Solid-phase enzyme immunoassay of terminal deoxynucleotidyl transferase evaluated.

1987 ◽  
Vol 33 (2) ◽  
pp. 293-296 ◽  
Author(s):  
M Fleisher ◽  
R Stankievic ◽  
D Schwartz ◽  
M K Schwartz
Keyword(s):  
Bone Marrow ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
Solid Phase ◽  
Linear Regression Analysis ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Human Leukemia ◽  
Solid Phase Immunoassay ◽  
Analysis Of Results ◽  
Good Concordance

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.

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

Blood ◽  
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.

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

Blood ◽  
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

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.

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

Blood ◽  
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

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.

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

Blood ◽  
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.

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

Blood ◽  
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.

scholarly journals Acquisition and Expansion of Adult Rat Bone Marrow Multipotent Mesenchymal Stromal Cells

2017 ◽  
Vol 61 (1) ◽  
pp. 15-22
Author(s):  
I. Šulla ◽  
V. Balik ◽  
M. Šarišský
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Mononuclear Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Human Leukemia ◽  
Sprague Dawley ◽  
Nestin Expression ◽  
Adult Rat ◽  
Standard Culture ◽  
Rat Bone

AbstractThis study was initiated in order to test a mini-invasive method of mesenchymal stem/progenitor cells (MS/PCs) isolation from a rat bone marrow (BM), and subsequently their expansion, differentiation, and evaluation of their immunophenotypic characteristics; and later their preservation as donor cells in an optimal condition for potential autotransplantation. The study group comprised of 6 adult male Sprague-Dawley (S-D) rats, weighing 480—690 g. The rats were anaesthetised by isoflurane with room air in a Plexiglas box and maintained by inhalation of a mixture of isoflurane and O2. Their femurs were surgically exposed and their diaphyses double-trephined. Then BM cells were flushed out by saline with heparin and aspirated into a syringe with a solution of DMEM (Dulbecco’s modified eagle’s medium) and heparin. The mononuclear cells from the BM were isolated by centrifugation and expanded in a standard culture medium supplemented with ES-FBS (es-cell-qualified foetal bovine serum), L-glutamine and rh LIF (recombinant human leukemia inhibitory factor). Following 14 days of passaging cultures, the cells were split into 2 equal parts. The first culture continued with the original medium. The second culture received additional supplementation with a human FGFβ (fibroblast growth factor beta) and EGF (epidermal growth factor). The populations of these cells were analysed by light-microscopy, then the mean fluorescence intensities (MFIs) of CD90 and Nestin were evaluated by a tricolour flow cytometry using monoclonal antibodies. The type of general anaesthesia used proved to be appropriate for the surgical phase of the experiments. All rats survived the harvesting of the BM without complications. The total number of mononuclear cells was 1.5—4.0 × 106per sample and the proportion of CD90/Nestin expressing cells was < 1 %. Following 14 days of expansion, the cells became larger, adherent, with fibrillary morphology; the proportion of cells expressing CD90/Nestin increased to almost 25 %, i. e. they earned basic phenotypic characteristics of MSCs. Throughout the further cultivation a gradual decrease of the CD90/Nestin expression occurred. This suggested that the suitability of rat bone marrow derived MS/PCs for replacement therapy would probably be the highest between days 12—15 of cultivation and then would diminish.

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

Blood ◽  
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

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.

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

Blood ◽  
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

scholarly journals P-24: a human leukemia-associated and lymphohemopoietic progenitor cell surface structure identified with monoclonal antibody.

1981 ◽  
Vol 153 (3) ◽  
pp. 726-731 ◽  
Author(s):  
J H Kersey ◽  
T W LeBien ◽  
C S Abramson ◽  
R Newman ◽  
R Sutherland ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Surface ◽  
Surface Structure ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Human Leukemia ◽  
Cell Surface Structure

This study was directed at surface structures that are found on human lymphohemopoietic progenitor cells in normal and leukemic bone marrow. A monoclonal antibody was produced against an acute lymphoblastic leukemia (ALL) cell line of the pre-B phenotype; this antibody (BA-2) was used to demonstrate a cell surface polypeptide of approximately 24,000 mol wt that migrates similarly in both reduced and nonreduced form. This polypeptide, p24/BA-2, was shown by immune precipitation and gel electrophoresis and cell distribution studies to be different from HLA-DR and gp 100/cALLa. p24/BA-2 was present on the surface of 77% (54/70) of cases of non-T, non-B ALL; BA-2 staining was less bright or nondetectable in surface Ig+ (SIg+) chronic lymphocytic leukemia (CLL) and T ALL and nondetectable on peripheral T and B lymphocytes. Approximately 3% of bone marrow mononuclear cells were p24/BA-2+, and these cells were E rosette-, surface (SIg-), and nonphagocytic. Marrow TdT+ progenitor cells were frequently p 24/BA-2+. Results suggest that p24/BA-2 represents a surface structure present on lymphohemopoietic bone marrow progenitor cells and that most common types of ALL bear the p25/BA-2 structure.

Sign in / Sign up

Export Citation Format

Share Document