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PubMed1994

[DNA ploidy and prognosis of cardia and stomach carcinomas. A comparative flow and image cytometric analysis pf paraffin-embedded tumor sections].

The prognostic relevance of DNA stem line ploidy was ascertained by comparative DNA analysis performed by flow cytometry (FCM) and by Feulgen image cytometry (ICM) of histologic slides on paraffin-embedded resection material from 221 carcinomas of the cardia and stomach. While flow cytometric detection of DNA stem line aneuploidy was rather insecure in smaller or diffuse carcinomas, the methodological restrictions of ICM were seen in a reduced measuring accuracy varying from one case to the other. This resulted in a lower security in distinguishing mere peridiploidy from real hyperdiploid DNA aneuploidy. Despite the difference of methodology, the results were principally concurring. The assessment of DNA stem line ploidy offered no appreciable information about the prognosis of Lauren's diffuse carcinomas. For intestinal carcinomas, however, there was a significant positive correlation between DNA stem line aneuploidy and the evidence of regional nodal metastases, which might explain to a considerable extent the significantly unfavorable clinical course of tumor cases with recorded DNA aneuploidy. The correlation between DNA stem line ploidy, nodal status, and clinical outcome was distinctly less strong in intestinal carcinomas of the cardia than in those of the stomach, because nodal metastatic spread of cardia carcinomas is strongly influenced by local conditions of lymph drainage and the special mechanical strain.

Aneuploidy↗
PubMed1978

A method for the use of immunofluorescence on paraffin-embedded tissues.

Immunofluorescence is increasingly used for the visualization of antigens and antibodies in various tissues. The commonly used frozen sections present several disadvantages, which can be avoided by the present adaptation of paraffin-embedded sections for immunofluorescence. In this method, Bouin's solution achieves fast, deep fixation which preserves well the capacity of antigens to react with specific FITC-labeled antibodies. The method gives superior resolution of morphology, lower nonspecific fluorescent background, and permanent sections suitable for indefinite storage and retrospective studies. It has been successfully applied thus far to the study of immunofluorescence of lymphoid, thyroid, renal, pulmonary, and intestinal tissues.

Fluorescent Antibody Technique↗
PubMed1991

Immunohistochemical characterization of Ki-M6 monoclonal antibody in Bouin-fixed, paraffin-embedded sections of normal and neoplastic human tissues.

A monoclonal antibody (Ki-M6) against the CD 68 antigen, which labels cells of the monocyte/macrophage system, was tested on Bouin-fixed, paraffin-embedded samples of normal, reactive and neoplastic tissues by an avidin-biotin-peroxidase complex method, with the aim of establishing its value in diagnostic pathology. In normal human tissues, Ki-M6 reactivity was confined to the so-called resident macrophages populating normal organs under physiological conditions. Moreover, restricted reactivity against cells of macrophage lineage was observed in reactive and inflammatory lesions. Granulocytes, monocyte/macrophage-related immune accessory cells, and other analysed normal tissue structures did not reveal any reactivity. Ki-M6 was strongly reactive with the cases of benign (4/4) and malignant (15/15) fibrous histiocytomas, in addition to the true histiocytic lymphomas (3/3). Cases of granular cell tumour (2/3) showed strong reactivity with Ki-M6, whereas only few immunoreactive cells, with weak staining, were seen in the other Ki-M6-positive neoplasms [neurofibroma (3/3), benign schwannoma (1/2), ganglioneuroma (1/1), malignant schwannoma (5/9), melanoma (9/28), dermatofibrosarcoma protuberans (1/1), myelomonocytic leukaemia (3/3)]. Among the epithelial malignancies tested (47 cases), Ki-M6 was positive only in renal cell carcinoma (11/14). Malignant lymphomas of the Hodgkin (56 cases) and non-Hodgkin type (67 cases) were uniformly non-reactive. From these data, Ki-M6 appears to be an excellent marker of monocyte/macrophage-related cells and appears to be a reliable indicator for fibrous histiocytomas and true histiocytic malignancies. The availability of this additional antibody capable of staining routinely processed tissue is of practical interest.

Antibodies, Monoclonal↗
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↗
PubMed1996

Immunohistochemical detection of Ki-67 in epithelial skin tumors in formalin-fixed paraffin-embedded tissue sections using a new monoclonal antibody (MIB-1).

