Biomedical subjects

O Babusíková

Publications and source records attributed to O Babusíková.

At least 37 records · Page 2Linked to original sources

PubMed1999

Flow cytometric analysis of some activation/proliferation markers on human thymocytes and their correlation with cell proliferation.

We immunophenotyped cells from ten human thymuses with emphasis on expression of the CD38 and CD71 antigens. These antigens play role in activation cells and increased expression of them was observed in some leukemia. Simultaneously, certain attention has also been devoted to some further activation markers, e.g. CD25, CD26 and HLA-DR. The classification of leukemia is based on comparison of normal and pathological cells. The study of expression of CD38, CD71 and other markers on thymocytes simultaneously with DNA analysis can be useful for answer if expression of CD38 and CD71 on pathologic cells is a sign of their proliferative ability, a part of immature phenotype in some leukemia, or it is a case of aberrant immunophenotype. In our study, 94% thymocytes were CD38+ and only 16% were CD71+. From our immunophenotypic results including MESF (molecules of equivalent soluble fluorochrome) values and analysis of the cell cycle, the conclusion could be drawn that antigen CD71 can participate in regulation of thymocyte development and presence of both -CD38 and CD71 on pathologic cells will be in all probability the case of aberrant phenotype. We observed a clear correlation of the percentage and MESF values of CD71-positive cells with the cell proliferation only after in vitro thymocytes stimulation with PHA and IL-2. In summary, a strong parallelism was observed regarding the positive relationship between the proliferative rate (assessed by the number of S-phase cells) of stimulated thymocytes and the quantitative (% and MESF) values of some markers - CD71, CD25, CD26 and HLA-DR and negative one with CD38 marker values.

Antigens, CD↗

PubMed1999

Flow cytometry of p53 protein expression in some hematological malignancies.

p53 is a tumor suppressor gene encoding a nuclear phosphoprotein that plays an important role in the control of normal cell proliferation. We have tried to establish the value of the p53 protein expression in peripheral blood (PB) and/or bone marrow (BM) cells of patients with some hematological malignancies. A recently developed fixation/permeabilization method was modified for flow cytometric assessment of p53 protein expression using two anti-p53 monoclonal antibodies. p53 quantitation expressed as molecules of equivalent soluble fluorochrome per cell (MESF) providing valuable data contributing to a more precise definition of leukemic cells, was also applied. Our findings showed higher percentage of p53 expression in cells of AML patients at the time of diagnosis opposite to the controls. These data, in association with immunophenotype of cells, accompanied diagnosis of relapse or definition of remission after allogeneic BM transplantation. We observed also elevated levels of p53 protein at initial diagnosis of early B-ALL. According to our results quantitation of p53 protein allows better characterization of selected population of BM cells and should be used for the monitoring of blast persistence during and after therapy and might also be one of the methods to indicate early relapse. Percentage of p53 protein positivity varied in our group of B-CLL patients tested in connection with progression of disease. We documented also one case of Burkitt's lymphoma with high percentage of p53 positivity. Measurement of p53 protein expression by flow cytometry may be of clinical importance by indicating levels of positivity. Our results suggest, that p53 alteration is frequently involved at initial diagnosis of AML, in some T-cell disorders and on the contrary more frequently during early B-ALL relapse, in advanced stages of B-CLL and in Burkitt's lymphoma. p53 protein quantitation is of value to ascertain malignancy and provides additional parameter suitable for the evaluation of residual disease and for the monitoring of therapy.

Adult↗

PubMed1998

The single cell gel electrophoresis: a potential tool for DNA analysis of the patients with hematological malignancies.

