Immunophenotype characterization of hematopoietic stem cells, progenitor cells restricted to myeloid lineage and their leukemia counterparts.

The aim of this review is to evaluate recent immunophenotype knowledge of hematopoietic stem cells and their restricted progenies committed to myeloid lineage - common myeloid progenitors, myelo-monocytic progenitors, megakaryo-erythroid progenitors and granulocyte progenitors up to mature neutrophil granulocyte. This study evaluates also recent knowledge of immunophenotype of leukemic stem cells and their more differentiated progeny committed to myeloid lineage - acute myeloid leukemia blast cells with regard to their phenotypic similarity to normal stem and granulocyte committed progenitor cells. Improved knowledge of normal stem and progenitor cells phenotypes, identifying new leukemia-specific markers, searching for aberrant marker expression and evaluation of aberrant intensity or combination of various marker expressions is important for distinguishing normal cells from their malignant counterpart in view of the diagnostics of leukemias or follow-up of minimal residual disease.

Possible prognostic value of nucleolar morphology in pathologic cells of B-chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) represents a heterogeneous disease with a very variable outcome. The reliable prognosis of this disease at the time of initial diagnosis is difficult to predict. The purpose of this preliminary study was to utilize the nucleolar morphology and to investigate the incidence of main nucleolar types in leukemic lymphocytes in B-CLL patients to assess their possible predictive value for the disease outcome, in correlation with immunophenotype parameters. The evaluation of nucleolar morphology of pathologic lymphocytes was performed at diagnosis and during the course of disease. Median follow up period of patients was 16.4 months (range from 2 to 32 months) from diagnosis. The nucleoli were visualized by a simple cytochemical demonstration of RNA and the proportion of main nucleolar types in pathologic lymphocyte population infiltrating bone marrow of 84 patients suffering from B-CLL was analyzed. The presence of ring shaped and compact nucleoli in leukemic lymphocytes divided patients into two subgroups with different outcome of the disease. Malignant lymphocytes of the majority of patients (Group 1, 71 patients, 84.5%) mostly contained ring shaped nucleoli. These patients were in stable phase and did not require any treatment during the follow up. The population of leukemic cells of a small group of B-CLL patients (Group 2, 13 patients, 15.4%) was characterized by the presence of various proportions of pathologic lymphocytes with one large compact nucleolus.Different response to the therapy discriminated the B-CLL patients whose leukemic lymphocytes revealed evident compact nucleoli at presentation, to next two subsets. Four of these patients (Group 2, 4/13, 31%) appeared to be resistant to chemotherapy, others (9/13, 69%) showed response to therapy, though the response time was variable. Leukemic cells with compact nucleolus morphologically resembled prolymphocytes, but hematologically and immunophenotypically did not fulfill the diagnostic criteria for prolymphocyte population. None of our B-CLL patients had the signs of transformation to prolymphocytic or other type of B cell neoplasms during the follow up. Our results indicate the possibility of relationship between the presence of malignant lymphocytes with compact nucleoli and unfavorable outcome in patients with B-CLL. The simplicity and utility of the nucleolar test as a possible prognostic parameter may help to identify the subset of patients with early B-CLL disease that will run a more progressive course.

Immunophenotyping parameters as prognostic factors in T-acute leukemia patients.

The main aim of this study represents the extension of our studies using multiparametric flow cytometry analysis for exact definition of membrane and intracellular (cytoplasmic and nuclear) markers of acute leukemia cells of T-phenotype. The study of blasts of each patient with all available monoclonal antibodies targeted to T-cell differential antigens and against possible marker coexistence from different lineages has been performed. The main aim was concerned to more proper T-ALL diagnosis and stage definition and identification of the prognostic factors and the useful markers for the follow-up of T-ALL in remission. New knowledge of the T-cell maturation stages of hematopoietic cells in bone marrow and thymus has been applied, as each T-acute leukemia clone is representative of one blocked stage through maturation. We evaluated 44 patients with T-ALL by multiparameter flow cytometry. Patients with more favorable prognosis (i. e. those of cortical stage) could have been already differentiated at diagnosis from those, allocated to pro-T stage, with very immature phenotypes and of an unfavorable clinical course. These patients had very distinctive immunophenotes, CD1a and CD8 markers completely negative, CD7 and cCD3 positive; CD5 was weakly expressed and myeloid markers CD33 and CD13 were coexpressed, or immature markers CD34, HLA-DR were coexpressed, together with myeloid markers CD13 and CD33 of weak positivity. The patients were either completely unresponsive to therapy or because of persistent MRD during continuation therapy, indicated for allogeneic hematopoietic stem-cell transplant. The results have been discussed with similar the most relevant immunophenotypic results of others and mainly with gene-expressing profiling associated with a significantly worse clinical outcome.