The expression of the Ki-67 antigen was investigated in 44 epithelial skin tumors using an immunohistochemical technique on formalin-fixed, paraffin-embedded tissue sections. Microwave oven heating was employed for retrieval of the antigen in these tissue sections. The staining patterns varied among the epithelial skin tumors. The assessment of immunohistochemical staining was based upon the growth fraction (GF), defined as the number of Ki-67 positive cells divided by the total number of tumor cells counted and expressed as a percentage. GF was 9.7 +/- 3.1% in seborrheic keratosis, 19.5 +/- 2.9% in keratoacanthoma, 23.1 +/- 4.9% in basal cell carcinoma, 18.5 +/- 6.3% in actinic keratosis, 37.1 +/- 6.0% in Bowen's disease, and 32.9 +/- 10.5% in squamous cell carcinoma. There was a significant difference in GF between the keratoacanthoma and squamous cell carcinoma (p < 0.01). Actinic keratosis showed a relatively low GF, whereas Bowen's disease showed a high one. Furthermore, the GF tended to increase with tumor cell differentiation in squamous cell carcinoma: 23.7% (+/- 5.0) in well-differentiated, 35.0% (+/- 6.2) in moderately-differentiated, and 47.6% (+/- 4.5) in poorly-differentiated squamous cell carcinomas. Immunohistochemistry with MIB-1 may give useful additional information in the differential diagnosis of KA and SCC.

Antibodies, Monoclonal↗
PubMed1989

Immunohistological localisation of rinderpest virus in formalin-fixed, paraffin-embedded tissues from experimentally infected cattle.

Six Freesian steers were subcutaneously inoculated with the virulent rinderpest virus strain Kabete "0" and sacrificed at the height of fever. Sections of formalin-fixed, paraffin-embedded tissues were stained according to the peroxidase anti-peroxidase (PAP) technique. Labelling of viral antigen, both in the cytoplasm and in the nuclei of infected cells, was observed in the epithelial cells of the upper and lower alimentary tract and in lymphoid organs, i.e. spleen, lymph nodes, pharyngeal tonsils, Peyer's patches and thymus. Electron microscopy studies confirmed the results.

Animals↗
PubMedDate not supplied

[Analysis of immunoglobulin heavy chain genes rearrangement by PCR from paraffin-embedded tissue in B-cell lymphomes in Tunisia].

OBJECTIVE: To study the lymphoid clonality on Tunisian B-cell lymphomas cases by polymerase chain reaction (PCR)-based techniques using DNA from paraffin-embedded tissues. MATERIAL AND METHODS: Here we conducted a retrospective PCR clonality study on 73 cases of B-cell lymphomas and 12 reactive lymphoid tissues. The quality of DNA extracted was tested by beta-globin PCR. Consensus primers directed at the FRIII-VH and FRII-VH regions of the immunoglobulin heavy chain (IgH) gene were used to detect clonality. RESULTS: The results showed that 52 of 73 (71%) B-cell lymphomas exhibited good quality of amplifiable DNA. Clonality was found in 77% of cases using the set of primers FRIIIa/LJH/VLJH and in 65.5% using the set of primers FRIIa/LJH/VLJH. Lymphomas derived from pregerminal centre showed a high rate detection of clonal IgH gene rearrangement (100%) compared to other group of tumors derived from germinal centre or postgerminal centre (74.5%). None of the polyclonal controls gave a clonal pattern. CONCLUSION: This is the first large series of PCR clonality study of IgH gene rearrangements on B-cell lymphoma from Tunisia. Our results were similar to other reports in terms of sensitivity and specificity of these techniques and confirm the interest of that PCR for detecting clonal IgH gene rearrangements in lymphoma.

Base Sequence↗
PubMed1999

Hydrolysis profiles of formalin fixed paraffin-embedded tumors based on IOD (integrated optical density) and nuclear texture feature measurements.

