Subtracted, unique-sequence, in situ hybridization: experimental and diagnostic applications

Nonrandom chromosomal aberrations, particularly in cancer, identify pathogenic biological pathways and, in some cases, have clinical relevance as diagnostic or prognostic markers. Fluorescence and colorimetric in situ hybridization methods facilitate identification of numerical and structural chromosome abnormalities. We report the development of robust, unique-sequence in situ hybridization probes that have several novel features: 1) they are constructed from multimegabase contigs of yeast artificial chromosome (YAC) clones; 2) they are in the form of adapter-ligated, short-fragment, DNA libraries that may be amplified by polymerase chain reaction; and 3) they have had repetitive sequences (eg, Alu and LINE elements) quantitatively removed by subtractive hybridization. These subtracted probes are labeled conveniently, and the fluorescence or colorimetric detection signals are extremely bright. Moreover, they constitute a stable resource that may be amplified through at least four rounds of polymerase chain reaction without diminishing signal intensity. We demonstrate applications of subtracted probes for the MYC and EWS oncogene regions, including 1) characterization of a novel EWS-region translocation in Ewing's sarcoma, 2) identification of chromosomal translocations in paraffin sections, and 3) identification of chromosomal translocations by conventional bright-field microscopy.     

In situ hybridization with riboprobes: an overview for veterinary pathologists

Although preliminary reports indicate that fatigue is a common symptom of human immunodeficiency virus (HIV) disease, little empirical research has focused on its prevalence or characteristics among patients with acquired immunodeficiency syndrome (AIDS). We assessed the frequency of fatigue and its medical and psychological correlates, in a cross-sectional survey of ambulatory AIDS patients. Ambulatory patients with AIDS who participated in a study of quality life (N = 427) were classified into fatigue/no fatigue groups based on their responses to fatigue items on the Memorial Symptom Assessment Scale (MSAS) and the AIDS physical symptom checklist. Self-report inventories were also administered to assess psychological distress, depressive symptoms, and overall quality of life. Medical information was elicited through clinical interview and review of medical chart. Fifty-four percent of the patients endorsed both of the fatigue items from the MSAS and the AIDS physical symptom checklists, and were classified as having fatigue. Women were significantly more likely to report fatigue than men (chi square = 5.28, df = 1, P < 0.03), and patients reporting homosexual contact as their transmission risk factor were significantly less likely to report fatigue than were patients reporting injection drug use or heterosexual contact (chi square = 5.13, df = 2, P < 0.03). The presence of fatigue was significantly associated with the number of current AIDS-related physical symptoms [t(425) = 8.00, P < 0.0001], current treatment for HIV-related medical disorders (chi square = 12.51, df = 1, P < 0.0001), anemia [t(174) = -2.35, P < 0.02], and pain (chi square = 36.36, df = 1 P < 0.0001). Patients with fatigue also had significantly poorer physical functioning ability [Karnofsky: t(422) = -6.27, P < 0.0001], as well as greater degree of overall psychological distress and lower quality of life [F(5,418) = 23.79, P < 0.0001], as measured by the Brief Symptom Inventory, Beck Depression Inventory, Beck Hopelessness Scale, Functional Living Inventory for Cancer (modified for AIDS), and the MSAS Psychological Distress Subscale. Fatigue is a common symptom in ambulatory AIDS patients and is associated with significant physical and psychological morbidity.     