Single cell gel electrophoresis (SCGE) was used to evaluate the level of DNA damage in peripheral blood (PB), bone marrow (BM), and lymphatic node (LN) cells of patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and non-Hodgkin's lymphoma (NHL). The level of DNA damage was compared with the level of basal DNA damage in control group, represented by healthy donors. Statistically significant increase of basal DNA damage was found in leukemia/lymphoma cells of patients suffered from AML, CML, ALL of T-cell subtype (T-ALL), and NHL, however, no difference in basal DNA damage was found in patients with ALL of early B-cell subtype (B-ALL) and CLL in comparison to control group. The mean basal DNA damage increased in the order CLL<early B-ALL<T-ALL<AML<CML<NHL (5.6, 7.2, 10.7, 11.9, 16.9, and 23.4% of tail DNA, respectively) what correlated with survival prediction of patients with particular hematological disease. A large heterogeneity was found in the level of basal DNA damage among patients with AML. By sorting this group according to the immunophenotypic markers and cell maturity a good correlation was found between the level of basal DNA damage and French-American-British (FAB) classification for AML (M1-M2 vs. M4-M5). Though T-ALL group manifested larger homogeneity in comparison with AML, the value of basal DNA damage was also dependent on T-cells maturity and coexpression of surface marker CD10. Chemotherapy resulted in a significant but variable increase of DNA damage in leukemia/lymphoma cells. No increase of DNA damage was repeatedly observed in leukemia/lymphoma cells of patient who did not respond to therapy. The level of DNA damage in cells of patients in remission decreased more or less to the basal level in control cells.

Adolescent↗

PubMed1998

The multidrug resistance in human leukemias. Minireview.

Leukemia/lymphoma cells, clinically refractory to therapy are often associated with expression of P-glycoprotein (P-gp), which is encoded by the multidrug resistance (MDR) gene, mdr1. Cell lines expressing mdr1 exhibit resistance to several structurally unrelated lipophilic drugs, such as anthracyclines, vinca alkaloids, and epopodophyllotoxins. This MDR can be conferred to drug-sensitive cells mdr1 cDNA transfer. In resistant cells, MDR is characterized by overexpression of P-gp and by the enhanced efflux, and P-gp fluorescence probe, rhodamine 123 (Rh 123). This can be circumvented by addition of certain non-cytotoxic drugs, such as verapamil and cyclosporin A.

Animals↗

PubMed1998

The relationship of HLA-DR, CD38 and CD71 markers to activation, proliferation and differentiation of some human leukemia and lymphoma cells.

We investigated the expression-percentage as well as MESF values ("molecules of equivalent soluble fluorochrom" that represent approximately the density of marker expression) of HLA-DR, CD71 and CD38 markers in some human leukemias (ALL, AML, CLL, CML) and lymphomas. They are non-lineage restricted and are supposed to be activation markers except for cases where they represent pathological phenotype like HLA-DR in pre B-ALL, CD38 in some M0 AML or in plasmocytoma or CD38 and CD71 in less mature T-ALL. We used flow cytometry, immunofluorescent staining, DNA staining by propidium iodide and quantification by calibration particles. We demonstrated increased MESF values of HLA-DR compared with controls in all investigated disorders, what could have a prognostic value. We demonstrated significantly higher MESF values of HLA-DR in cALL (37,300-46,000) in comparison with AML (9400-12,400), what could represent another important parameter when distinguishing between these two groups of leukemia. In cells of CML patients with lower CD38% and CD71% increased MESF values (5100 for CD38 and 7900 for CD71), were found while in some T-ALL, AML and cALL patients with high percentages of CD71 and CD38 there were lower MESF values what could indicate a possible connection of higher stage of cell maturation with increased density of CD38 and CD71 markers. We investigated possible relationship between percentage of expression of HLA-DR, CD38 and CD71 and proliferation rate by DNA analysis of the cell cycle. In a group of non-Hodgkin's lymphoma patients, there was no significant increase of proliferation index of malignant cells compared with control. The correlation between percentage of expression of mentioned parameters and proliferation index was not significant. In one patient with Burkitt's lymphoma we demonstrated significant increase of proliferation index of CD71+ subpopulation compared with CD71- one, what indicates that in aggressive form of NHL CD71 can be evaluated not only as activation but also as proliferation marker.

ADP-ribosyl Cyclase↗

PubMed1998

Phenotypic heterogeneity and aberrant markers expression in T-cell leukemia.

