Biomedical subjects

C López-Fernández

Publications and source records attributed to C López-Fernández.

At least 19 recordsLinked to original sources

PubMed2006

Sperm DNA fragmentation in a random sample of the Spanish boar livestock.

A collection of 180 chilled boar semen samples, randomly chosen from stocks currently used for routine characterization of standard seminal quality, were studied for DNA fragmentation status using the sperm chromatin dispersion test and the DNA fragmentation index (DFI: percent of abnormal cell versus normal cells for DNA fragmentation) was determined. Values for sperm motility, acrosome status, coiled tails and abnormal head morphology, including presence and position of cytoplasmic droplets were also obtained. The DFI in the whole sample presented a wide range of variation with values oscillating between practically 0% and 47.95% and do not fit to a normal distribution. The most frequent classes (83.3%) presented a DFI lower than a 5%. Significant correlations between sperm DNA fragmentation and sperm motility, acrosome status, frequency of distal droplets, coiled tails and abnormal head morphology, were not observed. However, the presence of proximal cytoplasmic droplets showed a significant correlation with the level of DNA fragmentation observed in the ejaculated spermatozoa.

Acrosome↗

PubMed2004

Telomeric and interstitial telomeric-like DNA sequences in Orthoptera genomes.

A (TTAGG)n-specific telomeric DNA probe was hybridized to 11 orthopteroid insect genomes by fluorescence in situ hybridization. Nine different genera, mainly distributed within two evolutionary branches with male chromosome numbers 2n = 23 and 2n = 17 were included in the analysis. Telomere sequences yielded positive signals in every telomere and there was a considerable number of interstitial telomeric-like sequences, mainly located at the distal end of some, but not all, subterminal chromosome regions. One of the species, Pyrgomorpha conica, showed massive hybridization signals associated with constitutive heterochromatin. The results are discussed along two lines: (i) the chromosomal evolutionary trends within this group of insects and (ii) the putative role that ITs may play in a genome when they are considered telomere-derived, but not telomere-functional, DNA sequences.

Animals↗

PubMed2001

Sau3A in situ digestion of human chromosome 3 pericentrometric heterochromatin. I. Differential digestion of alpha-satellite and satellite 1 DNA sequences.

In situ digestion with the restriction endonuclease (RE) Sau3A (Sau3A REISD) uncovers a polymorphism for the pericentromeric heterochromatin of human chromosome 3, which can be positively stained (3+) or not (3-), and has proven useful to differentiate donor and recipient cells after sex-matched bone marrow transplantation and to analyze the so-called hemopoietic chimerism. The aim of the present investigation was to obtain insight into the molecular basis of such polymorphism to optimize its use for chimerism quantification using methodological approaches other than REISD. To this end, fluorescence in situ hybridization (FISH) assays using probes for the satellite DNA sequences that mainly constitute chromosome 3 pericentromeric heterochromatin (alpha-satellite and satellite 1 DNA) were performed on control and Sau3A-digested chromosomes. The results obtained suggest that chromosome 3 alpha-satellite DNA is digested in all individuals studied, irrespective of the karyotype obtained by Sau3A REISD (3++, 3+-, 3--), and thus it does not seem to be involved in the polymorphism uncovered by Sau3A on this chromosome. Satellite 1 DNA is not digested in any case, and shows a polymorphism for its domain size, which correlates with the polymorphism uncovered by Sau3A in such a way that 3+ chromosomes show a large domain (3L) and 3- chromosomes show a small domain (3S). It seems, therefore, that the cause of the polymorphism uncovered by Sau3A on the pericentromeric region of chromosome 3 is a difference in the size of the satellite 1 DNA domain. Small satellite 1 DNA domains fall under the resolution level of REISD technique and are identified as 3-.

Centromere↗

PubMed2000

A PCR product derived from female DNA with regional localization on the Y chromosome.

