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PubMed1997

[Measurement of aspecific alkaline phosphatase in the intraoperative histological examination of the breast].

One hundred seventy two consecutive cases of female breast pathology have been intraoperatively processed by frozen sections (FS) in combination with aspecific alkaline phosphatase (AAF) histochemical evaluation. The corresponding histology on permanent embedded tissues (ET) was compared with both these techniques. The related theoretical and practical methodological aspects are presented and discussed. The consultations concerned the following histopathological types (WHO terminology): 136 malignant epithelial tumours(intraductal carcinoma: 11; invasive ductal carcinoma: 100; invasive lobular carcinoma: 8; medullary carcinoma: 3; mucinous carcinoma: 8; papillary carcinoma: 3; tubular carcinoma: 2); one cystosarcoma phyllodes; 19 benign tumours; 15 mammary dysplasias (fibrocystic disease); one tumour-like lesion(hamartoma); one fibromatous lesion. In terms of concordance/discordance between FS and ET diagnoses no false positive cases and only one false negative resulted. The diagnosis was deferred in 18 circumstances (10,5%). The AAF reaction in 64/153 (41,8%) cases (correctly interpreted on FS) displayed more histological evidence with special reference to the detection of the myoepithelial cells. The morphological implementation was particularly relevant as far as the differentiation between intraductal and microinvasive carcinoma, carcinomatous and papillomatous papillary proliferation, carcinomatous and non carcinomatous tubular structures, pseudocarcinomatous (florid and/or sclerosing) dysplasia and carcinoma is concerned. Analogously in 12/17 (70,6%) comparable deferred diagnoses as well as in the single false negative case the interpretation could have been correctly stated intraoperatively. The normal and pathological features as disclosed by the AAF reaction have been analytically examined. The AAF procedure seems to be a possible relevant tool in order to improve the classical FS performance also in respect to the increasingly systematic, precocious and conservative approach of the modern breast surgery.

Alkaline Phosphatase↗
PubMed1984

Freeze-dried paraffin-embedded human tissue for antigen labelling with monoclonal antibodies.

Conventional fixation and paraffin embedding of human tissue destroys most of the antigens detected with currently available antibodies, while cryostat sections give poor morphological detail and require freezer space for storage. However, when tissue was freeze-dried before being embedded in paraffin S then stained for a wide range of antigens recognised by monoclonal antibodies (including T-cell and B-cell antigens), morphological preservation was clearly better, and the labelling reactions were of equal (or greater) intensity, than with cryostat sections. As the number of diagnostically important monoclonal antibodies continues to increase the use of freeze-dried paraffin sections should facilitate the introduction of immunohistological techniques for both routine histopathological diagnostic work and immunological research.

Antibodies, Monoclonal↗
PubMed2000

Cytogenetical diagnosis in paraffin-embedded fetoplacental tissue using comparative genomic hybridization.

Comparative genomic hybridization (CGH) is a FISH-related technique used to assess global chromosomal aberrations in a variety of human tumours. Recently CGH has been applied to cytogenetic analysis of fresh frozen fetoplacental tissues. Here we report the application of CGH to paraffin-embedded placental samples. Ten samples from paraffin-embedded blocks of 6 control placentas and fetoplacental tissue from 10 aneuploidies, and 2 unbalanced aberrations were evaluated. Balanced karyotype profiles were obtained from samples of healthy placentas and all samples from the same placenta appeared to have similar confidence intervals. CGH analysis of four cases of trisomy 21, three cases of trisomy 18, one case of trisomy 13, one case of trisomy 15 and one case of trisomy 7 all showed overrepresentation of the respective trisomic chromosome. The CGH profile was also in accordance with the karyotyping of a case with isochromosome 21. The CGH profile of a case with der (2)t(2;6)(q37.3;q22.2) revealed partial trisomy for chromosome 6 between q21 and q27. CGH may be a useful adjunct in prenatal genetic diagnosis when retrospective diagnosis is needed from archival samples.

Chromosome Aberrations↗
PubMed1991

DNA image cytometry of keratoacanthoma and squamous cell carcinoma.