Normal maturation sequence of immunoglobulin light and heavy chains in hematogone stages 1, 2 and 3 in acute leukemia after treatment.

The cellular diversity of bone marrow samples was studied by using multi-dimensional cluster analysis of six-parametric flow cytometry data (four CD, forward scatter and side scatter), focusing mainly on acute leukemia blast cells and regeneration of normal B-cells, hematogones. This approach should enhance the ability to study normal hematopoiesis, and to identify and monitor hematopoietic disorders. The study was performed on a homogeneous group of patients (mainly children), all of them after finishing complete therapy for AL, mostly B-ALL. In all of these patients complete pattern of all three individual Hg stages was present. Maturation spectra of surface immunoglobulin kappa (sIgkappa) and lambda (sIglambda) light chains and IgM, IgA heavy chains in all three stages of Hgs are presented as reliable reports on sIgs as their incidence on Hgs are scarse and even contradictory. The Ig expression paralles CD20 expression. SIg of light (kappa,lambda) and heavy (IgM, IgA) chains were completally absent in stage 1 Hgs and their expression increased through stage 2 to 3; IgM was expressed similarly. Light Ig chains kappa/lambda were expressed in a polytypic way. The results completed information on normal maturation sequence of bone marrow stage 1, 2 and 3 hematogone regeneration in treated acute leukemia patients.

Increased myeloid precursors in regenerating bone marrow; implications for detection of minimal residual disease in acute myeloid leukemia.

Presented study is focused on exact immunophenotypic definition of myeloid precursors and their following stages in regenerating bone marrow during treatment of ALL/AML for correct interpretation of the immunophenotype results and proper distinction from minimal residual disease (MRD) by multiparameter flow cytometry. This study includes bone marrow samples from 36 controls, 27 patients with AML, 39 patients with B-ALL undergoing therapy who remained in complete remission after treatment and also 30 B-ALL patients one year after the end of therapy. We observed substantial expansion of immature bone marrow populations in the regenerating bone marrows, which were identified by expression of CD34 and/or CD117 markers by 4-color flow cytometry. Myeloid precursors were significantly increased after cessation of induction therapy cycle of B-ALL (1.27+/-2.04%, p=0.0064) and also AML patients (0.87+/-0.77%, p=0.001), but also during follow-up of B-ALL patients (1.42+/-2.36%, p=0.0001) when compared with non-treated controls (0.38+/-0.29%). Some cases where their frequencies achieved up to 12% reflect the massive regeneration of myeloid lineage in bone marrow after chemotherapy cycles. Especially in these cases accurate interpretation of such a high frequency of immature myeloid cells as myeloid precursors was very important to exclude incoming relapse or secondary leukemia. The myeloid precursors represented by CD34+ in regenerating bone marrow expressed CD45 (94.8+/-5.5%), CD117 (38.3+/-26.2%), CD38 (91.4+/-5.7%), HLA-DR (90.6+/-7.6%), CD13 (73.0+/-20.8%) and CD33 (85.2+/-15.6%), while CD90 (2.7+/-2.5%), CD133 (10.0+/-8.2%) and T or B lymphocyte markers were negative. Comparing immunophenotypes with control bone marrows, only difference in expression of CD33 marker was found (85.2+/-15.6% versus 63.0+/-17.4% p=0.024). In addition, according to expression of these markers three different subsets of myeloid precursor cells were identified in regenerating bone marrow samples: CD34+ CD117- HLA-DR+, CD34+ CD117+ HLA-DR+ and CD34- CD117+ HLA-DR-/+ without aberrant marker expression. In conclusion for the correct discrimination of MRD in acute leukemia it is indispensable to define the range of normality in myeloid differentiation by extensive studies of bone marrows not only from healthy donors but also from regenerating bone marrow of patients undergoing therapy.

