Digital morphometry of cytologic aspirate endometrial samples [Digitalna morfometrijska analiza citoloških uzoraka aspirata endometrija]

Unlike cervical cytology, morphological cytology criteria in the differential diagnosis of endometrium have not yet been clearly defined, and methods to allow for more precise evaluation of endometrium status have been searched for. The aim of the present study was to assess the value of morphometric nucleus analysis of cytologic aspirate endometrial samples in proliferative, hyperplastic and malignant endometrium by use of digital image analysis. Morphometric analysis was performed on archival cytologic aspirate endometrial samples (at least 10 per group) stained according to Papanicolaou (n=77) and May-Grünwald-Giemsa (MGG; n=80) with the following histopathologic diagnoses: proliferative endometrium, hyperplasia simplex, hyperplasia complex, hyperplasia complex atypica, and adenocarcinoma endometriodes endometrii (grade I, II and III). Interactive image analysis (nuclear area, convex area, perimeter, maximum and minimum radius, length and breadth, as well as nucleus form factor and elongation factor) was performed by use of the SFORM software (VAMSTEC, Zagreb) on at least 50 (Papanicolaou stain) and 100 (MGG stain) well preserved endometrial epithelial cell nuclei without overlapping, at magnification of ´1000. Statistical data analysis was done by use of the Statistica Ver. 6 statistical package. Multivariate analysis (ANOVA) distinguished malignant, hyperplastic and proliferative endometrium according to all morphometric variables with both staining methods (p0.05) from atypical hyperplasia, adenocarcinoma and proliferative endometrium only according to the nucleus form factor and elongation factor (Papanicolaou stain), whereas malignant and atypical hyperplastic endometrium (MGG stain) differed statistically significantly (p0.05). According to the cytologic staining method, morphometric parameters were considerably higher in MGG stained endometrial samples, reaching the level of statistical significance (p0.05) in the groups of hyperplasia simplex and complex, well differentiated adenocarcinoma (form factor) and atypical hyperplasia (elongation factor). A combination of cytomorphology and the morphometric variables assessed in this study can yield useful information on the cytologic state of endometrium, with special reference to the possible differentiation of the group of hyperplasia without atypia from the group of adenocarcinoma and atypical hyperplasia

Digital Morphometry of Cytologic Aspirate Endometrial Samples

Unlike cervical cytology, morphological cytology criteria in the differential diagnosis of endometrium have not yet been clearly defined, and methods to allow for more precise evaluation of endometrium status have been searched for. The aim of the present study was to assess the value of morphometric nucleus analysis of cytologic aspirate endometrial samples in proliferative, hyperplastic and malignant endometrium by use of digital image analysis. Morphometric analysis was performed on archival cytologic aspirate endometrial samples (at least 10 per group) stained according to Papanicolaou (n=77) and May-Grünwald-Giemsa (MGG; n=80) with the following histopathologic diagnoses: proliferative endometrium, hyperplasia simplex, hyperplasia complex, hyperplasia complex atypica, and adenocarcinoma endometriodes endometrii (grade I, II and III). Interactive image analysis (nuclear area, convex area, perimeter, maximum and minimum radius, length and breadth, as well as nucleus form factor and elongation factor) was performed by use of the Sform software (Vamstec, Zagreb) on at least 50 (Papanicolaou stain) and 100 (MGG stain) well preserved endometrial epithelial cell nuclei without overlapping, at magnification of ´1000. Statistical data analysis was done by use of the Statistica Ver. 6 statistical package. Multivariate analysis (ANOVA) distinguished malignant, hyperplastic and proliferative endometrium according to all morphometric variables with both staining methods (p0.05) from atypical hyperplasia, adenocarcinoma and proliferative endometrium only according to the nucleus form factor and elongation factor (Papanicolaou stain), whereas malignant and atypical hyperplastic endometrium (MGG stain) differed statistically significantly (p0.05). According to the cytologic staining method, morphometric parameters were considerably higher in MGG stained endometrial samples, reaching the level of statistical significance (p0.05) in the groups of hyperplasia simplex and complex, well differentiated adenocarcinoma (form factor) and atypical hyperplasia (elongation factor). A combination of cytomorphology and the morphometric variables assessed in this study can yield useful information on the cytologic state of endometrium, with special reference to the possible differentiation of the group of hyperplasia without atypia from the group of adenocarcinoma and atypical hyperplasia