For exact determination of lineage assessment there is a need of surface membrane and intracellular (cytoplasmic and nuclear) immunophenotyping performed by flow cytometry. We evaluated in detail the results of surface and intracellular immunophenotyping of 34 T-ALL cases. The great heterogeneity of T-cell differentiation markers has been observed which did not allow relevant subclassification of T-ALL according to the existing subclassification schemes and the proposed three-stage model of physiological T-cell differentiation. Therefore, a simplified classification based on the CD3 marker expression either on cell membrane or in cytoplasm has been created with allocation of T-ALL into two main phenotypic groups. From 34 in detail examined T-ALL cases a great deal-27 (79%) belonged to an immature phenotype (Stage I) and only 7 (21%) expressed more mature phenotype (Stage II). Simultaneously the presence of atypical/aberrant T-cell phenotypes has been studied. We showed that in T-ALL it was possible to specify some cases with leukemia-associated phenotype with coexistence of atypical markers which are absent in nonleukemic cells. In a majority of cases early B-lineage marker (CD10) and in a smaller proportion of them non-lineage associated marker (CD34) were observed. Myeloid marker CD13 was observed in one case of the immature T-ALL, together with CD10 and CD34. As these atypical markers were present through all differentiation stages of T-ALL we obtained a strong evidence that they might represent an abnormal rather than an immature phenotype. The prognostic significance of T-ALL subtypes and aberrant markers coexpression have been discussed. Simultaneously it was shown that quantitative immunofluorescence could provide an additional important diagnostic marker also in T-ALL cases.

Antibodies, Monoclonal↗

PubMed1998

Is T-cell acute lymphoblastic leukemia still a high risk leukemia in children?

In a group of 21 children with T cell ALL we compared the clinical picture and laboratory finding in a subgroup of 13 patients with less mature (mCD3 negative) and a subgroup of 8 patients with mature (mCD3 positive) phenotypical type and the therapeutic response in relation to the stage of thymic differentiation of the blastic cells as well. We could not find any significant differences concerning the presence or absence of mediastinal thymic mass, organomegaly, WBC count, morphology of the blasts and their acid phosphatase and PAS reaction between cases with less mature and mature type of thymic differentiation. Concerning the therapeutic response, children with mature type of T-ALL have shown at 5 years significantly higher event free survival rate, in comparison with the group of patients with less mature type of T cells. Overall survival rate was also higher in the first group, but statistically not significant.

Acid Phosphatase↗

PubMed1998

Expression of cytokine receptors on different myeloid leukemic cells.

We have studied the expression of cytokine receptors CD25 (IL-2Ra,55kD), CD116 (hGM-CSRF,145kD), CD117 (CSFR,145kD), CD120a (TNFR,55kD), CD120b (TNFR,75kD), CD121a (IL-1R, type I, 80kD), CD123 (IL-3R), CD124 (IL-4R, 140kD), CD126 (IL-6R, 80kD), CDw127 (IL-7R, 75kD), CDw128 (IL-8R), CD130 (gp130 subunit), CD131 (common beta), CD134 (OX40) and also CD95 (Fas antigen) on the myeloid leukemic cells. Cells from peripheral blood or bone marrow of 30 patients with disorders in myeloid lineage included mostly acute myeloid leukemias (with high leukocyte count and percentage of blasts) were analyzed for the expression of surface membrane molecules by indirect immunofluorescence method evaluated by flow cytometry. The findings indicate that some monoclonal antibodies have a reactivity against cytokine receptors of pathological cells in individual cases, but with very variable qualitative and quantitative (number copies/cell) expression (preliminary results). The leukemic cells demonstrate unique cytokine receptor profiles, which reveal the great diversity of immunophenotypes within the main functional characterization of blood malignancies. The immunophenotype heterogeneity of leukemic cells has proved to be much greater than to match with existing classification criteria. This fact could raise the necessity of further evaluation and specification of cytokine markers of the myeloid acute leukemias. On the other hand, detection of cytokine receptors on the leukemia cells is important for cytokine therapy.

Adolescent↗

PubMed1998

Expression of CD10, CD19 and CD34 markers in bone marrow samples of children with precursor B-cell acute lymphoblastic leukemia in clinical and hematological remission.

