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A Orfao

Publications and source records attributed to A Orfao.

At least 163 records · Page 9Linked to original sources
PubMed1995

Light scatter characteristics of blast cells in acute myeloid leukaemia: association with morphology and immunophenotype.

AIMS: To analyse the forward scatter/side scatter (FSC/SSC) distribution of acute myeloblastic leukaemia (AML) blast cells in order to assess whether it correlates with their morphology, immunophenotype, and clinical and biological disease characteristics. METHODS: FSC/SSC patterns were established upon taking into account the localisation of the residual T lymphocytes in the FSC/SSC dot plot as an internal biological standard. One hundred and seventy one newly diagnosed AML patients were analysed and five different FSC/SSC patterns were established. These five patterns could be grouped into two major categories taking into account the FSC/SSC distribution of normal cells in a bone marrow aspirate: immature patterns (1 and 2) and mature patterns (3, 4, and 5). These FSC/SSC patterns were correlated with different clinical and biological characteristics of AML patients. RESULTS: No significant associations were detected in relation to the clinical and haematological disease characteristics and the prognosis of these patients. By contrast there was a significant correlation between the FSC/SSC pattern of the AML blast cells and the FAB classification. An increased reactivity for the antigens associated with myeloid differentiation such as CD13, CD33, CD11b, CD15, CD14, CD4, CD56, and/or CD16 was detected among cases showing a mature FSC/SSC pattern (3, 4, and 5), both in the whole series and even within each of the FAB AML subtypes. By contrast, the reactivity for the CD34 precursor cell associated antigen was higher among those cases displaying an immature FSC/SSC pattern, this being observed even within each FAB subgroup. CONCLUSIONS: The FSC/SSC pattern distribution of AML blast cells not only provides an additional objective and reproductible system for the classification of these leukaemias but it may also represent a connection between the FAB morphological groups and the immunophenotypic classification of AML patients.

Acute Disease↗
PubMed1995

Hypothyroidism prevents postnatal changes in rat liver mitochondrial populations defined by rhodamine-123 staining.

The effect of hypothyroidism on the percentages of low fluorescence population (LFP) and high fluorescence population (HFP) rhodamine-123-stained mitochondria, respiratory parameters, and ATPase activity were studied in liver mitochondria from early newborn rats. Hypothyroidism prevented the decrease in the percentage of HFP and the subsequent increase in LFP that occurs immediately after birth. This effect coincides with the impairment of mitochondrial respiratory function, as shown by the low respiratory control ratio and the low activity of F0,F1-ATPase found in hypothyroid newborns. All of these changes were reversed by the administration of thyroid hormones. ATP in vitro promotes the conversion of HFP into LFP and increases the respiratory control ratio in hypothyroid newborns, although this effect was not observed after thyroid hormone treatment. The effect of thyroid hormones on both the postnatal changes in mitochondrial populations and in F0,F1-ATPase activity was prevented by cycloheximide, but not by streptomycin. Thus, the observed effects of thyroid hormones on neonatal mitochondria must be accomplished by the induction of the synthesis of some nuclei-coded protein, possibly involved in F0,F1-ATPase assembly.

Adenosine Triphosphate↗
PubMed1995

Adrenalectomy induces a decrease in the light scatter properties and amylase content of isolated zymogen granules from rat pancreas as analyzed by flow cytometry.

