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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.
Array-based comparative genomic hybridization (aCGH) is a powerful, high-throughput tool for whole genome analysis. Until recently, aCGH could only be reproducibly performed on frozen tissue samples and with significant tissue amounts. For brain tumors however, paraffin-embedded tissue blocks from small stereotactic biopsies may be the only tissue routinely available. The development of methods to analyze formalin-fixed, paraffin-embedded (FFPE) material therefore has the potential to impact molecular diagnosis in a significant way. To this end, we constructed a BAC array representing chromosomes 1, 7, 19, and X because 1p/19q deletion and EGFR gene amplification provide clinically relevant information for glioma diagnosis. We also optimized a two-step labeling procedure using an amine-modified nucleotide for generating aCGH probes. Using this approach, we analyzed a series of 28 FFPE oligodendroglial tumors for alterations of chromosomes 1, 7, and 19. We also independently assayed these tumors for 1p/19q deletion by fluorescence in situ hybridization and by loss of heterozygosity analyses. The concordance between aCGH, standard loss of heterozygosity and fluorescence in situ hybridization was nearly 100% for the chromosomes analyzed. These results suggest that aCGH could offer an improved molecular diagnostic approach for gliomas because of its ability to detect clinically relevant molecular alterations in small FFPE specimens.
The use of glycol methacrylate (GMA) avoids some technical artifacts, which are usually observed in paraffin-embedded sections, providing good morphological resolution. On the other hand, weak staining have been mentioned during the use of different methods in plastic sections. In the present study, changes in the histological staining procedures have been assayed during the use of staining and histochemical methods in different GMA-embedded tissues. Samples of tongue, submandibular and sublingual glands, cartilage, portions of respiratory tract and nervous ganglion were fixed in 4% formaldehyde and embedded in glycol methacrylate. The sections of tongue and nervous ganglion were stained by H&E. Picrosirius, Toluidine Blue and Sudan Black B methods were applied, respectively, for identification of collagen fibers in submandibular gland, sulfated glycosaminoglycans in cartilage (metachromasia) and myelin lipids in nervous ganglion. Periodic Acid-Schiff (PAS) method was used for detection of glycoconjugates in submandibular gland and cartilage while AB/PAS combined methods were applied for detection of mucins in the respiratory tract. In addition, a combination of Alcian Blue (AB) and Picrosirius methods was also assayed in the sublingual gland sections. The GMA-embedded tissue sections showed an optimal morphological integrity and were favorable to the staining methods employed in the present study. In the sections of tongue and nervous ganglion, a good contrast of basophilic and acidophilic structures was obtained by H&E. An intense eosinophilia was observed either in the striated muscle fibers or in the myelin sheaths in which the lipids were preserved and revealed by Sudan Black B. In the cartilage matrix, a strong metachromasia was revealed by Toluidine Blue in the negatively-charged glycosaminoglycans. In the chondrocytes, glycogen granules were intensely positive to PAS method. Extracellular glycoproteins were also PAS positive in the basal membrane and in the region occupied by the lamina externa and reticular fibers surrounding each smooth muscle cells of the blood vessels. In the epithelial cells of the respiratory tract, acid and neutral mucins were histochemically detected by AB and PAS methods, respectively. Moreover, granules containing acid and neutral mucins were revealed in purple by AB and PAS concomitantly. In the sublingual gland sections, a distinct affinity of acid mucins by AB (in turquoise-blue) and collagen fibers by Picrosirius (in red) was obtained when these methods were combined. Although some routine dyes used in paraffin sections have showed a weak stain in historesin sections, our results showed that different dyes could be applied in GMA sections if modified staining procedures were assayed. Therefore, appropriate staining contrast and, thus, detection of one or different substances in a same section can be acquired in association to the good morphological resolution provided by GMA.
AIMS: The study was undertaken to demonstrate wild-type p53 in normal paraffin-embedded tissues using two widely used antibodies, DO7 and 1801 and two different antigen retrieval buffer systems. METHODS AND RESULTS: Formalin-fixed paraffin-embedded normal tissue samples were obtained from the archives of the John Radcliffe Hospital, Oxford. Antigen retrieval was performed by microwaving using two different buffer systems: (i) the commercially available Dako target retrieval solution (Cat. no. 1699) (pH 9.8-9.9), (ii) freshly prepared buffer consisting of 0.1 m EDTA with 0.1% Tween pH 6.0, and (iii) freshly prepared buffer consisting of 0.1 m EDTA with 0.1% Tween pH 8.0. Staining was performed with DO7 and 1801 antibodies using the Dako Envision kit (peroxidase/DAB). DO7 antibody elicited strong nuclear staining in the mucosal cells of the small and large intestine, lymphoid cells, decidua, neurones such as Purkinje cells of the cerebellum, glandular epithelial cells and stromal cells of the prostate, cardiac myocytes and bronchial epithelial cells. Cytoplasmic staining was noted in Purkinje cells, glandular epithelium of prostate, exocrine pancreas and renal tubular epithelium. The 1801 antibody did not produce staining in any of these tissues. CONCLUSIONS: Our study demonstrates the presence of p53 in normal paraffin-embedded tissue with nuclear and/or cytoplasmic localization in some instances. In our view, DO7 appears to be better suited for such detection.
