Correlation of morphological FAB classification and immunophenotyping: value in recognition of morphological, cytochemical and immunological characteristics of mixed leukaemias.

Correlation between the FAB classification and immunophenotype was studied in 169 consecutive adult patients with acute leukaemia (AL). The lineage of leukaemic cells could be determined in the majority of cases, whereas 3 patients (1.8%) remained unclassified. In 22 out of 71 patients (31%) with acute myeloid leukaemia (AML) FAB M1 and M2 types, and in 5 out of 16 patients (31%) with chronic myeloid leukaemia (CML) in myeloid blast crisis, leukaemic cells did not express myeloid lineage-related markers, indicating asynchronous expression of cell markers in a substantial proportion of patients. Flow cytometric two-colour immunofluorescence revealed mixed AL immunophenotype in 6 out of 169 patients (3.4%). This group included five CD2+AML (5% of AML tested) and one undifferentiated AL expressing CD10(CALLA), CDw65(VIM-2). The former group included FAB M1, M2, M3 and M4 forms of AML with a single cell population, and an AML M2 patient with both cytochemically and immunologically two separate populations of leukaemic cells. This further illustrates the heterogeneity of the target cell(s) for leukaemogenesis and the level of differentiation of AML cells. However, there was no difference in the treatment response and the remission duration between AML patients and patients with mixed phenotype AML.

Two-color flow cytometric analysis of preterm and term newborn lymphocytes.

Immune system maturation proceeds postnatally in humans. Therefore, newborns, especially those of a lower gestational age, are not fully immunocompetent and are more likely to acquire perinatal infections. In order to investigate the neonatal immune system status, the major lymphocyte subpopulations were studied in newborns of different gestational age, comparing term newborns and adults. The cord blood from 66 newborns and the peripheral blood from 23 adults were analyzed using fluorochrome labelled monoclonal antibodies and two-color flow cytometry. The newborns were divided into three groups according to their gestational age. Ten prematures were under 32 weeks of gestation, 35 were of 32-37 weeks and there were 21 term newborns. The percentage of cytotoxic T lymphocytes (CD4 CD8+) was lower in term newborns as compared to the adult controls (17.8 versus 30.3%), and so were the percentages of activated T lymphocytes (CD3+Ia+; 0.3 versus 3.7%), cytotoxic non-MHC restricted T lymphocytes (CD3+CD16+CD56+; 0.2 versus 1.8%) and NK cells (CD3-CD16+CD56+; 4.8 versus 15.5%). On the contrary, the proportions of unlabelled cells were increased in term cord blood. The expression of CD45R0 marker on neonatal lymphocytes was very low (1%). In comparison to the higher-gestation newborns, the lower gestation prematures had reduced percentages of T lymphocytes (CD3+; 43 versus 65%), mostly helper T lymphocytes (CD4+CD8-; 35 versus 50%), and increased percentages of unlabelled cells. The percentages of NK cells (CD3+CD16+CD56+) and B lymphocytes (CD3-CD19+; CD3-Ia+) did not differ among the tested newborn groups. There were no significant differences in major lymphocyte subpopulations between the group of highest-gestation prematures and the group of term newborns that differed significantly when compared to adults. The lowest-gestation newborns showed the most immature lymphocyte phenotype with the highest percentages of unlabelled cells.

Relationship between differing volumes of bone marrow aspirates and their cellular composition.

We compared the cellular composition of the first 1.0 ml volume bone marrow aspirate with that of an aliquot from the total bone marrow harvest at the end of the procedure in 17 healthy bone marrow donors. Each sample was assayed for its content of red blood cells, nucleated cells, CD2+, CD4+, CD8+, CD19+, HLA-DR+, CD56+, CD13+, CD33+, CD34+ and KiM8+ cells and CFU-GM. On the basis of data obtained, we estimated that the first 1.0 ml samples had 8.0 +/- 5.2% (SD) and the transplant samples 20.8 +/- 8.5% contamination with nucleated blood cells. The calculation revealed that both types of bone marrow samples had 100% volume contamination with peripheral blood, i.e. that bone marrow cells were aspirated within blood fluid volume. Nucleated cell concentration was 3-fold, and CFU-GM concentration 10-fold lower in the transplant than in the first-puncture 1.0 ml bone marrow samples. Various marker-positive cells appeared in transplant samples in concentrations that depended on their abundance in the first-puncture 1.0 ml and blood samples. Taken together, our data suggest that bone marrow harvesting would be substantially improved if individual aspirates were small in volume and taken from bone puncture sites as distant as possible.