Long-term follow-up of the rate of CD10, CD19 and CD34 antigens expression compared with the percentage of lymphocytes and blastic cells in 196 bone marrow specimens of 91 children with early B-cell acute lymphoblastic leukemia has been performed. It was shown that during a five year period there were no significant differences concerning the percentage of CD markers and the percentage of lymphocytes and blastic cells except those in the 4th year of clinical and immunological follow-up--when increase of lymphocytes and CD19 marker percentage have been observed. Consensus, i.e. more than 20% CD markers positivity associated with more than 5% blasts (positive consensus) or less than 20% CD markers positivity combined with less than 5% blasts (negative consensus) was ascertained in 92.8% of cases. Disagreement, i.e. the rate of CD markers over 20% combined with less than 5% blasts was found in 14 (7.2%) patients. This finding present in the initial phase of the disease could be considered as a sign of not complete remission, in contrast to the finding in children who completed chemotherapy and were in long-term hematological remission, in which the increase of CD markers could be considered as a sign of increased regeneration activity of bone marrow after chemotherapy induced medullary aplasia. Anyway, the increase of CD10, CD19 and CD34 antigens in bone marrow samples over 20% should be evaluated with caution. In these cases, mainly in children with long-term hematologic remission the examination should be repeatedly performed and/or more sensitive methods for detection of minimal residual disease should be applied. In control group of 20 children without leukemia the rate of CD markers in the bone marrow was always under 20%.

Antigens, CD19↗

PubMed1998

Immunophenotypic characteristics of T-acute lymphoblastic leukemia cells in relation to DPP IV enzyme expression.

In the present study we have examined immunophenotypic characteristics ofT-acute lymphoblastic leukemia (T-ALL) cells in relation to the expression of enzyme dipeptidyl peptidase IV (DPP IV). Peripheral blood and bone marrow cells of T-ALL patients at diagnosis were estimated. Cell surface markers were detected by a standard immunofluorescence assay and FACStar flow cytometry using a broad panel of monoclonal antibodies to define T-cell immunophenotype. DPP IV activity was investigated in phenotypically defined T-lymphoblasts. Association between DPP IV expression and proliferation was monitored by the expression of CD71 and CD38, which could be considered as markers of activation and proliferation, and by the silver-staining of nucleolar organizer regions-related proteins (argyrophilic proteins). Lymphoblasts, divided according to the presence or absence of DPP IV activity revealed remarkable heterogeneity in the immunophenotypic features. The vast majority of DPP IV positive T-ALL cases expressed CD4, CD8, CD7, CD5, CD2 along with CD71 and CD38 antigens, but the cells were surface membrane CD3 antigen negative. The phenotype of DPP IV negative cases displayed membrane CD3 antigen and variable expression of CD4 and CD8. CD71 and CD38 were frequently negative. It appears, that DPP IV active cells form the population with immature phenotype, as evidenced by mCD3 antigen absence. Relation between DPP IV positive cells and proliferation activity of T-blasts was observed, given by the presence of CD71 and CD38 positivity and overexpression of argyrophilic proteins (AgNORs). In conclusion, our study indicates a close relationship between DPP IV activity and the features ofT-cell immaturity. Association among DPP IV, CD71, CD38 and AgNORs might reflect possible relationship between immature phenotype and proliferative ability of blast cells in T-ALL patients.

ADP-ribosyl Cyclase↗

PubMed1998

Intracellular markers in acute myeloid leukemia diagnosis.

In our study we used a new proposed system of CD45 monoclonal antibody in combination with the side scatter (SSC) parameter as a very useful gating method allowing myeloblast detection especially in cases with low blasts percentage in examined samples. Immunological demonstration of myeloperoxidase (MPO) in the cytoplasm of AML blasts is considered to be a reliable and highly sensitive marker. Using a direct single and double immunofluorescence staining method and flow cytometry we evaluated the intracellular expression of two granular constituents of myeloid cells--MPO and lactoferrin (LF) in leukemia cells from 18 patients at AML diagnosis, two patients in remission after allogenic bone marrow transplantation and in six controls. Two different fixation/permeabilization techniques were used: Fix&Perm, paraformaldehyde and saponin prior to monoclonal antibody staining in order to verify the sensitivity of two labeling methods for MPO. Although both reagents used in this study proved to be efficient tools for the fixation and permeabilization of leukemia cells, the second one was characterized by higher sensitivity in detection of MPO. By double staining of MPO and LF we were able to distinguish undifferentiated cells from the granulomonocytic maturation compartments in bone marrow, since LF is proposed to be selectively expressed from the myelocyte stage of differentiation onward. Cytoplasmic CD13 expression was detectable in AML blasts after their buffered-formaldehyde-acetone fixation/permeabilization. According to our results the detection of MPO and CD13 markers in the cytoplasm of leukemia cells is of great importance in the definition of FAB M0-M1 subtype of AML. Furthermore we described overexpression of CD34 antigen in AML and revealed the characteristic marker combination when CD34 was studied simultaneously with MPO. This finding also coincided with some atypical phenotypic features (CD15/MPO, CD7/cCD13, CD2/cCD13, CD33/cCD13, MPO/cCD13) contributing to the differential diagnosis and allowing the immunologic monitoring of patients for the presence of residual disease.