The distribution of different cells of the immune system has been studied in formalin-fixed paraffin-embedded tissues from conventionally reared healthy pigs, using immunohistological techniques. The samples collected were: lungs, tonsils, lymph nodes (mediastinal, mesenteric, inguinal and submandibular), pancreas, spleen, liver, kidney, adrenal gland, ileum and stomach. A total of six primary antibodies anti-CD3, anti-CD79alpha, Mac 387, anti-lysozyme, anti-CD45RA (3C3/9) and anti-SLA-II-DQ (BL2H5) were used with a standard avidin-biotin peroxidase (ABC) method. Anti-CD3 and anti-CD79alpha mAb-reacted, respectively with cells located in T cell areas and B cell areas. Mac 387 recognised circulating polymorphonuclear leukocytes, while anti-lysozyme-stained resident macrophages in all tissues. 3C3/9 and BL2H5, were assessed in formalin-fixed paraffin-embedded tissues for the first time. 3C3/9 identified B lymphocytes, in primary follicles and mantle zones, a subpopulation of T cells, especially located in the marginal zone of the spleen and a variable number of immunoblasts, in the germinal centres. BL2H5 reacted with B cells in the mantle zones of the follicles of lymphoid tissues, with dendritic and interdigitating cells in all studied lymphoid tissues and with a variable number of resting and activated T cells in the periarteriolar lymphoid sheath (PALs), marginal zone and red pulp of the spleen. Furthermore, it stained Kupffer and perivascular macrophages in the liver. This study represents a detailed histological study of the distribution of the most important subpopulations of immune system cells in conventional, healthy pigs. In our view, these tools should be useful for future comparative studies in disease conditions.
OBJECTIVE: Detection of aberrantly accumulated p53 protein by immunohistochemistry may have prognostic significance in many human neoplasms. We wished to identify a technique applicable to formalin-fixed, paraffin-embedded sections that would reliably yield results equivalent to frozen-section immunohistochemistry. DESIGN: We compared the frequency of p53 immunostaining obtained by applying monoclonal antibodies PAb1801, DO7, or DO1, a 1:1 PAb1801-DO7 cocktail, and a 1:1 PAb1801-DO1 cocktail to fresh-frozen and formalin-fixed, paraffin-embedded tissues from 36 lung and upper aerodigestive-tract carcinomas. With the formalin-fixed tissues, we compared pepsin predigestion with microwave irradiation in citrate buffer as means of enhancing the sensitivity of p53 detection. SETTING AND PATIENTS: All tissues were obtained from surgical resections of tumors, from patients who underwent surgery at the Minneapolis Department of Veterans Affairs Medical Center between 1990 and 1992. MAIN OUTCOME MEASURES: The sensitivity of different paraffin section techniques for immunohistochemical detection of accumulated p53 protein was determined in reference to the optimal frozen section method (defined as the method that yielded the greatest number of p53-positive cases in frozen tissue). RESULTS: Microwave antigen retrieval markedly enhanced staining with PAb1801 and DO7 in paraffin sections, as compared with pepsin predigestion and no pretreatment. This technique was 100% sensitive relative to the optimal frozen tissue method. In contrast, staining with DO1 alone was not enhanced by microwaving. CONCLUSIONS: Microwave pretreatment in conjunction with the use of either PAb1801 or DO7 is highly efficacious in the immunohistochemical detection of aberrant p53 expression in formalin-fixed, paraffin-embedded tissues.
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.
DNA content of 36-non-Hodgkin's lymphomas was analyzed by flow cytometry (FCM) and cytogenetics (CG), 21 in fresh and 15 in paraffin-embedded tissue. The results of both techniques were coincident in 60% of the fresh tissue samples and in 45% of the paraffin-embedded ones, the reason for this difference could be the poor resolution of DNA histograms from paraffin-embedded tissue. All samples judged as aneuploid by FCM were aneuploid also by CG. Some samples with a hyperdiploid population by CG gave a diploid population by FCM with a 'false' high DNA-synthesis (S) fraction. From a technical point of view, CG and FCM have to be performed on the same fresh tissue.
