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PubMed1993

Immunoblot analysis of the placental form of glutathione S-transferase in protein extracted from paraffin-embedded human glioma tissue.

Protein extracted from conventional formalin-fixed and paraffin-embedded tissue sections of human gliomas was examined for immunoblot analysis using antibody against the placental form of glutathione S-transferase (GST-pi). Four benign astrocytomas, five anaplastic astrocytomas and four glioblastomas were used in this study. The preliminary study demonstrated that immunoreactivity of GST-pi was well preserved in normal brain tissue and normal term placenta fixed in acetone, formalin or buffered formalin (pH 7.4). GST-pi in gliomas fixed in formalin also had a good immunoreactivity and showed clear bands on nitrocellulose membranes processed by the method of Western blotting using anti-GST-pi antibody. The results of immunoblot analysis for GST-pi indicate that the intensity of immunoreactivity of benign astrocytoma, anaplastic astrocytoma and glioblastoma increases with the advance of malignancy of these neoplasms. Western blot analysis for GST-pi can be performed using protein extracted from formalin-fixed and paraffin-embedded tissue sections, and the immunoreactive bands can be analyzed quantitatively by densitometric scanning.

Blotting, Western↗

PubMed1999

Multiple mutation analyses in single tumor cells with improved whole genome amplification.

Combining whole genome amplification (WGA) methods with novel laser-based microdissection techniques has made it possible to exploit recent progress in molecular knowledge of cancer development and progression. However, WGA of one or a few cells has not yet been optimized and systematically evaluated for samples routinely processed in tumor pathology. We therefore studied the value of established WGA protocols in comparison to an improved PEP (I-PEP) PCR method in defined numbers of flow-sorted and microdissected tumor cells obtained both from frozen as well as formalin-fixed and paraffin-embedded tissue sections. In addition, the feasibility of I-PEP-PCR for mutation analysis was tested using clusters of 50-100 unfixed tumor cells obtained by touch preparation of ten breast carcinomas by conventional sequencing of exon 7 and 8 of the p53 gene. Finally, immunocytochemically stained microdissected single disseminated tumor cells from bone marrow aspirates were investigated with respect to mutations in codon 12 of Ki-ras by restriction fragment length polymorphism (RFLP)-PCR after I-PEP-PCR. The modified I-PEP-PCR protocol was superior to the original PEP-PCR and DOP-PCR protocols concerning amplification of DNA from one cell (efficiency rate I-PEP-PCR 40% versus PEP-PCR 15% and DOP-PCR 30%) and five cells (efficiency rate I-PEP-PCR 100% versus PEP-PCR 33% and DOP-PCR 20%). Preamplification by I-PEP allowed 100% sequence accuracy in > 4000 sequenced base pairs and Ki-ras mutation detection in isolated single disseminated tumor cells. For reliable microsatellite analysis of I-PEP-preamplified DNA, at least 10 unfixed cells from fluorescence-activated cell sorting, 10 cells from frozen tissue, or at least 30 cells from formalin-fixed and paraffin-embedded tissue sections were required. Thus, I-PEP-PCR allowed multiple reliable microsatellite analyses suited for microsatellite instability and losses of heterozygosity and mutation analysis even at the single cell level, rendering this technique a powerful new tool for molecular analyses in diagnostic and experimental tumor pathology.

Bone Marrow Neoplasms↗

PubMed1992

Comparative lectinhistochemical studies on paraffin- and glycol methacrylate-embedded CNS tissue specimens from AIDS autopsies. Mistletoe lectin I (ML I) as cell-marker.

Brain tissue and spinal cord tissue from 12 patients who had died of AIDS was fixed in neutral formalin; then after the embedment of some of it in paraffin and some of it in glycol methacrylate, it was analyzed lectinhistochemically with mistletoe lectin I (ML I). Mistletoe lectin (ML I) is a reliable marker for microglia cells and macrophages and for special cell forms (polynuclear giant cells, so-called pericytes) belonging to this cell system. In both the embedding procedures used, the representation of the cells is very clear and subtly differentiated so that the preparations are very well suited to the study of AIDS-associated tissue damage in the CNS. Whereas the immunological macrophage markers (CD 68 markers) KPI, PG-M1 produce good results on paraffin-embedded material but are completely useless on material embedded in glycol methacrylate, the cells of the monocyte-macrophage system can be represented very well and reliably after being embedded in glycol methacrylate with mistletoe lectin I (ML I).

AIDS Dementia Complex↗

PubMed1997

Effects of gonadotrophins on the proliferative activity of somatic testicular cells in neonatal and prepubertal rats.

