Ethics committees and consensus in the post-totalitarian society.

Ethics committees are usually expected to reach and frame their decisions by consensus. In a post-totalitarian society the conception of consensus might somewhat differ from the one present in societies with a long-term, uninterrupted tradition of democratic development. In 1990s, post totalitarian societies of Central and East Europe have been walking difficult paths from the situation, when matters of public interest had been decided almost solely within the structures of a totalitarian power (usually a 'communist' party and by the party dominated governmental or municipal structures) and the political decisions implemented by more or less coercive exercise of that power (while requiring the broadest community attainment--'imposed consensus'); toward societies deciding their fate and matters of public interest via institutions of a modern, pluralistic, parliamentary democracy (subscribing implicitly or even explicitly to consensus, perceived mostly as 'overlapping' or 'procedural'). The paper gives an analysis of evolution of the notion of consensus in the post-totalitarian Slovakia as seen within the frame of the first decade of establishment and work of ethics committees in the institutions of biomedical research and health care. Examples of present challenges for ethics committees' consensual decision making are outlined.

Study of in vitro conditions modulating expression of MN/CA IX protein in human cell lines derived from cervical carcinoma.

In an effort to better understand the biological significance of MN/CA IX human tumor-associated protein, we have investigated its expression in human cervical carcinoma cell lines in vitro. SiHa cells that naturally express MN/CA IX were used as a model for expression study at the protein level. In addition, we have transfected MN/CA9 gene-negative but transcription-competent C33A cells with a plasmid carrying CAT reporter gene under a control of MN/CA9 promoter. By this way, we have generated a stable cell line C33A/MNP-CAT that was employed in analysis of MN/CA9 regulation at the level of promoter activity as estimated by CAT protein abundance. For the purpose of our study, we have chosen experimental conditions relevant to growth characteristics and phenotypic features of malignantly transformed cells. Both the level of MN/CA IX protein and the gene promoter activity were found to be substantially elevated either in culture of high density or when the adherent carcinoma cells grew in suspension, but were not markedly affected by diminished serum concentration and in the cell cycle progression. These observations support the involvement of MN/CA IX protein in aberrant cell-cell and cell-matrix interactions that facilitate loss of contact inhibition and anchorage independence of cancer cells.

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.

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.

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.

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.

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.

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.

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.

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.

[Evaluation of the clinical status of patients with gastrointestinal hemorrhage and acute pancreatitis using the APACHE II score]. / Hodnotenie klinického stavu pacientov s krvácaním do GIT a s akútnou pankreatitídou prostredníctvom Apache II skóre.

In the group of 35 patients with gastrointestinal bleeding and in the group of 15 patients with acute edematous pancreatitis before beginning of treatment Apache II score was calculated. The obtained score values ranging from 2 to 18 were subsequently compared with the duration of the interval from appearing of the clinical signs to the beginning of treatment, the way of treatment, duration of hospitalization and recurrence of the diseases during hospitalization. The relations were evaluated through correlation analysis and T-test. Although the studied relations were in our conditions statistically insignificant we suppose, that Apache II score contributes to detachment of clinical assessment of patient in correlation to its severity and prognosis.

Human hematopoietic cell lines: a model system for study of minimal residual disease detection technique in acute leukemia.

Double immunofluorescence studies using both surface and cytoplasmic antigens were performed on cells of some human hematopoietic lines. We tested several permeabilization protocols in order to optimize, improve and simplify flow cytometric assay to detect the combinations of two markers present in one cell which could be regarded as leukemia-related markers. It was found, that buffered formaldehyde-acetone (BFA) fixation renders the cell membrane permeable without destroying surface antigens so that intracellular and cell surface markers could be measured simultaneously by flow cytometry. Cell lines used for the experiments reported here included MOLT4 T cell line, mature B cell lines DAUDI and U-266, and early B cell line REH-6. Results from our studies demonstrated, that in the absence of CD3 antigen on the surface membrane of viable MOLT4 blast cells, double labeling of fixed, permeabilized cells revealed 97% mCD7+, cCD3+ double positive cells. Two color staining with anti-CD19 and anti-CD22 monoclonal antibodies (MoAbs) in DAUDI cells showed, that larger part of cCD22+ cells expressed mCD19 antigen. CD22 antigen was absent on DAUDI cell membrane. Of great interest was the finding, that the marker detected by anti-CD19 MoAb which was absent on the membrane of U-266 cells was detected in their cytoplasm. Double staining of these cells revealed, that the number of mCD22+, cCD19+ double positive cells was 80%. Cytoplasmic CD22 antigen along with surface membrane CD19 was used to define early B cell line REH-6 as well. Our results demonstrate majority of double positive cells among tested population (mCD19+, cCD22+). To our knowledge the presence of cytoplasmic IgM detectable by flow cytometry in REH-6 cells, which could be so regarded as a precise and adequate counterpart to pre-B acute leukemia cell phenotype in children, is an original finding. Immunological typing plays an important part in the multiple marker analysis of hematopoietic malignancies. Through these surface and cytoplasmic marker combinations minor neoplastic cell populations could be detected. Human hematopoietic cell lines could serve as a reliable model system for monitoring minimal residual disease in acute leukemia patients.