Acute Disease↗

PubMed1997

Quantitative immunocytofluorometry--new parameters for the definition of leukemia cells.

In our study we used for definition of leukemia/lymphoma cells a new parameter which allows the enumeration of mean fluorescence intensity expressed by the number of antigen molecules per cell. Quantitative immunofluorescence using calibration microbeads was performed in 36 patients with different acute and chronic lymphoid and myeloid leukemia and in 19 healthy volunteers. We showed that quantitative immunophenotyping allowed the definition of aberrant marker densities on neoplastic cells. We demonstrated under- and overexpression of CD8 marker in CD3/CD4/CD8 complex in T acute lymphatic leukemia and T non-Hodgkin's lymphoma and T leukemia of large granular lymphocytes as compared to normal counterparts. We pointed out that certain antigens (e. g. CD10, CD4, CD24) were expressed at different levels on different cell subsets (CD10 in early B-acute lymphatic leukemia and coexpressed in T-acute lymphatic leukemia, CD4 on T cells and monocytes, CD24 on B cells and granulocytes in chronic myeloid leukemia). We showed that quantitative immune fluorescence could provide new data contributing to a more precise definition of cell differentiation. We documented the significant difference between antigen density of early and late markers in B-cell and myeloid malignancies. Further, we demonstrated that quantitative immune phenotyping could help in determination of exact definition of pathologic clone in morphologically immature leukemia population and showed that parameters of this method are also convenient for cytoplasmic marker evaluation. In our study we were able to demonstrate that CD45 quantitative expression appeared to be a more informative parameter than its percentage of antigen-positive cells as a measure of antigen expression only and we pointed out that low and high CD45 densities enabled to differentiate between pathological clone and residual healthy population in examined sample. We showed that quantitative immune phenotyping could be another important parameter for definition of leukemia phenotype suitable for detection of minimal residual disease.

Antigens, CD↗

PubMed1997

The relationship between argyrophilic proteins and some immunophenotypic markers in acute leukemia cells.

This study reports the immunophenotypic features of a series of 62 selected acute leukemia patients with increased incidence of argyrophilic proteins (AgNORs) at the time of initial diagnosis. Peripheral blood and bone marrow cells of patients with T-ALL, B-precursor ALL and AML were studied. The method of silver staining was used to determine the number of AgNORs per cell. Cell surface markers were detected by a standard immunofluorescence assay. To demonstrate the relationship between AgNOR quantity and cell proliferation, the expression of activation and proliferation antigens CD38 and CD71 was investigated. To characterize the immunophenotype and the discrete stages of differentiation, the wide panel of antibodies against lymphoid, myeloid and non-lineage specific antigens was used. The number of AgNORs at diagnosis ranged from 3.05 to 6.70. Immunophenotypic analysis showed a variation in CD38 and CD71 expression among different leukemia subtypes. CD71 antigen was more expressed in T-ALL than in B-precursor ALL or in AML. Notable was the relationship between increased AgNOR quantity and antigens that characterize the immaturity of leukemic cells. The association with CD7, CD2, CD5 (without CD3 membrane expression) and CD34 in T blasts was evident. High positivity of CD19, CD10, CD34 and HLA-DR in relation to the increased amount of AgNORs in B-lineage ALL was observed. The vast majority of AML patients with high numbers of AgNORs simultaneously expressed CD13, CD33, CD34 and HLA-DR. One third of AML cases coexpressed T cell marker CD7. In conclusion, the presence of increased numbers of AgNORs at diagnosis might reflect the dependence on an early stage of leukemia cell differentiation.

