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Background: Among oral biopsies, small incisional tissues, have to be preserved all through the processing and embedding to ensure optimal visualization of all the mucosal layers without compromise. Optimal tissue orientation is the most critical step in tissue processing for demonstration of definitive morphology in the sections, which is often more challenging in cases of minute/small or thinner sections using routine paraffin techniques to evaluate accurate diagnosis. Some modification is needed to handle these samples to get a better result. Double embedding technique with some modification has been widely used for small/ thin/ multiple biopsies and gives excellent results in many other fields like general pathology and biotechnology. The double embedding technique though produced excellent and significant results in mucosal biopsies yet, it is of minimal interest among oral pathologists. To best of our knowledge, this is the first study to use double embedding technique for pulp tissues., Objective: The present study was aimed to evaluate and compare the ease of embedding and sectioning sections using Agar-Paraffin double embedding technique for small oral mucosal biopsies and thin pulp tissues., Material and Methods: A total of 40 oral tissue samples categorized into two groups were taken for the present study. Group I included 20 small oral mucosal biopsy samples of size ranging from 0.2 to 0.5 cm and Group II included 20 pulp tissues obtained from freshly extracted non carious tooth. 10 blocks were prepared by routine paraffin method and 10 blocks were prepared by modified double embedding method for each group. Scores were given by comparing all the criteria with that of the routine paraffin technique. Chi-square test was used for statistical analysis., Results: The average ease score for the Agar-Paraffin double embedded small/minute biopsies showed better scores than the pulp tissue with that of the routine technique. However, no statistically significant difference was seen among embedding and sectioning sections between the two groups., Conclusion: Modified double embedding method is simple and reliable alternative technique that helps in better orientation, processing and sectioning especially for oral small or thin biopsies and delicate pulp tissues.

The lack of specific symptoms for the early detection of gastric cancer leads to the fact that it is often diagnosed at a late stage, when the prognosis is unfavorable. The analysis of molecular markers in addition to standard diagnostic procedures is a promising approach for improving the preoperative diagnosis of both gastric cancer and precancerous changes in the mucosa. Therefore, the aim of our study was to analyze the diagnostic significance of using miRNA expression to diagnosis gastric cancer and precancerous conditions (dysplasia) in histological material. In this work, 122 samples of archival histological material in the form of paraffin blocks were used: 34 samples of gastric adenocarcinoma, 54 samples of gastric ulcers with dysplasia and 34 samples of normal gastric mucosa obtained from patients after bariatric surgery. The expression level of miRNA-145-5p, -150-5p, -20a-5p, -21-5p, -31-5p, -34a-5p, -375 was determined using real-time RT-PCR. Samples were stratified into different groups using the C-RT decision tree algorithm. All miRNAs, except miRNA-20a, were included in the decision tree, which allows stratification of samples for normal mucosa, dysplasia, and gastric cancer. Normal mucosa can be distinguished from gastric cancer only by miRNA-34a, -21, -375. Diagnostic characteristics for the detection of dysplasia: specificity - 97%, sensitivity - 87%; for the detection of gastric cancer: specificity - 91%, sensitivity - 93%. The sufficiently high values of the diagnostic characteristics for detecting dysplasia of the gastric mucosa and gastric cancer obtained in our study indicate the possibility of using expression data of a small amount of miRNAs for the effective separation of samples with tumor and precancerous changes in the stomach tissue., Competing Interests: The authors declare no conflict of interest.

