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Biomedical subjects

A K Raap

Publications and source records attributed to A K Raap.

At least 127 records · Page 7Linked to original sources
PubMed1990

Multiple fluorescence in situ hybridization.

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.

2-Acetylaminofluorene↗
PubMed1990

Early replication signals in nuclei of Chinese hamster ovary cells.

DNA replication sites generally known as replicon domains were resolved as individual replication signals in interphase nuclei of permeabilized Chinese hamster ovary cells by immunofluorescent microscopy. Biotin-11-dUTP was utilized as a tool to label newly replicated DNA in permeable cells and to study the distribution of nascent DNA in pulselabel and in pulsechase experiments. Active sites of DNA replication were visualized in exponentially growing cells and in synchronized cultures throughout the S phase. Fluorescent images of replication sites were analyzed by standard fluorescence microscopy and in three dimensions by confocal laser scanning microscopy. The rapid increase in number of discrete foci of newly replicated DNA is an indication that DNA synthesis starts at limited number of sites in mammalian nuclei rather than at thousands of foci at the same time.

Animals↗
PubMed1990

Three sensitive methods for the detection of cytomegalovirus in lung tissue of patients with interstitial pneumonitis.

Interstitial pneumonia after allogeneic bone marrow transplantation is frequently associated with human cytomegalovirus (HCMV) infection. However, in a considerable proportion of the cases, no infectious agent can be determined and the interstitial pneumonia is then classified as idiopathic. Hypothetically, idiopathic interstitial pneumonia could be caused by HCMV present in such small amounts or such conformation that the virus cannot be detected by routine histopathologic analysis or viral culture techniques. To check this hypothesis, three sensitive methods for HCMV detection (in situ hybridization, the polymerase chain reaction for HCMV-DNA detection, and immunohistochemistry for the detection of HCMV immediate early antigens) have been applied on lung tissue sections of bone marrow transplant patients who died with interstitial pneumonia. Three categories were distinguished: (1) patients with HCMV-related interstitial pneumonia (n = 5); (2) patients with idiopathic interstitial pneumonia (n = 10); and (3) patients with HCMV interstitial pneumonia who had been treated with antiviral therapy (n = 2). In the first category, all three techniques yielded clearly positive results, whereas these techniques indicated that one of the patients of the second category had HCMV-related pneumonia. In the third category no positive signals could be obtained. The presented data indicate that a direct involvement of HCMV in idiopathic interstitial pneumonia is unlikely. However, a PCR performed for Epstein-Barr virus (EBV) was positive in two patients with idiopathic interstitial pneumonia. These data indicate that the introduction of new sensitive techniques such as in situ hybridization, immunohistochemistry, and the polymerase chain reaction revives the interest for HCMV and other causative infectious agents in idiopathic interstitial pneumonia.

Bone Marrow Transplantation↗
PubMed1990

Combined GTG-banding and nonradioactive in situ hybridization improves characterization of complex karyotypes.

Nonradioactive in situ hybridization (ISH) using biotinylated centromere probes for chromosomes 1, 6, 7, 10, 16, 17, 18, and the X, respectively, was combined with GTG-banding to study cytogenetic changes in two different ovarian cancer cell lines. ISH was performed after GTG-banding on the same metaphase. The use of a low trypsin concentration (0.01%) in the banding procedure was essential for subsequent ISH. This combined approach allows the detection of subtle chromosomal rearrangements and appears to aid the identification of marker chromosomes.

Adult↗
PubMed1990

Detection of the Philadelphia chromosome in interphase nuclei.

Double fluorescence in situ hybridization was used to detect Philadelphia (Ph) chromosomes in interphase nuclei and metaphases of patients with chronic myeloid leukemia. Application of cosmid probes for 3' ABL and 5' BCR sequences gave better results than libraries for chromosomes 9 and 22. The present approach may provide an alternative method for monitoring minimal residual disease in Ph+ CML patients.

Cosmids↗
PubMed1990

Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITC- and biotin-labeled oligonucleotides.