Acute Disease↗

PubMed1996

Leukemia-associated phenotypes: their characteristics and incidence in acute leukemia.

Leukemia-associated phenotypes have been suggested to be a valuable tool for the detection of minimal residual disease in acute leukemia patients, as they allow to distinguish leukemic blasts from normal hematopoietic progenitor cells. The aim of the present study was to analyze the proportion of acute leukemia patients (both with lymphoid and myeloid leukemias) in which the immunological detection of leukemia-associated phenotypes was convenient for the distinction of leukemic and normal cells. For this purpose we have studied the blast cells from 186 acute leukemia patients at diagnosis with a large panel of monoclonal antibodies by flow cytometry using double staining combinations. From aberrant phenotypes on blast cells we followed lineage infidelity (coexpression of myeloid markers in lymphoid leukemia cells and vice versa, as well as the simultaneous expression of both, T and B cell markers in one lymphoid blast cell) and asynchronous marker expression (simultaneous expression of early and late markers in one cell). One hundred and five of the 186 acute leukemia cases analyzed (56%) showed the presence of leukemia-associated phenotypes. In 41 of the 90 ALL cases followed (46%) and in 40 of the 96 AML cases studied (42%) lineage infidelity was observed. Asynchronous antigen expression was detected in 24 followed cases (13%). Evaluation of the cell marker density by means of calibration microbeads demonstrated abnormal mean channel immunofluorescence and molecules of equivalent soluble fluorescein for CD8 in two patients with T cell malignancies at diagnosis. Abnormal CD8 density might thus represent a characteristic feature of malignant CD8-positive T cell clone. Quantitative marker evaluation therefore seems to be another important mean for the detection of aberrant phenotypes on leukemia cells suitable for the detection of minimal residual disease.

Acute Disease↗

PubMed1996

Some early differentiation markers detected in cytoplasm of pre-B cells by flow cytometry.

Immunofluorescent staining of cytoplasmic IgM (heavy chains) and CD24 as well as their simultaneous staining with surface B cell markers was used to study immunophenotype changes in B cell differentiation. Human hematopoietic B cell lines P3HR1 and RAJI were used. We found that IgM and CD24 cell markers while absent on cell membrane could be detected in their cytoplasm (c). The presence of cIgM in cell lines RAJI, P3HRI indicates their early pre-B differentiation stage. The presence of cCD24 simultaneously with mCD22 and cIgM is the evidence that hematopoietic cell lines or leukemias may not accurately reflect normal differentiation pathway. Combinations of cIgM, cCD24 with surface B cell markers CD10, CD19 on these cell lines can be considered as leukemia associated phenotypes. Some of them were shown in bone marrow and peripheral blood of pre-B ALL and B-CLL patients and can be used for the detection of minimal residual disease. Different fixation/permeabilization methods were tested in order to choose the optimal one for simple detection of cytoplasmic markers or their simultaneous detection with surface markers by flow cytometry. They included "one-component-methods" (methanol-M, saponin-S), methods combining these components with paraformaldehyde (P+M, P+S) or buffered formaldehyde acetone (BFA). The choice depended on individual marker detected. General parameters like the proportion of debris, cell aggregation, cell loss and the changes of scatter parameters FSC and SSC were taken into consideration. The priorities of combined methods P+S, P+M1 and BFA over one-component methods are demonstrated.

Antibodies, Monoclonal↗

PubMed1996

Flow cytometric detection of some activation and proliferation markers in human hematopoietic cell lines.