Biospectroscopy towards screening and diagnosis of cancer

Systems biology is an emerging science that combines high throughput investigation techniques to define the dynamic interplay between different biological regulatory systems in response to internal and external cues. Related technologies, genomics, epigenomics, transcriptomics, proteomics, metabolomics and toponomics have been applied to investigate models of carcinogenesis to identify committing initiating events. Vibrational spectroscopy has the potential to play an integral role within systems biology research approaches, as it is able to identify chemical bond alterations within molecules independent of where these molecules reside. Its integration with current “systems biology” methodologies can contribute in the identification of potential biomarkers of carcinogenesis and assist in their incorporation into clinical practice. Breast tissue undergoes cyclical and longitudinal molecular and histological alterations that are influenced by environmental factors. These factors may include diet and lifestyle in addition to parity, lactation and menopausal status and are implicated in carcinogenesis. Breast cancer may appear decades after the initial carcinogenic event. Available research in this area is limited to when early histological changes occur due to the difficulties imposed by the molecular and histological diversity of breast tissue. Vibrational spectroscopy in combination with powerful chemometric techniques has identified spatial and temporal mammary alterations in benign tissue. Prostate cancer is influenced by environmental factors. Its incidence is higher in populations adopting a Westernised lifestyle and diet and has increased over the past generation. This leads to the assumption that prostatic tissue composition may exhibit chronological alterations. Vibrational spectroscopy techniques were applied to matching prostatic tissues with benign prostatic hyperplasia collected from 1983 to 2013. Significant trans-generational segregation was identified. Spectral areas responsible for this segregation pointed towards epigenetic changes. Immunohistochemical studies for DNA methylation and hypomethylation supported these results. Vibrational spectroscopy techniques were also implemented to explore molecular changes between normal ovarian tissue, borderline ovarian tumours and malignant ovarian carcinomas. Different chemometric techniques were applied to discriminate cancers from controls. Similar techniques were able to segregate different types of epithelial ovarian carcinomas. The accurate diagnosis obtained using ATR-FTIR spectroscopy demonstrates its potential for development as an assisting tool for histopathological diagnosis. The endometrial-myometrial junction areas of benign uterine tissues were scrutinised by Synchrotron FTIR and FPA. These techniques in combination with multivariate analysis revealed clear segregation between the functionalis and basalis layers within the uterine crypts. The same techniques illustrated potential areas within these epithelial surfaces where different stem cell types may reside. Targeting the activation/ inactivation of these stem cells may have applications in the diagnosis and treatment of early uterine cancer

Evaluation of PD-L1 expression in various formalin-fixed paraffin embedded tumour tissue samples using SP263, SP142 and QR1 antibody clones

Background & objectives: Cancer cells can avoid immune destruction through the inhibitory ligand PD-L1. PD-1 is a surface cell receptor, part of the immunoglobulin family. Its ligand PD-L1 is expressed by tumour cells and stromal tumour infltrating lymphocytes (TIL). Methods: Forty-four cancer cases were included in this study (24 triple-negative breast cancers (TNBC), 10 non-small cell lung cancer (NSCLC) and 10 malignant melanoma cases). Three clones of monoclonal primary antibodies were compared: QR1 (Quartett), SP 142 and SP263 (Ventana). For visualization, ultraView Universal DAB Detection Kit from Ventana was used on an automated platform for immunohistochemical staining Ventana BenchMark GX. Results: Comparing the sensitivity of two different clones on same tissue samples from TNBC, we found that the QR1 clone gave higher percentage of positive cells than clone SP142, but there was no statistically significant difference. Comparing the sensitivity of two different clones on same tissue samples from malignant melanoma, the SP263 clone gave higher percentage of positive cells than the QR1 clone, but again the difference was not statistically significant. Comparing the sensitivity of two different clones on same tissue samples from NSCLC, we found higher percentage of positive cells using the QR1 clone in comparison with the SP142 clone, but once again, the difference was not statistically significant. Conclusion: The three different antibody clones from two manufacturers Ventana and Quartett, gave comparable results with no statistically significant difference in staining intensity/ percentage of positive tumour and/or immune cells. Therefore, different PD-L1 clones from different manufacturers can potentially be used to evaluate the PD- L1 status in different tumour tissues. Due to the serious implications of the PD-L1 analysis in further treatment decisions for cancer patients, every antibody clone, staining protocol and evaluation process should be carefully and meticulously validated