Chemiluminescence: a sensitive detection system in in situ hybridization

PURPOSE: To quantify, with magnetic resonance (MR) imaging, the in vivo changes in cartilage volume and thickness after physical exercise. MATERIALS AND METHODS: The patellae of eight volunteers were imaged six times at physical test by using a spoiled fat-suppressed gradient-echo sequence with an acquisition time of 4.10 minutes. The volunteers then performed 50 knee bends, and two more data sets were acquired 3-7 minutes and 8-12 minutes after exercise. The patellar cartilage volume was determined after three-dimensional reconstruction, and the thickness was assessed with a three-dimensional minimal-distance algorithm. RESULTS: Whereas repositioning had a small effect on the measurements (mean coefficient of variation, 1.4%), a statistically significant decrease in cartilage volume was observed 3-7 minutes (mean decrease, 6.0%; P < .05) and 8-12 minutes (mean decrease, 5.2%; P < .05) after exercise. The deformation was homogeneous throughout the joint surface. In one asymptomatic volunteer, a cartilage lesion became more pronounced after exercise. CONCLUSIONS: MR imaging can be used to investigate the response of articular cartilage to physical exercise in vivo. Patients or volunteers should be allowed a sufficient period of physical rest if quantitative measurements of cartilage volume and thickness are to be undertaken in longitudinal studies.     

Identification of marker chromosomes and translocations in man using a computerized diagnostic database and fluorescent in situ hybridization

BACKGROUND: Many species of bats have two sets of submandibular glands, principal and accessory. The accessory gland may resemble the principal one but more often shows wide morphological divergence. The free-tailed bat, Tadarida thersites, is very unusual in that it has two sets of parotid glands rather than binary submandibular glands. We studied the ultrastructure of the principal parotid gland to establish a baseline for comparison with the accessory parotid. METHODS: Two specimens of adult free-tailed bats, one male and one female, were live-trapped in western Kenya. Parotid glands were fixed for electron microscopy using a protocol expressly designed for field fixation and then embedded by conventional means. RESULTS: Histologically, the principal parotid is a typical serous gland. The secretory granules of the endpiece cells have an unusual substructure in that they contain variable numbers of lucent halos and one or several spherules. Intercalated duct cells contain a significant number of dense, serous-like granules. Striated ducts have the usual basal configuration of mitochondria and folded plasma membranes, but the supranuclear cytoplasm contains many small, dense granules, so that these ducts resemble the granular convoluted tubules found in the submandibular glands of many families of rodents. The apices of the duct cells have a peculiar contour--the luminal surfaces obliquely invaginate into the apical cytoplasm, so that in thin section the luminal membranes appear to be underlaid by a layer of vacuoles. CONCLUSION: Although the principal parotid gland of the free-tailed bat shows some distinctive, species-specific ultrastructural features, it basically is similar to the parotid gland in two other molossid bats, Tadarida brasiliensis and Molossus molossus. The distinctive features in the principal parotid gland of T. thersites might relate to its feeding on hard-bodied insects and perhaps to the production of lysozyme.     

Thermography as a diagnostic aid in tennis elbow

Thermal screening was used in order to distinguish between Tennis Elbow and entrapment of posterior interosseous nerve. Both conditions have a very similar clinical picture but there should be an altogether different approach concerning treatment. The differential diagnosis is therefore of great importance and thermography of the elbow is a valuable aid in this distinction.     

In situ hybridization analysis of stanniocalcin mRNA expressing cells in the mouse kidney

INTRODUCTION: At present quality of life has a significant place within health care and scientific research work. This interest is stimulated by the fact that people want to live, not just to survive. The problem of quality of life in persons whose lives could not be preserved, opens discussions concerning artificial subsistence of life, euthanasia etc. This is not a new topic. What is new is development of official ways to measure quality of life and their routine application. CONCEPT OF QUALITY OF LIFE: In general, quality of life can be defined as the level of well-being. It cannot be identified with health, but probably primarily with ability to conduct an economically and socially productive life. Quality of life refers to physical, psychological and social domains of health, being influenced by one's experience, beliefs, expectations and perceptions. There is no consensus concerning the concept of quality of life and according to the same authors it includes functional ability, level and quality of social interactions, physical welfare, somatic sensations and life satisfaction. Generally speaking concepts include numerous dimensions and possibilities occurring during life, to death itself (Table 1). Although objective dimension of health is important in assessment of patient's health, subjective estimation and expectations make, what is found to be an objective situation, experienced quality of life (Graph. 1). MEASURING QUALITY OF LIFE: The most difficult task is to present various segments of health into quantitative values. All data can be measured at nominal, ordinal, interval or ratio scales. The nominal scale uses numbers and other symbols in classification of characteristics. Categories cannot be classified according to volume and are mutually exclusive. Ordinal scales are used when measuring a limited number of categories classified according to quality. Interval scales measure an unlimited number of categories with equal intervals and they are without a real zero point. Ratio scales have all the characteristics of interval scales, but they have real zero points. CHOICE OF MEASURING INSTRUMENTS: There are different instruments to assess quality of life such as generic instruments, battery scales and modular instruments index methods and instruments. The measuring instruments should be reliable, valid, responsive and sensitive. QUALITY OF LIFE EVALUATION: Three design studies are most frequently used: cross-sectional or nonrandomized longitudinal studies, randomized studies of clinical interventions and cost-effective and cost benefit analyses.     

