Detection of parvovirus B19 DNA in bone marrow cells by chemiluminescence in situ hybridization.

A chemiluminescence in situ hybridization method was developed for the search of B19 parvovirus DNA in bone marrow cells, employing digoxigenin-labeled B19 DNA probes, immunoenzymatically detected with a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. The light emitted from the in situ-hybridized probe was analyzed and measured by a high-performance luminograph connected to an optical microscope and to a personal computer for the quantification of the photon fluxes from the single cells and for image analysis. The chemiluminescence in situ hybridization was applied to bone marrow cell smears of patients with aplastic crisis or hypoplastic anemia, who had been previously tested by in situ hybridization with colorimetric detection, dot blot hybridization, and nested PCR. The chemiluminescent assay provided an objective estimation of the data, proved specific, and showed an increased sensitivity in detecting B19 DNA compared with in situ hybridization with colorimetric detection.     

应用间期荧光原位杂交技术进行快速产前诊断

选用１８、Ｘ着丝粒探针及２１号染色体特异重复序列探针分别对未培养的羊水和绒毛细胞进行了间期荧光原位杂交。结果表明，常染色体探针出现３个及１个信号点的比例约为１％和６％。Ｘ染色体探针出现３点的比例也在１％左右，可为常染色体单体或三体以及Ｘ三体或多体提供诊断依据。同传统的细胞遗传学方法比较，间期荧光原位杂交具有简便、迅速、灵敏等优点，有一定临床应用价值     

Prenatal diagnosis with repetitive in situ hybridization probes.

We have used chromosome-specific repetitive sequences to detect the most common human aneuploidies prenatally. Together chromosome 21, 13, 18, X, and Y aneuploidy comprises 95% of the chromosome abnormalities that result in a high risk of abnormal phenotypes at birth. The X, Y, and 18 repetitive probes work reliably in multiple tissue types including directly examined and cultured amniocytes, chorionic villus cells, lymphocytes, and cultured fibroblasts. The probe that detects both chromosomes 13 and 21 routinely gives results in each cell type tested except directly studied amniocytes which can be interpreted in seven-ninths of the cases with protocol 1 and all tested samples with protocol 2. Our protocols diagnosed trisomy 21 in a 23-week fetus with low maternal serum AFP and a trisomy 18 in a direct chorionic villus sample 2 working days after the samples were obtained. Trisomy 21 also has been ruled out in a CVS karyotype first thought to be 47,XY, +21. These studies reflect the potential value of in situ hybridization to provide a more rapid, less expensive means to screen most at-risk fetal populations with less effort in first world cytogenetic laboratories, and to provide economical cytogenetic services in less developed countries.     

Analysis of human IgA subclasses by in situ hybridization and combined in situ hybridization/immunohistochemistry.

In this report we describe a method for the analysis of the cellular expression of the two human IgA subclasses by in situ hybridization (ISH). The technique permits the detection of specific mRNA in individual cells using 35S-labeled oligonucleotide probes without any detectable cross-hybridization between the subclasses. This method was applicable both on cytospin preparation of peripheral blood mononuclear cells as well as in formalin-fixed, paraffin-embedded tissue sections. Furthermore, we describe a combined ISH/immunohistochemistry technique for the simultaneous detection of IgA subclass mRNA and protein at the single cell level. Examination of tissue sections from tonsils revealed a striking localization of labeled cells within the germinal center of some of the lymphoid follicles. The implications of this novel finding and the development of the method are discussed.     

应用荧光原位杂交快速产前诊断唐氏综合征

目的建立稳定的荧光原位杂交(fluorescence in situ hybridization,FISH)技术并用于唐氏综合征的产前诊断.方法对126例孕17w～24w未培养羊水细胞进行FISH快速产前诊断,以培养羊水细胞常规G显带核型分析作为FISH检测结果对照.结果被检126例羊水未培养细胞均获得诊断结果,发现4例异常胎儿.3例为标准型唐氏综合征;另1例为嵌合体.FISH检测与常规核型分析结果一致.结论FISH用于唐氏综合征产前诊断具有快速、简便、所用样本量少的优势,结果准确可靠,可达到产前诊断要求,有较大临床应用价值.     

Rapid diagnosis of bacterial meningitis by real-time PCR and fluorescence in situ hybridization

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.     

Circovirus-infected geese studied by in situ hybridization.