Morfometrijski i kinetički parametri kao dijagnostički i prognostički čimbenici leukemijskih oblika kroničnih limfoproliferativnih bolesti

The clinical and morphological picture of the leukemic types of chronic lymphoproliferative disorders (CLLPD) shows considerable heterogeneity. Due to the unpredictable course of the disease, the clinical stages and other parameters used to date have been unable to identify patients at a high risk of disease progression. Therefore, the aim of the study was to find new morphometric, proliferative and/or kinetic parameters in different tumor mass compartments, i.e. bone marrow, peripheral blood and lymph node, which would be able to predict survival and disease progression, in order to define the proliferative kinetic index (PKI). In addition, the aim was to evaluate, in terms of survival, the diagnostic and prognostic value of the variables characterizing disease subtypes, clinical stages, tumor mass, time to tumor mass doubling, and lymphocyte count in total CLLPD population and its subgroups. Then, the proliferative and kinetic parameters were used in an attempt to assess the origin and distribution of malignant lymphatic cells in various clinical and morphological entities. Patients, materials and methods. The study included 155 patients diagnosed with CLLPD. A total of 657 puncture smears of bone marrow (n=236), lymph nodes (n=146) and peripheral blood (n=275) were analyzed. The analysis consisted of morphometric studies, assessment of the nucleolar organization region (AgNOR) characteristics, and image cytometry (ICM), performed in 71895 cells on a SFORM PC (VAMSTEC, Zagreb). The univariate and bivariate analyses were used on statistical data processing, and Kaplan-Meier method for the analysis of survival (Statistica 7.1). Results. Prognostic potential was demonstrated for many clinical, hematological, biochemical, immunophenotypic parameters, morphological characteristics of differential blood count and bone marrow, factors of disease progression, and morphometric, proliferative and kinetic parameters. The "single" and "multiple” programmed stops in the development of typical forms of leukemias and lymphomas, subacute and subchronic leukemias were hypothesized. Differentiation impairment may occur at any stage, and different "stop" locations result in different morphology and affinity to accumulation in bone marrow, peripheral blood and lymph nodes. The new parameters of modified analysis of diploid histogram were found to be appropriate for kinetic analysis by the method of image DNA cytometry. Also, the newly characterized types of AgNOR points, homogeneous, inhomogeneous, and annular, yielded a statistically significant correlation with DNA histogram properties and morphometric characteristics of the cell and nucleus, and were additionally found to play a role in the survival, type of tumor mass distribution, biological behavior of tumor disease, and morphological characteristics of lymphatic cells in bone marrow and peripheral blood. Correlation analysis of the morphometric, proliferative and kinetic characteristics revealed the low proliferative cells to possess small, homogeneous AgNOR, with the majority of cells in the peak of DNA histogram. The high proliferative cells had inhomogeneous AgNOR, mostly containing greater DNA amount than peak cells, or pathologic mitoses (DNA >4N), or the majority of cells being in the S-phase of the cell cycle. Cells with medium proliferative activity and annular AgNOR were in-between. Analysis of different tumor mass compartments showed the lymphatic cells with affinity to accumulation in bone marrow ("damaged" naïve B-lymphocytes) to regularly exhibit low proliferative activity (a lower percentage of cells in SFC and highest percentage of cells in the peak of the G0/G1 phase). The "programmed" cells with affinity to accumulation in lymph nodes migrated to lymph nodes, where they transformed to the stage of "programmed stop", exhibiting the characteristics of proliferative cells (an increased number of AgNOR, larger more proliferative inhomogeous AgNOR and lowest percentage of cells in the G0/G1 phase). The migration of cells from bone marrow to lymph nodes and between lymph nodes occurs in peripheral blood (a mixture of cells with low and high proliferative activity: a higher proportion of cells in SFC and at the same time in the G0/G1 phase of the cell cycle). Analysis of cell size and proliferative activity in different compartments of tumor mass revealed a regular pattern within total CLLPD population, and in the subgroups of B-chronic lymphocytic leukemia with variants (B-CLL+V) and typical B-chronic lymphocytic leukemia (B-CLL). Whereas the cells in bone marrow and peripheral blood did not differ substantially according to size and proliferative activity, an inverse pattern was observed between peripheral blood and lymph node. As small cells are inactive and larger cells more proliferative, the analysis quite unexpectedly showed the peripheral blood cells to be largest and most inactive, in contrast to lymph node where the cells were smallest and most active. Based on the most representative AgNOR and DNA characteristics (related to survival) in various tumor mass compartments, the PKI score was calculated for the CLLPD population as a whole and for B-CLL+V in separate. The CLLPD (p=0,00118) and B-CLL+V (p=0,03589) patients had a statistically significantly better prognosis when score of PKI was less than 4. Conclusion. Peripheral blood sample is inadequate and not representative for the study of disease progression or stability. The morphometric, proliferative and kinetic properties of tumor cells, now for the first time analyzed simultaneously in different tumor mass compartments, provide better assessment of the disease dissemination and progression. The morphometric characteristics of homogeneous, inhomogeneous and annular AgNOR, and characteristics of diploid histogram have confirmed the hypothesis that prognostically unfavorable/favorable subgroups could be identified and the course of the disease predicted even within the groups of neoplasms of relatively low malignancy such as CLLPD with subgroups

