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PubMed1994

Immunocytochemical detection of cyclin A and cyclin D in formalin-fixed, paraffin-embedded tissues: novel, pertinent markers of cell proliferation.

Cyclin proteins in association with cyclin-dependent protein kinase subunits represent a new class of potentially oncogenic serine/threonine protein kinases that function to execute critical cell cycle transitions in all eukaryotic cells. Characterized by dramatic fluctuations in abundance, which occur in accordance with the periodicity of the cell cycle, the expression patterns of specific cyclins provide a unique and relevant indicator of cellular activation and cell cycle progression. In this study, we introduce a series of monospecific antibodies that are selective for human cyclin A and cyclin D, respectively, and we assess the feasibility of utilizing these reagents for immunocytochemical analyses. Conditions were optimized for detecting cyclin A and cyclin D in formalin-fixed, paraffin-embedded sections of the postnatal human palatine tonsil, in which normal cell proliferation is well characterized. Subsequent studies demonstrated the performance of these antibodies in the examination of pediatric bone tumors, in which decalcification methods are additionally performed. In both cases, the proliferative status of individual cells was monitored with an exceedingly high degree of resolution. Taken together with the available biochemical data, the results of these studies reveal a novel means of characterizing the proliferative status of normal as well as neoplastic tissues. The demonstrated utility of these immunochemical reagents will potentially facilitate retrospective studies aimed at examining cell proliferation in a wide variety of archival histopathologic specimens.

Antibodies↗
PubMed1989

Morphologic and immunophenotypic characterization of primary brain lymphomas using paraffin-embedded tissue.

Primary lymphomas of the brain constitute about 1% of all primary intracranial neoplasms, but recent studies suggest an increasing incidence. Most cases are associated with an immunosuppressed state. We reviewed 29 cases of primary brain lymphoma from the Yorkshire Health Authority Region between 1970 and 1988 and found a striking increase in incidence over this period. No overt evidence of immunosuppression was found in any case. All were non-Hodgkin's in type and were classified morphologically using Kiel criteria and immunophenotypically using a panel of antibodies. Cryo-preserved tissue was available in five cases for parallel immunophenotyping. The majority of tumours were high-grade lymphomas together with three of lymphoplasmacytoid type. Thirteen tumours showed a striking pleomorphic morphology with plasmacytoid features. A reactive, predominantly perivascular monomorphic T-cell population was seen in all tumours. Most tumours were of B-cell lineage. No cases of Hodgkin's disease, T-cell or histiocytic lymphoma were present. Light chain restriction was present in only 46% of cases. The results of tumour immunophenotyping on cryostat sections were comparable with those from paraffin blocks. Our study emphasizes the value of a panel of antibodies reactive in paraffin-embedded tissue, allowing simultaneous evaluation of morphology and immunophenotype, and suitable for small biopsies received from stereotactic procedures.

Adolescent↗
PubMed1985

Purification and localization of human carbonic anhydrase. III. Typing of skeletal muscle fibers in paraffin embedded sections.

Three different isoenzymes of human carbonic anhydrase are now well characterized. Carbonic anhydrase I and II have been known for several years and are located in high amounts in red blood cells as well as in many other tissues. Carbonic anhydrase III, a protein showing CO2 hydratase and p-nitrophenylphosphatase activity was isolated from skeletal muscle some years ago. Earlier observations based on enzyme activity and radioimmunoassay studies have suggested that this protein is present in greater quantities in red skeletal muscles than in white ones. We have purified CA III from human soleus muscle and using obtained monospecific polyclonal antibody localized this protein in the same muscle fibers which show acid resistant ATPase activity. Using this protein as a marker for type I muscle fibers, fiber classification into type I and II could now be done also from paraffin embedded sections.

Carbonic Anhydrases↗
PubMed1996

Detection of t(11;22)(q24;q12) translocation breakpoint in paraffin-embedded tissue of the Ewing's sarcoma family by nested reverse transcription-polymerase chain reaction.