The aim of the study was to determine optimal hydrolysis time for the Feulgen DNA staining of archival formalin fixed paraffin-embedded surgical samples, prepared as single cell suspensions for image cytometric measurements. The nuclear texture features along with the IOD (integrated optical density) of the tumor nuclei were analysed by an automated high resolution image cytometer as a function of duration of hydrolysis treatment (in 5 N HCl at room temperature). Tissue blocks of breast carcinoma, ovarian serous carcinoma, ovarian serous tumor of borderline malignancy and leiomyosarcoma were included in the study. IOD hydrolysis profiles showed plateau between 30 and 60 min in the breast carcinoma and leiomyosarcoma, and between 40 and 60 min in the ovarian serous carcinoma and ovarian serous tumor of borderline malignancy. Most of the nuclear texture features remained stable after 20 min of hydrolysis treatment. Our results indicate that the optimal hydrolysis time for IOD and for nuclear texture feature measurements, was between 40 and 60 min in the cell preparations from tissue blocks of three epithelial and one soft tissue tumor.

Breast Neoplasms↗
PubMed1990

Macrophage histology in paraffin-embedded multiple sclerosis plaques is demonstrated by the monoclonal pan-macrophage marker HAM-56: correlation with chronicity of the lesion.

Using the monoclonal antibody HAM-56 with the avidin-biotin method on recent or archival paraffin-embedded tissue from multiple sclerosis brains, we have been able to distinguish between acute, chronic active and inactive lesions. HAM-56 stains all macrophages, monocytes and at least some microglia; it is the only pan-macrophage marker to our knowledge that can be satisfactorily used on conventional paraffin sections. A much narrower range of mainly perivascular macrophages in acute plaques of multiple sclerosis is stained with MAC-387, anti-muramidase and anti-alpha1-anti-trypsin. The acute plaques show HAM-56-stained macrophages throughout the lesion, and these macrophages exhibit profiles of phospholipid-rich myelinic bodies, which are also usually stainable with Luxol fast blue. Active ongoing lesions show a rim of macrophages at the edge of the lesion. These macrophages show profiles of large vacuoles, thought to represent the sudanophilic esterified cholesterol formed during demyelination. Inactive cases show none of these features; the few perivascular macrophages present often contain the end product of lipid peroxidation, ceroidlipofuscin.

Antibodies, Monoclonal↗
PubMed1990

Comparative study with in situ hybridization and immunocytochemistry in detection of HIV-1 in formalin-fixed paraffin-embedded cell cultures.

The sensitivities of three immunohistological techniques were compared in this study for detecting human immunodeficiency virus (HIV-1) in infected cultured human lymphocytes that had been formalin-fixed and paraffin-embedded. The techniques included in situ hybridization (ISH) with HIV-1 cDNA; immunocytochemistry with HIV-1 p24 monoclonal antibody (ICC-m); and immunocytochemistry with HIV-1 polyclonal antibody from a patient with acquired immunodeficiency syndrome (AIDS) (ICC-p). Procedures were optimized for enzyme digestion and for antibody reaction conditions. HIV-1--infected cells and noninfected control cells were tested. Noninfected controls were uniformally negative by all three methods. Infected cells had the highest positivity rate by the ISH method (p less than or equal to 0.0001), and the ICC-p method was more positive than the ICC-m (p less than or equal to 0.0001). Both the ICC-p and the ICC-m techniques were more positive with the cocultivated cell cultures than the ISH, which was more sensitive with the infected continuous cell line (P less than or equal to 0.0001). The ICC-p method had a lower standard deviation on positive results than either the ICC-m or ISH method. The variability observed with these test procedures, reagents, and specimens suggests that these are important technological parameters in detecting p24, with implications for detecting other HIV-1 markers in infected tissues.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Evidence for false aneuploid peaks in flow cytometric analysis of paraffin-embedded tissue.

