The validity of telepathological frozen section diagnosis with ISDN-mediated remote microscopy.

We investigated 109 randomly selected frozen section specimens from lung surgery patients in a retrospective blind mode using telepathology equipment. The telepathology system applied (HISTKOM) used one ISDN B-channel and telemicroscopy with a remotely operated robotic microscope. The performance of telepathological frozen section diagnosis was compared with that of conventional frozen section diagnosis. The false-positive rate achieved was identical for both methods. The sensitivity (P=0.03), but not the specificity, was significantly lower for the telepathological method. The time needed to establish a diagnosis with the remote microscope was too high; therefore, upgrading to multichannel technology is recommended. The quality of the images transmitted was judged to be sufficient by the pathologists involved in the study. In conclusion, with further technical improvements in telemicroscopy and additional experience in telepathology, remote diagnosis seems to be feasible.

Clinical trial of telepathology as an alternative modality in breast histopathology quality assurance.

Telepathology is a potential alternative to conventional histopathology. A clinical trial using a robotic telepathology system was conducted to assess the clinical and technical utility and effectiveness of telepathology in the U.K. breast screening pathology quality assurance program. Eighty-seven cases of breast disease were chosen at random from a series of 192 cases from the U.K. Breast Screening Pathology National Quality Assurance Scheme (NEQAS) collection. There were 20 benign, 23 carcinoma in situ (CIS), and 44 invasive malignant cases. The diagnostic accuracy of telepathology (TP) compared with conventional light microscopic (LM) diagnosis was 98.8%; this included a single case deferred for LM examination. The figure was similar when compared with expert consensus diagnosis (CD). In invasive tumor typing, TP accuracy was 95.4% (42/44 cases), the difference being attributable to slide color fading and would have had no impact on patient management. The accuracy of TP versus LM and expert consensus in tumor grading was 91.3% for carcinoma in situ (21/23 cases), a discordance with no relevance to patient management. TP grading of invasive tumor compared with LM diagnosis, had an accuracy of 86.4% (38/44) with a clinically significant accuracy of 97.7% (43/44). The time taken for TP diagnosis averaged 3.9 minutes per case by the end of the study. This data demonstrates that telepathology diagnostic accuracy is comparable to conventional microscopy and may therefore be envisaged as an alternative to conventional light microscopy for more rapid proficiency testing in breast screening (and perhaps other) quality assurance schemes.

Technical aspects of telepathology with emphasis on future development.

Pathology undergoes presently changes due to new developments in diagnostic opportunities and cost saving efforts in health care. Out of the wide field of telepathology the paper selects three prototype applications: telepathology in teleeducation, expert advice for preselected details of a slide and finally telepathology for remote diagnosis. The most challenging field for remote diagnosis is the application in the frozen section scenario. The paper starts with the mental experiment to map conventional procedures to counterparts in telepathology. Technical opportunities and economical restrictions of telepathology equipment are discussed with respect to the components: electronic camera, display devices, haptic sensors and displays, available telecommunication channels and telepathology software. As an example and for illustration of the state of the art for an advanced telemicroscopy system able to perform remote frozen section diagnosis, the HISTKOM equipment is presented in more details. The section concerning future developments regards the aspects of the acceptance by tentative users, legal aspects, costs and affordability of equipment, the market for equipment components and the adequate telecommunication services. Further is regarded the mutual influence of properties of existing systems and application experiences gained with them on the next generation of equipment and application software. Conclusions and references close the paper.

Reliability of telepathology for frozen section service.

One of the most promising applications of telepathology (pathology at a distance by electronic transmission of images in pathology) is frozen section diagnosis, especially because by means of this tool operations requiring an intraoperative histopathological diagnosis are feasible at hospitals without a pathologist on-site. For the introduction of this diagnostic tool into pathologist's daily practice the evidence of its diagnostic accuracy comparable to that of the conventional frozen section diagnosis is crucial. For this purpose the literature on the diagnostic accuracy of telepathological frozen section diagnosis was reviewed. In a metaanalysis these studies and reports, in which a total of more than 1290 cases had been examined, showed a slightly lower overall diagnostic accuracy (of the telepathological frozen section diagnosis) of about 0.91 than the conventional frozen section diagnosis with an average accuracy of about 0.98 found in an analysis of several studies (on frozen section diagnosis of different organs). This difference is at least predominantly caused by a higher rate of deferred and false negative frozen section diagnoses in the telepathological method, while the specificity of both methods, each more than 0.99 was not significantly different. In conclusion, the introduction of a telepathological frozen section diagnosis for hospitals without an acceptable access to a pathologist is justifiable already at the current state of the technological development especially when considering the advantages (time saving, reduction in costs) compared to the alternative of surgical interventions without access to an intraoperative diagnosis.

Quantitative immunohistochemical analysis of the glutathione S-transferase GSTM1: in situ phenotyping in archival material.

