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PubMed1998

The mitochondrial DNA A8344G mutation in Leigh syndrome revealed by analysis in paraffin-embedded sections: revisiting the past.

In 1975, we presented the results of a study on a family with a constellation of features that included a chronic spinocerebellar syndrome, neuropathologically proven Leigh syndrome, and sudden death in infancy or childhood affecting several members over three generations. Inheritance was thought to be autosomal dominant. Twenty years later, we reinterpreted the inheritance pattern as maternal. Mitochondrial DNA (mtDNA) extracted from paraffin-embedded brain samples from the proband revealed the A8344G myoclonic epilepsy and ragged-red fiber (MERRF) mutation as the molecular basis for this multifaceted neurological syndrome. This re-evaluation of archival material is an instructive example of "medical archeopathology."

Adolescent↗

PubMed1996

Immunohistochemical detection of p21waf1/cip1/sdi1 and p53 proteins in formalin-fixed, paraffin-embedded tissue sections of colorectal carcinoma.

Wild-type p53 protein is a critical participant in G1 cell cycle arrest through the induction of waf1/cip1/sdi1 gene product p21, but mutant p53 proteins have lost transcriptional activity. To know whether the view obtained from studies using cultured cells is adaptable to human solid tumors, the authors immunohistochemically stained p21 and p53 in formalin-fixed, paraffin-embedded tissue sections from 67 cases of colorectal carcinoma. Of these, 54 cancers showed positive staining of p53, whereas p21-positive cells were identified in 46 tumors. However, the proportion of cells positive for these proteins varied considerably among cases, and moreover wide variation in p21 immunoreactivity was noted within the same tumor. Although there was an inverse relationship between p21 and p53 staining in tumors, namely, p21-positive cells were p53 negative and vice versa, an intermingling of tumor cells expressing concomitantly both genes was noted in 34 (51%) of 67 tumors. In these tumors, both proteins were usually moderately stained, but tumor cells intensely coexpressing both were found in one sample. This study supports the notion that although p21 expression is regulated by p53 under physiological conditions, it can be regulated by an additional pathway(s) in solid malignant tumors.

Carcinoma↗

PubMed2001

The significance of bivariate cytokeratin and DNA flow cytometry in paraffin-embedded specimens of non-small cell lung cancer.

BACKGROUND: The presence of non-tumor cells inside cancer tissue is one of the causes of errors in cell cycle analysis by DNA flow cytometry. The recent establishment of bivariate cytokeratin and DNA flow cytometry has made feasible the accurate assessment of tumor proliferative activity. METHODS: Bivariate flow cytometry and immunohistochemistry examinations of paraffin-embedded specimens were performed in 92 patients with non-small cell lung cancer (NSCLC). Determination of the S-phase fraction by flow cytometry, with cytokeratin gating (CK-gated SPF) and without gating (ungated SPF), and the expression of proliferating cell nuclear antigen by immunohistochemistry (PCNA labeling index), were used to assess cancer cell proliferation. RESULTS: Two tumors had DNA histograms with a coefficient of variation of more than 8.0% and were excluded from the flow cytometric analysis. In DNA diploid tumors (n = 25), the ungated SPFs (8.7 +/- 3.6%) showed a lower distribution than the CK-gated SPFs (14.3 +/- 4.7%) (P < 0.0001). In DNA aneuploid tumors (n = 65), there was no difference in distribution between the ungated SPFs (15.0 +/- 8.3%) and the CK-gated SPFs (15.1 +/- 7.1%) (P = 0.94). The CK-gated SPF and the PCNA labeling index of an individual tumor had a good correlation (P < 0.0001), and this agreed with the result showing that DNA diploid and aneuploid tumors had equal proliferative activity (P = 0.64 and P = 0.63, respectively). CONCLUSION: The technique using CK-gating markedly improved the SPF measurement in DNA diploid tumors. This assessment showed no difference in proliferative activity between DNA diploid and aneuploid tumors in NSCLC. Bivariate cytokeratin and DNA flow cytometry is an accurate and objective method for cancer-specific analysis, and will surely be informative in clinical oncology.

