Depositing archived paraffin tissue core biopsy specimens in paraffin tissue microarrays using a paraffin tissue punch modified with a countersink

Paraffin tissue microarrays (PTMAs) introduced by Kononen et al in 1998 have become a widely used technique in routine pathology and even more so in research. Kononen used a tissue puncher/arrayer (Beecher Instruments, Sun Prairie, WI, USA) to take paraffin tissue core biopsy specimens (PTCBs) of 0.6-2 mm in diameter from routine paraffin tissue blocks and transfer them to another paraffin block with up to 1000 holes. As pointed out by Mengel et al, however, it is not possible to use the Kononen/Beecher system to construct PTMAs out of archived PTCBs. To overcome this drawback in the extremely popular Beecher system, the paraffin tissue punch was modified by incorporating a conical 4 mm deep countersink. This countersink was milled with a conical precision cutter that can be bought in an ordinary hardware store (cost <5 US dollars). The countersink facilitates the insertion of an archived PTCB into the paraffin tissue punch and enables the construction of PTMAs with previously archived PTCBs using the widely distributed Beecher system. Moreover, this paraffin tissue punch can be used for other systems to create PTMAs, such as the low-budget systems designed by Vogel.     

Flow cytometric analysis of cellular DNA content in paraffin wax-embedded specimens of canine mammary tumours

Flow cytometric analysis of DNA content was performed on 72 formalin-fixed, paraffin wax-embedded samples of canine mammary tumours. DNA content was correlated with histological appearance, stage of the tumour, and survival after mastectomy. All benign tumours (14 cases) were diploid. Amongst the malignant tumours, a higher incidence of aneuploid tumours (50 per cent) was observed in cases with lymph nodal metastases in comparison with those without metastases (26 per cent). All dogs with benign tumours survived for at least two years after mastectomy and so did 26 (81 per cent) of 32 DNA diploid malignant cases without metastases. A 67 per cent (eight of 12 cases) survival rate was found in the dogs with DNA aneuploid malignant tumours without metastases, while a very low survival rate (17 per cent) was observed in the group with malignant tumours with metastases, independently of the extent of ploidy.     

Currarino syndrome with pelvic neuroendocrine tumor diagnosed by post-mortem genetic analysis of tissue specimens

Currarino syndrome (CS) is an autosomal dominant disorder of embryonic development characterized by the triad of anorectal abnormalities, partial sacral agenesis, and presacral mass. Mutations of the HLXB9 gene have been identified in most CS cases, but a precise genotype-phenotype correlation has not been described so far. We report the clinical case of a 44-year-old Caucasian woman with malignant neuroendocrine transformation of a pre-sacrococcygeal mass combined with bicornuate uterus, dermoid cyst of the ovaries, and chronic constipation. After the patient died, a sacrococcygeal malformation and anterior meningocele were diagnosed in her 22-year-old son. CS diagnosis was then retrospectively confirmed by molecular analysis of normal and pathological tissue specimens of the mother, with identification of a HLXB9 mutation (c.727C>T; p.R243W). CS should be considered, and genetic counseling recommended, to all patients with presacral masses. Since malignant neuroendocrine transformation of presacral mass in CS is a possible complication, even thought rare, close follow up in these patients is advisable.     

Increasing the efficiency of paraffin tissue microarrays by packing the paraffin tissue core biopsies in a honeycomb pattern.

Paraffin tissue microarrays (PTMAs) are blocks of paraffin holding up to 1000 paraffin tissue core biopsies (PTCBs) for high throughput molecular analysis. The number of PTCBs in a PTMA depends on the surface area of the PTMA, the diameter of and the distance between the PTCBs and on their arrangement inside the assembled PTMA. The PTCBs are usually arranged in a rectangular x-y pattern of rows and columns. This design facilitates the construction of a PTMA because the operator simply turns the wheels of an x-y-table for a set, unchanging distance. The evaluation of the stained sections is also relatively easy. However, this rectangular arrangement means wasted space in the PTMA. To reclaim this space, the PTCBs could be arranged in a honeycomblike pattern. For every 8 rows in the conventional rectangular arrangement, 1 additional row of PTCBs can be packed. However, the researcher has to become accustomed to this uncommon arrangement when filling and evaluating the PTMA. Automatic slide readers and specially designed computer programs for the digital evaluation of the PTMAs can be helpful. In summary, the arrangement of PTCBs in a honeycomblike pattern increases the density and number of specimens stored in a PTMA, thereby enhancing its efficiency.     