Tumors of the Ewing's sarcoma family often present a major diagnostic challenge for the pathologist. In recent years, significant progress has been made in identifying characteristic chromosomal rearrangements associated with certain solid tumors. More than 85% of Ewing's sarcoma and related tumors present a specific t(11;22) (q24;q12) balanced translocation, which generates a fusion transcript of the EWS gene and the FLI-1 gene. The cloning of the t(11;22)(q24;q12) breakpoint has raised the possibility of using a reverse transcription-polymerase chain reaction (RT-PCR) based assay as a diagnostic tool. We report an improvement of the established method, which currently depends on fresh or snap-frozen tissue, so that it is possible to use formalin-fixed, paraffin-embedded tissue as a source of RNA. The described nested RT-PCR assay enables the pathologist to investigate retrospectively archival tumor samples or to confirm the diagnosis in cases where no fresh or frozen material is available.

Adult↗
PubMed1989

Detection of human papillomavirus type 16 DNA in cervical swabs and formalin-fixed, paraffin-embedded squamous cell carcinomas of non-genital tissues using a synthetic oligodeoxynucleotide probe.

The potential of using a chemically synthesized oligodeoxynucleotide as a diagnostic probe to detect human papillomavirus type 16 (HPV-16) in genital infections was evaluated by comparing it with a cloned full-length HPV-16 probe in dot-blot DNA hybridizations. An oligonucleotide sequence, 20 bases in length from the E6 region of HPV-16 (E6 oligo) and different from the DNA sequences of HPV types 6, 11 and 18 by at least 2 base pairs, was chosen for chemical synthesis. The oligoprobe, which was 5'-end labelled with [32P]dATP, was found to be specific, but approximately ten times less sensitive than the full-length radiolabelled probe of HPV-16, in dot-blot hybridizations with the DNA of HPV-6, -11, -16 and -18, HPV positive and negative cell-lines. From 36 cervical or vulval scrapes two samples were found positive with both cloned HPV-16 and oligoprobe hybridization. Of 21 samples of formalin-fixed, paraffin-embedded squamous cell carcinomas originating from anus, oesophagus, penis, colon, breast and skin only 4 anal squamous cell carcinomas were positives when hybridized with cloned HPV-16 DNA or with the oligoprobe. This study confirms that HPV-16, which is frequently associated with squamous cell carcinoma of the cervix is also strongly associated with squamous cell carcinoma of the anus.

Autoradiography↗
PubMed1993

Analysis of epidermal growth factor receptor gene expression in stained smears and formalin-fixed, paraffin-embedded cell pellets by reverse transcription intron differential polymerase chain reaction.

Previous studies have demonstrated quantitation of epidermal growth factor receptors (EGFR) to be of prognostic significance in breast, bladder, esophageal and other neoplasms. However, the relatively large quantity of unfixed tissue required for epidermal growth factor radioligand binding assays (RLBA) has precluded its application to cytologic specimens and small biopsy specimens. For this reason we evaluated reverse transcription intron differential polymerase chain reaction (RTIDPCR) as an assay of EGFR gene expression. Squamous cell carcinoma (A431 and SiHa), transitional cell carcinoma (HT1376, T24, RT4), mammary (MCF7) and endocervical (HeLa) adenocarcinoma, and leukemia (K562) cell lines were used to compare RTIDPCR and RLBA. RTIDPCR involved reverse transcription of RNA and amplification of cDNA using primers for beta-actin and EGFR. Good agreement was observed between the RLBA and RTIDPCR results. RNA extracted from fresh cells, Diff-Quik-stained smears and formalin-fixed, paraffin-embedded cell pellet sections yielded similar results. These data suggest that RTIDPCR may be useful in evaluating gene expression by cells processed as cytologic specimens.

Adenocarcinoma↗
PubMed1986

In situ hybridization for the detection of cytomegalovirus (CMV) infection. Application of biotinylated CMV-DNA probes on paraffin-embedded specimens.