1. GSTM1 is present in only approximately 50% of Caucasian individuals and deficiency of GSTM1 is associated with susceptibility to a growing number of diseases, especially cancer. Thus, a method that would allow accurate, retrospective determination of the GSTM1 phenotype in different patient populations would have many applications. 2. Developed, therefore, is a quantitative, image-analysis-based immunohistochemical technique for the analysis of GSTM1 protein in paraffin-embedded tissue samples. It was applied to the determination of the GSTM1 phenotype using liver biopsies taken from 70 patients. 3. Of the 70 cases (depending on the cut-off point), 51-54% were deficient in GSTM1. A single 27 kD band characteristic for GSTM1 was found in seven of 16 cases analysed by Western blotting using the same GSTM1 antibody as in the immunohistochemical analysis. There was a good correlation (r = 0.87) between the staining intensity of the GSTM1 band and the staining intensity evaluated by immunohistochemistry. 4. It is concluded that this quantitative immunohistochemical method permits accurate determination of the GSTM1 phenotype and is well suited for retrospective analysis of GSTM1 expression in specific tissues in situ.

European field tests with HISTKOM telepathology equipment.

HISTKOM telemicroscopy equipment for telepathology is designed for the most challenging application in telepathology: intraoperational frozen section diagnosis. Adapted to this application, it is also excellently suited for all other telepathology modes requesting less sophisticated equipment. The technical concept and user interface are oriented to routine daily pathology. HISTKOM underwent heavy field-tests at several locations. The field-tests designs and the results of five of these are reported in this paper. Telepathology will exploit its advantages in networks hosting participants requesting and offering services. The solution of the interoperability problem caused by different equipment from different suppliers within such a network will be a major task, the solution for which is in progress. The new generation of HISTKOM equipment and software is designed in a modularized concept, allowing the integration of various hardware components from different manufacturers; thus special configurations can be realized easily. HISTKOM is offered as complete turnkey system, but can also be installed in yet existing configurations of the customer if they meet specifications.

Breast carcinoma. Correlations between visual diagnostic criteria for histologic grading and features of image analysis.

OBJECTIVE: To investigate the relevance of image analysis for grading breast carcinomas. STUDY DESIGN: The results of histologic grading were correlated with 18 features of image analysis, including SD. "Simple" characteristics, like area and perimeter, shape indices, optical density and textural features of nuclei from cancer cells, were analyzed. Hematoxylin-eosin-stained tissue sections of 67 cancer specimens were routinely used for the study. RESULTS: We found statistically significant correlations between overall histologic grading and the sum of its subscores and features of image analysis, especially nuclear area, nuclear perimeter and the diameter of the circumscribing circle (diametercirc), including their SDs. The visually and therefore subjectively assessed subscore of the nuclear pleomorphism of histologic grading significantly correlated with the features of image analysis, like nuclear area, nuclear perimeter, diametercirc, integrated optical density and correlation (and their SDs). There were significant relationships between the absolute numbers of mitoses per 10 high-power fields and nuclear area, nuclear perimeter and diametercirc (and their SDs). We did not observe a significant correlation between the subscore of tubule formation of histologic grading and any of the features of the image analysis studied. Furthermore, the correlations between the features of image analysis and the subscores of the visual histologic grading system were analyzed with respect to each other. The subscore of nuclear pleomorphism of histologic grading correlated best with overall grading (r = .72), whereas no significant correlation could be found between the subscores of nuclear pleomorphism and mitotic activity. CONCLUSION: Image analysis provides objectivity and reproducibility to the grading of breast carcinomas and thus could contribute to more individualized prognostication of the disease.

Immunohistochemical assessment of human microsomal epoxide hydrolase in primary and secondary liver neoplasm: a quantitative approach.

1. Epoxide hydrolases form an enzyme family involved in the metabolism of a variety of xenobiotics including cytostatic drugs and carcinogens. Whether human microsomal epoxide hydrolase--one of the main members of the epoxide hydrolase family--is expressed in neoplasia of the liver has been the subject of a controversial discussion. 2. We therefore developed a quantitative immunohistochemical assay and monitored epoxide hydrolase expression in hepatocellular carcinomas (HCC, n = 20), cholangio-cellular carcinomas (CCC, n = 2) and liver metastases (n = 57) of tumours of various origins, and compared the expression intensities and patterns to normal liver tissue. 3. In normal liver tissue microsomal epoxide hydrolase displays expression of the constitutive type with non-zonal staining of all hepatocytes. 4. When using a quantitative immunohistochemical approach statistically significant differences in microsomal epoxide hydrolase expression were observed between normal tissue, hepatocellular carcinoma and liver metastases (mean optical density 2.35, 1.63 and 0.21 respectively, p = 2.9, 6.3 and 18.9). These data indicate differential expression in different types of liver neoplasm. 5. As microsomal epoxide hydrolase is involved in metabolism of different xenobiotics our findings may have implications for tumour progression.

Field test to evaluate telepathology in telemedicine.

Telepathology is currently performed only on prototype systems. The application of telepathology consultations differs from the conventional procedure because of the technical and cost restrictions. This paper outlines technical concepts and the principal hardware and software modules necessary for telemicroscopy equipment. A field test to gather information concerning the user needs and to evaluate parameters of telepathology consultation sessions is reported.

