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M Spanò

Publications and source records attributed to M Spanò.

At least 37 records · Page 2Linked to original sources
PubMed1997

Quality control study of the Italian Group of Cytometry on flow cytometry DNA content measurements: II. Factors affecting inter- and intralaboratory variability.

A multicentric national quality control study has been organized under the auspices of the Italian Group of Cytometry to find a possible influence of some procedural steps in DNA flow cytometry measurements on DNA index (DI) values and to identify the main parameters affecting the interlaboratory variability. To 40 participating laboratories we provided suspensions containing unknown mixture of different cell types: an homogeneous thymocyte population used to check instrument linearity; one mixture composed of two cell types characterized by DI = 1.00 and 1.10; and another composed of three different cell types with relative DIs of 1.00, 1.26, and 1.62, respectively. Possible effects due to staining protocols were studied, allowing the participants to stain cellular DNA according to the procedure routinely adopted in each laboratory, in addition to a standardized procedure with a fixed PI solution. As far as the influence of instrument linearity on DI values is concerned, we did not find any correlation with the DI variability observed, even if the use of a standardized staining protocol could lead to a sensible gain in interlaboratory DI reproducibility. Twenty-five of 40 (65%) laboratories were able to discriminate the near-diploid subpopulation, and a coefficient of variation of less than 4% was the minimum condition necessary to recognize the DI = 1.1 population. In samples containing two aneuploid subpopulations, 25 of 35 (71.4%) laboratories showed a high reproducibility with the standard staining protocol and 22 of 38 (57.9%) with the free staining protocol. However, a sensible improvement in interlaboratory reproducibility emerged with respect to the previous trial.

Animals↗
PubMed1996

Flow cytometric assessment of trophosphamide toxicity on mouse spermatogenesis.

The effects of trophosphamide on mouse reproductive cells have been investigated by flow cytometric analysis of testicular cell populations and alterations of sperm chromatin structure. Mice were treated with single intraperitoneal injections of TP, the doses ranging between 50 and 150 mg/kg, and were killed after 7, 14, 21, 28, 35, or 49 days. Dose-dependent reductions of tetraploid cells, round spermatids, and elongated spermatids were detected at 7, 21, and 28 days, respectively, reflecting cytotoxic damage to the differentiating spermatogonia compartment. The dose necessary to reduce the number of differentiating spermatogonia to half the control value was approximately 70 mg/kg. Stem cells were not affected by this treatment, and the normal spermatogenic process was restored after 7 weeks. In addition, cauda epididymal sperm were analyzed by the sperm chromatin structure assay, a flow cytometric measurement of the susceptibility of the sperm nuclear DNA to in situ acid denaturation; a statistically significant increase of sperm with altered chromatin structure was detected after a TP treatment of 150 mg/kg. Together with previous findings published in the literature, where the same doses induced heritable genetic damage, this study demonstrates a marked adverse cytotoxic effect of TP on the male reproductive integrity. All this information should be taken into consideration when TP is used in chemotherapeutic regimens.

Animals↗
PubMed1996

Flow cytometric and histological assessment of 1,2:3,4-diepoxybutane toxicity on mouse spermatogenesis.

The effects of diepoxybutane (DEB) on mouse reproductive cells have been investigated by flow cytometric and histological description of testicular cell populations and alterations of sperm chromatin packaging. Mice were treated with single intraperitoneal injections of DEB, with doses ranging between 8.5 and 78 mg/kg (100-900 microM), and were killed after 7, 14, 21, 28 or 35 d. Dose-dependent reductions of tetraploid cells, round spermatids, and elongated spermatids were detected at 7, 21, and 28 d, respectively, reflecting cytotoxic damage on the differentiating spermatogonia compartment. The dose necessary to reduce the number of differentiating spermatogonia to half the control value was estimated equal to 650 microM or 55 mg/kg. Stem cells were not affected by this treatment. Histological section of seminiferous tubules showed depletion of spermatids and reduction of the secondary spermatocyte layers. In addition, a high although not statistically significant frequency of sperm with altered chromatin packaging was detected after DEB treatment. DEB is one of the key metabolites of butadiene, which is a compound of high environmental and occupational concern. These results contribute to the assessment of the reproductive health impact of butadiene in humans.

Animals↗
PubMed1996

Visual outcome in children with congenital hemiplegia: correlation with MRI findings.