A 154-bp PCR product amplified from human female DNA mapped onto the Y chromosome under high-stringency in situ hybridization conditions. The female DNA sequence revealed an 89% homology with the HSDYZ1 sequence. When the same primers were used to amplify male DNA, a 154-bp DNA fragment was also obtained, showing a 98% homology with HSDYZ1. However, although the HSDYZ1 sequence is widely distributed along the long arm of the Y chromosome, both of these particular PCR products are di-regionally localized within this distal block of constitutive heterochromatin. In situ hybridization under lower stringency showed that these 154-bp sequences map both onto the autosomes and the Y chromosome. Overall, this paper shows (i) a new class of DNA sequences shared by the autosomes and the Y chromosome; and (ii) a substructured organization of some DNA repeats within the DYZ1 family that forms a large part of the constitutive heterochromatin of the Y chromosome.

Chromosome Mapping↗

PubMed2000

Fluorescence in situ hybridization evaluation of minimal residual disease on stem-cell harvests.

The usefulness of fluorescence in situ hybridization (FISH) analysis to detect minimal residual disease (MRD) in autologous bone marrow and peripheral blood stem-cell harvests has been tested in three patients with hematologic malignancies. Conventional cytogenetics and FISH were used to characterize the leukemic clones identifying the specific chromosomal abnormalities (monosomy 7 in a myelodysplastic patient and trisomy 8 in two acute myeloid leukemic patients). Such analysis was useful to monitor the MRD persistent after treating these patients with intensive chemotherapy. The myelodysplastic patient underwent eight peripheral blood-stem cell harvests in which FISH detected the persistence of monosomy 7 cells, precluding their use for autologous transplantation. This patient relapsed and died. In two acute myeloid leukemia patients who underwent an autologous marrow harvest, FISH did not show a significant proportion of trisomy 8 cells. Nevertheless, autologous transplantation was not performed, owing to an insufficient CD34 cell content in the harvests. One of these patients relapsed with the reappearance of trisomy 8 and died. The other patient, on the contrary, is alive in complete remission 3 years after the bone marrow harvest. The usefulness and applicability of MRD quantification in stem-cell harvests is discussed on the basis of the sensitivity of the methodology applied.

Adolescent↗

PubMed1999

Chimerism quantification after sex-matched BMT: how probable is it to find donor/recipient pairs with distinguishable cells?

Chimerism quantification (CQ) after sex-matched bone marrow transplantation (BMT) is based on the identification of autosomal differences distinguishable at the chromosomal level, such as variations within constitutive heterochromatin between the recipient and the donor. The probability of finding distinguishable recipient/donor pairs at the karyotypic level depends on the frequency of the chromosome variants or morphs in the population, on whether recipient and donor are related, and if so, their kinship relation. We have developed a population genetics-based method that allows the estimation of the percentage of post-BMT CQ expected to be informative using any autosomal polymorphic marker. This method has been developed for the most common transplant situations, such as sibling-matched recipient/donor pairs, haploidentical related (parental/filial) pairs, and unrelated pairs. The method developed was applied to a polymorphism of the pericentromeric region of chromosome 3. This polymorphism becomes evident after in situ digestion with the restriction endonuclease Sau3A, and can be successfully used for CQ. It has been estimated that approximately 59% of the cases of BMT from unrelated donors, 36% of those from sibling donors, and 42% from parental/filial donors, are expected to be distinguishable for post-BMT CQ using this approach.

Bone Marrow Transplantation↗

PubMed1999

Polymorphisms for the size of heterochromatic regions allow sex-independent quantification of post-BMT chimerism targeting metaphase and interphase cells.

