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Results for “Paraffin Embedding”

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PubMed1992

An experience of cell sorting using samples isolated from paraffin-embedded tissue of neuroblastoma.

Flow cytometric DNA content analyses were performed on samples from 72 patients with neuroblastoma. Nuclear suspensions obtained from paraffin-embedded samples were prepared using the method described by Schutte et al. Determination of DNA ploidy was possible in 62 out of 72 patients. DNA diploidy was detected in 23 (37.1%) and DNA aneuploidy was found in 39 (62.9%) of these 62 patients. Cell sorting was performed on two peak areas of the DNA histogram of samples with aneuploidy. The sorted cells of the first peak area were composed of small round cells and were considered to be normal lymphocytes. The sorted cells of the second peak area were composed of relatively large cells with obvious nucleoli and were considered to be neuroblastoma cells. Thus, two cell components could be distinguished by cell sorting. It is concluded that cell sorting may be of benefit in giving detailed information about DNA histograms, even in samples obtained from paraffin-embedded tissue.

Cell Separation↗

PubMed1990

Immunophenotyping of cutaneous lymphoid infiltrates in frozen and paraffin-embedded tissue sections: a comparative study.

A panel of antibodies reactive in routinely fixed, paraffin-embedded tissue sections was compared with a panel of antibodies reactive in frozen sections for the immunophenotyping of cutaneous lymphoproliferative disorders. Three T cell-associated markers (UCHL1, MT-1, MT-2, six B cell-associated markers (MB-1, MB-2, LN-1, LN-2, L-26, 4KB5), immunoglobulin heavy and light chains, anti-LCA antibody, two markers for Reed-Sternberg cells (Ber-H2, Leu-M1), one marker for macrophages (Mac-387) and anti-S-100 protein antibody were tested on normal skin, inflammatory skin diseases, and cutaneous lymphomas and pseudolymphomas. On the basis of the results in frozen sections, 12 inflammatory T cell diseases, 14 T cell lymphomas and pseudolymphomas, and 10 B cell lymphomas and pseudolymphomas were identified. In addition, two cases of specific skin infiltrates of Hodgkin's disease have been examined. Among T cell markers, the greatest sensitivity was exhibited by UCHL1, which stained all but one specimen of T cell infiltrate; it was negative in one specimen of mycosis fungoides that progressed into a T-immunoblastic lymphoma. The combined use of MB-2, LN-2, and 4KB5 identified all B cell proliferations. LN-1 marked germinal centers in all cases of follicular lymphoma and pseudolymphoma. Ber-H2 stained the Reed-Sternberg cells in both cases of Hodgkin's disease and the large cells in the histiocytic type of lymphomatoid papulosis. Mac-387 and anti-S-100 protein antibody recognized macrophages and T-zone histiocytes (Langerhans cells and interdigitating cells), respectively. A panel of antibodies reactive in routinely fixed, paraffin-embedded tissue sections is proposed that facilitates the identification of most B and T cell infiltrates in the skin.

Antibodies, Monoclonal↗

PubMed1992

p53 overexpression in formalin-fixed, paraffin-embedded tissue detected by immunohistochemistry.

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.

Antibodies, Monoclonal↗

PubMedDate not supplied

Correlation of P-glycoprotein overexpression and cellular prognostic factors in formalin-fixed, paraffin-embedded tumor samples from breast cancer patients.