The effect of glucocorticoid deprivation induced in male rats by adrenalectomy on the pancreatic zymogen granules was studied. Zymogen granules were purified from control, sham-operated and adrenalectomized animals studied 1, 3 and 7 days after surgery. The zymogen granules were characterized by flow cytometry, and in each granule the size (based on the forward or low angle light scatter (FSC) parameter), membrane complexity (based on side or 90 degrees light scatter (SSC) parameter) and amylase content were evaluated. Amylase content/DNA ratio in pancreatic homogenates was also analyzed. The zymogen granules of the control rats were found to be distributed in two populations: a major one-R1 (95.45 +/- 1.21%)-containing zymogen granules with a smaller mean size and complexity, and a minor population-R2 (4.45 +/- 0.24%)-the granules of which had a mean size which was larger and more complex. At day +1 after adrenalectomy the zymogen granules were significantly (P < 0.05) smaller than those of control animals. The R2 zymogen granules were similar to those from R1 as regards their size, but were more complex, suggesting that the immediate effect of glucocorticoid deprivation is to induce a depletion of the larger granules presumably belonging to the R2 population. The amount of amylase per granule did not vary at day +1 after adrenalectomy, although the amylase content/size ratio per granule was significantly (P < 0.001) increased. This mechanism could be explained in terms of the existence of a bypass defined in the adrenalectomized animals between the granular content and cytosolic enzymes. Prolongation of the adrenalectomy period to 3 and 7 days resulted in a progressive increase in zymogen granule size and complexity, both parameters showing similar characteristics to those of the controls at day +7 after adrenalectomy. However, the percentage of zymogen granules within the R1 and R2 populations was clearly different from that of controls since the R2 population was much more numerous (11.25 +/- 0.75% and 15.25 +/- 1.15% (adrenalectomized rats at days +3 and +7 respectively) versus 4.45 +/- 0.24% (controls)). An increase in the content of amylase per DNA was observed in adrenalectomized rats at day +1 although this transient effect cannot be related to glucocorticoid deprivation because it was also observed in sham-operated rats (day +1). However, a significant reduction, nearly 64%, in the amylase content/DNA ratio is produced by the absence of glucocorticoids 7 days after adrenalectomy and this is associated with a reduction in the content of amylase in each individual zymogen granule which reaches a minimum 3 days after adrenalectomy. It should be noted that, despite this, the enzyme concentration in each granule remains constant as there is a parallel decrease in the zymogen granule amylase content and size.

Adrenalectomy↗
PubMed1995

Flow cytometry in the diagnosis of cancer.

Flow cytometry has rapidly expanded from basic research to clinical laboratories mainly due to its unique characteristics regarding cell analysis. Among the clinical uses of flow cytometry cancer represents one of the most relevant. Several applications of flow cytometry can currently be applied to the study of cancer, including the detection of tumour cell DNA aneuploidy, the analysis of tumour cell proliferation and the immunophenotyping of leukemias. Although standardized flow cytometry protocols for these applications are scanty, the clinical value has been clearly established. The presence of DNA aneuploidy and a high proportion of S-phase tumour cells have been associated with tumour malignancy and a poor prognosis. The immunophenotype of leukaemia is of great help both for the diagnosis and classification of chronic lymphoproliferative disorders and acute leukaemias, especially in acute lymphoblastic leukemia cases and the M0, M3-variant, M6 and M7 acute myeloblastic leukaemia subtypes. In addition, it allows the identification of relatively rare leukemia cases such as the biphenotypic and the Nk-cell lineage leukemias. The development of flow cytometry is continuously bringing new applications into the clinical laboratory in the area of cancer diagnosis.

Flow Cytometry↗
PubMed1995

Phenotypic analysis of CD34 subpopulations in normal human bone marrow and its application for the detection of minimal residual disease.