The impact of cell heterogeneity and immunophenotypic changes on monitoring minimal residual disease in acute myeloid leukemia.

Monitoring of minimal residual disease (MRD) becomes increasingly important for the more accurate stratification of the therapy in acute leukemia. The purpose of this study was to characterize in detail the phenotypes of heterogeneous population in various AML subtypes and to identify the leukemia associated aberrant phenotype (LAP) in individual patients with AML for precise investigation of MRD. The impact of heterogeneity of pathological populations, the effectiveness of location AML blasts on CD45/SSC dot plots in AML patients during follow-up and phenotype changes on MRD monitoring were evaluated in the second step. Bone marrow samples from 63 patients with AML were analyzed at diagnosis, 33 were selected for monitoring of MRD during follow-up and 13 analyzed at relapse using a wide antibody panel in quadruple combinations by multiparameter flow cytometry. In 88% of AML patients at least one LAP was defined at diagnosis, two or more aberrancies coexisted in 60% of them. The total number of LAPs identified by application of various combinations of antibodies was 112 (mean = 2.04 LAP/patient) and the median percentage of blasts carrying the LAP was 53.57%. In half of the patients, we were able to detect the presence of at least two subpopulations, which not always shared the same aberrancy. Although AML cells often have light scattering properties similar to those of normal (myeloid and B-lymphoid precursors, basophiles etc.) in a fraction of cases we found also very useful the location on CD45/SSC dot plots for MRD discrimination. In 13 patients relapse occurred and although we found in 69% changes of phenotype when comparing diagnosis and the first relapse, at least one LAP was constant in 92%. According to our observations, in majority of patients with AML monitoring of MRD by multiparameter flow cytometry is feasible although in some cases could present some specific difficulties owing to their immunophenotypic heterogeneity, similarity with other cell subpopulations or shifts at relapse. In conclusion, investigation of MRD should be based on the phenotypic characteristics of each subpopulation even if it is present in low frequencies.

Amplification of AML1 gene in association with karyotype, age and diagnosis in acute leukemia patients.

The AML1 gene, located in the chromosomal band 21q22, belongs to the runt domain family of genes and encodes the subunit of the core-binding factor (CBF). AML1 is normally expressed in all hematopoietic lineages and is essential for the transcriptional regulation of a number of hematopoietic specific genes. In acute leukemia three types of abnormality of AML1 have been observed -- chromosomal translocations, point mutation and duplication or amplification of the unrearranged gene. The most common origin of extra copies of the AML1 gene is polysomy of chromosome 21 or a partial duplication of the long arm of chromosome 21, less frequently ring, isochromosome or the tandem repetition of chromosome 21. In the study 13 children and 5 adults with ALL and AML or MDS, respectively, have been included. Using standard G-banding and dual color FISH analyses, gain of AML1 originated in polysomy of chromosome 21 in each group of patients was proved. True high-level amplification was not observed but some uncommon changes in noteworthy association with other chromosome aberrations, age or diagnoses were presented.

The value of dot plot patterns and leukemia-associated phenotypes in AML diagnosis by multiparameter flow cytometry.

The immunophenotypic features in patients with acute myeloid leukemia (AML) were investigated at diagnosis using a wide antibody panel including progenitor-associated, myeloid and lymphoid markers in quadruple combinations. Analyzed were bone marrow samples from 37 adult and pediatric patients for exact identification of AML blasts according their localization on CD45/SSC dot plots and aberrant immunophenotypes in various subtype of AML. We found the localization of AML blasts on CD45/SSC dot plots, which in combination with immunophenotype profile of blasts allow discrimination of several AML subtypes (M0-M2, M3, M4/M5 and other types). In 27/37 AML patients (73%) at least one leukemia-associated phenotype (LAP) was found, two or more aberrancies coexisted in more than a half of them (78%). Asynchronous expression was the most frequent type of LAP (77.8%, 21/27) followed by coexpression of lymphoid-associated antigens, which occurred in 18/27 (66.7%) patients. Presented study showed that leukemic cells of each AML patient had a unique antigenic profile and could be discriminated from their normal counterparts based on typical light scatter profiles and aberrant antigen expression that could further be used for detection of minimal residual disease.