Fourteen children with congenital hemiplegia were studied with a detailed assessment of various aspects of vision (linear acuity, stereopsis, visual fields) and MRI. The aim of this study was to evaluate the effect of a congenital lesion on visual function. The results showed a very high incidence (78%) of children who had abnormal results on at least one of the visual tests. Visual abnormalities were not correlated with the clinical severity of hemiplegia or with a specific pattern of lesion on MRI. Similarly no constant association could be found between visual structures (optic radiations and primary visual cortex) and visual function. Finally, our results would suggest that all the children with congenital hemiplegia need to be investigated irrespective of the clinical severity or of the type or the extent of the lesion. This would help to identify children with minor visual abnormalities which can affect everyday life performance.

Adolescent↗
PubMed1995

Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis.

Flow cytometry can measure variations in DNA content and chromatin structure as well as dramatic changes in the mitochondria of germ cells during maturation from spermatogonia to elongated spermatids. Using 10-N nonyl acridine orange (NAO), an inner mitochondrial membrane dye, it is easy to follow mitochondria rearrangements. Mouse testis cells stained with the DNA fluorescent probe propidium iodide (PI) and analyzed by flow cytometry can be discriminated on the basis of their ploidy levels into five main regions corresponding to elongated spermatids, round spermatids, diploid, S-phase, and tetraploid cells. The simultaneous use of PI and NAO demonstrated the presence of cells having low and high mitochondrial content in the haploid, diploid, and tetraploid compartments. Eleven sorting windows were selected from the bivariate analysis (PI/NAO) and the corresponding cells were identified by microscopic observation. Cells were also discriminated by two parameter analysis of DNA content vs. cell diameter. The definition of seven different regions allowed us to determine NAO or rhodamine 123 (Rh 123) uptakes in each compartment. We observed that the ratio (Rh 123/NAO) dramatically changed according to the progression of cell differentiation which occurs during spermatogenesis.

Acridine Orange↗
PubMed1995

Mechanisms and evolution of the brain damage in neonatal post-hemorrhagic hydrocephalus.

There are three main mechanisms of poor outcome in children with post-hemorrhagic hydrocephalus: (1) brain injuries due to ventricular dilatation, (2) shunt-related complications, and (3) primary cerebral hypoxic-ischemic and hemorrhagic lesions. The authors give a short up-to-date report, focusing mainly on the third mechanism, with reference to personal studies.

Brain Injuries↗
PubMed1994

Acrylamide-induced chromosomal damage in male mouse germ cells detected by cytogenetic analysis of one-cell zygotes.

Within a project coordinated by the Commission of the European Communities for the detection of germ cell mutagens, the cytogenetic analysis of first-cleavage metaphases was carried out to detect chromosomal damage induced by acrylamide (AA) in meiotic and postmeiotic stages of mouse spermatogenesis. Male mice were intraperitoneally injected with single acute doses of 75 or 125 mg/kg or treated with five daily injections of 50 mg/kg and mated either 7 or 28 days after the end of treatment. Chromosomal aberrations were scored in C-banded metaphases prepared from one-cell zygotes by a mass harvest technique. AA treatment of late spermatids-spermatozoa resulted in significant increases of structural aberrations at all doses tested. The data could be fitted to a curvilinear regression and a doubling dose of 23 mg/kg was calculated. The large majority of observed aberrations were of the chromosome type, including dicentrics, rings and translocations, in agreement with a mechanism of chromosomal damage mediated through the alkylation of DNA-associated protamines. Even though the frequency of aberrations 28 days after treatment was not significantly higher than the control value, the presence of multiple rearrangements in two cells suggested that AA might also have a minor effect on spermatocytes. The results of the cytogenetic analysis of first cleavage metaphases agreed well both qualitatively and quantitatively with the outcome of dominant lethal and heritable translocation assays. AA-induced cytotoxicity was monitored by flow cytometric DNA content analysis of testicular cells. By this method, a dose-dependent depletion of mature spermatids after treatment of spermatogonia and a toxic effect upon primary spermatocytes were detected.

Acrylamide↗
PubMed1993

Quality control study of the Italian group of cytometry on flow cytometry cellular DNA content measurements.

A quality control study on DNA flow cytometry, extended to 43 national laboratories, has been carried out by the Italian Group of Cytometry, using defined fixed suspensions of cultured human leukemia K562 cells and human blood lymphocytes. The participating laboratories were allowed to follow their own staining and measurement protocols. Aliquots of cellular suspension had to be measured three times on the same day and two other times on different days. A large heterogeneity of procedures emerged among participants. The average of mean DNA index laboratory values, from 36 laboratories who sent evaluable data, was 1.68, with a range from 1.49 to 1.97. The coefficients of variation ranged from 2.35 to 9.39% and from 2.79 to 8.5% for diploid and aneuploid peaks, respectively. Statistical analysis of the results showed quite good intralaboratory reproducibility, but statistically significant differences were observed among laboratories, for both DNA indices and coefficients of variation. These differences appear to be consistent. For standardization, it is essential that efforts should be made to identify the main sources of variation and to control them.