Absolute lymphocytopenia as a diagnostic aid in bronchogenic carcinoma

Total lymphocyte count and lymphocyte percentage were calculated in a group of 26 patients of Bronchogenic carcinoma and compared with the values in 22 cases of pulmonary tuberculosis. Patients of Bronchogenic carcinoma had a significant lymphocytopenia as compared to patients of tuberculosis. It is inferred that this peripheral lymphocytopenia can be used as a possible diagnostic pointer in patients of Bronchogenic carcinoma.     

HL-A as a diagnostic aid in incomplete rheumatic syndromes

Incomplete Reiter's syndrome (ICRS) and incomplete Beh?et's disease (ICBD) have many similar clinical features, making the diagnosis difficult. The association of RS and BD with different HL-A antigens is a help in differentiating between the two diseases. Eight patients with incomplete rheumatic syndrome are described. The results of HL-A typing suggest that five patients had incomplete RS and the other three incomplete BD. The value of tissue typing in the correct diagnosis of incomplete rheumatic syndrome is stressed.     

Advances in fluorescence in situ hybridization

The techniques of in situ hybridization (ISH) are widely applied for analyzing the genetic make-up and RNA expression patterns of individual cells. This review focusses on a number of advances made over the last 5 years in the fluorescence ISH (FISH) field, i.e., Fiber-FISH, Multi-colour chromosome painting, Comparative Genomic Hybridization, Tyramide Signal Amplification and FISH with Polypeptide Nucleic Acid and Padlock probes.     

In situ hybridization analysis of leucomyosuppressin mRNA expression in the cockroach, Diploptera punctata

In the cockroach Diploptera punctata, sequencing of the cDNA for the insect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demonstrated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-related peptide to be encoded by this gene (Donly et al. [1996] Insect Biochem. Mol. Biol. 26:627-637). However, in the present study, high performance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-eluted with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D. punctata. Immunohistochemistry with a polyclonal antiserum generated against FMRFamide, but which recognizes extended RFamide peptides, demonstrated numerous RFamide-like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and processes of the brain and optic lobes, the stomatogastric nervous system, including the frontal and ingluvial ganglia, and the suboesophageal ganglion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, positively stained processes were found in the crop, midgut, and hindgut, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning the entire LMS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression was most abundant in the brain and optic lobes and in the frontal and suboesophageal ganglia. LMS mRNA was also apparent, although less intensely, in all other ganglia of the ventral nerve cord. Within the alimentary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of LMS mRNA in the central nervous system, stomatogastric nervous system, and midgut suggests that LMS may play a central role in Diploptera and may be associated with feeding and digestion.     

