD19, CD22, CD7, nor CD5, and 15% to 50% also lacked CD10. Thus, a major subset of CD34 + TdT + cells lack lineage- specific surface antigens. TdT expression may be the earliest available marker of lymphoid differentiation, and CD34 + TdT + cells are likely to include progenitor cells for both the B and T lineages.

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Anti-Erythroblast Antibodies and Defective Apoptosis in Bone Marrow of Early Myelodysplastic Syndrome Patients.

Blood ◽

10.1182/blood.v104.11.4705.4705 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4705-4705

Author(s):

Anna Zaninoni ◽

Francesca G. Imperiali ◽

Elisa Fermo ◽

Alberto Zanella ◽

Wilma Barcellini

Keyword(s):

Bone Marrow ◽

Whole Blood ◽

Caspase 3 ◽

Solid Phase ◽

Statistical Significance ◽

Refractory Anemia ◽

Apoptotic Markers ◽

Apoptotic Marker ◽

Bone Marrow Cultures ◽

Marrow Cultures

Abstract Autoimmune phenomena, particularly directed against RBC, and alteration of apoptosis have been reported in MDS. We recently described a new method for the detection of anti-RBC antibodies in mitogen-stimulated whole blood cultures, named mitogen-stimulated-DAT (MS-DAT), which is able to disclose a latent anti-RBC autoimmunity in different diseases (autoimmune hemolytic anemia in clinical remission and in B-CLL). We investigated MS-DAT positivity and apoptosis in peripheral blood and bone marrow cultures from 14 patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS), compared with controls (healthy volunteers and miscellaneous haematological conditions). MS-DAT was performed by stimulating whole blood and marrow cultures with PMA, and anti-RBC or anti-erythroblast antibodies were detected by competitive solid phase ELISA. As apoptotic markers we evaluated NF-kB and Caspase-3 activity by ELISA and enzymatic assay, respectively. As shown in table, the amount of anti-erythroblast antibodies was significantly greater in BM cultures of MDS versus controls, and the test was clearly positive in 8/14 patients (not shown). The anti-apoptotic marker NF-kB was significantly increased in MDS versus controls, and consistently, caspase-3 activity decreased, although not significantly. On the contrary, PB of MDS displayed no MS-DAT positivity and no significant alterations of apoptotic and anti-apoptotic markers investigated (not shown). These findings suggest the existence of an anti-erythroblast autoimmunity in bone marrow of early MDS patients. This could be related to the observed defective apoptosis, which in turn determines survival of auto-reactive marrow effectors. MDS Controls Medium PMA Medium PMA *denotes statistical significance versus controls. Values are mean±SE Anti-erythroblasts (pg/ml) 407±116* 487±146* 76±15 99±20 NF-kB (OD units) 294±50* 358±83 59±20 67±21 Caspase-3 (OD units) 132±24 145±26 209±53 186±46

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A new solid-phase immunoassay for terminal deoxynucleotidyl transferase: analysis of TdT antigen in cells, plasma, and serum

Blood ◽

10.1182/blood.v65.1.41.bloodjournal65141 ◽

1985 ◽

Vol 65 (1) ◽

pp. 41-45

Author(s):

MS Coleman ◽

ML Cibull ◽

GL Manderino

Keyword(s):

Bone Marrow ◽

Enzyme Activity ◽

Horseradish Peroxidase ◽

Peripheral Blood ◽

Solid Phase ◽

Terminal Deoxynucleotidyl Transferase ◽

Polystyrene Bead ◽

Secondary Antibody ◽

Terminal Transferase ◽

Solid Phase Immunoassay

A solid-phase immunoassay for terminal deoxynucleotidyl transferase has been developed using a primary antibody-coated polystyrene bead and secondary antibody conjugated with horseradish peroxidase. The immunoassay was compared with assays for enzyme activity and detection of antigen with immunofluorescence using cells from peripheral blood and bone marrow from patients with leukemia or lymphoma. In each instance, the solid-phase immunoassay correlated correctly with cellular samples judged to be positive by other tests. However, the level of detection of terminal transferase antigen in plasma or serum of patients with leukemia did not reflect accurately the level of terminal transferase in neoplastic cells. The solid-phase immunoassay was greater than 100-fold more sensitive than conventional assays for enzyme activity, rendering it potentially useful for quantitatively monitoring terminal transferase in patients with leukemia.

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A new solid-phase immunoassay for terminal deoxynucleotidyl transferase: analysis of TdT antigen in cells, plasma, and serum

Blood ◽

10.1182/blood.v65.1.41.41 ◽

1985 ◽

Vol 65 (1) ◽

pp. 41-45 ◽

Cited By ~ 8

Author(s):

MS Coleman ◽

ML Cibull ◽

GL Manderino

Keyword(s):

Bone Marrow ◽

Enzyme Activity ◽

Horseradish Peroxidase ◽

Peripheral Blood ◽

Solid Phase ◽

Terminal Deoxynucleotidyl Transferase ◽

Polystyrene Bead ◽

Secondary Antibody ◽

Terminal Transferase ◽

Solid Phase Immunoassay

Abstract A solid-phase immunoassay for terminal deoxynucleotidyl transferase has been developed using a primary antibody-coated polystyrene bead and secondary antibody conjugated with horseradish peroxidase. The immunoassay was compared with assays for enzyme activity and detection of antigen with immunofluorescence using cells from peripheral blood and bone marrow from patients with leukemia or lymphoma. In each instance, the solid-phase immunoassay correlated correctly with cellular samples judged to be positive by other tests. However, the level of detection of terminal transferase antigen in plasma or serum of patients with leukemia did not reflect accurately the level of terminal transferase in neoplastic cells. The solid-phase immunoassay was greater than 100-fold more sensitive than conventional assays for enzyme activity, rendering it potentially useful for quantitatively monitoring terminal transferase in patients with leukemia.

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