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PubMed1994

Detection of cytomegalovirus genome by in situ hybridization in paraffin embedded endomyocardial biopsy specimens of viral myocarditis.

Cytomegalovirus (CMV) genes were detected by in situ hybridization in 25 Chinese patients with viral myocarditis (VMC). The positive hybridization signals were found in cardiomyocytes (6 cases, 24%), capillary endothelial cells (4 cases, 16%) and interstitial cells (7 cases, 28%). The difference between VMC and control group (16 cases died of brain trauma and 10 cases of congenital heart diseases was statistically significant. There was no definite pathomorphological relationship between the detection of CMV genes and myocardial lesions. The results suggest that CMV infection may be one of the causes of myocarditis and chronic stimulation of the immune system induced by CMV may be a possible pathogenesis of this disease.

Adolescent↗
PubMed2000

PCR analysis of IgH-gene rearrangements in small lymphoid infiltrates microdissected from sections of paraffin-embedded bone marrow biopsy specimens.

The differentiation of benign lymphoid infiltrates from nodular infiltrates of B-cell lymphoma is difficult in bone marrow (BM) biopsy specimens taken from patients with non-Hodgkin's lymphoma (NHL). We investigated whether the determination of clonality by polymerase chain reaction (PCR) analysis of the immunoglobulin heavy chain (IgH) genes could be of help for the distinction of benign and malignant lymphoid infiltrates. BM biopsy specimens of 28 patients were studied, comparing PCR of entire bone marrow sections with microdissected nodular lymphoid infiltrates. Patients were divided into 4 groups according to morphologic criteria: group 1 (n = 12), positive for B-NHL infiltration; group 2 (n = 5), suspicious for infiltration by known B-NHL; group 3 (n = 5), morphologically benign infiltrates in patients with B-NHL; group 4 (n = 6), benign lymphoid infiltrates in patients without history of B-NHL. PCR products were analyzed using polyacrylamide gels and a fragment length analysis system (Genescan). PCR of whole sections showed clonal amplification products in all cases of group 1 and 1 case of group 2. PCR analysis from microdissected nodular infiltrates showed the presence of a clonal B-cell population in 5 additional cases of groups 2 and 4. In 3 of these cases, clonal rearrangements of corresponding size were obtained from the primary lymphoma biopsy specimens. None of the cases of group 3 showed evidence of a clonal population with either technique. The results indicate that microdissection of small nodular lymphoid infiltrates from paraffin-BM sections increases the sensitivity of IgH gene rearrangement analysis. To avoid detection of biologically irrelevant clonal populations, comparison of PCR products obtained from the BM and the primary lymphoma biopsy is advisable.

Biopsy↗
PubMed2002

Polymerase chain reaction in cutaneous tuberculosis: is it a reliable diagnostic method in paraffin-embedded tissues?

BACKGROUND: Most cutaneous tuberculosis lesions contain few bacilli, so identification of Mycobacterium tuberculosis in conventional laboratory tests is difficult. In vitro amplification of specific DNA sequences using polymerase chain reaction (PCR) has become a valuable tool in the rapid detection of slow-growing organisms like M. tuberculosis. AIM: To investigate the presence of M. tuberculosis DNA in cutaneous tuberculosis. METHODS: Twenty-two archival biopsy specimens diagnosed as cutaneous tuberculosis were investigated for the presence of M. tuberculosis DNA by PCR. Normal skin samples of 29 healthy patients were used as a control. RESULTS: Amplification of the M. tuberculosis DNA was observed in one of the cutaneous tuberculosis specimens and in a healthy control. CONCLUSION: Although PCR is a rapid diagnostic method, fixation procedures may decrease its sensitivity.

Adolescent↗
PubMed1996

[A study on methodology and the criteria for positive immunohistostaining of estrogen and progesterone receptors in paraffin embedded sections of breast cancer].

The expression of estrogen receptor (ER) and progesterone receptor (PR) in 200 breast cancer specimens were imvestigated by monoclonal antibody with S-P staining to evaluate and standardize the criteria for positive staining. The results revealed that the incidence of ER and PR were 73.5% and 65.5% respectively. The authors concluded that the immunostaining could demonstrate in situ ER and PR, the results were stable, highly sensitive, consistent and comparable with the well established and recognized techniques home and abroad. The authors further pointed out that the crucial point of the staining is antigen retrieval and reported the optimal temperature and the time of sodium citrate buffer treatment. In this report, the criteria for positive staining were discussed intensively. The observations indicated that the percentage of the positive cells is more reliable, representative and practical than that of positive staining intensity index.