Quick-FISH: a rapid fluorescence in situ hybridization technique for molecular cytogenetic analysis

OBJECTIVE: To evaluate the pregnancy results of an ovarian hyperstimulation protocol for IVF-ET that combines GnRH agonist down-regulation, cessation of GnRH agonist therapy with the onset of menstruation, and high-dose gonadotropin administration in low responders. DESIGN: Prospective analysis. SETTING: Academic IVF program. PATIENT(S): One hundred eighty-two low responders undergoing 224 IVF-ET cycles. INTERVENTION(S): Down-regulation was obtained with the administration of leuprolide acetate beginning in the midluteal phase and ending with the onset of menses. Daily administration of 6 ampules of FSH alone or in combination with hMG was initiated on cycle day 3. MAIN OUTCOME MEASURE(S): Stimulation characteristics and pregnancy rates (PRs) were compared between fresh cycles in which pure FSH alone was used and 35 cycles in which a combination of FSH and hMG was administered. RESULT(S): The clinical PR per transfer, the ongoing PR per transfer, and the implantation rate were 32%, 24%, and 9%, respectively. No differences were noted between cycles in which pure FSH alone was used in comparison with cycles in which a combination of FSH and hMG was administered. CONCLUSION(S): Short-term ovarian suppression begun in the luteal phase and discontinued with the onset of menses followed by high-dose stimulation with gonadotropins yields favorable pregnancy results in low responders.     

Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.     

In situ hybridization for the Y chromosome reveals a donor origin for a posttransplant lymphoproliferative disorder in a sex-mismatched hepatic allograft

We report a case of posttransplant lymphoproliferative disorder diagnosed within 4 weeks of orthotopic liver transplantation from a male donor to a female recipient. To determine whether the posttransplant lymphoproliferative disorder was of donor or recipient origin, nonisotopic in situ hybridization for the human Y chromosome was performed on formalin-fixed, paraffin-embedded sections of the donor liver using a digoxigenin-labeled probe. The lymphoid cells hybridized with the Y chromosome probe, indicative of a male genotype consistent with posttransplant lymphoproliferative disorder of donor origin. This case illustrates that nonisotopic in situ hybridization for the Y chromosome can discriminate between donor and recipient cells in sex-mismatched organ transplants.     

In situ nucleic acid hybridization of cytokines in primary biliary cirrhosis: predominance of the Th1 subset

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by destruction of the intrahepatic bile ducts. It is generally believed that cellular immune mechanisms, particularly involving T cells, result in this bile duct damage. The relative strength of Th1 and Th2 responses has recently been proposed to be an important factor in the pathophysiology of various autoimmune diseases. In this study, we have attempted to identify the Th subset balance in PBC, by detection of cytokines specific to the two T-cell subsets, i.e., interferon gamma (IFN-gamma) for Th1 cells and interleukin-4 (IL-4) for Th2 cells. We analyzed IFN-gamma and IL-4 messenger RNA (mRNA) positive cells in liver sections from 18 patients with PBC and 35 disease controls including chronic active hepatitis C, extrahepatic biliary obstruction (EBO), and normal liver, using nonisotopic in situ hybridization and immunohistochemistry. Mononuclear cells expressing IFN-gamma and IL-4 mRNA were aggregated in inflamed portal tracts in PBC livers, but were rarely present in extrahepatic biliary obstruction, alcoholic fibrosis, or normal liver sections. The IFN-gamma and IL-4 mRNA positive cells in PBC livers were detected in significantly higher numbers than in control livers (P < .01). Moreover, IFN-gamma mRNA expression was more commonly detected than IL-4 expression in PBC livers, and the levels of IFN-gamma mRNA expression were highly correlated with the degree of portal inflammatory activity. IFN-gamma mRNA-positive cells were detected primarily around damaged bile ducts that were surrounded by lymphoid aggregates. The data indicate that Th1 cells are the more prominent T-cell subset in the lymphoid infiltrates in PBC.     