Evaluation of different fixation-permeabilization methods for simultaneous detection of surface, cytoplasmic markers and DNA analysis by flow cytometry in some human hematopoietic cell lines.

Different fixation/permeabilization methods were investigated for their convenience for simultaneous detection of membrane marker/DNA staining or cytoplasmic marker/DNA by flow cytometry. Nine different methods were employed. The expression of membrane marker CD20 and cytoplasmic marker CD22 on BJAB and DAUDI cells and cytoplasmic CD3 on MOLT4 cells were measured. Optimal methods were those that combine paraformaldehyde and saponin (for membrane CD20/DNA staining and cytoplasmic CD3/DNA staining) or buffered formaldehyde-acetone (for membrane CD20/DNA staining and for cytoplasmic CD22/DNA staining). Special interest was focused on proliferation marker CD71 and nuclear antigen Ki67. We investigated CD71 in MOLT4 cells and Ki67 in MOLT4, DAUDI and BJAB cells. Both of these markers are closely associated with proliferation rate. The optimal method for detection of Ki67/DNA staining combines paraformaldehyde with Tween 20.

Analysis of the phenotype and cellular DNA content in some leukemias by flow cytometry.

Phenotype and cell cycle distribution in peripheral blood and bone marrow mononucleated cells was studied in patients with different leukemias: T-ALL, AML, CLL, CML and plasmocytoma. DNA flow cytometry with propidium iodide fluorescence was used. Differences in cell cycle between mononucleated cells from T-ALL and CML patients on one hand and normal controls on the other were seen in peripheral blood but not in bone marrow specimens. Patients with AML, CLL and plasmocytoma showed a cell cycle distribution of mononucleated cells similar to normal controls. DNA content analysis in some leukemias were discussed.

Flow cytometric determination of leukemia-associated marker combinations for the study of minimal residual disease.

To study the minimal residual disease in acute leukemia patients we used some marker combinations related either to the simultaneous surface membrane and cytoplasmic marker expression, or to the expression of atypical marker combinations, that are absent or extremely rare in normal hematopoietic cells. We investigated to which extent flow cytometric analysis of leukemia-associated marker combination may contribute to sensitive follow-up in patients with acute leukemia. For this purpose dilution experiments were performed, in which artificial mixtures of normal peripheral blood lymphocytes and leukemia cells from a patient with leukemia-associated phenotype were prepared and analyzed for double positive cells. Our results showed that the sensitivity of double color immunofluorescence assay was 3 in 10(4) cells. Sequential studies of residual disease were evaluated in five acute leukemia patients with leukemia-associated markers combinations at diagnosis. In three of them morphologic relapse was preceded by the immunologic detection of small amounts of leukemia cells, while in two other cases, in which no double positive cells for leukemia-associated markers were found, patients are still in hematologic remission. This approach to the study of minimal residual disease could be valuable in monitoring the efficiency of chemotherapy, as well as in evaluating the quality control of bone marrow before autografting. Furthermore, flow cytometric approach can efficiently complete other methods, which are used for more exact definition of remission in acute leukemia patients.

Cytoplasmic and surface membrane phenotypic markers in cells of B cell chronic lymphocytic leukemia.

Peripheral blood cells of twenty-six patients with B cell chronic lymphocytic leukemia (B-CLL) were characterized for their surface membrane and cytoplasmic marker profiles using flow cytometry and fluorescence microscopy. According to surface membrane marker analysis three distinct immunophenotypic subgroups of B-CLL were identified: group I (SIg+, MR+, CD5+, B Ag+, T Ag-; 19 cases), group II (SIg+, MR+, CD5+, B Ag+, TAg+; 3 cases), group III (SIg-, MR+, CD5+, B Ag+, T Ag-; 4 cases). Cells from all patients were positive for the CD19 antigen and at least one of other B cell antigens. Cells from all patients expressed also CD5 and HLA-DR antigens and formed mouse rosettes (MR). Great heterogeneity was found in the membrane and cytoplasmic marking by anti-CD22 MoAb. In four of 23 patients tested, CD22 antigen was expressed in the cytoplasm of CLL cells while it was absent on surface membrane of these cells. This finding was discussed from the point of certain cell heterogeneity in the followed B-CLL cases. Cytoplasmic immunoglobulin (CyIg) detection showed to be very important especially in group III of followed B-CLL cases with undetectable surface immunoglobulins (SIg). Cytoplasmic antigens and immunoglobulin determinations are useful in phenotyping every B-CLL patient, as well as in the immunological study of different maturation stages of B lymphocytes.