Adult↗

PubMed1997

Detection of integrins in formalin-fixed, paraffin-embedded tissues.

Integrins are heterodimeric transmembrane receptors, which are expressed in many cells. In vitro experiments have demonstrated that integrins may be important in tumor progression and organ development. The functions of integrins were previously studied in cell cultures and their tissue expression was detected by immunofluorescence or immunoperoxidase in frozen sections. The purpose of this study was to determine the optimal conditions for detection of integrins in formalin-fixed, paraffin-embedded tissues. We utilized microwave heating and enzyme digestion in routinely processed, surgically removed tissues. Our results demonstrate that integrins can be reliably detected in archival material. This approach will facilitate further investigation of the role played by integrins in human malignancies and in developmental processes.

Formaldehyde↗

PubMed1995

Detection of Clostridium chauvoei in formalin-fixed, paraffin-embedded tissues of sheep by the peroxidase-antiperoxidase (PAP) technique.

A peroxidase-antiperoxidase (PAP) technique was used to detect Clostridium chauvoei in tissue sections from sheep inoculated intramuscularly with a pure culture of this microorganism. Samples of various tissues were taken for bacteriology, histopathology and immunohistochemistry. A primary antiserum against C. chauvoei for use in the PAP technique was produced in rabbits. Formalin-fixed, paraffin-embedded sections of muscle samples were positively and specifically stained by the PAP technique. The results were consistent with those obtained by bacteriology, but the PAP test was simpler, quicker and less expensive than the bacteriological procedures. The use of the PAP technique would be appropriate for detecting clostridial infections without the constraints of conventional identification methods, especially where laboratory conditions for anaerobic procedures are not readily available.

Animals↗

PubMed1997

A rapid and nonisotopic method for the screening and sequencing of p53 gene mutations in formalin-fixed, paraffin-embedded tumors.

Evaluation of the clinical significance of p53 gene mutations requires molecular analysis of primary tumors from large series of patients with long follow-up periods. In this study, we describe a sensitive, rapid, nonisotopic and inexpensive procedure for the polymerase chain reaction (PCR)-single-stranded conformational polymorphism (SSCP) detection and subsequent sequencing of p53 mutations in formalin-fixed and paraffin-embedded tumor (PET) samples. To optimize this method, we used a panel of 34 mutations previously identified in this gene by isotopic PCR-SSCP analysis of frozen colorectal carcinomas. Identical SSCP band shifts were observed in PET sections from each of these frozen tumors, except for one case in which the tumor cell content was probably too low. All of the 34 mutations were detected by use of an optimized minigel SSCP/silver staining procedure. The total PCR-SSCP screening time with use of this protocol was less than 8 hours. An additional improvement was the use of multiplex SSCP to screen two p53 exons (5/7 and 6/8) simultaneously, thus effectively halving the amount of reagents and time required for mutation analysis. To identify the exact nucleotide alterations, mutant single-stranded DNA was excised from silver-stained SSCP gels, reamplified, and used as template in sequencing reactions. The PCR-SSCP procedure we describe can be performed in routine histopathology laboratories, requiring only a thermal cycler and minigel electrophoresis apparatus. Our results demonstrated the feasibility of conducting large retrospective analyses of p53 gene mutation status in archival tumor material. Studies of this kind should allow elucidation of the clinical role of p53 mutation in various human cancer types.

Carcinoma↗

PubMed1998

[The examination of EBV-DNase gene fragment in the paraffin embedded NPC, pre-cancerous and high risk population nasopharyngeal tissues].

