Automated Molecular Pathology : One Hour In Situ DNA Hybridization

Abstract

We describe a rapid automated technique for colorimetric in situ DNA hybridization using the Code-On Immuno-DNA Slide Stainer. We report recent advances in positively charged glass slide technology, rapid limolene/xylene dewaxing, stable ribonuclease and pepsin predigestion, heat cycling during hybridization, and alkaline phosphatase enhancement. These amplify the signal to noise ratio of colorimetric molecular hybridization to the point where complete one hour assays can be performed from dewaxing to counterstaining by automation. Both biotin labeled oligonucleotide and nick translated DNA probes can localize their viral targets in situ with this single, mechanized protocol at the rate of one slide per minute. (The J Histotechnol 14:219, 1991)     

Heterochromatin Discrimination in Aegilops Speltoides by Simultaneous Genomic In Situ Hybridization

Simultaneous genomic in situ hybridization with probe preannealing (SP-GISH) was used for discriminating Aegilops speltoides chromosome regions by their relatedness to DNA of other species. We used a hybridization mixture of two differently labelled DNAs, one from the species used for chromosome spread preparations and a second from species of different and varying affinity, thus creating a two-colour system showing chromosome regions where alien DNA hybridized. Genomic DNA from A. speltoides was labelled with biotin and preannealed with digoxigenin-labelled total genomic DNA from different accessions of Ae. speltoides, Ae. bicornis, Ae. tauschii and Hordeum spontaneum. The probe mixture was hybridized to mitotic chromosmes of Ae. speltoides. Chromosome regions of preferential hybridization of self-DNA were visualized as green, whereas regions of combined hybridization showed orange–yellow fluorescence. We observed GISH banding patterns with a different degree of green fluorescence along Ae. speltoides chromosomes that directly correlated with evolutionary distance. Small green bands were observed in subtelomeric and telomeric heterochromatic regions using DNA of a different accession of Ae. speltoides, whereas when using DNA of H. spontaneum most regions of the chromosomes, except pericentromeric regions, showed mainly green fluorescence. The resolution and application of the approach to the study of heterochromatin differentiation are discussed.     

一种能精确检测人间期细胞核中21号染色体拷贝数的DNA探针

目的制备能精确检测人类间期细胞核中２１号染色体拷贝数的ＦＩＳＨ探针。方法利用万能引物ＰＣＲ法,从定位于人２１ｑ１１的ＹＡＣ克隆８８１Ｄ２分离制备ＤＮＡ探针,并用于与８例正常人和５例２１三体患者的外周血淋巴细胞中期相和间期核,及经细胞松弛素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ）诱导的双核细胞进行ＦＩＳＨ分析。结果该探针具有以下特点：（１）长度集中于３５０～７５０ｂｐ;（２）其靶序列位于２１号染色体长臂上且紧靠着丝粒;（３）特异性强;（４）杂交信号明亮,容易辨认;（５）对中期相及间期细胞核中２１号染色体的检出率分别高达９９．６０％和９８．４０％。结论制备的ＤＮＡ探针能精确检测人类间期细胞核中２１号染色体的拷贝数,且适用于细胞分裂时２１号染色体分离情况的研究     

间期荧光原位杂交对骨髓增生异常综合征患者5号和7号染色体异常的检测

目的筛选经济、实用的探针组合提高MDS染色体异常的检出率；比较常规细胞遗传学分析（CCA）及荧光原位杂交（FISH）两种技术在MDS染色体异常检测中的灵敏度和特异性。方法采用CCA法和HSH法分析48例患者骨髓细胞的染色体异常情况。结果CCA检出染色体异常18例（37．5％），其中复杂异常4例（8．3％），＋8异常8例（16．6％）、-5／5q-异常5例（10.4％），-7／7q-异常5例（10.4％）、20q-异常2例（4．6％）、不一致的易位3例（6．2％）。FISH除证实CCA发现的-5／5q-和-7／7q-各5例外，还检出2例有5q-，5例有7q-，1例有-7，从而使-5／5q-和-7／7q-的检出率分别增至14．5％和22．9％。平均随访12个月，38例存活，10例死亡，5例转变为急性白血病。结论CCA结合FISH能提高MDS染色体异常的检出率，与CCA相比，采用组合探针的HSH更为敏感和特异。     

