A K Graham

Interphase cytogenetics using biotin and digoxigenin labelled probes I: relative sensitivity of both reporter molecules for detection of HPV16 in CaSki cells.

This study was undertaken to develop technology for the detection of nucleic acid using two different DNA probe reporter molecules, the ultimate aim being to differentially label two nucleic acids within the same nucleus. Digoxigenin and biotin were used to label DNA probes. The absolute and relative sensitivity of digoxigenin and biotin labelled DNA probes for detecting integrated human papilloma virus 16 (HPV16) was investigated in CaSki cells by non-isotopic in situ hybridisation (NISH). Several methods for the detection of labelled probes were also investigated. The optimal sensitivity of digoxigenin labelled probe was equivalent to that of biotin when alkaline phosphatase was used as the final detector. The median number of discrete viral signals discernible in each cell with the most sensitive detection system was seven to eight with both labelled probes. The average number of HPV16 genomes in each CaSki cell, derived by dot blot hybridisation, was about 270. The calculated absolute sensitivity of NISH for viral detection in this system is complex because of variation of signal size and number. Nevertheless, one signal per nucleus equates to as little as 30 to 40 viral copies, and probably much less. The ability to distinguish up to 15 discrete signals with both digoxigenin and biotin labelled probes in the nuclei of CaSki cells indicates that these methods will be useful in interphase cytogenetics in material routinely fixed in aldehyde.

Interphase cytogenetics using biotin and digoxigenin labelled probes: III. Increased sensitivity and flexibility for detecting HPV in cervical biopsy specimens and cell lines.

A monoclonal antibody to digoxin enabled sandwich techniques to be used for the detection of hybridised digoxigenin labelled probes in cultured cells and paraffin wax sections. This system has greater flexibility than alkaline phosphatase conjugated polyclonal antidigoxigenin antibody and permits the use of alternative detector enzymes, such as horseradish peroxidase and fluorescence labels. The APAAP detection system that does not require the use of biotin can also be used in situations where endogenous biotin is a problem. The low level of background staining combined with precise substrate deposition of the amplified peroxidase system gives higher sensitivity and resolution. This permits localisation of closely adjacent chromosomal loci in interphase nuclei. The most sensitive peroxidase based digoxigenin detection system visualises two and a half to 12 copies of human papillomavirus (HPV) per nucleus. This system is also suitable for the analysis of low copy number HPV infection of cervical tissues.

In situ evidence for HPV 16, 18, 33 integration in cervical squamous cell cancer in Britain and South Africa.

In a previous study three types of HPV signal were described in CIN. It was suggested that a type 1 signal represented episomal HPV while a type 2 signal represented integrated HPV; and a type 3 signal was indicative of both episomal and integrated HPV. To test this hypothesis 91 squamous cell cancers (SCC) of the cervix from Britain and South Africa were examined for HPV 6, 11, 16, 18, 31, 33, 35. Of the South African group (n = 69) 64% contained HPV types 16 (n = 29) and 18 (n = 15). The SCC in the British group (n = 22) contained HPV 16 and HPV 33 in 12 and three cases, respectively. Of the HPV positive biopsy specimens, 86% showed a type 2 signal in keratinising and non-keratinising tumours and the remainder a type 3 signal. Type 3 signal was present only in keratinising tumours. The presence of punctate signal in 100% of HPV containing SCC, together with localisation of HPV signal to sister chromatids in tumour cell mitotic figures in vivo, provides further evidence for type 2, and the punctate component of type 3 signal representing viral integration.

Interphase cytogenetics using biotin and digoxigenin labelled probes II: Simultaneous differential detection of human and papilloma virus nucleic acids in individual nuclei.

A method was developed for the simultaneous detection of viral and human DNA in contrasting colours in routine formalin fixed, paraffin wax embedded biopsy specimens. This was achieved by non-isotopic in situ hybridisation (NISH) with a biotinylated Y chromosome probe and digoxigenin labelled probe for human papilloma virus type 6 (HPV 6). The tissues studied were peripheral lymphocytes, tonsil, and penile warts. The hybridisation signals produced by biotinylated probes were visualised in red using streptavidin peroxidase and those produced by digoxigenin labelled probes as a blue/black colour using anti-digoxigenin alkaline phosphatase. In lymphocytes and tonsil 95-100% of cells had a detectable Y chromosome; in warts only 60-70% of infected keratinocytes near the skin surface had a demonstrable Y chromosome. This suggests that this chromosome is lost or occluded in cell maturation. In simultaneous double hybridisation with both probes, HPV and Y sequences were demonstrable within the same nucleus in penile warts. This technique permits the simultaneous differential detection of two nuclei acid sequences in interphase nuclei and will have application in analysis of putative dual HPV infections and in determining the intranuclear spatial relations between nucleic acids in interphase nuclei.

Discrimination of closely homologous HPV types by nonisotopicin situ hybridization: definition and derivation of tissue melting temperatures

SummaryIt is generally assumed that nucleic acid association duringin situ hybridization reactions is similar to that of nucleic acid association in solution. This assumption has been investigated by detecting closely homologous human papillomavirus types 6 and 11 byin situ hybridization as a model for the evaluation of stringency conditions in clinical biopsies.By examining matched and mismatched, labelled and target sequences under various stringency conditions, empirical DNA-DNA stability curves and their derivative equations for tissue melting temperatures (Tmt) were derived. The corresponding values for Tmt are 10–20°C higher than their solution equivalents. These data, supported by polymerase chain reaction experiments, demonstrate that closely homologous viral DNAs cross linked in tissue by formaldehyde fixation do not interact with the corresponding labelled probes as predicted from solution kinetic equations. This not only has theoretical implications but is also relevant to the accuracy of clinical diagnostic testing.

The discrimination of high-risk HPV types by in situ hybridization and the polymerase chain reaction

SummaryThe parameter Tmt has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tmt) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tmt is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tmt is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy.Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material.

Clinical trials of robotic interactive telepathology accuracy in the UK breast pathology external quality assurance scheme

There is noroutine telepathologyactivity inHongKong. At theChineseUniversityof HongKong(CUHK) wehave experimentedwithsystems andmethods for telepathology. Wehavetestedaremote-controlledmicroscope.Wehavesent andreceivedpathologyconsultationsviaemail. InApril1998, westartedaseriesofteleconsultationsessionsusingISDNlines withpathologists at the People’s LiberationArmyGeneral Hospital inBeijing. Anagreement has beensignedbetween thepathologists of CUHKandtheBeijingMedical University withthe GoldenHealthProject of theMinistryof Healthof Chinatotest satellite transmission. Wearecurrentlyworking onthe compatibility of the satellite systems. While theremotelycontrolledmotorized microscope providedadequate instantaneous images, thesystemwas costlyand there was sluggishness inimagetransmission. The combinationof audioandvisual features inconsultationand discussionsessions was helpful inarrivingat thecorrect diagnoses. Transmissionat 384kbit/s was thebare minimum for histopathologyimages. Thestabilityof the ISDNlines was essential. Wearecontinuingtosearchfortheoptimumsystemandat same time trainingpathologists innewworkinghabits.