Analysis of Variance↗
PubMed1993

Flow cytometric analysis for reproductive biology.

Flow cytometric studies of spermatogenesis have been advanced by the need for: i) rapid, sensitive, objective and multiparameter measurements of reproductive effects due to environmental, occupational, and therapeutic exposure to toxicants; and ii) assessment of fertility potential of human and animal sperm. As a consequence, various flow cytometric techniques are already available to identify germ cell subpopulations undergoing both proliferative and maturative processes in normal and perturbed conditions. Significant improvements have been introduced in order to investigate the spermatogenic complex differentiation pathway and the apparent uniformity of mature sperm. Flow cytometry (FCM) has been applied to the measurement of both testis and sperm cells in a variety of species, including man. End points considered in toxicology studies are: altered testicular germ cell ratios, DNA and RNA content, increase of the coefficient of variation, induction of diploid elongated spermatids and diploid sperm, altered nuclear morphology, sperm cell viability, mitochondrial function and sperm chromatin structure. Precise DNA content measurements allow accurate analysis to determine the proportion of X- and Y-chromosome bearing sperm and sorting of these subpopulations for gender preselection. FCM technology has reached a maturation level that allows its inclusion in the list of available and routine methods for reproductive studies in human and animal populations.

Animal Husbandry↗
PubMed1991

Comparison of gaussian and t-distribution densities for modeling fluorescence dispersion in flow cytometric DNA histograms.

In this paper, we have analyzed, on an experimental basis, the behaviour of the standardized t-distribution with three degrees of freedom versus the gaussian model, when employed for describing the fluorescence dispersion effect in flow cytometric DNA measurements. The comparison has been performed by employing two different objective criteria based on a computer analysis for fitting DNA histograms and on a parameter related to the peak distribution shape. The results indicate that, when the peak variance is not particularly small, the two models are substantially equivalent. In the case of experimental data with very sharp peaks, the t-distribution performs better than the gaussian density.

Animals↗
PubMed1991

Evaluation of 2-methoxyacetic acid toxicity on mouse germ cells by flow cytometry.

Flow cytometric (FCM) DNA content measurements were carried out on testicular monocellular suspensions obtained from mice exposed per os to a single dose of 50, 100, 300, 600, and 900 mg/kg body weight (b.w.) of 2-methoxyacetic acid (MAA) in order to investigate its cytotoxic action on germ cells. The effects of MAA were evaluated 2, 7, 14, 28, and 45 d after treatment in terms of altered cell type ratios in FCM fluorescence distribution histograms. Testis weight and histological tissue sections were also analyzed. MAA induced marked changes in the relative percentages of tetraploid and haploid cells, indicating the occurrence of cytotoxic damage on primary spermatocytes. Multiparameter FCM analysis showed that, besides its action on nucleic acid synthesis, MAA can also affect the cellular energy metabolism reflected in an altered mitochondrial mass distribution on round spermatids surviving the MAA treatment. This study demonstrates that rapid and unique FCM procedures can be usefully applied in reproductive toxicology.

Acetates↗
PubMed1989

Automatic analysis of flow cytometric DNA histograms from irradiated mouse male germ cells.

An automatic procedure for recovering the DNA content distribution of mouse irradiated testis cells from flow cytometric histograms is presented. First, a suitable mathematical model is developed, to represent the pattern of DNA content and fluorescence distribution in the sample. Then a parameter estimation procedure, based on the maximum likelihood approach, is constructed by means of an optimization technique. This procedure has been applied to a set of DNA histograms relative to different doses of 0.4-MeV neutrons and to different time intervals after irradiation. In each case, a good agreement between the measured histograms and the corresponding fits has been obtained. The results indicate that the proposed method for the quantitative analysis of germ cell DNA histograms can be usefully applied to the study of the cytotoxic and mutagenic action of agents of toxicological interest such as ionizing radiations.

Animals↗
PubMed1989

Cytotoxic effects of benzene on mouse germ cells determined by flow cytometry.

Flow cytometric (FCM) DNA content measurements were performed on testicular monocellular suspensions obtained from mice exposed per os to 0, 1, 2, 4, 6, and 7 ml/kg body weight of benzene in order to investigate its cytotoxic action on germ cells. The effects of benzene were measured 7, 14, 21, 28, and 70 d after treatment. Benzene had no effect on testis weight, but FCM analysis showed the relative percentages of some cell subpopulations (tetraploid and haploid cells) to be different from the control pattern, indicating the occurrence of some cytotoxic damage to differentiating spermatogonia. These data demonstrate that spermatogenesis is sensitive to benzene single exposures as evidenced by an altered cell ratio of testicular cell types.

Animals↗