It has recently been shown that bimodal histograms with false aneuploid peaks may be obtained by DNA flow cytometry from histologically normal tissue allowed to autolyze. To investigate if such peaks can be generated from surgically excised archival tissue, 198 paraffin blocks from 179 patients containing histologically normal spleen (n = 65), liver (n = 26), thyroid (n = 32), pancreas (n = 19), salivary gland (n = 49), or lymph node tissue (n = 7), obtained from the archives of two university pathology departments, were analyzed for nuclear DNA content. The great majority (n = 160, 83.8%) of the 191 interpretable histograms had a single symmetrical G1 peak; and 8 histograms, all produced from liver tissue had a tetraploid pattern. A slight or a prominent repeatable deviation in the G1 peak outline was present in 14 (7.3%) cases. A peak resembling an aneuploid G1 peak with a DNA index (DI) ranging from 1.14 to 1.38 was repeatedly produced from 9 (4.7%) blocks containing histologically normal or inflamed splenic (n = 3), pancreatic (n = 3), liver (n = 1), thyroid (n = 1), or lymph node (n = 1) tissue. The three abnormal peaks produced from pancreatic tissue were rounded in shape and resembled closely the ones that can be obtained from autolytic pancreatic tissue, and the six remaining extra peaks were all fused with the "diploid" peak. In conclusion, false peaks, probably caused by degradation of the nuclear contents during formalin fixation or before it, may rarely be obtained from surgical paraffin-embedded samples.

Aneuploidy↗
PubMed1995

Immunohistochemical detection of progesterone receptors and the correlation with Ki-67 labeling indices in paraffin-embedded sections of meningiomas.

Female sex steroids may play a role in the proliferation of meningiomas. We investigated the progesterone receptor (PgR) immunoreactivities and the Ki-67 labeling indices in the formalin-fixed, paraffin-embedded sections of meningiomas from 39 patients. After autoclave pretreatment of the sections (which were immersed in a citrate buffer), the sections were incubated with the monoclonal antibody for the PgR and the MIB-1 monoclonal antibody for the Ki-67 antigen. In the meningiomas studied, the immunoreactivity for the PgR was moderately to strongly positive in 51%, weakly positive in 21%, and negative in 28%. The nuclear staining for the PgR was clear, and no tumors were positive for the estrogen receptor. The Ki-67 labeling indices of the PgR-positive meningiomas (mean +/- standard deviation, 2.35 +/- 2.12%) were significantly lower than those of the PgR-negative meningiomas (6.53 +/- 4.83%) (P < 0.05). Two meningiomas that had recurred more than once showed high Ki-67 labeling indices and negative immunostaining for the PgR. These findings indicate that the PgR status may be closely related to the growth potentials of the meningiomas. Our results confirm that the immunodetection of the PgR and the Ki-67 antigen on the paraffin sections of meningiomas provides a practical tool for estimating the biological behavior of the meningiomas.

Adult↗
PubMed1992

Monoclonal antibody-based immunohistochemical detection of bovine viral diarrhea virus in formalin-fixed, paraffin-embedded tissues.

Thirty-two monoclonal antibodies directed against epitopes on bovine viral diarrhea virus proteins and glycoproteins were tested for immunohistochemical reactivity with bovine viral diarrhea virus in formalin-fixed and paraffin-embedded tissues from 45 cases of bovine viral diarrhea virus-associated mucosal disease. Only one antibody, designated 15C5, which reacts with the 48-kD glycoprotein of bovine viral diarrhea virus, detected an epitope preserved in these specimens. Monoclonal antibody 15C5 and a polyclonal antibody to bovine viral diarrhea virus successfully detected bovine viral diarrhea viral antigens in 44/45 cases of mucosal disease and did not react with formalin-fixed tissues from 30 uninfected cattle. Monoclonal antibody-based immunohistochemical detection of bovine viral diarrhea virus is routinely fixed tissue specimens has advantages over other currently available techniques in terms of the convenience of specimen submission, the relative ease of method standardization, and the rapidity of the test, and by enabling identification of the virus in association with specific tissues, cell types, and histologic lesions.

Animals↗
PubMed1993

Use of an immunoperoxidase technique to detect equine herpesvirus-1 antigen in formalin-fixed paraffin-embedded equine fetal tissues.

An indirect immunoperoxidase (IP) procedure using the avidin-biotin-peroxidase complex detection technique was developed to detect viral equine herpesvirus-1 (EHV-1) antigen in formalin-fixed paraffin-embedded tissues from aborted equine fetuses. The procedure was applied to liver, lung, and other tissues from 20 cases of confirmed or suspected EHV-1-induced abortions. Specific staining was observed in tissue sections from EHV-1-infected fetuses. Positive IP staining was present in tissues of 7 cases that were also positive by fluorescent antibody (FA) and virus isolation (VI) and that had typical histologic lesions. There was no IP staining in 7 cases that had no histologic lesions and negative FA and VI results. Five cases had typical histologic lesions and positive results in only 1 laboratory test; 3 were positive by VI and 2 by FA. Liver of 1 case was positive by IP, but tissues were too autolytic for other tests to be evaluated.