The distinction between the keratoacanthoma (KA) and the squamous cell carcinoma (SCC) can sometimes be difficult on the basis of histologic and clinical criteria. The possible diagnostic significance of DNA ploidy initiated the present study evaluating the DNA ploidy in paraffin-embedded tissue sections of 7 KA and 15 SCC, and fresh frozen tissue touch preparations of 15 of the same cases using the CAS 200 Image Analyzer. In paraffin-embedded tissue sections the main peak DNA index was based on normal epidermis, and ranged from 1.03 to 1.59 in KA and from 1.47-2.71 in SCC. The DNA Index (DI) discriminated KA from SCC in 17 of 22 cases (p less than 0.0007). The highest DNA content of single nuclei ranged from 9.0-18.0 picograms (pg) (DI 2.9-6.03) in KA and 14.8-38.6 pg (DI 4.0-11.03) in SCC. The highest DNA content discriminated KA from SCC in 16 of 22 cases (p less than 0.003). In fresh frozen tissue touch preparations from 15 of the same lesions, there was considerable overlap in DNA indices of KA (0.534-1.39) and SCC (0.464-1.41). Abnormal DNA peaks seen in histograms from three SCC in paraffin-embedded tissue sections were lost in the touch preparation histograms, probably due to inadequate sampling. Therefore, image analysis of paraffin-embedded tissue sections is better able to distinguish KA from SCC than touch preparations.

Carcinoma, Squamous Cell↗
PubMed1999

The detection of CD2+, CD4+, CD8+, and WC1+ T lymphocytes, B cells and macrophages in fixed and paraffin embedded bovine tissue using a range of antigen recovery and signal amplification techniques.

In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59.

Animals↗
PubMed1989

Monoclonal antibody to a proximal nephrogenic renal antigen: immunohistochemical analysis of formalin-fixed, paraffin-embedded human renal cell carcinomas.

An IgG1 murine monoclonal antibody (PN-15) to an epitope preserved in formalin-fixed, paraffin-embedded tissue sections of renal cell carcinomas was developed following immunization of mice with the microsomal fraction of human renal cortical tissue homogenates. An immunohistochemical screening procedure was utilized against fixed, embedded tissue sections of renal cell carcinomas. This antibody identified a Mr 200,000 determinant on immuno-Western blot analysis, and the recognized epitope was shown to reside in the carbohydrate domain of a Mr 200,000 glycoprotein (gp200). Analysis of its tissue distribution was performed by indirect immunoperoxidase staining of fixed, embedded tissue sections of the following: 36 different types of normal tissues; 45 primary and 38 metastatic renal cell carcinomas; and 124 cases of 39 different types of neoplasms. In the normal kidney, gp200 was localized along the brush border of the proximal tubule and the luminal surface of Bowman's capsule adjoining the outgoing proximal tubule. Shared tissue localization was also noted in breast lobules and ducts, parathyroid gland, and epididymis. The gp200 was expressed by 93% of primary and 84% of metastatic renal cell carcinomas, primarily on the cell surface membrane. Shared antigenic expression was noted in relatively few other tumors including: mammary carcinomas; teratocarcinomas; and parathyroid adenomas. The PN-15 monoclonal antibody holds promise as a useful reagent in retrospective investigations of fixed, embedded cases of renal cell carcinomas. The antibody may also be of practical utility in diagnostic immunohistochemistry by helping to retrospectively define the primary site of metastatic clear cell tumors, especially when fresh frozen tissue from such tumors is not available.

Antibodies, Monoclonal↗
PubMed2006

Embedding of bone samples in methylmethacrylate: a suitable method for tracking LacZ mesenchymal stem cells in skeletal tissues.

Considerable research has been focused on the use of bone marrow-derived mesenchymal stem cells (MSCs) for the repair of non-unions and bone defects. To date, the question of whether transplanted MSCs survive and engraft within newly formed tissue remains unresolved. The development of an easy and reliable method that would allow cell fate monitoring in transplant recipients is a pressing concern for the field of tissue engineering. To demonstrate the presence of transplanted cells in newly formed bone, we established a xenograft nude rat model allowing the detection of murine LacZ MSCs in vivo. MSCs were isolated from transgenic lacZ mice, seeded onto bioabsorbable collagen sponges, and transplanted to repair a calvarial defect in nude rats. As a preliminary step, the histological procedure was adapted to optimize the detection of LacZ cells in bone tissue embedded in methylmethacrylate (MMA). Four fixatives and four fixation times were evaluated. Among all the fixatives tested, 2% formaldehyde/0.2% glutaraldehyde at 4C for 4 days gave the best results for X-gal staining at pH 7.4 on both cell cultures and bone explants. All fixatives were effective for immunodetection of beta-gal. In the chimeric LacZ/nude rat animal model, MSCs were detected in vivo for up to 4 weeks after implantation and contributed to the repair and the neovascularization of the bone defect. LacZ is a suitable phenotypic marker to track MSCs in skeletal tissues embedded in MMA.