Five autopsy cases of cytomegalovirus (CMV) infections were studied. Conventional light microscopy disclosed characteristic cytopathic effects in lungs, kidneys, and brain. In one case, electron microscopy was carried out and revealed typical herpesvirus particles. In situ hybridization was done with biotin-labeled CMV-DNA probes and an avidin-alkaline phosphatase detection system. 4/5 cases were observed to contain hybridizing cells in different organs. Intensity of hybridization was related to the severity of CMV infection, roughly estimated by counting cytomegalic cells. In addition to cytomegalic cells, a high number of normal-looking epithelial and mesenchymal cell types were positive. These latter cells showed nuclear hybridizations in contrast to cytomegalic cells which hybridized both within the nuclei and the cell bodies. This modified in situ hybridization procedure is a rapid and valuable tool for the detection and final demonstration of virus infection, and will be of particular help for the examination of paraffin-embedded specimens.

Adult↗
PubMed1996

In situ detection of individual transplanted bone marrow cells using FISH on sections of paraffin-embedded whole murine femurs.

Studies of transplantation biology rely on the detection of donor hemopoietic cells in transplant recipients. Traditionally this has been achieved through ex vivo techniques, including flow cytometric analysis of cell surface markers to detect cells expressing specific epitopes, histochemical detection of cytoplasmic proteins, and the detection of Y chromosome-specific sequences by DNA hybridization. Studies using congenic models, such as the Ly5.1/5.2 mouse, or the utilization of fluorescent dyes, such as PKH-26, have allowed more in-depth analysis of transplantation, beginning to address key issues such as cell homing through cell tracking and elucidation of the "stem cell niche." However, these methods are limited by labeling sensitivity, specificity, crossreactivity and, in the case of PKH-26 labeling, the number of cell divisions the transplanted cells can make before the signal disappears. We have developed a fluorescent in situ hybridization (FISH) technique that utilizes a murine Y chromosome-specific "painting" probe to identify in situ individual transplanted male cells in paraffin-embedded sections of female whole bone marrow while maintaining good morphological integrity. This method is highly sensitive and specific, labeling more than 99% of male cells and no female cells, allowing each transplant to be assessed at the individual cell level. The technique provides unique opportunities to follow the path taken by transplanted cells, both during homing into the marrow and through their maturation and differentiation into mature, functional hemopoietic cells.

Animals↗
PubMed1982

Demonstration of AA-protein in formalin-fixed, paraffin-embedded tissues.

AA-protein was identified by SDS-acrylamide electrophoresis in amyloid fibrils fixed in formalin after isolation from fresh-frozen tissues obtained from patients with familial Mediterranean fever (FMF) amyloidosis and idiopathic AA-amyloidosis and, following deparaffination, rehydration and homogenization of embedded formalin-fixed tissues of old autopsy cases of the hereditary amyloidosis of FMF and amyloidosis acquired in association with tuberculosis, bronchiectasis, and rheumatoid arthritis. That AA-protein is unaltered by formalin was firmly established by agar gel diffusion using specific rabbit anti-AA serum. By contrast, AL proteins could not be demonstrated either in formalin-fixed amyloid fibrils derived from fresh-frozen tissues of a patient with presumably AL-amyloidosis dominated by cardiomegaly and one with AL-kappa amyloidosis or in blocks of cases of familial neuropathic amyloidosis, multiple myeloma, and idiopathic amyloidosis with cardiopathy. AA-protein is not denatured by formalin and retains its typical electrophoretic, chromatographic, and immunologic characteristics even 30 years after fixation and paraffin-embedding.

Amyloid↗
PubMed1996

[The determination of monoclonality in B-cell lymphomas using the polymerase chain reaction technic on paraffin-embedded material].

