In Situ Hybridization Technique Research Articles

Neuroglobin, a novel member of the globin family, is expressed in focal regions of the brain.

Hemoproteins are widely distributed among unicellular eukaryotes, plants, and animals. In addition to myoglobin and hemoglobin, a third hemoprotein, neuroglobin, has recently been isolated from vertebrate brain. Although the functional role of this novel member of the globin family remains unclear, neuroglobin contains a heme-binding domain and may participate in diverse processes such as oxygen transport, oxygen storage, nitric oxide detoxification, or modulation of terminal oxidase activity. In this study we utilized in situ hybridization (ISH) and RT-PCR analyses to examine the expression of neuroglobin in the normoxic and hypoxic murine brain. In the normoxic adult mouse, neuroglobin expression was observed in focal regions of the brain, including the lateral tegmental nuclei, the preoptic nucleus, amygdala, locus coeruleus, and nucleus of the solitary tract. Using ISH and RT-PCR techniques, no significant changes in neuroglobin expression in the adult murine brain was observed in response to chronic 10% oxygen. These results support the hypothesis that neuroglobin is a hemoprotein that is expressed in the brain and may have diverse functional roles.

Changes in CD4(+), CD8(+), CD4(+) CD8(+), and immunoglobulin M-positive peripheral blood mononuclear cells of postweaning multisystemic wasting syndrome-affected pigs and age-matched uninfected wasted and healthy pigs correlate with lesions and porcine circovirus type 2 load in lymphoid tissues.

Forty-one 8- to 12-week-old wasted pigs were selected from several conventional farms with histories of postweaning multisystemic wasting syndrome (PMWS) and classified into two groups according to their porcine circovirus type 2 (PCV2) infection status, as determined by in situ hybridization (ISH). Twenty-four pigs tested positive for PCV2 (PCV2-positive group), while 17 pigs tested negative for PCV2 (PCV2-negative group). In addition, eight uninfected healthy pigs from an experimental farm were used as controls. Heparinized blood samples were taken to obtain peripheral blood mononuclear cells. The CD4(+), CD8(+), CD4(+) CD8(+) (double-positive [DP]), and immunoglobulin M-positive (IgM(+)) cell subsets were analyzed by flow cytometry with appropriate monoclonal antibodies. Histopathological studies were done to evaluate the apparent degrees of lymphocyte depletion in different lymphoid organs (superficial inguinal and mesenteric lymph nodes, Peyer's patches, tonsils, and spleen) and to determine the viral load of the PCV2 genome by using an ISH technique. Animals of the PCV2-positive group showed a significant downshift of the CD8(+) and DP cell subsets compared to the other groups (P < 0.05). Moreover, in PCV2-positive pigs, the amount of PCV2 genome in lymphoid tissues was related to the degree of cell depletion in those tissues (P < 0.05) as well as to the relative decrease in IgM(+) and CD8(+) cells in peripheral blood. These data support the notion that PCV2-positive pigs might have an impaired immune response.

Expression of 5-HT 1A receptor mRNA in rat lumbar spinal dorsal horn neurons after peripheral inflammation

The present study observed the expression of the 5-hydroxytryptamine (5-HT) 1A receptor mRNA in the lumbar spinal dorsal horn neurons following carrageenan inflammation using in situ hybridization (ISH). We also studied the co-localization of 5-HT 1A receptor mRNA and γ-amino butyric acid (GABA) or enkephalin (ENK) immunoreactivities using a combined fluorescent ISH and immunofluorescent histochemical double-staining technique. The finding of this study demonstrated that 5-HT 1A receptor mRNA was widely distributed in the spinal dorsal horn with the highest density in laminae III–VI. Following carrageenan-induced inflammation, the 5-HT 1A receptor mRNA expression in all layers of ipsilateral dorsal horn was significantly enhanced, and the peak occurred after 8 h. Furthermore, the number of 5-HT 1A receptor mRNA and GABA or ENK immunoreactive double-labeled cells was also markedly increased 8 h after carrageenan injection. These findings suggested that following peripheral inflammation, the synthesis of 5-HT 1A receptor was increased in the lumbar spinal dorsal horn neurons, especially in spinal GABA and ENK neurons.