Myeloid enzymes profile related to the immunophenotypic characteristics of blast cells from patients with acute myeloid leukemia (AML) at diagnosis.

The purpose of this study was to assess the possible relationship between the cytochemical enzyme profile and immunophenotypic characteristics of distinct acute myeloid leukemia (AML) subtypes in discrete stages of leukemic cells maturation. As the proportion of leukemic blast cells is critical for exact cytochemical analysis, study was restricted to the evaluation of 48 adult and pediatric patients with newly diagnosed AMLs with 80% or more blasts in analyzed samples. The cytochemical investigation of myeloperoxidase (MPO), Sudan black B (SBB), chloroacetate esterase (CAE), alpha-naphthyl butyrate esterase (ANBE), alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AP) in peripheral blood and/or bone marrow was performed. The immunophenotype was examined for the maturation dependent myeloid antigens CD13, CD33, CD11b, CD14, CD15, CD65, CD36, cytoplasmic MPO, non-lineage associated CD34 and HLA-DR antigens, lymphoid- associated antigens CD7, CD4, CD38 as well as natural killer cell associated marker CD56. Flow cytometry by double marker staining and visualization of pathologic cells in dot plots reflected immunophenotypic aberrancy and degree of cell maturation. The patients were classified into AML subtypes M0- M2, M3, M4 and M5 according to the main morphological, cytochemical and immunophenotypical features. The variable combinations of MPO, SBB, CAE and ANBE were identified in relation to immunophenotype. The cytochemical profile of blasts was in concordance with immunophenotype, particularly in more differentiated AML subtypes, M3, M4 and M5. The findings of myeloid antigens expression and cytochemical features in poorly differentiated AML subtypes showed no practical relevance of cytochemical analysis. Notwithstanding that the cytochemical analysis of AML subtypes not sufficiently identifies the distinct aberrancies in heterogeneous leukemic blast cell populations, evaluation of the cytochemical profile in connection with immunophenotyping may help to classify the AML patients to relevant subtypes with more accuracy.

The knowledge on the 3rd type hematogones could contribute to more precise detection of small numbers of precursor B-acute lymphoblastic leukemia.

Bone marrow hematogones (benign B-lymphocyte precursors) may cause diagnostic problems due to their morphologic and immunophenotypic similarities with neoplastic lymphoblasts. Hematogone populations in presented study containing 358 bone marrow specimens of 251 individuals always exhibited a continuous and complete maturation spectrum of antigen expression typical for normal evolution of B-lineage precursors; lacking aberrant or asynchronous antigen expression. In contrast lymphoblasts of 19 bone marrows of precursors B-ALL patients showed maturation arrest and exhibited several immunophenotypic aberrancies. Hematogones were identified by 4-color flow cytometry using optimal antibody combinations in many bone marrow samples. They were more commonly found in higher numbers in children, and there was found a general decline in hematogones with increasing age. Bone marrow hematogones were separately assessed as hematogones 1 population of early stage and hematogones 2 of mid-stage precursor B-cells, respectively. In some (about 30%) of hematogones a third type hematogones could be assessed in bone marrow samples. This small B-cell subpopulation was defined by CD10-positivity, coexpressing more mature markers CD19,CD20,CD22 and CD45bright. These cells obviously blended with those of mature B-lymphocytes (CD10-negative) on CD45/SSC, and could be better recognized on CD10-gating. Quantitative immunophenotyping of this study completed the percent antigen expression data in two main hematogone subtypes and lymphocytes in 16 bone marrow specimens and precursor B-ALL lymphoblasts in some samples. Increased information on benign B-lymphocyte precursors, especially that of existence of the 3rd type hematogones could provide a basis for better discrimination of B-leukemia cells even in a very small amounts.