The proliferative activity of the different testicular somatic cell lineages was studied in neonatal and prepubertal rats after treatment with recombinant FSH (recFSH) and human chorionic gonadotrophin (hCG). Male rats were treated on days 5-9 or 15-19 with 1 or 3 IU/rat.day, respectively, of recFSH, 10 IU/rat.day of hCG or vehicle and sacrificed the day following the last injection. Proliferation was studied by the immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine (BrdU) in both paraffin- and Epon-embedded tissues. No differences existed for the labeling indices in paraffin- or Epon-embedded tissues, but the identification of some cell types were easier in these latter one. At 10 days of age, no significant differences were found for the proliferative activity of the different cell types, except for the labeling index of foetal Leydig cells that was increased in hCG-treated animals. At 20 days of age, treatment with recFSH increased the testicular weight and tubule diameter, as well as the size of adult type Leydig cells and the labeling index of macrophages. Treatment with hCG increased the testicular weight and the number and size of adult-type Leydig cells, as well as the labeling indices of mesenchymal, adult-type Leydig cells and macrophages. These results indicate that the effects of gonadotrophin treatment on the different somatic testicular cell types are dependent on both the type of cells and their developmental stage.

Analysis of Variance↗

PubMed1996

Comparison of two monoclonal antibodies for the detection of estrogen receptors in primary breast carcinomas.

A new monoclonal antibody against estrogen receptor (ID5, Dako) has shown promising results when applied to formalin fixed, paraffin embedded tissue. In order to determine whether this antibody can compete in specificity and sensitivity with the ER-ICA antibody (Abbott Laboratories), a comparative, prospective study of the two antibodies was carried out on paraffin embedded and fresh frozen tissue in three laboratories. Two hundred and fifteen breast carcinomas were examined. Formalin fixed, paraffin embedded tissue was available from 215 tumors, and fresh frozen tissue from 189 of the tumors. Of these, 124 tumors were also investigated by the enzyme-linked immunosorbent assay. The results from each of the three laboratories correspond with those obtained for the whole material. The percentage of tumors positive for estrogen receptors within the different methods, was as follows: 71% by ID5 on paraffin sections, 50% by ER-ICA on paraffin sections, 65% ER-ICA on frozen sections and 88% by the EIA. When comparing the different immunohistochemical results and the EIA in 2 x 2 tables, agreement was reached in 69% to 91% of the cases. The best agreement (91%) was found between results obtained with the ID5 antibody used on formalin fixed, paraffin embedded tissue and the ER-ICA kit used on fresh frozen tissue. The advantages of using the ID5 antibody are associated with its applicability to formalin fixed, paraffin embedded tissue: improved morphology, reproducibility, retrospective studying and low costs. Finally, it is reproducible not only within the same laboratory but also among different laboratories.

Antibodies, Monoclonal↗

PubMed1996

Loss of heterozygosity in tuberous sclerosis hamartomas.

We have previously described in tuberous sclerosis (TSC) hamartomas the phenomenon of loss of heterozygosity (LOH) for DNA markers in the region of both the TSC2 gene on chromosome 16p13.3 and the TSC1 gene on 9q34. We now describe the spectrum of LOH in 51 TSC hamartomas from 34 cases of TSC. DNA was extracted from leucocytes or normal paraffin embedded tissue, and from frozen paraffin embedded hamartoma tissue from the same patient. The samples were analysed for 11 markers spanning the TSC1 locus and nine markers spanning the TSC2 locus. Twenty-one of 51 hamartomas showed LOH (41%). There was significantly more LOH on 16p13.3, with 16 hamartomas showing LOH around TSC2, and five in the vicinity of TSC1. No hamartoma showed LOH for markers around both loci. All the areas of LOH on chromosome 9 were large, but the smallest region of overlap lay between the markers D9S149 and D9S114, providing independent evidence for the localisation of the TSC1 gene. These data show that LOH is a common finding in a wide range of hamartomas, affecting the same TSC locus in different lesions from the same patient but not affecting both loci. These data support the hypothesis that both the TSC genes act as tumour suppressors and that the manifestations of TSC in patients with germline TSC mutations rise from "second hit" somatic mutations inactivating the remaining normal copy of the TSC gene.

Angiomyolipoma↗

PubMed1994

[DNA cytometry using paraffin embedded tumor tissues. Comparison of flow cytometry (FCM) and image cytometry (ICM)].