Southern blot analysis of DNA extracted from formal-saline fixed and paraffin wax embedded tissue

Model experiments were designed to assess whether DNA could be recovered from formol-saline fixed peripheral blood lymphocytes and tonsil tissue for use in Southern blot gene analysis. Lymphocytes were fixed for 30 min and tonsil for 6 and 24 h, then paraffin embedded. High molecular weight DNA was extracted by prolonged digestion (2-7 days) with proteinase K or protease XXIV in the presence of 1 per cent sodium dodecyl sulphate. Restriction, transfer and hydridization were possible without modification of standard procedures. Multiple copy sequences were demonstrated using Mspl and Bst Nl restriction and hybridization for the Y chromosome (pHY 2.1 probe), single copy genes using EcoRI and BamHl restriction for the T-cell receptor beta chain (T beta probe), and Bgl II and Hind III for the immunoglobulin heavy chain (JH probe). Identical banding to unfixed tissue was achieved except when 24 h fixed extracts were used. With these, demonstration of the 24 KB Bam Hl/T beta and 9.2 KB Hind III/JH bands was not obtained. These findings suggest that as the fixation time is extended, alterations to DNA will limit the available range of restriction enzyme/probe combinations. However, with careful choice of these the extraction of DNA from formalin fixed and paraffin embedded pathological tissue for Southern blotting should be profitable.     

Use of paraffin wax embedded bone marrow trephine biopsy specimens as a source of archival DNA.

AIMS: To evaluate the use of DNA extracted from paraffin wax embedded trephine biopsy specimens as a source of archival nucleic acid for Southern hybridisation studies and polymerase chain reaction (PCR) amplification. METHODS: DNA was extracted simultaneously from paraffin wax embedded bone marrow trephine and lymph node biopsy specimens after incubation of tissue sections for one to five days in lysis mix and proteinase K with periodic sampling. DNA from 10 trephine biopsy specimens was subjected to PCR amplification using HLA-DPB primers to determine whether the extracted nucleic acid was of sufficient quality to permit amplification. RESULTS: For most specimens the greatest yield of high molecular weight DNA was seen after five days' incubation. Unlike lymph node material the quality of extracted nucleic acid and the quantity obtained from trephines was insufficient for Southern blot analysis. PCR amplification using HLA-DPB primers yielded positive results in six out of 10 trephine biopsy specimens. CONCLUSIONS: DNA extracted from paraffin wax embedded trephine biopsy specimens is largely degraded and unsuitable for Southern analysis but serves as a useful source of archival nucleic acid for PCR amplification.     

Diagnosis of nasopharyngeal tuberculosis by detection of tuberculostearic acid in formalin fixed, paraffin wax embedded tissue biopsy specimens.

The use of gas chromatography and mass spectrometry with selected ion monitoring detected tuberculostearic acid (TBSA) in 10 of 12 formalin fixed, paraffin wax embedded nasopharyngeal and head and neck biopsy specimens from patients with confirmed tuberculosis and carcinoma, and in one of 50 control specimens (giving a sensitivity of 83% and a specificity of 98%). The two false negative cases had very small tissue fragments and the patient with a false positive result may have had pulmonary tuberculosis. Tuberculostearic acid (TBSA) was also detected in nine of 16 specimens from the head and neck region with non-caseating granulomas suspected, but not confirmed, to be tuberculosis. It is concluded that nasopharyngeal tuberculosis is relatively common in Hong Kong and should be considered when biopsy specimens show granulomas. The detection of TBSA in tissue biopsy specimens is a useful, rapid method for the diagnosis of tuberculosis and other mycobacterial infections, and can be conveniently performed within two days on formalin fixed and paraffin wax embedded material.     