BACKGROUND AND OBJECTIVE: Fully quantitative cytological techniques for the analysis of hemopoietic chimerism are very limited and largely restricted to sex-chromosome detection after sex-mismatched bone marrow transplants (BMTs). The aim of the present investigation was to assess the usefulness of autosomal polymorphisms for the size of heterochromatic regions in the identification of donor and recipient cells and therefore in the quantification of the hemopoietic chimerism after sex-matched BMT. DESIGN AND METHODS: Hemopoietic chimerism was followed up in 3 transplanted patients targeting a polymorphism for the size of the pericentromeric heterochromatin (PCH) of chromosome 9, uncovered by restriction endonuclease (RE) in situ digestion (REISD) with the RE Sau3A, to differentiate donor and recipient cells on conventional bone marrow chromosome preparations. RESULTS: The polymorphism for the size of the PCH of chromosome 9 allowed differentiation of donor and recipient cells targeting both metaphase and interphase nuclei. The misidentification error for the polymorphism for the size of HPC of chromosome 9 was estimated as 1% for metaphases and 6-11% for interphases. The 3 cases studied showed complete chimerism in the first post-BMT sample analyzed, which was maintained in 2 of them. One patient relapsed and showed transient mixed chimerism. One month later, this patient achieved a second complete remission, showing complete chimerism again. In this patient, who received a sex-mismatched BMT, chimerism was also quantified by sex-chromosome identification using established methods, such as conventional cytogenetics and FISH, and the results obtained were similar to those rendered by Sau3A-REISD. INTERPRETATION AND CONCLUSIONS: The polymorphism for the size of the PCH of chromosome 9 uncovered by Sau3A-REISD allows accurate quantification of the hemopoietic chimerism after sex-matched BMT.

Bone Marrow Transplantation↗

PubMed1998

Conventional cytogenetics and FISH evaluation of chimerism after sex-mismatched bone marrow transplantation (BMT) and donor leukocyte infusion (DLI).

BACKGROUND AND OBJECTIVES: Sensitive and quantitative cytogenetic methods to better assess the biological significance of post-BMT chimerism have been recently developed. In this study, we compared the results of chimerism analysis and evolution employing conventional cytogenetics and fluorescence in situ hybridization (FISH) in 16 patients after sex-mismatched BMT, and in 5 patients after donor lymphocyte infusion (DLI) to treat post-BMT relapse. DESIGN AND METHODS: FISH studies were performed using separate digoxigenin labeled centromeric DNA probes for the X (pDMX1) and Y (DYZ1/DYZ3) chromosomes. To this purpose, different types of samples were used: bone marrow (BM) and peripheral blood (PB) slides processed for conventional cytogenetics, and routine BM and PB smears. RESULTS: Results of chimerism studies performed on different types of samples showed no significant differences. No significant differences in the ability to identify the sex of each cell with both pDMX1 and DYZ1/DYZ3 probes were found and the results obtained from independent experiments showed a high linear correlation. Chimerism analysis by FISH showed initial mixed chimerism after BMT in 10 patients. Seven of these patients were also studied by conventional cytogenetics and 2 of these showed mixed chimerism. Seven of the former 10 patients evolved to complete donor chimera. 6 patients showed cytogenetic or hematologic bone marrow relapse, 3 of which were preceded by mixed chimaerism as revealed by FISH studies. FISH studies permitted an easy and accurate monitorization of the response to DLI in 5 relapsed patients, showing an increase in the proportion of donor cells in 4 patients as they reached a new complete remission. INTERPRETATION AND CONCLUSIONS: Both FISH and conventional cytogenetics are quantitative methods to assess chimerism. However, FISH is more sensitive, accurate and can even be applied on routine BM and PB smears. Furthermore, its combination with immunophenotyping approaches to quantify chimerism on cell subpopulations, will help to clarify post-BMT chimerism significance.

Adolescent↗

PubMed1997

Dynamics of Sau3A in situ digestion of human chromosomes analyzed with computerized imaging.

The differential DNA removal obtained after in situ digestion of human metaphase chromosomes with the restriction endonuclease Sau3A was analyzed on complete and partially digested nuclei using computerized imaging. The results obtained permit the discrimination of Sau3A-resistant chromosome regions (pericentromeric constitutive heterochromatin on chromosomes 3 and 9) from those partially digested (Yq12 and pericentromeric heterochromatin on other chromosomes), according to the digestion dynamics deduced from the grey intensity profile along each chromosome. This approach permits an accurate labelling of chromosome markers for the identification of genomes from different individuals. This is of special interest for the analysis of the chimeric status found in patients after allogenic bone marrow transplantation.

Cells, Cultured↗

PubMed1997

Image analysis of murine chromatin fiber structure after in situ digestion with restriction endonucleases.