In the past, studies on CD34+ cells have been based on the use of monoclonal antibodies conjugated with different fluorochromes that show different fluorescence intensity and yield variable results. Moreover, most of these studies have neither specifically focused on adult human BM samples nor have they used combinations to explore specifically the phenotype of myeloid committed CD34+ cells. The aim of the present study has been to characterize the normal human CD34+ precursor cells from adult BM in order to identify missing or extremely rare phenotypes that can be used for detecting minimal residual disease (MRD) in patients with AML. For this purpose we have utilized the fluorochrome conjugates that provide the most sensitive signals for identifying low antigenic expression, and the technique has been adapted to the characterization of cells present at very low frequencies. Normal human BM samples from 13 adult healthy volunteers have been analyzed using triple stainings at flow cytometry. The mean percentage of CD34+ cells detected was 0.72 +/- 0.33%; these cells displayed an heterogeneous light-scatter distribution. Most CD34+ cells coexpressed CD38 (96.7 +/- 5.7%), HLADR (81.6 +/- 14.0%), CD33 (84.7 +/- 18.3%), CD13 (84.6 +/- 16.2%) and CD71 antigens (65.5 +/- 9.1%). In addition, almost half of CD34+ cells were CD117+ (60 +/- 26.8%). Only a small proportion of CD34+ cells coexpressed CD4 (15.5 +/- 11.7%, CD36 (31.7 +/- 6.2%), CD61 (16.3 +/- 12.9%), CD41 (6.5 +/- 5.5%) or the lymphoid associated markers CD10 (18.6 +/- 11.8%) and CD19 (12.3 +/- 13.2%). Reactivity for the CD15 antigen was observed in a small population of CD34+HLADR+ cells (11.6 +/- 11.2%) although its intensity of expression was lower than that of the more mature granulocytic cells. No CD34+ cells displayed CD14, CD65, CD20, strong CD22, CD3 and CD56 antigens. Accordingly, most adult bone marrow CD34+ cells appeared to be committed to the myeloid lineage (CD13+/CD33+) and displayed an intermediate-to-large FSC/SSC while the lymphoid-committed CD34+ cells (CD19+, CD10+) were in a minority with low FSC/SSC values. By triple marker stainings several phenotypes of CD34+ precursor cells were found to be either undetectable or present at very low frequencies (< 1 x 10(-3)) in the normal human adult bone marrow. These data may be of great value for defining leukemia 'associated' phenotypes used to detect minimal residual disease in adult acute leukemia patients.

Adult↗
PubMed1995

Flow cytometric DNA analysis of fine-needle aspirates of prostatic benign lesions.

Fine-needle aspiration samples from 54 consecutive patients suffering from prostatic benign lesions were analyzed for both cytological diagnosis and DNA cell content, by means of standard cytological criteria and flow cytometry, respectively. Of these patients, 13 displayed prostatitis: three being classified as acute prostatitis, 7 as chronic and 3 as granulomatosous prostatitis. All cases had a diploid DNA cell content, with a mean percentage of S-phase cells of 1.8 +/- 2.7%. Although no major differences were detected between patients with and without prostatitis, the former group had a slightly higher proportion of S-phase cells (1.4 +/- 2.2% vs 2.7 +/- 3.7%). Moreover, within those patients with prostatitis, acute prostatitis cases showed the highest proliferation rates although differences did not reach statistical significance.

Biopsy, Needle↗
PubMed1995

Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL).

Criteria for the immunological classification of acute leukemias are proposed by a recently established European group designated EGIL. The main aims of EGIL are to establish guidelines for the characterization of acute leukemias based on marker expression and provide a uniform basis for the diagnosis of the various types of these hemopoietic malignancies which should be helpful for future multinational clinical and laboratory investigations. Within the two major types (B and T cell lineage) of acute lymphoblastic leukemia (ALL), several groups are delineated according to the degree of cell differentiation. Within the acute myeloid leukemias (AML), only three subtypes as defined by the FAB classification: M0-AML, M6-AML and M7-AML, can be unequivocally defined by immunological markers; prospective studies are undertaken to see whether characteristic immunological profiles are associated with particular AML subtypes defined by specific cytogenetic abnormalities. Criteria for the definition of biphenotypic acute leukemia (BAL) are devised and a scoring system is outlined aimed to distinguish BAL from those acute leukemias with expression of a marker from another lineage. In addition, an uncommon subset of acute leukemias with no evidence of lymphoid or myeloid differentiation is recognized and the useful panel of markers to investigate and establish the cell nature of the acute leukemias is outlined. EGIL will focus in the future on testing the reproducibility of the proposed guidelines, particularly those for BAL, assessing their clinical value within a framework of multicentric trials and setting up uniform methodological criteria.