AgNORs predictive value of prognosis in non-Hodgkin's lymphoma: comparison with flow cytometric cell cycle analysis.

Paraffin-embedded histopathologic specimens, taken before the commencement of therapy from 14 low-grade and 21 high-grade malignant lymphoma patients, and 9 normal lymph nodes were utilized to analyze six cell DNA-related parameters. The flow cytometry technique was used to determine cell-cycle G0/G1, S and G2/M phases, and silver staining to enumerate nuclear organized regions (AgNORs); nucleus surface area was determined by an image-analyzing system. The six parameters and natural logarithm of cell proportion in the S-phase (LS) were determined according to the histologic tumor type and achievement of the first complete remission (CR). All parameters except cell proportion in G1/M cycle phase differed significantly with respect to histologic cell type, but were not related to the achievement of first CR. Inasmuch as the parameters significantly correlated with each other, multivariate discriminant analysis and proportional hazard regression were applied to estimate their discriminant/predictive values with respect to tumor malignancy. AgNORs proved to be far superior in all three clinical parameters, S-phase was significantly predictive for the achievement of first CR, and LS for tumor histology type. The statistical model applied narrowed down the analysis of seven parameters to two with respect to tumor histology type (AgNORs and LS) and achievement of first CR (AgNORs and S), but only to one for overall patient survival (AgNORs). Only the model for tumor histology type discrimination was statistically significant (R2 = 0.904, p < 0.001). It appears that AgNORs may be of utmost predictive importance for the clinical outcome in NHL.

Analysis of data on leukocyte surface markers for recognition of leukemia/lymphoma phenotype pattern.

OBJECTIVE: This study was designed to test the algorithm for the recognition of leukemia/lymphoma pattern, based on cell immunophenotype assessed using specific monoclonal antibodies and measured using flow cytometry. DESIGN AND METHOD: Analysis was performed by comparing phenotyping data with reference data, followed by scoring of such comparisons. Output of the recognition was designed as a report list of possible diagnoses (defined as objects in the informatic system). Reference data were compiled from the respective literature. RESULTS: From 57 blood and bone marrow samples tested in this study, accurate recognition of the real diagnosis (object) appeared on the first four places of the report list in 54 (94.7%) samples. CONCLUSION: The list of the objects recognized by the use of algorithm appeared to be helpful in making a differential diagnosis, occasionally pointing to the states that the physician had not in mind at the start of the analysis.

Molecular mechanisms involved in the antiproliferative action of protein tyrosine phosphatase inhibitor potassium bisperoxo(1,10-phenanthroline)oxovanadate.

Potassium bisperoxo(1,10-phenanthroline)oxovanadate, bpV(phen), a powerful protein phosphotyrosine phosphatase inhibitor and a potent insulinomimetic, influenced three fundamental cellular processes in HL-60 human leukemic cells: 1) inhibition of proliferation, 2) induction of differentiation and 3) apoptotic cell death. In the presence of micromolar concentrations of bpV(phen) cell number and DNA synthesis decreased progressively with time of incubation. A single treatment with bpV(phen) (3 microM) activated a differentiation program; after 6 days of incubation 82% of cells were differentiated, but differentiation started already within the first 24 h. Concentrations of 5-10 microM bpV(phen) caused the characteristic DNA ladder pattern, starting after 4.5 h. Differentiation in HL-60 cells appear to be associated with activation of extracellular signal-regulated kinase while apoptosis is connected with phosphorylation and activation of both extracellular signal-regulated kinase and c-Jun N-terminal kinase in a concentration and time-dependent manner. The antiproliferative and apoptotic action of bpV(phen) could be exploited in combination chemotherapy in leukemia.