Oligonucleotides labeled with FITC or biotin were applied for detection of specific mRNAs in microscopic preparations by in situ hybridization. The oligonucleotides were labeled with one FITC or biotin molecule at the 5' end or with a tail of biotin molecules at the 3' end. The target sequences were mRNAs coding for an ovulation hormone (CDCH) in the caudodorsal cells (CDC) of the pond snail Lymnaea stagnalis and a molluscan insulin-like peptide (MIP) in the light green cells (LGC) of the same organism. The hybridized oligonucleotides were detected either directly after the hybridization procedure by fluorescence microscopy or indirectly after an immunocytochemical procedure to visualize the biotin or FITC moiety. The results indicate that the detectability of the mRNA sequences is at least partially dependent on the accessibility of the target sequences for the immunocytochemical detection systems. The positive hybridization results obtained with oligonucleotides containing different labels enabled us to perform double hybridization experiments for simultaneous detection of CDCH and MIP mRNAs in one tissue section. Using FITC- and biotin-labeled oligonucleotides, we also demonstrated simultaneously different sequences on the same mRNA molecule.

Animals↗
PubMed1989

Detection of chromosome aberrations in interphase tumor nuclei by nonradioactive in situ hybridization.

In a blind study, chromosome aberrations in tumor cells were analyzed by conventional cytogenetic techniques (G banding) and nonradioactive in situ hybridization with chromosome-specific probes. The material was obtained directly from patients with hematologic diseases and from colon tumor derived cell lines. The cytogenetic data obtained with G banding were in accord with those obtained by in situ hybridization to metaphase chromosomes. Most importantly, in situ hybridization to interphase nuclei gave reliable results and even allowed detection of cell subpopulations that were not detected by analyzing metaphase chromosomes. Furthermore, in retrospect, even structural aberrations could be detected in interphase nuclei; abnormal cells with either an i(1q) or a translocation der(1)t(1;7) could be identified. Our results show that the application of in situ hybridization in combination with routine cytogenetic techniques offers significant advantages for cytogenetic analysis of solid tumors and hematologic malignancies.

Adenocarcinoma, Mucinous↗
PubMed1989

Three-color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences.

A method is described for visualizing three nucleic acid sequences simultaneously by in situ hybridization using a new blue immunofluorescent label, amino methyl coumarin acetic acid (AMCA), in combination with green and red fluorescing FITC and TRITC. Three chromosome-specific repetitive probes labeled with either amino acetyl fluorene (AAF), mercury, or biotin were hybridized simultaneously to metaphase chromosomes prepared from human blood lymphocytes or to interphase tumor nuclei. Conditions for the combined use of three immunocytochemical affinity systems as well as the optimal spectral separation of the three fluorescing labels have been determined. Three-color in situ hybridization was applied to the study of numerical chromosome abnormalities as occur in human solid tumors. Further applications of this method in prenatal diagnosis for the detection of aneuploidy of the most frequently involved autosomes, as well as for the quantification of gene copy number and mRNA expression, are discussed.

Base Sequence↗
PubMed1989

Somatic pairing of chromosome 1 centromeres in interphase nuclei of human cerebellum.

Interphase nuclei isolated from paraffin-embedded tissue of four normal brains were hybridized with biotinated repetitive DNA probes specific for the (peri)centromeric regions of chromosomes 1 and 7. Hybridization results were visualized with a peroxidase-DAB system after which the number of specific signals per nucleus was counted using bright field microscopy. Using the probe specific for chromosome 7 (p7t1), both the cerebral and the cerebellar samples showed 2 spots in 82% and 83%, respectively, of the nuclei. In situ hybridization with the chromosome 1 probe (pUC1.77) showed two spots in 69% of the cerebral nuclei. In cerebellar samples, hybridization with pUC1.77 resulted in only one large spot per nucleus in 82% of the cells. The average spot size in nuclei with one signal was about 1.6 times as large as that in nuclei with two signals. These observations suggest that the single large spot in the cerebellar cells is not the result of monosomy of chromosome 1 but that it reflects somatic pairing of the two chromosome 1 centromeres. Based on the size and the fraction of nuclei with one large spot, the small granular neuron is the most likely candidate. The difference between cerebral and cerebellar samples indicates that this somatic pairing of chromosome 1 is a cell-type-dependent phenomenon.