OBJECTIVE: To examine if EB virus enters nasopharyngeal epithelial cells in the course of the tumorigenesis in nasopharyngeal carcinoma (NPC). METHODS: Based on the annual investigation of Si-Hui county, a cancer-prevention base of the Sun Yet-sen University of Medical Sciences and Tumour Hospital outpatient clinic, more than 450 paraffin-embedded tissues with NPC, and with pre-cancerous lesions in high risk populations were collected for the detection of EBV-DNase gene fragment by an optimized PCR method. RESULTS: In 145 of the 149(97.3%) cases with invasive NPC, and 2 of 4 cases with in situ NPC, EBV-DNase gene was detected. However, in Sihui, Canton, a high-risk county for NPC, only 2 of the 155 subjects with nasopharyngeal precancerous state was the gene positive, and none of the 47 cases examined in the out-patient department of the Tumor Hospital with precancerous lesions in the nasopharynx was positive. CONCLUSION: EBV is seldomly detected in the non-malignant and precancerous lesions in the nasopharynx in high-risk populations. It implies that EBV is probably not etiologically involved in the genesis of NPC.

Deoxyribonucleases↗

PubMed1978

Histochemical identification of T and B areas in paraffin-embedded lymphoid tissues by demonstration of alpha-naphthyl acetate esterase (ANAE) activity.

A strong activity of acid alpha-naphthyl acetate esterase (ANAE) in T lymphocytes and macrophages within mouse lymphoid organs is demonstrable even after paraffin-embedding, if a rapid embedding procedure with acetone as dehydrating agent is used. The lymphoid tissue structure is better preserved in paraffin sections procured after this rapid embedding procedure than in frozen sections commonly used for histochemical demonstration of ANAE. In the paraffin sections the pattern of the ANAE reaction allows a clear delineation of the T and B areas in the lymphoid organs, and the intracellular location of the focal ANAE activity in T lymphocytes is readily discernible.

Animals↗

PubMed2002

DNA ploidy and S-phase fraction analyses of hyperplastic, atypical and cancerous endometrium using flow cytometry from paraffin-embedded tissues.

Atypical hyperplasia of the endometrium is an entity that needs to be clearly distinguished from other florid hyperplastic states on the one hand and from well-differentiated adenocarcinomas on the other. This may at times be difficult on pure morphological grounds. In this study, DNA ploidy and S-phase fraction of hyperplastic, atypical and cancerous endometrium were evaluated using flow cytometry from paraffin-embedded tissues. In total, 72 cases (24 hyperplastic, 24 atypical and 24 adenocarcinomas) were selected. All hyperplastic endometria showed a diploid stemline, while 2/24 atypical hyperplasias showed aneuploid (Near-diploid) peaks. Both of these cases were severely atypical and one of these, on hysterectomy, showed early invasive carcinoma. There was no significant difference in mean S-phase fractions of hyperplastic vs. atypical endometria. DNA aneuploidy was significantly more common with much higher S-phase fractions in poorly and moderately differentiated carcinomas than in well-differentiated ones. It was concluded that aneuploid (near-diploid) peaks, if ever present in atypical hyperplasias, may indicate an aggressive disease/neoplastic transformation.

Adenocarcinoma↗

PubMed2000

Molecular cytogenetic studies in three patients with partial trisomy 2p, including CGH from paraffin-embedded tissue.

We report on three cases of partial trisomy 2p in which the identification and exact localization of the duplicated chromosome segment was possible only by application of molecular cytogenetic techniques. These included fluorescence in situ hybridization by use of wcp2, N-myc, and subtelomeric 2p probes and comparative genomic hybridization with DNA isolated from blood samples, frozen fetal tendon, and formalin fixed, paraffin-embedded fetal lung tissue. Two of the cases concerned fetuses of gestational week 20 and 24 with duplication of nonoverlapping terminal (2pter-->p24) and more proximal (2p25-->p23) segments and with distinctly different phenotypes. The third case was due to a de novo inverted duplication of 2p25-->p23, with loss of the subtelomeric region of 2p. This 53-month-old girl was a Bloom syndrome carrier. The patient had prenatal growth failure, borderline microcephaly, dilated lateral horns of the cerebral ventricles, transient cortical blindness, myopia, muscle hypotonia, and dilatation of the left renal collecting system. Dermal cysts were found on the glabella, the soles of both feet, and the vocal cord, causing respiratory embarrassment. Previously reported cases of pure trisomy 2p are reviewed, in an attempt to correlate clinical findings to overlapping regions in 2p. These cases illustrate the effectiveness of molecular cytogenetic methods in resolving subtle chromosomal aberrations in order to coordinate more accurately a chromosome regionspecific phenotype.