Breast Neoplasms↗
PubMed1997

The combination of in situ hybridization and immunohistochemical analysis: an evaluation of Her2/neu expression in paraffin-embedded breast carcinomas and adjacent normal-appearing breast epithelium.

The combination of in situ hybridization and immunohistochemical techniques can successfully identify viral DNA/RNA in specific subsets of cellular populations. We recently modified this method to evaluate amplification of the oncogene Her2/neu and overexpression of its protein c-erbB-2 in a series of 15 breast carcinomas. This combination allows the simultaneous evaluation of the oncogene and its corresponding protein expression in single cells and specific cellular populations in histologic tissue sections. Double staining demonstrated heterogeneity within breast carcinomas. In addition, both nuclear and cytoplasmic signals were often detected in morphologically normal-appearing adjacent breast epithelium. The ability to view both the oncogene and its corresponding protein in single cells offers a unique look at the biology of c-erbB-2.

Breast↗
PubMed1990

Effect of sonication on paraffin-embedded tissue preparation for DNA flow cytometry.

Since the publication of paraffin block extraction procedures, flow cytometric analysis of DNA ploidy and S-phase of tumor specimens has been widely applied. DNA aneuploidy, DNA tetraploid (elevated G2/M), and elevated S-phase are clinically significant in some tumor systems. True DNA tetraploid cell lines will contain a large 4c population and perhaps an 8c population; samples with cell aggregates will also contain a 6c population. Microscopic examination of samples having a 6c peak revealed nuclei with adhering debris and doublets, triplets, and larger nuclear aggregates. After sonication, a uniform suspension of single nuclei without adherent debris was seen. In addition to reducing the percent of G2/M cells, sonication also reduced S-phase percent such that it was closer to the bromodeoxyuridine labeling index. The DNA ploidy classification of specimens was also compared pre- and post-sonication. Four of 96 breast cancer samples changed classification; all were specimens in which the histogram became cleaner and a small DNA aneuploid peak became apparent after sonication.

Breast Neoplasms↗
PubMed1993

Differentiation of reactive from neoplastic small-cell lymphoid aggregates in paraffin-embedded marrow particle preparations using L-26 (CD20) and UCHL-1 (CD45RO) monoclonal antibodies.

The pathologic findings in 1,390 consecutive patients who had bone marrow examinations at Nashville Veterans Administration Hospital from 1977 through 1979 were reviewed. Seventy-three patients who did not meet diagnostic criteria for a small-cell lymphoid neoplasm (SCLN) and 11 patients with SCLN had, on marrow particle preparations: (1) at least three lymphoid aggregates and (2) suitable material available for immunoperoxidase studies with monoclonal antibodies UCHL-1 (CD45RO, pan T cell) and L-26 (CD20, pan B cell). Staining with UCHL-1 was difficult to interpret due to high background positivity in myeloid elements. With L-26, three distinct patterns of lymphocyte marking were identified within aggregates: (1) homogeneous--uniform marking of almost all lymphocytes; (2) mixed--even distribution of marking and nonmarking lymphocytes; (3) and focal homogeneous--collections of uniformly marking lymphocytes either surrounding or surrounded by nonmarking lymphocytes. A homogeneous marking pattern was the predominant pattern in 8 of 11 patients (73%) with SCLN. Only 6 of 73 patients without overt SCLN marked in a homogeneous pattern, and these were always associated with aggregates with other staining patterns. All patients with apparently non-neoplastic lymphoid infiltrates had mixed (67 of 73) or focal homogeneous (32 of 73) patterns of aggregate marking, whereas only 5 of 11 patients (45%) with extramarrow SCLN had aggregates with these patterns. The size and number of aggregates with a homogeneous marking pattern further helped discriminate between the patients with SCLN and those with apparently non-neoplastic lymphoid aggregates. These findings suggest that a homogeneous pattern of lymphoid aggregate staining with L-26 is more common in patients with SCLN than in those patients with lymphoid aggregates and no evidence of neoplasia. Paraffin immunoperoxidase staining with L-26 may be a helpful adjunct to histopathologic examination in evaluating marrow lymphoid aggregates.