BACKGROUND: P-glycoprotein overexpression seems to play a prognostic role independent from its association with multidrug resistance. We have been able to verify this previously on fresh-frozen tumor samples, but up to now P-glycoprotein detection on formalin-fixed, paraffin-embedded material has generally been unsatisfactory. METHODS: Two distinct groups of previously untreated mammary carcinomas were studied for P-glycoprotein expression by means of immunohistochemistry, using the NCL-p-GLYp polyclonal antibody, carried out on slides from archival, paraffin-embedded material. The first group was composed of 15 tumors expressing at least three out of four factors indicative of a worse prognosis: c-erb-B2, mutant p53, cathepsin-D and PCNA (proliferating cell nuclear antigen). The control group was composed of 16 tumors lacking expression of any of these proteins. RESULTS: There was a statistically significant difference in survival (Fisher's exact test, p = 0.0036) between both groups. All four patients who died during the follow-up period (mean 55.1 months) belonged to the first group. Three of these patients harbored tumors with the highest level of expression of P-glycoprotein. This association between P-glycoprotein overexpression and prognosis was also statistically significant (Fisher's exact test, p = 0.0059). CONCLUSION: These results, obtained from a group of mostly operable tumors stratified for cellular risk factors, complement those previously reported by us for locally advanced, inoperable breast cancer only, using fresh-frozen tissue. P-glycoprotein again seems to play a prognostic role, independent of its involvement in multidrug-resistance. The technique used by us yielded useful results on formalin-fixed, paraffin-embedded archival tumor samples. This opens the possibility for larger studies on populations with a closed follow-up.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Immunohistochemical detection of VHS virus in paraffin-embedded specimens of rainbow trout (Oncorhynchus mykiss): the influence of primary antibody, fixative, and antigen unmasking on method sensitivity.

The influence of the primary antibody, the fixative, and the antigen unmasking technique on the method sensitivity of immunohistochemistry as a method for the identification of viral hemorrhagic septicemia (VHS) virus in paraffin-embedded specimens of naturally infected rainbow trout (Oncorhynchus mykiss) was examined. Fish (200-300 g) were collected during an outbreak of VHS. Parallel specimens from liver, spleen, kidney, and brain were fixed by immersion in 10% phosphate-buffered formalin, periodate-lysine-paraformaldehyde (PLP), Bouin's fluid, or absolute ethanol. Virus cultivation was also performed on parallel specimens, and the virus titer (TCID50/ml) was determined. Purified nucleocapsid protein (N-protein) of the virus was incorporated in an artificial antigen substrate polymerized bovine serum albumin), fixed as described above, and embedded in paraffin wax. Microwave unmasking was performed on formalin-, PLP-, and Bouin's fluid-fixed specimens. The presence of virus peptides in situ or N-protein in the artificial antigen substrates was visualized using an immunohistochemical method based on alkaline phosphatase or peroxidase and one polyclonal and five monoclonal polypeptide-specific antibodies. VHS virus was identified in situ in specimens with high virus titers (10(7-8) TCID50/ml) regardless of the fixative and without the need of an unmasking procedure. A pronounced masking effect was observed for the cross-linking formalin and PLP fixatives. Regardless of the primary antibodies used, there was a significantly higher epidemiologic sensitivity (the proportion of virus positive samples that tested positive by immunohistochemistry) using ethanol and Bouin's fluid compared with formalin and PLP (P < 0.05). At 10(5) TCID50/ml, the average sensitivity reached 0.5, and at > or = 10(6) TCID50/ml, sensitivity was 0.9. Unmasking procedures showed a moderate effect and did not result in significantly higher epidemiologic sensitivity (P = 0.17), There was great variation for the different monoclonal antibodies/antigens and fixatives. Sensitivity studies on antigen substrates were in accordance with results of in situ studies that showed the highest sensitivity for ethanol and Bouin's fluid. Virus cultivation was more sensitive than immunohistochemistry. This study showed that the fixative and the primary antibody both influence method sensitivity and that VHS virus antigens concealed during fixation are difficult to reexpose. Immunostaining for VHS virus should be performed with monoclonal antibodies specific for the N-protein, and tissue samples should be fixed in either ethanol or Bouin's fluid. Immunohistochemistry is specific but is less sensitive than virus cultivation. Immunostaining for VHS virus can be a valuable supplement to virus cultivation during acute outbreaks of disease.

Animals↗

PubMed2003

A case of primary esophageal tuberculosis diagnosed by identification of Mycobacteria in paraffin-embedded esophageal biopsy specimens by polymerase chain reaction.