Prognostic relevance of non-Hodgkin's lymphomas cell cycle data.

Determination of proliferative activity of non-Hodgkin's lymphomas (NHL), aimed at improving the prediction of their clinical behavior, has gained considerable attention in the recent years. Flow cytometry has allowed rapid measurement of the cellular DNA content in terms of ploidy and proliferative activity. Flow cytometric DNA analysis was performed on paraffin embedded biopsy specimens taken from 125 patients with NHL. In 90 of them, proliferative index (PI) could be accurately measured and correlated with histology grade of the Working Formulation (WF). Intermediate and high grade NHL (54 patients) were analyzed together as HG-NHL. With the discrimination point for PI of 10%, the survival of high and low proliferative lymphomas was compared in the whole NHL group and within the WF prognostic groups. The median PI was 5% in LG (low grade) NHL and 10% in HG (high grade) NHL group. Acturial survival in NHL with high proliferative activity (39 patients) was 31% at 5 years and 15% at 10 years, and in NHL with low proliferative activity (51 patients) 53% and 18%, respectively (p = 0.002). In HG-NHL, survival at 5 years for low proliferative cases was 55% and for high proliferative cases 28% (p = 0.065), whereas in the LG-NHL group it was 54% and 28%, respectively (p = 0.059). The survival at 10 years was nearly equal in all groups. Proliferative index was associated with the overall survival of NHL in the whole group, as well as within the LG and HG prognostic categories. PI could differentiate more and less aggressive NHLs both within LG-NHL and HG-NHL. A tendency of survival curves toward continuous relapse was observed in low proliferative NHL and a tendency toward "plateau" in high proliferative NHL, irrespective of the histology grade.

Etiology and clinical features of anterior uveitis in southern croatia (dalmatia).

Patients with endogenous uveitis represent 6.5+ of patients in University Hospital Split, which serves most of South Croatia. Within a four-year period 208 patients were treated for endogenous uveitis. Results of clinical-laboratory examinations and treatment of 112 subjects suffering from anterior uveitis are presented and compared. Acute anterior uveitis (AAU) was the commonest form of uveal inflammation. It was present in 49+ of all uveitis cases and in 91.1+ of all anterior uveitis cases (AU). 67.6+ of the subjects with AAU had and 32.4+ did not have the HLA B(27) antigen. The inflammatory pattern in B(27)(+) patients was typical of B(27)(+) AAU. Patients with B(27)(+) AAU exhibited the same inflammatory pattern as those with B(7)(+) AAU. B(27)(+) AAU patients had significantly more systemic/rheumatic diseases (p>0.05), while patients with B(27)(-) AAU had significantly more infectious diseases (p>0.05). Forty percent of the patients with chronic anterior uveitis suffered from juvenile rheumatoid arthritis. The authors observed the rise in peripheral blood IgG, IgA, IgM, CD(2)(+), CD(4)(+) and B cells during the acute phase of AAU. Normalization of B cells (CD(20)(+)) was observed in early remission of anterior uveitis, about eight weeks after the onset of the disease.

Mutual predictive value of c-erbB-2 overexpression and various prognostic factors in ductal invasive breast carcinoma.