In order to examine the correlation of cytometric DNA ploidy in paraffin-embedded tumour material, a comparison was made of flow (FCM) and image cytometry (ICM); in the case of ICM FEULGEN-stained tissue sections (8-10 microns) and in some cases nuclear suspensions were used. Tumour tissue from seminomas (FCM: n = 50; ICM/section: n = 28), oral squamous carcinomas (FCM: n = 110; ICM/section: n = 50) and adenocarcinomas of the gallbladder (FCM: n = 50; ICM/section: n = 46; ICM/nuclear susp.: n = 40) was studied. The concordance regarding diploid/non-diploid characteristics was 96.5% in the case of seminomas, 87% in oral carcinomas and 100% in gallbladder carcinomas. A statistically significant correlation of DNA indices of FCM with ICM histograms was found in all three organ tumours examined. 6% of oral and up to 18% gallbladder carcinomas, however, could not be evaluated by means of FCM due to poor quality of the histograms (coefficient of variation (CV) > 6%). In the determination of DNA indices and resolution of multiploid stemlines, FCM analysis proved to be superior. Regional intratumoural differences and rare tumour populations were best detected by ICM using sections. Without correction factors being used, these sections did not show any significant deviation of DNA indices from the other methods used. Our study has revealed that in paraffin-embedded tumour material FCM and ICM usually provide well-correlating results, when standardised and controlled preparation and measuring procedures are applied.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗

PubMed1997

The effect of decalcification on in situ hybridization.

In situ hybridization (ISH) for mRNA polyadenylated sequences was performed on 25 non-neoplastic and neoplastic decalcified, paraffin-embedded tissues using a poly d(T) oligonucleotide probe to assess the efficacy of this molecular diagnostic tool on decalcified tissue samples. Three commercially available decalcifying agents were used, including one EDTA-based solution (Versenate) and two hydrochloric acid-based solutions (S/P Decal, RBD). Before decalcification, the tissues were fixed in formalin for 6, 24, and 72 hours, respectively. The results of ISH performed on decalcified tissues were compared with the results from the nondecalcified control samples for each tissue using a numeric scoring system (0, negative; 4, strong positivity equal to control; 5, stronger than control). There was generally excellent reactivity when using Versenate (mean, 4.15), good reactivity with S/P Decal (mean, 3.17), and fair-to-poor reactivity with RBD (mean, 1.69) (all P values < .0001). The length of time in formalin did not affect the outcome of ISH on these tissues. We conclude that ISH can be performed with success on decalcified, paraffin-embedded tissues when using Versenate, an EDTA-based agent. Although accurate results might be obtained with S/P Decal and RBD, caution should be exercised when using these two hydrochloric acid-based solutions because they might produce false-negative results.

Chelating Agents↗

PubMed1994

Androgen receptor immunostaining and its tissue distribution in formalin-fixed, paraffin-embedded sections after microwave treatment.

We used microwave (MW) oven heat treatment to unmask human androgen receptor (AR) immunostaining in formalin-fixed, paraffin-embedded tissue. Prostate tissue was used as an AR-positive control. Tissue sections were boiled in citrate buffer in a conventional MW oven for 30 min, followed by immunostaining with a validated murine monoclonal antibody (MAb), F39.4.1, raised against a peptide included in the N-terminal domain of the 100 KD human AR. AR immunostaining was localized to the nuclei of prostate secretory epithelial cells but was weak or absent in basal cells and of variable intensity in the stromal cells. Slides exposed to less than 10 min of MW heat treatment or none at all manifested no AR immunoreactivity. Tissue morphology was well preserved. Immunohistochemical determination of AR status in a wide variety of human tissues was consistent with that previously reported by others using frozen sections. MW heat treatment of tissue samples in an excellent method of localizing AR antigenicity, enabling immunohistochemical evaluation of AR status in formalin-fixed, paraffin-embedded material.

Adrenal Glands↗

PubMed1983

Staining plastic sections: a review of problems, explanations and possible solutions.

Many of the difficulties of staining plastic embedded tissues for light and electron microscopy derive from physical exclusion of hydrophilic staining reagents by hydrophobic embedding media. Structures which stain most intensely with hydrophilic reagents usually contain less hydrophobic plastic than do non-staining structures. Such incomplete infiltration is apparently caused by exclusion of viscous, hydrophobic monomers by physically dense and/or well hydrated tissue elements. In keeping with this, generalized staining of tissues embedded in hydrophobic media does occur when hydrophobic reagents are used. Staining of plastic-free structures with single hydrophilic reagents or with sequences of such reagents, is, however, largely rate-controlled. The surprising similarity of hydrophilic and hydrophobic plastic embedding media is discussed. Limits of this simple model are explored, with a consideration of the roles of fixative and of monomer-tissue reactions.