Cell Nucleus↗
PubMed1989

Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes.

Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF- or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidin-biotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV16 DNA and haptenized HSV2 DNA.

DNA Probes, HPV↗
PubMed1989

An improved immunocytochemical method for the detection of human cytomegalovirus antigens in peripheral blood leucocytes.

A recently described immunoperoxidase method for the detection of nuclear human cytomegalovirus (HCMV) immediate early antigen (IEA) directly on peripheral blood leucocytes suffers from the drawback that the antigen is vulnerable to endogenous peroxidase inactivation procedures. To solve this problem a procedure is developed in which endogenous peroxidase is inactivated after binding and immobilization of the primary antibody with 4% formaldehyde. In combination with this procedure, three types of inactivation were investigated: glucose/glucose oxidase, hydrochloric acid and methanol/H2O2. Of these three, the first gives optimal results, especially in combination with methanol/acetic acid (20/1 v/v) as the primary fixative. This procedure results in preparations which allow for a more objective evaluation and enable automated examination using bright field microscopy. As a second improvement we developed a simple adherence method in order to diminish the risk of infection for the laboratory staff during processing of unknown blood samples. The protocol described shows great clinical potential for the diagnosis of HCMV infections.

Antigens, Viral↗
PubMed1989

Polymerase chain reaction and viral culture techniques to detect HSV in small volumes of cerebrospinal fluid; an experimental mouse encephalitis study.

A technique is described for the detection of HSV-DNA in very small volumes (5-10 microliters) of cerebrospinal fluid (CSF). The method was evaluated in CSF samples of 4-6-week-old mice inoculated with HSV-1 via the corneal route. The sensitivity of the PCR assay was compared with results of spin-amplified viral culture with immunofluorescent visualization (SAC/IF), routine viral culture (RVC) and radioactive dot-blot hybridization (DBA) in CSF samples obtained from other mice. The results show the PCR to be superior over the other techniques: infectious virus or viral DNA in CSF was demonstrated by PCR, SAC/IF, RVC and DBA in 68, 55, 20 and 2.5%, respectively. These results show the feasibility of the PCR for a rapid, non-invasive diagnosis of human HSV-encephalitis.

Animals↗
PubMed1989

Rapid detection of human parvovirus B19 DNA by dot-hybridization and the polymerase chain reaction.

The results of a comparison of three DNA-detection methods for human parvovirus B19 DNA are described. The sensitivity of detection of virus from hybridization assays using 32P-radiolabeled DNA and RNA probes was compared with a method for enzymatically amplifying specific target DNA sequences (polymerase chain reaction). B19 virus DNA was detected using a radiolabeled DNA probe at serum dilutions of 10(-3), equivalent to approximately 3 pg of viral DNA. Using radiolabeled RNA probes even 0.3 pg of viral DNA was detectable. The polymerase chain reaction was more sensitive than the hybridization assays: 100 fg of viral DNA was easily detectable by electrophoresis on agarose and after subsequent hybridization with a radiolabeled probe approximately 10 fg of B19 DNA was detected. The sensitivity of the PCR, combined with the simplicity and reduced time scale, demonstrates the potential of this technique as an additional method for routine diagnosis of B19 infections.

Base Sequence↗
PubMed1989

Rapid detection of human cytomegalovirus DNA in peripheral blood leukocytes of viremic transplant recipients by the polymerase chain reaction.