Liver metastasis from carcinoid: diagnostic imaging

Our study was aimed at describing the diagnostic imaging patterns of carcinoid liver metastases. Six patients with liver metastases secondary to carcinoid tumor were examined. The metastases were histologically proved in each patient. All patients were examined with ultrasonography (US), Computed Tomography (CT), Magnetic Resonance Imaging (MRI) and Digital Subtraction Angiography (DSA). All patients were treated with transcatheter embolization of liver metastases. Diagnostic imaging methods showed ten to many dozen metastases in each patient. Tumor size ranged 0.5 to 14 cm. US showed hypoechoic, hyperechoic and isoechoic carcinoid liver metastases with a hypoechoic halo. Large lesions had anechoic central areas due to colliquative necrosis. Hypoechoic patterns were the most frequent ones. Precontrast CT showed hypodense metastases; very small lesions were isodense relative to surrounding liver. CT during contrast agent injection showed that the metastases were hyperdense in the arterial phase; contrast enhancement was poorer in the portal phase. Large lesions showed a hypodense central area due to necrosis which remained hypodense in the late phase. The metastases were hypointense on T1-weighted and hyperintense on T2-weighted MR images. Gradient echo dynamic imaging with Gd-DTPA showed high-signal metastases in the arterial phase and lower signal intensity in the portal phase. DSA, an essential exam before embolization, showed tortuous and elongated intra- and extrahepatic arteries and tumor neovascularization with no malignant abnormalities. In the capillary phase, tumor uptake and inhomogeneous hypervascular patterns were shown. Portal veins were only displaced and compressed, but never infiltrated by the metastases. All the techniques we used contributed to assess liver involvement by carcinoid metastases. DSA must be used only before treatment; both CT and MRI showed the hypervascular patterns of the metastases, but no technique could predict their nature.     

In situ hybridization analysis of presenilin 1 mRNA in Alzheimer disease and in lesioned rat brain

Presenilin-1 (PS-1) gene mutations are responsible for the majority of the early onset familial forms of Alzheimer disease (AD). Neither PS-1's anatomic distribution in brain nor expression in AD have been reported. Using in situ hybridization in the rat forebrain, we show that PS-1 mRNA expression is primarily in cortical and hippocampal neurons, with less expression in subcortical structures, in a regional pattern similar to APP695. Excitotoxic lesions lead to loss of PS-1 signal. A neuronal pattern of expression of PS-1 mRNA was also observed in the human hippocampal formation. AD and control levels did not differ. PS-1 is expressed in brain areas vulnerable to AD changes more so than in areas spared in AD; however, PS-1 expression is not sufficient to mark vulnerable regions. Collectively, these data suggest that the neuropathogenic process consequent to PS-1 mutations begins in neuronal cell populations.     

In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease

PURPOSE: The common finding of thrombi between the bifoil balloons when they were extracted after mitral dilation prompted us to look for evidence of minor brain embolisms using the sensitive technique of BMRI (brain magnetic resonance T2-weighted imaging). METHODS: BMRI was performed within 48 hr before and after a percutaneous mitral balloon commissurotomy (PMBC) in each of the 63 patients in this study. RESULTS: There was evidence (hyperintensity foci: HI) of a previous asymptomatic brain embolism in 38 of 63 patients before PMBC and a new HI appeared in 18 of 63 patients after the procedure. New HI signals were found exclusively in the white matter in 8 of 18 patients and in only 3 of 18 were HI signs larger than 1 cm. One patient, with an HI signal >1 cm in the thalamus and another <1 cm in the brain stem, presented diplopia accompanied by other minor clinical signs. The differences in HI rate among four subgroups (1, older vs younger than 43 years; 2, sinus rhythm vs atrial fibrillation; 3, echo score <8 vs >8; 4, patients from western countries vs the others) were not statistically significant, probably because the number of patients in each subgroup was low. Patients in atrial fibrillation had slightly more (not significant) HI before PMBC (15/20, 75%) than patients in sinus rhythm (23/43, 53%), but after PMBC their HI frequencies were similar (atrial fibrillation: 5/20, 25%; sinus rhythm: 13/43, 30%). CONCLUSION: Brain microembolism is frequent during PMBC, but is often anatomically limited and free from clinical signs in most cases. Brain embolism seems to be related mainly to the procedure itself and not the features of the patient.