Antibodies, Monoclonal↗
PubMed1996

Characterization of novel monoclonal antibodies raised against formalin-fixed, paraffin-embedded human ameloblastoma.

To obtain monoclonal antibodies reactive with odontogenic but not other types of epithelium, mice were immunized with homogenates of fixed ameloblastoma tissues, and monoclonal antibodies Y4 and M11 were produced. Y4 reacted immunohistochemically with odontogenic epithelial components but not with those of squamous differentiation, while M11 reacted with odontogenic epithelial components and a part of keratotic epithelial tissues. Immunoglobulin isotypes of both antibodies were IgM as determined by Ouchterlony immunodiffusion and enzyme-linked immunosorbent assays. Western blotting revealed that the antigen recognized by Y4 had a molecular mass of approximately 66 kDa; however, the antigen reactive with M11 was not identified by Western blotting in spite of various attempts in changing reaction conditions. These antibodies may be beneficial to histological analyses of odontogenic tissues and their related lesions.

Ameloblastoma↗
PubMed2004

The expression of CD44 in archival paraffin embedded interface tissues of failed orthopaedic implants.

CD44 hyaluronan receptor is present on large number of different cell type. It acts as one of the adhesion proteins, binding to hyaluronan and is known to play a part in cell migration from vessels in inflammation. The aim of this study was to examine the presence and distribution of CD44 in interface membrane in aseptic loosening. Immunohistochemistry (IHC) using human anti-mouse CD44 antibody studied 20 aseptically loosened interface samples. Extracted protein from all cases was examined by Western blot and RT-PCR. CD44 was detected in 85% of interfaces by IHC and the presence of protein confirmed by blotting and RT-PCR, which showed the mRNA level for CD44. CD44 was expressed by macrophage, multinucleated giant cells, mast cells and lymphocytes. Further studies are needed to characterise the role of this molecule in the inflammatory response to wear debris in aseptic loosening.

Aged↗
PubMed1992

Immunohistochemical and histochemical profile of Aschoff bodies in rheumatic carditis in excised left atrial appendages: an immunoperoxidase study in fresh and paraffin-embedded tissue.

We have evaluated the nature of Aschoff cells within Aschoff bodies seen in 35 of 100 excised left atrial appendages from cases of rheumatic mitral stenosis who underwent closed mitral valvotomy. These were tested using a panel of monoclonal and polyclonal antisera by the indirect immunoperoxidase staining for leucocyte common antigen, macrophage, desmin, vimentin, alpha-1-antitrypsin, alpha-1-antichymotrypsin, lysozyme, acid phosphatase and nonspecific esterase. The Aschoff cell gave strong reactivity with monoclonal antisera to vimentin, macrophage and variable reaction with polyclonal antisera known to recognise macrophages/histiocytes in tissues, namely alpha-1-antitrypsin, alpha-1-antichymotrypsin and lysozyme. These were also strongly positive for acid phosphatase and nonspecific esterase. The Aschoff cell lacked affinity for desmin and only an occasional cell in 4 out of 20 and 6 out of 35 cases showed a weak reaction with myoglobin and leucocyte common antigen, respectively. Intense consistent reactivity with several histiocytic markers affirms the genesis of these cells from macrophages/histiocytes and not muscle cells; a controversy which must be laid to rest!

Antibodies, Monoclonal↗
PubMed1994

Detection of proliferating cell nuclear antigen in paraffin-embedded specimens is dependent on preembedding tissue handling and fixation.

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA), a cell cycle-related protein used to estimate tumor growth fraction, is variable in formalin-fixed compared with methanol-fixed tissue specimens. This is assumed to result from conformational changes in the antigenic epitope induced by formaldehyde; therefore, to be susceptible to retrieval in archival specimens. In this study, formalin fixation reduced the intensity of staining and the number of positive cells to approximately 25% of those in methanol-fixed material. The washing of tissue specimens prior to methacarn fixation also reduced PCNA staining. Loss of staining was not restored after use of a commercial retrieval kit recommended for PCNA immunohistochemistry. Immunoblotting of formalin fixatives and saline washings after removal of tissue specimens consistently demonstrated the presence of PCNA-like activity in solution. We conclude that the exceptional solubility of PCNA is responsible for reduced immunostaining in formalin-fixed material, that the loss is irreversible, and that methanol or methacarn is the fixative of choice for PCNA immunohistochemistry.