In situ hybridization on organotypic slice cultures

Publisher Summary Slice cultures of nervous tissue represent an in vitro preparation that is thin enough to use for experimental manipulations and visualization of individual cells, yet retains much of the basic structural and connective organization of its tissue of origin. In culture, the slices become thinned to a quasi-monolayer, facilitating study of the single cells that form the intricate neuronal network. Organotypic neuronal cultures can be derived from various brain regions. The cytoarchitectural organization of the parent slice is retained and the spatial distribution of synaptic connections, which develop de novo during the first weeks in culture, forms much as it would in situ . As an in vitro analog of axonal connectivities between brain areas, slices from regions that are anatomically remote but interconnected in the brain can be co-cultured, providing a powerful model for studying the development and function of neuronal projections. Organotypic cultures thus offer several advantages over other preparations, such as cultures of dissociated cells or acute slices, and are well suited for acute and long-term pharmacological, electrophysiological, and morphological studies. In situ hybridization (ISH) allows the visualization of gene expression in cells at the mRNA level. Each cell represents a living single unit within a highly organized network, which can be investigated under standard culture conditions and also after a variety of experimental manipulations. This chapter discusses the use of the ISH technique, adapted for organotypic neuronal cultures.

Detection of Circovirus Infection in Pigeons by in Situ Hybridization Using Cloned DNA Probes

Degenerate primers were designed based on known sequence information for the circoviruses psittacine beak and feather disease virus and porcine circovirus and applied by polymerase chain reaction (PCR) to known virus-infected bursa of Fabricius (BF) from a pigeon. A 548-bp DNA fragment was amplified and shown to be specific to a novel circovirus, named pigeon circovirus (PiCV), and was used to produce sensitive and specific probes for detection of circovirus DNA by in situ hybridization (ISH). Using ISH on BF from 107 pigeons submitted for necropsy, infection was detected in 89%, compared with a histologic detection rate of 66%. Using the ISH technique, infected cells were also found in liver, kidney, trachea, lung, brain, crop, intestine, spleen, bone marrow, and heart of some birds. Large quantities of DNA were present in some of these tissues, and in the absence of BF, liver in particular is identified as a potentially useful organ to examine for presence of PiCV. This high prevalence of infection in diseased birds is noteworthy, emphasizing the need for studies to determine the precise role of this virus as a disease-producing agent.

人乳头状瘤病毒16 型DNA 在乳腺癌组织中表达的研究

Objective To investigate the etiologic relationship between human papillomavirus (HPV) type 16 infection and human breast cancer.Methods In situ hybridization (ISH) technique was used to detect the expression of HPV type 16 DNA in 50 cases of breast cancer and 30 cases of normal breast tissues.Results ISH technique showed the positive rate of HPV type 16 DNA in cancer group and in normal group were 52% and 20.0%,respectively.HPV type 16 infection was confirmed in muliple breast cancer tissues types and mainly belonged to integrated infection. Conclusion HPV type 16 infection is comn in breast cancer tissues from Liuzhou area,HPV 16 type infection is closely related to carcinogenesis of breast cancer in Liuzhou area.

Expression of 5-HT1A receptor mRNA in rat dorsal raphe nucleus and ventrolateral periaqueductal gray neurons after peripheral inflammation.

In the present study we observed the expression of 5-hydroxytryptamine (5-HT)1A receptor mRNA in the dorsal raphe nucleus (DRN) and ventrolateral periaqueductal gray (vlPAG) neurons, especially in 5-HT immunoreactive neurons (5-HT-IR), using in situ hybridization (ISH) and double staining with fluorescent ISH (FISH) and immunohistochemical (FIH) techniques. The findings of this study demonstrated that 5-HT1A receptor mRNA was expressed with moderate to high level in the DRN and vlPAG neurons. Following carrageenan inflammation, the expression of 5-HT1A receptor mRNA in the DRN and bilateral vlPAG neurons was significantly increased. The peak occurred at 3-8h followed by a clear decrease at 24 h, which basically corresponded to the time-course of behavioral hyperalgesia. Moderate 5-HT1A receptor mRNA and 5-HT immunoreactive (5-HT-IR) double-labeled cells were observed in the DRN and vlPAG, suggesting that some of 5-HT1A receptors in the DRN and vlPAG may be autoreceptors. Eight hours after carrageenan injection, the number of the double labeled cells was significantly increased. These results suggest that the synthesis of 5-HT1A receptors, including autoreceptors, is increased in the DRN and vlPAG during peripheral inflammation.