The false negative rate in cervical cytology. Comparison of monolayers to conventional smears.

OBJECTIVE: To compare cervical monolayers prepared by a density gradient technique to conventional smears. STUDY DESIGN: The study evaluated 2,863 patient samples. After conventional smears were prepared, the residual samples (collected in preservative fluid) were processed by the monolayer preparation system, which disaggregates the cells, removes nonclinical debris and layers the diagnostic material onto a microscopic slide. Slides were screened in Germany and the United States in masked studies. RESULTS: In the U.S. study, the false negative rate for the conventional smears was 9.4% when endocervical components were present and 16.7% when absent. The false negative rate for the monolayer preparation was 4.0% when endocervical components were present and 5.7% when absent. A comparison of the diagnostic results showed that the monolayer made possible the detection of 48 cases of low grade disease or higher that were not found on the conventional smear. The German study was independent but used the same samples. The results, although based upon the Papanicolaou classification, were similar. CONCLUSION: Cervical samples that are preserved in a liquid suspension and prepared using the density gradient monolayer technology have many advantages over conventional cervical cytologic smear preparations. The quality of cell preservation and presentation is superior. Elimination of obscuring material, homogeneity of the sample and standardized quality work together to produce preparations that can be effectively and reliably screened by cytotechnologists. These preparations should greatly enhance the reliability of machine-based screening in the future.

Quantitation in immunohistochemistry. A research method or a diagnostic tool in surgical pathology?

Quantification of immunohistochemical methods is feasible by two ways, namely microscope photometry and TV densitometry. The rapid progress of computer technology argues largely in favour of the second system. From the immunohistochemical methods either ABC, the PAP or the APAAP method should be chosen. Diaminobenzidine is the H2-donor of choice for the peroxidase mediated systems, whereas Fast Red TR or Neufuchsin can be used with the APAAP system. The test efficiency, and the precision of measurements should be investigated in each laboratory doing quantitative immunohistochemistry. Low levels of coefficients of variation (CV) should be reached for one field of interest. Examples of introducing the method of quantitative immunohistochemistry in a routine laboratory are discussed. The demonstration of either a steroid receptor or a proliferation marker, such as PCNA or Ki67, are seemingly of the greatest clinical interest.

Telemicroscopy stations for telepathology based on broadband and ISDN connections.

Telemicroscopy equipment is a key tool to perform Telepathology successfully. It allows the local separation of the microscope with the tissue samples from the investigating pathologist. The Telemicroscopy stations presented provide the user with a full access to the functions such as scanning stage, focus, illumination and magnification selection of a remote microscope. One system applies as communication link a broadband video conference net of the German Telekom with full realtime capabilities and color TV-image quality. As this network provides adequate interfaces for video and computer net signals, it is well suited to establish within a short time interval a very comfortable Telemicroscopy connection without the development of additional equipment. As the broadband network is expensive the application is economically restricted to special applications. The other system relies on the narrow band connections of the ISDN telephone network. This design is inexpensive with respect to data transmission and is available nearly worldwide everywhere. But on the other hand this strategy is limited concerning realtime capabilities. To reduce these limitations to an acceptable level intelligent coding and operation concepts of the stations have to be developed.

Aspects of standardization in telepathology.

Effective telepathology is only possible, if the partners are supplied by a broad variety of equipment connected by far-spread communication lines. These prerequisites can only be fulfilled if technical and medical standards are available obligate for all participants. The technical and medical aspects of those standards are described in general and include procedures and notations in pathology, standards of the image source, image sampling, clinical information, classification of diagnosis, communication channels, hard- and software equipment. New standards should only be developed for those not existing, or if the already existing ones are not appropriate. The standardization classes should allow flexible formats and be developed as "open modules" allowing "individual" peripheral work stations and progressive development in the future.

Telemicroscopy. Design considerations for a key tool in telepathology.

The paper introduces telemicroscopy as a mean to establish online telepathology services. It is differentiated between specialized point to point connections with only a few restrictions on one side, and telecommunication links available inexpensive worldwide in the form of the public telephone net on the other side with the restriction of a limited channel capacity. The restrictions imposed by the application of the telephone net (preferentially the ISDN service) are discussed and strategies outlined to overcome these restrictions. It is concluded, that if dedicated broadband connections are available, realtime telemicroscopy links can be realized immediately with available equipment. If a switched and general available telemicroscopy service applying public telecommunication links is favoured, for effective and acceptable ("online") links, computer intelligence has to be introduced to allow effective strategies for data reduction and data transmission. In this field design and development efforts are still necessary.

Quantitative immunohistochemistry: standardization and possible application in research and surgical pathology.

We describe three different methods of calibration of quantitative immunohistochemistry, two of them based on self-made dye-agarose or antigen-agarose substrates. Quantification of immunostained objects (immunosignals) can be done by (1) microscope photometry (plug method) or scanning-photometry; (2) and densitometry of a television transformed microscope image. Both approaches are subjected to the different methods of calibration and three examples of application.