Animals↗
PubMedDate not supplied

Investigation of Epstein-Barr virus DNA in formalin-fixed and paraffin- embedded breast cancer tissues.

OBJECTIVE: To investigate etiological role of Epstein-Barr virus (EBV) DNA in breast cancer. MATERIALS AND METHODS: The presence of EBV DNA in 57 breast cancer tissues was investigated with a sensitive PCR assay. The breast cancer tissues were from invasive ductular (n=28), lobular (n=20) and other miscellaneous carcinomas (n=9). Tissues from normal breasts and patients with various benign breast diseases (n=55): fibrocystic disease (n=34), fibroadenoma (n=16), hyperplasia, and granulomatous mastitis (n=5), were used as control samples. RESULTS: EBV DNA was detected in 13 (23%) cancerous tissues (7 ductular, 4 lobular, 2 other carcinoma) and 19 (35%) in the control tissues. The difference between EBV presence in malignant and benign tissues was not statistically significant (p>0.05). CONCLUSION: The presence of EBV DNA was detected almost equally in both breast cancer and normal tissues, which indicates no etiological role for EBV in breast cancer. We suggest further etiological studies.

Adolescent↗
PubMedDate not supplied

Immunohistochemical detection of C-100 hepatitis C virus antigen in formaldehyde-fixed paraffin-embedded liver tissue. Correlation with serum, tissue and in situ RT-PCR results.

We localized HCV C-100 protein in liver biopsies of 15 patients with chronic hepatitis C using immunohistochemistry. The results were compared to serum, tissue extract analysis of HCV RNA and in situ RT-PCR described in a previous study. HCV was detected in 80% of the sera tested, in 40% of the tissue extracts and in 80% and 60% of the tissue sections tested by immunohistochemistry and in situ RT-PCR respectively. Compared to the serum positive cases, 83% and 67% of the cases were respectively positive with immunohistochemistry and in situ RT-PCR and 41% were positive with tissue extract detection. Compared to the tissue extract positive cases, 25% and 50% of the cases were respectively positive with immunohistochemistry and in situ RT-PCR. Finally, 75% of the cases positive by immunohistochemistry were also positive by in situ RT-PCR. These results underline the complementarity of the different methods for the precise diagnosis of hepatitis C.

Formaldehyde↗
PubMed1993

A new proliferation-associated nuclear antigen detectable in paraffin-embedded tissues by the monoclonal antibody Ki-S1.

A monoclonal antibody (Ki-S1) has been raised that reacts with the nuclei of proliferating cells. The antigen recognized is resistant to formalin fixation and can be detected in frozen tissues as well as in routinely processed specimens. In immunohistochemistry, nuclear staining can be seen in those tissues and cellular compartments known to be actively proliferating. Peripheral blood lymphocytes are negative but show a strong increase in antigen expression after mitogen stimulation. Flow cytometric determination of DNA content and antigen expression revealed negativity of G0 cells and positivity of G1 to G2/M cells. A cytoplasmic co-reactivity, not associated with proliferation, was confined to Langerhans islands of the pancreas. The nuclear localized antigen has a molecular mass of 160 kd and therefore seems to be different from all other known immunohistochemical markers of proliferating cells. We conclude that the monoclonal antibody Ki-S1 might provide a useful tool for studying cell proliferation in situ under normal and pathological circumstances.

Animals↗
PubMed2003

Estrogen and progesterone receptor assay in paraffin-embedded breast cancer--reproducibility of assessment.