Animals↗
PubMed2004

Molecular typing of Pneumocystis jirovecii found in formalin-fixed paraffin-embedded lung tissue sections from sudden infant death victims.

Previous studies have provided histological evidence of an association between primary Pneumocystis infection and sudden infant death syndrome (SIDS). The aim of this work was to determine the species of clustered Pneumocystis organisms found in formalin-fixed paraffin-embedded (FFPE) lung tissue sections from Chilean sudden infant death (SID) victims. This approach needed first to optimize a DNA extraction method from such histological sections. For that purpose, the QIAamp DNA Isolation from Paraffin-Embedded Tissue method (Qiagen) was first tested on FFPE lung tissue sections of immunosuppressed Wistar rats inoculated with rat-derived PNEUMOCYSTIS: Successful DNA extraction was assessed by the amplification of a 346 bp fragment of the mitochondrial large subunit rRNA gene of the Pneumocystis species using a previously described PCR assay. PCR products were analysed by direct sequencing and sequences corresponding to Pneumocystis carinii were found in all the samples. This method was then applied to FFPE lung tissue sections from Chilean SID victims. Pneumocystis jirovecii was successfully identified in the three tested samples. In conclusion, an efficient protocol for isolating PCR-ready DNA from FFPE lung tissue sections was developed. It established that the Pneumocystis species found in the lungs of Chilean SID victims was P. jirovecii.

Animals↗
PubMed1983

[Method of double embedding of tissue in paraplast-piccolyte].

A histological method of double embedding of the tissue in paraplast-piccolyte used for light microscopy is described. The method requires no special equipment, is simple and suitable for all methods of staining of sections obtained upon paraffin embedding, including immunofluorescent and immunoperoxidase procedures.

Bicyclic Monoterpenes↗
PubMed1994

Nuclear morphometry and DNA flow cytometry as prognostic methods for endometrial carcinoma.

In a series of 227 women with endometrial carcinoma stages I-IV the prognostic value of nuclear morphometry and DNA analysis was evaluated prospectively. The tumors were also classified according to histologic subtype, degree of differentiation (FIGO), and nuclear grade. The DNA analysis (ploidy and S-phase fraction) was made using flow cytometry on fresh-frozen tissue. The morphometric measurements and the grading of the tumors were done on both fresh-frozen tissue and on formalin-fixed and paraffin-embedded tissue. The rate of recurrence for the complete series was 14% with 2.2% vaginal metastases. The overall 5-year survival rate was 68% and the corrected survival rate 76%. The minimum nuclear diameter and the standard deviation (SD) of the maximum nuclear diameter were significant and independent prognostic variables in a Cox multivariate analysis when analyzed on paraffin-embedded tissue. The nuclear grade was the single most prognostically informative variable. A corresponding analysis on fresh-frozen tissue showed that the S-phase fraction and the FIGO grading of the tumor were significant variables. The morphometric variables and the nuclear grading were non-significant prognostic variables. Morphologic changes in tumor cell nuclei during the freezing and thawing procedures may explain the loss of prognostic information. A combination of DNA index (fresh-frozen tissue) and the mean values of the shortest nuclear diameter and the SD of the longest diameter (paraffin-embedded tissue) gave the best prognostic information vis-a-vis cancer-related death in an all-possible-subsets regression analysis.

Journal Article↗
PubMed1995

[Hormone receptor determination in breast carcinoma tissue. Comparison of recent immunohistologic techniques with biochemical receptor testing].