OBJECTIVE: To examine the alteration and significance of the DPC4 gene in paraffin-embedded tissues of pancreatic carcinomas. METHODS: Polymerase chain reaction and single-strand conformation polymorphism analysis were used to search for deletions and mutations in the DPC4 gene in 46 cases of pancreatic carcinomas. RESULTS: Thirteen of forty-six (28.3%) cases were found to have homozygous deletions in exon 1, 2, 3, 4, 8 and 11. One was in exon 11, one in exon 1 and 11, one in exon 2 and 3, one in exon 3 and 8, one in exon 1, 2 and 8, one in exon 2, 4 and 11, one in exon 3, 4 and 11, three in exon 3, 4 and 8, one in exon 2, 3, 4, and 8, one in exon 2, 3, 8 and 11, one in exon 2, 3, 4, 8 and 11. Intragenic mutations were found in 10 of 46 cases (21.7%). One case was in exon 1, one in exon 2, three in exon 8, four in exon 11, and one in exon 4 and 11. The total frequency of intragenic changes of DPC4 in paraffin-embedded tissues was 45.6% (21/46). CONCLUSION: Inactivation of tumor-suppressor gene DPC4 may play an important role during the tumorigenesis of pancreatic carcinomas.
The aim of the work was a selection of the best method of DNA extraction from soft tissues, and than application of this method to the fixed and paraffin wax embedded tissue. Isolated DNA was amplified using PCR method. Amplification was done for amelogenin locus (sex specific sequences) and some polymorphic systems like mini- and microsatellites. It was found that it is possible to amplify 788 and 977 bp long amelogenin fragments, using DNA isolated from fixed and paraffin wax embedded tissue stored for two years. The case of identification of unknown person (victim of fire at Gdańsk Shipyard show room) on basis of DNA isolated from soft tissue was presented.
The ability to isolate DNA from archived human serum, plasma and paraffin-embedded human tissues enhances opportunities to study breast, lung and other cancer risk factors. We report herein a simple and fast protocol for the extraction of genomic DNA from these sources. Using a phenol-based extraction method, the recovery for DNA is quantitative and reproducible. DNA yields in serum (250 microl) were between 162 and 1060 ng (n = 18 subjects), in plasma (250 microl) were between 165 and 375 ng (n = 5 subjects) and in embedded tissues (5-microm thick sections for ethanol fixed, and between 5- and 20-microm sections for formaldehyde fixation) were between 1 microg and 11.7 microg (n = 32 subjects). The extraction method was combined with newly designed PCR-based assays for cancer susceptibility marker genes such as CYP1A1 (exon 7), CYP2E1 (Dra1, Rsa1), GSTM1 and NAT2 [NAT2*5A (C481T), NAT2*6A (G590A), NAT2*7A (G857A)]. Genotyping results from the serum and paraffin-embedded tissues compared favorably to results from archived freshly frozen tissues, where concordance was 98% for serum, 100% for ethanol-fixed embedded tissues, and 97% for formaldehyde-fixed and paraffin-embedded tissues. This facile method will allow for the use of archived tissue samples of prospective cohort and other studies where intact DNA was not previously available.
We have developed a simple, rapid method for isolating cells from a block of formalin-fixed, paraffin-embedded tissue specimen for laser scanning cytometric (LSC) DNA analysis by using a grater. The scraping-like tissue samples were obtained by grating a paraffin-embedded tissue block. The grated samples were collected, put into a small plastic tube, and deparaffinized with xylene. Subsequently, the samples were immersed in 100% ethanol to remove the xylene. After using a syringe with a 26-gauge needle and filtering through 40-microm nylon mesh, the cells suspended in ethanol were dropped directly onto a glass slide. As a result, isolated cells adhered tightly to the glass slide. The slides mounted with isolated cells were treated with 0.1% pepsin in 0.1 N HCl for 1 h at 37 degrees C and then 0.1% RNase for 10 min at room temperature. The slides were dipped in propidium iodide (25 microg/ml) to stain DNA and sealed with nail varnish. The coefficients of variation for histograms were small enough to detect an aneuploid peak close to the diploid peak.
A method is described for the preparation of monolayer smears from paraffin-embedded tissue. The smears are suitable for automated image analysis and DNA measurements while still allowing interpretation of nuclear morphology. The proposed technique uses enzyme treatment and syringing for cell dispersal. The preparation of cell monolayers is performed by cytocentrifugation. After staining the specimens with gallocyanin, nuclear DNA can be measured. Automated DNA measurements using the Leyden Television Analysis System (LEYTAS) showed coefficients of variation of 4.5% for the diploid cell population of suspended benign tissue. After DNA measurements, the specimens are counterstained using orange G and eosin. Since gallocyanin has spectral properties similar to those of hematoxylin, the obtained end product is comparable to specimens stained according to the routinely used Papanicolaou procedure. Using this technique, image cytometry can be applied to paraffin-embedded tissue in combination with conventional cytomorphologic study of the cells.
