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Successful extraction of DNA from formalin-fixed Paraffin embedded tissues is important to DNA typing for forensic purpose. In the research, 26 samples were analyzed by method of PM and HLA-DQA1 loci typing. 24 of these samples were successful. It showed that this technique and method could be applied to individual identification and paternity test by use of these sorts of samples.
Estrogen receptor (ER) was estimated immunohistochemically in formalin-fixed and paraffin-embedded tissue from the primary breast cancer in 349 postmenopausal patients with a high risk of recurrence and compared with the results of dextran-coated charcoal assay. There was a highly significant correlation between the ER classification obtained by the two methods (p less than 10(-6)). Patients ER positive according to immunohistochemical estimate had a significantly longer disease-free survival (p less than 0.001) and survival (p less than 0.001) than ER negative patients. The DCC assay showed an advantage of ER positive patients of the same magnitude. The patients, who were followed for a median of 86 months, were a subset of 1,700 patients participating in the Danish Breast Cancer Cooperative Group's randomized trial of adjuvant tamoxifen (TAM) treatment. In the presently analyzed subset of patients there were no statistically significant difference in disease-free survival (p = 0.52) or survival (p = 0.54) between patients who received adjuvant TAM and the controls. The same was true for receptor-defined subgroups regardless if the ER receptor was estimated in paraffin-embedded tissue or by the dextran-coated charcoal method. The analyzed subset might have been too small for demonstrating a positive effect of adjuvant TAM treatment.
Seven monoclonal antibodies (Moabs), recognizing melanoma-associated antigens in human tissues, were evaluated for their ability to immunohistochemically stain formalin-fixed, paraffin-embedded canine melanomas. Only 2 Moabs, designated human melanosome-specific antigen (HMSA)-1 and HMSA-5, stained routinely processed canine melanomas, staining 21/35 (60%) and 24/35 (69%), respectively. Twenty-nine of 35 (83%) melanomas tested were stained if results of the 2 Moabs were combined. Monoclonal antibody HMSA-1 also stained neoplastic cells of 10/35 (29%) tumors of nonmelanocytic origin and some neurons and salivary gland epithelial cells in normal canine tissues. However, Moab HMSA-1 staining in the nonmelanocytic tumors, consisting of small, discrete periodic acid-Schiff-positive cytoplasmic droplets, was readily distinguishable from the diffusely granular, cytoplasmic staining of melanocytic tumors. In addition to melanomas, Moab HMSA-5 stained melanocytes and some melanin-containing tumor cells of a pigmented basal cell tumor and melanocytes in normal canine skin. Monoclonal antibodies HMSA-1 and HMSA-5 immunohistochemically identified the majority of canine melanomas, had limited and distinguishable staining in normal tissues and nonmelanocytic tumors, and therefore may be a useful adjunct for the diagnosis of canine melanoma in formalin-fixed, paraffin-embedded tissues.
Paraffin embedded and formalin fixed needle biopsies of prostate cancer (PC) were used to immunocytochemically detect the p120 nucleolar protein in relation to the Gleason histological gradings (GHG), the labelling indices of proliferating nuclear immunocytochemical markers (PCNA/Cyclin, Ki-67/MIB1) and the argyrophilic nucleolar region (AgNOR) rate. The twenty-six cases of PC (6 from large histological samples and 20 from needle biopsies) were equally distributed into low (< or = 6) or high (> or = 7) GHG groups. The p120 nucleolar protein immunocytochemical reaction was randomly expressed in large histological sections but uniformly distributed without gaps in needle biopsy sections. Only on the latter were quantitative values of PCNA/Cyclin (23.2 in low and 45.3 in high GHG), Ki-67/MIB1 (13.8 in low and 43.3 in high GHG) and AgNOR (5.0 in low and 7.5 in high GHG) related to those of p120 nucleolar protein (0.8 in low and 3.8 in high GHG). The values of all these cell cycle markers increased from low to high GHG of PC, all four reaching high statistical significance between the two groups (ANOVA-two tailed p < 0.0001). The PCNA/Cyclin index showed a higher positivity than the Ki-67/MIB1 index in PC with low GHG but not in PC with high GHG. In conclusion, paraffin embedded and formalin fixed PC needle biopsies exhibit a higher diagnostic PCNA/Cyclin than Ki-67/MIB1 index for cases presenting differentiated features, whereas p120 nucleolar protein detection seems to be a suitable marker of poorer outcome of PC.
