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PubMed1991

A monoclonal antibody to human leukocyte common antigen, SHL-1, and its use for formalin-fixed, paraffin-embedded tissues.

The authors describe a newly characterized murine monoclonal antibody to the human leukocyte surface antigen, SHL-1. The antigen belongs to the leukocyte common antigen (LCA) family, and its molecular weight is about 180,000 daltons, which is similar to that of some previously characterized LCAs. The SHL-1 antigen is resistant to conventional tissue-fixation and embedding procedures. This antibody can therefore be used in the immunohistochemical staining of paraffin-embedded tissue sections. Wide screening with a sufficient number of both fresh and routinely processed paraffin-embedded tissues was done with indirect immunoperoxidase technique. With this procedure, SHL-1 labeled the majority of normal leukocytes and hematopoietic malignancies. Some B-cell malignancies were not stained with this antibody. The non-hematologic malignancies posing diagnostic problems of differentiation from lymphomas or leukemias were completely negative to SHL-1. The immunoreactivity to SHL-1 of samples from 24 leukemic patients and 15 human tumor cell lines was determined by the immunofluorescence method. Of 24 leukemic preparations, 23 were strongly reactive to this antibody. One case of B-cell leukemia did not react with SHL-1. No immunoreactivity was demonstrated in non-hematopoietic tumor cell lines. The overall reaction pattern of SHL-1 proved its usefulness in both diagnostic and research practice in hematological disorders. This antibody detected cell surface antigens of the T cell series more effectively than those of the B-cell series in terms of the positive number of cells and mean fluorescence intensity.

Antibodies, Monoclonal↗

PubMed1987

The formaldehyde-fixed and paraffin-embedded tissues for diagnostic transmission electron microscopy: a retrospective and prospective study.

The fine structures of tissues processed routinely for light microscopy were studied retrospectively in 44 tissues and tumors and prospectively in 13 tissues and tumors. In the prospective study, we fixed tissues in an ample amount of fixatives, carefully avoiding crushed and air-dried portions, and processed them by five methods. The fine structures of the retrospective cases were mostly poor or passable, whereas those of the prospective cases were generally good. All formaldehyde-fixed tissues showed varied degrees of tissue extraction, notably of lipids, membranous structures, ribosomes, glycogen, and other loose cellular and intercellular matrix materials, that was related to the status and type of tissue as well as the kind and duration of fixation, dehydration, and tissue clearance. Paraffin embedding per se appeared to cause little alteration of the fine structure. Transmission electron microscopy of the paraffin-embedded tissue appeared most useful to identify infective agents, foreign particles, and densely packed organelles or structures, usually in the differential diagnosis of neoplasms. Although many factors are difficult to control, initial careful thin slicing, judicious selection, and fixation of the tissue (even by regular phosphate-buffered formaldehyde solution) can improve the fine structure of paraffin-embedded tissues and be useful in the direct correlation of light and electron microscopic findings.

Bone Marrow Diseases↗

PubMed1986

Ia antigens in plastic-embedded tissues: a post-embedding immunohistochemical study.

The aim of the present study was to establish a plastic embedding technique that makes possible the immunohistochemical demonstration of class II major histocompatibility complex (MHC) antigens (Ia antigens) in undecalcified joint tissues. Therefore a series of fixatives and dehydrating agents was tested for saving Ia immunoreactivity by post-embedding immunostaining of thin sections (2 microns) of rat tissues that had been embedded in glycol methacrylate (GMA), and by comparing with cryostat sections. An indirect immunoperoxidase and the avidin-biotin complex (ABC) technique were used. Combined with fixation by 4% formaldehyde, dehydration with GMA was found to give the best preservation of Ia antigenicity, followed by dehydration with ethylene glycol. The thinness of tissue sections facilitated the association of Ia antigens with different subcellular compartments in distinct cell populations. These various patterns are described.

Animals↗

PubMed1989

Measurement of S-phase fractions in lymphoid tissue comparing fresh versus paraffin-embedded tissue and 4',6'-diamidino-2 phenylindole dihydrochloride versus propidium iodide staining.