Abortion, Induced↗

PubMed2006

Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization.

Gene expression in diseased tissues can indicate the contribution to a disease process and potentially guide therapeutic decision-making. Archival tissues with associated clinical outcome may be useful to discover or validate the role of a candidate gene in a disease process or the response to therapy. Such archival tissues are commonly formalin-fixed and paraffin-embedded, restricting the methods available for gene expression analysis. Obviously, the detection of proteins in tissues requires adaptation for each protein and the detection of secreted proteins can prove difficult or of reduced value since the protein detected may not reflect the total amount produced. Thus, we describe here a reliable method for the detection of mRNA in archival tissues. The method for mRNA in situ hybridization (ISH) was adapted by us for >15 different genes and applied to several hundred tissue microarrays (TMAs) and full sections generating >10,000 expression data points. We also discuss the utility of TMAs to simultaneously analyze several hundred tissue samples on one slide to minimize variability and preserve valuable tissue samples. Experimental protocols are provided that can be implemented without major hurdles in a typical molecular pathology laboratory and we discuss quantitative analysis as well as advantages and limitations of ISH with a special focus on secreted proteins. We conclude that ISH is a reliable and cost effective approach to gene expression analysis in archival tissues that is amenable to screening of series of tissues or of genes of interest.

Animals↗

PubMed2000

In situ-PCR for the detection of Mycobacterium paratuberculosis DNA in paraffin-embedded tissues.

Molecular biological techniques have permitted the rapid and sensitive detection of the Mycobacterium paratuberculosis genome in infected tissues, most commonly by polymerase chain reaction amplification of sequences in the IS900 DNA insertion sequence. The aim of this work was the detection of M. paratuberculosis DNA in ovine tissues by in situ-polymerase chain reaction, which is sensitive and localises the signal within the tissue sample. Paraffin embedded tissues from three acid-fast positive ovine guts with classical lesions of paratuberculosis, and from negative control samples were tested. A 413-bp fragment of the IS900 sequence was amplified in-situ and hybridised to an internal PCR-synthesised digoxygenin-labelled probe. The samples from sheep affected by paratuberculosis clearly showed cell-specific cytoplasmic signals in mucosal and submucosal macrophages. This technique could be useful both in the diagnosis and study of the pathogenesis of infections in which involvement of M. paratuberculosis is suspected.

Animals↗

PubMed2003

Feasibility of T-cell receptor gamma (TCRgamma) gene rearrangement on formalin-fixed, paraffin-embedded tissues by PCR assays.

INTRODUCTION: T- and B-lymphocytes are involved in recognition of foreign antigen by the specificity of their surface T-cell receptor and immunoglobulin, generated by gene rearrangement. Each T- and B-lymphocyte carries unique rearranged TCR or immunoglobulin gene, which has been applied to detect clonal from non-clonal T- and B-cell proliferation. METHODS: Paraffin-embedded biopsy tissues of 85 T-, 24 B-cell non-Hodgkin's lymphomas (NHL) of various subtypes, and seven reactive lymphoid hyperplasia were retrieved from the archives for determining the feasibility of TCRgamma gene rearrangement analysis by PCR assays in our laboratory. DNA was extracted by Proteinase K digestion. The analyses were performed by five PCR assays, and analysed on polyacrylamide gel. RESULTS: Clonal TCRgamma gene rearrangement was demonstrated in 69/85 (81.2%) of the cases. Selective rearrangement of specific Vgamma segment was observed, especially in peripheral T-cell lymphoma-unspecified and nasal NK/T-cell lymphoma. Clonal TCRgamma rearranged band was also demonstrated in 4/24 (16.7%) and 2/7 (28.6%) of B-NHL and reactive lymphoid tissues respectively. CONCLUSION: PCR assays were able to demonstrate clonal TCRgamma gene rearrangement in a high proportion of T-NHL. However, the PCR results should be interpreted carefully. A neoplasm should only be considered as T-cell type if it does not express any B-cell marker because TCRgamma is not lineage specific as shown by the presence of clonal TCRgamma gene rearrangement in B-NHL. Hence, the results for TCR gene rearrangement should always be interpreted in conjunction with histology and immunophenotyping.