We present a case of primary esophageal tuberculosis diagnosed by polymerase chain reaction (PCR) of paraffin-embedded esophageal biopsy specimens. A 42-year-old Japanese woman visited our clinic because of dysphagia. Radiologic and endoscopic examinations revealed a stenotic lesion with reddish mucosa and multiple ulcers in the middle esophagus. There was no associated lesion outside the esophagus. Histological and bacteriological studies of esophageal biopsy specimens and gastric aspirates did not give a definitive diagnosis. However, mycobacterial DNA was detected by PCR of paraffin-embedded esophageal biopsy specimens. She then was diagnosed as having primary esophageal tuberculosis. The esophageal mucosal lesion almost healed after 1 month of antituberculosis medication with residual annular stenosis which was resolved later by endoscopic balloon dilation.

Adult↗

PubMed1996

Immunoglobulin light chain staining in paraffin-embedded tissue using a heat mediated epitope retrieval method.

We have compared light chain immunohistochemistry in reactive lymphoid tissue and a series of paraffin-embedded B-cell lymphomas using standard trypsin digestion with a heat mediated epitope retrieval method. Fifty-seven B-cell lymphomas (18 high grade, 29 low grade and 10 cases of nodular lymphocyte predominance Hodgkin's disease), two reactive lymph nodes and eight tonsils fixed for known times between 12 h and 2 years were studied. Paraffin-embedded tissue was stained with polyclonal anti-kappa and anti-lambda antibodies. For each antibody staining was performed on two sections, one treated with trypsin digestion and one with microwave heating. Sections were scored from 0 to + + + with 0 representing poor staining and + + + excellent staining. A score of ++ was considered satisfactory. Light chain restriction was recorded if present. Satisfactory staining was obtained in 34/59 cases using trypsin digestion and 56/59 cases using heat retrieval. Light chain restriction was demonstrated in 32/57 (56%) B-cell lymphomas using trypsin digestion and 52/57 (91%) using heat retrieval. Satisfactory staining was obtained in tonsils fixed for up to 48 h using trypsin digestion and up to 2 years using heat retrieval. We have shown that for light chain immunostaining a heat mediated epitope retrieval method produces more consistent and satisfactory results and is effective over a greater range of fixation times than traditional trypsin digestion.

Epitopes↗

PubMed1993

Identification of Toxoplasma gondii in formalin-fixed, paraffin-embedded tissue by polymerase chain reaction.

We investigated the use of polymerase chain reaction (PCR) for diagnosis of toxoplasmosis in formalin-fixed, paraffin-embedded tissue, using seven cases in which the diagnosis was certain based on histologic examination and eight cases in which histologic findings only suggested Toxoplasma gondii infection. After amplification the PCR products were subjected to agarose gel electrophoresis followed by Southern blotting with a 32P-labeled oligonucleotide probe. In six of seven proven cases of toxoplasmosis, organisms were detected using DNA extracted from 1/50 to 3/50 of a 6-microns tissue section. In addition, organisms were detected in one of eight cases in which infection was suspected, but not proven by identification of organisms. We conclude on the basis of this study that PCR may be useful for detection of T. gondii in a few cases in which routine histologic examination or immunoperoxidase staining is not definitive. Since PCR has 1000-fold higher sensitivity when applied to fresh, rather than paraffin-embedded, tissues, we expect PCR to be more sensitive in fresh tissue.

Animals↗

PubMed1993

Detection of immunoglobulin gene rearrangement of B cell non-Hodgkin's lymphomas and leukemias in fresh, unfixed and formalin-fixed, paraffin-embedded tissue by polymerase chain reaction.