AIMS AND BACKGROUND: Breast carcinoma is a heterogeneous disease, the prognosis of which correlates with various prognostic factors. The aim of this study was to assess the prognostic significance of c-erbB-2 overexpression in breast carcinoma patients in association with other known prognostic factors. METHODS & STUDY DESIGN: The relationship between immunohistochemical expression of the c-erbB-2 oncoprotein and various established prognostic factors such as tumor size, axillary node status, estrogen and progesterone receptor status, DNA ploidy, proliferation index, cathepsin D expression and histological grade in invasive ductal breast carcinoma is presented in this study. RESULTS: Of the 93 ductal invasive carcinomas 22 (23.7%) were grade I, 51 (54.8%) grade II, and 20 (21.5%) grade III, and the majority (78: 83.9%) were 2-5 cm in diameter. Tumor metastases were identified in one or more lymph nodes in 55 (59.1%) patients, the remaining 38 (40.9%) patients being lymph node negative. According to the DNA histograms 40 (43.0%) tumors were aneuploid and 53 (57.0%) were diploid, and the majority of tumors had more than 4% of cells in the S phase of the cell cycle (83.9%). Expression of c-erbB-2 as shown by immunohistochemical intense membrane staining was present in 49 (52.7%) tumors. Cathepsin D-positive cytoplasmic granular staining and cathepsin D-positive stromal macrophages were found in 60 (64.5%) and 72 (77.4%) tumors, respectively. Univariate analysis showed that overall survival correlated significantly with axillary lymph node involvement and with estrogen and progesterone receptor status for each of the receptors separately and for their coexpression, and only marginally with c-erbB-2 overexpression. In mulitivariate analysis only axillary lymph node metastases and coexpression of estrogen and progesterone receptors were found to be independent and significant prognostic factors. CONCLUSIONS: When patients were stratified according to c-erbB-2 expression it was shown that those with c-erbB-2 overexpression and grade II tumors, tumor size greater than 2 cm, high content of aneuploid cells and cathepsin D-positive stromal macrophages had a shorter long-term survival than c-erbB-2 negative patients.

[Analysis of lymphocyte immunophenotypes in neonates]. / Analiza imunofenotipova limfocita novorodencadi.

Immune system development is not completed at the end of gestation, so newborns are not fully immunocompetent. In order to evaluate the neonatal immune system status and investigate the reasons for increased neonatal susceptibility to infections, the major lymphocytes subpopulations were studied in newborns comparing the results to adult controls. The cord blood from 21 term-newborns and the peripheral blood from 23 adults were analyzed using fluorochrome labelled monoclonal antibodies and two-color flow cytometry. The percentage of T lymphocytes was lower in newborns (64.9 versus 72.8% in adults), as well as the percentage of NK cells (4.8 versus 15.5%). On the contrary, the proportions of unlabelled cells were increased in term cord blood. The percentage of cytotoxic T lymphocytes was significantly lower in term-newborns as compared to the adult controls (17.8 versus 30.3%), and so were the percentages of activated T lymphocytes (0.3 versus 3.7%) and cytotoxic non-MHC restricted T lymphocytes (0.2 versus 1.8%). The expression of CD45R0 marker on neonatal lymphocytes was very low (1%). These characteristics of newborn lymphocytes phenotype are the result of inexperienced and partly undeveloped immune system.

Mechanisms involved in resistance of preirradiated Chinese hamster V79 cells to cytotoxic drugs are multifactoral.

We found previously that Chinese hamster V79 cells irradiated with multiple fractions of gamma rays (0.3 Gy of gamma rays daily, five times per week over 12 weeks) become resistant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and vincristine sulfate (VCR), sensitive to methotrexate and cis-dichlorodiammineplatinum (II), but exhibit no change in their sensitivity to gamma rays and ultraviolet light. The aim of this study was to elucidate the mechanisms by which these cells acquire the resistance to MNNG and VCR. Flow cytometric analysis shows this induced resistance is not the result of parasynchronization, i.e. the selective killing of the cells in the sensitive part of the cell cycle. The levels of protective molecules, glutathione, and metallothioneins were significantly increased in V79 cells irradiated with multiple fractions of gamma rays. Addition of verapamil reverses the resistance of these cells to VCR, suggesting the involvement of plasma membrane-associated P-glycoprotein in acquiring resistance to VCR. We infer that mechanisms of resistance to MNNG and VCR are multifactoral, involving changes in the plasma membrane as well as an increase in the cellular levels of glutathione and metallothioneins. Both mechanisms may be responsible for the non-effectiveness of the treatment for cancer, in which radiotherapy is used during or before chemotherapy.

Prognostic factors in squamous cell carcinoma of the larynx.