Objective: to assess mutational events in exons 5, 7, and 8 of the p53 gene and to reveal mutant p53 protein in verified cases of morphologically altered (proliferative and precancerous changes, lung cancer) and histologically unaltered, lung tissues in workers exposed to occupational radiation., Material and Methods: The investigation used formalin-fixed paraffin-embedded unaltered and altered lung tissue blocks (FFPBs) obtained from the human radiobiological tissue repository. The shelf-life of FFPBs was 5-31 years. An immunohistochemical technique using mouse antibodies against p53 protein (<>, Denmark), stained with diaminobenzidine (DAB) chromogen, was employed to determine p53 protein. DNA was isolated from lung tissue FFPBs with QIAmp DNA FFPE Tissue Kit, (<>, USA). Polymerase chain reaction (PCR) was performed to amplify the p53 gene exons 5, 7, and 8 selected for examination, by applying the sequences of genes and primers, the specificity of which was checked using the online resource (http://www.ncbi.nlm.nih.gov/blast). PCR products were detected by temporal temperature gradient gel-electrophoresis and the Sanger sequencing method. The obtained DNA fragments were analyzed on a sequencer ABI Prism 3100 Genetic Analizer (<>, USA). Computer-aided DNA analysis was made using the BLAST program. A package of applied Statistica 6.0 programs was employed for statistical data processing. Results. Immunohistochemical analysis showed that mutant p53 protein was absent in the cells of unaltered lung tissue and the number of cells with mutant p53 protein increased in all the patients with proliferative and precancerous changes and lung cancer, suggesting p53 protein dysfunction. The total number of p53 gene mutations in exons 5, 7, and 8, if there were proliferative and precancerous lung tissue changes and lung cancer, were 25, 20, and 40%, respectively. All the found mutations were transversions (the substitution of purine for pyrimidine or, conversely), indicating the action of exogenous mutagens., Conclusion: The results of this investigation have confirmed other investigators' data showing that p53 gene mutations in lung cancer are observed in 40-70% of cases. The differences in the number of cases of altered lung tissue with mutations in the p53 gene (not more than 40%) and in those of p53 protein expression were found in 100%, suggesting the regulation of p53 gene function in the cell at multiple levels.

Optimal conditions were defined for DNA isolation from formalin-fixed and paraffin-embedded (FFPE) archived autopsy lung tissue slices. The quality of DNA preparations isolated from the FFPE archived slices was assessed and their suitability for further molecular genetic analysis estimated. DNA isolated from the FFPE slices stored less and more than 10 years was suitable for molecular genetic studies in 100 and 66.7% of cases, respectively.

High resolution melting analysis (HRMA) using special "saturating" fluorescent dyes is a new and very effective approach to genotyping and mutation scanning. HRMA, which is carried out usually just after PCR without any intermediate manipulations (the "closed tube" format), is simple and high-throughput method excluding sample cross-contaminations. The "closed tube" format makes, however, HRMA dependent on PCR mixes and, as such, limits its capability. The "open tube" format (post-PCR amplicon shortening and optimization of the ionic medium) proposed by us earlier, although somewhat more laborious, significantly increases sensitivity of the method and makes it possible to scan mutations in the short amplicons using conventional SYBR Green I dye and a standard (not adapted specifically for HRMA) real-time PCR instrument. Detection of mutant K-RAS in DNA of clinical specimens (tumor tissues, formalin-fixed paraffin-embedded samples) reveals equal, at least, sensitivity of this method as compared with the HRMA and much higher as compared with Sanger sequencing. The problem of false-negative results in mutation scanning of K-RAS, which is highly important in some forms of cancer, is discussed.

Experiments on 55 male chinchilla rabbits with model widespread purulent peritonitis have been performed for determinig structural changes in adrenal glands with the aid of optical microscopy. The introduction of aerobic-anaerobic culture of E. Coli and B. Fragilis into the abdominal cavity causes expressed structural changes in parenchyma of adrenal glands within 6 hours. It is established for the first time that the administration of metabolic drugs citoflavin (containing succinic acid) and neoton (containing creatine phosphate) prevents the development of pathological structural changes in adrenal glands under conditions of experimental widespread purulent peritonitis.