Granular acute lymphoblastic leukemia in a 15-year-old boy.

We report a case of a boy with acute lymphoblastic leukemia expressing granular inclusions in the cytoplasm of blastic cells. Granular lymphoblasts had mostly L2 morphology but azurophilic granules were also present in part of the cells with L3 morphology. Immunophenotyping clearly indicated a lymphoid origin of the blasts and showed positivity of HLA-DR, CD10, CD19 and CD24 markers. Cytogenetic analysis brought normal karyotype 46 XY. Molecular genetic analysis showed immunoglobulin heavy chain rearrangement (IgH R/R) and T-cell receptor gamma rearrangement pattern (TCR-gamma C/R). TCR-beta and TCR-delta did not show rearrangements. The course of the disease was favourable. The patient achieved initial complete remission within 4 weeks of protocolar treatment and the remission remains until now, 5 years from the diagnosis and three years after finishing the treatment.

Multiparametric flow cytometry in detection of minimal residual disease in acute lymphoblastic leukemia of early B-cell phenotype.

Leukemic cells can be distinguished from normal hematopoietic cells on the basis of chromosomal or molecular abnormalities, antigen receptor gene rearrangements and immunophenotype. Set of 3-, 4-combination of monoclonal antibodies was used for exact definition of immunophenotypic characteristics of B-cells populations from healthy donors and aberrant, asynchronous, over/under-expressed phenotypes and detection changes in intensity expression of markers that characterized pathological leukemic B-cells at diagnosis. These differences in normal and abnormal cell patterns were very important and could be utilized for analysis of minimal residual disease. On the basis of these findings we were able to clearly distinguish residual leukemic cells from hematogones (healthy B-lymphocyte precursors) too. We also verified that in some cases the CD58 marker is overexpressed on CD10+, CD34+ blast cells at diagnosis and can be feasible used for detection of minimal residual disease (MRD).

Hairy cell leukemia: early immunophenotypical detection and quantitative analysis by flow cytometry.

UNLABELLED: The abnormal coexpression of the so-called 'HCL-restricted' markers (CD22+CD11c, CD25 and CD103) identified on monotypic, slightly large B-lymphocytes in the large cell-gate of dot-plots has previously been shown to be highly characteristic of hairy cell leukemia (HCL). The main aim of our present study was to determine if patterns with low levels of neoplastic cells in bone marrow (BM) or peripheral blood (PB) are of a value the early diagnosis and/or detection of minimal residual disease (MRD) in HCL. Next we wished to determine if quantitative immunophenotyping given by molecules of equivalent soluble fluoresceine (MESF) could help to distinguish pathologic B-lymphocytic pool from that of normal residual B-cells also in patients with low numbers of HCL cells. The abnormal immunophenotypes were studied in 174 specimens from 19 patients with suspect HCL or during follow-up of already treated patients. For evaluation of marker density fluorescent calibration microbeads were used. In 12 HCL patients (67%) permanent complete remission was observed after treatment. In 6 patients (33%) transient MRD+ phenotype was identified but the clinical manifestation of relapse was followed till now in only three patients. One patient was phenotyped just only at diagnosis. The pathological cells in low levels were found in 5 patients at diagnosis (in the range from 2 to 12%) and in patients with MRD+ phenotype they were recognized repeatedly in the range from 2 to 8%. Furthermore, we observed in hairy cells significantly higher values of molecule numbers of some B-cell markers, comparing to that of residual B-cells in nonleukemic lymphocyte gate of the same sample. We found profound and persistent CD4+ lymphopenia in all but one studied patients after CdA treatment. CONCLUSIONS: Flow cytometric immunophenotyping of PB and BM is highly sensitive and specific method and is capable to detect low levels of malignant cells in HCL. Quantitative analysis of MESF values of pathological B-cells comparing to normal residual B-cells seems to be another new marker of HCL in common, which is reliable detecting also small cell numbers in examined sample. A long-term decline of CD4+ T-cells correlated with the relatively low incidence of clinical progression of HCL.

NK-cell activity affected by some cytostatic drugs and their additives.