Sixty-three patients with squamous cell carcinoma of the larynx were included in a retrospective study examining the influence of the following prognostic indicators: localization, size of primary tumor, presence or absence of neck metastases, disease stage and histologic grade of differentiation. Flow cytometric analysis of the cell cycle, DNA ploidy and proliferative activity as direct prognostic indicators of tumor aggression was performed on paraffin-embedded blocks of specimens taken from 36 patients. Supraglottic tumor localization (p = 0.008), greater tumor size (p = 0.0064), local neck metastases (p = 0.00009), higher clinical disease stage (p = 0.0030), DNA aneuploidy (p = 0.0091), higher overall activity (p = 0.0001), and higher overall proliferative activity of diploid tumors (p = 0.0017) were found to be significant single unfavorable prognostic indicators of overall survival, whereas the histological grade of differentiation was not found to be a reliable prognostic indicator (p = 0.988). Only a higher overall proliferative activity of tumor cells was confirmed by the multivariate analysis as a reliable unfavorable prognostic indicator (p = 0.013). Cellular DNA content (ploidy, overall proliferative activity and overall proliferative activity of diploid tumors) correlated significantly with primary localization and size of the tumor, the presence of local metastases in the neck and the disease stage.

Alterations in lymphocyte phenotype of infected preterm newborns.

The cord blood lymphocytes from 44 premature newborns were analyzed using two-color monoclonal antibodies and flow cytometry. Depending on whether or not there was an infection at birth, the newborns were divided into two groups and the immunophenotypes of infected and uninfected newborns were compared. The percentage of T lymphocytes (CD3+) was significantly lower in the infected prematures. The percentage of both helper and cytotoxic T lymphocytes was lower. The proportion of activated T lymphocytes, cytotoxic non-MHC-restricted T lymphocytes, NK cells and B lymphocytes did not differ between the group of infected and the group of uninfected prematures. The percentage of memory helper T lymphocytes (CD45RO+CD4+) was very low in premature newborns regardless of whether or not they were infected and could not be used as a marker of bacterial infection at this age.

Leukocyte membrane markers on cell populations defined by six flow cytometric bitmaps.

Peripheral blood samples from healthy adults, patients with transplanted bone marrow, and healthy children were analyzed for the proportions of cells positive for CD2, CD4, CD8, CD19, CD56, CDw65, and KiM8 cell membrane markers. Six cell gates were defined on granularity vs. cell size (L90 degrees LSx-FALS) flow cytometric display for each blood sample: large lymphocytic, small lymphocytic, monocytic, mononuclear, granulocytic, and all-cell gate, and the listed markers were determined in each of them. Considerable differences in marker-positive cell proportions were found between healthy adults and children, and particularly between healthy adults and patients with transplanted bone marrow. Marker-positive cell frequencies corresponded to biologic distribution of three leukocyte populations within the defined gates, in a general agreement with known specificities of the antibodies used. A considerable degree of variations in the positions of gates drawn for different samples and the numbers of cells counted in them was observed. Still it appeared that all six gates, otherwise commonly used in flow cytometric analysis, could be precisely defined and yielded reproducible data. large and small lymphocytic gates yielded very similar marker frequencies, revealing that in cases where three leukocyte populations were not clearly delineated on (90 degrees LSxFALS) display, a smaller lymphocytic gate could be safely drawn in order to avoid contamination with monocytes.

Clinical, histopathological and flow-cytometric properties of incidental renal cell carcinomas.

Medical records of 63 patients operated on for renal cell carcinoma (RCC) between 1986 and 1996 in the Karlovac General Hospital were studied retrospectively. In 23 (36.5%) patients, the tumor was incidentally detected. The median patient age was 62 in the incidental group and 64 years in the symptomatic group (P > 0.05). Ultrasonography was the leading technique for incidental detection of RCC. The median tumor diameter was 6 cm in the incidental group and 9 cm in the symptomatic group (P < 0.001). Incidental carcinomas had a lower stage (P = 0.022) and a lower nuclear grade (P < 0.001) than the symptomatic ones. The incidental cases were associated with a more favorable ploidy status (P = 0.027) and a lower proliferative activity (P = 0.005). The 5-year survival rate was significantly higher in incidental (81.4%) than in symptomatic cases (44.3%) (P = 0.020). Univariate analysis showed that tumor stage, ploidy status, and proliferative activity were good prognostic parameters, while patient age, tumor size, and nuclear grade were not. Tumor stage was the only independent prognostic parameter in multivariate analysis. In conclusion, the incidentally detected RCC show more favorable clinical, histopathological, and flow-cytometric characteristics and their prognosis is significantly better than in symptomatic cases.