B-Lymphocytes↗

PubMed2002

Comparison of the effects of microwave heating and high pressure cooking for antigen retrieval of human and rat Bc1-2 protein in formaldehyde-fixed, paraffin-embedded sections.

Immunohistochemical detection of expression of the anti-apoptotic Bcl-2 protein is widely studied as a putative prognostic and predictive factor in various types of cancer. For that purpose, heating for 10 min by microwave (MW) up t o 100 degrees C in citrate buffer, pH 6.0, prior to immunostaining is often used to retrieve Bcl-2 antigens in archival formalin-fixed, paraffin-embedded tissue. We recently reported that Bcl-2 is not only a cytoplasmic protein, but that it is present also in interphase nuclei and that it strongly associates with mitotic chromosomes. Furthermore, we showed that binding of the monoclonal antibody (MAb) #124 with nuclear/chromosomal epitopes is diminished by formaldehyde-based fixatives and cannot be restored by MW treatment for 10 min. Here we report that prolonged MW heating or heating up to 130 degrees C in a high pressure cooker (HPC), despite improved cytoplasmic immunostaining, fails to retrieve nuclear/chromosomal Bcl-2 epitopes recognized by the MAb #124 in human tissues. In contrast, these procedures can retrieve nuclear/chromosomal Bcl-2 epitopes detected by polyclonal #15616E antibodies in rat tissues. The specificity of these epitopes was confirmed by Western blot analysis of tissues treated by MW heating or HPC.

Animals↗

PubMed1996

Comparison of fluorescence in situ hybridization analysis of isolated nuclei and routine histological sections from paraffin-embedded prostatic adenocarcinoma specimens.

Fluorescence in situ hybridization (FISH) is a powerful tool for quantitative analysis of chromosomes and genes and can be applied in a variety of specimens, including cell cultures, isolated nuclei from fresh and fixed tissues, and histological tissue sections. However, the results of FISH analysis of isolated nuclei in prostate cancer have not been previously compared with those from histological sections from the paraffin-embedded tissue blocks. To compare these methods, we studied isolated nuclei derived from 50-microns sections and adjacent 5-microns tissue sections from 10 cases of benign nodular hyperplasia of the prostate and 16 cases of prostatic carcinoma. FISH analysis employed centromere-specific probes for chromosomes 7, 8, 11, and 12. In benign tissue, the percentage of nuclei with three or more signals for chromosomes 7, 8, 11, and 12 was less than 3% for both isolated nuclei and tissue sections. However, the percentage of nuclei with no and one signals was less than 8% for isolated nuclei and more than 24% for tissue sections. In prostatic carcinoma, numeric chromosomal anomalies were found in 75% of cases by both FISH methods. However, isolated nuclei had more chromosomal tetrasomy than tissue sections (mean, 9.2 to 11.0% versus 5.1 to 5.6%, respectively). Conversely, intratumor heterogeneity of chromosomal anomalies was identified in 5 cases by FISH analysis of tissue sections but not in isolated nuclei. Cancer ploidy analysis by FISH correlated well with ploidy analysis by flow cytometry, although FISH was more sensitive for aneuploidy. We conclude that FISH analysis of isolated nuclei and histological tissue sections from paraffin blocks are reliable methods for detection of chromosomal anomalies in archival tissue of prostate cancer, although each method has advantages and disadvantages.