OBJECTIVE: To determine and quantify the differences produced in chromatin structure after selective in situ DNA digestion with restriction endonuclease (RE). STUDY DESIGN: Chromatin fiber structure from a murine cell line was analyzed under light and electron microscopy before and after in situ digestion with AluI, HinfI and HaeIII using digital image analysis (DIA). The proposed DIA-based method entails the generation of a binary image and a skeleton characteristic of the chromatin fiber. Morphologic features (surface, perimeter, shape, number of triple points and Euler number) of the chromatin structure can then be quantified from the digitized images. The results of these experiments were compared with those obtained from direct digestion of naked DNA using the same endonucleases in an attempt to correlate the distribution of restriction sites, according to the DNA fragment size obtained, with the extent of chromatin disorganization after RE in situ digestion. RESULTS: Homologous chromatin fiber regions are differentially affected by the action of each RE, although they are indistinguishable under light microscopy. CONCLUSION: The proposed analytical routine constitutes a valuable tool for uncovering subtle alterations in the structure of chromatin fiber that has been modified under different experimental conditions.

Animals↗

PubMed1996

Restriction endonuclease in situ digestion (REISD) and fluorescence in situ hybridization (FISH) as complementary methods to analyze chimerism and residual disease after bone marrow transplantation.

The efficiency of restriction endonuclease in situ digestion (REISD) with Sau3A to analyze chimerism and residual disease (RD) has been tested before and after an allogenic bone marrow transplant (BMT) in an acute lymphoblastic leukemia (ALL) patient. The combined results obtained with REISD and FISH using the appropriate probes for detecting chromosome rearrangements have proven to be useful for the identification and quantification of both the hemopoietic chimerism achieved after BMT and the RD persistent in the patient. The sensitivity of REISD has been determined to be around 95%, i.e., similar to that obtained by FISH. REISD with Sau3A was particularly useful in the analysis of chimerism since this enzyme revealed the polymorphic status of constitutive heterochromatin in human chromosome 3 and thus allowed discrimination of cells derived from donor and recipient. The method itself seems promising since neither a donor/recipient sex mismatch nor a cytogenetic disease marker are needed for its application.

Adult↗

PubMed1996

Digital image analysis of chromatin fibre phenotype after "in situ' digestion with restriction endonucleases.

Restriction Endonucleases (REs) may recognize, cleave and remove DNA from fixed chromatin producing specific chromosome banding patterns. However, the modifications produced in the chromatin fibre are not easy to evaluate and compare. The aim of the present investigation was to visualize differences resulting in the texture of the chromatin fibre from metaphase chromosomes after each digestion using digital image analysis (DIA) facilities. To this purpose, metaphase chromosomes derived from a L-929 mouse cell line were digested with different REs (AluI, HpaII and HaeIII). Since light microscopy does not permit the observation of the chromatin fibre, DIA was performed on digitalized images of metaphase chromosomes under electron microscopy. The application of a LUT (Look Up Table) within the DIA software assigns a colour to each grey level of a digital image. The results obtained using a particular LUT, which permits the discrimination of specific chromatin fibre phenotypes resulting from each digestion, are reported and compared with those obtained under the light microscope.

Animals↗

PubMed1996

Quantification of C-ERB-B2 gene amplification in breast cancer cells using fluorescence in situ hybridization and digital image analysis.

Fluorescence in situ hybridization (FISH) allows detection of the intercellular heterogeneity of C-ERB-B2 gene amplification in uncultured breast cancer cells. Nevertheless, because high levels of amplification result in coalescence of signals, direct microscopy quantification is restricted to cells wih low levels of amplification or with dispersed signals. A methodology of digital image analysis, using surface and grey-level FISH signals as parameters that permit a rapid, objective, and accurate estimation of gene copy number, is presented. This procedure is independent of the signal overlapping and results in a more accurate quantification and characterization of tumor cell heterogeneity.

Breast Neoplasms↗

PubMed1996

Differential sensitivity of some human alphoid and classical satellite DNA regions from lymphocyte chromosomes to in situ exonuclease III digestion.