BACKGROUND: The majority of B cell nonHodgkin's lymphomas (NHLs) are composed of a genotypically identical cell population characterized by a unique immunoglobulin (Ig) VDJ gene rearrangement which is customarily documented by Southern blot hybridization analysis of fresh tissue. Sometimes, however, this approach cannot be used because of an insufficient quantity of tissue or the unavailability of fresh tissue. Therefore, alternative strategies should be designed in order to overcome these limitations. EXPERIMENTAL DESIGN: One possible alternative is the identification of Ig VDJ products of normal and neoplastic B cells by polymerase chain reaction (PCR) using mixed oligonucleotide primers recognizing the framework III region or Ig variable heavy chain leader sequences and universal Ig heavy chain joining region (JH) oligonucleotide primers. To determine whether the respective DNA samples are suitable for PCR amplification, control and unrelated genes should also be investigated (exon 5 of the p53 gene). In this study, genomic DNA was extracted from a well characterized panel of 139 human B cell lymphoid leukemias and NHLs derived from fresh (84) and/or paraffin-embedded (55) tissue, 19 normal peripheral lymphoid tissues, 9 Epstein-Barr virus infected lymphoblastoid cell lines and, as negative controls, 11 T cell LLs. Clonal Ig gene rearrangement products were assessed for the presence of a distinct PCR fragment after framework III-JH PCR amplification and electrophoretic separation and by DNA sequencing of the cloned PCR-Ig fragments. RESULTS: Eighty-eight of the 139 (63%) B-NHLs consisting of 53/84 (63%) fresh, unfixed and 35/55 (64%) formalin-fixed, paraffin-embedded samples, exhibited distinct PCR bands. Using this approach we were able to identify a single clonal B cell population mixed with 1,000 nonB cells or 5 polyclonal B cells. There was no difference in the detection of monoclonality among different B-NHL categories. PCR fragments were not identified in any of 27 normal lymphoid tissues or 11 T-lymphoid leukemias. To detect a larger number of Ig gene rearrangement products, genomic DNA of 12 B-NHL/lymphoblastoid cell lines were investigated using VH-specific leader and JH oligonucleotides by PCR. A single PCR product was obtained in 9 of 12 (75%) cases and their clonality was documented by DNA sequencing of the cloned PCR fragments. The clonality of 11 of the 12 (92%) cases could be demonstrated using both PCR approaches. CONCLUSIONS: Our results suggest that the monoclonality of human neoplastic B cells can be efficiently evaluated by PCR equally well from fresh, unfixed and formalin-fixed, paraffin-embedded tissues. This technique should prove to be a powerful tool in clinical diagnosis and research as well as in the retrospective analysis of archival pathologic specimens.

Base Sequence↗

PubMed2000

The use of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections for the study of microchimerism.

We describe here a simple and versatile method of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections with specific application in the study of microchimerism, that is, the presence of intact foreign cells within an individual. This is accomplished through the use of X and Y chromosome-specific probes to identify the presence of male nuclei within a tissue section from a female, and vice versa. This technique requires only minor modification if at first the hybridization does not yield fluorescent signals of high quality. Analysis of a wide variety of tissue types is possible with this method, and multiple tissue types from one or more individuals can be processed in the same hybridization reaction. This robust FISH method has been used successfully in our laboratory to investigate fetal cell microchimerism in the following paraffin-embedded tissue types: skin, lung, thyroid, adrenal gland, lymph node, heart, spleen, liver, pancreas, kidney, and intestine.

Cell Nucleus↗

PubMed1998

RNA quantitative analysis from fixed and paraffin-embedded tissues: membrane hybridization and capillary electrophoresis.

Fixed and paraffin-embedded tissues from pathology department archives are available for RNA expression analysis. We describe a general method for quantitation of specific RNA sequence extracted from single 6-8-micron human histological tissue sections cut from paraffin blocks. For each specific mRNA, the range of linear relationship between the log of the initial total RNA concentration and the log of the specific product after reverse transcription (RT)-PCR must be established. We usually perform RT with avian myeloblastosis virus (AMV)-RT, using specific antisense primers and a variable number of cycles of PCR amplification. The number of cycles must be adjusted within the range in which a linear relationship exists between the log of the amount of amplification product and the number of cycles. The quantity of specific product is standardized relative to beta-actin mRNA to normalize for the degree of RNA degradation, which can be quite different among samples. The amplification products were quantified by dot blot and 32P-labeled hybridization probe or by capillary electrophoresis with a laser-induced fluorescence detector. The intratest variation range was for the dot blot mean +/- 10% standard deviation (SD) and for the capillary electrophoresis mean +/- 3% SD.

Actins↗

PubMed1993

Detection of aneuploidy and possible deletion in paraffin-embedded rhabdomyosarcoma cells with FISH.