Histological Techniques↗

PubMed1996

Oligosaccharide release from frozen and paraffin-wax-embedded archival tissues.

Altered glycosylation is a feature of many solid tissue diseases such as ulcerative colitis, Crohn's disease, cancer, and connective tissue disorders. Conventionally, oligosaccharide changes have been studied by immunohistochemical techniques. We have adapted existing techniques, developed for purified protein preparations, to allow the release of intact oligosaccharides from archival tissues, so that the oligosaccharides may be structurally characterized. In our study, sections cut from paraffin-wax blocks were dewaxed and oligosaccharides were released using hydrazine and labeled with 2-aminobenzamide. Sialylated oligosaccharides were compared by passing through a GlycoSep C divinylbenzene anion exchange resin column and neutral oligosaccharides were compared by passing through a BioGel P4 column. The oligosaccharide profiles obtained from the same fresh frozen versus archival paraffin-wax-embedded, normal liver, and tumor tissues showed remarkable similarity in terms of their sialylated and neutral structures. Matrix-assisted laser desorption ionization mass spectrometry has shown that the oligosaccharides are not affected by fixation in formalin and storage in paraffin wax. The results indicate that while the proteins themselves may be denatured, oligosaccharides are not adversely affected by fixation in formalin and storage in paraffin wax. By applying these methods, oligosaccharides from archival tissues, where the natural history of the disease has been followed, may now be liberated and structurally characterized.

Animals↗

PubMed1993

Mucin-producing pancreatic tumors: a study of nuclear DNA content by flow cytometry.

Nuclear DNA content in eight surgically resected mucin-producing pancreatic tumors (MPPT) consisting of two mucinous intraductal adenocarcinomas (MIDAC), two mucinous intraductal adenomas (MIDA), one mucinous cyst-adenocarcinoma (MCAC), and three mucinous cystadenomas (MCA) were measured by flow cytometry using paraffin-embedded tissue samples. The technique of Shutte was used for the preparation of paraffin-embedded tissue into single dissociated nuclei, while the method of Vindelov was used for staining the isolated nuclei with propidium iodine. Clinicopathologically, the four patients with MIDAC or MIDA were all male and had cystic lesions with a dilated pancreatic duct at the head of the pancreas, while the four patients with MCAC or MCA were all females and had cystic tumors at either the body or tail of the pancreas. All eight patients with MPPT had no metastasis to the regional lymph nodes and were all still alive without recurrence. In an analysis of nuclear DNA content, seven of eight patients had DNA diploid tumors while one patient with a MIDAC perforating the duodenum and choledochus had a DNA aneuploid tumor. Thus, these findings suggest that DNA diploid patterns in MPPT might be associated with a favorable prognosis in MPPT although some patients whose MPPT invaded the surrounding organs might have DNA aneuploid tumors.

Adenocarcinoma, Mucinous↗

PubMed1989

Immunophenotypic diagnosis of non-Hodgkin's lymphoma in paraffin sections. Co-expression of L60 (Leu-22) and L26 antigens correlates with malignant histologic findings.

The recent availability of monoclonal antibodies immunoreactive to T- or B-lineage antigens in formalin-fixed, paraffin-embedded tissue has permitted the adaptation of frozen-section immunodiagnostic criteria to paraffin-embedded tissue. Among a variety of reactive lymphoid processes, monoclonal antibody L26 showed a pattern of staining consistent with pan-B reactivity. Antibodies L60 (Leu-22) and UCHL-1 showed a pan-T and T-subset pattern of reactivity, respectively. In benign processes, L26 and L60 (or UCHL-1) were not coexpressed. In contrast, among 77 B-lineage non-Hodgkin's lymphomas, 42% showed aberrant co-expression of L26 and L60. The L26+/L60+ phenotype was most common in small lymphocytic lymphomas (80%) but was also noted in one third of diffuse large cell lymphomas. Expression of UCHL-1 was not identified in B-lineage neoplasms but was found, along with L60, on four of five T-lineage lymphomas studied as controls. The authors conclude that the anomalous coexpression of L60 and L26 antigens is a unique feature of B-lineage lymphomas and can be used for the immunodiagnosis of these malignancies in routinely processed tissue.

Antibodies, Monoclonal↗

PubMed1982

Classification of plastic-embedded rat seminiferous epithelium prior to electron microscopy.