One hundred and thirty seven cases of paraffin-embedded tissue specimens biopsied each from condylomata and pseudo-condylomata of vulva and vagina were examined. Type 6b 11 human papillomavirus (HPV) DNA was detected in condylomata by in situ hybridization using digoxigeninlabelled probes and polymerase chain reaction (PCR). The positive rate was 82.5%, 83.9% respectively. It was negative in the 137 cases of pseudo-condylomata by in situ hybridization, but positive in 3 cases by PCR. For a correct diagnosis, the necessity of combining the data of clinical finding, microscopic examination and in situ hybridization or PCR is emphasized. Our results showed that pseudo-condylomata represent dysplastic appearances of vulval and vaginal mucosa, which become more marked during pregnancy and fungal or trichomonal infections, and could rather easily be misdiagnosed as condylomata.
AIMS: Non-isotopic histone mRNA in-situ hybridization (HmRNA NISH) allows detection of cells in the S phase of the cell cycle in paraffin-embedded tissue. The aim of this study was to evaluate the technique in measuring the proliferative activity of astrocytic neoplasms, and to compare the results with other proliferation estimates and patient survival. METHODS AND RESULTS: The proliferative activity of 71 routinely fixed and paraffin-embedded astrocytomas was studied by light microscopic HmRNA NISH, proliferating cell nuclear antigen (PCNA), Ki67MIB-1 and mitoses. A significant correlation was found between the labelling indices of histone mRNA (the percentage of histone-positive tumour cells: HmRNA-LI), immunohistochemical proliferation marker labelling indices (PCNA-LI: r = 0.64 and Ki67MIB-1-LI: r = 0.44) and mitotic indices (r = 0.45). The results were reproducible as judged by intra- and interobserver agreement (HmRNA/10 HPF (high power fields): r = 0.91 and r = 0.75, respectively, and HmRNA-LI: r = 0.61 and r = 0.62, respectively). The fraction of the cells in the most active cell cycle phases, as suggested by the HmRNA/Ki67 and mitotic index/Ki67 ratios, increased significantly with malignancy grade. In the univariate analysis the association of HmRNA-LI with survival was highly significant (P < 0.0001). Multivariate survival analysis showed that HmRNA-LI was an independent prognostic marker. CONCLUSIONS: Non-isotopic histone mRNA in-situ hybridization assay offers an alternative and reproducible method for measuring proliferative activity (S phase) in tumours under morphological control.
Twenty-five reactive lymph nodes, 10 palatine tonsils, and 72 B-cell non-Hodgkin's lymphomas (NHLs) of supposed follicular origin were investigated in an immunohistologic study of fixed, paraffin-embedded tissues using a panel of monoclonal antibodies reactive with antigens resistant against fixation and paraffin-embedding techniques together with polyclonal antibodies. The results concerning the microenvironmental organization of reactive lymphoid follicles confirmed that the distribution of CD21+ and CD23+ dendritic reticulum cells, vimentin+ fibroblastic reticulum cells, and CD68+ tingible-body macrophages is heterogeneous with reference to their immunostaining patterns and topographic localization within the germinal center and mantle zone. Moreover, a close microenvironmental similarity between the follicular lymphomas of supposed germinal center or mantle zone origin and their normal counterparts was noted. The study of the microenvironment of the B-zone small lymphocytic lymphoma cases, showing the same distribution patterns for the nonlymphoid cells as seen in mantle zone lymphomas, corroborated the supposed follicular origin of this unusual variant of small lymphocytic lymphoma. In conclusion, this study shows that monoclonal antibodies recognizing CD21, CD23, and CD68 antigens may be valuable additions to vimentin, S-100 protein, laminin, and type IV collagen antibodies for investigating the microenvironmental organization of lymphoid tissues in both normal and neoplastic conditions.