The direct effect of selected cytostatic drugs on natural killer (NK) cell activity was evaluated. Peripheral blood mononuclear cells from healthy donors were tested for their cytolytic activity in vitro in the presence of adriamycin, methotrexate, leucovorin, vincristin, cytosine arabinoside and teniposide. Most of the tested cytostatic drugs did not show to be active at concentrations comparable to their plasma level. However, diluents of some preparations (cytosine arabinoside, teniposide) containing organic solutions and stabilizing additives (e.g. benzyl alcohol) suppressed the NK activity more than chemotherapeutic agents alone. Thus teniposide, containing such additives, inhibited NK activity already at 5 mg/ml, while its peak plasma concentration was 23.8 mg/ml. The inhibitory concentrations of teniposide did not affect the target binding of effector cells and the expression of 14 tested leukocyte differentiation markers. This implies that a postbinding step of the lytic process was altered by the preparation. Likewise, no inhibition of lectin dependent cellular cytotoxicity by teniposide and its diluent was observed, suggesting that the lectin may substitute the missing lytic signal.

Sequential development of chronic lymphocytic leukemia in a patient with polycythemia vera.

A patient with a seven-year history of polycythemia vera treated by repeated phlebotomies and intermittent busulfan administration developed gradually lymphocytosis accompanied by thrombocytopenia in peripheral blood and in the bone marrow. A marked pathological monoclonal proliferation of the B-cell population was detected. The sequential development of chronic lymphocytic leukemia in the patient with polycythemia vera could be considered as a coincidence because there is no reliable explanation of this event at present.

The value of multiparameter flow cytometry of cerebrospinal fluid involved by leukemia/lymphoma cells.

The usefulness of multiparameter flow cytometric (FC) analysis of cerebrospinal fluid (CSF) was evaluated in leukemia/lymphoma patients having central nervous system (CNS) involvement of the disease. In 12 specimens of 8 patients with different types of leukemia/lymphoma (one case of T-ALL, 3 cases of early B-cell ALL, one case of AML, and 3 proven or suspicious NHL cases) the presence of pathological clone in CSF has been confirmed or excluded. The phenotypic patterns of CSF cells were defined according to those of bone marrow (BM)/peripheral blood (PB) at diagnosis or during follow-up of the same patients. Furthermore, in one case of suspicious CNS infiltration of NHL, the pathological clone was characterized as a highly suspicious of solid tumor and was proved to be a lung cancer metastasis. The definition was made on the basis of CD45 (common leukocyte antigen) and other studied CD markers negativity. The exact comparison of immunophenotypic profiles of specimens from different sites (CSF, BM, PB) of the same patient has been performed and no phenotypic changes were found. In some CSF specimens, where no cells of suspicious pathological clone were detected, in 4-color analysis only normal lymphocyte population was found even in small cell samples (even if the cellularity was < than 0.3x10-6). In these populations the high values of T-cells (CD3+) predominated and the high prevalence of CD4+ over CD8+ cells, and an almost total lack of B-lymphocytes was found. Our results suggest that positive CSF immunology is a useful indicator of malignancy and reflects leptomeningeal involvement. Simultaneously we demonstrated that FC analysis of CSF in the aim to detect possible CSF seeding of leukemia/lymphoma is a reliable and quick technique.

Analysis of surface and cytoplasmic immunoglobulin light/heavy chains by flow cytometry using a lysed-whole-blood technique: Implications for the differential diagnosis of B-cell malignancies.