Acute Disease↗
PubMed1995

In vivo bromodeoxyuridine (BrdU)-labelling index of rat thymus: influence of different BrdU doses and exposure times as analyzed both in tissue sections and in single cell suspensions.

Tissue sections and single cell suspensions of rat thymus were analyzed by flow and image cytometry to study the effects of different doses of bromodeoxyuridine (BrdU) (10, 50 and 100 mg/kg and different labelling periods (15, 30 and 60 min) on the BrdU-labelling index. The proportions of BrdU-labelled cells, as analyzed by flow cytometry, were similar in the different groups of our study. However, the quality of the results was not exactly the same, since when the BrdU dose decreased, the separation between BrdU-positive S-phase cells and both the G0/G1 and the G2/M BrdU-negative cells on a DNA/BrdU histogram became less clear. On the other hand, the proportion of BrdU-labelled cells in tissue sections was greater in the groups of animals that received doses of 100 mg/kg and were killed 15, 30 or 60 min later than in the sections obtained from the remaining groups. Our results show that both dose and exposure time to BrdU may influence the final results when cell proliferation is assessed, the variations obtained clearly depending on the technique used for the immunological detection of BrdU-positive S-phase cells.

Animals↗
PubMed1995

Analysis of isolated zymogen granules from rat pancreas using flow cytometry.

Rat pancreatic zymogen granules were analyzed using flow cytometry to determine their heterogeneity with respect to different characteristics such as size (FSC), internal complexity (SSC) and membrane permeability to lipophilic and cationic dyes using rhodamine-123 as probe. Differences in the chemical composition of the membrane were determined using FITC-labeled lectins (concanavalin A, Wheat germ agglutinin (WGA) and Tetragonolobus purpureus) displaying specific binding for different carbohydrates (D-mannose, N-acetyl D-glucosamine and L-fucose, respectively). Finally, the amylase content of zymogen granules was also analyzed using an anti-amylase antiserum. Our results show the existence of two populations of zymogen granules that can be identified on the basis of their FSC and SSC characteristics: a minor population of approximately 5% all zymogen granules with larger size (FSC) and internal complexity (SSC), and a major population clearly differentiable on the basis of a lower FSC and SSC. Rhodamine-123 uptake was similar in both subpopulations of zymogen granules. By contrast, labeling with fluoresceinated lectins and anti-amylase antiserum showed the existence of a higher content on both amylase and the monosaccharide residues analyzed for the Z2 zymogen granules. However, it is shown that those differences are strongly dependent on the FSC (size) of the granules suggesting that although the carbohydrate contents (D-mannose, N-acetyl D-glucosamine and L-fucose) and the amount of amylase differed from one granule to another, their concentration per granule was similar.

Animals↗
PubMed1994

Postnatal changes in rhodamine-123 stained mitochondrial populations are sensitive to protein synthesis inhibitors but mimicked in vitro by ATP.

The incubation of term fetus mitochondria with ATP mimicked in vitro the increase in the respiratory control index and in the percentage of the rhodamine-123-low fluorescence population that occurred in vivo immediately after birth, suggesting that both phenomena are closely associated. The administration of streptomycin inhibited the increase in the percentage of the low fluorescence population that occurred immediately after birth, while the administration of cycloheximide even reversed these changes. These results suggest that the in vivo interconversion between mitochondrial forms depends on both cytosolic and mitochondrial protein synthesis.

Adenosine Diphosphate↗
PubMed1994

The phenotype of L-CFU and its correlation with the immunological characteristics of the blast cell population in AML.