The t(X;18)(p11;q11) translocation has been shown to be the specific alteration for synovial sarcomas. The translocation leads to production of chimeric protein SYT/SSX by fusion of SYT and SSX genes involved. The expression analysis of SYT/SSX1 and SYT/SSX2 chimeric transcripts was performed in formalin-fixed soft tissue tumour specimens and the diagnostic validity of immunohistochemistry, FISH and RT-PCR methods was compared. The chimeric transcripts were detected in 12 from 16 synovial sarcomas: 7 SYT/SSX1 and 5 SYT/SSX2 fusion variants; by fluorescence hybridization in situ (FISH) the translocation was found in 13 from 16 sarcoma samples. As synovial sarcoma represents a diagnostically challenging group, genetic analysis of translocations and chimeric transcripts is an extremely useful confirmatory diagnostic tool providing higher sensitivity than immunohistochemistry markers do.

Analysis of the results of examining 57 primary and metastatic soft tissues tumors has shown the principal capabilities of immunophenotyping in the cytosmears for differential diagnosis, the determination of histogenesis, and the assessment of their prolferative activity. Its major benefit is short response time and easiness to use. All results were assessed by comparison with the data of immunohistochemical study on paraffin sections.

Multifunctional granular mast cells (MCs) are involved in various pathological processes. The response of MC populations of myocardium, pericardium and lung to pulmonary hypertension (PH) has been studies 8 weeks after injection of monocrotaline. Five intact and five experimental rats were used. The density of MCs of different maturity was estimated on paraffin sections stained with Alcian blue and Safranin. Expressiveness of PH was estimated by functional parameters with the help of echocardiograms and by morphological markers. The MC density in myocardium of the intact and experimental rats was relatively low: 2 to 4 cells/mm2. MC density in the pericardium of intact rats was 14 times higher than in myocardium and increased 3 times for PH. The mature Safranin-positive cells predominated (70-80%) in myocardium and pericardium of intact and experimental rats. The MC density in the lungs of intact rats was about 30 cells/mm2; 98% of these cells were immature Alcian-positive cells. The mean density of MCs in the lungs of rats with PH increased 5.6 times. The mature Safranin-positive cells appeared in the lungs of rats with severe pathology. The greatest number of MCs in lungs was in the rats with the most pronounced disorders of myocardium function and marked histological damages (injuries) of myocardium and lungs. The finding show active response of MC population to monocrotaline-induced PH that stimulates migration of immature MCs into pericardium and lungs from the outside. Our data indicate the important role of MCs in the pathogenesis of PH.

Aim: To test feasibility of detection of translocations which are diagnostic for Berkitt's lymphoma with the method of fluorescence in situ hibridization (FISH) on histological sections of paraffin blocks., Material and Methods: FISH on histological sections for detection of t(8;14)(q24;q32) and variant t(2;8)(p12;q24) and t(8;22)(q24;q11) was performed on the material obtained from 53 patients with typical clinical, morphological and immunological picture. DNA probe LSI IgH/MYC, CEP 8 Tri-color, Dual Fusion Translocation Probe (Vysis, USA), for variant translocations DNA-probe LSI MYC, Dual color, Break Apart Rearrangement Probe (Vysis, USA) were used., Results: Histological material from 31 patients contained translocations characteristic for LB: in 29 (93.5%)--t(8;14)(q24;q32), in 2--variant rearrangements of locus of gene c-myc. Translocation t(8;14)(q24;q32) and its variants were not detected in 22 patients, the diagnosis was changed for diffuse large B-cell lymphoma (DLBCL)., Conclusion: Typical for BL clinical, morphological and immunological picture may present in extra-nodal diffuse large B-cell lymphoma with high proliferative activity. Differential diagnosis between BL and the latter lymphoma is possible only basing on detection of translocation t(8;14)(q24;q32) or its variants. If it is impossible to obtain native material, FISH on histological sections of parasffin blocks is the only possible method of differential diagnosis.