BACKGROUND: Clonal rearrangements of immunoglobulin are useful in the diagnosis of B-cell lymphomas. The value of the polymerase chain reaction (PCR) with consesuated primers in the variable (V) and joining (J) region is now well established. METHODS: Forty cases of B-cell lymphomas and 10 patients with reactive lymphoid proliferations have been studied. All cases were studied with two different semiacotated techniques, with similar primers in the J region (LJH and VLJH) and changing the primers of the V region (Fr2A and Fr3A). RESULTS: A 100% specificity and positive predictive value were obtained with both techniques. The sensitivity was 90% with Fr2A and 82% with Fr3A, but in 20% of the studies with Fr2A the extracted DNA was insufficient to perform the study while it could be done with Fr3A in all cases. The negative predictive value was 76% with Fr2A and 58% with Fr3A. CONCLUSIONS: Both techniques showed a good sensitivity, optimal specificity and positive predictive value, with low negative predictive value. Although the results are somewhat higher with Fr2A, this technique needs well preserved DNA and it can be an important set back when we deal with formaldehyde fixed, paraffin embedded tissues.

Base Sequence↗
PubMed2006

In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts.

BACKGROUND: Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. METHODS: We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. RESULTS: In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. CONCLUSION: We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.

Animals↗
PubMed2000

Detection of Streptococcus suis by in situ hybridization, indirect immunofluorescence, and peroxidase-antiperoxidase assays in formalin-fixed, paraffin-embedded tissue sections from pigs.

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1-31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.

Animals↗
PubMed1990

Monocytoid B-cell lymphoma in a patient with human immunodeficiency virus infection. Demonstration of human immunodeficiency virus sequences in paraffin-embedded lymph node sections by polymerase chain reaction amplification.

There is a significantly increased incidence of malignant lymphoma in patients with acquired immunodeficiency syndrome (AIDS). The lymphomas are usually of a high grade and of B-cell phenotype. While the frequent presence of reactive monocytoid B lymphocytes in patients with AIDS-related lymphadenopathy has recently been documented in several studies, to our knowledge, there are no reported cases of monocytoid B-cell lymphoma, the neoplastic counterpart of monocytoid B lymphocytes, in patients with AIDS. We now describe a human immunodeficiency virus (HIV)-positive patient with HIV-related lymphadenopathy in whom monocytoid B-cell lymphoma developed during the course of his disease. The morphologic and immunologic features of the lymphoma were characteristic of monocytoid B-cell lymphoma, and the involved lymph node exhibited a reversed CD4/CD8 ratio. Moreover, using the polymerase chain reaction, we were able to demonstrate HIV genome in DNA extracted from the lymph node tissue. To our knowledge, this is the first report of a case of monocytoid B-cell lymphoma occurring in an HIV-positive patient and in which we were able, by using a sensitive molecular biologic technique, to demonstrate HIV sequence in paraffin-embedded, fixed lymph node sections.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Immunophenotyping of hematologic neoplasms in paraffin-embedded tissue sections.

Several immunohistochemical methods are now available for the staining of neoplastic cells in tissue sections. The authors have found that the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method is sensitive and reliable. Murine monoclonal or nonmurine polyclonal antibodies can be used to label a variety of membranous and/or cellular constituents in tissues that have been routinely processed in a histopathology laboratory. The monoclonal antibody against leukocyte common antigen (CD45) can be used to differentiate hematologic from nonhematologic tumors. Monoclonal antibodies (L26, LN1, LN2, LN3, MB1, MB2) label B-cell lymphomas, whereas other monoclonal antibodies (UCHL1, MT1) more characteristically stain T-cell lymphomas. Polyclonal antibodies against CD3 specifically mark neoplastic cells from T-cell lymphomas and leukemias but as yet are not commercially available. Monoclonal antibodies Leu-M1 (CD15), Ber H2 (Ki-1; CD30), and LN2 label Reed-Sternberg cells from most cases of nodular sclerosis, mixed cellularity, and lymphocyte-depleted Hodgkin's disease. Monoclonal antibodies Mac 387, KP1 (CD68), and NP57 (antielastase), as well as polyclonal antibodies against lysozyme, help identify subtypes of acute myeloid leukemia and extramedullary myeloid cell tumors. Although there are now excellent reagents ready for use, there is still a significant need for more lineage-specific (particularly against CD epitopes) monoclonal antibodies capable of labeling neoplastic cells in paraffin-embedded tissue sections from patients with hematologic malignancies.