Fluorescent in situ hybridization of alphoid and classical satellite III DNA sequences was performed on fixed chromosomes from human lymphocytes that were previously digested in situ with exonuclease III to produce single-stranded DNA motifs. Digital image analysis showed that while labeled alphoid satellite DNAs produced signals of similar strength to thermally denatured chromosomes, those of classical satellite III DNAs of chromosomes 9 and Yq were around 50% weaker. This result shows a differential sensitivity of these satellite DNA regions to in situ exonuclease III digestion and suggests structural differences in the higher-order organization of both subchromosomal constitutive heterochromatic regions.

Chromosomes, Human↗

PubMed1996

Improving chimaerism quantification in bone marrow transplant recipients by image processing and analysis after restriction endonuclease in situ digestion (IPA-REISD).

Restriction endonuclease in situ digestion (REISD) with Sau3A of human metaphase chromosomes and interphase nuclei produces a conspicuous banding pattern involving pericentromeric regions of chromosomes 9 and 3. Constitutive heterochromatin of chromosome 9 is never digested by this enzyme while that of chromosome 3 is polymorphic, giving rise to three possible karyotypes: homozygous digested (3--), homozygous undigested (3++) or heterozygous individuals (3+-). Discrimination of this polymorphism between donor and recipient cells constitutes a rapid sex-independent method to monitor quantitatively the chimaerism achieved after bone marrow transplantation. An image processing and analysis (IPA)-assisted procedure which resolves residual fluorescent regions in metaphase chromosomes or interphase nuclei after REISD has been developed. IPA-REISD has interesting advantages over the basic REISD method by allowing a rapid, objective and precise discrimination of the polymorphism in large cell samples.

Bone Marrow↗

PubMed1996

Restriction endonuclease in situ digestion (REISD): a novel quantitative sex-independent method to analyze chimerism after bone marrow transplantation.

Restriction endonuclease (RE) in situ digestion (REISD) of human metaphase chromosomes and interphase nuclei may uncover cryptic polymorphisms. This technique can be applied to identify the individual origin of cells and thus analyze the hemopoietic chimerism that eventually results in leukemic patients after allogeneic bone marrow transplantation (BMT). In the current study, results of REISD with different REs are shown. In particular, the use of Sau 3A reveals a polymorphism for constitutive heterochromatin of chromosome 3 and may differentiate BMT donor (D) and recipient (R) cells. Once pre-BMT characterization shows a different Sau 3A digestion pattern of D and R cells, it is possible to monitor the development of hematopoietic cell populations in the R bone marrow after BMT. A panel of 24 patients who underwent BMT and their Ds were analyzed. The method presented here allowed cells from D and R to be distinguished, and therefore to quantify the post-BMT hemopoletic chimerism, in 6 (25%) of the cases. This quantitative and sex-independent genetic approach to the study of hemopoietic chimerism has already shown itself to be useful in patients with leukemia who require a BMT, but could also be extended to other transplant situations.

Bone Marrow Transplantation↗

PubMed1995

Digital image analysis of chromatin fibre phenotype after "in situ" digestion with restriction endonucleases.

Alum Compounds↗

PubMed1995

Difference in constitutive heterochromatin behaviour between human amniocytes and lymphocytes detected by a sequential in situ exonuclease III digestion-random primer extension procedure.

Fixed chromosomes from human amniotic fluid cells and peripheral blood lymphocytes were digested in situ with exonuclease III and the single stranded DNA obtained was used as template for an in situ random primer extension. Under these conditions an R banding pattern, more evident in lymphocytes than in amniocytes, was obtained. Nevertheless, constitutive heterochromatin of chromosomes 1, 16, Yq, and mainly the pericentromeric region of chromosome 9 was far more intensely labelled in amniocytes than in lymphocytes. Fluorescence in situ hybridisation with a specific classical satellite DNA probe, showed that this differential labelling was dependent on a greater sensitivity of chromosome 9 constitutive heterochromatin to exonuclease III digestion in amniocytes than in lymphocytes, thus indicating qualitative differences in this region between both human cellular materials.

Adolescent↗