Abortion, Veterinary↗
PubMed1990

Type-specific human papillomavirus detection in formalin-fixed, paraffin-embedded tissue sections using nonradioactive deoxyribonucleic acid probes.

DNA probes directly conjugated to horseradish peroxidase have been used successfully to detect human papillomavirus (HPV) types 6/11, 16, and 18 in formalin-fixed, paraffin-embedded tissue sections. By using silver enhancement of a heavy metal-modified diaminobenzidine precipitate, the sensitivity of human papillomavirus detection was significantly increased without compromising specificity. In studies comparing the specificity of the horseradish peroxidase-labeled probe/silver enhancement system to that of a biotinylated-DNA probe/streptavidin-alkaline phosphatase system, the former was found to be superior.

3,3'-Diaminobenzidine↗
PubMed1985

Leu-M1--a marker for Reed-Sternberg cells in Hodgkin's disease. An immunoperoxidase study of paraffin-embedded tissues.

Monoclonal antibody to Leu-M1, a granulocyte-related differentiation antigen, represents a highly effective reagent for detection of diagnostic Reed-Sternberg (R-S) cells and variants in paraffin-embedded tissues of Hodgkin's disease. In 69 of 73 cases of Hodgkin's disease (41 nodular sclerosis, 25 mixed cellularity, 4 lymphocyte predominance, and 3 lymphocyte depletion types), R-S cells were strongly immunoreactive for Leu-M1. Four cases of lymphocyte predominance Hodgkin's disease (nodular) were uniformly nonreactive for Leu-M1. In most of the positive cases (57/69, 83%), the majority (60-90%) of R-S cells and variants exhibited immunoreactivity for Leu-M1. A characteristic staining pattern included granular and/or vesicular cytoplasmic immunoreactivity, often with a prominent globular paranuclear reaction product, and membrane staining with highly irregular cytoplasmic borders. Evaluation of B-cell (37 specimens), T-cell (20 specimens), and true histiocytic (3 specimens) neoplasms and a case of mastocytosis revealed immunoreactivity for Leu-M1 only in 1 B-cell and 4 T-cell malignancies. The staining patterns in these cases, however, clearly differed from that observed for R-S cells. Studies of nonneoplastic lymphoid tissues (38 total) demonstrated that lymphoid cells were typically nonreactive; histiocytes revealed variable reactivity for Leu-M1. Occasional histiocytes of the sinusoidal network of lymph nodes, particularly in toxoplasmic lymphadenitis, exhibited a staining pattern (membranous/cytoplasmic/paranuclear) similar to that observed for R-S cells. Leu-M1 represents a potentially helpful diagnostic discriminant in the assessment of Hodgkin's disease and its distinction from non-Hodgkin's lymphomas and other lymphoid proliferations.

Antibodies, Monoclonal↗
PubMed1990

Enhanced immunocytochemical staining sensitivity in formalin-fixed and paraffin-embedded material by simultaneous use of PAP and ABC.

The simultaneous use of two immunoperoxidase (IP) methods; e.g. the PAP (Peroxidase-antiperoxidase) and ABC (Avidin-Biotin) to localize a single antigen enhances the sensitivity of polyclonal antibodies (FVIII/RAg and laminin) or monoclonal antibodies (MABs) (against human B-, T-cells and macrophages) in routinely formalin fixed and in paraffin embedded material. The increased sensitivity was not accompanied by loss of specificity. With antibodies against Factor VIII related antigen (FVIII/RAg) and laminin the increased staining intensity of blood vessels were demonstrated in experimental rat transplantation tumors. The similar enhancement of immunocytochemical reactions was observed with biopsies of human brain tumors, where the monoclonal antibodies against white blood cells were used. The staining results were of comparable intensity as in snapfrozen tissue or after special embedding procedure for immunocytochemical purposes acc. to Bolton and Mesnard formol sucrose gum, sucrose paraffin; FSGSP).

Animals↗