The aim of this study was to develop an optimal and easy protocol for immunocytochemical demonstration of nestin (an intermediate filament protein which is believed to be a neuronal stem cell marker) in paraffin sections of rat brain with target retrieval procedure being omitted for better preservation of the neural tissue structure. The influence of fixation, character of blocking of nonspecific antibody binding, temperature and time of incubation on the results of immunocytochemical reaction was studied. On the basis of the results obtained, we propose a processing protocol allowing distinct demonstration of nestin in rat brain cells together with the optimal preservation of the nervous tissue structure.

Tissue microarrays are an ordered array of tissue cores in a paraffin block. They permit immunohistochemical analysis of numerous sections of archival formalin fixed paraffin embedded tissues. Short information on the construction is given and the authors' modification is described. Sixteen tissue microarrays out of 252 archival cases of non-Hodgkin's lymphoma with diffuse small cell, diffuse large cell and blastic morphology were immunophenotyped. Leading technical problems are discussed. The design and precision in execution of a tissue microarray have a role to obtain valid results.

The study was undertaken to investigate the effect of various methods of fixation of the brain upon the demonstrability of catecholaminergic structures using tyrosine hydroxylase (TH) immunocytochemical reaction. It was established that most complete demonstration of TH-containing structures was achieved after perfusion fixation with 4% paraformaldehyde followed by post-fixation in the Carnois fluid and after immersion fixation in the IHC zinc fixative (BD Pharmingen, U.S.A.). Immersion fixation in 4% paraformaldehyde, ethanol-formaldehyde, Carnois and Bouin fixatives can be used for demonstration of perikarya of catecholaminergic neurons.

The aim of this study was to develop an optimal protocol of processing of rat brain sections permitting to minimize the shortcomings of the method of heat retrieval of antigens and preserving its high efficiency. The research was conducted using the methods of heat antigen retrieval, immunohistochemistry and light microscopy. It was shown that prolonged incubation in blocking solution, containing normal serum of an animal--source of secondary antibodies, eliminated false-positive reaction, demonstrated after heat antigen retrieval in paraffin sections of rat brain.

Amygdala belong to the system of basal nuclei of telencephalon. Amygdala are a complicated structure based on both principles of grey matter organization: nuclear and screen-type, on its territory. Authors investigate into the structure of Amygdala using theory of Zavarzin A. As the result of this study, a classification of components of Amygdala into nuclei, paleocortical and intermediate formations, has been developed.

The Bilshovsky method is often used for studies of the morphology of neuronal fibrillar system. The classical variant of this method requires sections made with a freezing microtome, which is sometimes inconvenient. Preparation of paraffin sections often gives unsatisfactory results. The authors managed to overcome this deficiency. Their modification consists in the following: after deparaffinization of paraffin sections glued to the slides they are repeatedly fixed in warm formalin and then treated with silver nitrate with heating. This modification is easily reproducible and essentially simplifies staining; no freezing microtome is needed.

Recovery of specific antigenic characteristics using microwave treatment of paraffin sections of tissues fixed by formalin allows to extend spectrum of antibodies for immunohistochemical diagnosis. Microwaves enable the reaction on the material prepared according to the standard technique, and macropreparations long stored in formalin and archive blocks.

The results of polymerization chain reaction (PCR) and morphological study are compared. The presence of cytomegalovirus (CMV) DNA in the organs of dead children is demonstrated by PCR in 13 of 16 cases. In 3 of 13 cases structural changes typical for CMV infection were absent; slight symptoms of the damage produced by DNA-virus were present in 4 cases. The results indicate that there exist variants of CMV infection with a minimum manifestation of the infectious process. The presence of mutations in the CMV genome was revealed in 6 cases.

Two accelerated chloroform-paraffin processings of materials with the use of ultrasound (US) and microwave (MW) irradiation in the stove "Electronica" as well as a combined method with US- and MW-irradiation are proposed to shorten drastically the duration of the prehistologic processing.