A method is described for the preparation of monolayer smears from paraffin-embedded tissue suitable for automated image analysis and DNA measurements. The proposed technique uses enzyme treatment and syringing for cell dispersal. Slide preparation is performed by centrifugal cytology. After Feulgen staining the quality of the monolayer smears is sufficiently high to enable visual morphologic evaluation. Automated DNA measurements using the Leyden television analysis system (LEYTAS) show coefficients of variation (CV) of 4.5% for the diploid cell population of the suspended tissue. This is approximately the same as the CV in fresh material from the same tumor. Formalin fixed trout red blood cells are used as reference cells. By applying image cytometry to paraffin-embedded tissue this method allows retrospective studies of, for instance, the significance of DNA content with regard to the behavior of a tumor.
Frozen and formalin-fixed paraffin-embedded tissue sections were studied concurrently in 17 cases of granulomatous lesions of different etiologies using antibodies recognizing either fixation-sensitive or fixation-resistant antigens. In fixed tissues, the antibodies 4KB5 and L26 for B cells and UCHL1 and MT1 for T cells gave results similar to those obtained in frozen tissues with anti-leu-12/leu-14 for B cells and T-3 for T cells. Paraffin-embedded sections from 35 additional cases of granulomatous lesions were studied retrospectively using the same markers. The combined results from all 52 cases show that granulomas can be divided into two main "families" according to the presence or absence of B cells within the granulomas: one is a B-cell-negative family of lesions to which sarcoidosis and mycobacterial infection belong; the other is a B-cell-positive family of lesions to which toxoplasmosis, granulomatous lesions of unknown significance and tumor-related sarcoid reactions belong.
OBJECTIVES: To compare the detection of HER-2 status by real-time PCR, on paraffin-embedded breast carcinomas, in respect to immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). DESIGN AND METHODS: Paraffin-embedded breast carcinomas collected from 85 patients diagnosed with early stage breast cancer were analyzed for HER-2 gene amplification by real-time PCR and CISH, as well as for HER-2 protein expression by IHC. RESULTS: HER-2 gene amplification was observed in 19 (22.4%) of 85 breast cancer patients by real-time PCR and in 19 (22.4%) of 85 patients by CISH. Strong (3+) HER-2 protein over-expression was observed in 13 (15.3%) out of 85 patients. Moreover, there were 4 out of 85 (4.7%) patients that had moderate (2+) HER-2 protein over-expression, while 68 out of 85 (80%) patients had no HER-2 protein over-expression by IHC. There were strong concordance rates between real-time PCR and IHC (79/85, 92.9%, p<0.0001) and real-time PCR and CISH (77/85, 90.6%, p<0.0001). The concordance rate between the three methods was 90.6% (p<0.0001). CONCLUSIONS: Our data show that the results obtained for amplification of HER-2 by real-time PCR on the LightCycler are comparable to those obtained by IHC and CISH.