Application of PCR amplification of DNA from paraffin embedded tissue sections to linkage analysis in familial retinoblastoma.

A family segregating for the retinoblastoma predisposition gene has been analysed using the polymerase chain reaction to exclude their son as being an affected gene carrier. The unusual feature of this family is that the affected child, who would ordinarily have been used to establish phase in a linkage study, died as a result of developing a second tumour some years ago. The only tissue available from this child was a paraffin embedded, formalin fixed histopathological specimen from the second tumour. It was possible to isolate DNA from this tissue and amplify the DNA flanking two polymorphic restriction enzyme sites to establish alleles which cosegregated with tumour predisposition. Archival material can now be used to offer families such as this prenatal screening to provide informed genetic counselling.     

Localisation of enteropathogens in paraffin embedded tissue by immunoperoxidase

An indirect immunoperoxidase technique has been used to identify enteropathogens in formol-sublimate fixed paraffin embedded sections of calf intestine. Infections with bovine rotavirus, bovine coronavirus, Newbury agent SRV -1, and K99+ Escherichia coli have been detected in the intestines from experimentally infected and conventially reared diarrhoeic or normal calves. The ability to visualize enteropathogenic agents in histological sections resulted in the demonstration of virus infected cells at sites not previously shown to be infected using the immunofluorescence technique.     

Warthin's tumor with malignant lymphoma. DNA analysis of paraffin-embedded tissue

The authors report a case of malignant lymphoma, small lymphocytic type, involving the lymphoid stroma of a Warthin's tumor of the parotid gland. This was confirmed by the presence of a monoclonal immunoglobulin heavy chain gene rearrangement, demonstrated by Southern blot hybridization of DNA extracted from paraffin-embedded tissue. Similar techniques showed only germline immunoglobulin gene bands in two control cases of Warthin's tumor.     

Chromaticity analysis of immunostained tumor specimens.

In order to evaluate the correlation between immunohistochemical and morphometric data on the same histological sections, we have developed a flexible color image analyzer (Microcomputer-Assisted Picture Processing System type II, MAPPS-II), and established an effective method to analyze the immunostained colorectal neoplasms based on the color recognition theory of human visual system. Colorectal adenomas and adenocarcinomas were stained with a monoclonal antibody C 12, which recognizes abnormal H antigen, using Avidin-Biotin method and diaminobenzidine (DAB, brown dye). Nuclei were stained with Hematoxylin (blue dye). Density and colorimetric analyses revealed two results: (A) Separation of immunostained brown area from blue nuclei was best performed by plotting the representative sample areas on a standard chromaticity diagram, which displays the hue and saturation of colors simulating color of human visual system. (B) After separation of immunostained areas, usual density analysis was useful for the assay of nuclear morphometric information. Using these programs, normal mucosa was negative for C 12, and showed low nuclear/cytoplasmic ratio (NCR). Adenoma was occasionally focally positive for C 12, and showed medium NCR. Carcinomas were C 12 positive, and showed high NCR. Our method permits nuclear counterstaining by hematoxylin instead of low contrast methyl green, which will widen the field of combined immunohistochemical and morphometric study.     

Genome‐wide DNA methylation analysis of formalin‐fixed paraffin embedded colorectal cancer tissue

Formalin fixation and embedding of clinical tissue samples in paraffin is a common method for archiving biological material. These samples are often well annotated and provide an invaluable resource for research. However, this process of fixation and storage of tissue leads to DNA damage and fragmentation. The use of DNA from formalin fixed, paraffin‐embedded (FFPE) tissue to interrogate methylation levels on a genome‐wide scale can pose challenges. We compared fresh and matched FFPE tissue DNA samples using the Illumina Infinium HD Human Methylation 450K BeadChip platform with a companion application for repair and “restoration” of DNA from FFPE tissue. Our results showed good correlation between fresh and FFPE sample data. FFPE DNA captured 99% of the CpG sites on the array on average. Significant cancer subgroups based on the CpG island methylator phenotype (CIMP) were clearly distinguished for both fresh and FFPE sample sets with cluster and scaling analysis. The DNA methylation status for the five standard CIMP panel genes which was evaluated for all samples by the MethyLight assay was correctly assigned in both fresh and FFPE samples by the array data. We conclude that the “restoration” method followed by assay on the Infinium HD Human Methylation 450K microarray can produce good quality data for DNA from FFPE samples. © 2014 Wiley Periodicals, Inc.     