Leukemia-associated marker combinations in acute leukemia suitable for detection of minimal residual disease.

In the absence of truly leukemia-specific antigen, antigen combinations were identified in leukemia cells that are absent or extremely rare among normal hemopoietic cells. Some of the studied combinations related to the simultaneous surface and cytoplasmic marker expression, others, expressed mainly on cell surface membrane, represented atypical or aberrant combinations. Comparing membrane (m) and cytoplasmic (c) antigen expression (followed in 23 acute leukemia cases), we observed that CD3 could be detected in cytoplasm in the majority of T-ALL cells, while was absent on cell surface membrane where simultaneous expression of more immature T cell markers, such as CD7 and CD5, could be detected. Combination of mCD7/cCD3 could be regarded as a suitable marker of individual T-ALL cells. In cases of B-precursors of acute leukemia cells, leukemia-related combination of mCD19/cCD22 was found, which could characterize a single leukemia cell. The cells in one of 11 AML followed cases were positive for CD13 in cytoplasm, but not on cell surface membrane, where CD33 and other myeloid antigens were expressed. The cells in another two AML cases were positive for CD11 in cytoplasm but not on cell surface membrane, where CD13 or CD33 were expressed. Again, marker combinations of mCD33/cCD13 and mCD13 or mCD33/cCD11, respectively, represent a leukemia-related feature, suitable for tracing single leukemia cells in double immunofluorescence. Acute leukemia defined by the coexpression on most blast cells of antigens classically attributed to different lineages (referred as atypical/aberrant marker combinations) remains a rare event. We isolated a series of 27 (12%) such cases of 225 acute leukemia patients whose cells were immunophenotyped at diagnosis. Myeloid markers were present in T-ALL of two cases, T and B markers were coexpressed in 13 cases, markers of B and myeloid lineage were associated in one case, and T cell and myeloid antigens were found in 10 AML cases; in one AML case (M3 according to FAB classification) an aberrant nuclear coexpression of TdT was observed. In one case of the last group an interesting antigen combination of CD4/CD34 present in AML with monocytic differentiation was observed. When 5 patients with leukemia-associated (aberrant) markers were again analyzed at relapse, the relevant antigen combinations were retained in all of them. In summary, 44 of 50 cases (88%) from our acute leukemia series studied for leukemia-associated antigen combination, both with surface membrane and cytoplasmic marker combinations and those with aberrant markers coexpression allow the detection of minimal residual disease.

Detection of cytoplasmic and surface membrane markers in cells of some human hematopoietic cell lines.

The cells of some human leukemia-lymphoma T cell lines (JURKAT, MOLT4), B cell lines (DAUDI, U-266) and of myeloid U-937 cell line were characterized for their surface membrane and cytoplasmic marker profiles. The usefulness of some fixation and permeabilization methods of cell membrane for detection of cytoplasmic markers by flow cytometry was studied. The methods of cell fixation in suspension were found to be more sensitive than the methods of cell fixation in smears. With the very short buffered formaldehyde-acetone (BFA) fixation used in this study an optimal penetration of the monoclonal antibodies (MoAbs) through the plasma membrane and specific binding to the appropriate structures were achieved. CD22 antigen was detected in cytoplasm but not on membrane of DAUDI cells. In another B cell line, U-266, CD22 antigen was present both in cell membrane and cytoplasm. The marker corresponding to anti-CD19 MoAb was detected in cytoplasm but was absent on membrane of U-266 cells. Furthermore, the antigen estimated by anti-CD3 MoAb could be detected intracellularly in cells of both T cell lines tested, while it was absent on cell membrane of these cells. The phenotypic study of U-937 cells showed that the majority of cells expressed myeloid associated antigens. In our study the CD14 marker detected on cell surface membrane of U-937 cells was missing in their cytoplasm. The surface antigens remained intact after BFA fixation enabling a simultaneous detection of membrane and cytoplasmic markers in double immunofluorescence studies. Through this combination of markers minor cell populations could be detected. Human hematopoietic cell lines could serve as a reliable model system for a rapid and quantitative immunodiagnosis.

Flow cytometry (FCM) of early radiation response in epidermoid carcinoma of uterine cervix.

DNA flow cytometry (FCM) investigation of tumor specimens before and after 30 Gy 137Cs radiation treatment was performed in 33 cases of epidermoid uterine cervix carcinoma. Distinct differences in the type of FCM response to radiation were seen when the results of DNA index (DI) in diploid and aneuploid tumors and proliferation index (PI) values in diploid tumors from pretreatment and 30 Gy irradiated specimens were compared. We observed partial or total reduction of PI in 12 of 17 diploid and near diploid tumors, and total reduction of the aneuploid population in 14 of 16 aneuploid tumors. No significant correlation was found between the type of FCM response and clinical stage of the disease or the histological degree of differentiation.