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Results for “Paraffin Embedding”

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PubMed1987

Ploidy and proliferative activity of human gastric carcinoma: a cytofluorometric study on fresh and on paraffin embedded material.

Flow cytometric DNA analysis was performed on both fresh and on paraffin embedded samples obtained by gastroscopic biopsies in 5 patients with histologically normal gastric mucosa (20 specimens) and by radical gastrectomies in 9 cases of human gastric cancer (36 specimens). Ploidy and the distribution of cells in the different cell cycle phases were estimated. Results from fresh tissue were compared with those from paraffin embedded material. All the samples of normal mucosa showed a diploid modal DNA content. Unimodal DNA distribution was found both in fresh and paraffin embedded material of 3 well differentiated gastric adenocarcinomas, whereas the remaining 6 tumors (1 moderately differentiated, 4 poorly differentiated and 1 undifferentiated adenocarcinomas) showed aneuploidy. The percentage of cells in S phase in normal tissue and in tumors were respectively 9.0% and 11.9% in the fresh material, and 10.0% and 15.0% in the paraffin embedded material. No statistically significant differences were found between fresh and paraffin embedded samples, whereas the proliferative activity was, in both cases, statistically higher in tumors than in normal mucosa (p less than .01 and p less than .001 respectively). The quality of DNA flow cytometry from paraffin embedded material was comparable with that from fresh samples.

Aged↗

PubMed1990

Immunohistochemical detection of c-myc oncoprotein in paraffin embedded tissues.

In almost all studies using paraffin embedded tissue, c-myc protein has been found in the cytoplasm of cells. Since the protein is normally localized in the nucleus it is difficult to determine which histochemical observations are real and which are artefactual. The study designed here evaluated several different methods of fixation prior to paraffin embedding in an attempt to identify which would prevent the diffuse of the protein out of the nucleus. Using various fixation procedures (formalin, paraformaldehyde, B-5, Zamboni and AMeX) we found that fixation in cold acetone (-20 degrees C) overnight followed by 2x15 min fixation in acetone at +4 degrees c and at room temperature, cleared in methyl benzoate and xylene (AMeX procedure) gives reproducible nuclear staining when a variety of normal and tumor tissues are treated with an anti c-myc protein antibody. This method was then compared to frozen sections. While there was no cytoplasmic staining in same tissue specimens in both AMeX processed and frozen sections, the tissue architecture was much better preserved in AMeX processed samples. Our data strongly suggest that AMeX fixation, originally developed for T and B lymphocyte antigens, should be used for immunolocalization of c-myc oncoprotein in paraffin embedded tissues.

Cell Nucleus↗

PubMed1988

Flow cytometric analysis of ploidy in solid neoplasms: comparison of fresh tissues with formalin-fixed paraffin-embedded specimens.

The DNA content of both fresh and formalin-fixed, paraffin-embedded tissues from 30 solid neoplasms was analyzed by flow cytometry. Cells from a single paraffin block of each tumor were disaggregated according to the method of Hedley et al. Cells were sampled from multiple sites of each fresh specimen. Fresh and deparaffinized cells were stored frozen and prepared for cytometric analysis by the technique of Vindelov et al. Thirteen fresh specimens showed DNA diploidy/tetraploidy and 17 showed DNA aneuploidy. Twenty-six of the 30 paraffin-embedded samples were concordant; aneuploid peaks present in histograms from fresh tissue specimens were absent in the 4 paraffin-embedded specimens that were discordant. For 2 of the discordant cases, a hyperdiploid peak that was absent in the initial paraffin-embedded samples was evident on analysis of cells from additional paraffin blocks, indicating ploidy heterogeneity. Peridiploid aneuploid peaks present in the other 2 fresh samples were not observed in initial or additional deparaffinized specimens. Although fresh tissues are preferred for ploidy examination by flow cytometry, formalin-fixed paraffin-embedded specimens, despite a few inherent pitfalls, are generally useful for retrospective studies that compare the ploidy status of solid neoplasms to therapeutic response and prognosis.

DNA, Neoplasm↗

PubMed2003

Genotyping for polymorphic drug metabolizing enzymes from paraffin-embedded and immunohistochemically stained tumor samples.