For evaluation of the hormone receptor status in breast cancer tissues two methods are mainly used: immunohistochemical detection by monoclonal antibodies on frozen sections (ER-ICA, PgR-ICA) and the biochemical radioligand-binding assay (DCC) of fresh tissue. Using new antibodies makes it possible to evaluate the estrogen and progesterone receptor status in formalin-fixed and paraffin-embedded tissue. In the present retrospective study, tissues from 223 primary breast carcinomas or breast carcinoma recurrences were re-evaluated with the three methods mentioned above and the results were compared. We used antibody 1D5.26 reacting with the estrogen receptor and mPR1 specific for the progesterone receptor in paraffin-embedded tissue. The agreement of positive and negative cases between these two immunohistochemical procedures was 97.8% for the estrogen receptor and 85.7% for the progesterone receptor. Comparison of immunohistochemistry on paraffin-embedded tissue and biochemical evaluation showed an agreement of 74.7% for the estrogen receptor and 68.7% for the progesterone receptor. These results are comparable to the correspondence between ER-ICA and PgR-ICA and the DCC method. This study proves that the prognostically and therapeutically important hormone receptors can be reliably determined in formalin-fixed and paraffin-embedded tissues. These results are not only important for the evaluation of hormone receptors of a small breast carcinoma that is not found in the frozen section, but for the considerable difference in costs among the different methods.

Antibodies, Monoclonal↗
PubMed2001

Tissue microarray profiling of cancer specimens and cell lines: opportunities and limitations.

The implementations of high-throughput genetic technologies, such as oligonucleotide microarrays, generate myriad points of data. The identified potential candidate genes need to be further characterized and selected using a large number of well-characterized tumors and stringent criteria. Tissue microarrays allow for such high-throughput expression profiling of tumor samples, providing, in addition, information at the microanatomical level. Different techniques could be applied for identification of specific phenotypic (immunohistochemistry and in situ hybridization) or genotypic (fluorescence in situ hybridization) alterations, holding strong potential for translational research. Tissue microarrays consisting of 0.6-mm biopsies of paraffin-embedded tissues are well validated and have been used for various clinicopathological studies. This review discusses the technical considerations for construction of such arrays from paraffin-embedded tissues and cell lines and outlines their potential for clinical research applications. The use of paraffin-embedded tissues has some limitations with regard to analysis of RNA or certain proteins. To overcome such limitations, we have developed a cryoarray strategy allowing for the processing of multiple frozen tissue specimens and/or cell lines on a single tissue block. These approaches offer the opportunity to conduct pilot and validation studies of potential targets using clinical samples linked to clinicopathological databases.

Animals↗
PubMed1990

Detection of hepatitis B virus DNA in formalin-fixed, paraffin-embedded liver tissue by the polymerase chain reaction.

DNA isolated from formalin-fixed, paraffin-embedded liver tissues from nine patients with hepatocellular carcinoma and six control patients was screened for hepatitis B virus (HBV) DNA with surface (S) and core (C) gene-specific primers by a modification of the polymerase chain reaction--southern blot technique (PCR-SB). PCR-SB results were correlated with histologic, immunohistochemical, and serologic findings. All cases with an established HBV etiology were positive by PCR-SB, as were three cases with negative immunohistochemistry and serology. Often there was selective amplification with one primer set and, in two cases, smaller than expected HBV amplification products suggesting internal deletions. The presence of a potent PCR inhibitor in nucleic acid preparations from tissue blocks that can be removed by Sephadex G-50 chromatography was confirmed. PCR-SB will be a powerful method for the diagnosis and follow-up of patients with HBV infection and may provide new insights into viral hepatocarcinogenesis.

Base Sequence↗
PubMed2001

Immunohistochemical detection of cell growth fraction in formalin-fixed and paraffin-embedded murine tissue.