地高辛标记探针原位杂交法检测肝病患者外周血单个核细胞中TTV-DNA

为检测肝病患者外周血单个核细胞(PBMC)中TTV-DNA(输血传播病毒脱氧核糖核酸), 以TTV ORF1为模板, 应用地高辛(Dig)作为标记物, 经聚合酶链反应(PCR)制备探针, 建立原位杂交方法进行检测.结果显示: 血清TTV-DNA阳性组, 双链探针检测PBMC中TTV-DNA, 阳性检出率为58.06%(18/31); 血清TTV-DNA阴性组, 双链探针检测PBMC中TTV-DNA, 阳性检出率为27.59%(8/29).双链探针阳性者, 再以负链探针检测其复制情况, 阳性率为22.2%(4/18). 结论: TTV可感染PBMC并在PBMC中复制.     

In Situ hybridization and its diagnostic applications in pathology

In situ hybridization (ISH) is a technique by which specific nucleotide sequences are identified in cells or tissue sections. These may be endogenous, bacterial or viral, DNA or RNA. On the basis of research applications, the technique is now being translated into diagnostic practice, mainly in the areas of gene expression, infection and interphase cytogenetics. Diagnostic applications are most often based on short nucleotide sequences (oligomers) labelled with non-isotopic reporter molecules, and sites of binding may be localized by histochemical or immunohistochemical methods. The technique can be applied to routinely fixed and processed tissues; with some targets, it is even possible to obtain hybridization in autopsy material. ISH has been used to detect messenger RNA (mRNA) as a marker of gene expression, where levels of protein storage are low; for example, to confirm an endocrine tumour as the source of excess hormone production. Its application in infectious diseases has to date been mainly in viral infections, such as the typing of human papillomavirus (HPV) or the detection of Epstein–Barr virus by the presence of small nuclear RNAs (EBERs). The expression of mRNAs for histone proteins has been used to detect cells in S phase, and related methods may be applied to detect apoptotic cells. Using probes to chromosome-specific sequences, it is possible to detect aneuploidy, and to document changes in specific chromosomes, which may have prognostic significance in some tumours, such as B-cell chronic lymphatic leukaemia. Using sequence-specific probes, translocations can be identified, such as the t(11;12) of Ewing's sarcoma. This review presents an outline of the technique of in situ hybridization and discusses areas of current and potential diagnostic application. © 1997 John Wiley & Sons, Ltd.     

In Situ Hybridization Evidence for a Paracrine/Autocrine Role for Insulin-Like Growth Factor-I in Tooth Development

Abstract

Insulin-like growth factor-I(IGF-I) has both metabolic and growth-promoting activities in many cell and tissue types. Although IGF-I is present in serum, it is also thought to have important autocrine and paracrine functions. Immunohistochemistry for IGF-I and its receptor have shown that IGF-I is synthesised locally by the tooth forming cells which exhibit both the IGF-I and the growth hormone receptors. This concept required to be tested by in situ hybridization. Using a digoxigenin-labelled synthetic oligodeoxyribonucleotide probe for IGF-I, we investigated the distribution of IGF-I mRNA in the continuously erupting rat incisor by in situ hybridization. The distribution and intensity of the hybridization signal varied with the developmental stage of the rat incisor. The cells of the apical loop expressed a positive hybridization signal, but the earliest polarised odontoblasts and pre-ameloblasts did not show any positive signal. The onset of enamel secretion was accompanied by a strong hybridization signal in the secretory ameloblasts as well as the odontoblasts. Maturation ameloblasts also demonstrated IGF-I message in their cytoplasm as well as their nuclei. The cells of the pulp and the dental follicle were consistently negative. However, in the adjacent alveolar bone, the signal was high in the osteoblasts and osteoclasts. These findings support the notion of paracrine or autocrine function for IGF-I in tooth development.     