Systematic electron microscopic studies of the changes which take place during the cycle of the rat seminiferous epithelium require that each of the 14 stages of the cycle are identified in the light microscope in plastic-embedded material allowing each stage to be trimmed out separately before the electron microscopic study. The classification of the 14 stages has originally been based on the morphological characteristics seen in paraffin-embedded tissue after PAS-staining. PAS-staining is less effective in plastic-embedded material, and this may be a main reason why electron microscopic findings of the seminiferous epithelium relatively seldom are related to the stages of the cycle. In the present paper a method is described by which the 14 stages of the cycle can be easily identified in the light microscope in plastic-embedded tissue. Some of the stages could be subdivided and a total of 23 stages were distinguished. This constitutes a safe method allowing accurate classification prior to electron microscopic examination of the seminiferous epithelium.

Animals↗

PubMed1990

Histological features and histochemistry of the mucous glands in ventral skin of the frog (Rana fuscigula).

The glycoconjugate components of secretory granules were analyzed in cells of mucous glands in ventral skin from Rana fuscigula. The analysis was done with standard histochemical methods on semithin glycol methacrylate-embedded tissues. The staining patterns in semithin sections were comparable to those using paraffin-embedded tissue while the cytological detail was better preserved. The mucous glands contained at least two different types of secretory cells lining the lower two-thirds of the mature gland: a principal cell type filled with dense staining secretory granules and a solitary type containing paler staining, globular secretory granules. The principal type of cell contained variable amounts of acid glycoconjugates; predominantly carboxylated but also variably carboxylated and weakly sulfated glycoproteins. Other secretory cells contained mainly neutral glycoproteins. The results indicated that the mucus is a heterogeneous substance and that one cell type may produce different secretory products. We suggested that the variability in histochemical staining might be related to the sequence of biosynthesis of the secretory granule.

Animals↗

PubMed1984

Pemphigus in the dog: comparison of immunofluorescence and immunoperoxidase method to demonstrate intercellular immunoglobulins in the epidermis.

In 3 dogs with pemphigus vulgaris and 4 dogs with pemphigus foliaceus, intercellular immunoglobulins were demonstrated in the epidermal stratum spinosum. The immunofluorescence technique on cold ethanol-fixed and paraffin-embedded tissue sections was compared with the immunoperoxidase method on formalin-fixed paraffin-embedded tissue sections. The results of both methods were identical. However, the advantage of the unlabeled antibody-enzyme method was that the same formalin-fixed tissue specimens could be used for conventional light microscopy, as well as for immunohistologic studies.

Animals↗

PubMed1983

Immunohistological localization of human lymphocyte subsets.

The technical aspects of immunohistological detection of lymphocyte surface antigens were studied in frozen and fixed, embedded tissues employing several immunoperoxidase and immunofluorescent methods. It was found that routine fixation and embedding destroyed immunostaining of surface immunoglobulin and T-cell surface antigen. Cytoplasmic immunoglobulin was preserved in fixed, embedded tissues. A comparative study of nonspecific staining in various tissues using different techniques revealed that immunoperoxidase methods generally gave lower nonspecific staining than immunofluorescent methods. The labeled avidin biotin method achieved the lowest nonspecific staining and appears to be superior to the other methods studied. A preliminary study of T lymphocyte subsets in human B-cell lymphomas and reactive lymph nodes was also performed. It was found that T-cell immunostaining patterns were altered in B-cell lymphomas in comparison to reactive lymph nodes, suggesting a more generalized disturbance of immunoregulation in these tumors.

Antibodies, Monoclonal↗

PubMed1983

The complementary role of scanning electron microscopy in renal pathological diagnosis.

Techniques applied in SEM studies of a solid organ such as the kidney are reviewed. The tissue can be prepared by razor sectioning, ethanol cryofracture and ultraplanning of polyethylene glycol embedded tissue. Tissues embedded in paraffin can also be used. Glomeruli and tubuli can be isolated from renal biopsies. A new procedure for tubular isolation is based on sequential digestion by trypsin, pepsin and Pronase E. SEM examination has proved useful in a number of renal diseases, such as glomerular diseases, hypertensive renal disease, an tubular diseases, including medullary cystic disease, adult polycystic disease, and acute tubular necrosis. Particularly in human acute tubular necrosis, SEM was helpful. SEM has also contributed to the study of the physiologically important basal-lateral surfaces of human, dog, rat, rabbit and frog renal tubules, and in particular allowed the elucidation of patterns of processes on the basal-lateral surfaces of proximal S1, S2, and S3 tubular segments, thin limbs, distal ascending and convoluted limbs and collecting ducts in human tubules.

Cytodiagnosis↗