S-phase fractions for 62 lymphoid biopsies were calculated, by means of flow cytometry, from both fresh and paraffin-embedded tissue. The purposes of this study were to determine whether significant differences were seen between S-phase estimates obtained from fresh and fixed tissue and to compare results obtained with two DNA dyes, namely 4'-6'-diamidino-2 phenylindole dihydrochloride (DAPI) and propidium iodide (PI). The 62 cases consisted of 38 cases of non-Hodgkin's lymphoma (NHL), 19 reactive samples, and 5 cases of Hodgkin's disease. Fifty-four of the samples showed DNA diploid profiles. A good agreement between S-phase results from fresh and fixed tissue was seen, with technical factors accounting for around 20% of the total variance. Using a paired t test, no significant difference was seen between fresh and fixed tissue for diploid cases, but there was a trend for S-phase estimates from fixed tissue to be higher. If all cases (including the eight DNA aneuploid samples) were included in the analysis this difference just reached statistical significance (P less than .05). In a subgroup of 19 of the cases, a comparison was performed on both fresh and fixed tissue of staining with DAPI and PI. A good agreement between results with both DNA stains was found on fresh and fixed tissue, with no significant differences being apparent, and stain-related factors accounted for only 10% of the total variance.

Cell Cycle↗

PubMed1997

Immunohistochemical detection of hog cholera viral glycoprotein 55 in paraffin-embedded tissues.

Formalin-fixed, paraffin embedded tissues obtained from 40 pigs inoculated with a field isolate of hog cholera virus were examined for the presence of Gp55, a major structural protein of the virus envelope, using a monoclonal antibody-based immunohistochemical test with the avidin-biotin-peroxidase complex method. Immunoreactivity was detected in hog cholera virus-infected tissues but not in control pigs tissues, African swine fever virus-infected tissues, or bovine viral diarrhea virus-infected porcine or bovine tissues. The first positive reactions were seen in lymphatic tissues, digestive tract and skin on postinoculation day (pid) 4, respiratory and urinary tissues on pid 5, nervous tissues on pid 6, and endocrine tissues on pid 7. These staining reactions persisted until the last observation on pid 18. Hog cholera virus antigen was not detected in heart tissue at any time. The highest levels of antigen detection were found in tonsils, spleen, and pancreas, although the esophageal mucosa and skin epithelial cells were also intensely and widely stained. The cellular staining pattern of Gp55 had a ubiquitous distribution. It was found in epithelial cells, macrophages and circulating monocytes, endothelial cells, lymphoid cells, and glial cells. The results showed a high specificity and high sensitivity for detecting hog cholera Gp55 in formalin-fixed, paraffin embedded tissue samples. This method allows precise association of Gp55 with specific cells, tissues, and histologic lesions, making the technique suitable for use in routine diagnosis of hog cholera.

Animals↗

PubMed2005

An alternative protocol for DNA extraction from formalin fixed and paraffin wax embedded tissue.

BACKGROUND: DNA extraction from paraffin wax embedded tissue requires special protocols, and most described methods report an amplification success rate of 60-80%. AIMS: To propose a simple and inexpensive protocol consisting of xylene/ethanol dewaxing, followed by a kit based extraction. METHOD: Xylene/ethanol dewaxing was followed by a long rehydration step and a kit based DNA extraction step. RESULTS: This method produced a 100% amplification success rate for fragments of 121 to 227 bp for tamponated formalin fixed paraffin wax embedded tissue. CONCLUSION: This cost effective and non-laborious protocol can successfully extract DNA from tamponated formalin fixed paraffin wax embedded tissue and should facilitate the molecular analysis of a large number of archival specimens in retrospective studies.

DNA, Neoplasm↗

PubMed2003

Comparison of methods for DNA extraction from paraffin-embedded tissues and buccal cells.

Both paraffin-embedded tissue specimens and buccal cells are excellent resources for large-scale molecular epidemiological studies. In order to identify the optimal method for DNA extraction, we compared three methods: (1) modified phenol-chloroform protocol; (2) simple boiling method; and (3) DNA Extraction Mini Kit. For paraffin-embedded tissue specimens, amplification of the beta-globin gene sequence was successful in 30 of 34 (88.2%) by the simple boiling method, 29 of 34 (85.3%) samples using DNA extracted by the phenol-chloroform method, and 18 of 34 (52.9%) by the DNA Mini Kit. For buccal cells, amplification of the beta-globin gene sequence was successful in 16 of 17 (94.1%) DNA samples extracted by the phenol-chloroform method, 2 of 16 (12.5%) by the simple boiling method, and 12 of 16 (75%) by the DNA Mini Kit. Both the simple boiling method and the phenol-chloroform method are better methods for DNA isolation from paraffin-embedded tissue specimens, and the phenol-chloroform method is the best method for DNA extraction from buccal cells.