Comparative Analysis of Crossover Exchanges and Chiasmata in Allium Cepa × Fistulosum After Genomic In Situ Hybridization (GISH)

Genomic in situ hybridization (GISH) successfully differentiated homoeologous genomes in the interspecific hybrid Allium cepa × fistulosum, thus allowing the detection of reciprocal crossover events as label exchanges in separating anaphase I chromosomes. Three of the eight chromosome pairs were positively identified by fluorescence in situ hybridization (FISH) to rDNA sequences. There was a general similarity of the GISH-based label exchange frequencies and metaphase I chiasma frequencies, but with a 20% deficit of chiasmata. Reasons for this apparent deficit are discussed. The locations of chiasmata and label exchanges are in broad agreement.     

原位核酸分子杂交技术检测宫颈鳞状上皮病变HPV感染

目的:观察宫颈鳞状上皮病变过程中HPV6/11、HPV16/18的表达,探讨其测定的临床意义.方法:选择病理诊断为宫颈慢性炎症50例、鳞状上皮乳头状瘤样增生30例、尖锐湿疣30例、上皮内瘤变89例、鳞癌50例共249例石蜡包埋标本,应用原位核酸分子杂交技术对其进行HPV6/11,HPV16/18检测.结果:慢性炎症组HPV6/11阳性表达4%,HPV16/18阳性表达0%;乳头状瘤样增生组HPV6/11阳性表达10%,HPV16/18阳性表达3.3%;尖锐湿疣组HPV6/11阳性表达93.3%,HPV16/18阳性表达23.3%;CIN组中Ⅰ级HPV6/11阳性表达48.9%,HPV16/18阳性表达42.2%;Ⅱ级HPV6/11阳性表达39.1%,HPV16/18阳性表达69.6%;Ⅲ级HPV6/11阳性表达9.5%,HPV16/18阳性表达66.7%;鳞癌组HPV6/11阳性表达6%,HPV16/18阳性表达82%.结论:应用原位核酸分子杂交技术检测宫颈鳞状上皮病变细胞的HPV6/11、HFV16/18,为临床早期诊断、鉴别诊断及评估预后提供可靠的理论依据.     

原位杂交法检测柯萨奇B3病毒感染鼠各脏器内病毒核酸的动态分布

用柯萨奇Ｂ３病毒（ＣｏｘｓａｃｋｉｅｖｖｉｒｕｓＢ３，ＣＶＢ３）ｃＤＮＡ探针原位杂交法检测感染后１２ｄ内小鼠各脏器内病毒核酸的分布。于不同时期分别在心肌细胞、骨骼肌细胞、胰腺细胞、胸腺细胞、脾脏红髓细胞、直肠上皮细胞和肾小管上皮细胞中检测到病毒核酸。为ＣＶＢ３对多脏器的侵犯提供了直接的依据。     

Fixation/Permeabilization: New Alternative Procedure for Immunofluorescence and mRNA In Situ Hybridization of Vertebrate and Invertebrate Embryos

A new procedure is described to visualize the spatial pattern of expression of proteins and mRNAs in cryosections or whole‐mounted leech, Drosophila, zebrafish, and chick embryos. Our principal contribution is in the use of a nonconventional fixation/permeabilization procedure based on the use of formaldehyde or paraformaldehyde combined with a short C‐chain carboxylic acid. Detergents, methanol, and proteinases were omitted. Hybridization procedures were modified from those of routinely used protocols developed for the same embryos. Results showed that cytoskeletal and other cytoplasmic proteins, as well as different mRNAs, were clearly visualized in the expected regions of the embryos. Our procedure has several advantages over currently used protocols: is simpler, produces better general preservation of cells, yields reliable results, and can be used for embryos of different taxa at different developmental stages. It is hypothesized that short C‐chain aliphatic carboxylic acids modulate the cross‐linking effect of aldehyde fixatives on cell proteins. Developmental Dynamics 242:493–507, 2013. © 2013 Wiley Periodicals, Inc.     