Monoclonal antibody MIB-1 is a reliable tool for determining proliferating cells in human tissues, but does not react with the homologous mouse antigen and is therefore useless in experimental pathology using mice as model systems. Standard method for assessment of cellular proliferation in formalin-fixed, paraffin-embedded murine tissues is immunohistochemical detection of DNA synthesis using antibodies against exogenously injected 5-bromodeoxyuridine (BrdU), which is a tedious procedure and not useful for routine investigations. We tested monoclonal antibody MIB-5 and monoclonal and polyclonal anti-MCM3 antibodies as immunohistochemical proliferation markers for paraffin-embedded nonneoplastic and neoplastic tissues of wild-type and transgenic mice, compared to anti-BrdU immunostaining. Percentage of proliferating cells was determined with continuously decreasing antibody dilutions. Percentages of MIB-5 and anti-BrdU immunostained cells correlated strongly, as well as percentage of MIB-5-decorated cells and frequency of mitotic figures. Anti-MCM3 antibodies labeled significantly higher percentages of cells than anti-BrdU or MIB-5, and showed a linear decrease with increasing antibody dilutions. We conclude that MIB-5 detects reliably the cell growth fraction in formalin fixed, paraffin-embedded murine tissues, bypassing methodological drawbacks of BrdU. Anti-MCM3 antibodies are less useful for determination of proliferating cells although they might detect the fraction of cells remaining competent for proliferation.

Adenoma↗
PubMed1991

Variability in beta-globin and HPV DNA amplification by PCR from fixed tissues.

Paraffin-embedded tissues from a variety of sources and treated with different fixatives were tested for beta-globin and HPV amplification by polymerase chain reaction (PCR). In tests of tissues collected in the previous 2 to 3 yr, excellent rates (87% to 93%) for beta-globin amplification were obtained for specimens fixed in buffered formalin, Bouin's, and Hartmann's solutions. In contrast, the rate of beta-globin amplification was low for tissues fixed in Hollande's solution (7%) and in Hartmann's solution with eosin (33%). The results of beta-globin amplification from archival tissues stored for variable time periods showed no decrease in the amplification rate with longer periods of storage. Human papillomaviruses (HPVs) were identified in 17% of globin negative and in 43% of globin positive tissues. HPV-16 amplification was more efficient when the targeted DNA sequence was small. Variability in amplification depends not only on the type of fixative used, but also on other ill-defined factors. Therefore, conditions for optimal amplification should be determined before undertaking studies of archival material.

Blotting, Southern↗
PubMed1999

Comparison of allelic ratios from paired blood and paraffin-embedded normal tissue for use in a polymerase chain reaction to assess loss of heterozygosity.

BACKGROUND: One method to assess loss of heterozygosity (LOH) of various genes is the amplification of DNA from neoplastic tissue by using microsatellite markers. LOH can best be considered on a quantitative basis as a comparison of allelic ratios of neoplastic tissue to that of the normal control. We will illustrate through quantitative methods the importance of using the appropriate controls when determining allelic loss. METHODS AND RESULTS: DNA extracted from 28 paired blood and formalin-fixed, paraffin-embedded normal mucosal tissue was amplified using the DP1 microsatellite marker, consisting of a variable number of CA repeats. This marker is located within the D5S346 (DP1) region on chromosome 5 and is linked to the adenomatous polyposis coli gene. Allelic ratios were calculated after scanning autoradiographs on a densitometer. Ratio values approaching 1 were observed when the two alleles were close in molecular weight, whereas ratios less than 1 were detected when the two alleles had very different molecular weights. This discrepancy was more pronounced in paraffin-embedded tissue than with blood samples. CONCLUSION: For LOH amplification assays, it is best to use normal control samples that are of the same tissue source as the neoplastic sample being analyzed. When assessing LOH in neoplastic tissue, a quantitative value rather than visual assessment of the alleles should be considered. The values may be normalized by dividing the ratio of the two tumor alleles by the ratio of the two normal alleles.

Alleles↗
PubMed1988

Analysis of DNA sequences in forty-year-old paraffin-embedded thin-tissue sections: a bridge between molecular biology and classical histology.

DNA sequences from human tissues paraffin embedded 40 yr ago were studied using the in vitro gene amplification technique known as the polymerase chain reaction. Although significant DNA degradation was observed, single copy genomic sequences and viral segments were readily detected from single 5- to 10-microns tissue sections. This demonstrates that the world-wide collection of archival paraffin-embedded tissues may be used to study the association of biological agents (viral, bacterial, or parasitic) or endogenous DNA lesions with disease over time and to carry out retrospective studies on material where the clinical outcome has already been established. This will be especially valuable in studying rare cancers and other rare diseases.

Base Sequence↗