Demonstration of human papillomavirus types in paraffin processed tissue from human ano-genital lesions by in-situ DNA hybridisation

A sensitive in situ hybridization technique for the demonstration of human papillomavirus (HPV) employing a biotin-streptavidin polyalkaline phosphatase complex has been successfully applied to formalin-fixed, paraffin processed tissue obtained from a selected series of patients with ano-genital lesions. Benign condylomata from males and females showed the presence of HPV 6 and 11. Two cases of vulval intraepithelial neoplasia showed HPV 16. Four cases of squamous carcinoma of the anal canal also showed HPV 16 in the tumour or in the adjacent pre-invasive neoplastic epithelium. A case of malignant transformation in a cervical condyloma was associated with HPV 6 and 11. This technique permits the retrospective evaluation of routinely processed material thus widening the investigative spectrum for HPV.     

DNA-Feulgen-cytophotometric analysis of single cells isolated from paraffin embedded tissue

This study describes a method for the isolation of cells from paraffin embedded tissues for DNA-Feulgen-cytophotometric measurements. The relevance of the method used is demonstrated by the analysis of the DNA-distribution pattern of cells isolated from an alveolar sarcoma of the soft tissues of a 10-year old girl. The comparison between freshly prepared imprint preparations and specimens after pepsin--extraction clearly shows, that the preparation mode used did not influence the relative DNA-content. The mean values of DNA/nuclei did not show a decrease even after prolonged pepsin treatment (90 minutes). Therefore this method permits access to stored histopathological material for retrospective DNA-Feulgen-Cytophotometric investigations.     

Demonstration of Epstein-Barr virus in primary brain lymphoma by in situ DNA hybridisation in paraffin wax embedded tissue.

Tumour tissue from 29 patients with primary brain lymphoma was reviewed to determine if there was an aetiological association between Epstein-Barr virus and polyclonal and monoclonal lymphoproliferations. The morphology and immunophenotype in 24 patients for whom paraffin wax embedded tissue was available were studied. A high grade pleomorphic tumour morphology with plasmacytoid features was seen in 13 tumours. Because of the large number of pleomorphic lymphomas, all tumours were examined for the presence of the Epstein-Barr virus genome using in situ DNA hybridisation. A panel of three biotinylated probes to different sequences in the Epstein-Barr virus genome was used. Positive hybridisation with one or more probes was shown in tumours from 11 patients. The remaining tumours gave no hybridisation signal. There was no correlation between positive hybridisation and morphological subtype or clinical outcome.     

Direct multiplex amplification of DNA from a formalin fixed, paraffin wax embedded tissue section.

The extraction of DNA from formalin fixed, paraffin wax embedded tissue can be problematical, with long protocols producing low yields. This report describes a very simple and useful method for amplifying DNA from formalin fixed, paraffin wax embedded tissue without the need for prior DNA extraction. This method allows direct polymerase chain reaction (PCR) based molecular analysis of fixed tissue. It is an invaluable method if clinical biopsy specimens are to be investigated, because extraction of uncontaminated DNA from such small samples can be very difficult or even impossible. It will also facilitate the study of intratumour heterogeneity, with the analysis of multiple small areas from within a single tumour section. In addition, this method can be used for other samples where only a few tests are to be carried out and a stock of DNA is not required, thus shortening the analysis time.     

Identification of Candida species in formalin fixed, paraffin wax embedded oral mucosa by sequencing of ribosomal DNA

Aim—To identify Candida species in formalin fixed, paraffin wax embedded tissue by sequencing candidal rDNA.