In Situ Hybridization Protocols

2nd ed, edited by Ian A. Darby (Methods in Molecular Biology, vol 123), 343 pp, with illus, Totowa, NJ, Humana Press, 2000.The second edition of In Situ Hybridization Protocols has a new editor and is substantially different in its stated aims and intended audience. While the first edition tried to cover as many areas of the in situ hybridization (ISH) technique as possible, the goals of the second are to describe research methods for gene mapping and localization of messenger RNA expression in tissues. This is a slimmer volume than the first edition, with more emphasis on tissue techniques than on chromosomal techniques. There are 2 clinical application chapters at the end, compared with 5 in the first edition.New chapters include those on ISH to whole mount embryo specimens, ISH at the electron microscope level, and detection of DNA fragmentation in apoptosis. However, a fair amount of the material in the new edition is repeated from the 1994 edition without substantial change. For example, both chapters on ISH to polytene chromosomes are nearly the same as in the first edition, down to the illustrations and references, and both cover similar material. Another topic included in both editions is radioactive ISH for gene mapping. (Even in the early 1980s when this was the only technique available, many researchers found it entirely too tedious. It is hard to imagine that anyone would try to master this time-consuming procedure rather than pursue other alternatives.)When learning a new technique, figures demonstrating equipment or results, comparative tables, notes, and background information can be especially helpful to novices. As one would expect from a multiauthored anthology, the chapters vary considerably in this regard. The new chapter on ISH with electron microscopy has beautiful pictures of several applications, as well as detailed protocols, discussion, and notes. The chapter on localization of polymerase chain reaction–amplified DNA is copiously illustrated with examples of what can go wrong and contains extensive explanatory notes. However, the chapter on fiber fluorescence ISH doesn't contain a single figure, not even of a successful result.As the editor notes in the preface, ISH techniques span the fields of molecular biology and histology/cytology, making them difficult to master. In fact, most of the techniques described in this volume are not at all simple, requiring expertise not only in molecular biology and histology, but also in tissue culture, cytogenetics, electron microscopy, statistics, or other areas. Le Moine, the author of the chapter on in situ hybridization using radioactive probes to study gene expression, acknowledges that ”the entire procedure is quite complicated” (page 152). I know from personal experience how hard it was to get the radioactive ISH technique to work, starting with a good background in cytogenetics and autoradiography and a mediocre knowledge of molecular techniques. So I do have to wonder if it might not be easier and even cheaper, at least for your first foray into these complex protocols, to just go on a field trip to learn from an expert. If you're an intermediate looking to expand your ISH repertoire, you should be able to pick up some tips from this book.

Expression of neuronal nitric oxide synthase in several cell types of the rat gastric epithelium.

The aim of this study was to identify which cell types of the rat gastric epithelium express neuronal nitric oxide synthase (nNOS) because the results of the previous studies have been very divergent regarding this point. By the combination of immunohistochemical (IHC) and in situ hybridization (ISH) techniques, we detected expression of nNOS in chief and mucosecretory cells of the gastric epithelium. Moreover, some gastric endocrine cells were immunoreactive for nNOS, although they could not be distinguished in sections treated with ISH techniques. The strongest signal for all antibodies in IHC techniques was obtained when microwave (MW) heating was performed before the IHC procedure. Our results indicate that in the gastric epithelium a variety of cell types are able to produce NO. The NO produced by the different cell types (chief, mucous, and endocrine) may form a complex network of paracrine communication with an important role in gastric physiology.

The expression of mRNA of proinflammatory cytokines during skin wound healing in mice: a preliminary study for forensic wound age estimation (II).

This study examined the temporal expression of mRNA for the proinflammatory cytokines, interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor-alpha (TNFalpha) in incised wounds in mice using the reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) techniques. After incision, an increase in each cytokine mRNA level was observed by RT-PCR. The local IL-6 mRNA level peaked at 6 h, while the peak levels of mRNA for IL-1alpha, IL-1beta and TNFalpha occurred between 48 and 72 h. All cytokine mRNA levels were almost normalized after 240 h. In the early phase of wound healing, infiltrating polymorphonuclear cells were labeled with antisense probes for IL-1alpha, IL-1beta and TNFalpha mRNA by the ISH technique. Thereafter, infiltrating mononuclear cells and spindle-shaped mesenchymal cells showed positive signals for all the cytokines examined. Regenerating epidermal cells were also labeled with the antisense probes for IL-1alpha, IL-6 and TNFalpha mRNA, indicating that IL-1, IL-6, and TNFalpha are involved in skin wound healing and their local production by various cells involved in the healing process is suggested. From the viewpoint of forensic pathology, the temporal characteristics of the cytokine mRNA expression may have a potential to indicate wound age or wound vitality.

