Immunohistochemistry (IHC) plays an essential role in Pathology. In order to improve reproducibility and standardization of the results interpretation, IHC quantification methods have been developed. IHC interpretation based in whole slide imaging or virtual microscopy is of special interest. The objective of this work is to review the different computerbased programs for automatic immunohistochemistry and Fluorescence In Situ Hybridization (FISH) evaluation. Scanning solutions and image analysis software in immunohistochemistry were studied, focusing especially on systems based in virtual slides. Integrated scanning and image analysis systems are available (Bacus TMAScore, Dako ACIS III, Genetix Ariol, Aperio Image Analysis, 3DHistech Mirax HistoQuant, Bioimagene Pathiam). Other image analysis software systems (Definiens TissueMap, SlidePath Tissue Image Analysis) can be applied to several virtual slide formats. Fluorescence is the preferred approach in HistoRx AQUA, since it allows for a better compartmentalization of signals. Multispectral imaging using CRi Nuance allows multiple antibodies immunohistochemistry, and different stain unmixing. Most current popular automated image analysis solutions are aimed to brightfield immunohistochemistry, but fluorescence and FISH solutions may become more important in the near future. Automated quantitative tissue microarrays (TMA) analysis is essential to provide high-throughput analysis. Medical informatics standards in images (DICOM) and workflow (IHE) under development will foster the use of image analysis in Pathology Departments.

Abstract:We report the results of a study designed for assessment of the diagnostic accuracy and usability of internet-based digital microscopy: the dynamic real-time telepathology system (Coolscope) and the Virtual microscopy (Aperio Scan Scope) system, in the context of pulmonary pathology. The systems were implemented to the routine pulmonary pathology workflows and used for the intra-operative frozen-section primary diagnosis as well as for the secondary (consultative) diagnosis. The histological material presented for the teleconsultations included the samples of lung parenchyma, bronchial biopsy and resected lung/bronchi tumours. For the primary diagnosis 4 categories of material can be distinguished (304 samples): 1) the frozen sections of lung tumours, resected bronchial margins and lymph nodes; 2) fine needle aspiration [FNA] biopsies (TBNA; EBUS-TBNA, EUS-FNA; 3) oligobiopses of bronchus, oesophagus, skin; and 4) exfoliative cytology. The telepathology diagnoses compared with conventional light microscopy diagnoses showed very high concordance for the Coolscope and Aperio Virtual Slide modality: 87.5% and 100%, respectively -within the group of teleconsultations. For the frozen sections, the primary telediagnoses were concordant with the light microscopy paraffin sections diagnoses in 100% for Aperio; and in 97.5% for Coolscope. An excellent agreement (100%) was seen in the telediagnoses and conventional slides diagnoses for FNA, oligobiopsies and cytology -for both telepathology systems. These results provide some encouragement for the implementation of Coolscope and virtual slide-based telepathology (Aperio) system to the routine histopathological diagnostics.

Human chorionic gonadotropin (HCG), a classic trophoblastic marker, has been found recently in many nontrophoblastic tumors. Previously we have found elevated serum βHCG levels in 14% of small cell lung cancer patients. The aim of the present study was to assess the frequency and clinical significance of βHCG expression in non-small cell lung tumors and in the sera of patients. 153 non-small cell lung cancer patients entered into this study. The control group consisted of 85 patients with benign lung diseases. Serum βHCG elevation exceeding 5 mIU/ml was found in 3.5% of patients with benign lung diseases and in 12% of lung cancer patients (p = 0.03). Tumor analysis revealed the presence of βHCG positivity in 28% of resected lung specimens. βHCG positivity was found more often in adenocarcinoma than in squamous cell lung carcinoma both in tissue and in serum, the differences being not significant. Elevated serum βHCG values were found more frequently in stage IV patients than in the remainder (p = 0.03). Response to chemotherapy (partial or minor response) was obtained more often in the patients with normal serum βHCG than in those with serum βHCG elevation (p = 0.03). We suppose that the ability to produce βHCG is a rare but important biologic feature of lung carcinomas combined to some extent with chemoresistance.