Conventional cytogenetic studies of solid tumors are limited by the difficulty of culturing tumor cells, while in situ hybridization using paraffin sections of interphase cells results in too many truncated cells. To solve these problems, fluorescent in situ hybridization (FISH) technique was used on free nuclei isolated from formalin-fixed paraffin-embedded embryonal rhabdomyosarcoma (RMS) tissue using our modification of Hedley's method for isolation of nuclei. Biotinylated DNA probes for the centromeric regions of chromosomes 6, 8, 11, 12, 17, and 18, painting probes for chromosomes 8 and 11, and a cosmid probe for the HER-2/neu oncogene, were used. The centromeric probes worked well, demonstrating two copies of chromosomes 6, 17, and 18, but three copies of chromosome 11 in 52.9% of nuclei. Four copies of chromosome 8 were observed in 57.1% of nuclei and five or more in 17.1%. Chromosome 12 demonstrated 21.8% trisomy and 62.2% tetrasomy. Painting probes for chromosome 11 also worked well and matched the results of the centromeric probes, with no suggestion of structural aberration. However, the results of the painting probe for chromosome 8 yielded fluorescent areas of different sizes, suggesting that some of the extra chromosomes 8 could be deleted. The cosmid probe for the HER-2/neu oncogene also worked well, and revealed two signals in each nucleus without evidence of amplification. This study illustrates the successful use of a new technique for studying chromosomal aberration in paraffin-embedded solid tumors. The importance of this technique is that it has not been previously possible to use painting probes or cosmid probes on paraffin tissue sections. Use of this procedure will broaden the type of retrospective studies that can be performed to include detection of deletions or translocations.

Aged↗

PubMed1997

Sample preparation from paraffin-embedded tissue specimens for laser scanning cytometric DNA analysis.

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.

Aneuploidy↗

PubMed2005

Tissue microarrays from HOPE-fixed specimens allow for enhanced high throughput molecular analyses in paraffin-embedded material.

In this study, we describe a technique that allows for the production and the use of tissue microarrays (TMA) from HOPE-fixed, paraffin-embedded specimens. The combination of these two technologies unites the advantages of the high throughput aspects in TMA, with good preservation of nucleic acids, proteins, and morphology of HOPE-fixed tissues, thus substantially widening the capabilities presently available for molecular studies in paraffin-embedded tissues.

Biomarkers↗

PubMedDate not supplied

Prognostic value of flow cytometric DNA analysis in non-small-cell lung cancer: rationale of sequential processing of frozen and paraffin-embedded tissue.

The objective of this study was to determine the prognostic information provided by flow cytometric DNA analysis in non-small-cell lung cancer. Lung samples of 132 consecutive patients submitted to surgery were prospectively processed. When no aneuploid populations were detected in fresh frozen samples, the process continued as a second step in paraffin-embedded tissue, consuming all the tumor available. The influence of ploidy on the postoperative outcome was studied by both a univariate and a multivariate analysis. Aneuploidy was found in 81 patients (61.4%). Fourteen patients showed no aneuploidy in fresh frozen samples; and only after further analysis in paraffin-embedded tissue was abnormal DNA detected. Overall, the 36-month survival was 69% for the diploid group and 24% for the aneuploid group (p = 0.0006). Including subjects submitted to complete tumor removal (stages I, II, and IIIA) in a multivariate analysis adjusted for TNM stage and histologic type, bearers of aneuploid tumors exhibited a higher risk of relapse (hazard ratio 2.65; CI 95% 1.5-4.66;p = 0.004) or death (hazard ratio 2.17; CI 95% 1.08-4.39;p = 0.032) than patients with diploid tumors. DNA ploidy resulted an independent prognostic factor of survival and tumor relapse in completely resected non-small-cell lung cancer. Sequential analysis of fresh and paraffin-embedded samples can help avoid the bias due to intratumoral DNA content heterogeneity. DNA ploidy could be an useful parameter in any future multifactorial analysis of outcome in such tumors.

Aneuploidy↗

PubMed1993

Loss of heterozygosity detected in formalin-fixed, paraffin-embedded tissue of colorectal carcinoma using a microsatellite located within the deleted in colorectal carcinoma gene.