Peripheral blood leukocyte samples (n = 458) of 24 bone marrow transplant and 52 kidney transplant patients were examined weekly for the presence of human cytomegalovirus (HCMV) using an improved culture technique (DEAFF; detection of early antigen fluorescent foci). In total 5 (21%) bone marrow transplant and 11 (21%) kidney transplant patients developed a viremia. Patients' samples were investigated for the presence of HCMV DNA using an in vitro DNA amplification technique, the polymerase chain reaction (PCR). From the statistically evaluable viremic patients (n = 13), 110 blood samples were analyzed. In 5 of these patients, the DEAFF and PCR led to identical results. In 8 patients however the PCR was more sensitive, i.e. HCMV DNA was detected for a longer period of time. Applying statistical analysis using the McNemar test, this result was significant (P less than 0.05). The PCR applied on leukocyte samples did not detect HCMV DNA in viruric patients without viremia. Moreover, the current PCR never led to positive results with peripheral blood leukocyte samples of healthy seropositive or seronegative controls. Since the PCR can be performed in 6 hr, this technique will contribute to rapid detection of HCMV DNA in peripheral blood leukocytes and therefore to optimal clinical management of HCMV-infected transplant recipients.

Antigens, Viral↗
PubMed1989

Diagnosis of progressive multifocal leucoencephalopathy by hybridisation techniques.

In situ hybridisation with acetyl-aminofluorene (AAF) and 35S-labelled DNA probes for polyomaviruses, was used to detect JC virus DNA in brain necropsy material in a patient with progressive multifocal leucoencephalopathy (PML). In a second patient PML was diagnosed from a brain necropsy specimen using the same technique. The main infected cell type were oligodendrocytes; dot hybridisation was used to estimate the number of viral copies in each infected cell. Southern blot hybridisation for further analysis of the viral genome was also carried out. In situ hybridisation with non-radioactive labelled polyomavirus DNA provides a simple and specific means for studying viral DNA in formaldehyde fixed tissue sections from patients with suspected PML. Even in small biopsy samples hybridisation results can be correlated with standard histopathological, immunocytochemical, and electron microscopic findings.

Adult↗
PubMed1989

Detection of parvovirus B19 DNA in fetal tissues by in situ hybridisation and polymerase chain reaction.

Attempts were made to detect human parvovirus B19-DNA by in situ hybridisation and the polymerase chain reaction in placental and fetal tissues from a case of intrauterine fetal death. In the in situ hybridisation experiments radioactive and non-radioactive (labelled with 2-acetyl-aminofluorene, AAF) DNA probes were used. B19-DNA was detectable in paraffin wax embedded fetal tissue from the liver, heart, lung, brain and thymus. The resolution with the AAF-labelled probes was higher than with the radiolabelled DNA. Parvovirus B19 DNA sequences were also detected in these tissues by an enzymatic in vitro amplification technique--the polymerase chain reaction. Amplification of a B19-DNA sequence before detection increases the rapidity and sensitivity of detection. The rapid, specific, and sensitive analysis of parvovirus B19 in normal and diseased tissues using these techniques may contribute considerably to determining the role of this virus as a risk factor in the outcome of pregnancy.

DNA Probes↗
PubMed1989

Detection of cytomegalovirus antigens and DNA in tissues fixed in formaldehyde.

Immunohistochemical techniques with monoclonal antibodies against cytomegalovirus (CMV) immediate early (IEA) and early antigens (EA), and in situ hybridisation, were used to detect CMV infection in routinely obtained, formaldehyde fixed and paraffin wax embedded tissues taken from bone marrow transplant patients, who had died form interstitial pneumonia. To improve the rates of detection of CMV-IEA and EA the wax embedded material was pretreated with 0.4% pepsin/HCl at 37 degrees C for 30 minutes. This pretreatment was also advantageous for in situ hybridisation. In the patients with histological evidence of CMV infection or positive viral culture from the lung tissue, or both, viral proteins and nucleic acids were detected in lung, as well as in other organs. Immunohistochemical techniques proved superior in heavily infected but necrotic tissues. In control patients (patients who had died from interstitial pneumonia without any evidence of CMV, or with no interstitial pneumonia at all) in situ hybridisation showed no positive signal, while immunohistochemical techniques showed only a few positive cells in lung tissue of one of nine patients. In addition to CMV-DNA analysis, formaldehyde-fixed, paraffin wax embedded tissue is amenable to immunohistochemical analysis with CMV monoclonal antibodies.

Antigens, Viral↗