Epstein-barr virus early ribonucleic acids as a diagnostic adjunct for relapsed metastatic tumors in patients with cured primary undifferentiated nasopharyngeal carcinoma

Epstein-Barr virus (EBV) has been consistently shown to be associated with undifferentiated nasopharyngeal carcinoma (NPC). In this article, the authors attempt to detect Epstein-Barr virus in distant relapsed metastatic sites in undifferentiated NPC patients with cured primary cancer. In situ hybridization (ISH) technique is a reliable method to detect EBV early RNAs (EBERs) within NPC cells. We used a nonisotopical ISH technique to examine the presence of EBERs in paraffin-embedded tissues obtained from 1 paired specimen of primary NPC and its metastatic counterpart at liver and 2 metastatic specimens of retroperitoneal lymph nodes and bone. All the primary lesions and the metastatic tumors of NPC with undifferentiated histology contained EBERs that could be clearly detected in the nuclei of cancer cells. This article shows that EBERs can be successfully detected in cells of the distant relapsed metastatic sites. These results suggest that this nonisotopical ISH method of EBERs can be potentially used to diagnose NPC patients developing distant relapsed metastatic lesions with cured primary cancer early. It can offer quick information as to institute suitable salvage chemotherapy for these patients.

Effect of Bone Decalcification Procedures on DNA In Situ Hybridization and Comparative Genomic Hybridization: EDTA Is Highly Preferable to a Routinely Used Acid Decalcifier

Decalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on the use of these techniques on archival decalcified bony material. In this study we compared the effects of two commonly used decalcifiers, i.e. , one proprietary, acid-based agent (RDO) and one chelating agent (EDTA), in relation to subsequent DNA ISH and CGH to bony tissues (two normal vertebrae, six prostate tumor bone metastases with one sample decalcified by both EDTA and RDO). We found that RDO-decalcified tissue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal status of EDTA-decalcified material of both control and tumor material. Gel electrophoresis revealed that no DNA could be successfully retrieved from RDO-treated material. Moreover, in contrast to RDO-decalcified tumor material, we detected several chromosomal imbalances in the EDTA-decalcified tumor tissue by CGH analysis. Furthermore, it was possible to determine the DNA ploidy status of EDTA- but not of RDO-decalcified material by DNA flow cytometry. Decalcification of bony samples by EDTA is highly recommended for application in DNA ISH and CGH techniques.

In situ detection of Aspergillus 18S ribosomal RNA in invasive pulmonary aspergillosis.

We attempted to evaluate the usefulness of in situ hybridization (ISH) in the specific diagnosis of Aspergillus pulmonary infection. We used an ISH technique using a multiple digoxigenin-incorporating probe, which was constructed by means of the polymerase chain reaction (PCR) from the 18S ribosomal RNA of Aspergillus fumigatus. We studied twelve formalin-fixed, paraffin-embedded lung tissue sections from autopsy-confirmed invasive pulmonary aspergillosis (IPA) (5 acute myelocytic leukemias, 2 acute lymphocytic leukemias, 2 chronic myelocytic leukemias, 1 adult T-cell leukemia, 1 non-Hodgkin's lymphoma and 1 chronic obstructive pulmonary disease.), and 18 sections from other pulmonary infections as control. ISH using the probe and a low-viscosity hybridization buffer solution (LV) positively stained hyphal elements in 12 of 12 autopsy lung tissue specimens from subjects with IPA, while ISH using the probe and a high viscosity hybridization buffer solution (HV) positively stained the hyphal elements in 6 of 12. Specifically, ISH (LV) demonstrates hyphal elements of Aspergillus spp. in the center of Aspergillus abscess. While, ISH (HV) can detect hyphal elements located in the periphery of a suppurative abscess as well as those in the blood vessel. Conversely, ISH did not show positive results for any of the autopsy tissue specimens from subjects with other fungal pneumonia infections (Candida n=5, Mucor n=2, Cryptococcus n=2, and Pseudallescheria n=1), Pneumocystis carinii pneumonia (n=5), and cytomegalovirus pneumonia (n=3). Dual staining by means of ISH and immunohistochemistry (IHC) using anti-neutrophil elastase (NE) and anti-CD68 monoclonal antibodies showed that NE positive cells were localized at the edge of the radial growth of the organism, but CD68 positive cells were located around the center of the abscess. The accumulation of NE positive cells was rarely seen in half of the cases (6/12). In contrast, CD68 positive cells were routinely present in the center of the abscess (12/12). ISH in conjunction with IHC is a useful tool for differentiating Aspergillus spp. from other fungal genera in tissue sections from patients with IPA and may have a certain role in the evaluation of the interactions between organisms and recruiting inflammatory cells.