Adenocarcinoma↗

PubMed2004

The influence of immunohistochemistry on mRNA recovery from microdissected frozen and formalin-fixed, paraffin-embedded sections.

Laser-assisted microdissection (LAM) is now widely used to obtain specific cell populations from heterogeneous tissues. A major disadvantage of LAM is poor tissue morphology during microscopy, in part because coverslips are not used. Immunohistochemical labeling can improve identification of target cells but may affect the subsequent analysis of the microdissected tissue. We studied the effect of immunohistochemistry (IHC) on mRNA recovery from labeled cells after microdissection from both frozen and formalin-fixed, paraffin-embedded (FFPE) sections, using Melan-A and Ki-67 staining in lymph nodes with metastatic melanoma as a model. We developed rapid protocols for immunostaining in an attempt to limit loss of mRNA during procedures. A sensitive real-time quantitative reverse transcription-PCR was used to measure mRNA. We found a marked decrease in the mRNA yield from 500 microdissected cells from frozen and paraffin sections after immunostaining for both markers. Recovery of mRNA decreased by up to 89%, comparing the immunostained with the routinely stained sections. Interestingly, the ratio between mRNA for the two markers was similar in all stains, indicating that immunostained sections may be used for mRNA analysis. We also investigated the effect of storing membrane-mounted sections for microdissection under different conditions. Slides mounted with paraffin sections could be stored at room temperature for up to 90 days with no significant decrease in mRNA recovery.

Actins↗

PubMed2000

Pitfalls in the immunohistochemical localization of the cystic fibrosis transmembrane conductance regulator in paraffin embedded sweat glands.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the hereditary disease cystic fibrosis. The most frequent mutant deltaF508 has been shown in vitro to be retained in the endoplasmic reticulum. Ex vivo studies using immunohistochemical labelling in cryofixed skin biopsies have confirmed the mislocalization of deltaF508 CFTR in sweat glands. The purpose of this study was to test CFTR antibodies in paraffin-embedded skin biopsies to take advantage of the superior tissue preservation as compared to cryofixation. A panel of 7 CFTR antibodies was applied to skin sections of healthy controls and of cystic fibrosis patients homozygous for the deltaF508 mutation. Sweat gland labelling consistent with CFTR localization and different between control and cystic fibrosis tissue was obtained with 2 antibodies. Conventional staining controls confirmed the labelling specificity. The antibodies were subsequently tested in a series of 237 sections of 16 biopsy specimens. However, the sweat gland labelling pattern proved not to be dependent on CFTR genotype. This finding was the sole indicator of non-specificity of the staining which was revealed only by the size of our random sample. Our results emphasize that CFTR immunolabelling following formalin fixation has to be interpreted with the utmost caution.

Antibodies, Monoclonal↗

PubMed2005

A simple antigen retrieval method for the optimal demonstration of cyclin-D1 overexpression in formalin-fixed paraffin-embedded cases of mantle cell lymphoma.

Successful diagnosis of mantle cell lymphoma (MCL) by immunohistochemistry is largely dependent on the successful demonstration of cyclin D1 overexpression in cases that fit clinical, morphologic, and immunophenotypical profiles. Accurate diagnosis in these cases is important due to the aggressive progression and poor survival times associated with this type of lymphoma. However, demonstration of cyclin D1 by immunohistochemistry on formalin-fixed, paraffin-embedded tissue is often fraught with technical difficulties, chiefly poor staining intensity largely due to insufficient antigen retrieval. The authors report a simple antigen retrieval technique that combines both heat and enzymatic treatment to demonstrate cyclin D1 overexpression. Furthermore, it appears that the antigen retrieval method is the key factor in the demonstration of cyclin D1, as detection systems with differing sensitivities (labeled streptavidin biotin [ABC] and dextran polymeric conjugate [Envision]) fail to demonstrate any significant differences in their staining intensities. This isa simple, cost-effective method that can be performed manually or can easily be adapted to suit automated staining systems.

Antigens↗