OBJECTIVE: To investigate the feasibility of detecting SYT-SSX fusion gene in paraffin-embedded tissues and its diagnostic significance for synovial sarcoma (SS). METHODS: Formalin-fixed, paraffin-embedded samples of 38 cases of SS and 40 cases of control tumors (including spindle cell sarcoma and metastatic adenocarcinoma) were retrived from archival materials. SYT-SSX fusion transcripts were detected in all samples by RT-PCR. House-keeping gene Porphobilinogen Deaminase (PBGD) was used to detect the quality of mRNA. RESULTS: PBGD mRNA was detected in 64 of the 78 tumor cases (82.1%). SYT-SSX fusion transcripts was detected in 33 of the 38 synovial sarcoma specimens. No SYT-SSX mRNA expression was found in control tumors. SYT-SSX mRNA expression rate reached 89.2% (33/37) in synovial sarcomas after exclusion of 1 case which was negative for both SYT-SSX and PBGD. In 33 SYT-SSX positive synovial sarcomas, 22 had SYT-SSX1 and 6 had SYT-SSX2 fusion transcripts. In 5 cases, the fusion type could not be distinguished. There was an association between SYT-SSX fusion type and histologic subtype. All 10 biphasic synovial sarcoma cases had the SYT-SSX1 fusion, whereas 12 of 18 monophasic synovial sarcoma had the SYT-SSX1 and 6 had the SYT-SSX2 fusion gene (P < 0.05). CONCLUSIONS: (1) Detection of SYT-SSX fusion gene in paraffin-embedded tissues was found to be a sensitive and specific method for the diagnosis and differential diagnosis of synovial sarcoma. (2) There was an association between SYT-SSX fusion type and histologic subtype.SYT-SSX2 fusion transcript could only be found in monophasic synovial sarcomas.
Although many investigators have demonstrated a relationship between argyrophilic nucleolar organizer regions (AgNORs) and Ki-67 expression in solid tumors, no previous studies have simultaneously assessed the relationship between AgNOR and Ki-67 expression in paraffin-embedded tissue. We describe a method for simultaneous demonstration and quantitation of Ki-67 and AgNORs in routinely processed tissue. The Ki-67 equivalent monoclonal antibody MIB1, which can detect proliferative activity in routinely processed tissue with microwave heating, was employed. Fresh human tonsil tissue was fixed in formalin and embedded in paraffin for Ki-67/AgNOR dual staining. Image analysis was employed for quantitation of AgNOR staining in Ki-67-positive and Ki-67-negative nuclei. The double-staining procedure had no measurable effect on the individual parameters: Ki-67 labeling index, mean AgNOR number (NN), and NOR percentage nuclear area (NPNA). However, microwave processing for Ki-67 immunostaining significantly increased nuclear area (NA) and AgNOR area (AA). A significant difference was found between Ki-67-positive and Ki-67-negative cells for NN (p < 0.001), NA (p < 0.001), AA (p < 0.001), and NPNA (p < 0.001). These results suggest a direct relationship between AgNOR and Ki-67 in paraffin-embedded tissue.
We evaluated the effect of six different histologic stains on the productivity of PCR amplification of DNA isolated from paraffin-embedded tissue samples. The tissue was collected from glass slides by microdissection techniques, whereby tiny portions of tissue are visually identified through a microscope and selectively resected for subsequent DNA extraction and PCR amplification. We found that the success of PCR amplification depended on the type of histologic stain that was used to facilitate microscopic visualization of the undeparaffinized tissue section. The best results were obtained with methyl green and nuclear fast red, while Wright's stain yielded less PCR product. Two other stains, Evans blue and light-green SF yellowish (also known as the counterstain for geomori methenamine silver stain) yielded sufficient PCR products; however, their staining characteristics did not afford satisfactory visualization of nuclear chromatin to discriminate between benign and malignant cells. Our most significant finding was that a commonly used histologic stain, hematoxylin, failed to produce DNA templates that could be consistently amplified by PCR. In conclusion, it is prudent to avoid hematoxylin stains when preparing tissues as starting material for PCR. Among the remaining five stains that were evaluated, the best choice depends on the differential staining characteristics of the cells to be dissected.
Although several factors affecting the sensitivity of polymerase chain reaction (PCR) amplification from formalin-fixed tissues have been investigated mostly by experiments, the feasibility of archival formalin-fixed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2-3 days and 14% (4/28) of the samples fixed for 4-6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified p53 products from archival tissues using a non-isotopic method, temperature gradient gel electrophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding just after 1 day or less fixation for subsequent use in PCR amplification.
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