The study was aimed at the proper detection of surface and cytoplasmic clonal Ig light/heavy chains in the frame of multiparameter flow cytometry analysis of some B-cell malignancies. An exact direct evidence has been obtained that the leukemia cells following staining by antibodies to immunoglobulins will need to be washed to eliminate free plasma Igs. The results of proper Ig detection with simultaneous unaltered staining of further 2-3 markers on the cell surface after elimination of free plasma Ig in the whole blood sample are described. In differential diagnosis of some chronic B-cell malignancies and subclassification of some acute B-leukemias the detection of intracytoplasmic light/heavy chain Igs is required. The unique phenotypic structures of multiple myeloma (MM) cells have been utilized in our approach to detect cytoplasmic Ig light and heavy chains. A modified 2-step method for analysis of cytoplasmic immunoglobulin light chains by flow cytometry in MM patients was used and the method was extended for measurement of IgM heavy chain in B-ALL. For membrane staining in MM patients cells the combination of CD45-FITC and CD138-PE was used; the CD138 was found to be more specific than CD38 for MM cells. The whole blood cells were lysed, acquired on flow cytometry (first acquisition), then permeabilized by paraformaldehyde and saponin, and incubated with anti-kappa-FITC and anti-lambda-FITC antibodies and acquired again (second acquisition). In B-ALL patients cells in first step the combinations of CD45-FITC or CD22-FITC and CD10-PE have been successfully applied and after RBC lysis, acquisition and membrane permeabilization anti-IgA-FITC and anti-IgM-FITC were applied and cells were acquired again. The FITC fluorescence intensity of the second measurement was equal to the sum of surface CD45 or CD22 marker expression during the first step, and cytoplasmic clonal light or heavy chains expression during the second acquisition in both, MM and pre-B ALL patients, as well.

Antigen modulation followed by quantitative flow cytometry of B-chronic lymphocytic leukemia cells after treatment.

Presented study analyzed the immunophenotypic characteristics and antigen density of peripheral blood (PB) and bone marrow (BM) cells of 23 patients with B-chronic lymphocytic leukemia (B-CLL) and 10 control subjects using flow cytometry. The patients were subclassified into two groups. Group I formed 13 patients with B-CLL at the time of diagnosis and group II 10 patients with B-CLL after the therapy but not in remission. For definition of B-CLL cells we used immunological marker analysis of surface markers characteristic for B-CLL pattern: CD5, CD19, CD20, CD23 and HLA DR and enumeration of fluorescence intensity of these markers given by molecular equivalent of soluble fluorochrome--MESF. In group II of B-CLL patients, who were already treated, in PB and BM somehow lower MESF values for CD19, CD20 and CD23 markers and higher MESF values for CD5 marker (in PB and BM) than in group I patients have been detected. The MESF level of HLA DR marker was little higher in group II than in group I B-CLL patients. However in PB and BM the percentage expression of HLA DR and CD19 markers in both patients groups was approximately the same. The values of HLA DR, CD19, CD20, CD23 and CD5 markers (% expression and MESF values) in both patients groups with B-CLL were significantly higher versus controls (p<0.001 resp. p<0.01) even in PB and BM. In conclusion, in our study we observed that the patients with B-CLL after therapy (group II) had similar or a little smaller (except CD5) but nonsignificantly decreased expression level of markers characteristic for B-CLL, but the MESF values of some of them (CD19, CD23) were significantly (p<0.05) decreased when compared with untreated B-CLL patients (group I). The determination of antigen density (MESF values) may be an important marker to characterize the leukemic cells. Our results showed that chemotherapy did not influence in a significant level the antigen modulation of B-CLL cells, however, could influence MESF values of some characteristic markers. Quantitative analysis of some markers in B-CLL cells seems to offer valuable information concerning possible influence of some chemotherapeutics on antigen equipment of leukemic cells.

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.

Further evidence on lymphocyte resistance of tumor-bearing in vitro rats to phytohemagglutinin.

A study was made of the survival of peripheral blood lymphocytes in short-term cultures with a standard dose of phytohemagglutinin (PHA) in rats during the course of methylcholanthrene (MC)-induced carcinogenesis. Peripheral blood lymphocytes of (BDXLW)F1 hybrid rats were tested at various time intervals following MC application. The investigation involved a group of rats in which tumors had not appeared as yet after MC application, a further group with developed tumors of varying sizes, and a third group in which the tumors were extirpated, their peripheral blood lymphocytes being examined both before and at a definite time interval after the extirpation. Finally, another group of animals was investigated in which no tumor appeared up to 14 months after MC administration. The higher survival values of lymphocytes in PHA cultures in animals with primary MC-induced tumors in comparison with those of the control animals were found to be related to the presence of a progressing tumor. The results are discussed in terms of a possible change of T and B lymphocyte ratio in the peripheral blood in the presence of a tumor antigen.