OBJECTIVES: Paraffin-embedded tumor samples are valuable in the study of cancer for routine staging, tumor marker analysis, and in retrospective studies to test new prognostic and predictive biomarkers. Their utility in retrospective pharmacogenetic analysis of clinical trials has yet to be evaluated. We set out to establish genotyping methods for relevant genes from archival tumor samples and determine if fixation, processing or somatic changes in the tumor might affect our ability to identify germ-line polymorphisms. METHODS AND RESULTS: To establish the assays, paraffin blocks were made using pellets prepared from eight tumor cell lines. DNA was isolated from viable cells and from sections from these blocks, and genotyped for polymorphisms in CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A5 and MDR1 using conventional PCR-RFLP assays. This demonstrated that fixation and processing did not alter the genotypes obtained (100% concordance). Next, sections were obtained from paraffin-embedded archival breast samples from 10 patients for whom gDNA isolated from peripheral blood was available for comparison. Concordance was complete with the same genotype being obtained for 100% of the samples tested. Attempts to extend these methods for the study of hematoxylin/eosin or immunohistochemically stained sections were not successful since the staining inhibited the PCR reactions. Only 25 of 50 samples were successfully amplified and of those only 14 produced accurate genotypes. CONCLUSIONS: Accurate genetic testing for polymorphisms in several genes of pharmacogenetic importance can be obtained from archival paraffin-embedded tumor samples. Thus, pharmacogenetic analysis can be applied to existing cancer therapy trials to test associations between these polymorphisms and treatment response.

Base Sequence↗

PubMed2025

Extraction, Purification, and Next-Generation Sequencing (NGS) Analysis of DNA and RNA from Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue.

Formalin fixed paraffin embedded (FFPE) tissues have long been used for immunohistological analyses. FFPE tissues can be stored at room temperature for several years enabling analyses to be performed later. Ease of storage and transport makes these tissues an attractive source of biological material. However, formalin fixation results in chemical modifications of proteins and nucleic acids that poses a major challenge to any type of analysis. Recovery of nucleic acids for quantitative assays is rendered difficult due to degradation resulting from fixation and long-term storage, producing low usable yields. Extensive efforts in the last 20 years have led to significant improvements in use of FFPE tissues for DNA and RNA analyses and resulted in development of sensitive assays for a wide range of applications, including next-generation sequencing. In this chapter, we describe the optimization of methods for sequential extraction of DNA and RNA from FFPE tissue and subsequent preparation of DNA-seq and RNA-seq libraries for use with the Illumina platform using commercially available reagents/kits.

Paraffin Embedding↗

PubMed1994

Differential staining of nerve cells and fibres for sections of paraffin-embedded material in mammalian central nervous system.

A differential staining method is described of myelinated fibres and nerve cell bodies applicable to sections of mammalian, including human, central nervous system specimens embedded in paraffin wax. Experimental and human necropsy material fixed in acetic paraformaldehyde in phosphate buffer was used. Sections of 15-20 microns in thickness were obtained, attached to slides, deparaffinized and hydrated. After hydration, sections are oxidized (30 s) in 2% potassium permanganate, bleached (1 min) in 5% oxalic acid and rinsed in distilled water. Staining is for 2-5 h in the following solution: 0.06% thionin, 1% formaldehyde, 10% acetic acid in distilled water. Sections are subsequently washed in distilled water, dehydrated through 96% and absolute ethanol, cleared in eucalyptol and mounted in Eukitt. Using the method described in the present paper, a differential coloration of myelin and neurons is obtained. Myelinated fibres appear red, whereas nerve cell bodies and glial nuclei are stained blue. This procedure provides a high contrast between myelin and cells suitable for observation and photography of sections. Simultaneous and differential coloration of both myelin and cells is easily and directly obtained with constant and homogeneous results.

Animals↗

PubMed1994

Extraction of DNA from paraffin-embedded tissue for analysis by polymerase chain reaction: new tricks from an old friend.

For decades virtually all human tissues have been fixed in formalin and embedded in paraffin. The DNA present in fixed tissues is degraded in size but can be analyzed by the polymerase chain reaction (PCR) for specific genetic alterations. The original purpose of fixation, to optimally preserve complex histological and spatial features, is lost with bulk DNA extraction. Because the PCR allows the analysis of even a single molecule of DNA, it is possible to genetically analyze small groups of cells on a microscope slide with techniques such as selective ultraviolet radiation fractionation (SURF). The direct correlation of histological phenotype with genotype provides information that is difficult to obtain by other methods and allows a more precise translation between molecular studies and the current vast histological database.