Effect of elevated PCO2 on optical properties of the coccolithophorid Emiliania huxleyi grown under nitrate limitation

Side scatter and red fluorescence properties of the coccolithophore Emiliania huxleyi were investigated by flow cytometry when NO3-limited continuous cultures were submitted to a CO2 partial pressure (pCO2) increase from 400 to 700 ppm. Cultures renewed at the rate of 0.5 d-1 and were submitted to saturating light level. pCO2 was controlled by bubbling CO2-rich or CO2- free air in the cultures. Most of the analyses were repeated 5 times and the average SD were < 1.6%, 0.1 and 0.2% for counting, fluorescence and side scatter respectively. Considering the possible decalcification induced by the increase of CO2 in the chemostat atmosphere, the maximum variation that can be expected for side scatter is that provided by the coccolith depletion upon acidification of the cell suspension. The acidification induced a large (36%) decrease of the side scatter signal but had no detectable effect on the red fluorescence. A control was run with a non-calcifying species, Dunaliella tertiolecta, where acidification induced no detectable change, both on fluorescence and side scatter. During the time of the experiment, the decline of side scatter in chemostat 1 never approached the potential 36% change observed when coccoliths are fully dissolved. Interestingly, the specific chl a fluorescence of E. huxleyi slightly increased during the period of high CO2 level. At the end of the experiment this increase amounted to a significant 2.8% of the initial signal. Furthermore, it progressed linearly with time over the period of observation. However, the experiment did not last enough to know if the fluorescence increase had already reached its maximum value. The acidification experiment supported the use of side scatter as a relevant parameter to trace potential changes in calcification. Since the estimated 25% decrease in calcification induced by the rise in CO2 atmosphere did not result in dramatic changes in side scatter values, we can conclude that the number of cocoliths and the overall shape and granulosity of cells was not significantly affected by this decrease. Changes must have only affected tiny structure details of the coccoliths which is supported by scanning electron microscopy observations. The small but significant increase of the fluorescence signal can be considered as a physiological response to the CO2 rise. This suggests a more dynamic photosynthetic process that would result in a higher rate of organic matter production providing that the system is not nutrient limited as in the present situation

Feasibility study on bovine fetal sexing utilizing circulatory cell-free fetal DNA in maternal peripheral blood

