[Conventional paraffin embedding technique and double-embedding technique for minute oral biopsies and delicate pulp tissue - a comparative study]. / Traditsionnyi metod zalivki v parafin i metod dvoinoi zalivki dlya malen'kikh bioptatov polosti rta i myagkikh tkanei pul'py ­ sravnitel'noe issledovanie.

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.

Assessing the Quality of Long-Term Stored Tissues in Formalin and in Paraffin-Embedded Blocks for Histopathological Analysis.

Introduction: Formalin is the most commonly used fixative which enables for long-term storage of specimens and preserves morphologic features allowing the microscopic evaluation for future research analysis. Archival collections of the tissue serve as a reliable tool for diagnostic research purpose. They have an important role in on-going patient care, allows for evaluation of recurrent cases for diagnostic purpose and rare case specimens can also be used as an educational tool as well as for further biomedical research purposes. However, studies assessing quality and their usefulness for such purposes are scanty. Hence, the present study is aimed at evaluating and comparing the tissue changes after long-term storage in formalin as well as in paraffin-embedded blocks. Methodology: Three study groups include specimens stored in formalin for a minimum of 5 years (long-term fixed tissue) and their corresponding paraffin-embedded old tissue blocks along with freshly fixed tissues taken as controls which were subjected to routine histopathological procedures and were assessed for macroscopic and microscopic evaluation.Chi-square test and Z-proportion tests were considered for statistical analysis. Results: Prolonged storage of the tissues in formalin showed variation in color and consistency, difficulty in cutting during grossing with inadequate sectioning characters, loss of tissue integrity and architecture, and inadequate nuclear and cytoplasmic details. Conclusion: On histological analysis, prolonged formalin-stored specimens showed deleterious effects than archival blocks. Hence, it can be proposed that tissues are better preserved in paraffin blocks rather than in formalin for further biomedical research purposes.

Making Formalin-Fixed, Paraffin Embedded Blocks.

Paraffin embedding is a standard technique used in clinical and research laboratories to create a formalin-fixed, paraffin-embedded (FFPE) block of tissue. Formalin-fixed tissue undergoes tissue processing and then is embedded in paraffin (wax) to create a FFPE block or paraffin block. The paraffin block can be cut using a microtome to generate thin sections of tissue contained in paraffin to be stained or paraffin tissue ribbons suitable for nucleic acid extraction. In addition, the FFPE blocks can be stored at room temperature for years. Herein, we provide a basic knowledge, and introduce common methods of the paraffin embedding process.

Immunostaining by dual tumor tissue paraffin blocks increases the sensitivity of c-Met detection in gastric cancer.

BACKGROUND: Gastric cancer (GC) is the second most frequent cause of cancer deaths worldwide. c-Met, a receptor tyrosine kinase, transduces signals from extracellular growth factors. c-Met-targeted therapeutics hold a great potential in treating gastric and related cancers, and a precise evaluation of c-Met expression is a prerequisite for subsequent treatment. METHODS: We compared the sensitivity between one and two paraffin blocks in evaluating c-Met expression in GC subjects by immunohistochemistry (IHC). A total of 365 GC patients were divided into cohort 1 (n = 206) for the one tumor tissue paraffin block test and cohort 2 (n = 159) for the dual tumor tissue paraffin block test. In the dual blocks group, we investigated the results from two different paraffin blocks, then we used the higher one as the final score. RESULTS: Inconsistent c-Met expression in the dual paraffin blocks group occurred in 29 (18.2%) cases. The pooled data in cohort 1 and cohort 2 indicate that when using results from dual paraffin blocks, the c-Met positive (3+) rate of GC testing could be promoted. CONCLUSION: In GC, using dual tumor tissue paraffin blocks instead of one tumor tissue paraffin block is an efficient, economical and practical method of minimizing the false-negative rate of c-Met status assessment by IHC.

Clinical significance of assessing Her2/neu expression in gastric cancer with dual tumor tissue paraffin blocks.

One paraffin block is routinely used for human epidermal growth factor receptor 2 (Her2/neu) immunohistochemistry (IHC) assessment. Here, we investigated if picking 2 paraffin blocks for Her2/neu evaluation on 1 slide is an economical, efficient, and practical method, which may reduce false negativity of Her2/neu IHC assessment due to intratumoral heterogeneity. A total of 251 gastric cancer (GC) patients were divided into a cohort using 1 tumor tissue paraffin block (single-block group, n = 132) and a cohort using dual tumor tissue paraffin blocks (dual-block group, n = 119) when evaluating Her2/neu expression status by IHC. In dual-block group, we combined the results from 2 different paraffin blocks and used the higher one as the final score. The number of IHC 1+, 2+, and 3+ specimens in the single-block group was 31 (23.5%), 40 (30.3%), and 19 (14.4%), respectively. The combined final IHC score in the dual-block group of 1+, 2+, and 3+ was 26 (21.8%), 34 (28.6%), and 23 (19.3%), respectively. Inconsistent Her2/neu expression between blocks was found in 36 (30.3%) cases in the dual-block group. The pooled data in the single-block group and the dual-block group indicated that, when using dual blocks, the Her2/neu-positive (3+) rate of GC was higher compared to that in the single-block group. Our results implied that using dual paraffin blocks to assess Her2/neu expression of GC may help identify more patients with Her2/neu-positive GC who could benefit from targeted therapy, by reducing false-negative rate of Her2 status assessment. This is an efficient, economical, and practical method for Her2/neu evaluation of GC.