Comparison of Interleukin-1β Expression by In Situ Hybridization in Monoclonal Gammopathy of Undetermined Significance and Multiple Myeloma

AbstractWe investigated whether interleukin-1β (IL-1β) is differentially expressed in plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients because IL-1β appears to play a major role in the development of lytic bone lesions, the major clinical feature distinguishing MGUS from myeloma. In situ hybridization (ISH) for IL-1β was performed using bone marrow aspirates from 51 MM, 7 smoldering MM, 21 MGUS, and 5 normal control samples. Using the ISH technique IL-1β mRNA was detectable in the plasma cells from 49 of 51 patients with active myeloma and 7 of 7 patients with smoldering myeloma. In contrast, 5 of 21 patients with MGUS and 0 of 5 normal controls had detectable IL-1β message. Bone lesions were present in 40 of the 51 MM patients analyzed, and all 40 patients had IL-1β mRNA by ISH. These results show that greater than 95% of MM patients but less than 25% of MGUS patients are positive for IL-1β production. In the future, continued follow-up of IL-1β positive and negative MGUS patients should determine whether aberrant expression of plasma cell IL-1β is predictive of those MGUS patients that will eventually progress to active myeloma.

PCR In Situ Amplification and Catalyzed Signal Amplification. Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization.

The method of in situ hybridization (ISH) using non-radioactive detection systems has become a very important molecular tool in research and diagnosis. However, nonradioactive ISH techniques generally detect only relatively abundant DNA or RNA molecules. The methods of target amplification, which multiply target molecules, or the methods of signal amplification, which increase the sensitivity of visualization, have been introduced for the detection of rare targets. Polymerase chain reaction (PCR) has been employed in most approaches to target amplification. Although the general principle of PCR in situ amplification is simple, its practical methods have revealed poor amplification efficiency and reproducibility as well as target localization. We have evaluated PCR in situ amplification methods to detect single copies of viral DNA with an experimental model of cervical carcinoma cell lines, and compared them with catalyzed signal amplification methods that we have developed using hapten-conjugated tyramide. Both amplification methods demonstrated the capability of detecting a single copy of DNA. In this overview, the applications and limitations of both target and signal amplification are summarized.

Comparison of Interleukin-1β Expression by In Situ Hybridization in Monoclonal Gammopathy of Undetermined Significance and Multiple Myeloma

We investigated whether interleukin-1β (IL-1β) is differentially expressed in plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients because IL-1β appears to play a major role in the development of lytic bone lesions, the major clinical feature distinguishing MGUS from myeloma. In situ hybridization (ISH) for IL-1β was performed using bone marrow aspirates from 51 MM, 7 smoldering MM, 21 MGUS, and 5 normal control samples. Using the ISH technique IL-1β mRNA was detectable in the plasma cells from 49 of 51 patients with active myeloma and 7 of 7 patients with smoldering myeloma. In contrast, 5 of 21 patients with MGUS and 0 of 5 normal controls had detectable IL-1β message. Bone lesions were present in 40 of the 51 MM patients analyzed, and all 40 patients had IL-1β mRNA by ISH. These results show that greater than 95% of MM patients but less than 25% of MGUS patients are positive for IL-1β production. In the future, continued follow-up of IL-1β positive and negative MGUS patients should determine whether aberrant expression of plasma cell IL-1β is predictive of those MGUS patients that will eventually progress to active myeloma.

Ultrastructural Localization of Human Papillomavirus Sequences by In Situ Hybridization and Image Analysis

Genital infection caused by human papillomavirus (HPV) is of great sanitary importance because of the role it can play in the development of squamous intraepithelial lesions and carcinomas. Because not all lesions have a malignant transformation, it is important that clinical and biological parameters contributing to the evolution of a specific lesion be determined.We used CaSki cells with 400–600 integrated copies of HPV-16 genome for in situ hybridization (ISH) with electron microscopy (EM) to locate HPV sequences. Three biotinylated DNA probes for HPV types 6/11, 16/18 and 31/ 33/51 were used, and hybrid detection was done with an anti-biotin antibody conjugated with 15 nm gold colloids. Electron microphotographs with positive labeling were studied by image analysis to skeletonize them to highlight the heterochromatin fibers and to determinate the spatial localization of colloidal-gold particles. Electron microscopy and ISH technique revealed 2 different distribution patterns: colloidal-gold particles spread over the whole nuclear surface, individually or in small clusters, and large accumulations with many gold particles in the nuclear periphery, next to the membrane. Image analysis revealed there was a spatial association between the gold particles and heterochromatin fibers in all cases.We believe this association is the consequence of viral genome integration. With these results and knowledge that integration of viral DNA is linked to a worse prognosis, we propose the application of this methodology for the future study of HPV infected biopsies to obtain prognostic information in lesions caused by this virus. (The J Histotechnol 21: 295–298, 1998)