Simultaneous surface marker/DNA, cytoplasmic/DNA or nuclear/DNA staining was used to study proliferation of hematopoietic cell lines (MOLT4, BJAB, P3HR1). Different fixation/permeabilization methods (paraformaldehyde with metanol or Tween 20 or saponin, buffered formaldehyde-acetone) were used providing optimal results of the double stainings. There was a significant increase of S phase and proliferation index (PI) of CD71+ and Ki67+ MOLT4 cells in comparison with their negative counterparts. This indicates their close connection with proliferation. Unlike that, the correlation between the expression of CD38 and S phase or PI was not significant either in MOLT4 or in P3HRI cells. For cytoplasmic markers CD3 (in MOLT4 cells) and CD22 (in BJAB cells) statistically significant (cCD3) and not significant (cCD22) correlation was demonstrated between their expression and S phase or PI. Molecular equivalents of soluble fluorescein values for CD71 were always higher than for CD38. The density of these cell surface markers in addition to the percentage of their expression is of considerable significance for their evaluation as activation or proliferation markers.

Antigens, CD↗

PubMed1996

The study of AgNOR proteins in leukemias: diagnostic value and correlation to S-phase cell fraction.

The study assessed the diagnostic value of silver staining method and its possible relevance as an alternative to DNA analysis for the study of cellular proliferation in various leukemias (ALL, AML, CML). Silver staining of nucleolar organizer region-related proteins (AgNORs) was applied to peripheral blood and bone marrow cells. The analysis of S-phase cells was carried out using a FACStar flow cytometer. The mean number of AgNOR dots per nucleus and the percentage of S-phase cells varied according to immunophenotype of leukemic cells, depending on the time of initial diagnosis, remission or relapse. Peripheral blood and bone marrow cells of healthy subjects exhibited less AgNOR dots than leukemic cells. The number of AgNORs in bone marrow cells was higher than that of AgNORs in peripheral blood. Significant differences between ALL and AML, as well as AML and CML in AgNOR quantity were observed. Important increase in AgNOR values was evident in relapsed leukemias and in the CML blast crisis. DNA flow cytometry analyses provided results comparable to those of AgNOR enumeration. The correlation between AgNOR dots and proportion of S-phase cells prompted us to consider that AgNOR count reflects cell proliferation capacity of leukemic cells.

Acute Disease↗

PubMed1995

Chronic myeloid leukemia: correlation between purine metabolism enzyme activities and membrane immunophenotype.

Peripheral blood or bone marrow of 24 patients with chronic myeloid leukemia (CML) were characterized for their surface membrane marker profiles using flow cytometry and fluorescence microscopy. Purine metabolism enzyme activities were compared with membrane immunophenotype and cytochemical stains. CML subtypes were correlated with the expression of surface membrane antigens detected by the monoclonal antibodies. On the basis of immunophenotyping we found the following characteristic marker profiles: In stable phase of CML (CML-SP)-CD15, CD11b, CDw65, CD13, in accelerated phase of CML (CML-AP)-CD15, CDw65, CD11b, CD13 and CD33, in myeloid blastic phase of CML(CML-BP-M)-CD13, CD33, HLA-DR, CD11b, CD15, CDw65, in myeloid and lymphoid (mixed) blastic phase of CML (CML-BP-M+L)-CD13, CD33, CD34, HLA-DR, CD11b, CD10 and in chronic myelomonocytic leukemia (CMML)-CD14, CDw65, CD11b, CD33 and HLA-DR. Analysis of purine metabolism enzyme activities showed that there was a correlation between the values of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) and various types of CML. ADA levels in CML-SP, CML-AP and CMML were comparable with those in normal cells. In CML-BP-M, which represents proliferation of less mature myeloid cells (similar to less mature AML subtypes), ADA activity increased and PNP activity decreased. ADA activity was significantly different between control group and CML-BP-M (p < 0.01), between CML-SP and CML-BP-M (p < 0.05). The values of PNP activity were the highest in stable phase of CML (125 pkat. 10(-6) cells) and the lowest (23 pkat.10(-6) cells) in CML-BP-M+L. PNP activity in the other groups corresponded to control values. High ADA/PNP ratio was found in CML-BP-M and CML-BP-M+L (0.7 and 2.0, respectively) in comparison to CML-SP (0.2). It follows from our results that ADA/PNP ratio enables to discriminate between stable and blast phases of CML (p < 0.01). The level of the cytochemical enzymes (CHAE, MPO, SBB, ANAE and 5' NT) varied and reflected the degree of cell differentiation and maturation. CHAE and MPO were characteristic enzymes for CML, ANBE for CMML and 5' NT for CML-BP-lymphoid.

Adenosine Deaminase↗