The membrane phenotype of AML clonogenic cells (L-CFU) was analyzed in 19 AML patients using an in vitro culture technique after a complement-mediated lysis assay employing a panel of six monoclonal antibodies (McAb) -HLA-DR, FMC56 (CD9), FMC27 (CD9), CD14, CD15, CD41a-. Our results show that L-CFU has a heterogeneous but immature phenotype lacking on the expression of differentiation antigens (CD14, CD15, CD41a). In addition, we observed that the L-CFU phenotype is different from that of the whole blast cell population. Interestingly, L-CFU showed a higher expression of HLA-DR antigens with respect to their progeny. Upon analyzing whether the L-CFU phenotype was related to both the morphological and immunological features of AML blast cells, it was observed that, while there is no correlation with the FAB classification, there was a partial relationship between the immunological phenotype of AML blast cells and that of L-CFU. Accordingly, the more immature AML cases showed a more differentiated L-CFU phenotype (HLA-DR+, CD9+, FMC27+) when compared with cases with a more mature blast cell phenotype. These results suggest that those AML cases with a relatively immature myeloblastic phenotype may arise from a progenitor cell that has undergone partial differentiation and that is unable to acquire myeloid differentiation antigens, while those AML cases with mature blast cells might emerge from a very early L-CFU that has the capacity to undergo a greater degree of differentiation.

Antigens, CD↗
PubMed1994

Transrectal fine needle aspiration biopsy of the prostate combining cytomorphologic, DNA ploidy status and cell cycle distribution studies.

Fine needle aspiration (FNA) cytology of the prostate is becoming a common diagnostic procedure, and DNA flow cytometry (FCM) data have been shown to correlate with the pattern of evolution of prostatic carcinoma, thus emphasizing the importance of assessing both parameters together. The aim of the present paper is to analyze the presence of DNA aneuploidy, cell cycle distribution and their relationship with the cytologic grade in transrectal fine needle aspiration prostate biopsies from 78 consecutive patients. Herein we studied the DNA ploidy status, the cell cycle distribution and their relationship with cytologic grade in transrectal FNA biopsies of the prostate from 78 consecutive patients -47 benign hyperplasias and 31 carcinomas- as analyzed by a reproducible FCM method for single cell suspension preparations, data acquisition and analysis. The presence of DNA aneuploidy was detected in 39% of the carcinomas and it was found to be a specific marker for prostatic carcinoma since all benign hyperplasia cases were diploid. Moreover, the incidence of DNA aneuploidy increased progressively from well-differentiated to moderately-differentiated and poorly-differentiated carcinomas (p = 0.005). Regarding cell cycle distribution, carcinomas displayed a higher proportion of both S-phase (p = 0.0003) and G2/M-phase (p = 0.0006) cells with respect to benign hyperplasias. Aneuploid cases also showed a greater proliferation rate as compared to the diploid carcinomas, regardless of their cytopathologic grade (p = 0.00001). Despite the fore-mentioned results, these correlations were far from being absolute, suggesting that combined assessment of these parameters should give additional information for the clinical management of prostatic disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopsy, Needle↗
PubMed1994

The relationship between hyperthyroidism and the distribution of peripheral blood T, NK and B-lymphocytes in multinodular goiter.

Peripheral blood T, NK and B-cell subsets were analyzed in 18 patients with nontoxic multinodular goiter (NMG) and 11 patients with toxic multinodular goiter (TMG) in order to evaluate whether hyperthyroidism modifies the distribution of these cell populations. As a control group, 26 age and sex-matched healthy individuals were included in the study. Lymphoid subsets were analyzed with flow cytometry by double staining immunofluorescence techniques using a large panel of monoclonal antibodies. No differences were found in the absolute or relative numbers in any of the cell populations analyzed in both groups--NMG and TMG--, with the exception of a significant decrease in CD19+ cells in TMG. However, patients with multinodular goiter showed important abnormalities in the distribution of T, NK and B lymphocytes with respect to the control subjects. The pattern of abnormalities detected was characterized by a marked increase in the absolute and relative counts of activated T-lymphocytes (CD3+/HLA-DR+), cytotoxic T-cells (CD57+/CD8+) and of cells expressing NK-related antigens. None of these alterations were related to the serum levels of T3, T4 or TSH. Our results point to the existence of important abnormalities in the distribution of several lymphoid subsets in multinodular goiter, regardless of whether the subjects are euthyroid or hyperthyroid.

Adult↗
PubMed1994

Acute lymphoblastic leukemia (ALL): detection of minimal residual disease (MRD) at flow cytometry.