It has now been established that circovirus infection is common in farmed geese, but little is known about the clinicopathological significance of such infections. Ten clinically diseased geese suspected of being infected by circovirus were studied by in situ hybridization using a goose circovirus DNA probe. Circovirus DNA was demonstrated in the bursa of Fabricius (BF), spleen, thymus, bone marrow, liver, kidney, lung and heart, indicating that infection can be multisystemic. In some birds, virus DNA was present in very large quantities, most notably in the BF, liver and small intestine. With the exception of BF and thymus, there were no histological findings that would have suggested the presence of such quantities of circovirus DNA. In view of the very large quantities of virus DNA labelling present in some tissues, and by analogy to porcine circovirus type 2 infection and psittacine beak and feather virus infections, which are known to cause severe disease, and which have similar virus distribution to that found in our geese, it seems probable that the circovirus was important in the disease manifestations shown by the infected geese.     

Prenatal diagnosis with repetitive in situ hybridization probes

We have used chromosome-specific repetitive sequences to detect the most common human aneuploidies prenatally. Together chromosome 21, 13, 18, X, and Y aneuploidy comprises 95% of the chromosome abnormalities that result in a high risk of abnormal phenotypes at birth. The X, Y, and 18 repetitive probes work reliably in multiple tissue types including directly examined and cultured amniocytes, chorionic villus cells, lymphocytes, and cultured fibroblasts. The probe that detects both chromosomes 13 and 21 routinely gives results in each cell type tested except directly studied amniocytes which can be interpreted in seven-ninths of the cases with protocol 1 and all tested samples with protocol 2. Our protocols diagnosed trisomy 21 in a 23-week fetus with low maternal serum AFP and a trisomy 18 in a direct chorionic villus sample 2 working days after the samples were obtained. Trisomy 21 also has been ruled out in a CVS karyotype first thought to be 47,XY,+21. These studies reflect the potential value of in situ hybridization to provide a more rapid, less expensive means to screen most at-risk fetal populations with less effort in first world cytogenetic laboratories, and to provide economical cytogenetic services in less developed countries. © 1992 Wiley-Liss, Inc.     

Rapid diagnosis of Legionella infection by a nonisotopic in situ hybridization method

The authors report a nonradioactive adaptation of DNA hybridization technology for the direct detection of Legionella organisms in situ in routinely processed histologic specimens. The probe used consisted of synthetic oligodeoxynucleotides, complementary to the ribosomal RNA of all clinically relevant Legionella species, labeled with biotinylated dUTP at their 3' ends. By in situ DNA hybridization and detection with an avidin-alkaline phosphatase complex. Legionella was visualized by light microscopy within the alveoli of lung specimens in 9 of 13 direct fluorescent antibody- or culture-positive cases of Legionnaires' disease. No cross-hybridization was observed in lung specimens infected with Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or other pathogens. The authors' results illustrate a novel adaptation of in situ DNA hybridization techniques, usually used for viruses, to the detection of a bacterial organism. The method enables direct visualization of bacterial nucleic acid in infected tissues and may facilitate early diagnosis and treatment of legionellosis.     

Specific tissue expression and cellular localization of human apolipoprotein M as determined by in situ hybridization

Apolipoprotein M (apoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL), and in minor proportion in triglyceride-rich lipoprotein (TGRLP) and low-density lipoprotein (LDL). The gene coding for apoM has been detected in all mammal genomes. The function of apoM is unknown yet. In the present study, we demonstrated that apoM is exclusively expressed in a strong manner in adult liver and kidney, and is expressed weakly in fetal liver and kidney as detected with human multiple tissue expression array. Both immunohistochemical staining and apoM mRNA in situ hybridization demonstrated that apoM was exclusively expressed in hepatocytes in human liver and in tubular epithelial cells in human kidney. The present study helps to elucidate the pathophysiological functions of apoM in vivo.     

Ultrastructure of in situ hybridization.

Techniques for the ultrastructural localization of structures identified by in situ hybridization are being developed for both preembedding labeling and labeling on thin sections (postembedding). Successful labeling of both RNA and DNA sequences has been reported in recent years. Biotinylated nucleic acid probes are becoming increasing available. Colloidal gold is the only successful ultrastructural label with meaningful spatial localization, and the best results have been obtained with small (20-5 nm) gold particles. The link between biotinylated nucleic acid probes and gold has been protein A, antibiotin, or avidin binding. The size of the target nucleotide sequence, the size of the probe, and the number of gold particles attached to the labeling protein must be understood before there can be meaningful interpretation of micrographs. In addition, the spatial considerations depend on whether preembedding or postembedding is used.     