Background. This paper presents the study concerning hot-spot selection in the assessment of whole slide images of tissue sections collected from meningioma patients. The samples were immunohistochemically stained to determine the Ki-67/MIB-1 proliferation index used for prognosis and treatment planning. Objective. The observer performance was examined by comparing results of the proposed method of automatic hot-spot selection in whole slide images, results of traditional scoring under a microscope, and results of a pathologist's manual hot-spot selection. Methods. The results of scoring the Ki-67 index using optical scoring under a microscope, software for Ki-67 index quantification based on hot spots selected by two pathologists (resp., once and three times), and the same software but on hot spots selected by proposed automatic methods were compared using Kendall's tau-b statistics. Results. Results show intra- and interobserver agreement. The agreement between Ki-67 scoring with manual and automatic hot-spot selection is high, while agreement between Ki-67 index scoring results in whole slide images and traditional microscopic examination is lower. Conclusions. The agreement observed for the three scoring methods shows that automation of area selection is an effective tool in supporting physicians and in increasing the reliability of Ki-67 scoring in meningioma.

Abstract:Her-2/neu is overexpressed in 20-30% of breast cancer patients and is associated with a more aggressive disease. Identification of Her-2/c-erbB-2-neu overexpression is based on immunohistochemical [ihc] detection of protein and/or gene amplification in fluorescence in situ hybridization test (FISH). Also Estrogen receptors [ER] and Progesterone receptors [PR] are the prognostic and predictive biomarkers, recently analysed by ihc methods. Subjective, manual scoring of the ihc Her-2/neu expression and expression of the ER/PR reported as the percentage of immunopositive cells are the most common mode of interpretation among pathologists. Automated microscopy and computerised processing have provided increased accuracy in quantification and standardisation. The aims of our study were: to evaluate the scoring reproducibility of Her-2 /neu ihc expression tested by two automated systems: ACIS (Dako) and ScanScope (Aperio); to estimate the ER/PR expression in ihc staining methods with different anti-ER/anti-PR antibodies (the monoclonal and the ER/PR pharmDx TM Kit ) by the ACIS system. Her-2/neu ihc expression was measured in 114 primary invasive breast carcinomas by the manual and the automated scoring (ACIS and Aperio system). 106 slides stained ihc with two types of anti-ER/anti-PR antibodies entered the quantisation. The results of our investigations showed very high reproducibility of Her-2/neu scores in intra-and interobserver analysis by ACIS evaluation. The major concordance was present in strong 3+ ihc cases; very small discordance was shown by cases with low expression of Her-2/neu. The accuracy of scoring by the Aperio was little lower in comparison to ACIS but it might result from the smaller and variable series of samples analysed by Aperio. The concordance in scoring of two automated systems was 86.5% (p<0.0001; γ=0.887); the discordance was referred only to the lower expression of Her-2/neu. The concordance in manual scoring performed by the single observer and the panel was 84.2% (p<0.0001, γ = 0.99); the discordance comprised a few cases with strong expression (2+ vs 3+). Very high intra-and interobserver reproducibility of the ER/PR ihc measurements was present in the readers results (referred to the percentage of immunoreactive carcinomatous cell population in the breast carcinomas acc. to the ACIS algorithm). No differences were disclosed in the percentage of ER-immunoreactive and PR-immunoreactive carcinomatous cell populations when used 2 different type of antibodies, in the ACIS automated method.