荧光原位杂交技术对骨髓增生异常综合征患者染色体异常的检测分析

目的:分析荧光原位杂交(FISH)技术对骨髓增生异常综合征(MDS)5、7、8、20号染色体异常检出率,并与常规细胞遗传学分析(CCA)进行比较。方法:收集48例MDS患者骨髓标本,CCA采用骨髓短期培养法及G带核型分析。FISH采用间期FISH,选取针对5、7、8、20号染色体的不同探针组合。χ2检验比较两种方法检出率。结果:CCA对5、7、8、20号染色体异常检出率分别为8.33%、12.50%、6.25%、12.51%,总的异常检出率为39.58%;FISH对5、7、8、20号染色体异常检出率分别为12.50%、16.67%、10.42%、16.67%,总的异常检出率为56.25%。两法检出率差异无统计学意义(P>0.05),但FISH检测时间(2～3天)明显短于CCA(10～14天)。结论:FISH检测MDS患者5、7、8、20号染色体异常是CCA的重要补充,并明显缩短检测时间。     

Chromogenic in situ Hybridization is a Reliable Alternative to Fluorescence in situ Hybridization for Diagnostic Testing of 1p and 19q Loss in Paraffin‐Embedded Gliomas

Recent studies imply the importance of rapid and reliable diagnostic assessment of 1p/19q status in oligodendroglial tumors. To date, fluorescence in situ hybridization (FISH) is the most commonly applied technique. FISH, however, has several technical shortcomings that are suboptimal for diagnostic applications: results must be viewed in a fluorescence microscope, results are usually evaluated by a single investigator only, and signal fading excludes physical archiving. Also, in gliomas, the distinction of diffusely infiltrating tumor cells from reactively altered normal tissue may be challenging in fluorescence microscopy. Dual‐color chromogenic in situ hybridization (CISH) has started to replace FISH in some diagnostic tests performed in pathology. Here, we present the first single institute experience with a side‐by‐side analysis of 1p/19q FISH and CISH in a series of 42 consecutive gliomas. FISH and CISH produced identical results for 1p and 19q in 93% of cases (n = 39/42). Discrepant results were reevaluated by repeated FISH and a polymerase chain reaction (PCR)‐based microsatellite marker analysis for loss of heterozygosity. Reevaluation confirmed CISH data in all three cases. We conclude that CISH is a reliable alternative in 1p/19q testing in paraffin‐embedded tissues likely to be more sensitive to detect 1p/19q status than FISH analysis.     

DNA标记杂交技术筛选抗HBs-IFN-α融合蛋白表达载体

为在无抗药性改变 ,转化细胞无生物指示系统改变的克隆载体中扩大筛选范围 ,提高筛选阳性率 ,本文应用地高辛标记DNA检测系统筛选人源单抗片段与干扰素融合蛋白表达质粒 (Anti HBs IFN α ) ,取得了理想的效果。纯化制备载体的插入段DNA ,用地高辛标记系统进行化学标记 ,制成标记探针。挑取单克隆菌扩增 ,以溶菌酶加煮沸法批量制备载体DNA ,点样于硝酸纤维膜上并经 80℃烤 2h ,分别用预杂交液和标记探针进行 42℃ 2h、 42℃ 6h的膜预杂交和杂交反应 ,洗膜后用酶标抗地高辛抗体显色。未带插入片段的抗体表达载体为阴性对照 ,IFN α质粒DNA作阳性对照 ,在 40株转化细胞中筛选出 7个目的克隆。为进一步核实该法的筛选效率和可靠性 ,分别用单酶切和双酶切法对筛选出的阳性载体进行鉴定 ,琼脂糖电泳结果显示 ,两者的符合率为 10 0 %。该系统筛选结果可靠 ,尤其在克隆载体转化细胞后无抗药改变 ,无其他筛选特征的载体构建中有一定的优势     

神经生长因子mRNA在大鼠脊髓和脊神经节的表达——原位杂交研究

我们以 SD大鼠坐骨神经为材料 ,在 NGF- c DNA文库建立的基础上 ,人工合成神经生长因子引物 ,并用 PCR地高辛标记法标记 NGF探针 ,采用原位分子杂交组织化学方法 ( ISHH) ,观察 NGF- m RNA神经生长基因表达细胞在大鼠腰段脊髓和脊神经节内的分布。结果发现在大鼠坐骨神经损伤模型腰段脊髓横切面的前角、侧角及腰背根神经节均有 NGF基因的表达细胞 ,蓝色反应物弥散性分布于胞浆内 ,呈细小颗粒状或长柱状。损伤侧要强于未损伤侧 ,并对其杂交信号进行定量分析 ,结果显示在大鼠坐骨神经损伤模型术后第 5天、第 10天及第 15天 ,脊髓前角运动神经元 ,侧角交感神经元、背根节感觉神经元内的杂交信号增强 ,表明损伤的早、中期 NGF- m RNA表达量增加。讨论了神经再生的理化因素     