The authors describe a newly characterized murine monoclonal antibody to the human leukocyte surface antigen, SHL-1. The antigen belongs to the leukocyte common antigen (LCA) family, and its molecular weight is about 180,000 daltons, which is similar to that of some previously characterized LCAs. The SHL-1 antigen is resistant to conventional tissue-fixation and embedding procedures. This antibody can therefore be used in the immunohistochemical staining of paraffin-embedded tissue sections. Wide screening with a sufficient number of both fresh and routinely processed paraffin-embedded tissues was done with indirect immunoperoxidase technique. With this procedure, SHL-1 labeled the majority of normal leukocytes and hematopoietic malignancies. Some B-cell malignancies were not stained with this antibody. The non-hematologic malignancies posing diagnostic problems of differentiation from lymphomas or leukemias were completely negative to SHL-1. The immunoreactivity to SHL-1 of samples from 24 leukemic patients and 15 human tumor cell lines was determined by the immunofluorescence method. Of 24 leukemic preparations, 23 were strongly reactive to this antibody. One case of B-cell leukemia did not react with SHL-1. No immunoreactivity was demonstrated in non-hematopoietic tumor cell lines. The overall reaction pattern of SHL-1 proved its usefulness in both diagnostic and research practice in hematological disorders. This antibody detected cell surface antigens of the T cell series more effectively than those of the B-cell series in terms of the positive number of cells and mean fluorescence intensity.
AIM: To investigate whether spectrin can be used as an immunohistochemical marker for erythroid precursors in routinely processed paraffin embedded bone marrow sections. METHODS: Bone marrow biopsies and clot sections were stained with rabbit antihuman erythrocyte spectrin antibodies, specific for erythroid cells as shown by western blotting and bone marrow smears, and compared to sections stained with antiglycophorin monoclonal antibodies (JC159 and Ret49f). RESULTS: Antispectrin antibodies resulted in diffuse cytoplasmic staining of early erythroblasts and membranous staining of late erythroblasts as well as erythrocytes. In haematopathological samples, immature erythroid cell clusters were clearly identified. In contrast, antiglycophorin monoclonal antibodies resulted in only membranous staining of late erythroblasts, and faint staining of early erythroblasts. CONCLUSIONS: Spectrin may be a superior marker to glycophorin for the identification of erythroid precursors in paraffin embedded sections.
This is the first comparative study of DNA quantification of oesophageal squamous cell carcinoma by flow cytometry (FCM) and image cytometry (ICM) using formalin fixed paraffin embedded tissue. The potential advantages of ICM include the identification of a reliable control cell population; avoidance of non-tumour stromal and inflammatory cell nuclei, nuclear fragments, degenerate cell nuclei and doublets, triplets etc., which are not possible with FCM using archival tissue. Twenty-eight cases, all of the same stage (stage 2a) and similar grade (well or moderately differentiated) were analysed. The cases were separated into two groups, those that had succumbed to tumour in less than 18 months (group A) and those that were tumour free at least 18 months post-resection (group B). Using ICM all 28 tumours yielded interpretable histograms by comparison to 25 of 28 using FCM. Aneuploidy was identified in 100% of cases in group A using ICM (in comparison to 73% by FCM) and in 73% of group B using ICM (in comparison to 44% by FCM). Any tumour aneuploid by FCM was also aneuploid by ICM. Nine cases aneuploid by ICM were euploid by FCM. The mean 5C exceeding rate (% of cells whose nuclei contain a DNA mass equivalent to > 5 sets of 23 chromosomes) was 21% in group A and 14% in group B (P < 0.01). Euploidy was confined to tumours of those patients disease free for more than 18 months. The conclusions of this study are that: firstly, ICM is superior in its yield of interpretable histograms to FCM using formalin fixed paraffin embedded tissue; secondly, ICM is more sensitive in the identification of aneuploid stemlines than FCM; and thirdly, euploid tumours (as detected by ICM) appear to have a better prognosis than aneuploid tumours of similar stage and grade.
Human papilloma virus (HPV) DNA sequences have been detected in paraffin-embedded tissue using an enzymatic in vitro amplification technique known as the polymerase chain reaction. Amplification of a HPV DNA sequence before its detection with a cDNA probe significantly increases the rapidity as well as the sensitivity of detection such that a single 5-10-micron thick paraffin-embedded tissue section can be analyzed within 24 h. The assay specifically detected HPV 16 or 18 without crossreactivity with HPV 6 or 11. As few as 20 viral copies could be detected. The rapid and sensitive analysis of HPV in normal and pathological tissues using this technique may contribute significantly to identifying the role of HPV as a risk factor in carcinoma.