A novel xylene-free deparaffinization method for the extraction of proteins from human derived formalin-fixed paraffin embedded (FFPE) archival tissue blocks.

Protein detection methods in formalin-fixed paraffin embedded (FFPE) tissue blocks are widely used in research and clinical setting in order to diagnose or to confirm a diagnosis of various types of diseases. Therefore, multiple protein extraction methods from FFPE tissue sections have been developed in this regard. However, the yield and the quality of proteins extracted from FFPE tissues are significantly reduced in blocks stored for longer periods of time. Regardless the protein extraction method used, tissue sections must be first deparaffinized with xylene, and then washed in serial dilutions of ethanol in order to remove the toxic organic solvent "xylene" and rehydrate the tissue. The objective of this study was first to develop a method to deparaffinize FFPE blocks that excludes the use of toxic solvent "xylene". Second minimize the time required to perform the extraction. Here we describe a method where:•The entire paraffin embedded blocks are deparaffinized and rehydrated using only hot distilled water as a substitute for both xylene and ethanol•The entire procedure takes about 15 min•Deparaffinized blocks are immediately homogenized in lysis buffer, and the obtained lysate analyzed by Western blot. With this new modified technique, we were able to successfully detect actin and AKT proteins in lysates from blocks embedded in paraffin for up to 9 years.

Extracción de ADN de muestras incluidas en parafina sin el uso de xilol para la detección y tipificación de VPH / DNA extraction from paraffin embedded samples without using xylene for HPV detection and typing

En el presente trabajo se llevó a cabo la extracción de ADN de biopsias cervicales con informe citológico de lesión intraepitelial cervical, incluidas en bloques de parafina, utilizando el “QIAamp DNA mini kit” (QIAGEN, Alemania) y la detección del Virus de Papiloma Humano (VPH). En el procedimiento original se eliminó la incubación en xilol, reduciendo los lavados del material biológico; la calidad de los ADNs extraídos fue probada mediante PCR para la detección del gen de la β-globina y del VPH, obteniéndose 84,3% y 28,1% de positividad, respectivamente. Estos resultados fueron comparables a los de otros trabajos similares y evidencian la obtención de ADN celular amplificable por PCR a partir de muestras parafinadas, que pudo ser utilizado eficientemente en procedimientos moleculares, eliminando la toxicidad del xilol y disminuyendo la manipulación del material biológico al reducir los lavados.

In the present study, DNA from cervical biopsies with a cytological report of intraepithelial cervical lesion and embedded in paraffin blocks was extracted using the “QIAamp DNA mini kit” (QIAGEN, Germany), and the Human Papyloma Virus (HPV) was detected. In the original procedure, the xylene incubation was eliminated, therefore reducing the washes of the biological material; the quality of the DNA extracted was tested by PCR for detecting the β-globine gene and HPV, with 84.3% and 28.1% positivity, respectively. These results were comparable to those of other similar studies, and they demonstrate that it is possible to obtain PCR amplifiable cellular DNA from paraffin embedded samples which can be used efficiently for molecular procedures, eliminating xylene toxicity, and decreasing the manipulation of the biological material by reducing washes.

Existing data sources for clinical epidemiology: the Danish National Pathology Registry and Data Bank.

Diagnostic histological and cytological specimens are routinely stored in pathology department archives. These biobanks are a valuable research resource for many diseases, particularly if they can be linked to high quality population-based health registries, allowing large retrospective epidemiological studies to be carried out. Such studies are of significant importance, for example in the search for novel prognostic and predictive biomarkers in the era of personalized medicine. Denmark has a wealth of highly-regarded population-based registries that are ideally suited to conduct this type of epidemiological research. We describe two recent additions to these databases: the Danish National Pathology Registry (DNPR) and its underlying national online registration database, the Danish Pathology Data Bank (DPDB). The DNPR and the DPDB contain detailed nationwide records of all pathology specimens analyzed in Denmark since 1997, and an incomplete but nonetheless valuable record of specimens from some pathology departments dating back to the 1970s. The data are of high quality and completeness and are sufficient to allow precise and efficient localization of the specimens. We describe the relatively uncomplicated procedures required to use these pathology databases in clinical research and to gain access to the archived specimens.