Abstract:Many studies have emphasised the importance of Ki-67 labeling index (LI) as the proliferation marker in meningiomas. Several authors confirmed, that Ki-67 LI has prognostic significance and correlates with likelihood of tumour recurrences. These observations were widely accepted by pathologists, but up till now no standard method for Ki-67 LI assessment was developed and introduced for the diagnostic pathology. In this paper we present a new computerised system for automated Ki-67 LI estimation in meningiomas as an aid for histological grading of meningiomas and potential standard method of Ki-67 LI assessment. We also discuss the concordance of Ki-67 LI results obtained by presented computerized system and expert pathologist, as well as possible pitfalls and mistakes in automated counting of immunopositive or negative cells. For the quantitative evaluation of digital images of meningiomas the designed software uses an algorithm based on mathematical description of cell morphology. This solution acts together with the Support Vector Machine (SVM) used in the classification mode for the recognition of immunoreactivity of cells. The applied sequential thresholding simulated well the human process of cell recognition. The same digital images of randomly selected tumour areas were parallelly analysed by computer and blindly by two expert pathologists. Ki-67 labeling indices were estimated and the results compared. The mean relative discrepancy between the levels of Ki-67 LI by our system and by the human expert did not exceed 14% in all investigated cases. These preliminary results suggest that the designed software could be an useful tool supporting the diagnostic digital pathology. However, more extended studies are needed for approval of this suggestion.

Human chorionic gonadotropin (HCG)-like immunoreactivity has been found in many non-trophoblastic tumours, but the biological behaviour of HCG-producing cells has not been clarified yet. The aim of the study was to estimate the frequency of serum HCG beta subunit (s beta HCG) elevation in patients with small-cell lung cancer (SCLC) and to assess its possible prognostic role in this type of tumour. An attempt was also made to reclassify the histology in selected cases to see whether the elevated (s beta HCG) level is connected with any special subtype of small-cell lung cancer. A total of 156 SCLC patients entered the study: 93 men, 63 women, median age 58 years. s beta HCG activity was measured by immunoenzyme assay (Abbott EIA beta HCG 15-15) before treatment. s beta HCG elevation (above 5 mIU/ml) was found in 21 of 156 patients (14%). Response to treatment after chemotherapy (complete and partial response) was obtained in only 48% of those patients in whom elevated s beta HCG was found, in comparison to the 73% response rate observed in the remaining patients. Only 5% of patients with elevated s beta HCG survived 2 years, in comparison to 21% surviving for 2 years among the remaining patients. The prognostic significance of elevated s beta HCG and extent of disease were independent of each other (Cox's proportional-hazard model). Thus s beta HCG elevation in SCLC seems to be a marker of more resistant tumours and of poor prognosis. We have not found any connection between the subtype of small-cell lung cancer and elevated s beta HCG. Elevated s beta HCG was found in 2 out of 11 patients with oat-cell carcinoma, in 3 out of 10 patients with an intermediate cell type and in 5 out of 13 patients with small-cell lung cancer in which the assessment of the subtype was not possible.

Abstract:The Virtual Slide (VS) is an interactive microscope emulator that presents a complete digitized tissue section via the Internet. A successful implementation of VS has been observed for educational, research venues and quality control. VS acquisition for consultative pathology is not so common. The purpose of this study was to explore the efficacy and usability of VS in the consultative pulmonary telepathology. 20 lung tumors entered the study. The performance was programmed for 2 medical centers specialized in pulmonary pathology (beginner and advancer in telepathology). A high-quality VSs were prepared by Coolscope (Nikon, Eclipsnet VSL, Japan), and were evaluated via the Internet. The cases were reviewed for the second time with conventional light microscope. VS diagnostic accuracy and the interobserver variability were evaluated. Also the time taken by examiners to render the diagnoses and time needed to scan the microscopic slide were analyzed. Percentage concordance between original glass-slides diagnosis and diagnosis for VSs was very high. Pathologists found the download speed of VSs adequate; experience in telepathology reduced the time of VS diagnosis. VS implementation suggests advantages for teleconsulation and education but also indicate some technical limitations. This is the first Polish trial of VS implementation in telepathology consultative service.