Cheek↗

PubMed2004

Differentiation of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain.

OBJECTIVE: To find a staining method for formalin-fixed, paraffin-embedded tissue that would distinguish bone from surrounding soft tissues, including muscle, periosteal tissue and bone marrow. STUDY DESIGN: A variety of stains were tested and compared with hematoxylin-eosin. The potential value of any given stain was evaluated based on its ability to stain bone and soft tissues different colors or shades that could be readily identified in photomicrographs. Stains were evaluated using both endochondral (tibia) and intramembranous bone (calvaria) samples. RESULTS: In contrast to standard hematoxylin-eosin stain, which stains both bone and soft tissues pink, the methylene blue/acid fuchsin stain demonstrates remarkable contrast between bone and other tissues. Methylene blue/acid fuchsin stained bone bright pink and the surrounding soft tissues blue-purple. CONCLUSION: In addition to the superior staining properties of methylene blue/acid fuchsin, other benefits of this stain include its stability, ease of use and low cost. This stain has many potential applications in the study of erosive bone disease in humans and also in animal models for research.

Animals↗

PubMed1992

p53 overexpression in formalin-fixed, paraffin-embedded tissue detected by immunohistochemistry.

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.

Antibodies, Monoclonal↗

PubMed1987

Utilization of monoclonal antibody L26 in the identification and confirmation of B-cell lymphomas. A sensitive and specific marker applicable to formalin-and B5-fixed, paraffin-embedded tissues.

Immunophenotypic analysis of paraffin-embedded tissues of lymphoproliferative disorders has been facilitated by recent developments of monoclonal antibodies that react with epitopes that survive histologic processing. Leukocyte common antigen (LCA) antibody has made a significant contribution to the immunocytochemical separation of non-Hodgkin's lymphomas from nonlymphoid neoplasms. However, a small percentage of lymphomas, particularly some large cell or immunoblastic B-cell tumors, will not label with LCA antibody. Other antibodies, directed against B lymphocytes, experience problems of specificity and a lack of sensitivity when applied to formalin-fixed specimens. The authors recently investigated a monoclonal antibody (L26) that demonstrates excellent specificity and sensitivity for B lymphocytes, and tumors derived from them, in formalin- and B5-fixed, paraffin-embedded tissue. The avidin-biotin peroxidase complex (ABC) technique was utilized for immunostaining 95 cases of malignant lymphoproliferative disorders and a variety of normal and neoplastic nonlymphoid tissues. When applied to sections of benign lymphoid tissue, the L26 antibody labeled germinal center cells, mantle zone and scattered interfollicular lymphocytes, but not histiocytes or plasma cells. L26 marked 100% (44/44) of the large cell and immunoblastic B-cell lymphomas, along with 1 case of pre-B cell lymphoblastic lymphoma. This included 8 cases that were LCA-negative. None of the T-cell lymphomas or plasma cell tumors studied demonstrated L26 immunostaining. No normal, benign, or neoplastic nonlymphoid tissues examined stained with this antibody. L26 successfully labels B lymphocytes and B-cell lymphomas in routinely processed tissues, often with greater sensitivity and intensity than LCA. This antibody should prove invaluable in the investigation of atypical lymphoid proliferations and the identification of B-cell derived lymphomas, when fresh or frozen tissue is unavailable for analysis.

Antibodies, Monoclonal↗

PubMed1987

Monoclonal antibody L26: an antibody that is reactive with normal and neoplastic B lymphocytes in routinely fixed and paraffin wax embedded tissues.