Significance of aneuploidy in non-Hodgkin lymphomas.

The incidence of aneuploidy was determined by flow cytometric analysis in paraffin-embedded tumor samples of 125 patients with non-Hodgkin lymphoma (NHL). There were 48 low-grade (LG) and 77 high-grade (HG) tumors. Aneuploidy was found in 34 (27%) NHL, 15 (31%) LG, and 19 (25%) HG tumors. The analysis of patient survival showed a significantly better survival of patients with LG than HG NHL, but the presence of aneuploidy did not influence the survival on the level of all patients, patients with LG or patients with HG NHL. In patients with LG NHL a tendency of diploid vs. aneuploid patients to survive longer was observed, but only at P = 0.056 significance level.

Natural killer (NKH-1+) cell number and activity in health and disease.

The proportion of lymphocytes bearing the NKH-1 membrane marker (a natural killer-cell marker) was determined by flow cytometric analysis of the peripheral blood of 40 healthy individuals, 45 patients with multiple sclerosis and 19 with other neurological diseases. Natural killer (NK) cell activity was determined in parallel. It was shown that NKH-1-positive cells were more abundant in the lymphocytic than the mononuclear cell window on the flow-cytometric granularity versus cell size two-parameter display. NK-cell concentration did not correlate with NK-activity in any of the three groups of individuals studied. However, when the data for the three groups were compounded (i.e. when the number of samples analysed exceeded 100) significant correlations were disclosed. No correlation was found between age and NKH-1-positive cell number or activity in any of the three groups of subjects. High variability of the number and activity of natural killer cells in different individuals, and imprecision of phenotyping of the entire pool of NK-cells by means of a single marker, appeared to be responsible for poor correlations of NK-cell number and activity as observed in the study.

Immunological aspects of progeria (Hutchinson-Gilford syndrome) in a 15-month-old child.

A thorough analysis of the immunological status was conducted in a 15-month-old child with progeria (Hutchinson-Gilford syndrome). Total leukocyte and neutrophil counts were slightly increased, and monocytes were decreased. Percentage and numbers of CD4+ cells in the blood were mildly decreased as well as the CD4/CD8 cell ratio. CD20 (B-cell marker) bearing cells and cells bearing Ia-antigens were increased, as well as CD16 and CD56 marker-bearing cells (natural-killer cells, NK). Lymphocyte proliferation upon stimulation with phytohaemagglutinin and purified protein derivative were decreased, and with pokeweed mitogen increased. NK cell activity appeared increased, particularly at lower effector: target cell ratios.

Peripheral blood CD5+ B cell subset in the remission phase of systemic connective tissue diseases.

OBJECTIVE: To get a better insight into the level of circulating CD5+ B cells as related to the systemic connective tissue disease activity. METHODS: Peripheral blood CD5+CD19+ cells of patients in the remission phase of systemic lupus erythematosus (SLE) (n = 28), Sjögren's syndrome (SS) (n = 20), rheumatoid arthritis (RA) (n = 26), and 19 control healthy subjects were analyzed by 2-color flow cytometry. RESULTS: In comparison to control group, the patients with SLE had a significant increase in the relative CD19+CD5+ blood cell count (p < 0.0005); this count was also different from the finding in both RA (p < 0.005) and patients with SS (p < 0.05). In contrast, the proportion of B cells expressing CD5 (within an individual B cell population) was significantly increased in all the 3 diseases compared to healthy subjects (SLE, p < 0.0001; SS, p < 0.05; and RA, p < 0.01). In the multivariate discriminant analysis, a discriminant function defined by the CD19+CD5+ subset strongly discriminated SLE, SS and RA from the control, but also SLE from both SS and RA. CONCLUSION: Our findings demonstrated that, in relation to healthy control subjects, the blood CD5+ B subset tended to be elevated in the patients in the remission phase of systemic connective tissue diseases, particularly in SLE.