Chemical Fractionation↗

PubMed1997

Immunohistochemical detection of p21WAF1/CIP1 and p53 proteins in formalin-fixed paraffin-embedded tissue sections of squamous cell carcinoma of the skin.

Thirty-two cases of squamous cell carcinoma (SCC) of the skin were investigated as to the expression of p53 and p21 (WAF1/CIP1) using an immunohistochemical method. These cases were surgically resected or biopsied, tissue samples were then fixed in formalin and embedded in paraffin in the conventional way. Microwave heating was used for antigen retrieval. The primary monoclonal anti-p21 antibody and the monoclonal antibody against p53 were employed. The labeled-streptoavidin-biotin-peroxidase method was used for immunohistochemical staining. Of these, 30 cases showed overexpression of p53 staining, but normal epidermal cells were free of stain. p21 positive cells were detected faintly in the middle layer cells of normal epidermis. Of these, 30 cases showed overexpression of p21 staining. The staining pattern of p53 and p21 showed intratumoral heterogeneity in SCC. In general, there was the inverse relationship between p21 and p53 staining in tumors, namely p53 positive cells were p21 negative and vice versa. However, some of the tumor cells expressed both genes simultaneously. This study supports a hypothesis that p21 expression is regulated by p53, and that it is also regulated by an additional pathway(s) in SCC.

Antibodies, Monoclonal↗

PubMed2025

A Study on Differential Proteomics in Differentiated Gastric Adenocarcinoma With Low-grade Atypia Based on Paraffin-embedded Tissues.

In this study, we analyzed and characterized differentially expressed proteins in differentiated gastric adenocarcinoma with low-grade atypia for screening potential protein markers. We collected gastric tissue specimens from 90 patients treated at the Pathology Department of the First People's Hospital of Yunnan Province, China, between January 2019 and December 2022. These specimens had been fixed in 10% neutral-buffered formalin and embedded in paraffin. We classified these samples into 3 groups: the control group (normal gastric mucosa), the low-grade atypia group (differentiated gastric adenocarcinoma with low-grade atypia), and the high-grade atypia group (differentiated gastric adenocarcinoma with high-grade atypia), consisting of 30 cases in each group. We analyzed differential proteomes with the data-independent acquisition-mass spectrometry (DIA-MS) methodology and selected 4 differentially expressed proteins that were subjected to immunohistochemistry (IHC) staining for validation. A total of 4406 proteins were identified, among which 598 and 357 proteins were statistically different in the low-grade atypia group as compared with the control group and the high-grade atypia group, respectively. IHC staining showed that the expression of FHL3, CSRP2, and FCGR3A was significantly higher in the low-grade atypia group than in the control group ( P <0.05) and significantly higher in the high-grade atypia group than in the low-grade atypia group ( P <0.05). FHL2 expression was negative to weakly positive in the control and low-grade atypia groups and not significantly different between the 2 groups, whereas FHL2 expression in the high-grade atypia group was significantly higher than in the control and low-grade atypia groups ( P <0.05). Proteomic analysis is helpful for discovering new protein markers. Using a combination of FHL3, CSRP2, and FCGR3A can increase the accuracy of the pathologic diagnosis of differentiated gastric adenocarcinoma with low-grade atypia.

Humans↗

PubMed1993

Detection of Mycobacterium leprae DNA in formalin-fixed, paraffin-embedded samples from multibacillary and paucibacillary leprosy patients by polymerase chain reaction.

BACKGROUND: Diagnosis of paucibacillary leprosy is often difficult. A method that could confirm the diagnosis is the polymerase chain reaction (PCR) of M. leprae DNA. This reaction was applied to biopsied tissues of leprotic patients to determine the suitability and sensitivity of the reaction. METHODS: Biopsy samples were taken from previously untreated patients with multibacillary (5 patients) and paucibacillary (3 patients) leprosy, fixed in formalin, and embedded in paraffin. DNA was extracted from paraffin blocks and PCR applied. The sensitivity of the PCR method was tested by using the serially diluted DNA sample as the template. RESULTS: All eight patients showed a positive PCR for M. leprae DNA. The sensitivity was such that a single organism of M. leprae, as counted by staining of the acid-fast bacilli was identified by the PCR. CONCLUSIONS: The PCR method is simple, sensitive, specific, and does not require the use of radioisotopes. It can be applied to the unequivocal diagnosis of paucibacillary leprosy which is difficult by other means. The diagnosis can be obtained within 10 hours.