The studies conferred in this thesis were performed to establish an appropriate PCR methodology to target sex-chromosome specific genes for prenatal fetal sex determination in pregnant cows. The first study determined the effect of frozen and fresh plasma on cell-free fetal DNA (cffDNA) extraction for PCR-based fetal sexing and compared different DNA extraction methods on downstream PCR for fetal sexing in cows by detecting sex-chromosome-specific gene products. We found that none of the DNA extracts obtained from frozen plasma samples of cows (120-150 days pregnant) yielded any male fetus-specific PCR products, although all cows were carrying male fetii. In contrast, male fetus-specific amplicons were successfully amplified in 5/6 DNA extracts obtained by the phenol-chloroform method when fresh plasma samples were used. Although the smaller sample size was a limitation, we further found that DNA extraction methodologies compared in the present study (DNeasy Blood & Tissue Kit, QIAamp DSP Virus Kit, QIAamp DNA Blood Midi Kit, NucleoMag cfDNA Isolation Kit, MagMAX cfDNA Isolation Kit, KAPA Express Extract Kit, Salting-out protocol, and Phenol-chloroform method) appeared to show variability in their ability to isolate fetal DNA from freshly harvested maternal plasma/blood of pregnant cows. The second study determined the minimum concentrations of cffDNA that must be present per milliliter of maternal plasma for successful extraction and PCR. In addition, we also compared Y-specific sequence PCR-based fetal sexing results in the cows divided into two gestational stages. We concluded that the spiked serum and cellular DNA from aborted male fetii could be successfully re-extracted (Blood & Tissue Kit) from maternal plasma at all dilution rates (0.5-100%). Furthermore, the lowest concentration of spiked fetal DNA for successful extraction (from maternal plasma) followed by Y-specific PCR and bAML PCR was found to be >31.25 pg/ml and >2ng/ml, respectively. Besides, we also found that the proportion of cows with a positive Y-specific PCR outcome were higher (P<0.05) in 151-240 days pregnant cows (90%; CI: 55.5-99.75) than 60-150 days pregnant cows (33%; CI: 7.5-70.1). Lastly, we found that the lower limit (35 pg/ml) of spiked male fetal DNA could be successfully recovered from maternal plasma of advanced pregnant cows (using Blood & Tissue Kit, DSP Virus Kit, and NucleoMag cfDNA Isolation Kit) for Y-specific PCR. However, there was variability in the Y-specific PCR results when neat plasma samples from the same pregnant cows were processed for DNA extraction using these kits. In conclusion, fresh but not frozen plasma could be used for PCR-based prenatal fetal sexing. Moreover, due to variability between different DNA extraction methodologies, the PCR assay for fetal sex determination was unreliable for fetal sexing in pregnant cows. Besides, cffDNA could be consistently extracted from maternal plasma for Y-specific PCR when present at amounts ≥31.25 pg/ml. Although a significantly higher proportion of samples from advanced pregnant cows yielded Y-specific fetal DNA amplicons, the study needs to be replicated on a large dataset to confirm our observations

Medical-Data-Models.org:A collection of freely available forms (September 2016)

MDM-Portal (Medical Data-Models) is a meta-data repository for creating, analysing, sharing and reusing medical forms, developed by the Institute of Medical Informatics, University of Muenster in Germany. Electronic forms for documentation of patient data are an integral part within the workflow of physicians. A huge amount of data is collected either through routine documentation forms (EHRs) for electronic health records or as case report forms (CRFs) for clinical trials. This raises major scientific challenges for health care, since different health information systems are not necessarily compatible with each other and thus information exchange of structured data is hampered. Software vendors provide a variety of individual documentation forms according to their standard contracts, which function as isolated applications. Furthermore, free availability of those forms is rarely the case. Currently less than 5 % of medical forms are freely accessible. Based on this lack of transparency harmonization of data models in health care is extremely cumbersome, thus work and know-how of completed clinical trials and routine documentation in hospitals are hard to be re-used. The MDM-Portal serves as an infrastructure for academic (non-commercial) medical research to contribute a solution to this problem. It already contains more than 4,000 system-independent forms (CDISC ODM Format, www.cdisc.org, Operational Data Model) with more than 380,000 dataelements. This enables researchers to view, discuss, download and export forms in most common technical formats such as PDF, CSV, Excel, SQL, SPSS, R, etc. A growing user community will lead to a growing database of medical forms. In this matter, we would like to encourage all medical researchers to register and add forms and discuss existing forms