In the present study the usefulness of a method combining multiple staining direct immunofluorescence technique together with flow cytometry in order to predict relapse in ALL is analyzed in a group of 47 patients (11 T-ALL and 36 B-ALL). Results show that this method can be applied to at least two-thirds of all ALL patients being specially useful for the T-ALL cases (100% vs 56%) as this corresponding to the incidence of "aberrant" phenotypes. The detection of an increase in the percentage of bone marrow cells displaying "aberrant" phenotypes in two consecutive samples from the same patient is of great help on predicting relapse (sensitivity of 92% and specificity of 75%).

Adolescent↗
PubMed1994

CD4 enumeration in HIV infected individuals.

In the present study, two alternative methods for the enumeration of PB CD4+ lymphocytes are compared with conventional flow cytometry (FCM): the TRAX CD4 tm enzyme immunoassay test kit and a new method using an internal biological standard (liophilized lymphocytes) for the direct enumeration of total PB CD4+ T-cells. Both methods showed an overall high degree of correlation with conventional FCM. However, the TRAxCD4 tm enzyme immunoassay showed a significant overestimation of PB CD4+ T-cells that was not only due to monocyte-derived CD4 molecules and the use of liophilized lymphocytes as an internal biological standard showed a low level of correlation for patients displaying low CD4 counts.

CD4 Lymphocyte Count↗
PubMed1993

Acute lymphoid leukemias following either a previous chronic myelogenous leukemia or myelodysplastic syndrome: phenotypic and genomic differences.

We have analyzed the immunological and genomic characteristics of the lymphoid blast cells present in secondary lymphoid leukemias following either a previous chronic myelogenous leukemia (CML) or myelodysplastic syndrome (MDS). Twenty-one of 107 secondary leukemias analyzed displayed a lymphoid phenotype (15 after a CML and 6 after a MDS). Most of the lymphoid blast crises of CML (73%) correspond to pure lymphoid transformation, all of them having a common acute lymphoblastic leukemia (ALL) phenotype (CD10+). By contrast, in all MDS cases, lymphoid blast cells coexisted with another myeloid component (hybrid leukemias) and showed an early B phenotype. IgH and TCR-gamma gene rearrangements were detected in the CML-lymphoid blast crisis (86% of cases) more frequently than in the MDS transformations (33%). The TCR-beta gene was in germ line configuration in all cases while TCR-delta gene rearrangements were detected in four cases, all of them corresponding to a previous diagnosis of CML. These results show the existence of both immunophenotypic and genomic differences between the lymphoid transformations of either CML or MDS, which could reflect differences at the stage of maturation of the target cell in these transformations.

Blast Crisis↗
PubMed1993

Expression of silver-stained nucleolar organizer regions is coupled to cell cycle in rat thymic cells.

To directly analyze the relationship between the expression of silver-stained nucleolar organizer regions (AgNORs) and cell proliferation, thymic cells from newborn rats were separately sorted at the G0-G1-phase and early-mid and late-mid S-phase of the cell cycle according to their DNA content. Different AgNOR-derived parameters (mean area and numbers of AgNORs per cell and mean AgNOR-particle area) were evaluated after silver staining of cytospins. A linear correlation was observed between the mean area and numbers of AgNORs per cell, both parameters increasing progressively from G0-G1-phase to early-mid and late-mid S-phase. An increase of the mean AgNOR-particle area was also seen between G0-G1 and S-phase but this was not significant along the S-phase. A bias on the selection of S-phase cells linked to intrathymic maturation can be ruled out as S-phase cells labeled with bromodeoxyuridine were found throughout the thymus, and, moreover, the analysis of the frequency distribution of nuclear area did not show a bimodal pattern. It is concluded that the expression of AgNORs--evaluated as AgNOR area/cell or AgNOR numbers/cell--is causally or indirectly coupled to DNA synthesis and, thus, AgNORs can be considered as a cell proliferation marker.

Animals↗