Prenatal diagnosis of Charcot-Marie-Tooth disease type 1a by multicolor in situ hybridization

Genetic heterogeneity within the most common genetic neuropathy, Charcot-Marie-Tooth disease (CMT) result in about 70% slow nerve conduction CMT1 and 30% normal nerve conduction CMT2. Autosomal dominant CMT1A on chromosome 17p11.2 represents about 70% of CMT1 cases and about 50% of all CMT cases. Three different size CMT1A duplications with variable flanking breakpoints were characterized by multicolor in situ hybridization and confirmed by pulsed field gel electrophoresis and quantitative polymerase chain reaction (PCR) amplification. These different size duplications result in the same CMT1A phenotype confirming that trisomy of a normal gene region results in CMT1A. the smallest duplication does not include the 409 locus used previously to screen for CMT1A duplications. Direct analysis of interphase nuclei from fetuses and at-risk patients by multicolor in situ hybridization to a commonly duplicated CMT1A probe is informative more often than polymorphic PCR analysis, faster than pulsed field gel electrophoresis (PFGE), and faster, more informative, and more reliable than restriction enzyme analysis. CMT1B restriction enzyme analysis of CMT pedigrees without CMT1A is expected to diagnose another 8% of at-risk CMT1 patients (total: 78%). © 1993 Wiley-Liss, Inc.     

Fluorescence in situ hybridization in diagnosis of urinary bladder cancer

The paper presents preliminary results of a study of the diagnosis of urinary bladder cancer by fluorescence in situ hybridization (FISH). Specific chromosomal aberrations are shown to be detectable in both proper tumor tissue and urine from patients with urinary bladder cancer; and the pattern of these changes coincides. FISH may identify cells with genetic disorders in urine long before the clinical symptoms of a recurrence occur.     

荧光原位杂交技术在染色体病产前诊断中的应用及前景

荧光原位杂交是近年来发展起来的检测染色体异常的新型技术,在产前诊断中也得到了广泛的应用,成为传统细胞遗传学检测的一种重要补充。本文着重介绍了FISH技术的原理及探针的分类,FISH技术在染色体病产前诊断中的应用及前景。     

Position statement on interphase in situ hybridization prenatal diagnosis

Williams syndrome (WS) usually occurs sporadically. Few familial cases of Williams syndrome have been described, and those reports have often lacked photographic documentation. We describe 3 families, including a 3-year-old boy and his 34-year-old father, a 2-year-old girl and her 30-year-old mother, and a 3-year-old girl and her 31-year-old mother. None of these patients has supravalvular aortic stenosis or chromosome abnormalities. In all 3 families, the parent with Williams syndrome was diagnosed after the identification of the syndrome in the affected child. © 1993 Wiley-Liss, Inc.     

DNA sequence mapping by fluorescence in situ hybridization.

Various types of DNA probes, such as total genomic DNA, repetitive sequences, unique sequences, and composites of chromosome-specific DNA probes, can be used with fluorescence in situ hybridization (FISH) techniques to address research questions having to do with localization, mapping, and distribution of DNA in situ. FISH involves the formation of a heteroduplex between such DNA probes and chromatin targets on a microscope slide, which can be visualized with fluorescent reporter molecules. Three chromatin targets--metaphase chromosomes, somatic interphases, and zygote interphases--offer increasingly extended states of chromatin which can be strategically selected, individually or in combination, to address specific research questions of interest.     

Analysis of gastrinomas by immunohistochemistry and in situ hybridization histochemistry.

Gastrinomas from 25 patients were examined by immunohistochemistry (IHC) and in situ hybridization histochemistry (ISH). Most patients (84%) presented with the Zollinger-Ellison syndrome. Six had multiple endocrine neoplasia type I (MEN-I). Twelve patients (48%) had duodenal primaries and 11 of 12 of these had metastases to regional lymph nodes and/or liver in spite of the small sizes of the primary tumors (mean size of 0.9 cm). Five patients had pancreatic gastrinomas and eight patients had metastatic tumor in regional lymph nodes or liver at surgery but a primary was not found. IHC and ISH analyses showed that all cases were positive for gastrin protein and 24 of 25 (96%) expressed gastrin mRNA that was easily detected in formalin-fixed, paraffin-embedded tissue sections. Both benign and malignant tumors expressed alpha subunit of human chorionic gonadotropin protein (alpha-HCG). However, only malignant gastrinomas (29%) expressed adrenocorticotropic hormone protein or proopiomelanocortin (POMC) mRNA. ISH and Northern hybridization analysis revealed that chromogranin A mRNA was the most common member of the chromogranin/secretogranin (Cg/Sg) family which was expressed in both benign and malignant gastrinomas. These results indicate that duodenal gastrinomas are common in both sporadic and MEN-1-associated cases, and small duodenal primaries may be associated with extensive regional lymph node and liver metastases. Expression of ACTH/POMC protein and mRNA was consistently associated only with malignant gastrinomas while gastrin protein, gastrin mRNA and Cgs/Sgs mRNAs were readily detected in both benign and malignant gastrinomas.