Immunohistochemical (IHC) assays are routinely used for determining the estrogen receptor (ER) and progesterone receptor (PgR) status of women with breast cancer. The present knowledge of reproducibility of the subjective IHC assessment is limited. The purpose of this study was to evaluate the interobserver reproducibility in IHC assessment of 127 (ER) and 126 (PgR) cases of primary breast cancer. The slides were independently re-assessed by 22 pathologists from nine hospitals. A simple assessment protocol was used with a cut-off value > 10% stained nuclei, regardless of staining intensity. The concordance was high, with a kappa value of 0.78 for ER and 0.72 for PgR. Since some of the 22 pathologists considered one or several sections to be inevaluable, the evaluation comprised three groups (< or = 10% vs. > 10% vs. not evaluated). Although the percentage of positive cases varied among the 22 pathologists (ER: 57%-79%; PgR: 41%-66%), the overall concordance was good for both ER and PgR (kappa = 0.78 and 0.72, respectively). A further improvement in concordance should be possible to achieve through training.

Breast Neoplasms↗
PubMed1994

Fluorescence in situ hybridization on paraffin-embedded abortion material as a means of retrospective chromosome analysis.

A fluorescence in situ hybridization (FISH) procedure was used to detect chromosome abnormalities in archival abortion material. Nuclei were isolated from 50-microns-thick tissue blocks from 18 selected and karyotyped abortions. Five probes for repetitive centromeric sequences of chromosomes 1, 16, 18, X and Y were used. For each chromosome, at least 200 nuclei were scored blindly, i.e. without knowledge of the karyotype. The FISH results obtained were compatible with the cytogenetic data in 14 cases. There were four discrepancies. Two of these were observed for cases karyotyped as trisomy 16. Furthermore, FISH results showed trisomy 18 in two cases having normal chromosomes 18 and 18q+, respectively. The latter case was not discrepant if the structural rearrangement involved chromosome 18 material. The remaining discrepancies could be explained by chromosomal mosaicism. Admixture of normal maternal cells was also noted. It is concluded that FISH can be used to study retrospectively the presence of chromosome abnormalities in abortion material. However, the quality obtained after the use of fresh material is superior.

Abortion, Spontaneous↗
PubMed1996

The influence of salts on the reaction between hGH and several of its monoclonal antibodies in fixed, paraffin-embedded human pituitary as a model system.

A study of the influence of three salts of monovalent cations (Nacl, KCl, and CsCl) and three salts of divalent cations (MgCl2.6H2O, anhydrous CaCl2, and SrCl2.6H2O), on the specific reaction between human hGH and three of its monoclonal antibodies has been carried out, using human pituitary glands obtained at autopsy as a model system. This study is an attempt to find out the physico-chemical laws ruling the immunohistochemical reaction in the special circumstance of fixed specimens, where the antigen (Ag) is immobilized in the tissue substrate, while the antibody (Ab) is mobile. Our interpretation of the results points to a reaction of the salts with side groups of aminoacids in the paratope (specific saline effect) and a mass salt effect on the reaction kinetic proper that is dependent on the salt concentration. The high affinity coefficient of the in situ Ag-Ab reaction is shown, at the immunohistochemical level, by the high molarity necessary to make the reaction negative. Also, it seems there is a direct relationship between the critical molarity values obtained and the affinities of the monoclonal antibodies used in this study.

Antibodies, Monoclonal↗
PubMed1999

Immunohistochemical detection of hepatitis C virus antigen in paraffin embedded liver biopsies from patients with chronic liver disease.

A simple and reproducible immunohistochemical (IHC) staining method that adequately identifies and localizes hepatitis C virus (HCV) in human livers is still not available. We performed IHC staining using both a new monoclonal antibody against HCV and a polyclonal human anti-HCV IgG to 94 liver biopsies from HCV seropositive patients with chronic hepatitis and 15 control liver biopsies. Positive nuclear staining was consistently observed predominantly in hepatocytes and much less in lymphocytes with either antibody in 57% of anti-HCV seropositive cases but in none of the controls. However, the monoclonal antibody yielded a stronger positive reaction and in a greater proportion of hepatocytes. In 78% of the positive cases, more than a quarter of the hepatocytes showed nuclear staining. The degree of hepatic HCV load as revealed by intensity and extent of positive staining did not correlate with histological changes in the liver. The new monoclonal antibody against HCV appeared to be suitable for identifying HCV in tissues by a simple IHC stain and can be used to explore the pathogenesis of liver injury induced by this virus.

Antibodies, Monoclonal↗