Blotting, Southern↗

PubMed1993

Immunocytochemical localization of androgen receptor with polyclonal antibody in paraffin-embedded human tissues.

We investigated the immunohistochemical localization of androgen receptor (AR) using a polyclonal antibody for 55 KD recombinant human AR in human tissues fixed with 4% paraformaldehyde solution and embedded in paraffin. Immunoreactive AR was restricted to the nuclei of various tissues. Among the well-known androgen target organs, secretory cells and basal cells of the prostate, spermatogonia, spermatocytes, Sertoli cells and Leydig cells of the testis, epithelial cells of the rete testis, fibroblasts in the whole organ, squamous cells, sweat gland and hair follicle cells of the skin, and hepatocytes of the liver were positive for AR. In addition, smooth muscle cells of the prostate, uterus, urinary bladder, gastrointestinal tract, arteries, and arterioles were strongly immunoreactive for AR. Cardiac muscle and striated muscle of psoas were positive for AR. Acinar cells, ductal cells, and myoepithelial cells of the breast, endocervical and endometrial cells of the uterus, cyto- and syncytiotrophoblast of the chorionic villi, and tubules of the kidney were also positive for AR. Most FSH, LH, and some GH endocrine cells in the anterior and posterior lobes of the pituitary gland, follicular cells of the thyroid gland, and adrenocortical cells were positive for AR. Cells immunoreactive for AR were ubiquitously distributed throughout the entire body. The present study demonstrated the diversity of androgen effects on many target tissues.

Antibodies↗

PubMed1996

LacZ staining in paraffin-embedded tissue sections.

Femora and tibiae of rats carrying leukemia from a LacZ-marked acute promyelocytic leukemia-derived leukemic cell line (LT12NL15) were decalcified using EDTA and routinely embedded in paraffin. Sections were used to develop for the first time an immunostaining method for LacZ, employing catalyzed reporter deposition (CARD) based on the deposition of biotinylated tyramine. This method was used to study homing and adhesion of leukemic cells.

Animals↗

PubMed1995

[A molecular pathological study on the localization of the human papillomavirus using nested PCR and in situ hybridization methods in paraffin embedded tissues].

This study was designed to use nested polymerase chain reaction (nested PCR), in situ hybridization (ISH) and immunohistochemical techniques to identify human papillomavirus (HPV) in tissues fixed in formalin and embedded in paraffin. Eighty cases including 17 cases of condyloma acuminatum, 10 cases of cervical dysplasia, 6 cases of carcinoma in situ, 16 cases of squamous cell carcinoma of the uterine cervix, 14 cases of nasal and paranasal papilloma, 1 case of transitional cell papilloma and 16 cases of esophageal squamous cell carcinoma were examined. With the nested PCR method, the positive reaction rate of HPV was higher than with the single step PCR method. The detection rate was about ten-fold higher in condyloma acuminatum and two times higher in cervical dysplasia with the nested PCR method. In four cases of condyloma acuminatum, ISH was positive. Three of those cases were type 18 HPV and the another was type 11 HPV. Immunohistochemically, HPV was detected only in the cases of condyloma acuminatum. The nuclei of superficial epithelial cells, especially kilocytes reacted positively both by ISH and immunohistochemical methods. Nested PCR is not a very complicated technique, and the detection rate is higher than that of commonly used pathological techniques. These results suggest that nested PCR will be a useful test for HPV routine pathological diagnosis.

Base Sequence↗

PubMed1993

Immunohistochemical detection of alkaline phosphatase in formalin-fixed and paraffin-embedded rat liver.

Alkaline phosphatase (ALP) in rat liver was detected by means of immunohistochemical techniques in ordinary histologic specimens which were fixed with formalin and embedded in paraffin. The specimens were incubated in anti-ALP antibody at room temperature for a longer time, 3hr, than ordinary incubation time. Antigen detection sensitivity and contrast of reaction product of the immunogold silver staining were superior to those of the peroxidase-labeled antibody method. In rat livers which were partially hepatectomized 24 hr before, the more abundant enzyme localized around enlarged bile canalicular spaces.