Acute Disease↗
PubMed2003

PCR-based detection of canine Leishmania infections in formalin-fixed and paraffin-embedded skin biopsies: elaboration of a protocol for quality assessment of the diagnostic amplification reaction.

Diagnosis of the cutaneous form of canine leishmaniosis is mostly performed by histological or immunohistological examination of skin biopsies. In modern histology, the polymerase chain reaction (PCR) has gained increasing importance as a complementary tool to directly demonstrate the presence of parasite DNA in the tissue sections. For the present study, a previously described Leishmania-PCR has been further developed and optimised in view of its practicability for routine histological application. Since formalin-fixation of histological specimens causes partial DNA-destruction, which may hamper diagnostic PCR analysis, primers specific for the highly conserved alpha-actin gene sequences were used to pre-diagnostically assess the isolated sample-DNA for its functionality in a PCR-reaction. This alpha-actin-specific PCR detects DNA from a large variety of mammalian species and thus exhibits relevance for both human and veterinary medical application. A recombinant internal positive control was introduced to monitor possible sample-related inhibitory effects during the amplification reaction. We performed a retrospective evaluative study with 18 formalin-fixed samples from dogs with suspected or proven leishmaniosis. Six samples were PCR-incompatible. In turn, 9 of the other 12 samples were PCR-positive, and immunohistochemical results matched these findings. Based on these technical achievements, the Leishmania-PCR proved to be a valuable tool to complement conventional histological and immunohistological methods for diagnosis of cutaneous leishmaniosis in formalin-fixed, paraffin-embedded skin biopsies.

Actins↗
PubMed1997

Immunohistochemical detection of nucleolar protein p120 in paraffin-embedded tissues.

The monoclonal antibody FB-2 recognizes the antigen p120-kDa protein (p120), associated with the nucleolar matrix. p120 has originally been reported as expressed and detectable in malignant and non-neoplastic proliferating cells, but not in most normal resting tissues and benign tumours. In the present study, a reliable immunostaining method was used to detect p120 on formalin-fixed, paraffin wax-embedded tissue, testing it on 148 samples from different neoplastic and non-neoplastic tissues from different organs (breast, colon, lung, prostate, bladder, lymph nodes, skin, tongue and liver). The immunostaining was performed after the application of a specific antigen-unmasking protocol based on six consecutive cycles of microwave oven heating. Under these retrieval conditions, p120 antigen was clearly detectable, not only in hyperplastic and malignant cells, but also in stromal and normal non-proliferating cells of all the tissues evaluated. Our results show that the nucleolar protein p120 can be detected by routine immunohistochemistry in formalin-fixed, paraffin-embedded tissue and is expressed in all nucleated cells under any biological condition.

Antigens, Neoplasm↗
PubMed1983

A sample-grouping technique for paraffin embedments.

A technique is described which facilitates histological preparation of multiple tissue specimens for light microscopy. The procedure enables the investigator to separate and label identifiable subgroups from a larger number of specimens in one histological section. After standard fixation, murine esophagi were arranged longitudinally and secured within segments of murine intestine. Markers such as plant fibers and human hairs were threaded alongside the esophagi within each intestinal casing. After standard dehydration and infiltration, several segments of intestine were arranged parallel to each other and at right angles to the intended plane of sectioning and were embedded together in one paraffin block. This method made it possible to assemble onto one microscope slide cross sections of 42 individual esophagi in 6 identifiable subgroups, each containing 7 esophagi.

Animals↗
PubMed2002

Detection of hepatitis B virus antigens in paraffin-embedded liver specimens from the Amazon region, Brazil.

Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in difFerent other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4%) histological samples were HBsAg reactive and 5 (6.3%) were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens.

Adolescent↗