An Advanced Detection System for In Situ Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe

In situ hybridization (ISH) is a powerful method for detecting specific RNAs at the cellular level. Although conventional ISH using hapten-labeled probes are useful for detecting multiple RNAs, the detection procedures are still complex and required longer time. Therefore, we introduced a new application of fluorescence resonance energy transfer (FRET)-based molecular beacon (MB) probes for ISH. MCF-7 cells and C57BL/6J mouse uterus were used for ISH. MB probes for ERα mRNA and 28S rRNA were labeled with Cy3/BHQ-2 and 6-FAM/DABCYL, and conventional probes were labeled with digoxigenin. Fluorescence measurements revealed that of more-rapid hybridization kinetics compared to conventional probes. In MCF-7 cells, 28S rRNA was detected in nucleolus and cytoplasm of all cells, whereas ERα mRNA was detected in some nucleolus. In the uterus, 28S rRNA was clearly detected using complementary MB probe, but there were no signals in control slides. Moreover, 28S rRNA was detected in all cells, whereas ERα mRNA was detected mainly in the epithelium. Fluorescence intensity of 28S rRNA was decreased significantly in 1 or 2 base-mismatched sequences, that indicates highly specific detection of target RNAs. In conclusion, the FRET-based MB probes are very useful for ISH, providing rapid hybridization, high sensitivity and specificity.     

Viral DNA Detected by In Situ Hybridization in the Developing Mouse Brain Infected with Murine Cytomegalovirus

To investigate the pathogenesis of brain abnormalities caused by congenital cytomegalovirus (CMV) infection, we previously reported experimental murine models of brain damage induced by intraventricular injection of murine CMV (MCMV) at the late stage of gestation. In the present study, viral DNA-positive cells in the damaged brain at different postnatal stages detected by in situ hybridization were compared with viral antigen-positive cells detected by an immunoperoxidase method using a monoclonal antibody against the immediate early antigen. At birth, the number of viral DNA-positive cells almost equalled that of viral antigen-positive cells. Seven to ten days after birth, the number of viral DNA-positive cells in the brain of MCMV-injected mice was one-fifth that of viral antigen-positive cells. Viral DNA-positive cells were more numerous in the hippocampus than in the cortex, and their density was dependent on the presence of viral antigen-positive cells. Dotted reaction products were observed in the nuclei of viral DNA-positive cells. These cells were rarely detected in lesions of later stages such as atrophy of the cortex and hippocampus, or the wall of the cystic lesions. These results suggest that viral DNA-positive cells detected by in situ hybridization are infected cells in which viral DNA replication is occurring actively. Acta Pathol Jpn 41: 661–667, 1991.     

Polymorphism of the Nucleolus Organizer Regions (NORs) in Physalaemus Petersi (Amphibia, Anura, Leptodactylidae) Detected by Silver Staining and Fluorescence In Situ Hybridization

The nucleolus organizer regions (NORs) of both karyotypes I and II of Physalaemus petersi (Jiménez de la Espada, 1872) from the Brazilian Amazon were studied by Giemsa staining, and by the Ag- NOR method. Karyological group I specimens were also studied by the fluorescence in situ hybridization (FISH) technique. Multiple NOR-bearing chromosomes were detected in both karyotypes. The coincident results of the Ag-NOR and FISH methods rule out the occurrence of silent NORs in this anuran. There was no intraindividual NOR variability in either group, but interindividual variability of NORs was high in group I. Seven different patterns of active NOR distribution were definitely recognized among fifteen specimens. This was considered to be a NOR site polymorphism. These results, combined with the C-band polymorphism previously reported for P. petersi, demonstrate a high rate of chromosome evolution in this group.