Formalin fixed and paraffin wax embedded tissue from 85 well characterised cases of non-Hodgkin's lymphoma and Hodgkin's disease were studied using the avidin-biotin-peroxidase complex technique. Among the non-Hodgkin's lymphomas all cases of B cell lymphoma were reactive with L26, a monoclonal antibody which is as yet an unclustered pan B cell reagent, with the exception of pre-B cell acute lymphoblastic leukaemia and malignant lymphoma plasmacytic. Eighteen well characterised cases of T cell lymphoma, selected to include tumours previously shown to exhibit cross reactivity with antibodies to fixation resistant B cell related antigens, were similarly studied. Neoplastic cells in all but one case were unstained by L26. Twenty seven cases of Hodgkin's disease were also examined. In five cases all Reed-Sternberg cells and their variants were strongly stained by L26; only a proportion of Reed-Sternberg cells and their variants were recognised in a further five cases. Monoclonal antibody L26 promises to be a valuable reagent for the diagnosis of malignant lymphoma in routinely fixed and paraffin wax embedded tissues. Its advantage lies in its sensitivity and greater B cell specificity than any of the B cell related reagents currently available for the study of malignant lymphoma in fixed tissues.

Antibodies, Monoclonal↗

PubMed1986

An immunocytochemical study of T-cell lymphomas using monoclonal and polyclonal antibodies effective in routinely fixed wax embedded tissues.

Formalin fixed and paraffin wax embedded tissue from 24 cases of T-cell lymphoma diagnosed using immunocytochemistry on cryostat sections was examined using a panel of eight monoclonal and three polyclonal antisera. The monoclonal antibodies UCHL1 and MT1 proved to be comparable and reliable markers of neoplastic cells in T-cell lymphomas. The B-cell specific marker, MB1, strongly stained all cells in two cases of pleomorphic large cell T-cell lymphoma, large cells in two cases of pleomorphic mixed medium and large cell lymphoma, and isolated clusters of blast cells in four cases of T-zone and angioimmunoblastic lymphadenopathy-like T-cell lymphoma. The cells stained by MB1 expressed T suppressor/cytotoxic surface markers on frozen section. Epithelial membrane antigen, as detected by a polyclonal anti-EMA and the monoclonal antibody HMFG2, was expressed in 36% of tumours especially those of monomorphic large cell and pleomorphic large cell phenotype. Single granules or finely dispersed cytoplasmic granularity was seen in four tumours using the anti-granulocyte reagent Leu M1. Tumour cells in one case stained in a pattern identical to Reed-Sternberg cells in Hodgkin's disease. Granular alpha-1-antitrypsin staining was found in 10 cases of pleomorphic large cell and monomorphic large cell lymphoma. No staining was observed using anti-lysozyme or the monoclonal macrophage specific marker Mac411. Monomorphic and pleomorphic large cell lymphomas tended to show a common immunophenotype with the majority of cells co-expressing alpha-1-antitrypsin HLA-DR and epithelial membrane antigen. Scattered large transformed blast cells in cases of angioimmunoblastic lymphadenopathy-like T-cell lymphomas and T-zone lymphomas shared a similar immunophenotype with the large cell lymphomas. Using a panel of monoclonal antibodies effective in paraffin embedded tissue, diagnostically useful staining profiles which correlate with the morphological phenotype can be established in T-cell lymphomas.

Antibodies, Monoclonal↗

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↗

PubMed1990

Improved flow cytometric determination of proliferative activity (S-phase fraction) from paraffin-embedded tissue.

Recent studies suggest that proliferative activity (S-phase fraction [SPF]) may have greater prognostic significance than total nuclear DNA content; however, relatively few studies have examined SPF from paraffin-embedded tissue because of significant contamination of histograms with debris. In this study, cell cycle analysis was performed on 124 matched tissue specimens. Fresh tissue was divided into two equal portions; one portion was frozen, whereas the other portion was processed and embedded in paraffin. S-phase could be determined for both frozen and paraffin-embedded tissue in 81 cases. Correlation between SPF from frozen and paraffin-embedded tissue was demonstrated (r = 0.80) when debris was subtracted from histograms with the use of two new subtraction algorithms referred to as multicut and singlecut. Unlike other debris-subtraction algorithms, the quantity and distribution of debris calculated by these algorithms are dependent on the magnitude and position of histogram peaks. A lesser degree of correlation was demonstrated with the use of a standard exponential debris subtraction algorithm (r = 0.67). Correlation of SPF for aneuploid cases was greater when SPF was calculated as a percentage of the aneuploid cell population rather than as a percentage of the entire cell population. This was attributed to the observation that the proportion of aneuploid cells from paraffin-embedded tissue was less than that from frozen tissue. The results of this study indicate that SPF can be calculated from paraffin-embedded tissue with values comparable to those obtained from frozen tissue. The ability to calculate SPF reliably from paraffin-embedded tissue should allow additional evaluation of this parameter as a prognostic indicator.