Histology and immunohistochemistry are important tools in the study of human diseases and their respective animal models. The study of mouse models has been hampered by the absence of a large set of mouse-specific antibodies adapted to paraffin-embedded tissues. A total of 196 antibodies were tested on paraffin-embedded mouse tissues preserved in five different fixatives (Fekete's acid-alcohol-formalin, 10% neutral buffered formalin, 4% paraformaldehyde, IHC Zinc Fixative, and Bouin's fixative). The antibodies were targeted to proteins of the cytoplasm (n = 100), plasma membrane (n = 48), nucleus (n = 36), extracellular compartment (n = 5), cytoplasm/cell membrane (n = 4), and viral proteins (n = 3). A total of 83 antibodies provided an adequate signal to noise ratio. Of these, adequate labeling required heat-mediated epitope retrieval or enzymatic digestion for 32 and 8 antibodies, respectively. Epitope recognition was best for tissues fixed with Fekete's acid-alcohol-formalin. However, some proteins could be detected only in IHC Zinc Fixative, confirming that there is no single fixative suitable for the preservation of all epitopes. Four of 13 antibodies that failed to label their cellular targets on tissue sections successfully labeled whole-mount tissues, indicating that tissue processing plays an important role in epitope degradation. Regularly updated information on immunohistochemistry of normal and neoplastic mouse tissues is accessible online at (http://tumor.informatics.jax.org); links to antibody suppliers' web sites are provided.
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.
Analysis of clonality by X chromosome inactivation has proven to be a powerful strategy in the study of neoplastic and preneoplastic disorders (P. J. Fialkow, Biochim. Biophys. Acta, 458: 283-321, 1976; B. Vogelstein et al., Cancer Res., 47: 4806-4813, 1987). Recently, the gene for the androgen receptor has been shown to be a highly polymorphic locus in which methylation of DNA correlates with inactivation of one or the other X homologue (R. C. Allen et al., Am. J. Hum. Genet., 51: 1229-1239, 1992). Unlike other loci used for analysis of X inactivation, the methylation sites within the androgen receptor gene lie close to the region of DNA containing the polymorphism. Consequently, it should be possible to use methylation-sensitive restriction enzymes and polymerase chain reaction to study differential methylation among alleles of this gene in formalin-fixed and paraffin-embedded archival tissue specimens. To investigate this question, we performed clonal analysis on a variety of randomly selected, formalin-fixed, paraffin-embedded tumor tissues from 15 women. Thirteen of the women were found to be heterozygous for polymorphisms at the androgen receptor locus. Among these women, 11 tumors were clearly clonal in assays of methylation of the androgen receptor gene. Interpretation of results for the remaining two cases was complicated because of a skewed pattern of X chromosome inactivation found in normal control tissues. We conclude that analysis of methylation in the androgen receptor gene should allow study of clonality in most formalin-fixed, paraffin-embedded tissue specimens from women, including small preneoplastic lesions or rare conditions for which sufficient material is not available for analysis by other techniques.
Many techniques have been described for the extraction of DNA from paraffin-embedded tissues. Numerous efforts have been directed at simplification of these methods for rapid analysis using PCR. One disadvantage to some of the simpler procedures is inefficient PCR amplification, and for more involved ones using phenol/chloroform extraction, reduction in the yield of DNA. In the present study we report the use of a novel salting-out procedure that was utilized to extract DNA from 259 separate microdissection specimens of formalin-fixed, paraffin-embedded tissue sections. These sections were derived from 97 patients with tumors of the ampulla of Vater resected between 1965 and 1995 at our institution. The mean DNA yield was 22.75 micrograms (median 13.2 +/- 30.25) and the mean 260/280 absorbance ratio was 1.68 (median 1.70 +/- 0.25). All specimens (259/259) were successfully used to amplify K-ras exon 1 by a nested PCR technique. These results indicate that this DNA extraction method produces good yields of quality DNA, even from specimens several decades old.