Alkaline Phosphatase↗

PubMed1993

Immunohistochemical detection of alkaline phosphatase in formalin-fixed and paraffin-embedded rat organs by means of avidin-biotin peroxidase complex method.

The author attempted the detection of alkaline phosphatase (ALP) protein in various rat organs, which were fixed with formalin and embedded in paraffin, by means of the avidin-biotin peroxidase complex (ABC) method. Reaction products were detected after a usual incubation time of 30 min at room temperature in anti-ALP antibody. Positive reactions were observed in the kidney and liver but not in the small intestine, and this indicates that the antibody used in the present study reacts only with the tissue-nonspecific type of ALP.

Alkaline Phosphatase↗

PubMed1997

Immunoperoxidase staining for estrogen and progesterone receptors in archival formalin fixed, paraffin embedded breast carcinomas after microwave antigen retrieval.

Immunoperoxidase staining was performed for estrogen and progesterone receptors in 93 cases of primary breast carcinoma. Breast tumor samples were fixed in formalin and embedded in paraffin. Antigen retrieval was performed by microwave heating in citrate buffer, pH 6.0, using precisely defined and reproducible conditions. The cases studied included material from the current year and from paraffin blocks retrieved from archival storage dating back to 1981. In all cases, estrogen and progesterone receptor values determined by biochemical assay were available for comparison with the immunohistochemical results. We found 94% agreement of results between the two methods.

Adult↗

PubMed1996

Use of immunohistochemistry and polymerase chain reaction for detection of oncornaviruses in formalin-fixed, paraffin-embedded fibrosarcomas from cats.

OBJECTIVE: To determine whether there was intralesional infection or expression of FeLV or feline sarcoma virus in suspected vaccine-associated fibrosarcomas in cats. DESIGN: Prospective case series. SAMPLE POPULATION: 130 suspected vaccine-associated fibrosarcomas from cats and 1 multicentric fibrosarcoma from 1 cat. PROCEDURE: Excisional biopsy specimens were fixed in formalin and embedded in paraffin. Expression of FeLV antigen was assessed, using a polyclonal goat anti-FeLV glycoprotein 70 (gp 70) serum and an avidinbiotin immunoperoxidase staining technique. The FeLV genome was detected with a polymerase chain reaction (PCR), using primers targeted to a conserved sequence in the untranslated region of the long terminal repeat (LTR) of the FeLV. RESULTS: FeLV gp 70 and LTR sequence were detected in a multicentric fibrosarcoma. All 130 of the suspected vaccine-associated fibrosarcomas were FeLV gp 70 negative on the basis of immunohistochemical test results: 100 fibrosarcomas also were examined by use of PCR and were negative for FeLV LTR region. CLINICAL IMPLICATIONS: Exogenous retroviruses, FeLV, and feline sarcoma virus were not detected in these suspected vaccine-associated fibrosarcomas, using immunohistochemistry and PCR. Additional testing will be required to determine the nature of genomic alterations that are involved in the oncogenesis of vaccine-associated fibrosarcomas in cats.

Animals↗

PubMed1992

Molecular pathology of paraffin-embedded tissue. Current clinical applications.

Molecular biology techniques have been adapted to analyze paraffin-embedded tissues, expanding their potential clinical utility. The isolation of intact nucleic acids from tissue blocks is fundamental to the molecular pathology of paraffin-embedded tissues. In vitro amplification with the polymerase chain reaction (PCR) promises to be the most useful means of retrospective analysis since it can be performed successfully on DNA that has partially degraded during fixation, paraffin embedding, and the extraction process. Four clinical situations in which DNA analysis of paraffin-embedded tissues can be helpful are: (a) gene rearrangement analysis in lymphoproliferative disorders, where fresh tissue has not been obtained at the time of surgery; (b) identification of infectious agents, particularly viruses; (c) genetic testing of families with a putative inherited disease where the affected member has died; and (d) specimen identification. The PCR and other techniques of genetic analysis are powerful in sensitivity and specificity when performed and interpreted with appropriate precautions and controls. DNA analysis of paraffin-embedded tissues will likely become a fixed part of the future pathologist's diagnostic armamentarium.

DNA↗