We determined loss of heterozygosity from formalin-fixed, paraffin-embedded tissue of colorectal carcinoma using microsatellite polymorphism. The polymorphism was assayed based on DNA amplification by the polymerase chain reaction (PCR). The PCR-analyzed microsatellite method was applied to assay degraded DNA extracted from paraffin-embedded blocks with adenocarcinoma of colon. The DNA from 26 tumors as well as their corresponding normal tissue samples were successfully amplified using a dinucleotide microsatellite located within an intron of the deleted in colorectal carcinoma gene. Allele losses on this marker were detected in 33% of informative colorectal carcinomas. This study demonstrates that microsatellites provide a powerful set of DNA markers for loss of heterozygosity on archival specimens.

Adenocarcinoma↗

PubMed2004

[A study on the method of DNA extraction from unbuffered formalin-fixed and paraffin-embedded samples].

BACKGROUND & OBJECTIVE: Unbuffered formalin is widely used to fix resected specimens in China. The DNA in unbuffered formalin-fixed and paraffin-embedded tissues is usually degraded seriously, so the extraction of DNA from these samples is difficult. This study was conducted to seek an optimal method to extract DNA from these samples. METHODS: Fifteen blocks of esophageal carcinoma resected in Fourth Hospital of Hebei Medical University in 2000 were selected. The cells were lyzed by proteinase K digestion or heating under different pH values, then DNA was extracted by phenol:chloroform. After that, four parameters (deparaffined by xylene or histolene; digested for 48 h or 72 h at 37 degrees C or 56 degrees C; extracted by salting-out or phenol:chloroform) were optimized according to the principle of cross design. At last, the quality of obtained DNA was analyzed with electrophoresis and PCR amplification. RESULTS: The quality and quantity of DNA obtained by proteinase K digestion (the average yield is 17.88 microg) were better than that of heating under different pH (7-12)(P< 0.05). The quality and quantity of DNA digested at 56 degrees C were better than that at 37 degrees C, and similarly, digestion for 72 hours was better than that for 48 hours. The methods of deparaffin and extraction had no obvious influence on the quality and quantity of DNA. CONCLUSION: By means of NaCl salting-out after proteinase K digestion, more reliable quality of DNA can be obtained from unbuffered formalin-fixed and paraffin-embedded samples. Furthermore,digestion for three days at 56 degrees C is more likely to obtain DNA with high quality and quantity.

DNA↗

PubMed1999

Detection of PAX3-FKHR and PAX7-FKHR fusion transcripts in rhabdomyosarcoma by reverse transcriptase-polymerase chain reaction using paraffin-embedded tissue.

BACKGROUND: Alveolar rhabdomyosarcoma (RMS) is associated with a characteristic chromosomal translocation t(2;13)(q35;q14). The genes involved in this translocation are paired box (PAX)3 on chromosome 2 and forkhead in RMS (FKHR) on chromosome 13. An occasional variant translocation t(1;13)(p36;q14) affecting PAX7 and FKHR on chromosomes 1 and 13, respectively, has also been described. Chromosomal translocations in RMS are detected using conventional cytogenetic analysis, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) on fresh or frozen tissue samples. We describe the results of RT-PCR analysis of PAX3-FKHR and PAX7-FKHR chimeric messages in formalin-fixed, paraffin-embedded tissue samples from 17 RMS cases. METHODS: RNA was extracted from formalin-fixed, paraffin-embedded RMS tissue. Oligonucleotide primers corresponding to the regions of PAX3, PAX7 and FKHR were used for the detection of PAX3-FKHR and PAX7-FKHR chimeric messages. A seminested PCR of the PCR products was used to increase the sensitivity of detection. The amplified fragments were purified and directly sequenced to confirm the specificity of the methods. RESULTS: The PAX3-FKHR chimeric message was detected in all three cases of alveolar RMS but not in any of the 12 embryonal and two pleomorphic RMS cases. The PAX7-FKHR fusion transcript was detected in one case of embryonal RMS. CONCLUSIONS: The results indicate that the RT-PCR assay is a reliable method for the detection of the PAX3-FKHR fusion transcript of alveolar RMS in formalin-fixed, paraffin-embedded tissue. This simple method enables pathologists to identify chromosomal rearrangements in RMS as a diagnostic aid in cases where fresh or frozen tissue is not available.

DNA-Binding Proteins↗