Algorithms↗

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↗

PubMed1998

Mutations in BRCA1 from fixed, paraffin-embedded tissue can be artifacts of preservation.

DNA isolated from paraffin-embedded tissues has been used for analysis of DNA alterations in disease states. Use of archival tissue can expedite the gathering of large numbers of specimens from rare disease subtypes that would take years to accumulate prospectively. Therefore, archival tissues from 70 ovarian cancer cases diagnosed before or at age 40 were retrieved for analysis of BRCA1 mutations. DNA was isolated from paraffin-embedded tissue of 70 ovarian cancer cases diagnosed before or at age 40. BRCA1 mutation analysis was conducted by single-strand conformation polymorphism analysis and DNA sequencing. Fifty-eight BRCA1 mutations were found in 34 of the 70 ovarian cancer cases. Twenty-two cases had one mutation each and 12 cases had multiple mutations. Multiple mutations found in histologically normal tissue of 2 cases were not present in matched tumor tissue. For another case, DNA from two separate blocks of normal tissue contained different mutations. These observations were anomalous and suggested that mutations detected in fixed tissues may be artifacts of tissue preservation and not present in the original unfixed tissues. To test this suggestion, blood was obtained from 2 patients for whom mutations were found in fixed, normal tissue. DNA from their unfixed lymphocytes did not contain the mutations found in fixed normal tissue. Thus, mutations found in fixed, paraffin-embedded tissues can be artifacts of tissue preservation. The reliability of DNA sequence data derived from such tissues must be questioned in the absence of corroborating data from unfixed tissues. This severely limits the use of fixed tissues as a source of DNA for retrospective research and for clinical genetic testing in families for which a disease-affected member is not alive.

Adult↗

PubMed2002

Detection of angiotensin-converting enzyme gene insertion/deletion polymorphism from paraffin-embedded tissues: the Hisayama study.

Previous studies have suggested that archival materials from formalin-fixed paraffin-embedded blocks are unsuitable for most molecular techniques because the extracted DNA can be severely degraded. Therefore, the present study was designed to investigate the accuracy of genotyping for the insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene from paraffin-embedded tissues of autopsy cases from Hisayama Town, Japan. The genotype was determined using the double polymerase chain reaction method and to test the accuracy of the method, the polymorphism was investigated using paraffin-embedded tissues from 18 cases whose ACE genotypes (6 cases for each genotype) were known in advance from analysis of fresh-frozen tissue samples. Genotyping using paraffin-embedded tissues was then determined for 968 autopsy subjects. The genotype could be determined in 16 of the 18 test samples (88.9%) and there was no discrepancy with the results obtained from the fresh-frozen tissues. Of the 968 autopsy cases, the frequency of the DD, ID, and II genotypes was 12.4%, 47.3%, and 40.3%, respectively, a distribution that did not deviate from the Hardy-Weinberg equilibrium (chi(22df )= 0.67, p=0.72). These findings suggest the accuracy of the present method of ACE genotyping from paraffin-embedded tissues.

Autopsy↗

PubMed1987

[Flow cytometric DNA analysis of gynecologic malignant tumors using paraffin-embedded tissue].

We have studied flow cytometric DNA analysis of gynecologic malignant tumors using paraffin-embedded tissue for histopathology. A close correlation was observed between the DNA indices obtained from fresh unfixed tissue and from paraffin-embedded tissue (r = 0.993). The coefficient of variation (CV) for the diploid G0G1 peak of the paraffin-embedded tissue was one and half greater than that obtained from fresh tissue on the average. In the DNA analysis of 64 endometrial carcinomas, DNA aneuploidy was detected in 3% of nuclear grade 1, in 25% of nuclear grade 2 and in 63% of nuclear grade 3. That is, the more abnormal the nuclear findings, the higher the percentage of DNA aneuploidy. In the analysis of 48 ovarian tumors, DNA aneuploidy was detected in 79% of serous adenocarcinoma, in no of mucinous adenocarcinoma, in 17% of endometrioid carcinoma, in all of clear cell carcinoma and dysgerminoma, that is, its incidence was different among histological subtypes.

Aneuploidy↗