Ultrastructural Localization of Human Papillomavirus Sequences by In Situ Hybridization and Image Analysis

Genital infection caused by human papillomavirus (HPV) is of great sanitary importance because of the role it can play in the development of squamous intraepithelial lesions and carcinomas. Because not all lesions have a malignant transformation, it is important that clinical and biological parameters contributing to the evolution of a specific lesion be determined.We used CaSki cells with 400–600 integrated copies of HPV-16 genome for in situ hybridization (ISH) with electron microscopy (EM) to locate HPV sequences. Three biotinylated DNA probes for HPV types 6/11, 16/18 and 31/ 33/51 were used, and hybrid detection was done with an anti-biotin antibody conjugated with 15 nm gold colloids. Electron microphotographs with positive labeling were studied by image analysis to skeletonize them to highlight the heterochromatin fibers and to determinate the spatial localization of colloidal-gold particles. Electron microscopy and ISH technique revealed 2 different distribution patterns: colloidal-gold particles spread over the whole nuclear surface, individually or in small clusters, and large accumulations with many gold particles in the nuclear periphery, next to the membrane. Image analysis revealed there was a spatial association between the gold particles and heterochromatin fibers in all cases.We believe this association is the consequence of viral genome integration. With these results and knowledge that integration of viral DNA is linked to a worse prognosis, we propose the application of this methodology for the future study of HPV infected biopsies to obtain prognostic information in lesions caused by this virus. (The J Histotechnol 21: 295–298, 1998)

Diagnosis of cytomegalovirus infection in HIV-infected patients with respiratory disease

Background: The role of the cytomegalovirus (CMV) in the respiratory morbidity and mortality of HIV-infected patients remains unclear. This is due in part to difficulties in making an accurate and rapid diagnosis. There has been a limited number of studies, often with few or no AIDS patients, on the use of DNA–DNA in situ hybridization (ISH) and polymerase chain reaction to diagnose CMV respiratory infection directly on bronchoalveolar fluid samples. Objectives: To compare the centrifugation culture (CC), ISH, and nested-primer polymerase chain reaction (npPCR) techniques on bronchoalveolar fluid for the diagnosis of respiratory CMV infection. Study design: Samples were obtained prospectively from a group of 35 HIV-infected homosexual men evaluated for pneumonia at a university hospital. Sensitivity, specificity, and predictive values of the three techniques were measured and compared, using the conventional roller tube cell culture (CRTC) as the gold standard. Results: Sensitivity, specificity, positive and negative predictive values were as follows: 86%, 86%, 90%, and 80% for the CC; 5%, 100%, 100%, and 41% for ISH; and 86%, 57%, 75%, and 73% for npPCR. Of the six false positive samples by npPCR, two were positive by CC (none by ISH). If the latter were considered true positives, the specificity and positive predictive values of npPCR would increase to 67% and 83%, respectively. Conclusions: CC appeared to be the best of the three techniques compared in this study for diagnosis of respiratory CMV infection in HIV-infected patients. The sensitivity and predictive values of DNA–DNA ISH were very poor. Results with npPCR were acceptable, and this technique may be considered in situations when rapid diagnosis of CMV infection is necessary.

Application of in-situ hybridization techniques to study human preimplantation embryos: a review.

Although the first in-situ hybridization (ISH) experiments were performed more than two decades ago, it was not until 1987 that the technique was implemented in the study of preimplantation. Since then, several groups have explored the feasibility of using the ISH technique as a diagnostic tool to study the genomic constitution of human preimplantation embryos. Though a fair number of such ISH studies have been published and unravelling of the embryos' genomic secrets proceeds with great strides, there are still many challenges in this area of research. The scope of this review is to outline the research and diagnostic applications of single and multicolour (fluorescence) ISH to study the chromosomal content of human preimplantation embryos and to discuss the implications for human preimplantation genetic diagnosis. Current hybridization techniques are being discussed, including the capabilities and pitfalls of this approach when applied to human preimplantation embryos. Finally, future perspectives for the ISH technique in studying the genomic constitution of preimplantation embryos are reviewed.